1,3-Disubstituted benzazepines as neuropeptide Y Y1 receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(99)00087-5

A novel class of potent and selective non-peptide neuropeptide Y (NPY) Y1 receptor antagonists, having benzazepine nuclei, have been designed, synthesized, and evaluated for activity. Through a blind screening we found the compound 1-N-(3-(N' -(tert-butoxycarbonyl)amino)benzyl)-7-methoxy-3-(3)-methylureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (9: IC₅₀ =1.6 μM). Chemical modifications of 9 gave a potent NPY Y1 antagonist 3-(N-(4-hydroxyphenyl)-N0 -methylguanidino)-1-N-(3-(N'-(tert- butoxycarbonyl)amino)benzyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (14c: IC₅₀ =43 nM), which had no affnity for NPY Y2 and Y5 receptors. (C) 1999 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0968-0896(99)00087-5

Bioorganic & Medicinal Chemistry 7 (1999) 1703±1714
1,3-Disubstituted Benzazepines as Neuropeptide Y Y1 Receptor
Antagonists
Yasushi Murakami,* Sanji Hagishita, Tetsuo Okada, Makoto Kii, Hiroshi Hashizume,
Tatsuroh Yagami and Masafumi Fujimoto
Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553-0002, Japan
Received 11 January 1999; accepted 1 April 1999
AbstractÐA novel class of potent and selective non-peptide neuropeptide Y (NPY) Y1 receptor antagonists, having benzazepine
nuclei, have been designed, synthesized, and evaluated for activity. Through a blind screening we found the compound 1-N-(3-
(N0-(tert-butoxycarbonyl)amino)benzyl)-7-methoxy-(3-(3)-methylureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (9: IC50=1.6 mM).
Chemical modi®cations of 9 gave a potent NPY Y1 antagonist 3-(N-(4-hydroxyphenyl)-N0-methylguanidino)-1-N-(3-(N0-(tert-
butoxycarbonyl)amino)benzyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (14c: IC50=43 nM), which had no anity for NPY Y2
and Y5 receptors. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
our screening of the Shionogi compound library, we
found compound 9 which showed mmol order of bind-
ing anity for the NPY Y1 receptor. Compound 9 has a
benzazepine nucleus, which prompted us to pursue its
chemical modi®cation. In this paper, we report the
design, synthesis and discovery of a series of novel
potent and selective NPY Y1 receptor antagonists,
compound 1 (Fig. 1).
Neuropeptide Y (NPY), a 36-amino acid polypeptide
hormone was discovered in 1982 by Tatemoto et al.1
NPY is suggested to play diverse physiological roles in
the control of cardiovascular function,2 food intake,3
energy production, pain,4 and anxiety via a number of
G-protein coupled receptors,5±8 Y1,9 Y2,10 Y4/PP1,11
and Y5.3 Of the receptors, the Y1 receptor is best char-
acterized and has been demonstrated to mediate long-
lasting vasoconstriction in the periphery.12 On the other
hand, the Y1 receptor in the central nervous system
appears to participate in food intake because a selective
Y1 antagonist inhibits the increase in food intake after
intracerebroventricular injection of NPY.13 The recently
cloned Y5 receptor is also suggested to be involved in
feeding behavior,3 but further investigation is needed to
prove this suggestion. These observations revealed that
the NPY Y1 receptor may be very useful for targeted
therapeutic treatment, especially for congestive heart
failure, angina pectoris, hypertension, and obesity.
Chemistry
3-Aminobenzylalcohol, 2, was treated with (BOC)2O to
protect the amino group and then brominated with NBS
and triphenylphosphine to give a benzyl bromide deri-
vative, 4 (Scheme 1). N-Alkylation of 3-azidobenzaze-
pine, 7b,17 or 3-azido-7-methoxybenzazepine, 7a, which
was prepared from 7-methoxybenzazepine, 5,18 by the
same procedure described for the preparation of 7b,17
was achieved by reaction with 4 under phase transfer
conditions (Method A) and then the azide group was
reduced to amino compounds 8a and 8b. Urea deriva-
tives, 9 and 10a±s, were prepared by several methods as
outlined in Scheme 2. First, an amino compound, 8a or
8b, was treated with isocyanates to obtain urea deriva-
tives 9 and 10a±f (Method B). Second, 8b was treated
with carbonyldiimidazole and then with amines to
obtain 10g (Method C1). Alternatively, carbonyldiimi-
dazole and several protected amines were reacted initi-
ally to give imidazolylaminocarbonyl compound which
was then reacted with 8b, followed by deprotection to
To date, four non-peptide NPY Y1 receptor antago-
nists, BIBP3226 (Ki=7.2 nmol),13 SR120819A (Ki=15
nmol),14 PD160170 (Ki=48 nmol),15 and LY-357897
(Ki=0.75 nmol),16 have been reported. In the course of
* Corresponding author. Tel.: +81-06-6458-5861; fax: +81-06-6458-
0987.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00087-5

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Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
Structure±Activity Relationship and Discussion
The compounds prepared in this study were evaluated
as NPY Y1 antagonists by testing their potency to dis-
place [125I]peptide YY ([125I]PYY) binding to SK-N-
MC cells. To ®nd antagonists more active than 9, which
was found to be active by a blind screening, three posi-
tions for substituents, R, R1, and R3 of 1, were selected
for chemical modi®cation.
Figure 1. Substitutents R, R1, and R3 selected for chemical modi®ca-
tions.
We ®rst evaluated whether the OMe group at position 6
was indispensable for the activity of the NPY Y1 bind-
ing anity. Since the OMe group de®cient analogue 10a
had slightly stronger binding anity than 9, we carried
out the chemical modi®cation on unsubstituted com-
pounds at the benzene ring. When substituent R3 was
®xed to the 3-methyl ureido group, we introduced var-
ious kinds of bulky alkyl groups to R1, but none of
them were active (Table 1). Therefore, the variation of
R3 was examined when substituent R1 was ®xed as the
3-N-(tert-butoxycarbonylamino)benzyl group (Table 2).
As the bulkiness of the alkyl group at the urea moiety
increases, the binding anity decreased (10a±d). The
absence of hydrogen of the terminal urea eliminated
binding anity (10g versus 10a and 10b). This suggests
that the ureido hydrogen interacts with a hydrogen
bond acceptor in the binding site of the NPY Y1 recep-
tor. The compounds with aryl groups at the 3-position
of the urea group were more potent than 10a. The fact
that the phenyl urea group, but not the cyclohexyl one,
enhanced binding anity (10d versus 10e) indicates that
the aromaticity of the substituents at urea is favorable
for the NPY Y1 binding anity. The e€ects of intro-
ducing a substituent to the ortho-, meta-, or para-posi-
tion of the ureido benzene ring were examined next
(10h, 10i, and 10j). The para-substituted one 10j was the
most potent and thus was chosen for further modi®ca-
tion. Because the phenolic proton was acidic, we intro-
duced an acidic group such as a carboxyl group and a
tetrazolyl group at the para-position (10k, 10l and 10m)
but they were detrimental to the activity. We also
examined the e€ect of introducing heteroaromatic
groups. Basic groups, such as pyridine or pyrimidine
groups, had an unfavorable e€ect on the receptor bind-
ing anity (10n and 10o), and the tert-Bu group may be
too bulky a substituent (10q). The tert-Bu group may
cause an unfavorable steric interation with the receptor.
Only the compound 10s was more potent than 10e.
give ureas 10i and 10j (Method C2). Third, 8b was
treated with triphosgene and triethylamine, followed by
the reaction with amines to give ureas (10h, 10l, 10n,
10o, 10p, 10r, and 10s) (Method D). In a synthesis of
10q, 8b was reacted with the isocyanate which was gen-
erated in situ according to Method D from 5-amino-1-
tosyl-benzimidazole prepared from 5-nitro-1-tosyl-ben-
zimidazole19 by catalytic hydrogenolysis and then
hydrolyzed by treatment with methanolic KOH.
Compound 10k was obtained by the treatment of 8b
with 4-phenoxycarbonylaminobenzoic acid20 (Method
E). Compound 10j was converted to 10m by the treat-
ment with sulfur trioxide pyridine complex, followed by
treatment with aqueous NaOH.
7b was converted to compound 11a±n by the same pro-
cedure described for the preparation of 10a (Scheme 3).
8b was converted to an amide, 12, and a carbonate
derivative, 13, by treatment with acetyl chloride and
methylchlorocarbonate, respectively. 8b was further
converted to guanidine derivatives, 14a±c, by the reac-
tion with S-methylisothiourea hydroiodide salt, or 14d±
g by the reaction with isothiocyanate compounds, fol-
lowed by methylation at the sulfur position with
methyliodide and then by heating with amines, as shown
in Scheme 4.
3-(Phthalimido)-2,3-dihydro-1,5(5H)-benzothiazepine-4-
one, 15,21 was treated with hydrazine hydrate and then
with phenylisocyanate to give an urea derivative, which
was N-alkylated as above to a€ord 16. 3-(Cbz-amino)-
2,3-dihydro-1,5(5H)-benzoxazepine-4-one, 17,21 was N-
alkylated as above and after removal of the Cbz group
by hydrogenolysis, the amino compound was converted
to the urea derivative, 18 (Schemes 5 and 6).
