Synthesis and evaluation of [2-(4-quinolyloxy)phenyl]methanone derivatives: Novel selective inhibitors of transforming growth factor-β kinase

Article abstract of DOI:10.1021/jm701626z

We synthesized and evaluated various [2-(4-quinolyloxy)phenyl]methanone derivatives. These compounds had novel chemical structures that were distinct from those of previously reported inhibitors. Biological data suggested that these compounds inhibited transforming growth factor-β signaling by interacting with the ATP-binding pocket of the transforming growth factor-β type I receptor kinase domain. Here, we report on the synthesis and structure-activity relationships of the compounds in this series.

Full text of DOI:10.1021/jm701626z

3326
J. Med. Chem. 2008, 51, 3326–3329
Synthesis and Evaluation of [2-(4-Quinolyloxy)phenyl]methanone Derivatives: Novel Selective
Inhibitors of Transforming Growth Factor-ꢀ Kinase
Toshiyuki Shimizu,* Kaname Kimura, Teruyuki Sakai, Kazuki Kawakami, Tetsuko Miyazaki, Masayoshi Nakouji,
Akira Ogawa, Hitomi Ohuchi, and Kiyoshi Shimizu
KIRIN Pharma Co., Ltd., 6-26-1 Jingumae, Shibuya, Tokyo, 150-8011, Japan
ReceiVed December 25, 2007
We synthesized and evaluated various [2-(4-quinolyloxy)phenyl]methanone derivatives. These compounds
had novel chemical structures that were distinct from those of previously reported inhibitors. Biological
data suggested that these compounds inhibited transforming growth factor-ꢀ signaling by interacting with
the ATP-binding pocket of the transforming growth factor-ꢀ type I receptor kinase domain. Here, we report
on the synthesis and structure-activity relationships of the compounds in this series.
Introduction
suggested a requirement for a hydrogen bond acceptor at the
2-position to the quinolyloxy group on the phenyl ring. On the
basis of our experience and knowledge of kinase inhibitors and
6, we initiated a research program targeting selective inhibitors
of the TGF-ꢀ RI kinase.
Transforming growth factor-ꢀ (TGF-ꢀ) is a cytokine that
plays important roles in cell differentiation, wound healing,
and regeneration. TGF-ꢀ signaling is mediated by two types
of transmembrane receptor serine/threonine kinase: types I
(RI) and II (RII).1,2 TGF-ꢀ binding to RII recruits and
activates the TGF-ꢀ RI kinase (activin receptor-like kinase
5 (ALK5)). Activated ALK5 phosphorylates a subset of
downstream signaling molecules, Smad2 and Smad3, allow-
ing them to bind to the commonly mediated Smad4.1,2 This
complex is shuttled into the nucleus and regulates the transcrip-
tion of target genes.1,2 The deregulation of TGF-ꢀ signaling
has been implicated in the pathogenesis of various diseases,
including fibrosis,3 atherosclerosis,4 and cancer.5–7 The inhibition
of TGF-ꢀ signaling with a small-molecule compound is thus
expected to be a practical method for the treatment of the
diseases mentioned above. Some research groups have reported
on small-molecule inhibitors of the TGF-ꢀ receptor kinase.8 The
chemical structures of many of these inhibitors possess a similar
topology, comprising a five-membered azole scaffold attached
by two or three aryl groups.
We have previously reported on several inhibitors of receptor
tyrosine kinases, including the platelet-derived growth factor
receptor (PDGFR),9 fibroblast growth factor receptor 2 (FGF-
R2),10 and vascular endothelial growth factor receptor 2
(VEGFR-2)11 (Figure 1). Our initial screening for TGF-ꢀ
signaling inhibitors using the Smad2/3 reporter assay identified
some of our in-house quinoline compounds as potential hits.
Among these, 6 was selected as a seed compound after an in
vitro nonspecific toxicological study (Figure 1).
Chemistry
The representative processes used to synthesize [2-(4-
quinolyloxy)phenyl]methanone derivatives are illustrated in
Schemes 1 and 2. 4-Chloro-6,7-dimethoxyquinoline (7) was
prepared as previously reported,11 and a coupling reaction with
the phenols, purchased or synthesized, produced the phenoxy-
quinoline derivatives (Scheme 1). 2-(4-Quinolyloxy)benzalde-
hyde (9a) was prepared using 2-hydroxybenzaldehyde under
the same coupling reaction conditions. The Grignard reaction
followed by oxidation produced compounds with a range of
alkylphenylketones, such as 9b (Scheme 2).
