Synthesis and structure-activity relationship of diarylamide urea derivatives as selective inhibitors of the proliferation of human coronary artery smooth muscle cells.

Article abstract of DOI:10.1016/S0968-0896(02)00012-3

A series of diarylamide urea derivatives were synthesized and evaluated for their inhibitory activities against human coronary artery smooth muscle cells (SMCs) and human coronary artery endothelial cells (ECs). Compound 2o was superior to the lead compound, Tranilast, in terms of its potency of the inhibitory activity and cell selectivity.

Full text of DOI:10.1016/S0968-0896(02)00012-3

Bioorganic & Medicinal Chemistry 10 (2002) 1865–1871
Synthesis and Structure–Activity Relationship of Diarylamide
Urea Derivatives as Selective Inhibitors of the Proliferation of
Human Coronary Artery Smooth Muscle Cells
Haruhisa Ogita, Yoshiaki Isobe, Haruo Takaku, Rena Sekine,
Yuso Goto, Satoru Misawa and Hideya Hayashi*
Pharmaceuticals & Biotechnology Laboratory, Japan Energy Corporation, 3-17-35, Niizo-Minami,
Toda-shi, Saitama 335-8502, Japan
Received 22 October 2001; accepted 21 December 2001
Abstract—A series of diarylamide urea derivatives were synthesized and evaluated for their inhibitory activities against human
coronary artery smooth muscle cells (SMCs) and human coronary artery endothelial cells (ECs). Compound 2o was superior to the
lead compound, Tranilast, in terms of its potency of the inhibitory activity and cell selectivity. # 2002 Elsevier Science Ltd. All
rights reserved.
Introduction
selective inhibition of SMCs proliferation.7 According
to the report of Tranilast analogues by the Kissei
group8 and our research,7 methoxy groups substitution
was more potent than the other substituents such as
halogen, hydroxy, nitro, alkyl and amino groups. On
the other hand, Kirin Brewery research group also
reported a series of quinazoline analogues exhibiting potent
inhibition of PDGF receptor autophosphorylation.9,10
According to their reports, over 100-fold elevation of
the activity was observed when a ureide group replaced
the methoxy groups on the phenoxy moiety.10 In fact,
we actually evaluated the inhibitory activities of Kirin
compounds (3a–b) on PDGF-induced proliferation of
SMCs, and the elevation of the activity was also
observed (3a: IC50=0.5 mM, 3b: 55% inhibition @
0.016 mM). In view of the similarity of the structure
between their compounds and ours, we speculated that
the introduction of a ureide group to the B ring of our
diarylamide derivatives might make the inhibitory
activity more potent (Fig. 1). In this paper, we report
the results of our preliminary study on a series of these
diarylamide urea derivatives.
Proliferation of SMCs plays a cardinal important part
in restenosis and in the progression of atherosclerosis.1
Many growth factors have been reported to induce the
proliferation of SMCs in vitro and in vivo.2 Among them,
platelet-derived growth factor (PDGF) is a momentous
regulator of SMCs action.3 Recently, Tranilast ([N-(3,4-
dimethoxycinnamoyl]) anthranilic acid) was reported to
noticeably inhibit the proliferation of SMCs and ECs in
vitro.4 Besides, a double-blind, large-scale, multicenter
trial demonstrated the potent preventive effect of Tra-
nilast on restenosis [restenosis rate 14.7% (Tranilast 600
mg/day for 3 months, n=68) versus 46.5% (placebo,
n=71); P <0.001] after percutaneous transluminal cor-
onary angioplasty.5 However, some of the patients had
been liver dysfunction in this trial. We speculated that
these frequent and severe side effects may have been
caused by the high dose of Tranilast. In addition, the
selective inhibitiors of the proliferation of SMCs over
that of ECs was more preferable for the treatment of
restenosis than the non-selective inhibitors like Tranilast.6
Thus, we have tried to develop more potent and SMCs-
selective compounds by the modifying Tranilast.
Chemistry
Previously, we reported a series of diarylamide deriva-
tives, 1a and 1b (Fig. 1), exhibiting potent and highly
The synthesis of urea derivatives 2a–2s are outlined in
Scheme 1.
Acid-catalytic ester formation with sulfuric acid or
amide formation with thionyl chloride (SOCl2) of 4,5-
*Corresponding author. Tel.:+81-48-433-2194; fax:+81-48-433-1605;
e-mail: hhayashi@j-energy.co.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00012-3

1866
H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 1865–1871
Figure 1.
dimethoxy-2-nitro benzoic acid, followed by catalytic
hydrogenation with 5% Pd/C gave the corresponding
compounds 4a–b in a good yield (70–75%). Condensa-
tion of 4a–b with 4-nitrobenzoyl chloride in the presence
of triethylamine, followed by catalytic hydrogenation
with 5% Pd/C gave 5a–b in a good yield (70–90%), and
5c was quantitatively obtained by the condensation of
4b with 4-methylaminobenzoic acid by use of 1-ethyl-3-
[3-(dimethylamino)propyl]-carbodiimide hydrochloride
group substitution at the N atom in the ureide group (2c,
2d, 2e) resulted in less inhibition. The solubility of 2b in
various organic solvents as well as in water was very poor.
