Design and synthesis of 7-alkoxy-4-heteroarylamino-3-quinolinecarbonitriles as dual inhibitors of c-Src kinase and nitric oxide synthase

Article abstract of DOI:10.1016/j.bmc.2008.04.065

Because both c-Src and iNOS are key regulatory enzymes in tumorigenesis, a new series of 4-heteroarylamino-3-quinolinecarbonitriles as potent dual inhibitors of both enzymes were designed, prepared, and evaluated for blocking multiple signaling pathways i

Full text of DOI:10.1016/j.bmc.2008.04.065

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5890–5898
Design and synthesis of 7-alkoxy-4-heteroarylamino-3-
quinolinecarbonitriles as dual inhibitors of c-Src
kinase and nitric oxide synthase
Xin Cao,a Qi-Dong You,a,* Zhi-Yu Li,a Qing-Long Guo,a Jing Shang,b
Ming Yan,b Ji-Wang Chernc and Men-Ling Chenc
aJiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009, China
bNational Drug Screening Center, China Pharmaceutical University, Nanjing 210009, China
cSchool of Pharmacy, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan
Received 7 March 2008; revised 24 April 2008; accepted 25 April 2008
Available online 29 April 2008
Abstract—Because both c-Src and iNOS are key regulatory enzymes in tumorigenesis, a new series of 4-heteroarylamino-3-quino-
linecarbonitriles as potent dual inhibitors of both enzymes were designed, prepared, and evaluated for blocking multiple signaling
pathways in cancer therapy. All compounds were evaluated by two related enzyme inhibition assays and an anti-proliferation assay
in vitro. The results showed that most compounds could inhibit both enzymes, and several of them showed potent inhibition activity
against different cancer cell lines. The best compound 20 (CPU-Y020) showed the IC50 values of 6.58 and 7.61 lM toward colon
cancer HT-29 and liver cancer HepG2 cell lines.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
The Src family of protein tyrosine kinases (SFKs) plays
key roles in regulating signal transduction, including cell
growth, differentiation, cell shape, migration, and sur-
vival, and specialized cell signals.1 However, c-Src was
also identified as a proto-oncogene based on decades
of research on an avian RNA tumor (sarcoma) virus.
In some abnormal cases, such as mutation of the c-Src
or over-expression, these enzymes can become hyper-
activated, resulting in uncontrolled cell proliferation.2
Inhibition of Src kinase activity to block uncontrolled cell
growth has potential therapeutic implications in develop-
ing cancer treatments. This research has achieved major
progress in this regard, and several quinoline/quinazoline
compounds were identified as potent and selective inhib-
itors of PTKs. For example, gefitinib (Iressa, ZD-1839,
Astra Zeneca) was launched in 2003, and bosutinib
(SKI-606, Wyeth), a dual inhibitor of Src and Abl kinases
Figure 1. The structures of Iressa (ZD-1839) and Bosutinib (SKI-606).
based on a 3-quinolinecarbonitrile skeleton, is currently
in phase II clinical trials (Fig. 1).3–5
iNOS is an inducible isoform of nitric oxide synthase
(NOS), expressed in a wide range of cells and tissues
such as macrophages,6 Kupffer cells, colonic epithelium,
vasculature,7 and various neoplastic tissues.8 iNOS is
regulated by a variety of factors including bacterial
lipopolysaccharides (LPS), cytokines, and signal trans-
Keywords: Src; iNOS; Dual inhibitors; Anticancer.
*
Corresponding author. Tel./fax: +86 25 83271351; e-mail:
youqidong@gmail.com
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.065

X. Cao et al. / Bioorg. Med. Chem. 16 (2008) 5890–5898
5891
duction enzymes. iNOS highly expresses in various neo-
2. Chemistry
plastic diseases. The role of iNOS during tumor develop-
ment is highly complex, and incompletely understood.
But there is no doubt that the malignant transformation,
angiogenesis, and metastasis effects of tumors are all
modulated by iNOS.7
The synthetic pathways used to prepare this series of 7-
alkoxy-4-heteroarylamino-3-quinolinecarbonitrile com-
pounds are shown in Schemes 1 and 2. The hydroxyl
groups of 2-methyl-5-nitrophenol (1a) and 5-nitroguaia-
col (1b) needed to be protected during the quinoline syn-
thesis, and isopropyl was chosen as the protective group
for its good thermal stability in a high temperature quin-
olones cycling reaction and its convenient deprotection
method.
To avoid complications resulting from inhibition of nor-
mal physiological NO production and to dissect the nat-
ural pharmacological effects, compounds are needed
that selectively inhibit iNOS. Fortunately, many NOS
inhibitors, such as 2-aminothiazole and 2-aminopyridine
derivatives (Fig. 2), have been reported9,10 to exhibit po-
tent inhibition with variable selectivity for iNOS and its
isoforms.
Following the designed methods (Scheme 1),11,12 the 3-
cyano-2-hydro-quinolin-4 (1H)-ones (5a, 5b) were read-
ily made. The core quinolones were aromatized in
refluxing POCl3 and the key intermediate 3-cyano-4-
chloro-quinolines (6a, 6b) were produced.
The attempt to suppress two distinct cancer-related en-
zymes with a single agent was an attractive idea. It is
reasonable that the dual inhibitors of both Src and
iNOS were more effective than single inhibitors as block-
ing multiple signaling pathways in cancer therapy and
could be beneficial to overcome drug resistance. With
the goal of identifying such a new anti-cancer strategy,
a new class of dual inhibitors of both enzymes were de-
signed (Fig. 3). Using the potent Src inhibitor template
4-aniline-3-quinolinecarbonitrile as the leading skeleton,
the existing 4-anilines were replaced with specialized side
groups, such as 2-aminothiazole or 2-aminopyridine
derivatives, that have selective inhibition against iNOS.
As a result, a new series of 7-alkoxy-4-heteroarylamino-
3-quinolinecarbonitriles was designed.
The different 4-heteroarylaminos were brought into the
skeleton using NaH in anhydrous DMF. Then the 7-iso-
propyl of the intermediates was deprotected by HBr in
an acetic acid solution, yielding the 4-heteroarylamino-
7-hydroxyquinolines (10, 11a–d, and 12a–c) (Scheme
2). It was reported4 that a morpholinoalkoxy group
Scheme 1. 1a–6a: R1 = Me, 1b–6b: R1 = OMe. Reagents and condi-
tions: (a) 2-bromopropane, NaOH, DMF; (b) Fe, HOAc, MeOH; (c)
toluene, reflux; (d) liquid paraffin, 260 °C; (e) POCl3, reflux.
Figure 2. Some selective iNOS inhibitors.
