Discovery of 1,7-cyclized indoles as a new class of potent and highly selective human β3-adrenergic receptor agonists with high cell permeability

Article abstract of DOI:10.1016/j.bmc.2004.10.032

The synthesis and evaluation of a novel series of 1,7-cyclized indole-based human adrenergic receptor (β₃-AR) agonists are reported. The synthesis of a variety of 1,7-cyclized indole part was accomplished by the Mitsunobu reaction or a ring closing metathesis (RCM) reaction. SAR studies revealed that expansion of the ring size resulted in considerable selectivity against the β₁- and β₂-ARs. Compound 26, an eight-membered ring analogue with a double bond on its 1,7-linker portion, was found to be a potent β₃-AR agonist (EC₅₀ = 0.75 nM, IA = 90%) with extremely high selectivity for the β₃-AR over the β₁- and β₂-ARs.

Full text of DOI:10.1016/j.bmc.2004.10.032

Bioorganic & Medicinal Chemistry 13 (2005) 855–868
Discovery of 1,7-cyclized indoles as a new class of potent and
highly selective human b3-adrenergic receptor agonists
with high cell permeability
Kazuhiro Mizuno,a,* Masaaki Sawa,a Hiroshi Harada,a Ikuko Taoka,a
Haruhisa Yamashita,a Mayumi Oue,b Hiroshi Tsujiuchi,b Yukiyo Arai,c
Shinya Suzuki,c Yasuji Furutanib and Shiro Katoa
aChemistry Research Laboratories, Dainippon Pharmaceutical Co., Ltd, 33-94, Enoki, Suita 564-0053, Japan
bPharmacology and Microbiology Research Laboratories, Dainippon Pharmaceutical Co., Ltd, 33-94,Enoki, Suita 564-0053, Japan
cPharmacokinetics and Physico-Chemical Property Research Loboratories, Dainippon Pharmaceutical Co., Ltd, Enoki 33-94,
Suita 564-0053, Japan
Received 9 September 2004; revised 14 October 2004; accepted 14 October 2004
Available online 11 November 2004
Abstract—The synthesis and evaluation of a novel series of 1,7-cyclized indole-based human adrenergic receptor (b3-AR) agonists
are reported. The synthesis of a variety of 1,7-cyclized indole part was accomplished by the Mitsunobu reaction or a ring closing
metathesis (RCM) reaction. SAR studies revealed that expansion of the ring size resulted in considerable selectivity against the
b1- and b2-ARs. Compound 26, an eight-membered ring analogue with a double bond on its 1,7-linker portion, was found to be
a potent b3-AR agonist (EC50 = 0.75nM, IA = 90%) with extremely high selectivity for the b3-AR over the b1- and b2-ARs.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
OH
H
Cl
N
The b3-adrenergic receptor (b3-AR) has been shown to
mediate various pharmacological and physiological ef-
fects such as lipolysis, thermogenesis, and relaxation of
the urinary bladder. Activation of the b3-AR is thought
to be a possible approach for the treatment of obesity,
noninsulin dependent diabetes mellitus (NIDDM),1
and frequent urination.2 Therefore, the b3-AR is recog-
nized as an attractive target for drug discovery. On the
other hand, activation of the b1- or b2-AR would cause
undesirable side effects such as increased heart rate or
muscle tremors. Consequently, a number of recent ef-
forts in this field have been directed toward the design
of selective agonists for the b3-AR.3 During the past dec-
ade, several groups have reported the importance of an
acidic group on the right-hand side (RHS) of the etha-
nolamine pharmacophore on potency and selectivity of
the b3-AR agonists (Fig. 1). For example, Sher et al.
O
CO2H
A : BRL37344
OH
H
N
N
NH
S
O
O
O
N
N
N
N
B : L-770644
Me
H
N
OH
S
H
O
O
Cl
N
O
N
H
Keywords: b3-Adrenergic receptor; Agonist; 1,7-Cyclized indole.
Corresponding author. Tel.: +81 663375904; fax: +81 663376010;
e-mail: kazuhiro-mizuno@dainippon-pharm.co.jp
C
*
Figure 1.
