Synthesis of potent and selective inhibitors against the proliferation of human coronary artery smooth muscle cells.

Article abstract of DOI:10.1248/cpb.51.117

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 2h was much superior to Tranilast, in terms of both the potency of its inhibitory activity toward the proliferation of SMCs and the cell selectivity.

Full text of DOI:10.1248/cpb.51.117

February 2003
Chem. Pharm. Bull. 51(2) 117—121 (2003)
117
Synthesis of Potent and Selective Inhibitors against the Proliferation of
Human Coronary Artery Smooth Muscle Cells
Haruhisa OGITA, Yoshiaki ISOBE, Haruo TAKAKU, Rena SEKINE, Yuso GOTO, Satoru MISAWA, and
,1)
*
Hideya HAYASHI
Pharmaceuticals & Biotechnology Laboratory, Japan Energy Corporation; 3–17–35 Niizo-Minami, Toda, Saitama
335–8502, Japan. Received July 10, 2002; accepted November 28, 2002
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 2h was much superior to Tranilast, in terms of both the potency of its inhibitory activity toward the
proliferation of SMCs and the cell selectivity.
Key words diarylamide urea; proliferation; coronary artery smooth muscle cell; coronary artery endothelial cell
Proliferation of smooth muscle cells (SMCs) plays an im- linker between these rings against the proliferation of SMCs.
portant role in restenosis and in progression of atherosclero- In this paper, we report the results of the further modification
sis.2) Tranilast has been shown to inhibit platelet-derived of these urea derivatives.
growth factor (PDGF)-induced migration and proliferation in
both SMCs3) and endothelial cells (ECs). And the multicen- Chemistry
ter, randomized, double-blinded placebo-controlled trials
The syntheses of desired urea derivatives 2c—s are out-
demonstrated the potent preventive effect on restenosis after lined in Chart 1. The condensation of 38) with corresponding
percutaneous transluminal coronary angioplasty (PTCA) in acid halides was performed in the presence of triethylamine
Japanese patients.4,5) [at the phase III trial,5) restenosis rate to provide 4a—c. Acid halide 10 was prepared from 9 with
18.8% (Tranilast 600 mg/d for 3 months, nϭ112) versus 44.1% thionyl chloride, which was obtained by the condensation of
(placebo, nϭ127); pϭ0.00005] But some patients had suf- 8 with ethyl bromoacetate followed by basic hydrolysis.
fered liver dysfunction in these trials. We speculated that Then, catalytic hydrogenation, followed by the condensation
these frequent and severe side effects in these trials might of 5a—c with the corresponding isocyanates gave the desired
have been caused by the high dose of Tranilast. In addition, urea derivatives 2c—s. Compound 2e was obtained by the
the selective inhibitors of the proliferation of SMCs over that condensation of 3 with 7 by using 2-chloro-1,3-dimethylimi-
of ECs was more preferable for the treatment of restenosis dazolidium hexafluorophosphate (CIP)9,10) as a condensation
than the non-selective inhibitors like Tranilast.6) So, we had reagent.
been trying to search more potent and SMCs-selective com-
pounds by modification of Tranilast.
We previously reported a series of diarylamide derivatives,
Biology
A series of these compounds were evaluated for their in-
exhibiting potent and highly selective inhibition against hibitory activities on PDGF-induced proliferation of human
SMCs proliferation induced by PDGF-BB.7) We conducted coronary artery SMCs and fetal bovine serum (FBS)-induced
that structure–activity relationship (SAR) study of the sub- the proliferation of human coronary artery ECs. Inhibition of
3
stituents at the A and B rings, and found potent inhibitor, 1a, the proliferation of these cells was determined by H-thymi-
which had about 30-fold stronger activity than Tranilast. In dine incorporation as previously reported method.8)
that case, the linker between A and B rings was not so influ-
enced on the inhibitory activities (1a—c). In order to find Results and Discussion
more potent inhibitor, we also reported that the introduction
The inhibitory activities of 2c—s on PDGF-induced pro-
of a ureido group to the B ring of our diarylamide derivatives liferation of SMCs and FBS-induced proliferation of ECs
make the inhibitory activity more potent and keep the selec- were shown in Tables 1 and 2.
tivity for SMCs and 2b showed the most potent inhibitory ac-
Table 1 presented the results of the SAR study on the
tivity for SMCs (IC50 = 40 nM), which was 20- and 600-fold linker X between A and B rings. A methylene (2c) or vinyl
stronger than 1a and Tranilast respectively.8) On the basis of (2e) groups at X position showed almost the same activity
these results (Fig. 1), we tried to confirm the effect of the with 2a. This tendency was easily expected from the result of
Fig 1. Structure of Tranilast and Diarylamide Derivatives
* To whom correspondence should be addressed. e-mail: hayashih@sumitomopharm.co.jp
© 2003 Pharmaceutical Society of Japan

118
Vol. 51, No. 2
Chart 1. Synthesis of the Compounds 2a—s
Reagents and conditions: a) (4-NO2–Ph)–X–COCl, Et3N, CH2Cl2; b) H2, 5% Pd/C, EtOH; c) RNCO, DMAP, THF; d) EtOH, H2SO4; e) NaOH, H2O, MeOH; f) CIP, HOBt,
iPr2NEt, CH2Cl2 then 3; g) BrCH2CO2Et , K2CO3, DMF; h) SOCl2.