Figure 3. The design of the dual inhibitors of Src and iNOS.

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X. Cao et al. / Bioorg. Med. Chem. 16 (2008) 5890–5898
Scheme 2. Reagents and conditions: (a) heteroaromatic amines, NaH, anhydrous DMF; (b) HBr, HOAc, 120 °C; (c) 1-bromo-3-chloropropane or 1-
bromo-2-chloroethane, K2CO3, DMF; (d) secondary amine (piperazine, piperidine, or morpholine, etc), K2CO3, DMF.
substituted on the 7-position of 4-anilinoquinolines can
greatly increase potency against Src kinase; therefore we
incorporated morpholinoalkoxy and its bioisosteric
groups at the 7-position and the target molecules were
achieved (Table 1).
3. Results and discussion
Compounds 16 to 28 were synthesized smoothly, and
their water solubilities were improved prominently com-
pared with their intermediates. In order to evaluate the
Table 1. The structures of compounds 16–28 and their enzyme inhibition activities against c-Src and iNOSa
No.
R1
n
R2
Ar
c-Src, IC50 (nM)
iNOS, IC50 (lM)a,b
16
17
Me
Me
3
3
3
3
3
3
3
3
3
2
2
2
3
N-Me-piperazine
Morpholine
Piperidine
2-Pyridine
2-Benzothiazole
2-Benzothiazole
75.9
172
209
646
15.4
111
>1000
186
178
371
>1000
9.23
133
1.2
18.6
139
412
12.7
313
16.3
15.5
98
18
19
Me
Me
N-Me-piperazine
Piperidine
2-(6-Me-benzothiazole)
2-(6-Me-benzothiazole)
2-(6-TFMeO-benzothiazole)
2-Benzimidazole
20
21
Me
Me
N-Me-piperazine
N-Me-piperazine
N-Me-piperazine
N-Me-piperazine
Piperidine
22
23
Me
OMe
OMe
OMe
OMe
OMe
OMe
2-Benzothiazole
24
25
2-(6-TFMeO-benzothiazole)
2-Benzothiazole
2-Benzothiazole
65.6
44.8
26.7
2.18
18.1
—
26
27
Morpholine
N-Me-piperazine
N-Me-piperazine
2-Benzothiazole
2-Benzimidazole
28
Bosutinib
L-Canavanine
2-Aminothiazole
—
—
60
18
a The two enzyme inhibition assay procedures are listed in the Supplementary Material. The IC50 values were means of triplet experiments, and the
variabilities were within 10%.
b iNOS of mouse macrophage ANA-1.

X. Cao et al. / Bioorg. Med. Chem. 16 (2008) 5890–5898
5893
Src kinase inhibition activities, bosutinib was also syn-
thesized as the reference molecule according to the re-
ported method. Besides, the commercially available
compounds L-canavanine and 2-aminothiazole were
chosen as the positive iNOS inhibitors. Then the target
molecules described above were tested in two related en-
zyme assays: a PTK inhibition assay and an iNOS inhi-
bition assay (see Supplementary Material). The IC50
values are shown in Table 1.
pound 21’s activity against Src improved to 111 nM
compared with that of the corresponding 6-methoxy
compound 24, while compound 19’s activity was about
5-times weaker than that of 21. When there was a benz-
imidazole-2-amine at C-4 position, the Src inhibition
activity of compound 22 was lost. This may imply that
the benzimidazole-2-amine was not suitable for C-4 po-
sition. In the meanwhile, their inhibition activities
against iNOS kept relatively stabilized between 10 and
20 lM. Interestingly, compound 19 showed the best
activity with an IC50 value of 12.7 lM and was 1.5-times
more potent than that of 2-amniothiazole.
The data in Table 1 clearly showed that most com-
pounds exhibited moderate inhibition activities toward
both Src and iNOS. In general, the 4-heteroarylamino
groups substituted compounds including 4-(benzothia-
zole-2-amine), 4-(6-methylbenzothiazole-2-amine), and
4-(pyridine-2-amine) as the 4-anilino headpieces showed
to be active for Src kinase, though their IC50 values
against Src were weaker than bosutinib. For iNOS, the
inhibition activities of the target molecules were rela-
tively weak compared with the inhibition activities
against Src kinase, but some of the compounds were
more potent than the inhibition activities of L-canavan-
ine and 2-aminothaizole. Several compounds inhibited
both enzymes well. Compound 27 showed the best inhi-
bition activities with the IC50 values of 9.23 nM and
2.18 lM toward Src and iNOS, respectively. Some of
the compounds exhibited good activity against only
one enzyme. For example, compounds 16, 19, and 21
inhibited iNOS well with the IC50 values of 18.6, 12.7,
and 16.3 lM, but their Src inhibition IC50 values were
only 75.9, 646, and 111 nM, which were much lower
than that of 27. The opposite situation was found in
compound 20 with the IC50 values of 15.4 nM and
313 lM against Src and iNOS, respectively.
Further investigation of the C-7 alkaline side chains was
carried out with 4-(benzothiazole-2-amine) substituted
compound 23. When n changed from 3 to 2, the new
compound 27’s inhibition activity against Src increased
about 20-times (IC50 = 9.23 nM), and the inhibition
activity against iNOS also increased about 40-times
(IC50 = 2.18 lM) compared with compound 23. But
unfortunately, when R2 of 27 was changed to piperidine
or morpholine, the new compounds 25 or 26’s inhibition
activities against both enzymes decreased a lot. Their Src
inhibition activities were even weaker than that of 23.
The iNOS inhibition activities of 25 and 26 were those
between L-canavanine and 2-aminothiazole.
The changes of 7-alkaline groups were also carried out
with their 6-methyl analogs. [Both the piperidine and
morpholine substituted 4-(benzothiazole-2-amine) com-
pounds 17 and 18 showed weak inhibition activities to-
ward Src and iNOS.] But when changed the N-
methylpiperazine of 19 to piperidine, the new compound
20 showed more potent activity against Src kinase with
an IC50 value of 15.4 nM while its iNOS inhibition activ-
ity deceased a lot.
With respect to different substituted groups, including
R1, R2, n, and several 4-heteroarylaminos, the last one
played the most important role in the different IC50
trends against the two enzymes. As shown in Table 1,
when the C-6 position was a methoxyl group, n was 3
and the alkaline group was N-methylpiperazine, all their
substituents except the 4-heteroarylamino headpieces of
compounds 23, 24, and 28 were the same with bosutinib.