Replacement of the urea with amide or carbonate moi-
ety resulted in two and fourfold decrease in anity (12
and 13 versus 10a). However, replacement of the urea
with monosubstituted guanidine moiety (14a-c) gave a
2.7±8.4 times more potent compound than the corres-
ponding urea compound. The N-methylated com-
pound 14d was 2.8-fold more potent than 14c and was
10.7-fold more potent than 10e, but other N-dis-
ubstituted guanidine and N-trisubstituted ones (14f±g)
lost the binding anity (Table 3).
Other modi®cations were examined, such as introduction
of hetero atoms in the lactam ring (16, 18). Compound
16, having a benzothiazepine nuclei, was as potent as
Scheme 1. Reagents: (a) (BOC)2O; (b) NBS, PPh3, CH2Cl2.

Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
1705
Scheme 2. Reagents: (a) PCl5; (b) H2, 5% Pd-C, MeOH; (c) NaN3, DMF; (d) Method A: 4, KOH, Bu4N+Br , THF; (e) H2, 10% Pd-C, MeOH; (f)
0
Method B: R3 NCO; Method C1: (i) Imd2CO, CH3CN; (ii) amines; Method C2: (i) Imd2CO, amines, THF; (ii) deprotection; Method D:
(CCl3O)2CO, TEA, amines, CH2Cl2; or Method E: 4-phenoxycarbonylaminobenzoic acid, DMF. (g) (i) SO3+pyr, pyridine; (ii) NaOH aq.
Scheme 3. Reagents: (a) H2, 10% Pd-C; (b) MeNCO; (c) R1X (X=Cl, Br, or I) (Method A).
the parent compound 10e, but compound 18, having a
benzoxazepine nuclei, dramatically reduced NPY Y1
binding anity compared to compound 10e (Table 4).
not shown). Thus our compounds may be selective NPY
Y1 receptor antagonists.
Scatchard plot analysis of the saturation binding
experiment is shown in Figure 2. The presence of the
compound 10s or 14c reduced the Kd value without
alteration of maximum binding, suggesting competi-
tive antagonism. The compounds (10s and 14c) were
subjected to other NPY receptor (Y2 and Y5) binding
assays for assessing Y1 selectivity. There was no anity
for either receptor (IC50>10000 nM). Finally, the e€ect
of the compounds on the functional activity of NPY
was examined. Either 10s or 14c (1 mM) almost com-
pletely abolished the NPY (100 nM)-induced increase in
cytosolic free Ca2+ concentration. In contrast, either
compound scarcely a€ected basal Ca2+ levels or the
endothelin-1 (10 nM)-induced Ca2+ mobilization (data
Conclusion
We have discovered novel 1,3-disubstituted-2,3,4,5-tetra-
hydro-1H-1-benzazepin-2-ones which are potent and
selective NPY Y1 receptor antagonists. SAR studies
revealed that introducing the 3-(N-(tert-butoxy-
carbonyl)amino)benzyl group at R1 and 3-((benzothia-
zol-6-yl)urea) and 3-(N-(4-hydroxyphenyl)guanidino)
groups at R3 in structure 1 was advantageous for NPY
Y1 receptor binding. However, the NPY Y1 binding
anity of the compounds, 10s and 14c, is not satis-
factory, and further pharmacological evaluations and
further chemical optimization, particularly at the R1
position, may be needed.

1706
Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
0
0
.
Scheme 4. Reagents: (a) MeCOCl, Et3N or ClCOOMe, Et3N, THF; (b) R3 NHC(SMe)=NH HI, EtOH, ~: or (i) R3 NCS; (ii) 1. MeI, 2. K2CO3;
(iii) amines, ~.
Scheme 5. Reagents: (a) N2H4, EtOH; (b) PlNCO; (c) Method A.
Scheme 6. Reagents: (a) (i) Method A; (ii) H2, 5% Pd-C, MeOH; (b) PhNCO.
Experimental
3-(N-(tert-Butoxycarbonyl)amino)benzyl alcohol (3).
This compound was prepared from 3-amino benzyl-
alcohol (2.5 g, 20.3 mmol) and (BOC)2O (7.2 g, 33.0
mmol) in the usual manner. 96% yield: mp 85±86ꢀC;
1H NMR (CDCl3) d: 1.52 (9H, s), 1.76 (1H, s), 4.66 (2H,
s), 6.52 (1H, brs), 7.04 (1H, m), 7.18±7.32 (2H, m), 7.44
General methods
Melting points are not corrected. 1H NMR spectra
were recorded on a Varian VXR-200 and VXR-300
FT±1H NMR spectrometer with tetramethylsilane (TMS)
as an internal reference. Fast atom bombardment mass
spectra (FABMS) and high-resolution (HR)±FABMS
were determined using m-nitrobenzyl alcohol as a
matrix. Silica gel used for column chromatography was
Kiesegel 60 (Merck). Preparative thin layer chromato-
graphy (PLC) was carried out on E. Merck 60F-
254 precoated plate (0.5 mm thickness). Unless other-
wise stated, all reactions were carried out under nitro-
gen atmosphere with anhydrous solvents that had been
dried over 4 A molecular sieves. After extraction of the
reaction mixture, the solution was washed with water,
dried over Na2SO4 or MgSO4 and concentrated at
reduced pressure. These procedures are simply indicated
by the phrase `the extracts were treated as usual' unless
otherwise noted.
.
(1H, brs). Anal. (C12H17NO3 0.1H2O); calcd: C, 64.04;
H, 7.70; N, 6.22. Found: C, 63.89; H, 7.65; N, 6.23.
3-(N-(tert-Butoxycarbonyl)amino)benzyl bromide (4). To
compound 3 (4.35 g, 19.5 mmol) in CH2Cl2 (20 mL)
were added PPh3 (5.82 g, 22.2 mmol) and NBS (3.95 g,
22.2 mmol). The mixture was stirred for 2 h and n-
hexane (250 mL) was added. The resulting precipitate
was ®ltered o€, and the ®ltrate was concentrated. The
residue was puri®ed by column chromatography
(EtOAc:n-hexane=1:10) and crystallized from CH2Cl2±
n-hexane to a€ord 4 (3.95 g, 71%) as crystals: mp 124±
125ꢀC; H NMR (CDCl3) d:1.52 (9H, s), 4.46 (2H, s),
1
6.51 (1H, brs), 7.06 (1H, m), 7.16±7.31 (2H, m), 7.51
(1H, t, J=1.8 Hz). Anal. (C12H16NO2Br); calcd: C,
50.37; H, 5.64; N, 4.89; Br, 27.92. Found: C, 50.47; H,
5.70; N, 4.94; Br, 27.83.
The following compounds were prepared by literature
methods: 2-(tert-butyldimethylsilyloxy)aniline,22 4-(2H-
tetrazol-5-yl)aniline,23 5-amino-benzotriazol,24 6-ben-
zothiazolamine,25 2-bromo-1-o-tolyl-ethanone.26
Method A. 3-Azide-1-N-(3-(N0-(tert-butoxycarbonyl)-
amino)benzyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one.
A suspension of compound 4 (3.26 g, 11.4 mmol), 7b

Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
1707
Table 1. In vitro NPY Y1 receptor binding anity (11a±n) Table 3. In vitro NPY Y1 receptor binding anity (12, 13, and 14a±g)
Compounds
R1
IC50 (mM)a
Compounds
R3
IC50 (mM)a
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
CH2COOtert-Bu
CH2CONHtert-Bu
(1-Naphthyl)methyl
(4-tert-Bu)benzyl
(2-Quinolinyl)methyl
benzhydlyl
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
12
13
NHCOMe
NHCOOMe
2.9
5.6
14a
14b
14c
14d
14e
14f
14g
NHC(-NH2)=NMe
NHC(-NH2)=NPh
NHC(-NH2)=NC6H4(4-OH)
NHC(-NHMe)=NPh
NHC(-NHOMe)=NPh
NHC(-NHOH)=NPh
NHC(-NMe2)=NPh
0.34
0.32
0.043
0.081
>10
1.4
(4,40-F2)benzhydlyl
(4-Ph)benzyl
(2-Ph)benzyl
>10
(4-Bn)benzyl
(4-OH)benzyl
(2-Tolyl)carbonylmethyl
(3-NO2)benzoylmethyl
N-Phthaloymethyl
a
Binding results are the means of two independent determinations.
Table 4. In vitro NPY Y1 receptor binding anity (16 and 18)
a
Binding results are the means of two independent determinations.
Compound
IC50 (mM)a
16
18
1.5
>10
Table 2. In vitro NPY Y1 receptor binding anity (9, 10a±s)
a
Binding results are the means of two independent determinations.
Compounds
R
R3
OMe NHCONHMe
IC50 (mM)a
9
1.6
1.3
4.9
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NHCONHMe
NHCONHiPr
NHCONHtert-Bu
NHCONH-cyclohexyl
NHCONHPh
NHCONHCH2Ph
>10
>10
0.87
1.3
>10
6.4
2.3
0.36
>10
2.6
4.5
2.4
>10
2.4
1.0
NHCONMe2
NHCONHC6H4(2-OH)
NHCONHC6H4(3-OH)
NHCONHC6H4(4-OH)
NHCONHC6H4(4-COOH)
NHCONHC6H4(4-tetrazolyl)
NHCONHC6H4(4-OSO3Na)
NHCONH(3-pyridinyl)
NHCONH(2-pyrimidinyl)
NHCONH(5-(3-tert-Bu-isoxazolyl))
NHCONH(5-benzimidazolyl)
NHCONH(6-benzotriazolyl)
NHCONH(6-benzothiazolyl)
0.7
0.18
a
Binding results are the means of two independent determinations.