Table 1. IC50 Values of the Diarylamide Derivatives for the Inhibition of the Proliferation of SMCs and ECs
No.
X
R
SMCs IC50 (nM)a)
ECs IC50 (nM)b)
[ECs]/[SMCs]
Tranilast
1a
1b
1c
2a
2b
2c
2d
2e
24500
850
800
1200
400
40
260
12
410
10000
19100
6000
4700
4100
1500
54000
290
14
1200
3800
0.8
7
6
4
4
140
1
1
3
0.4
—
CH2
(E)-CHϭCH
—
—
CH2
(CH2)2
(E)-CHϭCH
OCH2
3,4-(OMe)2
3,4-(OMe)2
3,4-(OMe)2
4-NHCONHPh
4-NHCONH(3,4,5-(OMe)3-Ph)
4-NHCONHPh
4-NHCONHPh
4-NHCONHPh
4-NHCONHPh
2f
a) Inhibitory activity against the proliferation of SMCs induced by PDGF-BB (20 ng/ml). b) Inhibitory activity against the proliferation of ECs induced by 5% FBS.
compounds 1a—c. But an ethylene group (2d) showed much need to be flexible to some extent. And we also evaluated the
more potent inhibition than 2a. Replacement of the ethylene effects of these compounds against the proliferation of ECs
group by methylenoxy group (2f) make it reduced the activ- but introducing the substituents such as the ethylene group at
ity. This result suggested that not only the distance but also X position makes the selectivity for SMCs lower. But in the
the orientation between these rings might be important fac- case of 2a and 2b, no substitution at X position, 3,4,5-
tors. Since we could not define the most stable conformation trimethoxyphenyl substitution at the urea moiety enhanced
from our MOPAC study (data not shown), the linker might the inhibitory activity and the selectivity as reported previ-

February 2003
119
Table 2. IC50 Values of the Compounds 2d, 2g—s for the Inhibition of the
Proliferation of SMCs and ECs
played the selectivity by one order of magnitude, That is to
say, the IC50 values of 2h for SMCs and for ECs were 0.1 nM
and 1.7 nM, respectively, displaying selectivity about 17-fold
greater for SMCs. In contrast, the IC50 values of Tranilast to-
ward SMCs and ECs were 25 mM and 19 mM, respectively, in-
dicating a selectivity of about 0.8-fold for SMCs. So, com-
pound 2h was much superior to Tranilast in the strength of
the activity and cell selectivity.
The mechanism of the cell selectivity between SMCs and
ECs and target molecules of our derivatives have been un-
known yet. In examining the mechanism of action of Trani-
last, several researchers showed that Tranilast increased the
levels of p21 and p53 protein and arrested SMCs at the
G0/G1 phase.11—14) On the basis of the structural resem-
blance between our compounds and Tranilast, the elucidation
of target molecules of our derivatives is still underway.
In conclusion, in order to optimize the structure of our di-
arylamide derivatives, we conducted the SAR study mainly
focused on the linker X between the A and B rings, and the
substituents at the urea moiety. We found that 2h had the
strongest inhibitory activity against the proliferation of
SMCs, which was 250000-fold more potent than Tranilast.
SMCs
ECs
[ECs]/
No.
R
IC50 (nM)a) IC50 (nM)b) [SMCs]
Tranilast
2d
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
25000
12
580
19000
14
1100
1.7
23
53
200
45
200
76
53
15
200
300
30
0.8
1
2
17
2
2
5
0.8
2
1
Ph
3,4,5-(OMe)3-Ph
4-NO2-Ph
3-NO2-Ph
2-NO2-Ph
4-OMe-Ph
3-OMe-Ph
2-OMe-Ph
4-NH2-Ph
4-Me-Ph
4-F-Ph
0.1
15
30
40
54
120
57
28
7
2
2
3
5
4-Ac-Ph
Bn
c-Hex
67
64
11
2s
3
Experimental
a) Inhibitory activity against the proliferation of SMCs induced by PDGF-BB
(20 ng/ml). b) Inhibitory activity against the proliferation of ECs induced by 5%
FBS.
Chemistry In general, reagents and solvents were used as purchased
without further purification. Column chromatography was performed on
FL60D (Fuji Silysia). Melting points were measured with a Yanako micro
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 tetramethylsilane. TOFMS
(Time-of-flight mass spectrometry) were recorded on a Kompact MALDI III
spectrometer. Elemental analyses were performed by the Toray Research
Center.
