Synthesis and structure-activity relationship of diarylamide derivatives as selective inhibitors of the proliferation of human endothelial cells

Article abstract of DOI:10.1016/S0968-0896(02)00258-4

A series of diarylamide urea derivatives were synthesized and evaluated for their inhibitory activities against human coronary artery endothelial cells (ECs) and human coronary artery smooth muscle cells (SMCs). Compound 5k was superior to Tranilast, in terms of both cell selectivity and the potency of its inhibitory activity toward the proliferation and angiogenesis of ECs.

Full text of DOI:10.1016/S0968-0896(02)00258-4

Bioorganic & Medicinal Chemistry 10 (2002) 3473–3480
Synthesis and Structure–Activity Relationship of Diarylamide
Derivatives as Selective Inhibitors of the Proliferation of
Human Endothelial 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 15 May 2002; accepted 18 June 2002
Abstract—A series of diarylamide urea derivatives were synthesized and evaluated for their inhibitory activities against human
coronary artery endothelial cells (ECs) and human coronary artery smooth muscle cells (SMCs). Compound 5k was superior to
Tranilast, in terms of both cell selectivity and the potency of its inhibitory activity toward the proliferation and angiogenesis of ECs.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
considered that the direct substituted carbonyl group
might be required to exert a potent activity. Therefore,
Tranilast [N-(3,4-dimethoxycinnamoyl) anthranilic acid],
an anti-allergic drug, has been clinically used for
patients with allergic diseases such as bronchial asthma,
allergic rhinitis, and atopic dermatitis.1aꢀc On the other
hand, it has been reported that Tranilast had inhibitory
activity toward angiogenesis of dermal vascular endo-
thelial cells in vitro and in experimental animal
models.2a,b Angiogenesis is a phenomenon involving
degradation of the membrane by proteolytic enzymes,
chemotaxis and proliferation of endothelial cells, tube
formation by endothelial cell differentiation, and reorgan-
ization of blood vessels, and is associated with various
diseases such as diabetic retinopathy, rheumatic arthri-
tis, psoriasis, cancer, and so on.3 So, we have had a
great interest in modifying Tranilast in order to develop
potent and cell-selective anti-angiogenetic compounds.
we introduced an acetyl group (5a) to the A ring of 1
instead of the ethoxycarbonyl group (Fig. 1). The inhi-
bitory activity of 5a toward the proliferation of SMCs
was weaker than that of 1; but unexpectedly and sur-
prisingly, the selectivity for SMCs had disappeared, or
rather this compound displayed selectivity for vascular
endothelial cells (ECs). That is, the acetyl group sub-
stitution increased the potency of inhibition against ECs
proliferation.
As selective inhibitors of ECs proliferation can be con-
sidered as a good drug candidate for diseases associated
with angiogenesis, and in view of our findings on 5a, we
sought to develop more potent and selective inhibitors
of ECs proliferation by modifying 5a. In this paper, we
report the results of the structure–activity relationship
(SAR) study on a series of these acetyl-type diarylamide
derivatives with respect to ECs proliferation.
Previously, we reported a series of diarylamide urea
derivatives (exemplified by 1) that exhibited a potent
and highly selective inhibition for vascular smooth
muscle cells (SMCs) proliferation.4a,b In the course of a
recent study on substitutions at the 2-position of the A
ring, we found that ethoxycarbonyl and carbamoyl
groups were suitable substituents for this position and
Chemistry
The synthesis of diarylamide derivatives 5a–x is out-
lined in Scheme 1. Condensation of 2-amino-4,5-dime-
thoxyacetophenone (2) with corresponding acid halides
in the presence of triethylamine, followed by catalytic
hydrogenation with 5% Pd/C gave 4a–d in a good yield
(70ꢁ90%) and 4e was obtained by reduction of 3c with
*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)00258-4

3474
H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 3473–3480
Fe/AcOH. Finally, condensation of 4a–e with the cor-
responding isocyanates or acid halides gave the desired
urea and amide derivatives 5a–x (except 5h). Compound
5h was obtained by the condensation of 4c with phenol
by using 1,1-carbonyldiimidazole (CDI) as a condensa-
tion reagent.
ECs are shown in Tables 1–3. Table 1 presents the
results of the SAR study on the effect of the distance
between the A and B rings. In the case of the diaryl-
amide derivatives in our previous work,4a the distance
did not much influence the inhibitory activities. But in
the case of these urea derivatives, the insertion of an
alkyl chain increased the activity; and an ethylene group
(5d) at the X position showed the most potent inhibition
of ECs proliferation. But the insertion of a vinyl group
(5c) instead of an ethylene group (5d) significantly
decreased the activity although the both had the same
number of carbon atoms. This result suggested that the
linker between these rings might need to be flexible to
some extent. So, further modification of 5d was con-
ducted.
Biology
A series of these compounds were evaluated for their
inhibitory activities toward platelet derived growth fac-
tor (PDGF)-induced proliferation of SMCs and fetal
bovine serum (FBS)-induced proliferation of ECs.4b The
inhibitory activity toward tube formation of human
umbilical vein endothelial cells (HUVECs) was deter-
mined by a previously reported method.2b
Table 2shows the results of the SAR study when the
urea moiety was changed to an alternate linkage. As we
expected, the ureido group (5d) was more suitable for
the activity than the others such as carbonylamino (5f–
g) and alkoxycarbonylamino (5h) groups. In our pre-
vious work,4b methylation at the N atom in ureido
Results and Discussion
The inhibitory activities of 5a–x toward PDGF-induced
proliferation of SMCs and FBS-induced proliferation of
Figure 1. Structure of Tranilast and diarylamide urea derivatives.