Their Src inhibition activities decreased much compared
with the reference compound, and all their IC50 values
were less potent than 100 nM. As for the inhibition of
iNOS, compound 23 and 24’s activities were also not
predominant with the IC50 values of 98 and 65.6 lM, re-
spectively, and were weaker than the selective iNOS in-
hibitor L-canavanine (IC50 = 60 lM). Compound 28
with a 4-(benzimidazole-2-amine) headpiece showed bet-
ter inhibition activity against iNOS with an IC50 of
18.1 lM, and the inhibition activity was similar to 2-
aminothiazole (IC50 = 18 lM).
In conclusion, some of the 4-heteroarylamino groups
showed to be active for Src kinase, especially com-
pounds 20 and 27 with the 4-(benzothiazole-2-amine)
and 4-(6-methylbenzothiazole-2-amine) groups showed
remarkable IC50 against Src kinase. This may suggest
that the hydrophobic pocket at the kinase binding site
may accommodate larger groups than the traditional
single ring arylamines. Although ATP did not directly
interact with the residues around this pocket, it was
important for inhibitors to bind compactly at the ATP
binding site.
For iNOS, the inhibition by the target molecules was rel-
atively weak. Guanidine moiety was a very crucial phar-
macophore for iNOS inhibition. Though 2-
aminobenzimidazole or 2-aminobenzthiazole derivatives
included the guanidine moiety or its bioisosteres, the 2-
amino group was linked to the quinoline C-4 position
directly and the other N-1, NH-3, or S-1, NH-3 atoms
were fixed by the constrained rings. The guanidine phar-
macophore in the designed compounds could not inter-
act with iNOS as the free ones. This may be the reason
for the weak inhibition of iNOS.
Different 4-heteroarylamino groups were also tested
with 6-methyl substituted compounds. When n was 3
and the alkaline group was N-methylpiperazine, com-
pounds 16, 19, 21, and 22 showed various activities to-
ward the target enzymes. Compound 16 with a
pyridine-2-amine substituent at the C-4 position exhib-
ited moderate inhibition activity toward both enzymes
with the IC50 values of 75.9 nM and 18.6 lM. Com-
To further investigate the anti-cancer activities of the
target molecules, an anti-proliferation SRB (Sulforho-

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X. Cao et al. / Bioorg. Med. Chem. 16 (2008) 5890–5898
Table 2. Anti-proliferation effects of compound 20 (CPU-Y020)
compared with bosutinib
5. Experimental
Assay parametersa (lM)
Compound 20
Liver, Colon, Liver,
HepG2 HT-29 HepG2 HT-29
Bosutinib
5.1. Chemistry
Colon,
All purchased starting materials and reagents were used
without further purification unless noted. 1H NMR
spectra were recorded on an ACF-300 Bruker, or an
ACF-500 Bruker instrument. Chemical shifts are ex-
pressed in parts per million (ppm, d units). Coupling
constants are in units of hertz (Hz). Splitting patterns
describe apparent multiplicities and are designated as s
(singlet), d (doublet), t (triplet), q (quartet), m (multi-
plet), or br s (broad singlet). Mass spectra were recorded
on a Shimadzu GC–MS 2010 (EI) or a Mariner Mass
Spectrum (ESI). HRMS spectra were recorded on a Agi-
lent HPLC–ESI/TOF. FT IR spectra were collected with
a Nicolet Impact 410 instrument (KBr pellet). The
HPLC spectra were recorded on a Jasco HPLC system.
Uncorrected melting points were determined in open
capillary tubes on a Mel-TEMP II melting point appara-
tus. TLCs and preparative thin-layer chromatography
were performed on silica gel GF/UV 254, and the chro-
matograms were performed on silica gel (200–300 mesh)
visualized under UV light at 254 and 365 nm. Most of
the reactions were carried out under an inert atmosphere
of nitrogen. The reported yields were for purified mate-
rials but were not optimized.
GI50
IC50
TGI
LC50
4.85
7.61
9.25
>10
5.59
6.58
>10
>10
1.15
3.91
7.66
>10
1.17.
3.87b
6.50
>10
a The anti-proliferation assay procedure is listed in the Supplementary
Material.
b The reported IC50 value in Ref. 5 is 1.5 lM.
damine B) assay was also carried out against five tumor
cell lines (lung cancer A549, stomach cancer AGS, liver
cancer HepG2, colon cancer HT-29, and prostate cancer
PC-3).13 According to the anticancer assay results, com-
pound 20 (CPU-Y020) exhibited good inhibition activi-
ties toward HepG2 and HT-29 cell lines with the IC50
values of 7.61 and 6.58 lM, and was the most potent
compound found (Table 2). Compounds 16, 24, 27,
and 28 also showed some anti-proliferation activities
in the preliminary screening, but their IC50 values were
all less potent than 10 lM.
Compound 20 exhibited the best anti-proliferation activ-
ity against several tumor cell lines. Specifically, it had an
IC50 value of 6.58 lM against colon cancer HT-29 cell
line, and this was similar to the anti-proliferative activity
of bosutinib with an IC50 value of 3.87 lM.5 The similar
result was also obtained against liver cancer Hep2 cell
line with an IC50 value of 7.61 lM compared with that
of bosutinib’s 3.91 lM.
Compounds 5b and 6b were previously reported in a dif-
ferent route.12 The synthesis of compounds 2a–6a and
2b–6b described in Scheme 1 are listed in the Supplemen-
tary Material.
5.1.1. 7-Isopropoxy-6-methyl-4-(pyridin-2-ylamino)-quin-
oline-3-carbonitrile (7). To a suspension of NaH (60% in
mineral oil, 100 mg, 2.5 mmol) in 15 ml of anhydrous
DMF, 2-aminopyridine (235 mg, 2.5 mmol) was added.
The mixture was heated to reflux for 10 min, then cooled
and 6a (460 mg, 2.0 mmol) was added. The mixture was
heated to reflux for 2 h then cooled to room temperature
and partitioned between water and ethyl acetate. The or-
ganic layer was washed with dilute aqueous ammonium
hydroxide, dried over MgSO4, filtered, and concentrated
in vacuum. Ethyl acetate and diethyl ether were added
to the residue and the solid was collected to provide
245 mg (42% yield) of 7 as a tan solid. Mp 243 °C; EI-
Compound 20 showed good inhibition activity toward
Src, with an IC50 value of 15.4 nM, but it did not show
good inhibition activity toward iNOS with an IC50 value
of 313 lM. This may imply that the inhibition mecha-
nism of compound 20 was mainly dominated by the
inhibition of Src kinase. Other factors besides enzyme
inhibitions, such as different degrees of cell penetration
and different polarization of the compounds, may also
play key roles in retarding the proliferation of tumor cell
lines, and this encouraging result might be expected.
Further evaluation of compound 20’s anticancer mecha-
nism was on the way.
