Orally active anti-proliferation agents: Novel diphenylamine derivatives as FGF-R2 autophosphorylation inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.12.019

(6,7-Disubstituted-quinolin-4-yloxy-phenyl)(4-substituted-phenyl)amine derivatives were synthesized and evaluated by a cellular autophosphorylation assay for FGF-R2 in the human scirrhous gastric carcinoma cell line, OCUM-2MD3. We also performed metabolic

Full text of DOI:10.1016/j.bmcl.2003.12.019

Bioorganic & Medicinal Chemistry Letters 14 (2004) 875–879
Orally active anti-proliferation agents: novel diphenylamine
derivatives as FGF-R2 autophosphorylation inhibitors
Toshiyuki Shimizu,* Yasunari Fujiwara, Tatsushi Osawa, Teruyuki Sakai, Kinya Kubo,
Kazuo Kubo, Tsuyoshi Nishitoba, Kaname Kimura, Terufumi Senga, Hideko Murooka,
Akemi Iwai, Kayoko Fukushima, Tetsuya Yoshino and Atsushi Miwa
Pharmaceutical Research Laboratories, Kirin Brewery Co., Ltd., 3 Miyahara, Takasaki, Gunma, 370-1295, Japan
Received 2 October 2003; revised 1 December 2003; accepted 3 December 2003
Abstract—(6,7-Disubstituted-quinolin-4-yloxy-phenyl)(4-substituted-phenyl)amine derivatives were synthesized and evaluated by a
cellular autophosphorylation assay for FGF-R2 in the human scirrhous gastric carcinoma cell line, OCUM-2MD3. We also
performed metabolic stability studies showing that substitutions at the 7-position of quinoline affect its biological stability. In this
study, we achieved a remarkable improvement in the solubility and metabolic stability of the diphenylamine derivative. The most
promising compound 15e showed a significant decrease in tumor volume when orally administered.
# 2003 Elsevier Ltd. All rights reserved.
Fibroblast growth factors (FGFs) are thought to play
important roles in angiogenesis, wound healing as well
as some malignancies.1 It has been reported that an
inappropriate expression of FGFs or their receptors
(FGFRs) could contribute to a variety of pathologies,
such as tumor neovascularization, diabetic retinopathy,
atherosclerosis and rheumatoid arthritis.2 FGFRs con-
sist of four related receptor tyrosine kinases, FGF-R1
(Flg), -R2 (Bek), -R3 and -R4. Among them, the over-
expression of FGF-R2 has been implicated with the poor
prognosis in some types of cancers, including scirrhous
gastric carcinoma.3 Some groups have reported small
molecule compounds that inhibit FGF-R1,4 but no
small molecule inhibitor of FGF-R2 has been reported.
synthesizing and evaluating some derivatives of 1, we
found that replacement of the benzophenone moiety of
1 into the diphenylamine moiety 2 increased kinase
inhibitory activity (Table 1). Furthermore, 2 did not
inhibit the EGFR kinase activity, which indicated 2 is
not a non-specific kinase inhibitor. The ex vivo inhibi-
tory effects on the autophosphorylation of FGF-R2
were tested using blood serum collected from mice 5 h
after the oral administration of these compounds. As
shown in Table 1, only the diphenylamine derivative 2
showed potent inhibitory activity. Encouraged by these
results, we investigated the potency of the diphenylamine
Table 1. Inhibition of FGF-R2 autophosphorylation
We previously reported5 the synthesis and biological
evaluation of 6,7-disubstituted-4-phenoxyquinolines
and 6,7-disubstituted-4-phenoxyquinazolines. In these
studies, we found novel selective inhibitors of PDGFR
kinase and showed their potency as novel therapeutic
agents. During the course of the investigation on
PDGFR tyrosine kinase inhibitors, we found that 1
inhibited the autophosphorylation of FGF-R2 and
PDGFR with about the same IC50 value.5c After
Compd
X
In vitro
(IC50, nM)
Ex vivo
(% inhibition at 100 mg/kg)
1
2
3
4
CO
NH
O
190
40
440
>1000
3
70
NT
5
Keywords: Kinase inhibitor; Quinoline; Anti-proliferation; FGF-R2;
Bek.
CH2
* Corresponding author. Tel.: +81-27-346-9966; fax: +81-27-346-
8418; e-mail: tshimizu@kirin.co.jp
NT, not tested.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.019