Scheme 1. Synthesis of compounds 5a–x. Reagents and conditions: (a) (4-NO2–Ph)-X-COCl, Et3N/CH2Cl2; (b) H2, 5% Pd/C, EtOH; (c) Fe, AcOH;
(d) RNCO, DMAP/THF; (e) Ph-Y-COCl, Et3N/CH2Cl2; (f) PhOH, CDI/THF then DBU.

H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 3473–3480
3475
group resulted in decreased activities. This result sug-
gested that the presence of two NH groups was essential
to exhibit the activity.
case, it decreased the activity toward not only ECs but
also SMCs (5s). Cyclohexyl (5x), n-butyl (5w), and 4-
pyridyl (5t) groups instead of the phenyl ring gave only
weak activities. However, the benzyl group (5u) affor-
ded almost the same activity as 5d. As a result, 5k was
the most potent (IC50=2.4 nM for ECs), and was about
8000-fold more potent than Tranilast. In terms of cell
selectivity, most of the evaluated compounds displayed
a low selectivity of with the [SMCs]/[ECs] value ranging
from 0.1 to 0.8 times for ECs. However 5k and 5u
showed good selectivity by one order of magnitude
toward ECs. We further evaluated the relation between
the inhibition of ECs proliferation and anti-angiogenic
activity of 5k, which showed the most potent inhibitory
activity toward ECs proliferation and had much higher
selectivity than Tranilast.
Table 3 presents the results of the SAR study on sub-
stituents of the urea moiety. First, we examined the
position of an electron-donating group (amino group)
and an electron-withdrawing group (nitro group) on the
phenyl ring and found that the para position was the
most suitable for the activity in both cases (5i–n). Next,
we determined the effect of substituents at the para
position of the phenyl ring and found the order of
potency to be nitro (5k)>amino (5n), fluorine (5o),
hydrogen (5d)>methyl (5q), methoxy (5r)>acetyl (5p).
We previously reported that in the case of the ethoxy-
carbonyl substitution at the A ring, the 3,4,5-tri-
methoxyphenyl compound showed the most potent
inhibitory activity toward SMCs.4b However, in this
To evaluate the activity for the angiogenesis, we used
the method of tube formation by HUVECs on Matrigel.
Before this analysis, we confirmed the inhibitory activity
toward HUVECs in the same manner as used with
SMCs and ECs. The IC50 of 5k was 0.7 nM, indicating a
little greater potency toward HUVECs than toward
ECs. As shown in Figure 2, Tranilast had marginal
inhibitory activity against the tube formation even at
100 mM. However, the tube formation was significantly
reduced by the treatment of compound 5k in a dose-
dependent manner, and it was completely inhibited at
10 mM of 5k. Thus, we confirmed the superior inhibi-
tory activity of 5k against angiogenesis, as we expected,
compared with that of Tranilast.
Table 1. IC50 values of the compounds 5a–e for inhibition of pro-
liferation of SMCs and Ecs
Compd
X
ECs
IC50 (mM)a
SMCs
IC50 (mM)b
[SMCs]/
[ECs]
Tranilast
19
1
0.43
1.8
0.0520.07
0.13
25
1.3
2.8
0.6
1.5
5a
5b
5c
5d
5e
—
CH2
2.8
0.26
2.7
1.3
0.05
Table 3. IC50 values of the compounds 5d, i–x for inhibition of pro-
liferation of SMCs and Ecs
(E)-CH¼CH
(CH2)2
(CH2)3
0.38
aInhibitory activity against the proliferation of ECs induced by 5%
FBS.
bInhibitory activity against the proliferation of SMCs induced by
PDGF-BB (20 ng/mL).
Compd
R
ECs
SMCs
[SMCs]/
[ECs]
Table 2. IC50 values of the compounds 5d, f–h for inhibition of pro-
liferation of SMCs and Ecs
IC50 (mM)a IC50 (mM)b
Tranilast
5d
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
19 25
0.0520.07
1.3
Ph
2-NO2–Ph
3-NO2–Ph
4-NO2–Ph
2-NH2–Ph
3-NH2–Ph
4-NH2–Ph
4-F–Ph
1.3
0.045
0.017
0.05
0.0024
0.21
0.22
0.074
0.073
1.1
2.6
0.78
11
0.29
0.28
0.039
0.026
0.061
0.061
7 0.020.2
0.031
0.35
0.42
0.32
4-Ac–Ph
4-Me–Ph
4-OMe–Ph
3,4,5-(OMe)3–Ph
4-Py
Compd
Y
ECs
IC50 (mM)a
SMCs
IC50 (mM)b
[SMCs]/
[ECs]
0.20.17
0.37
1.4
1.20.16
0.020.125
1.20.43
0.20.077
0.85
0.120.31
0.31
0.13
Tranilast
19
25
1.3
0.22
5d
5f
5g
5h
NH
—
CH2
O
0.0520.07
1.7
1.6
1.3
5t
5u
5w
5x
0.620.37
0.22
3.21.7
Bn
n-Bu
c-Hex
9
0.14
0.35
0.39
1.9
aInhibitory activity against the proliferation of ECs induced by 5%
FBS.
bInhibitory activity against the proliferation of SMCs induced by
PDGF-BB (20 ng/mL).
aInhibitory activity against the proliferation of ECs induced by 5%
FBS.
bInhibitory activity against the proliferation of SMCs induced by
PDGF-BB (20 ng/mL).

3476
H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 3473–3480
Figure 2. Phase-contrast micrographs of tube formation by HUVECs on Matrigel. Morphological features of HUVECs treated with medium alone
(a), 100 mM Tranilast (b), 1 mM 5k (c), or 10 mM 5k (d) were photographed at ꢂ40 magnification with a phase-contrast microscope.