Figure 2. Scatchard plot analysis. [125I]PYY binding to SK-N-MC
cells was performed in the absence (*) or presence of 10s (200 nM, *)
or 14c (50 nM, ~).
(2.0 g, 9.89 mmol) and tetrabutylammonium bromide
(0.33 g, 0.99 mmol) in THF (125 mL) was added to
powdered KOH (742 mg, 11.4 mmol) at 0ꢀC and stirred
at rt for 2 h. The reaction mixture was neutralized by
0.5 N HCl and extracted with EtOAc. The extracts were
treated as usual. The residue was puri®ed by column
chromatography (Et2O:n-hexane=1:2) to a€ord 3.80 g
powder: 1H NMR (CDCl3) d: 1.46 (9H, s), 2.21±2.66
(4H, m), 3.83 (1H, m), 4.72 (1H, d, J=15.3 Hz), 5.00
(1H, d, J=15.6 Hz), 6.76 (1H, d, J=7.2 Hz), 7.00±7.19
1
.
(96%) of the target compound as a powder: H NMR
(5H, m), 7.43±7.55 (2H, m). Anal. (C22H27N3O3
0.9H2O); calcd: C, 66.44; H, 7.30; N, 10.57. Found: C,
66.35; H, 7.02; N, 10.58.
(CDCl3) d: 1.50 (9H, s), 2.24±2.70 (4H, m), 3.76 (1H, t,
J=9.0 Hz), 4.85 (1H, d, J=14.7 Hz), 5.17 (1H, d, J=
14.7 Hz), 6.43 (1H, s), 6.86 (1H, d, J=7.5 Hz), 7.16±7.41
(7H, m).
Compound 8a was prepared from 7a according to a
similar procedure.
3-Amino-1-N-(3-(N0-(tert-butoxycarbonyl)amino)benzyl)-
2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (8b). The above
3-azide compound (3.79 g, 9.49 mmol) was hydro-
genated in MeOH (35 mL) with 10% Pd-C (370 mg)
under hydrogen atmosphere at rt overnight. After
removal of the catalyst by ®ltration, the ®ltrate was
evaporated to dryness to give 8b (4.09 g, quant.) as a
3-Amino-1-N-(3-(N0-(tert-butoxycarbonyl)amino)benzyl)-
7-methoxy-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (8a).
1H NMR (CDCl3) d: 1.50 (9H, s), 2.16±2.69 (4H, m),
3.75 (1H, dd, J=7.6 Hz, 15.3 Hz), 3.79 (3H, s), 4.76
(1H, d, J=14.4 Hz), 5.15 (1H, d, J=14.4 Hz), 6.44 (1H,
brs), 6.67 (1H, d, J=2.8 Hz), 6.78 (1H, dd, J=3.0 Hz,

1708
Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
8.8 Hz), 6.86 (1H, td, J=1.2 Hz, 7.7 Hz), 7.10 (1H, d,
J=8.8 Hz), 7.13 (1H, m), 7.17 (1H, t, J=7.8 Hz), 7.40
(1H, m). Anal. (C23H27N5O4); calcd: C, 63.14; H, 6.22;
N, 16.01. Found: C, 63.05; H, 6.33; N, 15.78.
1.24 (9H, s), 1.49 (9H, s), 1.93 (1H, m), 2.50 (2H, m),
2.70 (1H, m), 4.52 (1H, m), 4.65 (1H, s), 4.74 (1H, d,
J=15.9 Hz), 5.29 (1H, d, J=15.9 Hz), 5.35 (1H, d,
J=8.1 Hz), 6.85 (1H, d, J=7.5 Hz), 6.96 (1H, s), 7.06
(1H, d, J=7.5 Hz), 7.20 (9H, m), 7.59 (1H, d, J=7.5 Hz).
Anal. (C27H36N4O4); calcd: C, 67.48; H, 7.55; N, 11.66.
Found: C, 67.35; H, 7.73; N, 11.49.
Method B. Preparation of 1-N-(3-(N0-(tert-butoxycar-
bonyl)amino)benzyl)-3-(3-phenylureido)-2,3,4,5-tetrahydro-
1H-1-benzazepin-2-one (10e). A solution of phenyliso-
cyanate (45 mL, 0.412 mmol) in DMF (0.5 mL) was
added to a solution of compound 8b (150 mg, 0.393
mmol) in DMF (3 mL) at 0ꢀC. The mixture was stirred
at rt overnight and diluted with water and extracted
with EtOAc. The extracts were treated as usual. The
residue was puri®ed by column chromatography (Et2O:
n-hexane=3:2) to a€ord 10e (137 g, 70%) as a powder:
mp 139ꢀC (dec.); 1H NMR (CDCl3) d: 1.50 (9H, s), 1.99
(1H, m), 2.59 (2H, m), 2.80 (1H, m), 4.65 (1H, m), 4.79
(1H, d, J=15.6 Hz), 5.35 (1H, d, J=15.6 Hz), 6.54 (1H,
brs), 6.80±7.03 (8H, m), 7.35 (1H, brs), 7.62 (1H, brd,
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-cyclo-
hexylureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one
(10d). 78% yield: mp 198±201ꢀC; H NMR (CDCl3) d:
1
0.85±1.35 (4H, m), 1.49 (9H, s), 1.50±2.00 (7H, m),
2.40±2.80 (3H, m), 3.40 (1H, m), 4.55 (1H, dd, J=7.5,
11.1 Hz), 4.79 (1H, d, J=15.3 Hz), 5.25 (1H, d, J=
15.3 Hz), 5.60 (1H, brs), 6.86 (1H, d, J=8.1 Hz), 7.00
(1H, s), 7.09±7.40 (7H, m), 7.57 (1H, d, J=7.5 Hz).
Anal. (C29H38N4O4); calcd: C, 68.75; H, 7.56; N, 11.06.
Found: C, 68.54; H, 7.65; N, 11.01.
J=7.8 Hz). Anal. (C29H32N4O4 0.1H2O); calcd: C, 69.69;
H, 6.71; N, 10.87. Found: C, 69.77; H, 6.89; N, 10.83.
3-(3-Benzylureido)-1-N-(3-(N0 -(tert-butoxycarbonyl)-
amino)benzyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one
(10f). 99% yield: 1H NMR (CDCl3) d: 1.41 (9H, s),
1.71 (1H, m), 1.92 (1H, m), 2.48 (2H, m), 2.68 (1H, m),
4.08 (1H, m), 4.38 (1H, dd, J=6.4, 15.0 Hz), 4.67 (1H,
d, J=16.2 Hz), 5.20 (1H, d, J=15.6 Hz), 5.42 (1H, brs),
6.10 (1H, brs), 6.80 (1H, d, J=7.8 Hz), 7.05 (1H, d,
J=7.5 Hz), 7.12±7.21 (10H, m), 7.58 (1H, brd, J=
7.8 Hz). Anal. (C30H34N4O4); calcd: C, 70.02; H, 6.66;
N, 10.89. Found: C, 69.93; H, 6.88; N, 10.77.
.
Compounds 9 (from 8a) and 10a±d,f (from 8b) were
prepared according to a similar procedure.
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-7-meth-
oxy-3-(3-methylureido)-2,3,4,5-tetrahydro-1H-1-benzaze-
pin-2-one (9). 79% yield: mp 209±210ꢀC; 1H NMR
(CDCl3) d: 1.48 (9H, s), 1.94 (1H, dt, J=7.4 Hz,
11.1 Hz), 2.36±2.82 (3H, m), 2.61 (3H, s), 3.79 (3H, s),
4.55 (1H, m), 4.72 (1H, brs), 4.76 (1H, d, J=15.2 Hz),
5.18 (1H, d, J=15.2 Hz), 5.95 (1H, brs), 6.71 (1H, s),
6.73 (1H, dd, J=2.8 Hz, 8.6 Hz), 6.86 (1H, brd, J=
7.8 Hz), 7.00 (1H, dd, J=1.8 Hz, 7.2 Hz), 7.13 (1H, m),
7.17 (1H, brs), 7.23 (1H, t, J=7.8 Hz), 7.59 (1H, brd,
Method C1. Preparation of 1-N-(3-(N0-(tert-butoxy-
carbonyl)amino)benzyl)-3-(3-phenylureido)-2,3,4,5-tetra-
hydro-1H-1-benzazepin-2-one (10g). To a solution of
compound 8b (200 mg, 0.524 mmol) in CH3CN (6 mL)
was added carbonyldiimidazole (170 mg, 1.05 mmol) at
0ꢀC and the mixture was stirred at rt for 1 h. The reac-
tion mixture was poured into water and extracted with
EtOAc. The extracts were treated as usual. Dimethyl-
amine (1 M) in THF solution (0.25 mL) was added to the
residue. The reaction mixture was stirred at rt for 18 h,
acidi®ed with 10% HCl and extracted with EtOAc. The
extracts were treated as usual to give 10g (46%):
1H NMR (CDCl3) d: 1.49 (9H, s), 2.00 (1H, m), 2.48±
2.58 (2H, m), 2.73 (1H, m), 4.53 (1H, m), 4.83 (1H, d,
J=15.3 Hz), 5.19 (1H, d, J=15.6 Hz), 6.85 (1H, d,
J=7.8 Hz), 6.99 (1H, s), 7.07±7.27 (7H, m), 7.37 (1H,
.