Therefore, in view of this property, we concluded that
2b was not preferable as a drug candidate. On the
other hand, the solubility of 2a was good and the stability
of 2a in the assay medium was confirmed by HPLC
analysis although the ester bond was afraid to be
cleaved. Thus, the further examination of the substituents
of the urea moiety was performed by using 2a for
modification.
.
salt (WSC Cl) as a condensation reagent. Finally, con-
densation of 5a–c with the corresponding isocyanate or
N-phenyl-N-methylcarbamoylchloride gave the desired
urea derivatives.
Table 2 presents the results of the SAR study on sub-
stituents of the ureide moiety. At first, we examined the
effect of substituents at the para position of the phenyl
ring. Acetyl (2k) and amino (2h) groups there resulted in
increased activity. Earlier, Kubo et al. reported that 3-
methoxy group substitution on the phenyl ring resulted
in the most potent inhibition,10 and also we found that
methoxy group substitution on the B ring increased the
activity.7 Therefore, we examined the effect of the num-
ber and position of methoxy group substituents. As a
result, 3,4,5-trisubstituted compound (2o) was the most
potent (IC50=0.04 mM), and was about 600-fold more
potent than Tranilast. We also evaluated the effect of
most of the compounds against the proliferation of ECs.
Most of the evaluated compounds displayed selectivity
about 4ꢀ140 times greater for SMCs. However, benzyl
(2p), n-butyl (2q), cyclohexyl (2r) and 3-pyridyl (2s)
groups instead of the phenyl ring in the ureide moiety
showed low selectivity for SMCs in spite of almost the
same inhibitory activity for SMCs. In particular, the
IC50 values of 2o for SMCs and for ECs were 0.04 and
5.4 mM, respectively, displaying selectivity about 135 times
greater for SMCs. In contrast, the IC50 values of Tranilast
toward SMCs and ECs were 25 and 19 mM, respectively,
indicating a selectivity of about 0.76 times for SMCs.
Thus, compound 2o was superior to Tranilast, as were
Biology
A series of these compounds were evaluated for their
inhibitory activities on PDGF-induced proliferation of
SMCs and FBS-induced the proliferation of ECs. Inhi-
bition of proliferation of these cells was determined by
3H-thymidine incorporation as previously reported with
a minor modification.11
Results and Discussion
The inhibitory activities of 2a–s on PDGF-induced
proliferation of SMCs are shown in Tables 1and 2.
Table 1presents the result of the structure–activity
relationship (SAR) study on phenyl urea derivatives. As
we expected, the introduction of the ureide group (2a,
2b) to the B ring made the inhibitory activity more
potent than that of 1a and 1b. Similar to the case of
diarylamide derivatives 1a and 1b, the presence of the
ethoxy carbonyl group (2a) or the carbamoyl group (2b)
in the A ring results in almost the same activity. Methyl

H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 1865–1871
1867
our lead compounds 1a and 1b, in the strength of the
activity and cell selectivity.
other hand, in the case of quinazoline compounds, the
Kirin Brewery group had reported a potent inhibition of
PDGF receptor autophosphorylation.10 Therefore, we
also examined their effect on PDGF receptor autopho-
sphorylation but unexpectedly obtained a negative
result. Thus, the main mechanism of the action of these
urea derivatives is different from that of Tranilast and
quinazoline derivatives. Thus, the search for the main
In examining the mechanism of action of Tranilast,12
several researchers showed that Tranilast increased the
levels of p21and p53 protein and arrested SMCs at the
G0/G1phase. However, our compounds had no influ-
ence on the regulation of p21and p53 protein. On the
Scheme 1. Synthesis of compounds 2a–2s. Reagents and conditions: (a) EtOH, cH2SO4; (b) SOCl2 then NH3aq/CH2Cl2; (c) H2, 5% Pd/C, EtOH;
(d) 4-NO2–PhCOCl, Et3N/CH2Cl2; (e) RNCO, DMAP/THF; (f) PhNMeCOCl, iPr2NEt/THF; (g) 4-NHMe–PhCO2H, HOBt, Et3N, DMF,
WSCꢁ HCl.
Table 1. IC50 values of the compounds 2a–e for inhibition of proliferation of SMCs and ECs
No.
R1
R2
R3
SMCs IC50 (mM)a
ECs IC50 (mM)b
[ECs]/[SMCs]
0.76
Tranilast
25
0.5
19
n.t.
n.t.
6.0
>10
1.5
4.4
3a
3b
1a
1b
2a
2b
2c
2d
2e
<0.016
0.85
0.72
0.40
0.40
>2.0
>2.0
>2.0
7.1
>14
3.7
OEt
NH2
NH2
NH2
NH2
H
H
Me
H
H
H
H
Me
Me
11
n.t.
n.t.
n.t.
Me
n.t., not tested.
aInhibitory activity against the proliferation of SMCs induced by PDGF-BB (20 ng/mL).
bInhibitory activity against the proliferation of ECs induced by 5% FBS.

1868
H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 1865–1871
Table 2. IC50 values of the compounds 2f–s for inhibition of proliferation of SMCs and ECs
No.
R
SMCs
IC50 (mM)a
ECs
IC50 (mM)b
[ECs]/[SMCs]
0.76
Tranilast
3a
3b
1a
1b
2a
2f
2g
2h
2i
2j
2k
2l
2m
2n
25
0.5
19
n.t.
n.t.
6.0
>10
1.5
n.t.
n.t.