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-phenylcarbamoyl)amino)phenyl)-
propanoylamino)benzoate (2d) To a solution of 3 (0.10 g, 0.44 mmol) in
CH2Cl2 (10 ml), 4-nitrocinnamoyl chloride (0.11 g, 0.52 mmol) and triethyl-
amine (0.07 g, 0.70 mmol) were added, and the mixture was stirred for 4 h at
room temperature. The reaction mixture was poured into saturated NaHCO3
aq. and extracted with CH2Cl2, after which the organic layer was washed
ously.8) So, the further modification was conducted based on
2d.
Table 2 presents the result of the SAR study on the sub-
stituents at the urea moiety with the ethylene substitution at
X position. Considering the result of 2a and 2b,8) we evalu-
ated 2g that had the 3,4,5-trimethoxyphenyl group at the urea
moiety like 2b. But in this case, contrary to our expectation,
the trimethoxyphenyl substitution resulted in decreased the
activity. Therefore, we examined the effect of the substituents with brine, dried over MgSO4, filtered, concentrated and washed with
methanol to give 0.17 g (0.42 mmol) of 4b as a yellow solid with a yield of
96%. To a solution of 4b (0.17 g, 0.42 mmol) in tetrahydrofuran (THF)
(20 ml) was added 5% Pd/C (0.05 g), and the mixture was vigorously stirred
under a H2 atmosphere for 18 h. The reaction mixture was filtered and con-
at the urea moiety again.
At first, we examined the position of the substituent on the
B ring by introducing electron-donating group (methoxy
group) and electron-withdrawing group (nitro group), and
found that the para position was superior to the other posi-
tions in both cases (2h—j, 2k—m). Next, we tried to study
the effect of the substituents at the para position of the
phenyl ring and the order of potency was nitro (2h) Ͼ fluo-
rine (2p), hydrogen (2d) Ͼ methyl (2o), methoxy (2k) Ͼ
acetyl (2q), amino groups (2n). It seemed that the compact
electron-withdrawing group would be preferable to show the
potent activity. Benzyl group (2r) instead of the phenyl ring
gave weak activity. But, cyclohexyl group (2s) was almost
the same activity with 2d. These results suggested that the
size of the substituents might influence the activity as well as
the electronic effects. As a result, 2h showed the most potent
activity (IC50ϭ0.1 nM) with about 250000-fold more potent
than Tranilast and about 400-fold much more potent than
centrated to give 0.15 g (0.40 mmol) of 5b as a pale yellow solid with a yield
of 95%. To a solution of 5b (0.05 g, 0.13 mmol) and 4-dimethylaminopyri-
dine (DMAP) (0.02 g, 0.16 mmol) in THF (5 ml) was added phenyliso-
cyanate (0.05 g, 0.42 mmol), and the mixture was refluxed for 3 h. The reac-
tion mixture was poured into water and extracted with CH2Cl2, and the or-
ganic layer was then washed with brine, dried over MgSO4, filtered, and con-
centrated. The residue was purified by silica gel column chromatography
(CH2Cl2/methanolϭ100 : 1—20 : 1) and washed with methanol to give 0.04 g
(0.08 mmol) of 2d as a white solid with a yield of 63%. mp 210—212 °C.
1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz), 2.68 (2H, t, Jϭ7.3 Hz),
2.88 (2H, t, Jϭ7.3 Hz), 3.76 (3H, s), 3.81 (3H, s), 4.31 (2H, q, Jϭ7.0 Hz),
6.95 (1H, t, Jϭ7.3 Hz), 7.16 (2H, d, Jϭ8.6 Hz), 7.27 (2H, t, Jϭ8.1 Hz), 7.40
(5H, m), 8.15 (1H, s), 8.65 (1H, s), 8.69 (1H, s), 10.74 (1H, s). Anal. Calcd
for C27H29N3O6: C, 65.97; H, 5.95; N, 8.55. Found: C, 65.69; H, 5.99; N,
8.40.
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-phenylcarbamoyl)amino)phenyl)prop-
2-enoylamino)benzoate (2e) To a solution of 6 (1.12 g, 6.87 mmol) in
ethanol (100 ml) was added H2SO4 (1 ml), and the mixture was refluxed for
even 2b (40 nM), a second lead compound. We also evaluated 8 h. To the reaction mixture on ice added saturated NaHCO3 aq., adjust to
the pH 8 and extracted with CH2Cl2, after which the organic layer was dried
over MgSO4, filtered and concentrated. To the solution of the resulting
residue and DMAP (1.18 g, 9.66 mmol) in THF (30 ml) was added phenyliso-
cyanate (1.15 g, 9.65 mmol), and the mixture was refluxed for 3 h. The reac-
tion mixture was poured into water and extracted with CH2Cl2, and the or-
ganic layer was then washed with brine, dried over MgSO4, filtered, and con-
the effect of these compounds against the proliferation of
ECs and most of the evaluated compounds displayed a low
selectivity ranging from 2 to 5 times for SMCs. This result
showed that the ethylene group substitution at X position had
the detrimental effect for the selectivity. But only 2h dis-

120
Vol. 51, No. 2
centrated. The residue was washed with methanol to give 1.39 g (4.48 mmol) 9.12 (1H, s), 9.70 (1H, s), 10.74 (1H, s). Anal. Calcd for C27H28N4O8: C,
of a white solid. To the solution of the resulting solid (0.61 g, 1.97 mmol) in 60.44; H, 5.28; N, 10.44. Found: C, 60.22; H, 5.37; N, 10.17.