J=7.8Hz). Anal. (C25H32N4O5 0.2H2O); calcd: C, 63.60;
H, 6.92; N, 11.87. Found: C, 63.62; H, 6.97; N, 12.07.
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-meth-
ylureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (10a).
1
69% yield: H NMR (CDCl3) d: 1.47 (9H, s), 2.00 (1H,
m), 2.30±2.90 (3H, m), 2.53 (3H, s), 4.62 (1H, m), 4.68
(1H, d, J=16.2 Hz), 5.38 (1H, d, J=16.0 Hz), 6.19 (1H,
m), 6.86 (1H, d, J=7.8 Hz), 7.06 (1H, d, J=8.0 Hz),
7.10±7.30 (5H, m), 7.47 (1H, brs), 7.68 (1H, d, J=
.
7.8 Hz). Anal. (C24H30N4O4 0.2C6H6); calcd: C, 66.65;
H, 6.92; N, 12.34. Found: C, 66.31; H, 6.97; N, 12.15.
.
brd, J=7.5 Hz). Anal. (C25H32N4O4 0.3H2O); calcd: C,
65.57; H, 7.18; N, 12.23. Found C, 65.57; H, 7.17; N,
12.25.
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-iso-
propylureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one
(10b). 89% yield: mp 209±211ꢀC; H NMR (CDCl3) d:
1-N-(3-(N0 -(tert-Butoxycarbonyl)amino)benzyl)-3-(pyr-
imidin-2-yl)phenylureido)-2,3,4,5-tetrahydro-1H-1-benza-
zepin-2-one (10o). This compound was prepared
according to Method C1 from 8b and 2-aminopyr-
imidine instead of dimethylamine. 35% yield: mp 168±
1
0.95 (3H, d, J=6.3 Hz), 1.01 (3H, d, J=6.3 Hz), 1.48
(9H, s), 1.95 (1H, m), 2.50 (1H, m), 2.76 (2H, m), 3.79
(1H, dq, J=6.3, 14.4 Hz), 4.57 (1H, m), 4.70 (1H, brs),
4.73 (1H, d, J=15.9 Hz), 5.31 (1H, d, J=15.9 Hz), 5.64
(1H, d, J=8.1 Hz), 6.86 (1H, d, J=7.5 Hz), 7.06 (1H, d,
J=8.1 Hz), 7.20 (7H, m), 7.62 (1H, d, J=8.1 Hz). Anal.
(C26H34N4O4); calcd: C, 66.93; H, 7.34; N, 12.01.
Found: C, 66.91; H, 7.40; N, 12.01.
170ꢀC; H NMR (CDCl3) d: 1.49 (9H, s), 2.11 (1H, m),
1
2.56 (2H, m), 2.70 (1H, m), 4.50 (1H, dd, J=7.2Hz,
9.0 Hz), 4.82 (1H, d, J=15.0 Hz), 5.31 (1H, d, J=
15.0Hz), 6.86 (1H, d, J=9.0 Hz), 7.04 (1H, t, J=5.1 Hz),
7.13 (1H, t, J=9.0Hz), 7.10±7.40 (6H, m), 8.56 (2H,
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-tert-
butylureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one
d, J=5.1 Hz). Anal. (C27H30N6O4 0.2H2O); calcd: C,
64.07; H, 6.05; N, 16.60. Found: C, 63.99; H, 6.10; N,
16.72.
.
(10c). 84% yield: mp 202±205ꢀC; H NMR (CDCl3) d:
1

Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
1709
Method C2. Preparation of 1-N-(3-(N0-(tert-butoxy-
carbonyl)amino)benzyl)-3-(3-(4-hydroyxy)phenylureido)-
2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (10j)
pension of triphosgene (28 mg, 0.094 mmol) and 6-ben-
zothiazolamine (100 mg, 0.262 mmol) in CH2Cl2 (3 mL)
was cooled to 20ꢀC, and Et3N (40 mL, 0.287 mmol)
was added dropwise. The mixture was stirred for 30
min. Compound 8b (45 mg, 0.118 mmol) was added.
Et3N (77 mL, 0.552 mmol) was added dropwise, fol-
lowed by stirring at 20ꢀC for 2.5 h. Ice-water was
added. Extractive work up with EtOAc and PLC
(CHCl3:acetone=10:1) a€orded 10s (73 mg, 50%) as a
powder: 1H NMR (CDCl3) d: 1.51 (9H, s), 2.03 (1H, m),
2.60 (1H, m), 2.70 (1H, m), 2.86 (1H, m), 4.76 (1H, d,
J=15.9 Hz), 5.48 (1H, d, J=15.9 Hz), 6.81 (1H, d,
J=11.1 Hz), 6.89 (1H, d, J=8.4 Hz), 6.99 (1H, d,
J=8.4 Hz), 7.02 (1H, s), 7.19±7.35 (6H, m), 7.53 (1H, d,
J=9.0 Hz), 7.58 (1H, d, J=7.8 Hz), 7.71 (1H, s), 8.61
3-(3-(4-Benzyloxy)phenylureido)-1-N-(3-(N0-(tert-butoxy-
carbonyl)amino)benzyl)-2,3,4,5-tetrahydro-1H-1-benz-
azepin-2-one. A mixture of 4-benzyloxyaniline hydro-
chlroride (300 mg, 1.27 mmol) and carbonyldiimidazole
(207 mg, 1.27 mmol) in THF (10 mL) was stirred at rt
and Et3N (0.18 mL, 1.3 mmol) was added dropwise. The
mixture was stirred for 1.5 h and ®ltered. The ®ltrate
was washed with CHCl3 and evaporated. Compound 8b
(100 mg, 0.262 mmol) in DMF (3 mL) and Et3N (0.4
mL, 2.87 mmol) was added dropwise to the residue. The
mixture was stirrred for 16 h. Water was added and the
mixture was ®ltered. The residue was dried and puri®ed
by column chromatography (EtOAc:n-hexane=1:2) to
.
(1H, s). Anal. (C30H31N5O4S 0.4H2O); calcd: C, 63.79;
H, 5.67; N, 12.40; S, 5.68. Found: C, 63.82; H, 5.85; N,
12.36; S, 5.74.
1
a€ord 128 mg (80%) as a powder: H NMR (CDCl3) d:
1.49 (9H, s), 1.96 (1H, m), 2.49±2.63 (2H, m), 2.75 (1H,
m), 4.62 (1H, m), 4.80 (1H, d, J=15.9 Hz), 4.94 (2H, s),
5.29 (1H, d, J=14.7 Hz), 6.38 (1H, brs), 6,68 (2H, dd,
J=1.8 Hz, 8.7 Hz), 6.88 (3H, m), 7.12±7.39 (13H, m),
7.57 (1H, brd, J=8.7 Hz). Anal. (C36H38N4O5); calcd:
C, 71.27; H, 6.31; N, 9.23. Found: C, 71.10; H, 6.58; N,
9.03.
Compounds 10l, 10n, 10p, and 10r were prepared from
8b according to a similar procedure.
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-(4-
1H-tetrazol-5-yl)phenylureido)-2,3,4,5-tetrahydro-1H-1-
1
benzazepin-2-one (10l). 43% yield: H NMR (CDCl3+
CD3OD) d: 1.37 (9H, s), 2.05 (1H, m), 2.68±2.78 (2H,
m), 2.90 (1H, m), 4.59 (1H, m), 4.69 (1H, d, J=
16.2 Hz), 5.57 (2H, m), 6.76 (2H, brs), 7.01 (1H, brd,
J=7.2 Hz), 7.18±7.33 (9H, m), 7.59 (1H, s), 8.06 (1H,
brs). Anal. (C30H32N8O4 0.2C6H14 0.6H2O); calcd: C,
62.80; H, 6.08; N, 18.78. Found: C, 62.57; H, 6.04; N,
18.69.
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-(4-
hydroxy)phenylureido)-2,3,4,5-tetrahydro-1H-1-benz-
azepin-2-one (10j). The above O-benzyl compound
(128 mg, 0.211 mmol) was hydrogenated in MeOH (4.5
mL) with 10% Pd-C (18 mg) under hydrogen atmo-
sphere at rt for 4 h. After removal of the catalyst by ®l-
tration, the ®ltrate was evaporated to dryness. The
residue was puri®ed by column chromatography
(CHCl3:MeOH=48:1) to a€ord 10j (103 mg, 95%) as a
powder: 1H NMR (CDCl3) d: 1.45 (9H, s), 1.98 (1H, m),
2.54 (3H, m), 4.54 (1H, m), 4.87 (1H, d, J=15.6 Hz),
5.02 (1H, d, J=15.6 Hz), 6.38 (1H, d, J=7.8 Hz), 6.46
(1H, d, J=15.6 Hz), 6.80 (3H, m), 6.91 (1H, m), 7.34
(1H, d, J=8.6 Hz), 7.47±7.54 (1H, m), 7.04±7.54 (7H, m).