1
n.t.
>2.0
6.16.7
3.2
<0.016
0.85
0.72
0.40
1.7
7.1
>14
3.8
Ph
4-OMe–Ph
4-NO2-Ph
4-NH2–Ph
4-Me–Ph
4-F–Ph
0.50
0.10
0.33
0.40
0.090
0.23
0.28
0.094
0.040
0.10
0.23
0.34
0.19
10
>5.0
4-Ac–Ph
4-CO2Et–Ph
2-OMe–Ph
3-OMe–Ph
3,4,5-(OMe)3–Ph
Bn
14
5.4
17
135
12
4.8
4.1
0.16
1.51
1.6
5.4
1.2
1.1
1.4
0.030
2o
2p
2q
2r
n-Bu
c-Hex
3-Py
2s
n.t., not tested.
aInhibitory activity against the proliferation of SMCs induced by PDGF-BB (20 ng/mL).
bInhibitory activity against the proliferation of ECs induced by 5% FBS.
target molecules of our compounds and the further
structural optimization of our derivatives are underway.
To a solution of 3a (0.30 g, 1.18 mmol) in ethanol (20
mL) was added 5% Pd/C (0.06 g), and the mixture was
stirred at room temperature under H2 atmosphere for
14 h. The reaction mixture was then filtered and con-
centrated to give 0.26 g (1.16 mmol) of 4a as a white
solid with a yield of 98%. To a solution of 4a (0.26 g,
1.16 mmol) in CH2Cl2 (20 mL) 4-nitrobenzoyl chloride
(0.27 g, 1.47 mmol) and triethylamine (0.25 mL, 1.79
mmol) were added, and the mixture was stirred for 30 min
at room temperature. The reaction mixture was poured
into saturated NaHCO3aq and extracted with CH2Cl2,
after which the organic layer was washed with brine, dried
over MgSO4, filtered and concentrated. The residue was
redissolved in methanol (100 mL), 5% Pd/C (0.05 g) was
added to this solution and the mixture was stirred under
H2 atmosphere for 32 h. The reaction mixture was filtered,
and concentrated to give 0.28 g (0.81mmol) of 5a as a
yellow solid with a yield of 70%, by two steps.
Experimental
Chemistry
In general, reagents and solvents were used as purchased
without further purification. Column chromatography
was performed on Walkogel C-200 (WaKo, 70–150 mm) or
Walkogel C-300 (WaKo, 45–75 mm). Melting points were
measured with a BUCHI 535 melting point apparatus and
left uncorrected. Proton NMR spectra were recorded on a
JOEL GSX270 FT NMR spectrometer. Chemical shifts
were expressed in d (ppm) from internal standard tetra-
methylsilane. TOFMS were recorded on a Kompact
MALDI III spectrometer. Elemental analyses were per-
formed by the Toray Research Center and were within
0.4% of the calculated values unless otherwise noted.
To a solution of 5a (0.09 g, 0.26 mmol) and dimethyl-
aminopyridine (0.05 g, 0.41mmol) in THF (20 mL) was
added phenylisocyanate (0.05 g, 0.42 mmol), and the
mixture was refluxed for 18 h. The reaction mixture was
poured into water and extracted with CH2Cl2, and the
organic layer was then washed with brine, dried over
MgSO4, filtered, and concentrated. The residue was
purified by silica gel column chromatography (CH2Cl2/
methanol=100:1ꢀ20:1) and washed with methanol to
give 0.08 g (0.17 mmol) of 2a as a white solid with a
Ethyl 4,5-dimethoxy-2-((4-((N-phenylcarbamoyl)amino)-
phenyl)carbonylamino)benzoate (2a). To a solution of
4,5-dimethoxy-2-nitro benzoic acid (0.75 g, 3.30 mmol)
in ethanol (100 mL) was added H2SO4 (3 mL), and the
mixture was refluxed for 18 h. To the reaction mixture
on ice was added 5% NaOH aq, and adjustment of the
pH 8 to save a solid. The obtained solid was washed
with H2O to give 0.53 g (2.08 mmol) of 3a as a white
solid with a yield of 63%.
yield of 65%. Mp: 232–236 ꢂC; H NMR (DMSO-d6) d
1

H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 1865–1871
1869
ppm: 1.35 (t, J=7.2 Hz, 3H), 3.80 (s, 3H), 3.88 (s, 3H),
4.37 (q, J=7.2 Hz, 3H), 6.99 (t, J=7.3 Hz, 1H), 7.30 (t,
J=8.1Hz, 2H), 7.48 (d, J=7.5 Hz, 2H), 7.48 (s, 1H),
7.67 (d, J=7.3 Hz, 2H), 7.90 (d, J=8.9 Hz, 2H), 8.45 (s,
1H), 9.05 (s, 1H), 9.31 (s, 1H), 11.75 (s, 1H); MS (TOF)
m/z=464 (M++H). Anal. calcd for C25H25N3O6: C
64.8, H 5.5, N 9.1. Found: C 64.8, H 5.4, N 9.4.
