The design, synthesis and biological evaluation of 7-alkoxy-4-heteroarylamino-3-cyanoquinolines as dual inhibitors of c-Src and iNOS

Article abstract of DOI:10.1016/j.bmcl.2008.10.006

Because both c-Src and iNOS are key regulatory enzymes in tumorigenesis, a new series of 4-heterocycle amine-3-quinolinecarbonitriles as potent dual inhibitors of both enzymes were designed, synthesized and evaluated as multiple targets agents 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 inhibited c-Src and iNOS well. The best compound 8 inhibited both enzymes with the IC₅₀ values of 34.8 nM and 26.7 μM. Several compounds also showed moderate anti-proliferation at 10 μM against colon and liver cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2008.10.006

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6206–6209
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The design, synthesis and biological evaluation of 7-alkoxy-4-
heteroarylamino-3-cyanoquinolines as dual inhibitors of c-Src and iNOS
Xin Cao a,b,e, Qi-Dong You a,b, , Zhi-Yu Li a,b, Xiao-Rong Liu a,b, Dan Xu a,b, Qing-Long Guo a,
*
Jing Shang c, Ji-Wang Chern d, Meng-Ling Chen d
a Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009, China
b Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
c National Drug Screening Center, China Pharmaceutical University, Nanjing 210009, China
d School of Pharmacy, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan
e Department of Drug Research, Jiansu Simcere Pharmaceutical R&D Co, Ltd, No 699-18 Xuan Wu Avenue, Nanjing 210042, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Because both c-Src and iNOS are key regulatory enzymes in tumorigenesis, a new series of 4-heterocycle
amine-3-quinolinecarbonitriles as potent dual inhibitors of both enzymes were designed, synthesized
and evaluated as multiple targets agents 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 com-
pounds inhibited c-Src and iNOS well. The best compound 8 inhibited both enzymes with the IC50 values
Received 22 March 2008
Revised 11 September 2008
Accepted 1 October 2008
Available online 5 October 2008
of 34.8 nM and 26.7
colon and liver cancer cell lines.
lM. Several compounds also showed moderate anti-proliferation at 10 lM against
Keywords:
c-Src
iNos
Ó 2008 Elsevier Ltd. All rights reserved.
Dual inhibitor
Anti-cancer
Src family kinases (SFKs) belong to the non-receptor protein
tyrosine kinases (PTKs), and they transfer the -phosphoryl group
level of differentiation are all correlated with over-expressed
iNOS.14
c
from ATP to the hydroxyl group of specific tyrosine (Tyr) residues
on substrate proteins.1 SFKs express predominantly in differenti-
ated cell types and regulate many fundamental cellular processes,
including cell growth, differentiation, cell shape, migration, sur-
vival, and specialized cell signals.2 They were also found to be
over-activated in various human cancers, including colon,3 pancre-
atic,4 lung,5 and ovarian cancers.6 These observations have
prompted many investigations of c-Src inhibitors aimed toward
curing cancer, for example bosutinib (SKI-606, 1) of Wyeth has
been evaluating in a Phase III clinical trial in the treatment of
CML.7,8 CPU-Y020 (2) is another efficient Src inhibitor reported
by our group which is under preclinical evaluations (Fig. 1).9
iNOS is the inducible hypotype of nitric oxide synthase (NOS),
and is expressed in a wide array of cells and tissues, for example,
macrophages,10 Kupffer cells, hepatocytes,11 neutrophils, pulmon-
ary epithelium, colonic epithelium, and vasculature.12 Unfortu-
nately, iNOS also expresses highly in various neoplastic
diseases.13 The role of iNOS during tumor development is highly
complex and remains obscure. However, there is no doubt that
the tumor metastatic activity, host defense mechanisms, and the
The attempt to suppress two distinct cancer related-enzymes
with a single agent was an attractive idea. It is reasonable that
the dual inhibitors of both c-Src and iNOS were more effective
for blocking multiple signaling pathways in cancer intervention
* Corresponding author. Tel./fax: +86 25 83271351.
E-mail address: youqidong@gmail.com (Q.-D. You).
Figure 1. The structures of bosutinib (SKI-606, 1) and CPU-Y020 (2).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.006