1
MS (m/z): 290.1 (M+); H NMR (CD3OD, 500 MH) d
1.32–1.34 (d, J = 6 Hz, 6H), 2.41 (s, 3H), 4.60 (m, 1H),
7.24 (t, J = 7 Hz, 1H), 7.36 (s, 1H), 7.74 (d, J = 9 Hz,
1H), 7.94 (m, 1H), 8.61 (s, 1H), 9.01 (d, J = 7 Hz, 1H),
9.36 (s, 1H).
4. Conclusions
In this study, a series of new dual inhibitors of Src
kinase and iNOS as novel anticancer agents were pro-
posed. The synthesis of 6, 7-disubstituted-4-heteroaryla-
mino-quinoline-3-carbonitriles and their anticancer
activities were also described. Parts of these compounds
were effective against both enzymes, and they repre-
sented the culmination of our earlier efforts in designing
such dual inhibitors. One member of this series, com-
pound 20 (CPU-Y020), was the most promising com-
pound and was selected for additional studies to
determine its potential in the treatment of cancer.
5.1.2.
7-Isopropoxy-6-methyl-4-(benzothiazol-2-ylami-
no)-quinoline-3-carbonitrile (8a). To a suspension of
NaH (60% in mineral oil, 100 mg, 2.5 mmol) in 15 ml
of anhydrous DMF, 2-aminobenzothiazole (375 mg,
2.5 mmol) was added. The mixture was heated to reflux
for 15 min, then cooled and 6a (520 mg, 2.0 mmol) was
added. The mixture was heated at reflux for 3 h then
cooled to room temperature and partitioned between
water and ethyl acetate. The organic layer was washed
with dilute aqueous ammonium hydroxide, dried over
MgSO4, filtered and concentrated in vacuum. Ethyl ace-

X. Cao et al. / Bioorg. Med. Chem. 16 (2008) 5890–5898
5895
1
tate and diethyl ether were added to the residue and the
solid was collected to provide 345 mg (46%) of 8a as a
tan solid. Mp 234 °C; EI-MS (m/z): 374.1 (M+); 1H
NMR (DMSO-d6, 500 MHz) d 1.39–1.41 (d, J = 6 Hz,
6H), 1.41 (s, 3H), 2.37 (s, 3H), 4.90 (m, 1H), 7.41 (s,
1H), 7.53 (m, 2H), 7.99 (d, J = 8 Hz, 1H), 8.42 (s, 1H),
9.48 (d, J = 8 Hz, 1H), 9.59 (s, 1H), 10.00 (s, 1H).
(m/z): 276.1 (M+); H NMR (DMSO-d6, 500 MHz) d
2.33 (s, 3H), 7.25 (t, J = 7 Hz, 1H), 7.37 (s, 1H), 7.75 (d,
J = 9 Hz, 1H), 7.96 (m, 1H), 8.60 (s, 1H), 9.01 (d,
J = 7 Hz, 1H), 9.36 (s, 1H), 11.30 (s, 1H).
5.1.8. 7-Hydroxy-6-methyl-4-(benzothiazol-2-ylamino)-
quinoline-3-carbonitrile (11a). To a solution of acetic acid
(20 ml) and aqueous hydrobromic acid (47% yield, 40 ml)
heated at 120 °C was added 8a (300 mg, 0.80 mmol). Then
the mixture was heated at reflux for 12 h. After cooling,
the mixture was diluted with cold water (100 ml) and ex-
tracted with ethyl acetate. The organic layer was washed
with water and brine, dried over MgSO4, and evaporated.
The residue was purified by silica gel column to give
180 mg of product (68% yield). Mp 234 °C; EI-MS (m/
z): 332.1 (M+);1H NMR (DMSO-d6, 500 MHz) d 2.39
(s, 3H), 7.39 (s, 1H), 7.52 (m, 2H), 7.95 (d, J = 8 Hz,
1H), 8.41 (s, 1H), 9.48 (d, J = 8 Hz, 1H), 9.56 (s, 1H),
11.50 (s, 1H).
5.1.3. 7-Isopropoxy-6-methyl-4-(6-methylbenzothiazol-2-
ylamino)-quinoline-3-carbonitrile (8b). The compound
was prepared with a 65% yield according to the method
for 8a using 6a and 6-methyl-2-aminobenzothiazole. Mp
1
246 °C; EI-MS (m/z): 390.1 (M+); H NMR (DMSO-
d6,500 MHz) d 1.39–1.41 (d, J = 6 Hz, 6H), 2.31 (s,
3H), 2.43 (s, 3H), 4.89 (m, 1H), 7.39 (m, 2H), 7.81 (s,
1H), 8.42 (s, 1H), 9.32 (d, J = 9 Hz, 1H), 9.60 (s, 1H),
9.98 (br s, 1H).
5.1.4. 7-Isopropoxy-6-methyl-4-(6-trifluoromethoxybenzo-
thiazol-2-ylamino)-quinoline-3-carbonitrile (8c). The com-
pound was prepared with a 45% yield according to the
method for 8a using 6a and 6-trifluoromethoxy-2-amino-
benzothiazole. Mp 254–256 °C; EI-MS (m/z): 458.1
5.1.9. 7-Hydroxy-6-methyl-4-(6-methylbenzothiazol-2-
ylamino)-quinoline-3-carbonitrile (11b). The compound
was prepared with a 58% yield according to the method
for 11a using 8b. Mp 245 °C; EI-MS (m/z): 346.2 (M+);1H
NMR (DMSO-d6, 500 MHz) d 2.31 (s, 3H), 2.42 (s, 3H),
7.38 (m, 2H), 7.80 (s, 1H), 8.41 (s, 1H), 9.30 (d, J = 9 Hz,
1H), 9.60 (s, 1H), 9.99 (s, 1H), 11.80 (s, 1H).
(M+);1H NMR (CHCl3,300 MHz)
d 1.39–1.42 (d,
J = 6 Hz, 6H), 2.37 (s, 3H), 4.88–4.92 (m, 1H), 7.42 (s,
1H), 7.56–7.59 (d, J = 8.4 Hz, 1H), 8.17 (s, 1H), 8.41 (s,
1H), 9.55–9.58 (d, J = 9.6 Hz, 1H), 9.61 (s, 1H), 10.01 (s,
1H).