.
.
1-N-(3-(N0 -(tert-Butoxycarbonyl)amino)benzyl)-3-(3-
(pyridin-3-yl)phenylureido)-2,3,4,5-tetrahydro-1H-1-
benzazepin-2-one (10n). 26% yield: 1H NMR (CDCl3) d:
1.50 (9H, s), 1.97 (1H, m), 2.53±2.62 (2H, m), 2.74 (1H,
m), 4.60 (1H, m), 4.90 (1H, d, J=15.6 Hz), 5.23 (1H, d,
J=15.3 Hz), 6.54 (1H, d, J=7.5 Hz), 6.87 (1H, d,
J=7.5 Hz), 6.97 (1H, m), 7.11 (1H, brs), 7.23 (5H, m),
7.50 (2H, d, J=8.4 Hz), 7.64 (1H, brs), 8.10 (1H, d,
.
.
Anal. (C29H32N4O5 0.15C6H14 0.1H2O); calcd: C, 67.59;
H, 6.51; N, 10.54. Found: C, 67.66; H, 6.75; N, 10.51.
.
J=3.9 Hz), 8.29 (1H, brs). Anal. (C28H31N5O4 0.5H2O);
calcd: C, 65.87; H, 6.32; N, 13.72. Found: C, 66.16; H,
6.49; N, 13.53.
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-(3-
hydroxy)phenylureido)-2,3,4,5-tetrahydro-1H-1-benz-
azepin-2-one (10i). 1-N-(3-(N0-(tert-Butoxycarbonyl)-
amino)benzyl)-3-(3-(3-tetrahydopyranoxy)phenylureido)-
2,3,4,5-tetrahydro-1H-1-benzazepin-2-one was prepared
according to method C2 from compound 8b (100 mg,
0.26 mmol) and (3-tetrahydopyranoxy)phenol (65.6 mg,
0.34 mmol), obtained from 3-amino phenol, and then
removal of the THP group in the usual manner with p-
3-(3-((5-tert-Butyl)isoxazol-3-yl)phenylureido)-1-N-(3-
(N0 -(tert-butoxycarbonyl)amino)benzyl)-2,3,4,5-tetra-
hydro-1H-1-benzazepin-2-one (10p). 60% yield: 1H
NMR (CDCl3) d: 1.29 (9H, s), 1.48 (9H, s), 2.02 (1H,
m), 2.46±2.64 (3H, m), 4.54 (1H, m), 4.92 (1H, d,
J=14.7 Hz), 5.19 (1H, d, J=14.7 Hz), 6.10 (1H, s), 6.72
(1H, s), 6.83 (1H, d, J=7.8 Hz), 7.14±7.26 (6H, m), 7.48
(1H, d, J=7.2 Hz), 7.54 (1H, brs), 8.17 (1H, brs). Anal.
TsOH H2O to give 10i (35%): 1H NMR (CDCl3) d:
.
.
1.59 (9H, s), 2.02 (1H, m), 2.50±2.91 (3H, m), 4.59 (3H,
m), 6.30 (1H, d, J=9.4 Hz), 6.71 (3H, m), 6.93 (1H, brd,
J=7.8 Hz), 7.05 (1H, brs), 7.17±7.39 (6H, m), 7.51 (1H,
(C30H37N5O5 0.1H2O); calcd: C, 65.58; H, 6.82; N,
12.75. Found: C, 65.58; H, 6.85; N, 12.86.
.
d, J=8.2 Hz). Anal. (C29H32N4O5 0.3H2O); calcd: C,
66.73; H, 6.29; N, 10.73. Found: C, 66.69; H, 6.44; N,
10.81.
3-(3-(Bezotriazol-5-yl)phenylureido)-1-N-(3-(N0(tert-
butoxycarbonyl)amino)benzyl)-2,3,4,5-tetrahydro-1H-1-
1
benzazepin-2-one (10r). 41% yield: H NMR (CDCl3) d:
1.20 (9H, d, J=7.5 Hz), 2.11 (1H, m), 2.52±2.66 (2H,
m), 2.81 (1H, m), 4.66 (1H, m), 4.85 (1H, brs), 5.35 (1H,
brs), 6.63 (1H, brs), 6.94 (2H, d, J=15.9 Hz), 7.01 (1H,
brs), 7.22 (2H, m), 7.42 (2H, s), 7.51 (1H, br, s), 8.65
Method D. Preparation of 3-(3-(benzothiazol-6-yl)-
ureido)-1-N-(3-(N0 -(tert-butoxycarbonyl)amino)benzyl)-
2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (10s). A sus-

1710
Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
.
.
(2H, s). Anal. (C29H31N7O4 0.1C6H14 0.4H2O); calcd:
C, 63.79; H, 6.00; N, 17.58. Found: C, 63.87; H, 6.21; N,
17.67.
washed with CHCl3. The aqueous phase was extracted
with 1-butanol twice, and the extracts were treated as
usual to a€ord 290 mg (50%) of the target compound:
1H NMR (CDCl3) d: 1.49 (9H, s), 2.00 (1H, m), 2.36±
2.69 (3H, m), 4.35 (1H, m), 4.82 (1H, d, J=15.4 Hz),
5.27 (1H, d, J=14.8 Hz), 6.84 (1H, brd, J=7.4 Hz),
7.08±7.35 (11H, m). Anal. (C29H31N4O8Na 2.2H2O);
calcd: C, 52.91; H, 5.42; N, 8.51; S, 4.87. Found: C,
52.79; H, 5.35; N, 8.49; S, 5.26.
1-N-(3-(N0(tert-Butoxycarbonyl)amino)benzyl)-3-(3-(2-
hydroxy)phenylureido)-2,3,4,5-tetrahydro-1H-1-benz-
azepin-2-one (10h). 1-N-(3-(N0-(tert-Butoxycarbonyl)-
amino)benzyl)-3-(3-(2-tert-butyldimethylsilyloxy)phenyl-
ureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one was
prepared according to Method D from compound 8b
(100 mg, 0.262 mmol) and (2-tert-butyldimethylsilyl-
oxy)-phenol (39 mg, 0.174 mmol) and then removal of
the TBDMS group in the usual manner with n-
BuN+F gave 10h (58%): 1H NMR (CDCl3) d: 1.51
(9H, s), 2.00 (1H, m), 2.51±2.77 (3H, m), 4.56 (1H, m),
5.31 (2H, d, J=15.4 Hz), 6.62 (1H, m), 6.85 (4H, m),
7.08±7.23 (6H, m), 7.54±7.63 (2H, m). Anal. (C29H32
.
Compounds 11a±i, 11l±n were prepared according to
Method A from 7b.
1-N-(3-(N0 -(tert-Butoxycarbonyl)amino)benzyl)-3-(3-
methylureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one
(11a). 98% yield: mp 229±230ꢀC; 1H NMR (CDCl3)
d: 1.43 (9H, s), 2.00 (1H, m), 2.60 (2H, m), 2.74 (3H,
d, J=4.8 Hz), 3.35 (1H, m), 4.21 (1H, d, J=16.8 Hz),
4.45 (1H, m), 4.48 (1H, m), 4.69 (1H, d, J=17.1 Hz),
5.59 (1H, d, J=6.6 Hz), 7.12 (1H, d, J=8.4 Hz), 7.19±
.
.
N4O5 0.1C6H14 0.3H2O); calcd: C, 67.00; H, 6.46; N,
10.5. Found: C, 67.11; H, 6.75; N, 10.75.
3-(3-(Benzimidazol-5-yl)phenylureido)-1-N-(3-(N0-(tert-
butoxycarbonyl)amino)benzyl)-2,3,4,5-tetrahydro-1H-1-
benzazepin-2-one (10q). 1-N-(3-(N0 -(tert-Butoxy-
carbonyl)amino)benzyl)-3-(3-(p-tosyl[1H]-benzimidazol-
5-yl)phenylureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-
one was prepared according to Method D from com-
pound 8b (250 mg, 0.655 mmol) and 5-amino-1-(p-
tosyl)bezimidazole (180 mg, 0.72 mmol) and then the
tosyl group was removed in the usual manner with 10%
7.29 (3H, m). Anal. (C18H25N3O4 1.0H2O); calcd: C,
59.16; H, 7.44; N, 11.51. Found: C, 58.86; H, 7.18; N,
11.52.
.
1-N-(3-(N0-(tert-Butoxycarbamoylmethyl)amino)benzyl)-
3-(3-methylureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-
1
2-one (11b). 94% yield: H NMR (CDCl3) d: 1.19 (9H,
s), 2.14±2.31 (1H, m), 1.78±1.89 (1H, m), 2.50±2.58 (4H,
m), 3.17±3.28 (1H, m), 4.05±4.15 (1H, m), 4.19 (1H, d,
J=15.9 Hz), 4.43 (1H, d, J=16.2 Hz), 5.95±6.00 (1H,
m), 6.14 (1H, d, J=8.1 Hz), 7.17±7.35 (3H, m). Anal.