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.032

856
K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
showed that a negatively charged carboxylate group on
the RHS in compound A played a role in enhancing
selectivity for the b3-AR.4 Researchers at Merck dis-
closed a novel series of b3-AR agonists having an aryl-
sulfonamide on the RHS, represented by compound
B.5 In these compounds, the NH group of the sulfon-
amide function seems to act as an acidic group instead
of the traditional carboxyl group, and contributes to
the selectivity for the b3-AR. Workers at GlaxoSmith-
Kline also reported b3-AR agonists having an acylsul-
fonamide on the RHS as a carboxylic acid isosteres
(compound C).6 In addition, they suggested that the
steric bulk of the RHS substituent also contributed to
the potency and selectivity of the b3-AR agonists. How-
ever, it is thought that introduction of such hydrophilic
groups may generally cause low oral bioavailability due
in part to poor absorption.7
2. Chemistry
The general strategy adopted for the preparation of the
target compounds listed in Figure 2 and Tables 1 and 2
was a convergent route in which various tryptamine
derivatives were coupled with the common intermediate
4 as shown in Figure 3. Synthesis of the key intermediate
4 was carried out as depicted in Scheme 1. Asymmetric
reduction of the commercially available 3-nitrophenacyl
bromide 5 was accomplished by treatment with a BH3–
THF complex in the presence of CoreyÕs CBS-borane
reagent.9 After recrystallization from hexane/i-Pr2O,
the desired (R)-alcohol 610 was obtained in 78% yield
with an excellent enantiomeric purity (>99% ee). Pro-
tection of the secondary hydroxyl group in 6 was
accomplished with tert-butyldimethylsilyl chloride
(TBDMSCl) in the presence of an excess amount of
KBr to avoid the undesirable formation of the chloride
7b, which was derived by a halogen exchange reaction of
7a. The nitro compound 7a was then subjected to cata-
lytic hydrogenation in the presence of Pt on charcoal,
followed by treatment with 2-thiophenesulfonyl chloride
to provide the desired sulfonamide 4 in 91% yield from
the (R)-alcohol 6.
Previous work in our laboratory has demonstrated that
a series of tryptamine derivatives with a 2-thiophenesul-
fonamide group, represented by 1, showed strong agon-
istic activity for human b3-AR.8 The structure–activity
relationship (SAR) studies on the 7-position of the in-
dole ring indicated that introduction of an aliphatic sub-
stituent such as a methoxy group in 2 increased agonistic
activity, but modestly decreased subtype selectivity for
the b3-AR. Further SAR studies on the indole ring re-
vealed that substitution of the hydrogen at the 1-posi-
tion in 1 with an alkyl group significantly altered
subtype selectivity (3). Based on these results, we as-
sumed that substitutions at both the 1- and 7-positons
of the indole ring might induce subtype selectivity with-
out loss of potency for the b3-AR. Furthermore, these
1,7-disubstitutions seem to offer a potential advantage
in the drugÕs properties, since these compounds possess
no acidic group on the RHS, which would make oral
bioavailability of the compounds worse. In this study,
we report the synthesis and pharmacological evaluation
of a novel series of 1,7-disubstituted indole-based b3-AR
agonists with a 2-thiophenesulfonamide group on their
left-hand side (LHS).
The preparation of 1 and its N-ethyl derivative 3 is
shown in Scheme 2. (R)-a-Methyltryptamine 811 was
coupled with the bromide 4 in the presence of i-Pr2NEt
and KI, and subsequent deprotection of the TBDMS
group supplied 1 in 48% yield. Alternatively, the amino
group in 8 was protected with a Boc group, and subse-
quent N-ethylation of the indole ring with EtI in the
presence of NaH gave N-ethylated compound 10 in
76% yield from 8. The Boc group in 10 was then depro-
tected, and the resulting amine was coupled with the key
intermediate 4 in the presence of i-Pr2NEt and KI.
Finally, removal of the TBDMS group afforded the
desired compound 3 in 29% yield from compound 10.