Experimental
By the way, many factors, such as fibroblast growth
factor (FGF),5a transforming growth factor-b (TGF-b)5b
Chemistry
and hepatocyte growth factor (HGF),5c have been
reported to regulate angiogenesis. Among them, vas-
cular endothelial growth factor (VEGF) was known to
be a potent and specific angiogenic factor.6 In fact, the
prototype compounds of the Kirin Brewery group,7a,b
which we also used for modification in our previous
study,4b are a potent inhibitor of VEGF receptor tyr-
osine kinase.8a,b On the other hand, according to the
several studies on the mechanism of the Tranilast, this
drug inhibited protein kinase C-dependent signaling
pathway linked to angiogenetic activities and did not
affect on VEGF binding and tyrosine phosphoryla-
tion of the receptor.9 On the basis of the structural
resemblance between our compounds and these two
series of compounds, elucidation of the target mol-
ecules of our derivatives and the mechanism of the
cell selectivity between SMCs and ECs are still under-
way.
In general, reagents and solvents were used as pur-
chased without further purification. Column chromato-
graphy was performed on FL60D (Fuji Silysia). 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 spectro-
meter. Chemical shifts were expressed in d (ppm) from
internal standard tetramethylsilane. Time-of-flight mass
spectrometry (TOFMS) were recorded on a Kompact
Maldi III spectrometer. Elemental analyses were per-
formed by the Toray Research Center.
N-(2-Acetyl-4,5-dimethoxyphenyl)(4-((phenylamino)car-
bonylamino)phenyl)formamide (5a). To a solution of 2
(0.10 g, 0.67 mmol) in CH2Cl2 (10 mL) were added
4-nitrobenzoyl chloride (0.13 g, 0.70 mmol) and trie-
thylamine (0.13 g, 0.70 mmol), and the mixture was
stirred for 1 h at room temperature. The reaction mix-
ture was poured into saturated NaHCO3 aq and
extracted with CH2Cl2, after which the organic layer
was washed with brine, dried over MgSO4, filtered and
concentrated to give 0.08 g (0.23 mmol) of 3a as a yel-
low solid with a yield of 34%.
In conclusion, we established the lead compound 5a, a
selective inhibitor for ECs, by introducing acetyl group
to the A ring of 1 instead of the ethoxycarbonyl group.
From the SAR study of 5a, we found compound 5k to
be superior to Tranilast in terms of the strength of the
activity and cell selectivity. Compound 5k may thus be a
useful compound for the treatment of diabetic retino-
pathy, rheumatic arthritis, psoriasis and tumors, all of
which rely on angiogenesis.
To a solution of 3a (0.08 g, 0.23 mmol) in methanol (10
mL) and tetrahydrofuran (10 mL) was added 5% Pd/C
(0.03 g), and the mixture was stirred under a H2

H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 3473–3480
3477
1
atmosphere for 18 h. The reaction mixture was filtered,
concentrated and washed with methanol to give 0.05 g
(0.16 mmol) of 4a as a yellow solid with a yield of 69%.
steps. Mp: 218–219 ꢃC; H NMR (DMSO-d6) d ppm:
2.60 (s, 3H), 2.67 (t, J =7.6 Hz, 2H), 2.89 (t, J =7.6 Hz,
2H), 3.82 (s, 6H), 6.95 (t, J =7.3 Hz, 1H), 7.16 (d, J
=8.4 Hz, 2H), 7.27 (t, J =8.1 Hz, 2H), 7.35 (d, J =8.4
Hz, 2H), 7.43 (s, 1H), 7.44 (d, J =8.4 Hz, 2H), 8.24 (s,
1H), 8.59 (s, 1H), 8.64 (s, 1H), 11.67 (s, 1H); MS (TOF)
To a solution of 4a (0.05 g, 0.16 mmol) and 4-dimethyl-
aminopyridine (0.02g, 0.18 mmol) in tetrahydrofuran (5
mL) was added phenylisocyanate (0.05 g, 0.42mmol),
and the mixture was refluxed for 3 h. The reaction mix-
ture 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=100:1ꢁ20:1) and washed with
methanol to give 0.04 g (0.10 mmol) of 5a as a white
+
m/z=462(M + H). Anal. calcd for C24H24N4O5: C,
67.66; H, 5.90; N, 9.10. Found: C, 67.40; H, 6.07; N,
9.01.
N-(2-Acetyl-4,5-dimethoxyphenyl)-4-(4-((phenylamino)-
carbonylamino)phenyl)butanamide (5e). Compound 5e
was prepared from 2 in a manner similar to that descri-
bed for compound 5a with a yield of 60% by three
steps. Mp: 183–185 ꢃC;1H NMR (DMSO-d6) d ppm:
1.90 (5, J =7.3 Hz, 2H), 2.38 (t, J =7.3 Hz, 2H), 2.51
(t, J =7.3 Hz, 2H), 2.62 (s, 3H), 3.82 (s, 6H), 6.95 (t, J
=7.3 Hz, 3H), 7.12(d, J =8.9 Hz, 2H), 7.26 (t, J =7.8
Hz, 2H), 7.37 (d, J =8.4 Hz, 2H), 7.44 (s, 1H), 7.45 (d,
J =8.4 Hz, 2H), 8.27 (s, 1H), 8.80 (s, 1H), 8.85 (s, 1H),
11.68 (s, 1H); MS (TOF) m/z=476 (M+ + H). Anal.
solid with a yield of 65%. Mp: 234–236 ꢃC; H NMR
1
(DMSO-d6) d ppm: 2.70 (s, 3H), 3.86 (s, 3H), 3.89 (s,
3H), 7.00 (t, J =7.3 Hz, 1H), 7.30 (t, J =7.6 Hz, 2H),
7.48 (d, J =7.8 Hz, 2H), 7.50 (s, 1H), 7.67 (d, J =8.9
Hz, 2H), 7.92 (d, J =8.9 Hz, 2H), 8.56 (s, 1H), 8.92 (s,
1H), 9.20 (s, 1H), 12.76 (s, 1H); MS (TOF) m/z=434
(M+ + H). Anal. calcd for C24H23N3O5 0.8H2O: C,
.