Results and Discussion
All new compounds were tested in the Smad2/3 reporter
assay. Initial structure-activity relationship (SAR) studies were
started with the introduction of small groups to the benzophe-
none moiety. As shown in Table 1, the introduction of a methyl
group at R1 increased the potency of 9c. A similar tendency
was observed with halogen analogues 9d and 9e, although the
improvement in activity was not significant. Preliminary com-
putational modeling suggested that these small groups occupied
a hydrophobic pocket unique to TGF-ꢀ receptors. Interestingly,
the methoxy group of 9f led to a significant reduction in activity.
The introduction of small groups at R2 caused a slight decrease
in activity (9g, 9h, and 9i), while significantly reduced activity
was observed when a bulky group was attached at R3 in 9j.
In order to improve the druglike features of these compounds,
acetophenone rather than benzophenone derivatives were syn-
thesized and tested in the Smad2/3 reporter assay. Table 2 shows
the SARs of compounds with various acyl groups at the
2-position of the phenyl ring. Even small alkyl groups, such as
ethyl (9m), could replace the phenyl ring of 9c without a loss
of activity, while the benzaldehyde analogue 9k showed
comparatively little inhibition. Sterically hindered alkyl groups
(9n) and heterocyclic groups (9p and 9q) caused a slight
decrease in the activity. None of the amide derivatives, as
represented by 9o, showed significant inhibition.
The resulting series of compounds inhibited the TGF-ꢀ RI
kinase in an ATP-competitive manner,12 similar to all of the
quinoline derivatives previously reported by our group. Our
preliminary computational modeling using 6 and the kinase
domain of TGF-ꢀ RI suggested the presence of an essential
hydrogen bond between the nitrogen atom at the 1-position of
quinoline and the backbone N-H group in the hinge region of
the kinase domain. This interaction appeared to mimic the
binding of an ATP molecule to the pocket. Interestingly, the
reduction of the carbonyl group to an alcohol group or to an
alkyl group significantly reduced the potency. This observation
Next we examined the substituent effect at the 4-position and
5-position on the phenyl ring. The results are summarized in
* To whom correspondence should be addressed. Phone: +81-3-5485-
6294. Fax: +81-3-3499-6152. E-mail: tshimizu@kirin.co.jp.
10.1021/jm701626z CCC: $40.75
2008 American Chemical Society
Published on Web 05/15/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3327
Figure 1. Quinoline-derived kinase inhibitors.
Scheme 1. Synthesis of Phenoxyquinoline Derivativesa
a Reagents and conditions: (a) 4-dimethylaminopyridine, o-dichlorobenzene, 130-180 °C.
Scheme 2. Synthesis of Phenoxyquinoline Derivativesa
a Reagents and conditions: (a) 4-dimethylaminopyridine, chlorobenzene, 130 °C; (b) EtMgBr, THF, -78 °C; (c) N-tert-butylbenzenesulfinimidoyl chloride,
DBU, CH2Cl2, -78 °C, then 0 °C.
Table 3. We demonstrated that the methyl group at R1 is crucial
for the activity with acetophenone derivatives (9r). Large
functional groups are not permitted at R1 (9v, 9x, and 9y). R2
must be no bigger than the methyl group, and even the methoxy
analogues, 9aa and 9ad, showed decreased activity. Interest-
ingly, with regard to the acetophenone derivatives, the com-
pound with the methoxy group introduced at R1, which
significantly decreased the activity of the benzophenone deriva-
tives, retained its activity (9w). Among these acetophenone
analogues, the compounds with small substituents at R1 and
R2 showed the best potency (9ab and 9ac). Combined with the
SARs of the phenylpropanone derivatives (9b, 9m, and 9ae),
these observations shed light on the size of the binding pocket
and suggested that the whole ketone moiety should fit within
it.