ethylamine (0.46 g, 3.6 mmol) in THF (10 mL) was
added N-phenyl-N-methylcarbamoyl chloride (0.16 g,
3.6 mmol), and the mixture was refluxed for 16 h. The
reaction mixture was poured into water and extracted
with CH2Cl2, after which the organic layer was washed
with brine, dried over MgSO4, filtered, and con-
centrated. The residue was purified by silica gel column
chromatography (CH2Cl2/methanol=100:1ꢀ20:1) and
washed with methanol to give 0.05 g (0.15 mmol) of 2d
as a white solid with a yield of 42%. Mp: 196–198 ꢂC;
1H NMR (DMSO-d6) d ppm 3.29 (s, 3H), 3.81(s, 3H),
3.83 (s, 3H), 7.27 (t, J=6.8 Hz, 1H), 7.44 (m, 5H), 7.63
(m,3H), 7.80 (d, J=8.9 Hz, 2H), 8.30 (s, 1H), 8.52 (s,
4,5-Dimethoxy-2-((4-((N-phenylcarbamoyl)amino)phenyl)-
carbonylamino)benzamide (2b). To a solution of 4,5-
dimethoxy-2-nitro benzoic acid (0.66 g, 2.90 mmol) in
CHCl3 (40 mL) was added SOCl2 (5 mL), and the mix-
ture was then refluxed for 6 h. The reaction mixture was
concentrated and redissolved in CH2Cl2 (20 mL). To this
solution was added aqueous NH3 (20 mL) in an ice bath,
and the mixture was vigorously stirred for 10 min at room
temperature. After the organic layer had been concentrated
and the residue redissolved in methanol (50 mL), 5% Pd/C
(0.05 g) was added to this solution, which was then stirred
under H2 atmosphere for 19 h. The reaction mixture was
filtered and concentrated to give 0.55 g (0.28 mmol) of 4b as
a white solid with a yield of 96%, by two steps.
1H), 8.53 (s, 1H), 13.18 (s, 1H); MS (TOF) m/z=449
+
.
(M +H). Anal. calcd for C24H24N4O5 0.1H2O: C 64.0,
H 5.4, N 12.5. Found: C 63.7, H 5.5, N 12.5.
4,5-Dimethoxy-2-((4-(methyl(N-methyl-N-phenylcarba-
moyl)amino)phenyl)carbonylamino)benzamide (2e). Com-
pound 2e was prepared from 5c in a manner similar to
that described for compound 2d with a yield of 36%.
Mp: 176–178 ꢂC; H NMR(DMSO-d6) d ppm: 3.12 (s,
1
3H), 3.18 (s, 3H), 3.81 (s, 3H), 3.83 (s, 3H), 7.00 (m,
3H), 7.12 (m, 4H), 7.44 (s,1H), 7.68 (m, 3H), 8.32 (s,
1H), 8.49 (s, 1H), 13.18 (s, 1H); MS (TOF) m/z=463
(M +H). Anal. calcd for C25H26N4O5 1.1H2O: C 62.4,
H 5.7, N, 11.6. Found: C 62.2, H 5.7, N 11.5.
Compound 2b was prepared from 4b in a manner simi-
lar to that described for compound 2a with a yield of
24%, by three steps. Mp: 232–236 ꢂC; 1H NMR
(DMSO-d6) d ppm. 81(s, 3H), 3.84 (s, 3H), 7.00 (t,
J=8.1Hz, 1H), 7.30 (t, J=8.4 Hz, 2H), 7.44 (s, 1H),
7.47 (d, J=7.9 Hz, 2H), 7.64 (m, 3H), 7.87 (d, J=8.6
Hz, 2H), 8.31 (s, 1H), 8.53 (s, 1H), 8.87 (s, 1H), 9.13 (s,
1H), 13.21 (s, 1H); MS (TOF) m/z=435 (M++H).
+
.
Ethyl 4,5-dimethoxy-2-((4-((N-(4-methoxyphenyl)carba-
moyl)amino)phenyl)carbonylamino)benzoate (2f). Com-
pound 2f was prepared from 5a in a manner similar to
that described for compound 2a with a yield of 83%.
.
Anal. calcd for C23H22N4O5 0.6H2O: C 62.5, H 5.2, N
12.7. Found: C 62.7, H 5.1, N 12.9.
Mp: 244–247 ꢂC; H NMR (DMSO-d6) d ppm: 1.35 (t,
1
J=7.2 Hz, 3H), 3.73 (s, 3H), 3.80 (s, 3H), 3.88 (s, 3H),
4.37 (q, J=7.2 Hz, 3H), 6.89 (d, J=9.2 Hz, 2H), 7.38
(d, J=8.6 Hz, 2H), 7.48 (s, 1H), 7.65 (d, J=8.9 Hz,
2H), 7.89 (d, J=8.9 Hz, 2H), 8.45 (s, 1H), 8.73 (s, 1H),
9.11 (s, 1H), 11.75 (s, 1H); MS (TOF) m/z=494
(M++H). Anal. calcd for C26H27N3O7: C 63.2, H 5.5,
N 8.5. Found: C 63.2, H 5.5, N 8.9.
4,5-Dimethoxy-2-((4-(methyl(N-phenylcarbamoyl)amino)-
phenyl)carbonylamino)benzamide (2c). To a solution of
4b (0.04 g, 0.20 mmol), 1-hydroxybenztriazole (0.03 g, 0.20
mmol), 4-methylaminobenzoic acid (0.03 g, 0.20 mmol),
and triethylamine (0.02 g, 0.20 mmol) in DMF (3 mL) was
added 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide
hydrochloride (0.04 g, 0.20 mmol) in an ice-bath, and the
mixture was stirred at room temperature for 50 h. After the
reaction mixture had been concentrated, the residue was
poured into water, and extracted with CH2Cl2, and then
the organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated. The residue was
purified by silica gel column chromatography (CH2Cl2/
methanol=100:1ꢀ20:1) to give 0.07 g (0.18 mmol) of
5c as a white solid with a yield of 91%.