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T. Shimizu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 875–879
Scheme 1. (a) Chlorobenzene, 120 ꢀC, 32–100%; (b) Pd(OH)2, H2 gas, Et3N, DMF or Zn, NH4Cl, EtOH/H2O, 55-79%; (c) Cu(OAc)2, Et3N,
CHCl3, 4–79%; (d) NaBH(OAc)3, DMF, 11%.
derivatives as novel anti-tumor agents. In this report, we
describe the synthesis and biological evaluation of
the diphenylamine derivatives as FGF-R2 inhibitors.
We also present the in vivo data of the optimized
compound and its potency as an anti-proliferation agent.
Table 2 shows the inhibitory effects of these compounds
on the autophosphorylation of FGF-R2, assayed with
the cellular ELISAsystem. First, the substituent effects
on both phenyl rings of diphenylamine were investi-
gated. These results are shown in Table 2. The results of
2 and 8a–e strongly supported the necessity of a hydro-
phobic bulky group in R4 to retain the inhibitory
activity. This part of the molecules could be considered
to be recognized by FGF-R2 so strictly that a minor
change would not be allowed to provide any inhibitory
activity. Similar results were obtained regarding the R1
and R2 substituents. Although retention in the IC50
value was observed with the fluoro-substituted com-
pounds 8i and 8j, none of the compounds showed a
significant increase in inhibitory activity.
We previously established an excellent synthetic route of
the 4-chloroquinoline derivatives.6 We adopted this
route to prepare the 6,7-disubstituted chloroquinolines
5. Atypical synthetic Scheme for the diphenylamine
derivatives is shown in Scheme 1. Heating 5 with nitro-
phenols in chlorobenzene afforded the 4-nitrophenoxy
derivatives 6. The nitro groups of 6 were reduced in
DMF using palladium or zinc to give the anilines
7, which were reacted with boronic acids in the presence
of copper diacetate and triethylamine to afford the
diphenylamines 8. 11 was synthesized by the reductive
alkylation of the aniline 9.
After we found that substitutions on these phenyl rings
were severely restricted, our focus was shifted to
modification of the methoxy group at the 7-position of
quinoline.
Scheme 2 shows the synthetic route of the compounds
containing a substituted alkoxy group at the 7-position.
After the preparation of 12, the benzyl group was
removed using methanesulfonic acid in trifluoroacetic
acid. The treatment of 13 with bromochloroalkanes or
epibromohydrin, followed by nucleophilic substitution
with amines gave the amino-substituted alkoxy
compounds 15 and 17, respectively.
It has been demonstrated that the metabolic stability
is drastically changed depending on substituents at the
7-position on quinoline or quinazoline.8 For this reason,
we evaluated the stability in the presence of microsomes
at the same time. In addition to this issue, we needed to
increase the solubility of the compounds because 2 was
only slightly soluble in water. Therefore, we selec-
ted secondary and tertiary amines to introduce at
the 7-position in order to obtain compounds with both
good metabolic stability and good solubility. These
results are summarized in Table 3. During the investi-
gation, we observed a tendency that compounds with a
Biological evaluations of the diphenylamine derivatives
were carried out using OCUM-2MD3, a human scirrhous
gastric carcinoma cell line, established by Yashiro et al.7
This cell line is reported to constitutively express activated
FGF-R2, and to be tumorigenic in athymic animals.

T. Shimizu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 875–879
877
Scheme 2. (a) MsOH, TFA, DMF, 80–92%; (b) Br(CH2)nCl, K2CO3, DMF, 90–100%; (c) R5R6NH, K2CO3, DMF, 80 ꢀC, 47–94%; (d)
epibromohydrin, K2CO3, DMF; (e) R5R6NH, K2CO3, DMF, 80 ꢀC, 81–89% (2 steps).
Table 2. Inhibitory activity on autophosphorylation of FGF-R2
terminal hydroxyl group (15c–g) were metabolically
stable. Compound 15e had the best stability as well as
good inhibitory activity. Furthermore, 15e was stable
under incubation with human liver microsomes for 10
min (Table 4). It was noteworthy that 78 and 89% of 15e
existed after a 40-min incubation with rat and human
liver microsomes, respectively. Therefore, 15e was selec-
ted to investigate its potency as an anti-tumor agent.
Compd
R1
R2
R3
R4
IC50 (nM)
2
H
H
H
H
H
H
H
Ph
H
H
H
H
H
H
H
H
—
tBu
H
40
8900
4600
>10,000
780
8a
8b
8c
8d
8e
8f
8g
8h
8i
H
H
Ph
H
H
H
H
H
OMe
iPr
H
H
200
Table 5 shows the solubility of 15e. Adrastic improve-
ment in the solubility from the initial compound 2 was
achieved with 15e, which was soluble even into water
for injection (WFI; JP grade) at 108mg/mL.
Me
H
H
tBu
tBu
tBu
tBu
tBu
tBu
—
100
Me
Me
H
>10,000
130
Me
F
63
8j
H
F
54
8k
11
CF3
—
H
>10,000
3400
The kinase specificity profile of 15e was also demon-
strated, and these results are shown in Table 6. Auto-
phosphorylation of each receptor tyrosine kinase was
—
Table 3. Inhibition of candidates to FGF-R2 autophosphorylation assay and their metabolic stabilities
Compd
R1
R2
n
R5
—
R6
—
IC50 (nM)
Metabolizationa (%)
2
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
F
—
3
2
2
2
2
2
2
—
—
40
350
200
71
23
88
260
70
200
71
90
28
9
46
51
97
92
60
78
72
15a
15b
15c
15d
15eb
15f
15g
17a
17bb
–(CH2)5–
–(CH2)2O(CH2)2–
–(CH2)2OH
–(CH2)2OH
–(CH2)2CH(CH2OH)(CH2)2–
–(CH2)2OH
–(CH2)2OH
–(CH2)2OH
H
H
H
H
H
–(CH2)5–
–(CH2)2O(CH2)2–
a Percentage of parent compounds remaining after incubation with rat liver microsomes for 10 min at 37 ꢀC.
b Hydrochloride salt was used.