64.36; H, 5.54; N, 9.38. Found: C, 64.06; H, 5.44; N,
9.34.
.
calcd for C27H29N3O5 0.6H2O : C, 66.67; H, 6.26; N,
8.64. Found: C, 66.51; H, 6.15; N, 8.62.
N-(2-Acetyl-4,5-dimethoxyphenyl)-2-(4-((phenylamino)-
carbonylamino)phenyl)ethanamide (5b). Compound 5b
was prepared from 2 in a manner similar to that descri-
bed for compound 5a with a yield of 8% by three steps.
Mp: 208–210 C; H NMR (DMSO-d6) d ppm: 2.58 (s,
3H), 3.66 (s, 2H), 3.81 (s, 6H), 6.96 (t, J =7.3 Hz, 1H),
7.27 (m, 4H), 7.43 (m, 5H), 8.28 (s, 1H), 8.66 (s, 1H),
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(phenylcarbonyl-
amino)phenyl)propanamide (5f). To a solution of 4c
(0.05 g, 0.15 mmol) in CH2Cl2 (20 mL) were added
benzoyl chloride (0.03 g, 0.21 mmol) and triethylamine
(0.03 g, 0.30 mmol), and the mixture was stirred for 30
min at room temperature. The reaction mixture was
poured into saturated NaHCO3 aq and extracted with
CH2Cl2, after which the organic layer was washed with
brine, dried over MgSO4, filtered and concentrated. The
residue was purified by silica gel column chromato-
graphy (CH2Cl2/methanol=500:1ꢁ200:1) and washed
with methanol to give 0.03 g (0.07 mmol) of 5f as a
ꢃ
1
8.68 (s, 1H), 11.72(s, 1H); MS (TOF) m/z=448
+
.
(M +H). Anal. calcd for C25H25N3O5 0.2H2O: C,
66.56; H, 5.68; N, 9.31. Found: C, 66.43; H, 5.71; N,
9.26.
white solid with a yield of 45%. Mp: 211–213 ꢃC; H
1
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-((phenylamino)-
carbonylamino)phenyl)prop-2-enamide (5c). Compound
3c was prepared from 2 in a manner similar to that
described for compound 3a with a yield of 57%.
NMR (DMSO-d6) d ppm: 2.60 (s, 3H), 2.70 (t, J =7.6
Hz, 2H), 2.95 (t, J =7.6 Hz, 2H), 3.82 (s, 6H), 7.24 (d, J
=8.6 Hz, 2H), 7.43 (s, 1H), 7.56 (m, 3H), 7.68 (dd,
J1=8.6 Hz, J2=1.6 Hz, 2H), 7.93 (dd, J1=8.6 Hz, J2
=1.6 Hz, 2H), 8.24 (s, 1H), 10.19 (s, 1H), 11.68 (s, 1H);
MS (TOF) m/z=447 (M+ + H). Anal. calcd for
To a solution of 3c (0.13 g, 0.35 mmol) in AcOH (10
mL) was added Fe (0.23 g), and the mixture was heated
for 7 h at 70 ꢃC. The reaction mixture was concentrated
and the residue was purified by silica gel column chroma-
tography (CH2Cl2/methanol=300:1ꢁ50:1) and washed
with methanol to give 0.02g (0.06 mmol) of 4e as a
yellow solid with a yield of 17%.
.
C26H26N2O5 0.2H2O: C, 69.38; H, 5.91; N, 6.23. Found:
C, 69.11; H, 5.87; N, 6.09.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(2-phenylacetyl-
amino)phenyl)propanamide (5g). Compound 5g was pre-
pared from 4c in a manner similar to that described for
compound 5f with a yield of40%. Mp: 162–165 ꢃC; H
1
Compound 5c was prepared from 4e in a manner simi-
lar to that described for compound 5a with a yield of
NMR (DMSO-d6) d ppm: 2.58 (s, 3H), 2.66 (t, J =7.6
Hz, 2H), 2.88 (t, J =7.6 Hz, 2H), 3.61 (s, 2H), 3.82 (s,
6H), 7.18 (d, J =8.4 Hz, 2H), 7.31 (m, 5H), 7.41 (s, 1H),
7.49 (d, J =8.4 Hz, 2H), 8.21 (s, 1H), 10.10 (s, 1H),
11.65 (s, 1H); MS (TOF) m/z=461 (M+ + H). Anal.
calcd for C27H28N2O5: C, 70.42; H, 6.13; N, 6.08.
Found: C, 70.40; H, 6.13; N, 6.08.
ꢃ
1
75%. Mp: 291–292 C; H NMR (DMSO-d6) d ppm:
2.66 (s, 3H), 3.85 (s, 3H), 3.86 (s, 3H), 6.72 (d, J =15.4
Hz, 1H), 6.98 (t, J =7.3 Hz, 1H), 7.32(t, J =7.6 Hz, 2H),
7.53 (m, 6H), 7.67 (d, J =8.6 Hz, 2H), 8.43 (s, 1H), 8.87 (s,
1H), 9.06 (s, 1H), 11.89 (s, 1H); MS (TOF) m/z=459 (M+
.