5.1.10. 7-Hydroxy-6-methoxy-4-(benzothiazol-2-ylami-
no)-quinoline-3-carbonitrile (12a). A 390 mg (1.0 mmol)
quantity of 9a in 30% HBr in acetic acid (80 ml) was stir-
red at reflux for 12 h. After cooling, the mixture was di-
luted with cold water (100 ml) and extracted with ethyl
acetate. The organic layer was washed with water and
brine, dried over MgSO4, and evaporated. The residue
was purified by silica gel column to give 260 mg of prod-
uct (75% yield). Mp 238 °C (dec); EI-MS (m/z): 348.1
5.1.5. 7-Isopropoxy-6-methyl-4-(benzimidazole-2-ylami-
no)-quinoline-3-carbonitrile (8d). The compound was
prepared with a 34% yield according to the method for
8a using 6a and 2-aminobenzimidazole. Mp 223–
224 °C; EI-MS (m/z): 357.1 (M+);1H NMR (DMSO-d6,
300 MHz) d 1.31–1.33 (d, J = 6 Hz, 6H), 2.30 (s, 3H),
4.97–5.01 (m, 1H), 7.15–7.20 (m, 1H), 7.34–7.36 (m,
1H), 7.58 (s, 1H), 7.63–7.69 (m, 3H), 7.84–7.87 (d,
J = 8.1 Hz, 1H), 8.15 (s, 1H), 8.45 (s, 1H), 9.52 (s, 1H).
1
(M+); H NMR (DMSO-d6, 500 MHz) d 4.00 (s, 3H),
7.43 (s, 1H), 7.65 (m, 2H), 8.05 (s, 1H), 8.15 (d,
J = 8 Hz, 1H), 9.05 (d, J = 8 Hz, 1H), 9.35 (s, 1H),
10.52 (s, 1H).
5.1.6. 7-Isopropoxy-6-methoxy-4-(benzothiazol-2-ylami-
no)-quinoline-3-carbonitrile (9a). The compound was
prepared in 64% yield according to the method for 8a
using 6b and 2-aminobenzothiazol. Mp 234 °C; EI-MS
(m/z): 392.2 (M+);1H NMR (DMSO-d6, 500 MHz) d
1.43–1.45 (d, J = 6 Hz, 3H), 3.99 (s, 3H), 4.72 (m, 1H),
7.40 (s, 1H), 7.63 (m, 2H), 8.01 (s, 1H), 8.12 (d,
J = 8 Hz, 1H), 9.03 (d, J = 8 Hz, 1H), 9.32 (s, 1H).
[Similar procedures for preparing 11c, 11d, 12b, and 12c
were carried out following the preparation methods of
intermediates 11a, 11b, and 12a using 8c, 8d, 9b, and
9c as the stationary materials.]
5.1.11. 7-(3-Chloropropoxy)-6-methyl-4-(pyridin-2-ylami-
no)-quinoline-3-carbonitrile (13). A mixture of 10 (100 mg,
0.36 mmol), 1-bromo-3-chloropropane (150 ll, 1.5
mmol), and potassium carbonate (210 mg, 1.5 mmol) in
DMF (10 ml) was heated at 40 °C for 3 h. The mixture
was cooled, diluted with water, and extracted with ethyl
acetate. The organic extracts were combined, washed with
water and brine, and dried with MgSO4, and the volatiles
were removed by evaporation. The residue was chromato-
graphed on silica gel with MeOH–EtOAc to give 110 mg
(87% yield) of product. Mp212–214 °C (dec); EI-MS (m/
z): 352.2 (M+);1H NMR (DMSO-d6, 500 MHz) d 2.20
(m, 2H), 2.31 (s, 3H), 3.79 (t, J = 6 Hz, 2H), 4.24 (t,
J = 6 Hz, 2H), 7.25 (t, J = 7 Hz, 1H), 7.37 (s, 1H), 7.75
(d, J = 9 Hz, 1H), 7.96 (m, 1H), 8.60 (s, 1H), 9.01 (d,
J = 7 Hz, 1H), 9.36 (s, 1H), 11.30 (s, 1H).
Similarproceduresforpreparing9band9cwerecarriedout
following the preparation methods of intermediate 9a
using 6b and 2-amino-6-trifluoromethoxylbenzothiazole,
2-aminobenzimidazole as the reaction materials.
5.1.7. 7-Hydroxy-6-methyl-4-(pyridin-2-ylamino)-quino-
line-3-carbonitrile (10). To a solution of acetic acid
(20 ml) and aqueous hydrobromic acid (47%, 40 ml)
heated at 120 °C was added 7 (220 mg, 0.76 mmol). Then
the mixture was heated at reflux for 12 h. After cooling,
the mixture was diluted with cold water (100 ml) and ex-
tracted with ethyl acetate. The organic layer was washed
with water and brine, dried over MgSO4, and evaporated.
The residue was purified by silica gel column to give
115 mg product (55% yield). Mp 175–177 °C; EI-MS

5896
X. Cao et al. / Bioorg. Med. Chem. 16 (2008) 5890–5898
5.1.12. 7-(3-Chloropropoxy)-6-methyl-4-(benzothiazol-2-
ylamino)-quinoline-3-carbonitrile (14a). A mixture of 11a
(160 mg, 0.48 mmol), 1-bromo-3-chloropropane (150 ll,
1.5 mmol), and potassium carbonate (210 mg, 1.5 mmol)
in DMF (20 ml) was heated at 40 °C for 3 h. The mixture
was cooled, diluted with water, and extracted with ethyl
acetate. The organic extracts were combined, washed with
water and brine, and dried with MgSO4, and the volatiles
were removed by evaporation. The residue was chromato-
graphed on silica gel with CH3OH–EtOAc to give 135 mg
(68%) of 14a. Mp 243–245 °C (dec); EI-MS (m/z): 408.2
for 16 using 14a and morpholine. Mp 210 °C; EI-MS (m/
z): 460.1 (M+1)+; HRMS calcd for C25H27N5O2S
461.1641, Found 461.1658; 1H NMR (300 MHz, D2O): d
1.76 (br s, 2H), 2.14 (s, 3H), 3.16–3.21 (m, 4H), 3.43–3.57
(m, 4H), 3.78–3.86 (m, 2H), 4.11–4.15 (m, 2H), 6.36 (s,
1H), 7.42 (m, 3H), 7.65 (m, 1H), 8.11 (s, 1H), 8.72 (s,
1H); IR m (CN) 2187 cmꢀ1
.