The synthesis of 7-methoxy derivatives 2 and 16a,b was
carried out as shown in Scheme 3. The amino group in
1112 was protected with a Boc group, and the benzyl
group in 12 was then removed by hydrogenation to give
7-hydroxyindole derivative a in 96% yield from 11. O-
Alkylation of 13 was performed with MeI and K2CO3
to provide O-methylated compound 14 in 99% yield.
Deprotection of the Boc group in 14, coupling of the
resulting amine with the key intermediate 4, and subse-
quent removal of the TBDMS group afforded the de-
sired compound 2 in 50% yield from 14. Alternatively,
further alkylation of 14 with MeI or BnBr in the pres-
ence of NaH gave N,O-dialkylated indoles 15a,b in good
yield. Subsequent conversion of the tryptamines 15a,b
OH
H
N
H
N
S
S
O
O
Me
N
R1 R2
EC50 (nM)
β3 β1
0.88 66 21
0.55 29 6.6
0.61 >100 42
β2
1 : R1 = H, R2 = H
2 : R1 = H, R2 = OMe
3 : R1 = Et, R2 = H
Figure 2.
OTBDMS
OH
H2N
H
N
H
N
H
Br
N
S
S
S
O
S
Me
+
N
O
O
O
Me
R1 R2
N
R1 R2
4
Figure 3.

K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
OH
857
somehow unsuccessful. Compound 18f was finally ob-
tained in 32% yield, according to the procedures Hearny
and Ley outlined for the N-alkylation of an indole ring
(KOH in DMSO).13 The obtained tryptamines 18a–f
were converted into the desired products 19a–f by
removal of the Boc group, coupling with the sulfon-
amide 4, and deprotection of the TBDMS group.
O
O2N
Br
O2N
Br
a
b
5
6
OTBDMS
X
O2N
c, d
4
Next, we designed compound 22, containing a difluoro-
methylene unit on its 1,7-linker portion. Introduction
of the difluoropropyl unit in 20 was first attempted
by the Mitsunobu reaction (DEAD, PPh3) with
3-chloro-2,2-difluoro-1-propanol.14 However, the reac-
tion was considerably slow even at reflux temperature,
and prolonged reaction time led to the formation of
by-products. This result implied that the reactivity of
the difluoroalcohol toward the Mitsunobu reagent
would be low compared to the alcohols used in Scheme
4. Tsunoda et al. reported that cyanomethylenetributyl-
phosphorane (CMBP), an ylide-type Mitsunobu re-
agent,15 is very effective in the reactions of secondary
alcohols, which often show low reactivity. We therefore
adopted TsunodaÕs procedures for the preparation of 20.
As expected, with the use of CMBP, the reaction of 13
with the difluoroalcohol proceeded smoothly to provide
the desired compound in quantitative yield. Cyclization
of compound 20 was accomplished with KOH in
DMSO, affording 1,7-cyclized indole derivative 21 in
52% yield. Tryptamine 21 also afforded the final com-
pound 22 in 20% yield using the general three steps out-
lined in Schemes 2–4.
7a : X = Br
7b : X = Cl
Scheme 1. Reagents: (a) (R)-2-methyl-CBS-oxazaborolidine, BH3–
THF complex, THF; (b) TBDMSCl, KBr, imidazole, DMF; (c) 5%
Pt/C, H2, EtOAc; (d) 2-thiophenesulfonyl chloride, pyridine, CH2Cl2.
into the corresponding final products 16a,b was accom-
plished using the method already described.