+ H). Anal. calcd for C26H25N3O5 0.01H2O: C, 65.39; H,
5.70; N, 8.80. Found: C, 65.10; H, 5.46; N, 8.68.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(phenoxycarbo-
nylamino)phenyl)propanamide (5 h). To a suspension of
1,1-carbonyldiimidazole (0.34 g, 2.10 mmol) in tetra-
hydrofuran (5 mL) was added phenol (0.18 g, 0.18
mmol) at 4 ꢃC and the mixture stirred for 3 h at room
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-((phenylamino)-
carbonylamino)phenyl)propanamide (5d). Compound 5d
was prepared from 2 in a manner similar to that descri-
bed for compound 5a with a yield of 57% by three

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H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 3473–3480
temprature. The resulting solution was added to a sus-
pension of 4c (0.13 g, 0.38 mmol) and 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (0.06 g, 0.39 mmol) in
tetrahydrofuran (5 mL). After heating for 3 h at 50 ꢃC,
the mixture was concentrated and the residue 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=300:1ꢁ100:1) and washed with methanol to
give 0.03 g (0.06 mmol) of 5h as a white solid with a
14 h. The reaction mixture was filtered, concentrated
and washed with methanol to give 0.07 g (0.06 mmol) of
5l as a white solid with a yield of 66%. Mp: 184–186 ꢃC;
1H NMR (DMSO-d6) d ppm: 2.60 (s, 3H), 2.67 (t, J
=7.3 Hz, 2H), 2.88 (t, J =7.3 Hz, 2H), 3.82 (s, 6H),
4.76 (s, 2H), 6.56 (dt, Jd=1.4 Hz, Jt=7.3 Hz, 1H), 6.72
(dd, J1=1.4 Hz, J2=7.8 Hz, 1H), 6.83 (dt, Jd=1.4 Hz,
Jt=7.8 Hz, 1H), 7.15 (d, J =8.4 Hz, 2H), 7.36 (m, 3H),
7.43 (s, 1H), 7.71 (s, 1H), 8.24 (s, 1H), 8.69 (s, 1H),
11.66 (s, 1H); MS (TOF) m/z=476 (M+ + H). Anal.
calcd for C26H28N4O5: C, 65.53; H, 5.92; N, 11.76.
Found: C, 65.30; H, 5.97; N, 11.83.
yield of 11%. Mp: 180–182 ꢃC; H NMR (DMSO-d6) d
1
ppm: 2.58 (t, J=7.3 Hz, 2H), 2.59 (s, 3H), 2.76 (t, J
=7.3 Hz, 2H), 3.82 (s, 6H), 6.46 (d, J =8.1 Hz, 2H),
6.76 (m, 3H), 6.88 (d, J =8.1 Hz, 2H), 7.15 (t, J =8.1
Hz, 2H), 7.43 (s, 1H), 8.24 (s, 1H), 9.25 (s, 1H), 11.66 (s,
1H); MS (TOF) m/z=463 (M+ + H). Anal. calcd for
C26H26N2O6: C, 67.52; H, 5.67; N, 6.06. Found: C,
67.46; H, 5.71; N, 6.09.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((3-aminophenyl)-
amino)carbonylamino)phenyl)propanamide (5m). Com-
pound 5m was prepared from 5j in a manner similar to
that described for compound 5a with a yield of 73%.
Mp: 208–209 ꢃC; H NMR (DMSO-d6) d ppm: 2.60 (s,
1
3H), 2.67 (t, J =7.3 Hz, 2H), 2.88 (t, J =7.3 Hz, 2H),
3.82(s, 6H), 5.01 (s, 2H), 6.17 (d, J =9.5 Hz, 1H), 6.54
(d, J =8.6 Hz, 1H), 6.76 (s, 1H), 6.87 (t, J =7.8 Hz,
1H), 7.15 (d, J =8.1 Hz, 2H), 7.34 (d, J =8.1 Hz, 2H),
7.39 (s, 1H), 8.14 (s, 1H), 8.40 (s, 1H), 8.55 (s, 1H),
11.67 (s, 1H); MS (TOF) m/z=476 (M+ + H). Anal.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((2-nitrophenyl)-
amino)carbonylamino)phenyl)propanamide (5i). Com-
pound 5d was prepared from 4c in a manner similar to
that described for compound 5a with a yield of 52%.
Mp: 221–222 ꢃC; H NMR (DMSO-d6) d ppm: 2.60 (s,
calcd for C26H28N4O5 0.4H2O: C, 64.58; H, 5.96; N,
11.59. Found: C, 64.70; H, 5.71; N, 11.54.
1
.
3H), 2.68 (t, J =7.3 Hz, 2H), 2.90 (t, J =7.3 Hz, 2H),
3.82(s, 6H), 7.16 (m, 3H), 7.40 (m, 3H), 7.69 (dt,
Jd=1.6 Hz, Jt=8.4 Hz, 1H), 8.09 (dd, J1=1.4 Hz,
J2=8.4 Hz, 1H), 8.25 (s, 1H), 8.31 (d, J =8.4 Hz, 1H),
9.58 (s, 1H), 9.79 (s, 1H), 11.69 (s, 1H). Anal. calcd for
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((4-aminophenyl)-
amino)carbonylamino)phenyl)propanamide (5n). Com-
pound 5n was prepared from 5k in a manner similar to
that described for compound 5a with a yield of 26%.
.
C26H26N4O7 0.3H2O: C, 61.00; H, 5.24; N, 10.92.
Found: C, 61.05; H, 5.19; N, 10.88.
Mp: >300 ꢃC; H NMR (DMSO-d6) d ppm: 2.60 (s,
1
3H), 2.66 (t, J =7.6 Hz, 2H), 2.87 (t, J =7.3 Hz, 2H),
3.82(s, 6H), 4.57 (s, 2H), 6.49 (d, J =8.4 Hz, 2H), 7.05
(d, J =8.1 Hz, 2H), 7.13 (d, J =8.6 Hz, 2H), 7.32 (d, J
=8.6 Hz, 2H), 7.43 (s, 1H), 8.10 (s, 1H), 8.24 (s, 1H),
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((3-nitrophenyl)-
amino)carbonylamino)phenyl)propanamide (5j). Com-
pound 5j was prepared from 4c in a manner similar to
that described for compound 5a with a yield of 80%.