X. Cao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6206–6209
6207
compared with the single inhibitors and this would be beneficial to
overcome drug resistance. With the goal of identifying such a new
anti-cancer strategy, a new class of dual inhibitors of c-Src and
iNOS were designed following scaffold hopping principles.9
We chose the potent c-Src inhibitor template 4-aniline-3-quin-
olinecarbonitrile as the leading scaffold, and made functional hop-
ping from the conventional 4-aniline group to some
multifunctional heterocycle amines, like 2-aminothiazole and its
derivatives, etc (3–6, Fig. 2), which had been demonstrated as
selective inhibitors against iNOS previously.15–17 In order to retain
both the 4-aniline substituent as the molecule heads of Src inhib-
itors and 2-aminothiazole’s structural characters, 2,6-diam-
inobenzothiazole and 2,5-diaminobenzimidazole were chosen as
the hopped groups that were introduced into the leading scaffold
(Fig. 3). The relatively more active 60-amino group was connected
to the 4-position of 3-quinolinecarbonitrile and the 20-amino group
was directed out of the molecule.
The preparation of the designed compounds was carried out
smoothly. 2,6-Diaminobenzothiazole 7a and 2,5-diaminobenzimi-
dazole 7b were synthesized by selective nitration at 6-position of
2-aminobenzothiazole and 5-position of 2-aminobenzimidazole,18
and the nitro group intermediates were reduced by iron powder
(Scheme 1) to achieve the heterocycle diamine 7. The 3-quinoline-
carbonitrile core was prepared using the modified cyclic method as
described by us previously.9 Then the two main fragments were
connected in refluxing isopropanol. After that, the protective group
was removed and an alkaline side chain at C-7 position were intro-
duced (Scheme 2).
The compounds were tested in two related enzyme inhibition
assays to determine their inhibition activities on Src and iNOS: a
Protein Tyrosine Kinase Inhibition Assay (c-Src, and Tyrosine
Kinase Assay Kit, Invitrogen) and a iNOS Inhibition Assay (mouse
macrophage ANA-1, and Nitric Oxide Synthase Assay Kit, Beyo-
time China) were carried out following the reported meth-
ods.19–21
The IC50 values of compounds 8–18 (Table 1) clearly showed
that most compounds inhibited c-Src and iNOS with the IC50 val-
ues range in the domains of 20–80 nM and 10–50 lM, respec-
tively. However, the Src inhibition activities turned to be less
potent than SKI-606 (1, Table 1)7 for all their IC50 values were
more than 20 nM. The iNOS inhibition activities were similar
to those typical iNOS inhibitors, such as 2-aminothiazole (3)16
and
iNOS ranged 10–50
L
-canavanine (4, Table 1).17 Most of the IC50 values towards
l
M.
When a 20,60-diaminobenzothiazole group was at C-4 position,
and a methoxyl group was at C-6 position, compound 18 showed
the best Src inhibition activity with an IC50 of 20.7 nM while its
iNOS inhibition activity was only 105 lM. But when the C-6 meth-
oxyl group was changed into a methyl group, the compound 11’s
Src inhibition activity decreased to 129 nM while its iNOS inhibi-
tion activity increased to 17.3 lM. When n equaled 3 and the ter-
minal alkaline group piperazine was changed to 30,50-
Figure 2. The selective iNOS inhibitors and scaffolds.
dimethylpiperazine, compound 13 showed the IC50 values of 36.6
nM and 89.1 lM towards Src and iNOS.
Other C-7 alkaline substituted compounds like compounds 9, 10
and 12’s Src inhibition activities were 3- to 5-fold weaker than
compound 13 while their iNOS inhibition activities were more po-
tent than compound 13. When n equaled 2 and the terminal alka-
line group was piperidine, compound 8 showed IC50 values of
34.8 nM and 26.7 lM towards the two enzymes and that was bet-
ter compound 13.
The changed 4-heteroaromatic amines of these compounds af-
fected the inhibition activity towards Src kinase. When the 4-ani-
lino group was changed into 20,50-diaminobenzoimidazole,
compound 16 and 17 only showed weaker inhibition against Src
kinase with IC50 values of 143 and 159 nM. But it was interesting
that compounds 16 and 17 showed better inhibition activities
against iNOS with the IC50 values of 13.2 and 24.8 lM compared
with the 20,60-diaminobenzothiazole substituted compounds.
In general, most compounds could inhibit both enzymes in vitro
screening, while compound 8 showed the best activities against
both enzymes with the IC50 values of 34.8 nM and 26.7 lM, respec-
tively. The new compounds were more potent against Src than
Figure 3. The design of the dual inhibitors of Src and iNOS.
their activities against iNOS, as indicated by the IC50 values in
Scheme 1. Reagents: (a) HNO3, H2SO4; (b) Fe, HCl, ethanol.