Preparation of 1,7-cyclized indole derivatives 19a–f was
conducted starting from 7-hydroxyindole derivative 13
and commercially available chlorohydrins in five steps
as shown in Scheme 4. O-Alkylation of 13 was con-
ducted using appropriate chlorohydrins under the gen-
eral conditions of the Mitsunobu reaction (diethyl
azodicarboxylate (DEAD), Ph3P, room temperature),
except dimethyl derivative 17e, which required reflux
temperature. The resulting chloroalkyl compounds
17a–f were treated with strong bases such as NaH and
KOH to afford cyclized compounds 18a–f. Cyclization
of 17a–d proceeded easily by treating with NaH at room
temperature, yielding six- to nine-membered simple
rings in a good to moderate yield, although the yield
of 17d was not satisfactory (18%). Cyclization of 17e re-
quired heating due to the steric bulk of the dimethyl
group. Several attempts to obtain compound 18f, con-
taining two ether oxygen atoms in the linker part, were
Synthesis of compound 26, where a double bond was
incorporated into the 1,7-linker portion, is depicted in
Scheme 6. For the construction of an unsaturated ring
system, we employed the ring closing metathesis
(RCM) reaction. Thus, O-allylation of 13 with allyl
H
H
N
H2N
N
e, a, b
a, b
c
d
Boc
Boc
1
3
Me
Me
Me
N
N
N
H
H
Et
9
10
8
Scheme 2. Reagents: (a) 4, KI, i-Pr2NEt, MeCN; (b) 4M HCl–EtOAc, EtOH; (c) (Boc)2O, CHCl3; (d) EtI, NaH, THF; (e) 4M HCl–EtOAc.
H
H
H
N
H2N
N
N
g
a
c
b
Boc
Boc
Boc
Me
Me
Me
Me
N
H
N
N
N
H
H
H
O
Ph
OMe
O
Ph
OH
11
12
14
13
d, e, f
OH
H
N
H
H
N
N
d, e, f
S
Boc
S
O
O
Me
Me
2
N
N
R1
R1 OMe
OMe
16a : R1 = Me
16b : R1 = Bn
15a : R1 = Me
15b : R1 = Bn
Scheme 3. Reagents: (a) (Boc)2O, EtOAc; (b) 5% Pd–C, MeOH; (c) MeI, K2CO3, acetone; (d) 4M HCl–EtOAc; (e) 4, KI, i-Pr2NEt, MeCN; (f) 4M
HCl–EtOAc, EtOH; (g) MeI or BnBr, NaH, DMF.

858
K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
H
H
N
N
a
Boc
Boc
b or c
d, e, f
13
Me
Me
N
N
H
O
O
X
Cl
X
17a : X = none
18a : X = none
17b : X = -CH2-
17c : X = -(CH2)2-
17d : X = -(CH2)3-
17e : X = -C(Me)2-
18b : X = -CH2-
18c : X = -(CH2)2-
18d : X = -(CH2)3-
18e : X = -C(Me)2-
18f : X = -CH2OCH2-
17f : X = -CH2OCH2-
OH
H
H
N
N
S
S
O O
Me
N
O
X
19a : X = none
19b : X = -CH2-
19c : X = -(CH2)2-
19d : X = -(CH2)3-
19e : X = -C(Me)2-
19f : X = -CH2OCH2-
Scheme 4. Reagents: (a) HOCH2–X–CH2Cl, DEAD, Ph3P, THF; (b) NaH, DMF; (c) KOH, DMSO; (d) 4M HCl–EtOAc; (e) 4, KI, i-Pr2NEt,
MeCN; (f) 4M HCl–EtOAc, EtOH.
OH
H
H
H
H
N
N
N
N
b
c, d, e
S
S
Boc
Boc
a
O O
13
Me
Me
Me
N
N
N
H
O
O
O
F
F
21
F
22
Cl
F
F
20
F
Scheme 5. Reagents: (a) HOCH2CF2CH2Cl, Bu3P@CHCN, toluene; (b) KOH, DMSO; (c) 4M HCl–EtOAc; (d) 4, KI, i-Pr2NEt, MeCN; (e) 4M
HCl–EtOAc, EtOH.