9.40 (s, 1H), 11.67 (s, 1H); MS (TOF) m/z=476 (M+
+
Mp: 188–190 ꢃC; H NMR (DMSO-d6) d ppm: 2.60 (s,
H). Anal. calcd for C26H28N4O5: C, 63.60; H, 6.08; N,
11.41. Found: C, 63.62; H, 5.92; N, 11.31.
1
3H), 2.69 (t, J =7.3 Hz, 2H), 2.90 (t, J =7.3 Hz, 2H),
3.82(s, 6H), 7.19 (d, J =8.9 Hz, 2H), 7.40 (m, 3H), 7.56
(t, J =8.1 Hz, 1H), 7.69 (d, J =8.4 Hz, 1H), 7.80 (dd,
J1=1.9 Hz, J2=8.4 Hz, 1H), 8.24 (s, 1H), 8.56 (m, 1H),
8.78 (s, 1H), 9.20 (s, 1H), 11.68 (s, 1H). Anal. calcd for
C26H26N4O7: C, 61.65; H, 5.17; N, 11.06. Found: C,
61.56; H, 5.11; N, 11.05.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((4-fluorophenyl)-
amino)carbonylamino)phenyl)propanamide (5o). Com-
pound 5o was prepared from 4c in a manner similar to
that described for compound 5a with a yield of 43%.
Mp: 210–211 ꢃC; H NMR (DMSO-d6) d ppm: 2.60 (s,
1
3H), 2.68 (t, J =7.6 Hz, 2H), 2.89 (t, J =7.6 Hz, 2H),
3.82(s, 3H), 3.84 (s, 3H), 7.09 (d, J =7.3 Hz, 2H), 7.15
(d, J =8.6 Hz, 2H), 7.35 (d, J =8.6 Hz, 2H), 7.43 (m,
3H), 8.24 (s, 1H), 8.61 (s, 1H), 8.70 (s, 1H), 11.67 (s,
1H); MS (TOF) m/z=480 (M+ + H). Anal. calcd for
C26H26FN3O5: C, 65.13; H, 5.47; N, 8.76. Found: C,
65.12; H, 5.49; N, 8.72.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((4-nitrophenyl)-
amino)carbonylamino)phenyl)propanamide (5k). Com-
pound 5k was prepared from 4c in a manner similar to
that described for compound 5a with a yield of 92%.
Mp: 195–196 ꢃC; H NMR (DMSO-d6) d ppm: 2.51 (s,
1
3H), 2.68 (t, J =7.6 Hz, 2H), 2.90 (t, J =7.3 Hz, 2H),
3.85 (s, 6H), 7.19 (d, J =7.3 Hz, 2H), 7.40 (m, 3H), 7.68
(d, J =9.5 Hz, 2H), 8.20 (m, 3H), 8.86 (s, 1H), 9.42 (s,
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((4-acetylphenyl)-
amino)carbonylamino)phenyl)propanamide (5p). Com-
pound 5p was prepared from 4c in a manner similar to
that described for compound 5a with a yield of 98%.
.
1H), 11.68 (s, 1H). Anal. calcd for C26H26N4O7 0.1H2O:
C, 61.43; H, 5.20; N, 11.02. Found: C, 61.23; H, 5.17; N,
11.11.
Mp: 210–212 ꢃC; H NMR (DMSO-d6) d ppm: 2.50 (s,
1
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((2-aminophenyl)-
amino)carbonylamino)phenyl)propanamide (5l). To
solution of 5i (0.11 g , 0.22 mmol) in tetrahydrofuran
(20 mL) was added 5% Pd/C (0.02 g), which was then
stirred at room temperature under a H2 atmosphere for
6H), 2.68 (t, J =7.6 Hz, 2H), 2.89 (t, J =7.6 Hz, 2H),
3.82(s, 6H), 6.18 (d, J =8.6 Hz, 2H), 7.37 (d, J =8.6
Hz, 2H), 7.43 (s, 1H), 7.57 (d, J =8.6 Hz, 2H), 7.90 (d,
J =8.6 Hz, 2H), 8.24 (s, 1H), 8.78 (s, 1H), 9.13 (s, 1H),
11.67 (s, 1H); MS (TOF) m/z=504 (M+ + H). Anal.
a

H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 3473–3480
3479
8.50 (s, 1H), 11.66 (s, 1H); MS (TOF) m/z=476 (M+
+
H). Anal. calcd for C27H29N3O5: C, 68.19; H, 6.15; N,
8.84. Found: C, 67.98; H, 6.21; N, 8.82.
.
calcd for C28H29N3O6 0.1H2O: C, 66.54; H, 5.82; N,
8.32. Found: C, 66.36; H, 5.85; N, 8.24.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((4-methylphenyl)-
amino)carbonylamino)phenyl)propanamide (5q). Com-
pound 5q was prepared from 4c in a manner similar to
that described for compound 5a with a yield of 74%.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-((butyl amino)car-
bonylamino)phenyl)propanamide (5w). Compound 5w
was prepared from 4c in a manner similar to that
described for compound 5a with a yield of 47%. Mp:
182–184 ꢃC; 1H NMR (DMSO-d6) d ppm: 0.88 (t,
J=7.0 Hz, 3H), 1.32 (m, 4H), 2.60 (s, 3H), 2.70 (t,
J=7.3 Hz, 2H), 2.85 (t, J=7.3 Hz, 2H), 3.07 (q, J=6.5
Hz, 2H), 3.82 (s, 6H), 6.08 (t, J=5.6 Hz, 1H), 7.08 (d,
J=8.6 Hz, 2H), 7.27 (d, J=8.6 Hz, 2H), 7.42 (s, 1H),
Mp: 185–190 ꢃC; H NMR (DMSO-d6) d ppm: 2.23 (s,
1
3H), 2.60 (s, 3H), 2.66 (t, J=7.6 Hz, 2H), 2.88 (t, J=7.6
Hz, 2H), 3.84 (s, 6H), 7.07 (d, J=8.6 Hz, 2H), 7.15 (d,
J=8.6 Hz, 2H), 7.31 (d, J=6.5 Hz, 2H), 7.34 (d,
J=6.5 Hz, 2H), 7.43 (s, 1H), 8.24 (s, 1H), 8.55 (s, 1H),
8.57 (s, 1H), 11.67 (s, 1H); MS (TOF) m/z=476
+
.