methanol (50 ml) was added 5% NaOH aq. (50 ml) and the mixture was
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-(3-nitrophenyl)carbamoyl)amino)-
stirred for 18 h. The reaction mixture was extracted with CH2Cl2, and the phenyl)propanoylamino)benzoate (2i) Compound 2i was prepared from
aqueous layer was acidified with 1 N HCl aq. to save a solid. The resulting
solid was washed with water to give 0.50 g (1.77 mmol) of 7 as a white solid
5b in a manner similar to that described for compound 2d with a yield of
45%. mp 219—220 °C. 1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz),
with a yield of 59% by 3 steps. To the suspension of 7 (0.10 g, 0.35 mmol) 2.69 (2H, t, Jϭ7.4 Hz), 2.89 (2H, t, Jϭ7.3 Hz), 3.77 (3H, s), 3.82 (3H, s),
and N-hydroxybenzotriazole (HOBt) (6 mg, 0.04 mmol) in CH2Cl2 (30 ml) 4.31 (2H, q, Jϭ7.0 Hz), 7.18 (2H, d, Jϭ8.4 Hz), 7.39 (1H, s), 7.40 (2H, d,
was added diisopropylethylamine (0.36 g, 2.79 mmol) on ice. Then the addi- Jϭ5.9 Hz), 7.55 (1H, t, Jϭ8.4 Hz), 7.72 (1H, d, Jϭ9.2 Hz), 7.79 (1H, d,
tion of 2-chloro-1,3-dimethylimidazolidium hexafluorophosphate (CIP) Jϭ9.2 Hz), 8.14 (1H, s), 8.57 (1H, s), 9.04 (1H, s), 9.48 (1H, s), 10.74 (1H,
(0.20 g, 0.72 mmol) to the mixture was followed by that of 3 (0.13 g, 0.58 s). Anal. Calcd for C27H28N4O8: C, 60.44; H, 5.28; N, 10.44. Found: C,
mmol). After stirring for 18 h at room temperature, the reaction mixture was 60.21; H, 5.35; N, 10.21.
poured into the saturated NaHCO3 aq. and extracted with CH2Cl2. The or-
ganic layer was then washed with 1 N HCl aq. and brine, dried over MgSO4,
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-(2-nitrophenyl)carbamoyl)amino)-
phenyl)propanoylamino)benzoate (2j) Compound 2j was prepared from
filtered, and concentrated. The residue was purified by silica gel column 5b in a manner similar to that described for compound 2d with a yield of
chromatography (CH2Cl2/methanolϭ100 : 1) and washed with methanol to 69%. mp 109—110 °C. 1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz),
give 0.06 g (0.12 mmol) of 2e as a pale yellow solid with a yield of 21% 2.69 (2H, t, Jϭ7.3 Hz), 2.89 (2H, t, Jϭ7.3 Hz), 3.77 (3H, s), 3.82 (3H, s),
1
from 3. mp 294—298 °C. H-NMR (DMSO-d6) d: 1.35 (3H, t, Jϭ7.0 Hz), 4.34 (2H, q, Jϭ7.0 Hz), 7.21 (3H, m), 7.39 (3H, m), 7.69 (1H, t, Jϭ7.8 Hz),
3.79 (3H, s), 3.85 (3H, s), 4.35 (2H, q, Jϭ7.0 Hz), 6.76 (1H, d, Jϭ15.7 Hz), 8.08 (1H, d, Jϭ8.4 Hz), 8.14 (1H, s), 8.28 (1H, d, Jϭ8.4 Hz), 9.63 (1H, s),
6.98 (1H, t, Jϭ7.3 Hz), 7.29 (2H, t, Jϭ8.4 Hz), 7.53 (6H, m), 7.67 (2H, d, 9.82 (1H, s), 10.74 (1H, s). Anal. Calcd for C27H28N4O8·1.0H2O: C, 58.47;
Jϭ8.6 Hz), 8.33 (1H, s), 8.91 (1H, s), 9.10 (1H, s), 10.97 (1H, s). Anal. H, 5.45; N, 10.11. Found: C, 58.44; H, 5.47; N, 9.81.
Calcd for C27H27N3O6: C, 66.25; H, 5.58; N, 8.58. Found: C, 65.88; H, 5.64;
N, 8.51.
