Discovery of 6-benzyloxyquinolines as c-Met selective kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.12.109

A novel quinoline derivative that selectively inhibits c-Met kinase was identified. The molecular design is based on a result of the analysis of a PF-2341066 (1)/c-Met cocrystal structure (PDB code: 2wgj). The kinase selectivity of the derivatives is discussed from the view point of the sequence homology of the kinases, the key interactions found in X-ray cocrystal structures, and the structure-activity relationship (SAR) obtained in this work.

Full text of DOI:10.1016/j.bmcl.2009.12.109

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1405–1409
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 6-benzyloxyquinolines as c-Met selective kinase inhibitors
Hiroki Nishii a, Takashi Chiba a, Kenji Morikami b, Takaaki A. Fukami b, Hiroshi Sakamoto c, Kwangseok Ko d,
Hiroshi Koyano a,
*
a Department of Chemistry Research 2, Chugai Pharmaceutical Co, Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
b Department of Discovery Platform Technology, Chugai Pharmaceutical Co, Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
c Department of Pharmaceutical Research 2, Chugai Pharmaceutical Co, Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
d C&C Research Labs, 146-141, Annyeong-dong, Hwaseong-si, Gyeonggi-do, 445-380, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel quinoline derivative that selectively inhibits c-Met kinase was identified. The molecular design is
based on a result of the analysis of a PF-2341066 (1)/c-Met cocrystal structure (PDB code: 2wgj). The
kinase selectivity of the derivatives is discussed from the view point of the sequence homology of the
kinases, the key interactions found in X-ray cocrystal structures, and the structure–activity relationship
(SAR) obtained in this work.
Received 4 November 2009
Revised 28 December 2009
Accepted 29 December 2009
Available online 4 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
6-Benzyloxyquinolines
c-Met
Kinase inhibitor
c-Met is a prototypic member of a subfamily of receptor tyro-
sine kinases (RTKs). c-Met is structurally distinct from other RTKs
and is the only known high-affinity receptor for hepatocyte growth
factor (HGF), also known as the scatter factor.1,2 c-Met and HGF are
dysregulated in human cancers and also contribute to dysregula-
tion of cell proliferation, tumor invasion, and metastasis.3,4 c-
Met-activating point mutations in the kinase domain have been
implicated as the cause of hereditary papillary renal carcinoma
and also have been detected in sporadic papillary renal carcinoma
and head and neck cancers.5,6 Furthermore, amplification of the c-
Met gene locus has been detected in patients with gastric and met-
astatic colorectal cancer.7,8 These findings suggest that c-Met
would be an attractive target for cancer therapy.
PF-2341066 (1) has been identified as an ATP-competitive c-
Met inhibitor.9 We analyzed the structure deposited in PDB (PDB
code: 2wgj) and identified that the 2-aminopyridine bound to
the hinge region of c-Met in a bidentate manner with the primary
amine hydrogen-bonding to the carbonyl oxygen of P1158 and the
pyridine nitrogen to the amide NH of M1160. Furthermore, we
Discovery Ltd, London, UK) revealed a hydrophobic space around
the hinge region.
While exploring the scaffold of c-Met selective inhibitor, we
hypothesized that the hydrophobic interactions were essential
and the binding affinity from one of the hinge-interacting hydro-
gen bonds could be compensated by filling the hydrophobic space
with the core structure. At the same time we found that no hydro-
gen bond was observed around the substituent that faced to the
solvent-accessible region (green part in Fig. 1). We assumed that
this moiety was primarily intended to adjust the physicochemical
properties.10 Based on these hypotheses, we designed core struc-
ture using molecular design software Moloc.11
We identified 6-benzyloxyquinoline as the scaffold to form the
three interactions: (1) the hydrogen bond with the hinge region
M1160
P1158
N
O
H
N
found that 2,6-di-chloro-3-fluorophenyl ring formed a
p–p inter-
action with Y1230 at 0.40 nm, and a hydrophobic interaction with
M1211 at 0.40 nm (Fig. 1). These hydrophobic interactions may
contribute to the selective inhibition of c-Met since the two amino
acids are conserved among only three (c-Met, AXL, MER) of the 491
kinases analyzed. In addition, GRID calculation (ver. 22a, Molecular
Solvent-
accessible
region
HN
OH
N
N
F
Y1230
S
Cl
M1211
* Corresponding author. Tel.: +81 467 47 6065; fax: +81 467 47 2248.