Ethyl 4,5-dimethoxy-2-((4-((N-(4-nitrophenyl)carbamoyl)-
amino)phenyl)carbonylamino)benzoate (2g). Compound
2g was prepared from 5a in a manner similar to that
described for compound 2a with a yield of 63%. Mp:
235–236 ꢂC; 1H NMR (DMSO-d6) d ppm: 1.35 (t,
J=7.2 Hz, 3H), 3.80 (s, 3H), 3.88 (s, 3H), 4.37 (q,
J=7.2 Hz, 3H), 7.48 (s, 1H), 7.71 (m, 4H), 7.92 (d,
J=8.9 Hz, 2H), 8.22 (d, J=9.2 Hz, 2H), 8.43 (s, 1H),
9.40 (s, 1H), 9.65 (s, 1H), 11.76 (s, 1H). Anal. calcd for
.
Compound 2c was prepared from 5c in a manner simi-
lar to that described for compound 2a with a yield of
47%. Mp: 214–218 ꢂC; 1H NMR (DMSO-d6) d ppm
3.35 (s, 3H), 3.81(s, 3H), 3.85 (s, 3H), 6.97 (t, J=8.5
Hz, 1H), 7.25 (t, J=8.4 Hz, 2H), 7.45 (m, 5H), 7.68
(s,1H), 7.94 (d, J=8.1Hz, 2H), 8.33 (s, 1H), 8.53 (s,
C25H24N4O8 1.6H2O: C 55.9, H 5.1, N 10.4. Found: C
56.1, H 5.0, N 10.7.
Ethyl 2-((4-((N-(4-aminophenyl)carbamoyl)amino)phenyl)-
carbonyl amino)-4,5-dimethoxybenzoate (2h). To a solu-
tion of 2g (0.09 g, 0.18 mmol) in ethanol (10 mL) was
added 5% Pd/C (0.02 g), which was then stirred at room
temperature under a H2 atmosphere for 14 h. After the
reaction mixture had been filtered and concentrated, the
residue was purified by silica gel column chromato-
graphy (CH2Cl2/methanol=100:1ꢀ20:1) and washed
with methanol to give 0.03 g (0.06 mmol) of 2 h as
a light pink solid with a yield of 33%. Mp: 202–206;
1H), 8.59 (s, 1H), 13.32 (s, 1H); MS (TOF) m/z=449
+
.
(M +H). Anal. calcd for C24H24N4O5 2.6H2O: C 58.2,
H 5.9, N 11.3. Found: C 58.0, H 5.7, N 11.5.
4,5-Dimethoxy-2-((4-((N-methyl-N-phenylcarbamoyl)-
amino)phenyl)carbonylamino)benzamide (2d). To
solution of 5b (0.11 g, 0.36 mmol) and N,N-diisopropyl-
a

1870
H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 1865–1871
1H NMR (DMSO-d6) d ppm: 1.35 (t, J=7.2 Hz, 3H),
3.80 (s, 3H), 3.87 (s, 3H), 4.37 (q, J=7.2 Hz, 3H), 4.80
(s, 2H), 6.52 (d, J=8.1Hz, 2H), 7.10 (d, J=8.9 Hz,
2H), 7.48 (s, 1H), 7.63 (d, J=8.9 Hz, 2H), 7.87 (d,
J=8.9 Hz, 2H), 8.22 (d, J=9.2 Hz, 2H), 8.42 (s, 1H),
4.37 (q, J=7.2 Hz, 3H), 7.00 (m, 3H), 7.48 (s, 1H), 7.66
(d, J=8.4 Hz, 2H), 7.90 (d, J=8.9 Hz, 2H), 8.13 (dd,
J=1.6, 7.3 Hz, 1H), 8.41 (s, 1H), 8.45 (s, 1H), 9.75 (s,
1H), 11.76 (s, 1H); MS (TOF) m/z=493 (M++H).
Anal. calcd for C26H27N3O7 1.3H2O: C 60.4, H 5.7, N
8.1. Found: C 60.2, H 5.6, N 8.1.
.
8.45 (s, 1H), 9.03 (s, 1H), 11.74 (s, 1H); MS (TOF)
+
.
m/z=479 (M +H). Anal. calcd for C25H26N4O6 1.2H2O:
C 60.0, H 5.7, N 1 1 .2. Found: C 60.0, H 5.5, N 1 1 .1 .
Ethyl 4,5-dimethoxy-2-((4-((N-(3-methoxyphenyl)carba-
moyl)amino)phenyl)carbonylamino)benzoate (2n). Com-
pound 2n was prepared from 5a in a manner similar to
that described for compound 2a with a yield of 82%.