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T. Shimizu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 875–879
measured in intact cells.9 15e showed a decent inhibitory
activity for the autophosphorylation of KDR, PDGF-
Rb, c-Kit as well as FGF-R2. On the contrary, no activ-
ity was observed against the EGFR, IGF-1R and c-Met
tyrosine kinases at 10 mM. These results indicate that 15e
possesses a favorable specificity for the PDGF-R family.
manner. It is noteworthy that 15e did not inhibit the
proliferation of IEC-6, the rat ileac epithelial cell line,
which indicates that the inhibitory effect of 15e is selec-
tive among cell lines.
With these encouraging data in hand, the anti-tumor
effects of compound 15e were evaluated in a nude mice
xenograft model bearing OCUM-2MD3.11 2 did not
show any anti-tumor activity at 30 mg/kg, presumably
due to its poor solubility. On the contrary, as shown in
Figure 2, the oral administration of 15e, once daily for
five consecutive days, potently inhibited the growth of
established tumors in a dose-dependent manner. In the
The effects of 15e on the proliferation of cultured cells
were evaluated using the WST-1 colorimetric assay sys-
tem10 after a three-day incubation of cells with 10%
FBS. As shown in Figure 1, 15e potently inhibited the
proliferation of the human scirrhous gastric carcinoma
cell line OCUM-2MD3 in a concentration-dependent
Table 4. Metabolic stability profiles of 15e to human or SD rat
microsomes
Species
Rat
Time (min)
Metabolizationa (%)
10
40
10
40
97
78
99
89
Human
a Percentage of parent compounds remaining after incubation with rat
or human liver microsomes at 37 ꢀC.
Table 5. Comparison of solubilities
Compd
WFI (mg/mL)
pH1.2a (mg/mL)
2
15e
0
108
3
802
a An aqueous solution of NaCl (0.2%, w/v) and HCl (0.07N) was used.
Table 6. Kinase specificity of 15e
RTK
IC50 (nM)
FGF-R2
KDR
88
83
PDGFR
c-Kit
100
480
EGFR
IGF-1R
c-Met
>10,000
>10,000
>10,000
Figure 1. Inhibitory effects of 15e on the proliferation of cultured
cells: (1) Abs. (450-650) values were in proportion to the number of
cells. (2) Hydrochloride salt of 15e was used.
Figure 2. Anti-tumor effects of 15e: (1) hydrochloride salt of 15e was used.

T. Shimizu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 875–879
879
Figure 3. Anti-tumor effects of 15e: (1) hydrochloride salt of 15e was used.
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25 mg/kg and 50 mg/kg treatment groups, the anti-
tumor effects of 15e were sustained with a tendency of
tumor regression for nearly one week after discontinua-
tion of the administration. In the 25 mg/kg treatment
group, the administration of 15e was restarted 24 days
after the discontinuation of the initial administration
(Fig. 3). Re-administration of 15e, 25 mg/kg once daily
for five consecutive days, induced tumor regression.
This effect was sustained for nearly one week after the
discontinuation of the re-administration. In all treatment
groups, no abnormal body weights were detected. These
data suggest that the oral administration of 15e may be
able to decrease the volume of tumors in cancer patients.
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In summary, we described the synthesis of diphenyl-
amine derivatives and their inhibitory activity on the
autophosphorylation of FGF-R2. We also showed that
15e possessed anti-tumor activity in vivo. As shown in
Table 5, 15e possesses kinase inhibitory activity against
other receptor tyrosine kinases, which have been repor-
ted to correlate with cell proliferation.12 With its
encouraging anti-tumor effects data in a nude mice
xenograft model bearing OCUM-2MD3, 15e can be
expected to be an effective therapeutic agent against a
variety of tumors. Investigations on its potency will be
separately reported.
6. Osawa, T.; Kubo, K.; Murooka, H.; Nakajima, T.
WO9847873.
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M.; Chung, Y. S.; Nishimura, S.; Inoue, T.; Sowa, M.
Clin. Exp. Metastasis 1996, 14, 43.
Acknowledgements
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The authors wish to thank Dr. K. Hirakawa and Dr. M.
Yashiro (Department of Surgical Oncology, Graduate
School of Medicine, Osaka City University, Japan) for
kindly providing the OCUM-2MD3 cell line.
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