5.1.16. 6-Methyl-7-[3-(piperidin-1-yl)-propoxy]-4-(ben-
zothiazol-2-ylamino)-quinoline-3-carbonitrile (18). The
compound was prepared with a 44% yield according
to the method for 16 using 14a and piperidine. Mp
193 °C; EI-MS (m/z): 458.1 (M+1)+; HRMS calcd for
C26H29N5OS 459.1849, Found 459.1862; 1H NMR
(300 MHz, D2O): d 1.75 (br s, 2H), 2.02 (s, 3H), 3.15–
3.20 (m, 4H), 3.43 (m, 2H), 3.52–3.56 (m, 2H), 3.78–
3.87 (m, 2H), 4.11–4.15 (m, 2H), 6.34 (s, 1H), 7.40–
7.45 (m, 3H), 7.64 (m, 1H), 8.10 (s, 1H), 8.71 (s, 1H);
1
(M+); H NMR (DMSO-d6, 500 MHz) d 2.20 (m, 2H),
2.39 (s, 3H), 3.79 (t, J = 6 Hz, 2H), 4.24 (t, J = 6 Hz,
2H), 7.39 (s, 1H), 7.52 (m, 2H), 7.95 (d, J = 8 Hz, 1H),
8.41 (s, 1H), 9.48 (d, J = 8 Hz, 1H), 9.56 (s, 1H), 11.50
(s, 1H).
Similar procedures for preparing 14b–d and 15a–c were
carried out according to the preparation for intermedi-
ates 13 and 14a using 11b–d and 12a–c as different start-
ing materials.
IR m (CN) 2227 cmꢀ1
.
5.1.17. 6-Methyl-7-[3-(4-methylpiperazin-1-yl)-propoxy]-
4-(6-methyl-benzothiazol-2-ylamino)-quinoline-3-carboni-
trile (19). The compound was prepared with a 43% yield
according to the method for 16 using 14b and N-methyl-
piperazine. Mp 272 °C; EI-MS (m/z): 487.1 (M+1)+;
HRMS calcd for C27H32N6OS 488.2114, Found
5.1.13. 7-(3-Chloroethoxy)-6-methoxy-4-(benzothiazol-2-
ylamino)-quinoline-3-carbonitrile (15d). A mixture of 12a
(200 mg, 0.57 mmol), 1-bromo-2-chloroethane (125 ll,
1.5 mmol), and K2CO3 (210 mg, 1.5 mmol) in DMF
(20 ml) was heated at 40 °C for 3 h. The mixture was
cooled, diluted with water, and extracted with ethyl ace-
tate. The organic extracts were combined, washed with
water and brine, and dried with MgSO4, and the vola-
tiles were removed by evaporation. The residue was
chromatographed on silica gel with MeOH–EtOAc to
give 176 mg (75% yield) of product. Mp 234 °C (dec);
EI-MS (m/z): 410.2 (M+); 1H NMR (DMSO-d6,
500 MHz) d 3.91 (t, J = 6 Hz, 2H), 4.00 (s, 3H), 4.62
(t, J = 6 Hz, 2H), 7.43 (s, 1H), 7.65 (m, 2H), 8.05 (s,
1H), 8.15 (d, J = 8 Hz, 1H), 9.05 (d, J = 8 Hz, 1H),
9.35 (s, 1H), 10.52 (s, 1H).
1
488.2148; H NMR (300 MHz, D2O): d 1.98 (s, 3H),
2.27 (s, 5H), 3.05 (s, 3H), 3.43 (m, 2H), 3.73 (m, 8H),
3.85 (m, 2H), 6.69 (s, 1H), 7.10–7.13 (d, 1H,
J = 8.2 Hz), 7.36 (s, 1H), 7.64 (s, 1H), 7.90–7.93 (d,
1H, J = 8.4 Hz), 8.93 (s, 1H); IR m (CN) 2213 cmꢀ1
.
5.1.18. 6-Methyl-7-[3-(piperidin-1-yl)-propoxy]-4-(6-
methylbenzothiazol-2-ylamino)-quinoline-3-carbonitrile
(20). The compound was prepared with a 48% yield
according to the method for 16 using 14b and piperi-
dine. Mp 270 °C; EI-MS (m/z): 471.0; HRMS calcd
for C27H29N5OS 471.2093, Found 471.2098; 1H NMR
(300 MHz, DMSO-d6): d 1.75 (m, 2H), 1.82 (m, 4H),
2.31 (m, 2H), 2.32 (s, 3H), 2.33 (s, 3H), 3.02 (m, 2H),
3.25–3.27 (m, 2H), 3.47–3.51 (m, 2H), 4.29–4.33 (m,
2H), 7.47–7.50 (d, 1H, J = 8.5 Hz), 7.58 (s, 1H), 7.93
(s, 1H), 8.56 (s, 1H), 8.78–8.81 (d, 1H, J = 8.5 Hz),
5.1.14. 6-Methyl-7-[3-(4-methylpiperazin-1-yl)-propoxy]-
4-(pyridin-2-ylamino)-quinoline-3-carbonitrile (16).
A
mixture of compound 13 (100 mg, 0.28 mmol), N-meth-
ylpiperazine (200 mg, 2.0 mmol), and a catalytic amount
of NaI in 10 mL of DMF was heated at 80 °C for 6 h.
The reaction mixture was then poured into ice water
and the pH was adjusted to 8–9 by the addition of sat-
urated NaHCO3. The solids were collected by filtration
to provide 75 mg of crude product. The crude product
was purified by column chromatography eluting with
30% MeOH in ethyl acetate to provide 55 mg (47%
yield) of compound 16 as a light yellow solid: Mp
233 °C; EI-MS (m/z): 416.1 (M+); HRMS calcd for
C24H28N6O 416.2325, Found 416.2334; 1H NMR
(300 MHz, D2O): d 2.01 (s, 3 H), 2.29–2.31 (m, 2 H),
2.95 (s, 3 H), 3.38–3.43 (br s, 4 H), 3.45–3.49 (m, 2 H),
3.54–3.64 (bs, 4 H), 3.97-4.00 (t, 2 H, J1 = 6 Hz,
J2 = 6 Hz), 6.36 (s, 1H), 6.74–6.76 (m, 1H), 6.8–6.89
(d, 1H, J = 9 Hz), 7.23 (s, 1H), 7.64–7.66 (m, 1H),
10.01 (s, 1H), 10.37 (br s, 1H); IR m (CN) 2212 cmꢀ1
.
5.1.19. 6-Methyl-7-[3-(4-methylpiperazin-1-yl)-propoxy]-
4-[6-(trifluoromethoxy)-benzothiazol-2-ylamino]-quino-
line-3-carbonitrile (21). The compound was prepared
with a 64% yield according to the method for 16 using
14c and N-methylpiperazine. Mp 243 °C; EI-MS (m/z):
556.2 (M+); HRMS calcd for C27H27F3N6O2S
1
556.1868, Found 556.1843; H NMR (300 MHz, D2O):
d 2.13 (s, 3H), 2.39 (m, 2H), 3.05 (s, 3H), 3.50–3.51
(m, 4H), 3.57–3.59 (m, 2H), 3.63–3.69 (m, 4H), 4.10
(m, 2H), 6.51 (s, 1H), 7.38 (m, 2H), 7.43 (s, 1H), 7.45
(s, 1H), 8.22(s, 1H); IR m (CN) 2146 cmꢀ1
.