E-mail address: koyanohrs@chugai-pharm.co.jp (H. Koyano).
Figure 1. The interactions found in a PF-2341066 (1)/c-Met cocrystal structure and
the hydrophobic space (cyan) identified by GRID analysis.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.109

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H. Nishii et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1405–1409
Table 1
Effects of configuration on inhibitory activity (IC50/nM) against c-Met
N
N
N
N
NH2
O
O
Cl
O
Cl
O
Cl
HN
Cl
N
N
F
F
F
F
Cl
Cl
Cl
Cl
2
(racemic)
(R
)-2
(S)-2
PF-2341066 (1)
Compound
IC50 (nM)
KDR
c-Met
Src
1a
(R)-2
(S)-2
2
8
130
1200
453
>10,000
23,000
>50,000
32,000
Not available
22,000
20,000
>50,000
(M1160), (2) the hydrophobic and
p
–
p
interactions, and (3) hydro-
vent-accessible region of c-Met improved inhibitory activity. Thus,
acryl amide substituted compound 3 (racemic) showed one order
magnitude more potent c-Met inhibition retaining high selectivity
than nonsubstituted compound 2 and potent cytotoxicity against a
c-Met amplified gastric cancer cell line (MKN45). N-Monomethy-
lated amide 4 had the similar kinase inhibitory activity and the
cytotoxicity but N,N-dimethyl amide 5 showed weaker activities
than 3. Saturation of the double bond or removal of the carbonyl
group of the acryl amide (compounds 6 and 7, respectively) and
the corresponding carboxylic acid 8 showed less inhibitory activi-
ties (Table 2).
phobic space-filling with a quinoline skeleton. 6-Benzyloxyquino-
line derivatives were prepared by treatment of commercially
available 6-hydroxyquinoline with the corresponding alcohols pre-
pared according to a patent procedure12 under Mitsunobu condi-
tions.13 As
a primary evaluation of kinase selectivity, we
measured the inhibitory activities against kinase insert domain
receptor (KDR) and Src kinase as a counter assay. The inhibition
of KDR is reported to cause hypertension14 and the inhibition of
Src may link to the inhibition of other 10 kinases of Src family.15
An optically active (R)-2 showed inhibitory activity with a sub-
micromolar IC50 value with high selectivity against KDR while its
enantiomer (S)-2 showed 10-fold less potent activity (Table 1). This
result validates our hypotheses supporting the molecular design.
Substitution at position 3 of the quinoline directed towards the sol-
A cocrystal of c-Met was obtained with 4 (a racemic compound,
IC50 = 28 nM)16 and its X-ray structure analysis revealed that R-
enantiomer bound to the ATP binding site (M1160 backbone
nitrogen–quinoline nitrogen = 0.28 nm) in a mode similar to that
Table 2
SAR for racemic quinoline derivatives
N
R3
O
Cl
F
Cl
Compound
R3
c-Met inh. (IC50/nM)
20
KDR inh. (IC50/nM)
26,000
Src inh. (IC50/nM)
>50,000
Cell growth inh. (MKN45) (IC50
/lM)
H2N
3
0.35
O
O
H
N
4
5
28
59
>50,000
>50,000
>50,000
>50,000
0.24
0.61
N
O
O
H2N
6
7
8
205
368
94
50,000
33,000
>50,000
>50,000
46,000
4.4
4.3
H2N
HO
>50,000
10.2
O

H. Nishii et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1405–1409
1407
formed a
p–p interaction with Y1159 (distance between double
bond and aromatic ring, 0.42 nm) and an amide hydrogen of 4
formed a hydrogen bond with K1161 (0.29 nm) but no interaction
was found with the amide carbonyl oxygen of 4. We assumed that
the acryl amide moiety fixed the hydrogen donor at the appropri-
ate position to form the hydrogen bond to K1161. These observa-
tions and assumptions correlate with the results showing the
potency of saturated compound 6 and de-oxo compound 7 to be
less than 3. Because N,N-dimethyl acrylamide 5 has no NH group
and carboxylic acid 8 is supposed to be deprotonated, these two
compounds do not form hydrogen bond between the backbone
carbonyl group of K1161. The contribution of the hydrogen bond
to the inhibitory activity is small because the difference of inhibi-
tory activity among compound 3, 5 and 8 is not significant.