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-(4-methoxyphenyl)carbamoyl)amino)-
phenyl)propanoylamino)benzoate (2k) Compound 2k was prepared from
Ethyl 4,5-Dimethoxy-2-(2-(4-((N-phenylcarbamoyl)amino)phenoxy)- 5b in a manner similar to that described for compound 2d with a yield of
acetylamino)benzoate (2f) To a solution of 8 (0.30 g, 2.16 mmol) and 29%. mp 217—220 °C. 1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz),
K2CO3 (0.18 g, 1.30 mmol) in N,N-dimethylformamide (DMF) (10 ml), ethyl 2.67 (2H, t, Jϭ7.3 Hz), 2.88 (2H, t, Jϭ7.3 Hz), 3.71 (3H, s), 3.77 (3H, s),
bromoacetate (0.44 g, 2.63 mmol) was added, and the mixture was stirred for 3.81 (3H, s), 4.30 (2H, q, Jϭ7.0 Hz), 6.85 (2H, d, Jϭ8.9 Hz), 7.15 (2H, d,
18 h at room temperature. The reaction mixture was evaporated and poured Jϭ8.6 Hz), 7.35 (5H, m), 8.14 (1H, s), 8.47 (1H, s), 8.54 (1H, s), 10.74 (1H,
into water to save a solid. The resulting solid was washed with water to give s); MS (TOF) m/z: 522 (MϩϩH). Anal. Calcd for C28H31N3O7·0.5H2O: C,
0.46 g (2.04 mmol) of a white solid. To the solution of the resulting solid 63.38; H, 6.08; N, 7.92. Found: C, 63.08; H, 6.04; N, 7.64.
(0.20 g, 0.89 mmol) in methanol (10 ml) was added 5% NaOH aq. (10 ml)
and the mixture was stirred for 18 h. The reaction mixture was extracted phenyl)propanoylamino)benzoate (2l) Compound 2l was prepared from
with CH2Cl2, and the aqueous layer was acidified to pH 4 with 1 N HCl aq.
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-(3-methoxyphenyl)carbamoyl)amino)-
5b in a manner similar to that described for compound 2d with a yield of
and extracted with CH2Cl2. The organic layer was concentrated to give 0.17 45%. mp 185—188 °C. 1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz),
g (0.86 mmol) of 9 as a white solid with a yield of 92% by 2 steps. 9 (0.14 g, 2.68 (2H, t, Jϭ7.3 Hz), 2.88 (2H, t, Jϭ7.3 Hz), 3.72 (3H, s), 3.77 (3H, s),
0.71 mmol) was added to SOCl2 (15 ml) and refluxed for 4 h. The reaction 3.81 (3H, s), 4.30 (2H, q, Jϭ7.0 Hz), 6.53 (1H, dd, J1ϭ2.4 Hz, J2ϭ8.1 Hz),
mixture was concentrated to give 10 as brown oil. To a solution of 3 (0.11 g, 6.92 (1H, d, Jϭ8.4 Hz), 7.16 (4H, m), 7.37 (3H, m), 8.14 (1H, s), 8.67 (1H,
0.49 mmol) in CH2Cl2 (10 ml), this residue 10 and triethylamine (0.08 g, s), 8.73 (1H, s), 10.74 (1H, s). MS (TOF) m/z: 522 (MϩϩH). Anal. Calcd for
0.79 mmol) were added, and the mixture was stirred for 1 h at room tempera-
ture. The reaction mixture was poured into saturated NaHCO3 aq. and ex-
C28H31N3O7: C, 64.48; H, 5.99; N, 8.06. Found: C, 64.36; H, 6.05; N, 7.94.
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-(2-methoxyphenyl)carbamoyl)amino)-
tracted with CH2Cl2, after which the organic layer was washed with brine, phenyl)propanoylamino)benzoate (2m) Compound 2m was prepared
dried over MgSO4, filtered, concentrated and washed with methanol to give from 5b in a manner similar to that described for compound 2d with a yield
1
0.19 g (0.47 mmol) of 4c as a yellow solid with a yield of 96%.
of 74%. mp 207—208 °C. H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz),
Compound 2f was prepared from 4c in a manner similar to that described 2.68 (2H, t, Jϭ7.3 Hz), 2.88 (2H, t, Jϭ7.3 Hz), 3.76 (3H, s), 3.81 (3H, s),
for compound 2d with a yield of 83% by 2 steps. mp 286—289 °C. 1H-NMR 3.87 (3H, s), 4.30 (2H, q, Jϭ7.0 Hz), 6.93 (3H, m), 7.15 (2H, d, Jϭ8.4 Hz),
(DMSO-d6) d: 1.34 (3H, t, Jϭ7.0 Hz), 3.79 (3H, s), 3.85 (3H, s), 4.35 (2H,
7.35 (3H, m), 8.13 (3H, m), 9.24 (1H, s), 10.74 (1H, s). MS (TOF) m/z: 522
q, Jϭ7.0 Hz), 4.68 (2H, s), 6.94 (1H, t, Jϭ7.3 Hz), 7.04 (2H, d, Jϭ8.9 Hz), (MϩϩH). Anal. Calcd for C28H31N3O7·0.1H2O: C, 64.25; H, 6.01; N, 8.03.