Ethyl 4,5-dimethoxy-2-((4-((N-(4-methylphenyl)carbamoyl)-
amino)phenyl)carbonylamino)benzoate (2i). Compound
2i was prepared from 5a in a manner similar to that
described for compound 2a with a yield of 98%. Mp:
236–239 ꢂC; 1H NMR (DMSO-d6) d ppm: 1.35 (t,
J=7.0 Hz, 3H), 2.25 (s, 3H), 3.80 (s, 3H), 3.88 (s, 3H),
4.37 (q, J=7.0 Hz, 3H), 7.10 (d, J=8.4 Hz, 2H), 7.38
(d, J=8.4 Hz, 2H), 7.48 (s, 1H), 7.67 (d, J=8.9 Hz,
2H), 7.89 (d, J=8.9 Hz, 2H), 8.45 (s, 1H), 9.09 (s, 1H),
Mp: 200–201 ꢂC; H NMR (DMSO-d6) d ppm: 1.35 (t,
1
J=7.2 Hz, 3H), 3.74 (s, 3H), 3.80 (s, 3H), 3.88 (s, 3H),
4.37 (q, J=7.2 Hz, 3H), 6.58 (dd, J=2.4, 8.1Hz, 1H),
6.96 (d, J=9.5 Hz, 1H), 7.20 (m, 2H), 7.48 (s, 1H), 7.66
(d, J=8.6 Hz, 2H), 7.90 (d, J=8.9 Hz, 2H), 8.44 (s,
1H), 8.97 (s, 1H), 9.21 (s, 1H), 11.75 (s, 1H); MS (TOF)
m/z=493 (M++H). Anal. calcd for C26H27N3O7: C
63.2, H 5.5, N 8.5. Found: C 63.1, H 5.5, N 8.5.
9.43 (s, 1H), 11.75 (s, 1H); MS (TOF) m/z=478
+
.
(M +H). Anal. calcd for C26H27N3O6 0.2H2O: C 64.9,
H 5.7, N 8.7. Found: C 64.7, H 5.6, N 8.7.
Ethyl 4,5-dimethoxy-2-((4-((N-(3,4,5-trimethoxyphenyl)-
carbamoyl)amino)phenyl)carbonylamino) benzoate (2o).
Compound 2o was prepared from 5a in a manner simi-
lar to that described for compound 2a with a yield of
45%. Mp: 225–227 ꢂC; 1H NMR(DMSO–d6) d ppm:
1.35 (t, J=7.2 Hz, 3H), 3.61(s, 3H), 3.76 (s, 6H), 3.80
(s, 3H), 3.88 (s, 3H), 4.37 (q, J=7.2 Hz, 3H), 6.83 (s,
2H), 7.48 (s, 1H), 7.67 (d, J=8.4 Hz, 2H), 7.90 (d,
J=8.9 Hz, 2H), 8.44 (s, 1H), 8.93 (s, 1H), 9.19 (s, 1H),
11.74 (s, 1H); MS (TOF) m/z=554 (M++H). Anal.
Ethyl 2-((4-((N-(4-fluorophenyl)carbamoyl)amino)phenyl)-
carbonylamino)-4,5-dimethoxybenzoate (2j). Compound
2j was prepared from 5a in a manner similar to that
described for compound 2a with a yield of 73%. Mp:
243–244 ꢂC; 1H NMR (DMSO-d6) d ppm: 1.35 (t,
J=7.2 Hz, 3H), 3.80 (s, 3H), 3.88 (s, 3H), 4.37 (q,
J=7.2 Hz, 3H), 7.14 (t, J=6.2 Hz, 2H), 7.48 (s, 1H),
7.49 (dd, J=3.8, 8.6 Hz, 2H), 7.67 (d, J=8.6 Hz, 2H),
7.89 (d, J=8.9 Hz, 2H), 8.44 (s, 1H), 9.12 (s, 1H), 9.34
(s, 1H), 11.75 (s, 1H). Anal. calcd for C25H24FN3O6: C
62.4, H 5.0, N 8.7. Found: C 62.1, H 5.1, N 8.7.
.
calcd for C28H31N3O9 0.1H2O: C 60.6, H 5.7, N 7.6.
Found: C 60.3, H 5.6, N 7.3.
Ethyl 4,5-dimethoxy-2-((4-((N-benzylcarbamoyl)amino)-
phenyl)carbonylamino)benzoate (2p). Compound 2p
was prepared from 5a in a manner similar to that
described for compound 2a with a yield of 86%. Mp:
231–233 ꢂC; 1H NMR (DMSO-d6) d ppm: 1.34 (t,
J=7.2 Hz, 3H), 3.80 (s, 3H), 3.87 (s, 3H), 4.37 (m, 4H),
6.99 (t, J=6.5 Hz, 1H), 7.28 (m, 5H), 7.47 (s, 1H), 7.61
(d, J=8.6 Hz, 2H), 7.84 (d, J=8.9 Hz, 1H), 8.44 (s,
1H), 9.18 (s, 1H), 11.72 (s, 1H). Anal. calcd for
Ethyl 2-((4-((N-(4-acetylphenyl)carbamoyl)amino)phenyl)-
carbonylamino)-4,5-dimethoxybenzoate (2k). Compound
2k was prepared from 5a in a manner similar to that
described for compound 2a with a yield of 47%. Mp:
240–241 ꢂC; 1H NMR (DMSO-d6) d ppm: 1.35 (t,
J=7.2 Hz, 3H), 3.80 (s, 3H), 3.88 (s, 3H), 4.37 (q,
J=7.2 Hz, 3H), 7.48 (s, 1H), 7.62 (d, J=8.9 Hz, 2H),
7.68 (d, J=8.9 Hz, 2H), 7.93 (m, 4H), 8.44 (s, 1H), 9.34
.