H
N
The RCM reaction of 24 was successfully achieved using
Cl2(PCy3)2Ru@CHPh16 as a catalyst to give 25 in 64%
yield. Tryptamine derivative 25 was subjected to the
general three steps as outlined in Schemes 2–5 to afford
the final compound 26 in 23% yield.
b
a
Boc
13
Me
N
H
O
23
An alternative synthetic method shown in Scheme 7 was
developed to prepare 29 and 33, which have a methyl
group in the 1,7-linker unit. In this route, the order of
cyclization step was reversed. Namely, initial alkylation
at the nitrogen atom and the subsequent intramolecular
Mitsunobu reaction provided the tryptamine derivatives
28 and 32. For the synthesis of 28, the nitrogen of the
indole ring in compound 12 was alkylated with an excess
amount of propylene oxide in the presence of t-BuOK to
give 27 in 44% yield. The benzyl group in 27 was re-
moved by Pd catalyzed hydrogenation, and the resulting
7-hydroxyindole derivative was subjected to the intra-
molecular Mitsunobu reaction. As expected, cyclization
proceeded successfully and the desired compound 28
was obtained in 30% overall yield from compound 12.
Similarly, compound 32 was prepared as follows. N-
Alkylation of 12 with methyl 2-bromopropionate in
the presence of NaH gave 30 in 90% yield, and then,
alkaline hydrolysis of the ester, followed by reduction
of the resulting carboxylic acid with a BH3–THF com-
plex gave primary alcohol 31 in 47% yield. Removal of
the benzyl group and subsequent intramolecular
H
H
N
N
Boc
Boc
c
Me
Me
N
N
O
O
25
24
OH
H
H
N
N
d, e, f
S
S
O O
Me
N
O
26
Scheme 6. Reagents: (a) allyl iodide, K2CO3, acetone; (b) allyl
bromide, NaH, DMF; (c) Cl2(PCy3)2Ru@CHPh, CH2Cl2; (d) 4M
HCl–EtOAc; (e) 4, KI, i-Pr2NEt, MeCN; (f) 4M HCl–EtOAc, EtOH.
iodide and K2CO3 gave O-allyl derivative 23 in 82%
yield, and subsequent N-allylation with the use of allyl
bromide and NaH gave N,O-diallyl compound 24 in
97% yield, which was subjected to the RCM reaction.

K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
859
H
N
H
N
Boc
b, c
Boc
a
d, e, f
Me
Me
HO
OH
12
N
N
H
H
N
N
O
O
Ph
S
S
27
28
O
O
Me
Me
g
Me
N
3
O
d, e, f
29: 2-Me
33: 3-Me
Me
2
H
N
H
H
N
N
b, c
Boc
Boc
Boc
h, i
Me
MeO
Me
HO
Me
N
N
N
Ph
Ph
O
O
O
Me
Me
Me
31
30
32
O
Scheme 7. Reagents: (a) propylene oxide, t-BuOK, THF; (b) 10% Pd–C, H2, EtOH; (c) DEAD, Ph3P, THF; (d) 4M HCl–EtOAc; (e) 4, KI, i-Pr2NEt,
MeCN; (f) 4M HCl–EtOAc, EtOH; (g) methyl 2-bromopropionate, NaH, DMF; (h) 1M NaOHaq, MeOH; (i) BH3–THF complex, THF.
Mitsunobu reaction afforded desired compound 32 in
75% yield. As with the case described above, trypt-
amines 28 and 32 were coupled with the sulfonamide
part 4, giving the desired compounds 29 and 33.
initially synthesized 7-methoxy derivative 2 and its 1-
methylated analogue 16a. As expected, 16a showed
remarkable improvement in subtype selectivity against
both the b1- and b2-ARs (>160-fold and 57-fold, respec-
tively) compared to that of 2 (53-fold and 12-fold,
respectively) while maintaining good agonistic activity
for the b3-AR (EC50 = 0.61nM). However, the presence
of an aromatic group at the 1-positon of the indole ring,
such as a benzyl group in 16b, was not tolerated by the
b3-AR (EC50 = 4.4nM). Next, 1,7-cyclized indole-based
compound 19a was designed and synthesized. Surpris-
ingly, inclusion of a ring at this position did not result
in a dramatic change in either potency or selectivity.
Namely, the tricyclic compound 19a exhibited an almost
identical in vitro profile with the noncyclized parent
compound 16a. These results encouraged us to further
investigate the possibility of the other 1,7-cyclized indole
derivatives as b3-AR agonists.