Although an acryl amide moiety is a good substituent in terms
of kinase inhibitory activity, the drawbacks of 3 such as a low sol-
ubility and a potential risk of toxicity originated from Michael
acceptor structure need to be eliminated. We designed a cyclopro-
pyl moiety that avoids the Michael acceptor and maintains the po-
sition of the amide functional group. An optically active acrylamide
(R)-3 and cyclopropyl analog 14 were synthesized from known
compound 917 as shown in Scheme 1. Cyclopropanation of the ac-
ryl amide double bond was well tolerated, as compound 14 re-
tained the kinase inhibitory activity and cytotoxicity against
MKN45 and showed satisfactory solubility in fasted state simu-
lated intestinal fluid (FaSSIF) (Table 3).
Figure 2. X-ray crystal structure of the c-Met (green)/compound (R)-4 (pink)
complex showing key interactions and the hydrophobic space (light blue) identified
by GRID analysis.
of 1 (Fig. 2). As expected, the hydrophobic side chain formed the
hydrophobic interaction with M1211 (distance between sulfur
atom and aromatic ring, 0.45 nm) in addition to the
p–p interac-
tion with Y1230 (distance between centers of the aromatic rings,
0.42 nm). The right ring of quinoline occupied the hydrophobic
space found in the GRID analysis. The double bond of acryl amide
N
N
N
OH Cl
H2N
O
N
iii
O
Cl
O
Cl
i
ii
F
O
Cl
Br
+
F
O
F
O
F
32%
96%
82%
Br
OH
Cl
Cl
Cl
Cl
97%ee*
9
10
11
12
(R)-3
iv 49%
N
N
O
H2N
O
Cl
iii
O
Cl
F
F
O
O
94%
Cl
Cl
13
14
Scheme 1. Preparation of (R)-3 and 14. Reagents and conditions: (i) 1-(2,6-Dichloro-3-fluorophenyl)-ethanol, diisopropyl azodicarboxylate, PPh3, THF, rt; (ii) tert-butyl
acrylate, 10 mol % Pd(OAc)2, tetrabutylammonium iodide, K2CO3, DMF, 100 °C; (iii) trifluoroacetic acid, CH2Cl2, rt and then water-soluble carbodiimide, hydroxybenzotriazole,
*
diisopropylethylamine, DMF, rt; (iv) trimethylsulfoxonium iodide, NaH, DMSO, rt. Chiral HPLC conditions: column, CHIRALPAK AD–H, 4.6 Â 250 mm (Daicel); eluent, 4/1
0.1% diethylamine–EtOH/0.1% diethylamine–hexane; flow rate, 0.7 mL/min; column temp, 35 °C; detection, 334 nm. tR = 8.0 min [(R)-isomer], tR = 6.7 min [(S)-isomer].