6208
X. Cao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6206–6209
Scheme 2. Reagents and conditions: (a) AlCl3, CH2Cl2, 0–5 °C; (b) 1-bromo-3-chloropropane or 1-bromo-2-chloropropane, K2CO3, DMF; (c) 2,6-diaminobenzothiazole or 2,5-
diaminobenzimidazole, isopropanol, pyridine hydrochloride, reflux; (d) N-methylpiperazine, K2CO3, DMF or similar amines and reaction conditions.
Table 1
The structures of compounds 8–18 and their enzyme inhibition activities against c-Src and iNOS
R3
n
R4
IC50 (nM) c-Src
IC50 , (lM) iNOS
a
a b
Compound
X
8
9
10
11
12
13
14
15
S
S
S
S
S
S
S
S
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
MeO
2
3
3
3
3
3
3
3
3
3
3
Piperidine
N-methylpiperazine
Morpholine
34.8
50.6
81.1
129
53.4
36.6
42.0
267
143
159
20.7
1.8c
15.4
–
26.7
37.1
31.8
17.3
12.7
89.1
46.8
10.5
13.2
24.8
105
—
Piperazine
4-Methylpiperidine
3,5-Dimethylpiperazine
Diethylamine
3,5-Dimethylpiperidine
Dimethylamine
Pyrrolidine
16
17
18
NH
NH
S
Piperazine
1 (Bosutinib)
2 (CPU-Y020)
3 (2-Aminothiazole)
313
20d
4 (
L
-Canavanine)
–
65d
a
b
c
The IC50 values were means of triplet experiments, and the variabilities were within 10%.
iNOS of mouse macrophage ANA-1.
According to Ref. 7, the IC50 value of bosutinib for Src was 1.2 nM in an ELISA assay.
d
According to Refs. 16 and 17, the IC50 values of 2-aminothiazole and L-canavanine for iNOS from mouse macrophage ANA-1 were 18 and 60 lM, respectively.
the nanomolar range. This result may be explained as that the scaf-
fold of the series was mainly designed to fit in the hinge region
near the ATP binding site in Src kinase domain, and what is more,
the agents only possess some inhibition groups against iNOS. Thus,
there were no breakthrough findings with respect to iNOS
inhibitors.
However, there were no predominant compounds discovered
according to the assay results. All compounds were less active
than SKI-606 in the anti-proliferation screening assay according
to the percentages of survival cancer cells at 10 lM treated by
the compounds. Only a few compounds, such as compounds 8,
9 and 15, exhibited moderate inhibition activities towards the co-
The anticancer activities of these compounds were also evalu-
lon cancer HT-29 and liver cancer HepG2 cell lines at 10
in vitro assays (Table 2).
lM
ated by a SRB (Sulforhodamine B) anti-proliferation assay.22
Table 2
The percentages of survival cells at 10
lM treated by compounds 8, 9, 15 and bosutinib (1) against 5 different human cancer cell lines
Compound
Percentage of survival (%)
Liver HepG2
Lung A549
Stomach AGS
Colon HT-29
Prostate PC-3
8
9
15
55.39
64.14
53.64
9.32
61.01
68.13
65.46
3.80
37.10
40.45
42.16
9.20
42.70
31.60
60.59
2.84
77.68
59.45
62.99
7.81
1 (Bosutinib)

X. Cao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6206–6209
6209
The discrepancy between enzymatic and cell activity is likely
References and notes
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and 15 also showed moderate inhibition activities on colon
and liver cancer cells in vitro screening. However, the anti-prolif-
eration activities of these compounds decreased as compared
with SKI-606. Some further modifications with different 4-het-
eroaromatic amines are underway.
Acknowledgment
Acknowledgments are made to National Natural Science Foun-
dation of China (NSFC Grant No. 30371676) for the financial
supports.
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