7.26 (2H, t, Jϭ7.8 Hz), 7.43 (5H, m), 8.42 (1H, s), 8.74 (1H, s), 8.80 (1H, Found: C, 64.05; H, 5.99; N, 7.80.
s), 11.83 (1H, s). Anal. Calcd for C26H27N3O7·0.3H2O: C, 62.59; H, 5.58; N,
8.42. Found: C, 62.84; H, 5.55; N, 8.09.
Ethyl 2-(3-(4-((N-(4-Aminophenyl)carbamoyl)amino)phenyl)propano-
lamino)-4,5-dimethoxybenzoate (2n) To solution of 2h (0.17 g,
y
a
Ethyl 4,5-Dimethoxy-2-(2-(4-((N-phenylamino)carbonylamino)phenyl)-
acetylamino)benzoate (2c) Compound 2c was prepared from 3 in a man-
0.32 mmol) in THF (20 ml) was added 5% Pd/C (0.05 g), which was then
stirred at room temperature under a H2 atmosphere for 18 h. After the reac-
ner similar to that described for compound 2d with a yield of 18% by 3 tion mixture had been filtered, concentrated and washed with methanol to
steps. mp 188—190 °C. 1H-NMR (DMSO-d6) d: 1.31 (3H, t, Jϭ7.0 Hz), give 0.14 g (0.28 mmol) of 2n as a pale yellow solid with a yield of 86%. mp
1
3.67 (2H, s), 3.76 (3H, s), 3.80 (3H, s), 4.28 (2H, q, Jϭ7.0 Hz), 6.96 (1H, t,
Jϭ7.3 Hz), 7.27 (4H, m), 7.43 (5H, m), 8.18 (1H, s), 8.74 (1H, s), 8.75 (1H,
294—300 °C. H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz), 2.67 (2H, t,
Jϭ7.3 Hz), 2.87 (2H, t, Jϭ7.3 Hz), 3.77 (3H, s), 3.81 (3H, s), 4.30 (2H, q,
s), 10.80 (1H. s). Anal. Calcd for C26H27N3O6·0.4H2O: C, 64.42; H, 5.78; N, Jϭ7.0 Hz), 4.75 (2H, s), 6.49 (2H, d, Jϭ8.9 Hz), 7.05 (2H, d, Jϭ8.6 Hz),
8.67. Found: C, 64.60; H, 5.73; N, 8.60.
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-(3,4,5-trimethoxyphenyl)carbamoyl)-
7.13 (2H, d, Jϭ8.1 Hz), 7.32 (2H, d, Jϭ8.1 Hz), 7.39 (1H, s), 8.12 (1H, s),
8.14 (1H, s), 8.43 (1H, s), 10.74 (1H, s). Anal. Calcd for C27H30N4O6·
amino)phenyl)propanoylamino)benzoate (2g) Compound 2g was pre- 0.4H2O: C, 63.12; H, 6.04; N, 10.91. Found: C, 63.47; H, 6.13; N, 10.54.
pared from 5b in a manner similar to that described for compound 2d with a
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-(4-methylphenyl)carbamoyl)amino)-
phenyl)propanoylamino)benzoate (2o) Compound 2o was prepared from
yield of 79%. mp 198—202 °C. 1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0
Hz), 2.68 (2H, t, Jϭ7.3 Hz), 2.88 (2H, t, Jϭ7.3 Hz), 3.60 (3H, s), 3.70 (6H, 5b in a manner similar to that described for compound 2d with a yield of
s), 3.76 (3H, s), 3.81 (3H, s), 4.30 (2H, q, Jϭ7.0 Hz), 6.79 (2H, s), 7.15 (2H, 46%. mp 223—226 °C. 1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz),
d, Jϭ8.9 Hz), 7.35 (2H, d, Jϭ8.9 Hz), 7.39 (1H, s), 8.14 (1H, s), 8.58 (1H, 2.23 (3H, s), 2.67 (2H, t, Jϭ7.3 Hz), 2.87 (2H, t, Jϭ7.3 Hz), 3.76 (3H, s),
s), 8.63 (1H, s), 10.74 (1H, s). MS (TOF) m/z: 582 (MϩϩH). Anal. Calcd for 3.81 (3H, s), 4.30 (2H, q, Jϭ7.0 Hz), 7.07 (2H, d, Jϭ8.4 Hz), 7.15 (2H, d,
C30H35N3O9: C, 61.95; H, 6.07; N, 7.22. Found: C, 61.78; H, 6.07; N, 7.06.