(s, 1H), 9.38 (s, 1H), 11.76 (s, 1H); MS (TOF) m/z=506
+
C26H27N3O6 0.2H2O: C 65.4, H 5.7, N 8.8. Found: C
65.2, H 5.7, N 8.6.
.
(M +H). Anal. calcd for C27H27N3O7 1.0H2O: C 61.9,
H 5.6, N 8.0. Found: C 62.0, H 5.5, N 8.0.
Ethyl 2-((4-((N-butylcarbamoyl)amino)phenyl)carbonyla-
mino)-4,5-dimethoxybenzoate (2q). Compound 2q was
prepared from 5a in a manner similar to that described
for compound 2a with a yield of 27%. Mp: 218–219 ꢂC;
1H NMR (DMSO-d6) d ppm: 0.90 (t, J=6.7 Hz, 3H),
1.27 (m,4H), 1.34 (t, J=7.2 Hz, 3H), 3.10 (q, J=5.7 Hz,
2H), 3.80 (s, 3H), 3.87 (s, 3H), 4.37 (m, 4H), 6.45 (t,
J=5.4 Hz, 1H), 7.47 (s, 1H), 7.59 (d, J=8.9 Hz, 2H),
7.83 (d, J=8.6 Hz, 1H), 8.45 (s, 1H), 8.98 (s, 1H), 11.72
(s, 1H); MS (TOF) m/z=444 (M++H). Anal. calcd for
Ethyl2-((4-((N-(4-ethoxycarbonylphenyl)carbamoyl)amino)-
phenyl)carbonylamino)-4,5-dimethoxybenzoate (2l). Com-
pound 2l was prepared from 5a in a manner similar to
that described for compound 2a with a yield of 88%.
Mp: 204–205 ꢂC; 1H NMR (DMSO-d6) d ppm: 1.32 (m,
6H), 3.80 (s, 3H), 3.88 (s, 3H), 4.33 (m, 4H), 7.48 (s,
1H), 7.62 (d, J=8.4 Hz, 2H), 7.68 (d, J=8.6 Hz, 2H),
7.91 (m, 4H), 8.44 (s, 1H), 9.29 (s, 1H), 9.34 (s, 1H),
.
11.76 (s, 1H). Anal. calcd for C28H29N3O8 1.0H2O: C
60.8, H 5.6, N 7.6. Found: C 60.5, H 5.6, N 7.6.
.
C23H29N3O6 0.3H2O: C 61.5, H 6.7, N 9.4. Found: C
61.7, H 6.6, N 9.3.
Ethyl 4,5-dimethoxy-2-((4-((N-(2-methoxyphenyl)carba-
moyl)amino)phenyl)carbonylamino)benzoate (2m). Comp-
ound 2m was prepared from 5a in a manner similar to
that described for compound 2a with a yield of 48%.
Mp: 132–134 C; H NMR (DMSO-d6) d ppm: 1.35 (t,
J=7.2 Hz, 3H), 3.80 (s, 3H), 3.88 (s, 3H), 3.89 (s, 3H),
Ethyl 2-((4-((N-cyclohexylcarbamoyl)amino)phenyl)car-
bonylamino)-4,5-dimethoxybenzoate (2r). Compound 2r
was prepared from 5a in a manner similar to that
described for compound 2a with a yield of 68%. Mp:
231–234 ꢂC; 1H NMR (DMSO-d6) d ppm: 1.20 (m, 6H),
ꢂ
1

H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 1865–1871
1871
1.34 (t, J=7.2 Hz, 3H), 1.65 (m, 4H), 3.48 (m, 1H), 3.79
(s, 3H), 3.87 (s, 3H), 4.37 (m, 4H), 6.42 (d, J=7.8 Hz,
1H), 7.47 (s, 1H), 7.57 (d, J=8.9 Hz, 2H), 7.83 (d,
J=8.9 Hz, 1H), 8.45 (s, 1H), 8.88 (s, 1H), 11.72 (s, 1H);
MS (TOF) m/z=470 (M++H). Anal. calcd for
C25H31N3O6: C 64.0, H 6.7, N 9.0. Found: C 63.9, H
6.7, N 9.1.
4 h at 37 in 5% CO2–95% air. Then the cells were
harvested by trypsinization, and radioactive thymidine
incorporation into DNA was determined by scintilla-
tion counting.
Determination of DNA synthesis in endothelial cells
ECs from the second passage were seeded into 96-well
plates (3Â103 cells/well) in the above complete medium,
and allowed to attach to plates for 4 h at 37 in 5% CO2–
95% air. Then various concentrations of test com-
pounds were added to the complete medium, and the
cells were cultured at 37 in 5% CO2–95% air. After 3
days, DNA synthesis was determined during the last 4 h
of the 3-day culture.