Table 3
In vitro properties of compounds (R)-3 and 14
N
R3
O
Cl
F
Cl
Compound
R3
c-Met inh. (IC50/nM)
23
Cell growth inh. (MKN45) (IC50
/
l
M)
Solubility in FaSSIF (
<13
lg/mL)
H2N
(R)-3
0.27
O
O
H2N
14
9.3
0.093
345

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H. Nishii et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1405–1409
Table 4
Supplementary data
Inhibitory activity (IC50/lM) of quinoline derivatives against various kinases
(R)-2
(R)-3
14
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.12.109.
Tyrosine kinase
c-Met
RON
AXL
EphA2
KDR
LTK
FGFR2
Flt3
Fyn
IGF1R
lck
0.13
3.6
14
>50
>50
>50
24
0.023
0.45
0.57
4.4
11
18
6.9
32
28
>50
14
>50
>50
>50
>50
26
0.0093
0.23
0.43
5.2
9.7
10
12
15
25
27
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6.4
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9. (a) Zou, H. Y.; Li, Q.; Lee, J.; Arango, M.; McDonnel, S. R.; Dussell, C.; Stempniak,
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273; (b) Zou, H. Y.; Li, Q.; Lee, J. H.; Arango, M. E.; McDonnell, S. R.; Yamazaki,
S.; Koudriakova, T. B.; Alton, G.; Cui, J. J.; Kung, P.; Nambu, M. D.; Los, G.;
Bender, S. L.; Mroczkowski, B.; Christensen, J. G. Cancer Res. 2007, 67, 4408.
10. Cui, J. J.; Funk, L. A.; Jia, L.; Kung, P.; Meng, J. J.; Nambu, M. D.; Pairish, M. A.;
Hong, S.; Tran-Dube, M. B. U.S. Patent 2006046991; Chem. Abstr. 2006, 144,
274297.
cdk2
PKC alpha
PKC beta1
PKC beta2
Table 5
Growth factor effect on HUVEC anti-proliferative activity of compound 14
Growth factor
HUVEC anti-proliferative activity (IC50/lM)
11. For details of Moloc, see: http://www.moloc.ch/.
HGF
VEGF
ECGS
0.3
6.7
9.9
12. Kung, P.-P.; Martinez, C. A.; Tao, J. Appl. Int. WO2006021885, 2006; Chem.
Abstr. 2006, 144, 252788. Enantiomeric excess was determined by HPLC
analysis: column, CHIRALPAK AD-H, 4.6 Â 250 mm (Daicel); eluent, isopropyl
alcohol/hexane = 5/95; flow rate, 1.0 mL/min; column temp, 35 °C; detection,
210 nm. The retention times were 6.6 and 6.1 min for (R)- and (S)-isomer,
respectively.
13. The enantiomeric excess of the products was determined by chiral HPLC
analysis. HPLC conditions: column, CHIRALPAK AD–H, 4.6 Â 250 mm (Daicel);
eluent, 0.1% diethylamine–EtOH; flow rate, 0.4 mL/min; column temp, 35 °C;
detection, 210 nm.
14. Facemire, C. S.; Nixon, A. B.; Griffiths, R.; Hurwitz, H.; Coffman, T. M.
Hypertension 2009, 54, 652.
Compound 14 showed the most potent inhibitory activity
against c-Met among 19 tyrosine kinases18 and 8 serine/threonine
kinases19 (IC50 ratios >20, Table 4) and was less cytotoxic against c-
Met non-amplified 17 cell lines [colorectal cancer (HCT116, WiDr,
COLO205), lung cancer (NCI-H460, A549, Calu-6), pancreatic can-
cer (AsPC-1, Capan-1, BxPC-3), prostate cancer (DU145, PC3,
22Rv1), breast cancer (MDA-MB-231, T47D, MCF7), gastric cancer
15. Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science
2002, 298, 1912.
16. Cocrystals were obtained in 2 days under the crystallization conditions of
4 mg/ml protein, 5 (1 mM), 14% (w/v) PEG MME 5000, 12% (v/v) 2-methyl-2,4-
pentanediol, 5% (v/v) 2-propanol and 0.1 M Tris–Cl (pH 7.5) with streak
seeding, incubated at 12 °C. The structure was solved at 2.7 Å and the
coordinates have been deposited in the Protein Data Bank, http://
www.rcsb.org/pdb/home/home.do (PDB ID: 3a4p).