(M +H). Anal. calcd for C27H29N3O5 0.6H2O: C,
8.23 (s, 1H), 8.31 (s, 1H), 11.66 (s, 1H); MS (TOF) m/
+
.
z=442(M +H). Anal. calcd for C24H31N3O5 0.2H2O:
66.68; H, 6.26; N, 8.64. Found: C, 66.83; H, 6.14; N,
8.48.
C, 64.76; H, 7.11; N, 9.44. Found: C, 64.76; H, 7.18; N,
9.30.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((4-methoxyphe-
nyl)amino)carbonylamino)phenyl)propanamide (5r). Com-
pound 5r was prepared from 4c in a manner similar to
that described for compound 5a with a yield of 56%.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-((cyclohexylami-
no)carbonylamino)phenyl)propanamide (5x). Compound
5x was prepared from 4c in a manner similar to that
described for compound 5a with a yield of 59%. Mp:
204–206 ꢃC; 1H NMR (DMSO-d6) d ppm: 1.32(m,
10H), 2.59 (s, 3H), 2.64 (t, J=7.3 Hz, 2H), 2.85 (t,
J=7.3 Hz, 2H), 3.43 (m, 1H), 3.82 (s, 6H), 6.03 (d,
J=7.8 Hz, 1H), 7.08 (d, J=8.4 Hz, 2H), 7.26 (d, J=8.4
Hz, 2H), 7.42 (s, 1H), 8.21 (s, 1H), 8.23 (s, 1H), 11.65 (s,
1H); MS (TOF) m/z=468 (M++H). Anal. calcd for
Mp: 209–212 ꢃC; H NMR (DMSO-d6) d ppm: 2.60 (s,
1
3H), 2.70 (t, J=7.6 Hz, 2H), 2.88 (t, J=7.6 Hz, 2H),
3.71 (s, 3H), 3.84 (s, 6H), 6.85 (d, J=9.2Hz, 2H), 7.15
(d, J=8.6 Hz, 2H), 7.34 (d, J=8.9 Hz, 4H), 7.43 (s,
1H), 8.24 (s, 1H), 8.50 (s, 1H), 8.56 (s, 1H), 11.67 (s,
+
1H); MS (TOF) m/z=492(M
+ H). Anal. calcd for
C27H29N3O6: C, 65.97; H, 5.95; N, 8.55. Found: C,
65.95; H, 5.98; N, 8.59.
.
C26H33N3O5 0.3H2O: C, 66.02; H, 7.16; N, 8.89. Found:
C, 65.96; H, 7.16; N, 8.83.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((3,4,5-trimeth-
oxyphenyl)amino)carbonylamino)phenyl)propanamide (5s).
Compound 5s was prepared from 4c in a manner simi-
lar to that described for compound 5a with a yield of
Primary culture of smooth muscle cells and endothelial
cells
23%. Mp: 201–203 ꢃC; H NMR (DMSO-d6) d ppm:
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 (2ng/mL),
gentamicin (50 mg/mL), and amphotericin-B (50 pg/
mL). ECs were cultured in basal medium (EBM)
containing 5% fetal bovine serum (FBS), human epi-
dermal 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
3–5 days in culture at 37 ꢃC in 5% CO2–95% air,
both cell types were subcultured by trypsinization and
propagated in each complete medium described
above.
1
2.51 (s, 3H), 2.67 (t, J=7.6 Hz, 2H), 2.89 (t, J=7.6 Hz,
2H), 3.60 (s, 3H), 3.74 (s, 6H), 3.82 (s, 6H), 6.78 (s, 2H),
7.15 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz, 2H), 7.43 (s,
1H), 8.24 (s, 1H), 8.54 (s, 1H), 8.60 (s, 1H), 11.68 (s,
1H); MS (TOF) m/z=552(M ++H). Anal. calcd for
C29H33N3O8: C, 63.15; H, 6.03; N, 7.62. Found: C,
62.91; H, 6.09; N, 7.54.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-(((4-pyridyl)ami-
no)carbonylamino)phenyl)propanamide (5t). Compound
5t was prepared from 4c in a manner similar to that
described for compound 5a with a yield of 74%. Mp:
201–202 ꢃC; H NMR (DMSO-d6) d ppm: 2.60 (s, 3H),
1
2.68 (t, J=7.3 Hz, 2H), 2.90 (t, J=7.3 Hz, 2H), 3.82 (s,
6H), 7.19 (d, J=8.4 Hz, 2H), 7.40 (m, 5H), 8.24 (s, 1H),
8.34 (d, J=6.5 Hz, 2H), 8.83 (s, 1H), 9.11 (s, 1H), 11.66
(s, 1H); MS (TOF) m/z=463 (M+ + H). Anal. calcd
.
for C25H26N4O5 0.1H2O: C, 64.67; H, 5.69; N, 12.07.
Found: C, 64.51; H, 5.69; N, 11.98.
Determination of DNA synthesis in smooth muscle cells
SMCs from passage 1-3 were seeded into 96-well plates
(3 ꢂ 104 cells/well) in the complete medium described
above, and cultured for 16–18 h at 37 ꢃC in 5% 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, USA)
and various concentrations of the test compounds.