The assigned structures of the final compounds were
fully confirmed by MS, 1H NMR, and elemental
analysis.
3. Results and discussion
All compounds listed in Tables 1 and 2 were evaluated
in vitro for their ability to stimulate cAMP accumul-
ation in CHO cells expressing the cloned human b1-,
b2-, and b3-ARs.17 The binding affinity of the com-
pounds listed in Table 2 was also determined for human
b1-, b2-, and b3-ARs in competition with the radioligand
(ꢀ)-[125I]-iodocyanopindolol. Compounds with a high
potency for the b3-AR were further evaluated for their
ability to cross Caco-2 cell monolayers to predict their
oral absorption potential.
To examine the available space in the binding sites, we
initially synthesized seven- to nine-membered simple
ring analogues 19b–d, and the results are shown in Table
2. Despite the expansion of the ring size, all compounds,
19b–d, showed potent agonistic activity for the b3-AR
(EC50 = 1.0, 1.4, and 1.4nM, respectively). However,
the selectivity against the b1- and b2-ARs was signifi-
cantly affected by the ring size. Namely, the agonistic
activity of the eight- and nine-membered ring analogues,
19c and 19d, for the b1- and b2-ARs was dramatically
decreased, whereas the seven-membered ring analogue
19b was still active for these receptors. While the origin
of the high agonistic activity of 1,7-cyclized indoles for
the b3-AR is unknown, it is speculated to be the result
of a positive hydrophobic interaction with the binding
site and a more favorable conformational change of
the receptor induced by the rigid ring structure. These
tricyclic compounds, 19a–d, also showed similar ten-
dency in the binding assay with regard to subtype selec-
tivity. The binding affinity for the b3-AR was quite
insensitive to the changes in the ring size (Ki = 3.2–
7.7nM), however the expansion of the ring size signifi-
cantly attenuated the binding affinity for the b1- and
b2-ARs. These data strongly suggested that the selecti-
vity concerning the agonistic activity was elicited by
steric hindrance of the ring moiety, and not caused by
its antagonistic property.
To assess the possibility of the 1,7-disubstituted indole
derivatives as potent and selective b3-AR agonists, we
Table 1. Agonistic activity of substituted indole derivatives for human
b-ARs
OH
H
N
H
N
S
S
O O
Me
N
R1 OR2
Compd
R1
R2
EC50, nMa (IA, %)b
b3
b1
b2
2
H
Me
Me
Me
0.55 (101)
0.61 (86)
4.4 (80)
29 (36)
>100 (57c)
32 (65)
>88 (79c)
6.6 (67)
35 (57)
22 (64)
36 (42)
16a
16b
19a
Me
Bn
–(CH2)2–
0.92 (85)
a Agonistic activity was assessed by measuring cAMP accumulation in
CHO cells expressing human b-ARs.
b Values in parentheses represent the intrinsic activity (IA) given as
percentage of maximal stimulation with isoproterenol.
c % Activity at 1000nM.

860
K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
Table 2. In vitro data for 1,7-linked tricyclic indole derivatives: agonistic activity and binding affinity for human b-ARs, and Caco-2 permeability
OH
H
N
H
N
S
S
O O
Me
N
O
X
Compd
X
EC50, nMa (IA, %)b
Binding Ki, nMe
Caco-2 Permeabilityf
Class
b3
b1
b2
b3
7.7
4. 8
b1
b2
71
19a
19b
19c
19d
26
–(CH2)2–
–(CH2)3–
0.92 (85)
1.0 (81)
1.4 (98)
1.4 (79)
0.75 (90)
3.3 (77)
1.2 (95)
0.66 (94)
11 (84)
>88 (79c)
53 (70)
36 (42)
190
120
540
560
550
560
230
270
160
370
high
high
high
high
high
ntg
44 (40)
59
170
280
150
340
89
–(CH2)4–
–(CH2)5–
>100 (50c)
>100 (49c)
>100 (72c)
>100 (55c)
>100 (63c)
>100 (56c)
>100 (48c)
>100 (52c)
ndd (23c)
ndd (22c)
ndd (27c)
>100 (48c)
36 (51)
3.2
5.5
3.1
3.9
9.0
8.9
–CH2CH@CHCH2–
–CH2CH2OCH2CH2–
–CH2CH(Me)–
–CH(Me)CH2–
–CH2C(Me)2CH2–
–CH2CF2CH2–
19f
29
high
high
ntg
ntg
33
19 (42)
ndd (8c)
ndd (15c)
55
19e
22
23
10
140
96
3.4 (92)
a Agonistic activity was assessed by measuring cAMP accumulation in CHO cells expressing human b-ARs.