Some of the representative compounds were tested for
selectivity against the c-Src kinase (Table 4). Generally, the
acetophenone derivatives showed higher selectivity than the
benzophenone derivatives. The methyl group at the R1 position
increased the selectivity against the c-Src kinase. This observa-
tion was supported by the preliminary computational docking
model, which indicated that the methyl group occupied the
hydrophobic pocket and that a small alkyl group at this position
contributed to the activity and the selectivity.

3328 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Table 1. SARs of Benzophenone Derivatives
Brief Articles
Table 4. Selectivity Profile and ALK5 Kinase Inhibition of
Representative Compounds
compd
R1
R2
R3
Smad2/3 reporter assay, IC50 (µM)
IC50 (µM)
6
H
Me
Cl
Br
MeO
H
Me
Cl
Me
H
H
H
H
H
H
H
H
H
4.7
0.64
2.7
9ca
9d
9e
9f
Smad2/3 reporter
c-Src
ALK5
kinase
compd
R
R1
R2
assay
kinase
3.2
6
Ph
Ph
Me
Me
H
H
H
H
Me
4.7
0.64
2.3
4.5
1.7
8.4
13
16
H
>10
9ca
9l
Me
Me
Me
2.7
4.4
0.63
9g
9ha
9i
MeO
Me
Me
H
H
H
4.9
1.4
2.1
8.5
9ac
0.37
H
9j
tBu
a Hydrochloride salt was used.
a Hydrochloride salt was used.
Table 2. SARs of Acyl Derivatives
compd
R
Smad2/3 reporter assay, IC50 (µM)
9ca
9k
9l
9m
9n
9o
9p
9q
Ph
H
Me
Et
c-Pen
1-piperidinyl
2-furyl
5-isooxazoyl
0.64
>10
2.3
0.62
2.2
>10
Figure 2. Inhibition of Smad2 phosphorylation. Footnote “a” indicates
that the hydrochloride salt was used.
To confirm that the series of compounds inhibited TGF-ꢀ
signaling, we examined their effects against Smad2 phospho-
rylation by TGF-ꢀ in vitro using cultured cells. Smad2 or
Smad3, which is rapidly phosphorylated by TGF-ꢀ RI bound
to TGF-ꢀ, is known to function as a signal transducer of TGF-ꢀ
signaling. Our results showed that 9c and 9ac inhibited Smad2
phosphorylation by TGF-ꢀ in a dose-dependent manner (Figure
2). The inhibitory activity (IC50) against Smad2 phosphorylation
was around 0.5 µM, which was similar to the values seen in
the reporter assay. Because we observed rapid cellular events
30 min after TGF-ꢀ stimulation, these results also support the
hypothesis that both of the compounds directly interacted with
TGF-ꢀ RI or TGF-ꢀ RII.
2.7
2.5
a Hydrochloride salt was used.
Table 3. SARs of Acetophenone Derivatives
compd
R
R1
R2
Smad2/3 reporter assay, IC50 (µM)
9l
9r
9s
9t
9u
9v
9w
9x
9y
9z
9aa
9ab
9ac
9ad
9m
9b
9ae
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Me
H
F
Et
Pr
Bu
MeO
EtO
PrO
H
H
H
H
H
H
H
H
H
2.3
>10
3.8
Conclusion
We discovered a novel series of TGF-ꢀ signaling inhibitors,
the members of which appeared to inhibit the TGF-ꢀ RI kinase
in an ATP-competitive manner. Using the unique hydrophobic
pocket of the TGF-ꢀ RI kinase domain, we found that the
compounds in this series showed good inhibitory activity and
good selectivity against the c-Src kinase when appropriate
substituents were attached to the phenyl ring. The representative
compounds 9c and 9ac inhibited Smad2 phosphorylation by
TGF-ꢀ in a dose-dependent manner. These compounds are
expected to show therapeutic efficacy against the diseases areas
that are reported to correlate with abnormality of TGF-ꢀ
signaling.
0.83
0.54
2.2
0.59
5.6
H
>10
Me
MeO
Me
Me
MeO
H
5.5
8.8
H
Cl
0.42
0.37
>10
0.62
1.1
Me
MeO
Me
MeO
H
Et
Et
H
MeO
1.8
Experimental Section
Table 4 also shows the results of the ALK5 kinase inhibition
assay, which were consistent with those of the Smad2/3 reporter
assay. These observations supported the notion that the com-
pounds in this series possessed similar cell permeability.