1
KOH/MeOH to give 10q (26%): H NMR (CDCl3) d:
1.48 (9H, s), 2.01 (1H, m), 2.49±2.61 (2H, m), 2.77 (1H,
m), 4.60 (1H, m), 4.77 (1H, m), 5.28 (1H, brs), 6.69 (1H,
brs), 6.88 (2H, brd, J=8.4 Hz), 7.10±7.26 (8H, m), 7.34
(1H, s), 7.47 (1H, m), 7.67±7.81 (1H, m). Anal. (C30H32
N6O4 1.2H2O); calcd: C, 64.09; H, 6.17; N, 14.95.
Found: C, 64.03; H, 6.00; N, 15.02.
.
(C18H26N4O3 0.15CHCl3); calcd: C, 59.83; H, 7.23; N,
15.38. Found: C, 59.77; H, 7.25; N, 15.52.
.
3-(3-Methylureido)-1-N-((1-naphthyl)methyl)-2,3,4,5-
1
tetrahydro-1H-1-benzazepin-2-one (11c). 83% yield: H
NMR (CDCl3) d: 1.87 (1H, m), 2.34 (1H, m), 2.51 (2H,
m), 2.71 (3H, d, J=5.1 Hz), 4.39 (1H, m), 4.54 (1H, m),
5.35 (1H, d, J=15.3 Hz), 5.49 (1H, d, J=8.1 Hz), 5.73
(1H, d, J=15.3 Hz), 7.06±8.09 (11H, m). Anal. (C23H23
N3O2 0.15H2O); calcd: C, 73.44; H, 6.24; N, 11.17.
Found: C, 73.41; H, 6.34; N, 11.17.
Method E. 1-N-(3-(N0-(tert-Butoxycarbonyl)amino)-
benzyl)-3-(3-(4-carboxyphenylureido)-2,3,4,5-tetrahydro-
1H-1-benzazepin-2-one (10k). To a solution of com-
pound 8b (70 mg, 0.183 mmol) and 4-phenoxy-
carbonylaminobenzoic acid (50 mg, 0.195 mmol) in
DMF (3 mL) was added Et3N and the mixture was
stirred at rt for 18 h. 10% HCl was added to the mix-
ture and the resulting precipitation was ®ltered. The
residue was puri®ed by column chromatography
.
1-N-(4-(tert-Butyl)benzyl)-3-(3-methylureido)-2,3,4,5-
tetrahydro-1H-1-benzazepin-2-one (11d). 93% yield: mp
247±249ꢀC; 1H NMR (CDCl3) d: 1.28 (9H, s), 1.98 (1H,
m), 2.44±2.60 (3H, m), 2.68 (3H, d, J=4.5 Hz), 4.48
(1H, brs), 4.91 (1H, d, J=14.7 Hz), 5.09 (1H, d, J=
15.0Hz), 5.55 (1H, brs), 7.12±7.30 (8H, m). Anal.
(C23H29N3O2); calcd: C, 72.79; H, 7.70; N, 11.07.
Found: C, 72.69; H, 7.76; N, 11.04.
1
(CHCl3:MeOH=20:1) to a€ord 10k (46%): H NMR
(CDCl3) d: 1.45 (18H, s), 1.91 (2H, m), 2.31±2.61 (6H,
m), 4.37 (2H, m), 4.84 (2H, d, J=14.8 Hz), 5.16 (2H, d,
J=15.2 Hz), 6.15 (2H, brs), 6.75 (2H, d, J=7.6 Hz),
6.97±7.16 (14H, m), 7.52 (2H, brd, J=7.8 Hz). Anal.
.
.
(C45H52N6O7 0.1C6H14 0.8H2O); calcd: C, 67.45; H,
6.83; N, 10.35. Found: C, 67.34; H, 6.85; N, 10.12.
3-(3-Methylureido)-1-N-((2-quinolinyl)methyl)-2,3,4,5-
tetrahydro-1H-1-benzazepin-2-one (11e). 83% yield:
1H NMR (CDCl3) d: 1.93±2.03 (1H, m), 2.57±2.68 (5H,
m), 3.14±3.27 (1H, m), 4.47 (1H, brs), 4.53±4.62 (1H,
m), 5.27 (1H, d, J=15.9 Hz), 5.40 (1H, d, J=15.9 Hz),
5.51 (1H, brs), 7.12±7.23 (4H, m), 7.46 (1H, d,
J=8.4 Hz), 7.51 (1H, t, J=8.1 Hz), 7.68 (1H, td, J=1.5,
8.1 Hz), 7.79 (1H, d, J=7.5 Hz), 7.99 (1H, d, J=8.4 Hz),
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-(4-
sulfonyloxy)phenylureido)-2,3,4,5-tetrahydro-1H-1-benz-
azepin-2-one sodium salt (10m). To a solution of com-
pound 10j (100 mg, 0.193 mmol) in pyridine (4 mL) was
added sulfur trioxide pyridine complex (154 mg, 0.97
mmol). The mixture was stirred for 110 h and then
concentrated and water was added. The mixture was
®ltered and then concentrated. The residue was dis-
solved in 1 N NaOH (3.5 mL) and the solution was
.
8.13 (1H, d, J=8.4 Hz). Anal. (C22H23N4O3 0.15C6
.
H14 0.3H2O); calcd: C, 69.85; H, 6.58; N, 14.23. Found:
C, 69.64; H, 6.30; N, 13.94.

Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
1711
1-N-(Benzhydryl)-3-(3-methylureido)-2,3,4,5-tetrahydro-
1H-1-benzazepin-2-one (11f). 87% yield: 1H NMR
(CDCl3) d: 1.85±1.93 (1H, m), 2.36±2.55 (3H, m), 2.60
(3H, m), 4.52±4.60 (1H, m), 4.56 (1H, brs), 5.70 (1H,
brs), 7.00±7.17 (10H, m), 7.31±7.38 (5H, m). Anal.
7.67±7.81 (4H, m); HR-FABMS m/z 391.1565 (calcd for
C21H21N4O4 m/z 393.1562).
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-methyl-
carbonylamino-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one
(12). To a solution of compound 8b (84 mg, 0.22 mmol)
in THF (2 mL) were added Et3N (0.1 mL, 0.72 mmol)
and acetylchloride (50 mL, 0.703 mmol) at 0ꢀC. The
mixture was stirred at rt for 15 h and partitioned
between EtOAc and water. The aqueous phase was
extracted with EtOAc and combined organic extracts
were washed with brine, dried over Na2SO4, and con-
centrated in vacuo. The residue was puri®ed by column
chromatography (EtOAc:n-hexane=60:1) to a€ord 12
(94%): 1H NMR (CDCl3) d: 1.50 (9H, s), 1.86±1.95
(1H, m), 1.98 (3H, s), 2.46±2.69 (3H, m), 4.48±4.57 (1H,
m), 4.95 (1H, d, J=14.7 Hz), 5.05 (1H, d, J=15.3 Hz),
6.49 (2H, m), 6.86 (1H, d , J=7.8 Hz), 7.16±7.25 (5H,
m), 7.38 (1H, d, J=6.9 Hz). Anal. (C29H38N4O4
.
.
(C25H25N3O2 0.15C6H14 0.3H2O); calcd: C, 74.45; H,
6.68; N, 10.06. Found: C, 74.60; H, 6.87; N, 9.91.
1-N-((4,40 -Di¯uoro)benzhydryl)-3-(3-methylureido)-2,3,
4,5-tetrahydro-1H-1-benzazepin-2-one (11g). 72% yield:
mp 235±238ꢀC; 1H NMR (CDCl3) d: 1.83±1.91 (1H, m),
2.39±2.48 (3H, m), 2.70 (3H, d, J=5.1 Hz), 4.37 (1H,
m), 4.48±4.56 (1H, m), 5.36 (1H, d, J=8.1 Hz), 6.86
(2H, t, J=8.7 Hz), 6.95 (1H, d, J=7.5 Hz), 7.03±7.11
(7H, m), 7.28±7.32 (2H, m). Anal. (C25H23N3O2F2);
calcd: C, 68.95; H, 5.32; N, 9.65; F, 8.45. Found: C,
69.00; H, 5.58; N, 9.80; F, 8.73.
1-N-(p-Biphenylmethyl)-3-(3-methylureido)-2,3,4,5-tetra-
hydro-1H-1-benzazepin-2-one (11h). 97% yield: 1H
NMR (CDCl3) d: 1.76±1.99 (1H, m), 2.43±2.71 (3H, m),
2.60 (3H, d, J=5.0 Hz), 4.45±4.58 (1H, m), 4.85 (1H,
brs), 4.89 (1H, d, J=15.6 Hz), 5.16 (1H, d, J=15.8 Hz),
5.90 (1H, m), 6.81 (1H, d, J=5.8 Hz), 7.00±7.20 (6H,
m), 7.26±7.40 (5H, m), 7.49 (1H, d, J=7.4 Hz). Anal.
.
.
0.2C6H14 0.4H2O); calcd: C,67.57; H, 7.34; N, 9.38.
Found: C, 67.53; H, 8.10; N, 10.84.
Compound 13 was prepared from 8b according to the
same procedure.
(C25H25N3O2 0.1C6H6 0.4H2O); calcd: C, 74.04; H,
6.60; N, 10.01. Found: C, 74.06; H, 6.58; N, 10.07.