Jϭ8.4 Hz), 7.35 (5H, m), 8.14 (1H, s), 8.57 (1H, s), 8.59 (1H, s), 10.74 (1H,
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-(4-nitrophenyl)carbamoyl)amino)- s). Anal. Calcd for C28H31N3O6: C, 66.52; H, 6.18; N, 8.31. Found: C, 66.26;
phenyl)propanoylamino)benzoate (2h) Compound 2h was prepared from H, 6.19; N, 8.20.
5b in a manner similar to that described for compound 2d with a yield of
Ethyl 2-(3-(4-((N-(4-Fluorophenyl)carbamoyl)amino)phenyl)propano-
lamino)-4,5-dimethoxybenzoate (2p) Compound 2p was prepared from
68%. mp 209—211 °C. 1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz),
y
2.69 (2H, t, Jϭ7.3 Hz), 2.90 (2H, t, Jϭ7.3 Hz), 3.77 (3H, s), 3.82 (3H, s), 5b in a manner similar to that described for compound 2d with a yield of
4.31 (2H, q, Jϭ7.0 Hz), 7.19 (2H, d, Jϭ8.4 Hz), 7.39 (1H, s), 7.40 (2H, d, 75%. mp 218—221 °C. 1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz),
Jϭ8.9 Hz), 7.69 (2H, d, Jϭ9.2 Hz), 8.14 (1H, s), 8.18 (2H, d, Jϭ9.2 Hz), 2.68 (2H, t, Jϭ7.3 Hz), 2.88 (2H, t, Jϭ7.3 Hz), 3.77 (3H, s), 3.81 (3H, s),

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CO2–95% air. Then the complete medium was replaced with basal medium
(SmBM) containing 20 ng/ml human PDGF-BB (Carbiochem Corp; San
Diego, CA, U.S.A.) and various concentrations of the test compounds. After
4.30 (2H, q, Jϭ7.0 Hz), 7.12 (4H, m), 7.43 (5H, m), 8.14 (1H, s), 8.71 (1H,
s), 8.79 (1H, s), 10.74 (1H, s). Anal. Calcd for C27H28FN3O6: C, 63.65; H,
5.54; N, 8.25. Found: C, 63.42; H, 5.35; N, 8.22.
3
24 h, 1 mCi/ml H-thymidine was added to the medium, and the cells were
Ethyl 2-(3-(4-((N-(4-Acetylphenyl)carbamoyl)amino)phenyl)propano-
cultured for 4 h at 37 °C in 5% CO2–95% air. Then the cells were harvested
by trypsinization, and the amount of radioactive thymidine incorporated into
the DNA was determined by scintillation counting.
ylamino)-4,5-dimethoxybenzoate (2q) Compound 2q was prepared from
5b in a manner similar to that described for compound 2d with a yield of
58%. mp 202—204 °C. 1H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz),
2.69 (2H, t, Jϭ7.3 Hz), 2.90 (2H, t, Jϭ7.3 Hz), 3.77 (3H, s), 3.82 (3H, s),
4.30 (2H, q, Jϭ7.0 Hz), 7.18 (2H, d, Jϭ8.4 Hz), 7.38 (2H, d, Jϭ8.4 Hz),
7.39 (1H, s), 7.58 (2H, d, Jϭ8.6 Hz), 7.89 (2H, d, Jϭ8.6 Hz), 8.14 (1H, s),
8.82 (1H, s), 9.17 (1H, s), 10.74 (1H, s). Anal. Calcd for C29H31N3O7: C,
65.28; H, 5.86; N, 7.88. Found: C, 65.11; H, 5.74; N, 7.87.
Determination of DNA Synthesis in Endothelial Cells ECs from the
second passage were seeded into 96-well plates (3ϫ103 cells/well) in the
above complete medium, and allowed to attach to the plates for 4 h at 37 °C
in 5% CO2–95% air. Then various concentrations of test compounds were
added to the complete medium, and the cells were cultured at 37 °C in 5%
CO2–95% air. After 3 d, DNA synthesis was determined during the last 4 h
of the 3 d culture.
Ethyl 4,5-Dimethoxy-2-(3-(4-((N-benzylcarbamoyl)amino)phenyl)-
propanoylamino)benzoate (2r) Compound 2r was prepared from 5b in a
manner similar to that described for compound 2d with a yield of 46%. mp
1
References
200—203 °C. H-NMR (DMSO-d6) d: 1.32 (3H, t, Jϭ7.0 Hz), 2.66 (2H, t,
1) Present address: Sumitomo Pharmaceuticals Co., Ltd.; 3–1–98 Kasu-
gade Naka, Konohana-ku, Osaka 554–0022, Japan.
2) Ross R., Nature (London), 362, 801—809 (1993).
3) Fukuyama J., Miyazawa K., Hamano S., Ujiie A., Can. J. Physiol.
Pharmacol., 74, 80—84 (1996).