17. (a) Zymalkowski, F.; Tinapp, P. Ann. Chem., Justus Liebigs 1966, 699, 98; (b)
Crowley, P. J.; Salmon, R. Appl. Int. WO2004047538, 2004; Chem. Abstr. 2004,
141, 2846.
18. The inhibitory activity against tyrosine kinase was measured by quantitative
analysis of the phosphorylation of the substrate peptides by recombinant
enzyme proteins in the presence of test articles using a europium-labeled
antiphospho-substrate antibody (PerkinElmer). Five micro liter of solutions of
(MKN28, NCI-N87)] (IC50 >1 l
M) than MKN45.20 The selective c-
Met inhibition of 14 was properly reflected in an HGF-dependent
anti-proliferative activity of HUVEC (Table 5).
Although a quinoline skeleton, as well as quinazoline and ind-
olinone, is used as a scaffold in many kinases inhibitors,21 the
use of the 6-oxygen-functionalized quinoline scaffold is unique.
This skeleton was designed to occupy the hydrophobic region adja-
cent to the hinge binding site found in the PF-2341066/c-Met
cocrystal structure. The utility of this skeleton was demonstrated
by the highly selective and potent c-Met inhibitor 14 which
showed selective cytotoxicity reflecting selective c-Met inhibition.
compound, 10
lL of the substrate/ATP solution, and 5 lL of the enzyme
solution were mixed sequentially and incubated for 90 min at 30 °C. The
quantity of enzyme and ATP and the kind of substrate and cation species added
in each assay are given in Supplementary Table 1. After the reaction was
stopped by the addition of 10 lL of Stop solution, 20 lL of TR-FRET reagent
(PerkinElmer) was added and incubated for 5 min at room temperature. After
incubation, time-resolved fluorescence was measured by EnVison HTS (Model
2101, PerkinElmer, Inc.).
Acknowledgments
19. The inhibitory activity against various kinds of enzymes was measured by
quantitative analysis of the phosphorylation of the substrate peptides by the
recombinant enzyme proteins in the presence of test articles using an IMAP FP
Screening Express Kit (Molecular Devices). Five micro liter of solutions of
The authors acknowledge H. Shirai for the enantiomeric excess
determination, Dr. T. Matsuura and Dr. S. Kadono for protein pro-
duction and its crystallization, and Y. Tachibana, K. Sakata and T.
Fujii for their technical assistance with the kinase and cytotoxicity
assays.
compound, 10
lL of the substrate/ATP solution, and 5 lL of the enzyme
solution were mixed sequentially and incubated for 90 min at 30 °C. The

H. Nishii et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1405–1409
1409
quantity of enzyme and ATP and the kind of substrate and cation species added
in each assay are given in Supplementary Table 1. Sixty micro liter of IMAP
reagent was added and the mixtures were incubated for 5 min at room
temperature. After incubation, fluorescence polarization was measured by
EnVision HTS.
(R&D systems), endothelial cell growth Supplement (ECGS), (BD Biosciences).
Cells were treated with various concentrations of compound for 96 h.
Cytotoxicity of compound was determined using a WST-8 cell counting kit
(Dojin Laboratories).
21. (a) Buijsman, R. In Chemogenomics in Drug Discovery; Kubinyi, H., Müller, G.,
Eds.; Wiley-VCH: Weinheim, 2004. Chapter 7; (b) Liao, J. J.-L. J. Med. Chem.
2007, 50, 409.
20. All cancer cell lines were purchased from American Type Culture Collection.
HUVEC were treated with compound in the presence of 20 ng/mL hepatocyte
growth factor (HGF) (R&D systems), vascular endothelial growth factor (VEGF)