b Values in parentheses represent the intrinsic activity (IA) given as percentage of maximal stimulation with isoproterenol.
c % Activity at 1000nM.
d nd = not determined.
e Binding affinity is reported as Ki, the binding inhibition constant, determined by inhibition of 125I-iodocyanopindolol binding.
f According to the degree of permeability across Caco-2 monolayers, the compounds were classified for their absorption potential as follows: high,
Papp ꢁ 1.0 · 10ꢀ6 cm/s; moderate, 0.5 · 10ꢀ6 cm/s < Papp < 1.0 · 10ꢀ6 cm/s; low, Papp ꢂ 0.5 · 10ꢀ6 cm/s.
g nt = not tested.
Based on the good potency of the 1,7-cyclized indoles,
additional eight- and nine-membered ring analogues
were synthesized as shown by 26 and 19f. Incorporation
of a double bond into the eight-membered ring (26)
resulted in a 1.9-fold improvement in potency for the
b3-AR (EC50 = 0.75nM) compared to the saturated
eight-membered ring analogue 19c. This preference
for the alkenyl moiety might be due to either its
electron-donating property, which would strengthen
the binding ability to the receptor, or a more favorable
conformational bias imparted by the double bond.
In contrast, the insertion of one ether oxygen, as in
19f, resulted in a moderate decrease in potency for the
b3-AR. As can be seen from Table 2, these compounds
exhibited excellent selectivity against the b1- and b2-
ARs both in an agonistic assay and in the binding test
as expected.
fluoro group (22) resulted in improving the potency
for the b3-AR (EC50 = 3.4nM) without affecting potency
against the b1- and b2-ARs.
As a first step toward understanding the overall pharma-
cokinetic profile of the 1,7-cyclized indole-based deriva-
tives, an attempt was made to determine the
permeability of compounds with high potency for the
b3-AR (EC50 < 3.0nM). As shown in Table 2, all the
compounds examined showed high cellular permeability
(Papp ꢁ 1.0 · 10ꢀ6 cm/s) as expected from their struc-
ture. These data indicate that the 1,7-cyclized-based in-
dole series of compounds represent a leading group of
b3-agonists with markedly improved bioavailability.
4. Conclusions
Next, we examined the effect of substituents on the lin-
ker portion of 1,7-cyclized indoles. The ability to intro-
duce this substitution at the linker portion not only
provides an increase in potency and/or selectivity but
also affords the possibility of increasing the metabolic
stability against metabolic enzymes such as Cytochrome
P450 proteins.18 Therefore, we synthesized six-mem-
bered ring analogues with a methyl group (29 and 33)
and a seven-membered ring analogue with a dimethyl
group (19e), and the results are shown in Table 2. The
methyl group at either carbon atom in the linker was
acceptable to b3-AR (29 and 33), while geminal substitu-
tion, as in the dimethyl analog 19e, attenuated potency
for b3-AR, possibly due to steric effects. However, these
mono-methyl substitutions did not result in enhance-
ment of selectivity, especially against the b2-AR.