N-(2-Acetyl-4,5-dimethoxyphenyl)-3-(4-((benzylamino)-
carbonylamino)phenyl)propanamide (5u). Compound 5u
was prepared from 4c in a manner similar to that
described for compound 5a with a yield of 93%. Mp:
189–190 ꢃC; H NMR (DMSO-d6) d ppm: 2.59 (s, 3H),
1
2.65 (t, J=7.3 Hz, 2H), 2.86 (t, J=7.3 Hz, 2H), 3.81 (s,
6H), 4.27 (m, 2H), 6.60 (t, J=7.3 Hz, 1H), 7.10 (d,
J=8.4 Hz, 2H), 7.30 (m, 9H), 7.42 (s, 1H), 8.23 (s, 1H),
3
After 24 h, 1 mCi/mL H-thymidine was added to the
medium, and the cells were incubated for 4 h at 37 ꢃC in

3480
H. Ogita et al. / Bioorg. Med. Chem. 10 (2002) 3473–3480
5% CO2–95% air. Then the cells were harvested by
trypsinization, and the amount of radioactive thymidine
incorporated into the DNA was determined by scintil-
lation counting.
References and Notes
1. (a) Azuma, H.; Banno, K.; Yoshimura, T. Br. J. Pharma-
col. 1976, 8, 483. (b) Suzawa, H.; Kikuchi, S.; Arai, N.; Koda,
A. Jpn. J. Pharmacol. 1992, 60, 91. (c) Suzawa, H.; Kikuchi,
S.; Ichikawa, K.; Koda, A. Jpn. J. Pharmacol. 1992, 60, 85.
2. (a) Isaji, M.; Miyata, H.; Ajisawa, Y. PCT Patent Publica-
tion, WO 97/29744. (b) Isaji, M.; Miyata, H.; Ajisawa, Y.;
Yoshimura, N. Br. J. Pharmacol. 1997, 122, 1061.
Determination of DNA synthesis in endothelial cells
ECs from the second passage were seeded into 96-well
plates (3 ꢂ 103 cells/well) in the above complete med-
ium, and allowed to attach to the plates for 4 h at 37 ꢃC
in 5% CO2–95% air. Then various concentrations of
test compounds were added to the complete medium,
and the cells were cultured at 37 ꢃC in 5% CO2–95%
air. After 3 days, DNA synthesis was determined during
the last 4 h of the 3-day culture.
3. Timar, J.; Dome, D.; Fazakas, K.; Janovics, A.; Paku, S.
Pathol. Oncol. Res. 2001, 7, 85.
4. (a) Ogita, H.; Isobe, Y.; Takaku, H.; Sekine, R.; Goto, Y.;
Misawa, S.; Hayashi, H. Bioorg. Med. Chem. Lett. 2001, 11, 549.
(b) Ogita, H.; Isobe, Y.; Takaku, H.; Sekine, R.; Goto, Y.;
Misawa, S.; Hayashi, H. Bioorg. Med. Chem. 2002, 10, 1865.
5. (a) Kandel, J.; Bossy-Wetzel, E.; Radvanyi, F.; Klagsbrun,
M.; Folkman, J.; Hanahan, D. Cell. 1991, 66, 1095. (b) Pep-
per, M. S.; Belin, D.; Montesano, R.; Orci, L.; Vassalli, J. D.
J. Cell. Biol. 1990, 111, 743. (c) Bussolino, F.; Di Renzo, M. F.;
Ziche, M.; Bocchietto, E.; Olivero, M.; Naldini, L.; Gaudino,
G.; Tamagnone, L.; Coffer, A.; Comoglio, P. M. J. Cell. Biol.
1992, 119, 629.
6. Ferrara, N.; Davis-Smyth, T. Endocr. Rev. 1997, 18, 4.
7. (a) Kubo, K.; Shimizu, T.; Ohyama, S.; Murooka, H.;
Nishitoba, T.; Kato, S.; Kobayashi, Y.; Yagi, M.; Isoe, T.;
Nakamura, K.; Osawa, T.; Izawa, T. Bioorg. Med. Chem. Lett.
1997, 7, 2935. (b) Kubo, K.; Shimizu, T.; Ohyama, S. PCT
Patent Publication, WO 97/17329.
8. (a) Kubo, K.; Fujiwara, Y.; Isoe, T. PCT Patent Publica-
tion, WO 00/43366. (b) Kubo, K.; Fujiwara, Y.; Iwakubo, M.;
Murooka, H.; Iwai, A.; Nakamura, K.; Hasegawa, K.; Kobaya-
shi, Y.; Takahashi, N.; Takahashi, K.; Shibuya, M.; Osawa, T.;
Isoe, T. Am. Assoc. Cancer Res. 2002, 43, 182(abstract 913).
9. Isaji, M.; Miyata, H.; Ajisawa, Y.; Yoshimura, N. Br. J.
Pharmacol. 1997, 127, 537.
HUVECs microcapillary assay
The formation of capillary-like tubular structures was
assessed in Matrigel-coated multiwell plates. Aliquots of
Matrigel (Becton Dickinson Labware; 100 mL) were
distributed as a thin layer in 48-well cell culture plates
(Becton Dickinson Labware) and left to polymerize at
37 ꢃC for 30 min. Human umbilical vein endothelial
cells (HUVECs, Clonetics; 4 ꢂ 104/500 mL) were seeded
on the Matrigel and cultured in EGM-2medium (Clo-
netics) containing various concentrations of test com-
pounds at 37 ꢃC for 24 h in a humidified atmosphere
containing 5% CO2. After incubation, the tubular for-
mation was observed with a phase-contrast microscope
and microphotographed at ꢂ40 magnification.