5.1.20. 6-Methyl-7-[3-(4-methylpiperazin-1-yl)-propoxy]-
4-(1H-benzimidazole-2-ylamino)-quinoline-3-carbonitrile
(22). The compound was prepared with a 61% yield
according to the method for 16 using 14d and N-methyl-
piperazine. Mp 236 °C; EI-MS (m/z): 473.1 (M+1)+;
HRMS calcd for C26H28N6OS 472.2045, Found
7.75–7.78 (m, 1H), 8.15 (s, 1H); IR m (CN) 2192 cmꢀ1
.
5.1.15.6-Methyl-7-(3-morpholinopropoxy)-4-(benzothiazol-
2-ylamino)-quinoline-3-carbonitrile (17). The compound
was prepared with a 46% yield according to the method

X. Cao et al. / Bioorg. Med. Chem. 16 (2008) 5890–5898
5897
472.2056;1H NMR (300 MHz, D2O): d 2.00 (s, 3H), 2.29–
2.30 (m, 2H), 2.95 (s, 3H), 3.38–3.42 (m, 4H), 3.46–3.50
(m, 2H), 3.95–3.99 (m, 2H), 6.35 (s, 1H), 7.21–7.31 (m,
C5-H), 7.61–7.63 (m, 2H), 7.85–7.88 (m, 1H), 8.39–8.49
(m, 1H), (s, 1H), 9.21 (s, 1H); IR m (CN) 2086 cmꢀ1
.
4H), 7.55 (s, 1H), 8.11 (s, 1H); IR m (CN) 2217 cmꢀ1
.
5.1.26. 6-Methoxy-7-[3-(4-methylpiperazin-1-yl)-propoxy]-
4-(1H-benzimidazol-2-ylamino)-quinoline-3-carbonitrile (28).
The compound was prepared with a 45% yield according
to the method for 16 using 15c and N-methylpiperazine.
Mp 225 °C; EI-MS (m/z): 472 (M+1)+; HRMS calcd for
C26H29N7O2 471.2383, Found 471.2378; 1H NMR
(300 MHz, D2O): d 3.01 (s, 3H), 3.43–3.46 (m, 6H),
3.58–3.65 (m, 6H), 3.85 (s, 3H), 4.15 (m, 2H), 6.87 (s,
1H), 7.37 (s, 1H), 7.47 (m, 1H), 7.69 (m, 1H), 8.24 (m,
5.1.21. 6-Methoxy-7-[3-(4-methylpiperazin-1-yl)-pro-
poxy]-4-(benzothiazol-2-ylamino)-quinoline-3-carbonitrile
(23). The compound was prepared with a 47% yield
according to the method for 16 using 15a and N-methyl-
piperazine. Mp 202 °C; EI-MS (m/z): 488.1 (M+);
HRMS calcd for C26H28N6O2S 488.1994, Found
488.1987;1H NMR (300 MHz, DMSO): d 1.95–1.97
(br s, 2H), 2.25 (s, 3H), 2.48–2.49 (m, 10H), 3.98 (s,
3H), 4.17–4.22 (m, 2H), 7.42 (s, 1H), 7.46–7.57 (m,
2H), 7.91 (s, 1H), 7.97–7.99 (d, 1H, J = 6 Hz), 9.46–
9.48 (d, 1H, J = 6 Hz), 9.52 (s, 1H), 9.98 (s, 1H); IR m
1H), 9.02 (s, 1H); IR m (CN) 2198 cmꢀ1
.
5.2. Biology
(CN) 2220 cmꢀ1
.
5.2.1. Src kinase assay. Src kinase activity was carried out
in an ELISA format by measuring the fluorescence polar-
ization of the reaction system (Pan Vera’s Protein Tyro-
sine Kinase Red Assay kit, Invitrogen Corporation). Src
(human recombinant, histidine-tagged 1 U/reaction;
Invitrogen Corporation), reaction buffer (20 mM
HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 50 lM
Na3VO4), poly (Glu, Tyr = 4:1) (3200 ng/mL, Sigma, St.
Louis, MO, USA) substrate were added to the compound.
The reaction was started by the addition of ATP
(1,000 nM, Sigma) to a total volume of 20 ll and incu-
bated at 37 °C for 30 min. Then it was stopped by addition
of EDTA. Then antibody and tracer were added and the
mixture was incubated at 37 °C for another 30 min. The
fluorescence polarization was measured by a Magellan
TECAN instrument with a Safire2 workstation. Instruc-
tions from the manufacturer were followed for subse-
quent steps. Compounds were tested in triplet and the
value given is an average of three determinations.
5.1.22. 6-Methoxy-7-[3-(4-methylpiperazin-1-yl)-propoxy]-
4-[6-(trifluoromethoxy)-benzothiazol-2-ylamino]-quinoline-
3-carbonitrile (24). The compound was prepared with a
34% yield according to the method for 16 using 15b
and N-methylpiperazine. Mp 262 °C; EI-MS (m/z):
572.1 (M+); HRMS calcd for C27H27F3N6O3S
1
572.1817, Found 572.1835; H NMR (300 MHz, D2O):
d 3.03 (s, 3H), 3.60 (m, 6H), 3.67 (m, 6H), 3.88 (s,
3H), 4.18 (m, 2H), 6.91 (s, 1H), 7.40 (s, 1H), 7.49 (bs,
2H), 7.71–7.73 (d, 1H, J = 4.6 Hz), 8.28 (s, 1H), 9.05(s,
1H); IR m (CN) 2238 cmꢀ1
.
5.1.23. 6-Methoxy-7-[2-(piperidin-1-yl)-ethoxyl]-4-(ben-
zothiazol-2-ylamino)-quinoline-3-carbonitrile (25). The
compound was prepared with a 52% yield according to
the method for 16 using 15d and piperidine. Mp
210 °C; EI-MS (m/z): 460.1 (M+1)+; HRMS calcd for
C25H27N5O2S 461.1641, Found 461.1646; 1H NMR
(300 MHz, D2O): d 1.47–1.51 (m, 2H), 1.70–1.80 (m,
2H), 1.95–1.99 (m, 2H), 2.97–3.05 (m, 2H), 3.42 (m,
2H), 3.50–3.54 (m, 2H), 3.59 (s, 3H), 3.97 (m, 2H), 6.59
(s, 1H), 6.92 (s, 1H), 7.30–7.32 (d, 1H, J = 6.6 Hz),
7.54–7.56 (d, 1H, J = 6.9 Hz), 7.92 (s, 1H), 8.63 (s, 1H);
5.2.2. iNOS assay. Inducible NO synthetase activity was
measured by monitoring the NO concentration gener-
ated by the induced iNOS in murine macrophages
ANA-1 stimulated by exogenous LPS using a nitric
oxide synthase assay kit (Beyotime Corporation).