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-meth-
oxycarbonylamino-2,3,4,5-tetrahydro-1H-1-benzazepin-
2-one (13). 94% yield: mp 183±184ꢀC; 1H NMR
(CDCl3) d: 1.49 (9H, s), 1.96 (1H, m), 2.46±2.69 (1H,
m), 3.36 (1H, s), 4.32 (1H, m), 5.00 (2H, s), 5.66 (1H,
brd, J=7.8 Hz), 6.66 (1H, s), 6.85 (1H, brd, J=7.5 Hz),
7.12±7.22 (2H, m), 7.45 (1H, d, J=8.4 Hz). Anal.
.
.
1-N-(o-Biphenylmethyl)-3-(3-methylureido)-2,3,4,5-tetra-
hydro-1H-1-benzazepin-2-one (11i). 94% yield: 1H
NMR (CDCl3) d: 1.91 (1H, m), 2.41±2.62 (3H, m), 2.72
(3H, d, J=4.6Hz), 4.38(1H, brs), 4.47±4.54 (1H, m),
4.93 (1H, d, J=15.2 Hz), 5.22 (1H, d, J=15.2Hz), 5.44
(1H, d, J=7.8 Hz), 7.16±7.57 (11H, m). Anal. (C25H25
.
(C24H29N3O5 0.15H2O); calcd: C, 65.19; H, 6.68; N,
9.50. Found: C, 65.19; H, 6.74; N, 9.54.
.
.
N3O2 0.1CHCl3 1.1H2O); calcd: C, 69.91; H, 6.38; N,
9.74. Found: C, 69.95; H, 6.10; N, 9.84.
Preparation of 1-N-(3-(N0-(tert-Butoxycarbonylamino)-
benzyl)-3-(N-phenylguanidino)-2,3,4,5-tetrahydro-1H-1-
benzazepin-2-one (14b)
3-(3-Methylureido)-1-N-((2-(o-methylphenyl-oxo)ethyl)-
2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (11l). 79%
yield: 1H NMR (CDCl3+CD3OD) d: 2.07 (1H, m), 2.41
(3H, m), 2.41±2.70 (3H, m), 2.70 (3H, s), 3.45 (2H, m),
4.50 (1H, brs), 4.86 (1H, d, J=19.2 Hz), 5.42 (1H, d,
J=19.4 Hz), 6.00 (1H, brs), 7.13±7.45 (6H, m), 7.70
(1H, d, 7.2 Hz). Anal. (C21H23N3O3); calcd: C, 69.02; H,
6.34; N, 11.50. Found: C, 68.82; H, 6.41; N, 11.60.
S-Methyl-phenylisothiourea hydroiodide salt. A suspen-
sion of the 1-phenyl-2-thiourea (1.5 g, 9.85 mmol)
and MeI (1.25 mL, 20.0 mmol) in CH3CN (10 mL)
was stirred under re¯ux for 16 h. After cooling, the
deposited crystals were collected by ®ltration and crys-
tallized from Et2O to give 2.31g (77%) of the target
compound: 1H NMR (DMSO) d: 2.69 (3H, s), 7.30±7.60
(5H, m).
3-(3-Methylureido)-1-N-((2-(m-nitrophenyl-oxo)ethyl)-
2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (11m). 12%
yield: 1H NMR (CDCl3+CD3OD) d: 1.97±2.18 (2H,
m), 2.53±2.73 (1H, m), 2.71 (3H, m), 3.42±3.60 (1H, m),
4.48±4.57 (1H, m), 4.54 (1H, d, J=17.4 Hz), 5.08 (1H,
d, J=17.4 Hz), 7.14±7.28 (4H, m), 7.73 (1H, t, J=
8.0 Hz), 8.31 (1H, d, J=7.8 Hz), 8.47 (1H, d, J=7.8 Hz),
1-N-(3-(N0 -(tert-Butoxycarbonyl)amino)benzyl)-3-(N-
phenylguanidino)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-
one (14b). A mixture of 8b (382 mg, 1.0 mmol) and S-
methyl-phenylisothiourea hydroiodide salt (294 mg, 1.0
mmol) in EtOH (1 mL) was heated to 100ꢀC with stir-
ring in a sealed tube for 5 h. After removal of the sol-
vent, EtOAc and 10% aqueous Na2CO3 were added to
the residue. The EtOAc layer was washed with water,
dried over Na2SO4, and concentrated in vacuo. The
residue was washed with Et2O, puri®ed by PLC
(CH3CN:MeOH=3:1), and recrystallized from Et2O to
a€orded 47c (72 mg, 14%) as a crystal: mp 132±135ꢀC;
1H NMR (CD3OD) d: 1.49 (9H, s), 2.00 (1H, m), 2.56
(2H, m), 2.69 (1H, m), 4.45 (1H, dd, J=7.8 Hz,
12.3 Hz), 4.95 (1H, d, J=15.3 Hz), 5.15 (1H, d, J=
.
.
8.81 (1H, s). Anal. (C20H20N4O5 0.1CHCl3 0.1H2O);
calcd: C, 58.86; H, 4.99; N, 13.66. Found: C, 58.86; H,
5.10; N, 13.78.
3-(3-Methylureido)-1-N-((N0 -phthaloyl)methyl)-2,3,4,5-
tetrahydro-1H-1-benzazepin-2-one (11n). 6% yield:
1H NMR (CDCl3) d: 1.89±2.03 (1H, m), 2.43±2.79 (3H,
m), 2.75 (3H, d, J=4.6 Hz), 4.36±4.49 (1H, m), 4.42
(1H, d, J=13.6 Hz), 4.62 (1H, brs), 5.61 (2H, t, J=
8.0 Hz), 6.33 (1H, d, J=13.6 Hz), 7.13±7.45 (4H, m),

1712
Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
15.3 Hz), 6.85 (1H, d, J=8.0 Hz), 6.90±7.10 (5H, m),
.
7.10±7.40 (7H, m). Anal. (C29H33N5O3 0.5Et2O); calcd:
C, 69.38; H, 7.14; N, 13.05. Found: C, 69.25; H, 7.10; N,
12.95.
1-N-(3-(N0 -(tert-Butoxycarbonyl)amino)benzyl)-3-(N-
methyl-N0 -phenylguanidino)-2,3,4,5-tetrahydro-1H-1-
benzazepin-2-one (14d). To 1-N-(3-(N0-(tert-butoxy-
carbonyl)amino)benzyl)-3-(3-(S-methylphenylisothio-
ureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one hydro-
iodide salt (300 mg, 0.455 mmol) was added 40%
methylamine in MeOH solution (1.2 mL) and the mix-
ture was heated to 80ꢀC with stirring in a sealed tube for
4 h. After removal of the solvent, the residue was pur-
i®ed by PLC (1-BuOH:AcOH:H2O=5:1:1) to a€ord
Compounds 14a and 14c were prepared from 8b
according to a same procedure.
1-N-(3-(N0 -(tert-Butoxycarbonyl)amino)benzyl)-3-(N-
methylguanidino)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-
1
1
one (14a). 34% yield: H NMR (CD3OD) d: 1.49 (9H,
14d (206 mg, 88%) as a foam: H NMR (CD3OD) d:
s), 2.14 (1H, m), 2.54 (2H, m), 2.67 (1H, m), 2.81 (3H,
s), 4.22 (1H, dd, J=7.5 Hz, 12.5 Hz), 4.90 (1H, d,
J=14.6 Hz), 5.35 (1H, d, J=14.6 Hz), 6.81 (1H, d,
J=7.5 Hz), 7.11 (1H, t, J=7.2 Hz), 7.30 (3H, m), 7.40
(3H, m). Anal. (C24H31N5O3 2.0H2O); calcd: C, 60.87;
H, 7.45; N, 14.79. Found: C, 60.54; H, 7.22; N, 14.89.
1.48 (9H, s), 2.20 (1H, m), 2.40±2.80 (3H, m), 2.92
(3H, m), 4.31 (1H, dd, J=7.6, 11.4 Hz), 4.84 (1H, d,
J=14.8 Hz), 5.23 (1H, d, J=14.8 Hz), 6.80 (2H, d, J=
7.4 Hz), 7.00±7.40 (12H, m). Anal. (C30H35N5O3 2.3H2O);
calcd: C, 64.92; H, 6.79; N, 12.80. Found: C, 64.92; H,
7.19; N, 12.62.
.
.
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(N-(4-
hydroxyphenyl)guanidino)-2,3,4,5-tetrahydro-1H-1-benz-
Compounds 14e±g were prepared from 8b according to
a similar procedure.
azepin-2-one (14c). 17% yield: mp 180±183ꢀC; H NMR
1
(CD3OD) d: 1.48 (9H, s), 2.05 (1H, m), 2.50±2.80 (3H,
m), 4.35 (1H, m), 4.84 (1H, d, J=14.8 Hz), 5.30 (1H, d,
J=14.8 Hz), 6.8 (3H, m), 7.00±7.40 (9H, m). Anal.
1-N-(3-(N0 -(tert-Butoxycarbonyl)amino)benzyl)-3-(N-
methoxy-N0-phenylguanidino)-2,3,4,5-tetrahydro-1H-1-
1
benzazepin-2-one (14e). 22% yield: H NMR (CD3OD)
.
.