Ethyl 2-((4-((N-(3-pyridyl)carbamoyl)amino)phenyl)car-
bonylamino)-4,5-dimethoxybenzoate (2s). Compound 2s
was prepared from 5a in a manner similar to that
described for compound 2a with a yield of 88%. Mp:
224–226 ꢂC; 1H NMR (DMSO-d6) d ppm: 1.35 (t,
J=7.2 Hz, 3H), 3.80 (s, 3H), 3.88 (s, 3H), 4.37 (q,
J=7.2 Hz, 2H), 7.34 (m, 1H), 7.48 (s, 1H), 7.69 (d,
J=8.6 Hz, 2H), 7.90 (d, J=8.9 Hz, 2H), 7.97 (d, J=8.9
Hz, 1H), 8.20 (d, J=4.3 Hz, 1H), 8.44 (s, 1H), 8.66 (s,
1H), 9.50 (s, 1H), 9.70 (s, 1H), 11.75 (s, 1H); MS (TOF) m/
+
References and Notes
.
z=465 (M +H). Anal. calcd for C24H24N4O6 0.2H2O: C
61.6, H 5.3, N 12.0. Found: C 61.5, H 5.3, N 12.1.
1. Ross, R. Nature 1993, 362, 801.
2. Reidy, M. A. Arch. Pathol. Lab. Med. 1992, 116, 1276.
3. Ross, R.; Raines, E. W.; Bowen-Pope, D. F. Cell 1986, 46,
155.
4. (a) Tanaka, K.; Honda, M.; Kuromachi, T.; Morioka, S.
Atherosclerosis 1994, 107, 179. (b) Isaji, M.; Miyata, H.; Aji-
sawa, Y.; Takehata, Y.; Yoshimura, N. Br. J. Pharmacol.
1997, 122, 1061.
5. Tamai, H.; Katoh, O.; Suzuki, S.; Fujii, K.; Aizawa, T.;
Takase, S.; Kurogane, H.; Nishikawa, H.; Sone, T.; Sakai, K.;
Suzuki, T. Am. Heart. J. 1999, 138, 968.
6. Sakomura, Y.; Nagai, R. In Kekkan Remodeling to
Shushokuinshi; Yazaki, Y., Ed.; Medical Review Co. Ltd:
1997; p 295.
7. Ogita, H.; Isobe, Y.; Takaku, H.; Sekine, R.; Goto, Y.; Mis-
awa, S.; Hayashi, H. Bioorg. Med. Chem. Lett. 2001, 11, 549.
8. Harada, H., Kusama, H., Nonaka, Y. and Kamata, K.
PCT Patent Publication, WO 97/09301.
9. Kubo, K.; Shimizu, T.; Ohyama, S.; Murooka, H.; Nishi-
toba, T.; Kato, S.; Kobayashi, Y.; Yagi, M.; Isoe, T.; Naka-
mura, K.; Osawa, T.; Izawa, T. Bioorg. Med. Chem. Lett.
1997, 7, 2935.
Primary culture of smooth muscle cells and endothelial
cells
Human coronary artery smooth muscle cells (SMCs),
human coronary artery endothelial cells (ECs), and
their culture kits were obtained from Clonetics Corp.
(San Diego, CA, USA). SMCs were cultured in basal
medium (SmBM) containing 5% fetal bovine serum,
human epidermal growth factor (0.5 ng/mL), insulin (5
mg/mL), human fibroblast growth factor (2 ng/mL),
gentamicin (50 mg/mL) and amphotericin-B (50 pg/mL).
And ECs were cultured in basal medium (EBM) con-
taining 5% fetal bovine serum, human epidermal
growth factor (10 ng/mL), hydrocortisone (1 mg/mL),
bovine brain extract (12 mg/mL), gentamicin (50 mg/mL)
and amphotericin-B (50 pg/mL). After 3 to 5 days in
culture at 37 in 5% CO2–95% air, both cells were sub-
cultured by trypsinization and propagated in each com-
plete medium described above.
10. Kubo, K.; Shimizu, T.; Ohyama, S. PCT Patent Publica-
tion, WO 97/17329.
11. (a) Hishikawa, K.; Nakaki, T.; Hirahashi, J.; Marumo, T.;
Saruta, T. Eur. J. Pharmacol. 1995, 291, 435. (b) Wiernicki,
R. T.; Bean, S. J.; Dell, C.; Williams, A.; Wood, D.; Kauff-
man, F. R.; Singh, P. J. Pharmacol. Exp. Ther. 1996, 278,
1452.
12. (a) Koyama, S.; Takagi, H.; Otani, A.; Suzuma, K.;
Nishimura, K.; Honda, Y. Br. J. Pharmacol. 1999, 127, 537.
(b) Miyazawa, K.; Hamano, S.; Ujiie, A. Br. J. Pharmacol.
1996, 118, 915. (c) Kusama, H.; Kikuchi, S.; Tazawa, S.; Kat-
suno, K.; Baba, Y.; Zhai, Y. L.; Nikaido, T.; Fujii, S. Athero-
sclerosis 1999, 143, 307. (d) Takahashi, A.; Taniguchi, T.;
Ishikawa, Y.; Yokoyama, M. Circ. Res. 1999, 84, 543.
Determination of DNA synthesis in smooth muscle cells
SMCs from passages 1–3 were seeded into 96-well
plates (3Â104 cells/well) in the complete medium
described above, and cultured for 16–18 h at 37 in
5% CO2–95% air. Then the complete medium was
replaced with the basal medium (SmBM) containing 20
ng/mL human PDGF-BB (Carbiochem Corp; San
Diego, CA, USA) and various concentration of test
3
compounds. After 24 h, 1 mCi/mL H-thymidine was
added into the medium, and the cells ware incubated for