Jϭ7.3 Hz), 2.85 (2H, t, Jϭ7.3 Hz), 3.76 (3H, s), 3.81 (3H, s), 4.31 (4H, m),
6.60 (1H, t, Jϭ7.3 Hz), 7.09 (2H, d, Jϭ8.1 Hz), 7.30 (7H, m), 7.39 (1H, s),
8.13 (1H, s), 8.50 (1H, s), 10.73 (1H, s). MS (TOF) m/z: 506 (MϩϩH). Anal.
Calcd for C28H31N3O6: C, 66.52; H, 6.18; N, 8.31. Found: C, 66.27; H, 6.22;
N, 8.08.
4) Tamai H., Katoh K., Yamaguchi T., Hayakawa H., Kanmatsuse K.,
Haze K., Aizawa T., Nakanishi S., Suzuki S., Suzuki T., Takase S.,
Nishikawa H., Katoh O., Am. Heart. J., 143, 506—513 (2002).
5) Tamai H., Katoh O., Suzuki S., Fujii K., Aizawa T., Takase S., Kuro-
gane H., Nishikawa H., Sone T., Sakai K., Suzuki T., Am. Heart. J.,
138, 968—975 (1999).
6) Rosanio S., Tocchi M., Patterson C., Runge M. S., Thromb. Haemost.,
82, 164—170 (1999).
7) Ogita H., Isobe Y., Takaku H., Sekine R., Goto Y., Misawa S., Hayashi
H., Bioorg. Med. Chem. Lett., 11, 549—551 (2001).
8) Ogita H., Isobe Y., Takaku H., Sekine R., Goto Y., Misawa S., Hayashi
H., Bioorg. Med. Chem., 10, 1865—1871 (2002).
9) Akaji K., Kuriyama N., Kimura T., Fujiwara Y., Kiso Y., Tetrahedron
Lett., 33, 3177—3180 (1992).
10) Akaji K., Kuriyama N., Kiso Y., Tetrahedron Lett., 35, 3315—3318
(1994).
11) Koyama S., Takagi H., Otani A., Suzuma K., Nishimura K., Honda Y.,
Br. J. Pharmacol., 127, 537—545 (1999).
12) Miyazawa K., Hamano S., Ujiie A., Br. J. Pharmacol., 118, 915—922
(1996).
Ethyl 2-(3-(4-((N-Cyclohexylcarbamoyl)amino)phenyl)propano-
ylamino)-4,5-dimethoxybenzoate (2s) Compound 2s was prepared from
5b in a manner similar to that described for compound 2d with a yield of
1
46%. mp 209—213 °C. H-NMR (DMSO-d6) d: 1.19 (5H, m), 1.32 (3H, t,
Jϭ7.0 Hz), 1.76 (5H, m), 2.68 (2H, t, Jϭ7.3 Hz), 2.88 (2H, t, Jϭ7.3 Hz),
3.33 (1H, m), 3.76 (3H, s), 3.81 (3H, s), 4.30 (2H, q, Jϭ7.0 Hz), 6.05 (1H, d,
Jϭ8.4 Hz), 7.09 (2H, d, Jϭ8.4 Hz), 7.26 (2H, d, Jϭ8.4 Hz), 7.39 (1H, s),
8.13 (1H, s), 8.24 (1H, s), 10.73 (1H, s); MS (TOF) m/z: 498 (MϩϩH).
Anal. Calcd for C27H35N3O6: C, 65.17; H, 7.09; N, 8.44. Found: C, 64.79; H,
7.08; N, 8.19.
Primary Culture of Smooth Muscle Cells and Endothelial Cells
SMCs, ECs and their culture kits were obtained from Clonetics Corp. (San
Diego, CA, U.S.A.). SMCs were cultured in basal medium (SmBM) contain-
ing 5% FBS, human epidermal growth factor (0.5 ng/ml), insulin (5 mg/ml),
human fibroblast growth factor (2 ng/ml), gentamicin (50 mg/ml), and am-
photericin-B (50 pg/ml). ECs were cultured in basal medium (EBM) con-
taining 5% FBS, human epidermal growth factor (10 ng/ml), hydrocortisone
(1 mg/ml), bovine brain extract (12 mg/ml), gentamicin (50 mg/ml), and am-
photericin-B (50 pg/ml). After 3 to 5 d in culture at 37 °C in 5% CO2–95%
air, both cell types were subcultured by trypsinization and propagated in
each complete medium described above.
13) Kusama H., Kikuchi S., Tazawa S., Katsuno K., Baba Y., Zhai Y. L.,
Nikaido T., Fujii S., Atherosclerosis, 143, 307—313 (1999).
14) Takahashi A., Taniguchi T., Ishikawa Y., Yokoyama M., Circ. Res., 84,
543—550 (1999).
Determination of DNA Synthesis in Smooth Muscle Cells SMCs
from passage 1—3 were seeded into 96-well plates (3ϫ104 cells/well) in the
complete medium described above, and cultured for 16—18 h at 37 °C in 5%