(C29H33N5O4 0.2EtOAc 2.6H2O); calcd: C, 61.65; H,
6.93; N, 11.98. Found: C, 61.69; H, 6.49; N, 12.01.
d: 1.98 (1H, m), 2.30±2.70 (3H, m), 3.66 (3H, s), 4.10
(1H, m), 4.85 (1H, d, J=15.0 Hz), 5.07 (1H, d, J=
15.0 Hz), 6.80±7.40 (13H, m). Anal. (C30H35N5O4);
calcd: C, 68.03; H, 6.66; N, 13.22. Found: C, 67.84; H,
6.71; N, 13.04.
Preparation of 1-N-(3-(N0-(tert-Butoxycarbonyl)amino)-
benzyl)-3-(N-methyl-N0-phenylguanidino)-2,3,4,5-tetra-
hydro-1H-1-benzazepin-2-one (14d)
1-N-(3-(N0 -(tert-Butoxycarbonyl)amino)benzyl)-3-(N-
hydroxy-N0 -phenylguanidino)-2,3,4,5-tetrahydro-1H-1-
benzazepin-2-one (14f). 27% yield: mp 150±153ꢀC;
1H NMR (CDCl3) d: 1.50 (9H, s), 1.95 (1H, m), 2.50±
2.80 (3H, m), 4.30 (2H, m), 4.70 (1H, d, J=20.0 Hz),
5.32 (1H, d, J=20.0 Hz), 6.80±7.40 (13H, m). Anal.
1-N-(3-(N0 -(tert-Butoxycarbonyl)amino)benzyl)-3-(3-
phenylthioureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-
one. To a solution of compound 8b (1.0 g, 2.62 mmol)
in CH3CN (10 mL) was added phenyl isothiocyanate (343
mL, 2.88 mmol) at 0ꢀC and the mixture was stirred at rt
for 1 h. The deposited crystals were collected by ®ltra-
tion, washed with Et2O twice and recrystallized from
CH3CN to obtain the target compound 930 mg (68%)
.
(C29H33N5O4 0.2Et2O); calcd: C, 67.65; H, 6.63; N,
13.00. Found: C, 67.84; H, 6.65; N, 13.20.
as crystals: mp 132±135ꢀC; H NMR (CD3OD) d: 1.15
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(N,N0-
dimethyl-N00-phenyl)guanidino)-2,3,4,5-tetrahydro-1H-1-
1
(6H, d, J=6.3 Hz), 1.95 (1H, m), 2.50±2.70 (3H, m),
3.86 (1H, m), 4.88 (1H, d, J=15.0 Hz), 5.03 (1H, dd,
J=7.2, 10.1 Hz), 5.17 (1H, d, J=15.0 Hz), 6.86 (1H, d,
J=7.5 Hz), 7.13 (1H, t, J=8.1 Hz), 7.20±7.40 (11H, m).
1
benzazepin-2-one (14g). 43% yield: H NMR (CD3OD)
d: 1.49 (9H, s), 1.95 (1H, m), 2.15 (1H, m), 2.34 (1H, m),
2.52 (1H, m), 2.89 (6H, s), 3.99 (1H, dd, J=7.5,
11.5 Hz), 4.77 (1H, d, J=15.0 Hz), 5.09 (1H, d, J=
15.0 Hz), 6.61 (2H, d, J=7.2 Hz), 6.69 (2H, d, J=
7.2 Hz), 6.77 (1H, d, J=7.2 Hz), 6.90±7.30 (9H, m).
.
Anal. (C29H32N4O3S 0.1CH3CN); calcd: C, 67.35; H,
6.25; N, 11.03; S, 6.16. Found C, 67.18; H, 6.29; N,
11.36; S, 6.27.
.
Anal. (C29H33N5O4 0.2H2O); calcd: C, 70.09; H, 7.10;
N, 13.18. Found: C, 70.03; H, 7.12; N, 13.00.
1-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-(S-
methyl-phenylisothioureido)-2,3,4,5-tetrahydro-1H-1-
benzazepin-2-one hydroiodide salt. A suspension of the
above thiourea compound (1.0 g, 1.93 mmol) and MeI
(0.24 mL, 3.85 mmol) in CH3CN (3 mL) was stirred at
rt for 16 h and then MeI (0.24 mL, 3.85 mmol) was
added. The mixture was heated to 40ꢀC with stirring for
2.5 h. After cooling, the reaction mixture was con-
centrated to a€ord 290 mg (94%) of the target com-
pound as a foam, which was used directly in the next
step: 1H NMR (CD3OD) d: 1.15 (6H, d, J=6.6 Hz),
2.13 (1H, m), 2.30 (3H, s), 2.40±2.70 (3H, m), 3.85 (1H,
m), 4.51 (1H, dd, J=7.0, 10.2 Hz), 4.97 (1H, d, J=
15.0 Hz), 5.08 (1H, d, J=15.0 Hz), 6.73 (2H, d, J=
7.5 Hz), 5.90±7.00 (3H, m), 7.10±7.30 (8H, m).
Preparation of 5-N-(3-(N0-(tert-butoxycarbonyl)amino)-
benzyl)-3-(3-phenylureido)-2,3-dihydro-5H-1,5-benzothi-
azepin-4-one (16)
3-Phenylureido-2,3-dihydro-5H-1,5-benzothiazepin-4-one.
A mixture of 3-phthalimido-2,3-dihydro-5H-1,5-benzo-
thiazepin-4-one (250 mg, 0.77 mmol) and hydrazine
monohydrate (119 mg) in EtOH (8.5 mL) was stirred
under re¯ux for 1 h. After cooling, the reaction mixture
was partitioned between EtOAc and H2O. The organic
layer was washed with brine, dried over Na2SO4 and
concentrated in vacuo. The residue was washed with
n-hexane and no further puri®cation was done. The

Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
1713
resulting amine was N-alkylated by the procedure
according to Method B to give the target compound in
59% yield: mp 233ꢀC (dec.) 1H NMR (CDCl3+
CD3OD) d: 2.73 (3H, brs), 2.95 (1H, t, J=10.2 Hz), 3.85
(1H, dd, J=6.6 Hz, 11.0 Hz), 4.69 (1H, m), 6.23 (1H,
m), 6.96±7.38 (10H, m), 7.65 (1H, d, J=6.2 Hz). Anal.
(C16H15N3O2S 0.1H2O); calcd: C, 60.97; H, 4.86; N,
13.33; S, 10.17. Found: C, 61.16; H, 4.86; N, 13.41; S,
9.93.
receptor binding was carried out using membranes from
CHO cells expressing cloned human NPY Y5, as
described previously.29 Cytosolic free Ca2+ concentra-
tion in SK-N-MC cells was measured ¯uorometrically
using the Ca2+-sensitive ¯uorescent dye fura-2 with a
spectro¯uorometer (CAF-100, Japan Spectroscopy Inc.,
Tokyo, Japan) as described previously.27
.
References
5-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-phe-
nylureido)-2,3-dihydro-5H-1,5-benzothiazepin-4-one (16).
This compound was prepared according to Method A
from compound 4 and 3-phenylureido-2,3-dihydro-5H-
1,5-benzothiazepin-4-one in 56% yield: 1H NMR
(CDCl3) d: 1.49 (9H, s), 2.92 (1H, t, J=11.5 Hz), 3.79
(1H, dd, J=6.8 Hz, 11.2 Hz), 4.79 (2H, m), 4.89 (1H, d,
J=15.6 Hz), 5.26 (1H, d, J=16.2 Hz), 6.65 (2H, brs),
6.95 (2H, m), 7.08±7.26 (8H, m), 7.30±7.45 (2H, m), 7.46
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5-N-(3-(N0-(tert-Butoxycarbonyl)amino)benzyl)-3-(3-phe-
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was prepared according to Method A from compound
4 and 3-benzyloxycarbonylamino-2,3-dihydro-5H-1,5-
benzothiazepin-4-one, followed by removal of the Cbz
group by hydrogenolysis using 5% Pd-C. The resulting
amine was converted to the target compound according
to Method B using phenylisocyanate (37% yield):
1H NMR (CD3OD) d: 1.48 (9H, s), 4.30 (1H, m), 4.55
(1H, m), 4.89 (1H, dd, J=7.8 Hz, 11.4 Hz), 4.97 (1H, d,
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.
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NPY Receptor Binding Assay
NPY Y1 and Y2 receptor binding assays were con-
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of [125I]NPY because the former nonspeci®c binding
was lower than the latter. SK-N-MC and SMS-KAN
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Y2 receptor binding assays, respectively. After 2 days,
the medium was removed and the cells were washed
with HEPES (20 mM)-bu€ered Hank's solution (pH
7.4) containing 1% bovine serum albumin (binding
bu€er). For competitive binding experiments, the cells
were incubated with 0.1 nM [125I]PYY and varying
concentrations of unlabeled compounds in 0.5 mL of
binding bu€er at 37ꢀC for 60 min. For Scatchard plot
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[
125I]PYY. The incubation was stopped by removing the
assay mixture and the cells were washed twice with 1
mL of ice-cold binding bu€er, lysed with 1.5 mL of 1 N
NaOH and transferred to test tubes. The radioactivity
was counted with a g counter. Nonspeci®c binding was
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Chem. Commun. 1992, 57, 531.
6
determined in the presence of 10 M NPY. NPY Y5

1714
Y. Murakami et al. / Bioorg. Med. Chem. 7 (1999) 1703±1714
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