Mouse macrophages ANA-1 (Institute of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sci-
ences, Chinese Academy of Sciences) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% heat-inactivated FBS and L-glutamine
(2 mM). A total of 105 cells/mL were prepared the day
before each experiment, and the cells were incubated
at 37 °C in a humidified incubator under 5% CO2.
IR m (CN) 2218 cmꢀ1
.
5.1.24. 6-Methoxy-7-(2-morpholinoethoxy)-4-(benzo-
thiazol-2-ylamino)-quinoline-3-carbonitrile (26). The
compound was prepared with a 54% yield according
to the method for 16 using 15d and morpholine. Mp
236 °C; EI-MS (m/z): 461.0 (M+); HRMS calcd for
C24H23N5O3S 461.1522, Found 461.1535;1H NMR
(300 MHz, D2O): d 1.47–1.55 (m, 2H), 1.95–2.00 (m,
2H), 2.97–3.05 (m, 2H), 3.42 (m, 2H), 3.50–3.54 (m, 2H),
3.58 (s, 3H), 3.96 (m, 2H), 6.59 (s, 1H), 6.91 (s, 1H),
7.29–7.31 (d, 1H, J = 6 Hz), 7.52–7.55 (d, 1H, J = 6 Hz),
Cells were then stimulated by lipopolysaccharide (20 lM;
LPS, Sigma) for 24 h, and the cultured cells were collected
and transferred to a 96-well plate. Assay buffer, the L-argi-
nine substrate, and the tracer DAF-FM DA (3-amino-4-
aminomethyl-20,70-difluorescein diacetate) were added,
followed by addition of 0.1 mM NADPH to the reaction
mixtures to initiate the reactions. The plate was incubated
at 37 °C for 30 min, centrifuged, and washed three times
in PBS. Then the fluorescence of the plate was measured
by a Varioskan instrument. Instructions from the manu-
facturer were followed strictly, and each compound was
tested in triplet; the value given is an average of three
determinations.
7.92 (s, 1H), 8.62 (s, 1H); IR m (CN) 2116 cmꢀ1
.
5.1.25. 6-Methoxy-7-[2-(4-methylpiperazin-1-yl)-ethoxy]-
4-(benzothiazol-2-ylamino)-quinoline-3-carbonitrile (27).
The compound was prepared with a 49% yield according
to the method for 16 using 15a and N-methylpiperazine.
Mp 241 °C; EI-MS (m/z): 475.1 (M+1)+; HRMS calcd
1
for C25H28N6O2S 476.1750, Found 476.1751; H NMR
(300 MHz, D2O): d 3.17 (s, 3H), 3.78 (m, 6H), 3.80 (m,
6H), 4.01 (s, 3H), 4.35 (m, 2H), 7.07 (s, 1H), 7.55 (s, 1H,

5898
X. Cao et al. / Bioorg. Med. Chem. 16 (2008) 5890–5898
5.2.3. SRB proliferation assays. Five tumor cell lines
which were associated with overexpression of Src or
iNOS were selected, including lung cancer A549, stom-
ach cancer AGS, liver cancer HepG2, colon cancer
HT-29, and prostate cancer PC-3 lines. Sulforhodamine
B, trichloroacetic acid, trizma base, and acetic acid were
purchased from Sigma Chemical Co. (St. Louis, USA).
Lung cancer A549, stomach cancer AGS, colon cancer
HT-29, and prostate cancer PC-3 cell lines were cultured
in RPMI 1640 medium, supplemented with 10% fetal
bovine serum at 37 °C in humidified air under 5%
CO2. Liver cancer HepG2 cell line was cultured in
MEM medium supplemented with 10% fetal bovine ser-
um at 37 °C in humidified air containing 5% CO2.
References and notes
1. Parsons, S. J.; Parsons, J. T. Oncogene 2004, 23, 7906.
2. Martin, G. S. Oncogene 2004, 23, 7910.
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E.; Curry, B. J.; Barker, A. J.; Gibson, K. H. Cancer Res.
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4. Boschelli, D. H.; Ye, F.; Wang, Y. D.; Dutia, M.;
Johnson, S. L.; Wu, B.; Miller, K.; Powell, D. W.;
Yaczko, D.; Young, M.; Tischler, M.; Arndt, K.;
Discafani, C.; Etienne, C.; Gibbons, J.; Grod, J.;
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2001, 44, 3965.
5. Golas, J. M.; Lucas, J.; Etienne, C.; Golas, J.;
Discafani, C.; Sridharan, L.; Boghaert, E.; Arndt, K.;
Ye, F.; Boschelli, D. H.; Li, F.; Titsch, C.; Huselton,
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Med. Chem. 2004, 12, 4101.
A pilot-screening operation was initiated in which the
panel lines were inoculated onto a series of standard
96-well plates on day 0, in the majority of cases at
20,000 cells/well, and then pre-incubated in the absence
of drug for 24 h. Test agents were then added in 10-fold
dilutions starting from the highest soluble concentra-
tion, and incubated for a further 48 h. Following this,
the cells were fixed in situ, washed, and dried. SRB
was added, followed by further washing and drying of
the stained, adherent cell mass. The inhibition of cell
proliferation was assessed by measuring changes in total
optical density after a culture of each cell line that was
subjected to 48 h of drug treatment. The results were ob-
tained in one independent experiment run in triplicate.
10. Webber, R. K.; Metz, S.; Moore, W. M.; Connor, J. R.;
Currie, M. G.; Fok, K. F.; Hagen, T. J.; Hansen, D. W.,
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Acknowledgment
11. Wissner, A.; Berger, D. M.; Boschelli, D. H.; Floyd,
M. B.; Greenberger, L. M.; Gruber, B. C.; Johnson,
B. D.; Mamuya, N.; Nilakatan, R.; Reich, M. F.;
Shen, R.; Tsou, H. R.; Upeslacis, E.; Wang, Y. F.;
Wu, B.; Ye, F.; Zhang, N. J. Med. Chem. 2000, 43,
3244–3256.
Acknowledgments are made to the National Natural
Science Foundation of China (Grant No. 30271543
and 30371676) for financial supports.
12. Boschelli, D. H.; Wang, Y. D.; Johnson, S.; Wu, B.; Ye,
F.; Barrios Sosa, A. C.; Golas, J. M.; Boschelli, Frank. J.
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Supplementary data
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