Smad2/3 Reporter Assay. The TGF-ꢀ inhibitory activity of the
compounds was evaluated according to the method previously
described.13 A luciferase reporter plasmid (p(SBE)4-Luc) driven
by a promoter comprising four tandem binding sequences of

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3329
Smad2/3 was used to detect TGF-ꢀ signaling. The reporter plasmid
was transduced into human lung cancer epithelial cells (A549,
American type Culture Collection [ATCC]) to establish a stable
cell line expressing the reporter gene. A test compound and TGF-
ꢀ1 (2 ng/ml) were added to the cells, and the mixture was cultured
for 4 h. The luciferase activity of the cells was then measured by
a chemiluminescence method (Steady Glo luciferase assay system,
Promega).
6.39 (d, J ) 5.1 Hz, 1H), 7.07-7.15 (m, 2H), 7.39 (d, J ) 2.2 Hz,
1H), 7.45 (s, 1H), 7.57 (s, 1H), 8.49 (d, J ) 5.1 Hz, 1H); MS
(ESI) m/z 368 (M + 1)+; purity 99% (method D, tR ) 6.71 min),
98% (method I, tR ) 8.23 min).
Acknowledgment. We thank Atsuko Kobayashi for her
technical support with HPLC analysis.
Supporting Information Available: Details on general experi-
mental methods, biological assays, the synthesis of 9c-ae, and their
analytical data. This material is available free of charge via the
Internet at http://pubs.acs.org.
{2-[(6,7-Dimethoxy-4-quinolyl)oxy]phenyl}(phenyl)methanone (6).
7 (58 mg), 2-hydroxybenzophenone (271 mg), and 4-dimethylami-
nopyridine (166 mg) were mixed in o-dichlorobenzene (5 mL) and
then stirred at 160 °C for 4 h. After cooling to room temperature,
the mixture was concentrated. Chloroform was added, and the
solution was washed with 1 N NaOH solution and brine and then
dried over sodium sulfate. The solution was concentrated, and the
residue was purified by column chromatography (elution with
chloroform, followed by acetone/hexanes) to give 6 (100 mg,
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1-{2-[(6,7-Dimethoxy-4-quinolyl)oxy]-5-methoxyphenyl}-1-pro-
panone (9b). 7 (2.23 g), 8a (6.08 g), and 4-dimethylaminopyridine
(4.88 g) were mixed in chlorobenzene (40 mL) and then stirred at
130 °C overnight. After the mixture was cooled to room temper-
ature, H2O was added. The mixture was extracted with AcOEt, and
the organic layer was washed with H2O and brine and then dried
over sodium sulfate. The solution was concentrated, and the residue
was purified by column chromatography (elution with AcOEt/
hexanes) to give 9a (2.72 g, yield 80%). 9a (140 mg) was dissolved
in tetrahydrofuran (2 mL), and the solution was cooled to -78 °C.
A solution of 0.96 M ethylmagnesium bromide in tetrahydrofuran
(0.7 mL) was slowly added to the solution. After the mixture was
stirred at -78 °C for 1 h, an aqueous solution of ammonium
chloride was added, and the mixture was extracted with AcOEt.
The organic layer was washed with brine and then dried over
magnesium sulfate. The solution was concentrated, and the residue
was purified by column chromatography (elution with acetone/
hexanes) to give 10 (75 mg, yield 49%). 10 (66 mg) was dissolved
in dichloromethane (2 mL), and 1,8-diazabicyclo[5.4.0]-7-undecene
(72 mg) was added. The mixture was cooled to -78 °C, and a
solution of N-tert-butylbenzenesulfinimidoyl chloride (71 mg) in
dichloromethane was added. The mixture was stirred at -78 °C
for 30 min and then at 0 °C for 30 min. H2O was added, and the
mixture was extracted with chloroform. The organic layer was
washed with H2O and then dried over sodium sulfate. The solution
was concentrated, and the residue was purified by column chro-
matography (elution with acetone/hexanes) to give 9b (56 mg, yield
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1
86%). H NMR (CDCl3, 400 MHz) δ 1.05 (t, J ) 7.3 Hz, 3H),
2.88 (q, J ) 7.3 Hz, 2H), 3.89 (s, 3H), 4.06 (s, 3H), 4.06 (s, 3H),
JM701626Z