Replacement of the dimethyl group with a smaller di-
In this study, we have disclosed the synthesis and biolog-
ical evaluation of a new series of 1,7-linked tricyclic in-
dole derivatives as potent and highly selective human b3-
AR agonists. The synthesis of a variety of tricyclic in-
dole parts was accomplished by the construction of the
1,7-linker portion, through either the Mitsunobu reac-
tion or a ring closing metathesis (RCM) reaction. All
the 1,7-linked tricyclic indole analogues synthesized
showed potent agonistic activity for the b3-AR with
excellent subtype selectivity. SAR studies for the linker
portion revealed that expansion of the ring size resulted
in considerable selectivity against the b1- and b2-ARs
(19a vs 19d). These observations are explained in terms
of poor binding affinity for the b1- and b2-ARs due to
steric effects. Introduction of a double bond into the
linker portion resulted in further improvement in the
agonistic activity for the b3-AR. These 1,7-cyclized

K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
861
indole-based b3-agonists showed high cellular permea-
bility (Papp ꢁ 1.0 · 10ꢀ6 cm/s) in the Caco-2 cell mem-
brane assays. These results revealed the potential of
1,7-linked tricyclic indole-based derivatives as a new
and leading set of candidates for human b3-AR agonists
with good oral bioavailability.
Pr2O (500mL · 2). The combined organic layers were
washed with brine, dried over MgSO4, and concentrated
under vacuum. The residue was chromatographed on
silica gel, eluting with hexane/EtOAc = 50/1 to 20/1
to give 7a (69g, 95%) as a light yellow solid. Mp:
1
43ꢁC; H NMR (CDCl3): ꢀ0.06 (s, 3H), 0.14 (s, 3H),
0.91 (s, 9H), 3.44 (dd, J = 10.4, 5.3Hz, 1H), 3.50 (dd,
J = 10.4, 6.8Hz, 1H), 4.96 (dd, J = 6.8, 5.3Hz, 1H),
7.54 (t, J = 8.0Hz, 1H), 7.71 (d, J = 7.7Hz, 1H), 8.17
(m, 1H), 8.25 (t, J = 2.0Hz, 1H).
5. Experimental
5.1. Chemistry
5.1.3. (R)-N-{3-[2-Bromo-1-(tert-butyldimethylsilyloxy)-
ethyl]phenyl}(2-thiophene)sulfonamide (4). A mixture of
7a (131g, 364mmol), 5% Pt/C (4g), and EtOAc (1.3L)
was hydrogenated under H2 for 6h. The catalyst was
then removed by filtration, and the filtrate was concen-
trated under reduced pressure. After the oily residue
and pyridine (35mL, 433mmol) were then dissolved in
CH2Cl2 (1.5L), 2-thiophenesulfonyl chloride (66g,
363mmol) was added portionwise at 0ꢁC. The mixture
was stirred overnight at room temperature, 1M aqueous
HCl was added, and the organic layer was separated,
washed with brine, dried over MgSO4, and concentrated
under vacuum. The residue was chromatographed on
silica gel, eluting with hexane/EtOAc = 10/1 to give 4
(166g, 96%). 1H NMR (CDCl3): ꢀ0.13 (s, 3H), 0.07
(s, 3H), 0.87 (s, 9H), 3.34 (dd, J = 10.3, 5.0Hz, 1H),
3.39 (dd, J = 10.3, 7.0Hz, 1H), 4.78 (dd, J = 7.0,
5.0Hz, 1H), 6.68 (s, 1H), 6.99 (dd, J = 5.0, 3.8Hz,
1H), 7.07–7.16 (m, 3H), 7.27 (m, 1H), 7.47 (dd,
J = 3.8, 1.3Hz, 1H), 7.52 (dd, J = 5.0, 1.3Hz, 1H).
Melting points were determined using a Yanagimoto
micromelting point apparatus and are uncorrected. H
1
NMR spectra were recorded on a JEOL JNM-LA300
(300MHz) spectrometer. Chemical shifts were expressed
as d (ppm) value from tetramethylsilane as an internal
standard. Elemental analyses were performed on an Ele-
mentar Vario EL analyzer. Mass spectra were recorded
on a Shimadzu LCMS-QP8000A spectrometer (APCI).
5.1.1. (R)-2-Bromo-1-(3-nitrophenyl)ethanol (6).10 To a
stirred solution of 3-nitrophenacyl bromide (5) (100g,
410mmol) and (R)-2-methyl-CBS-oxazaborolidine (1M
in toluene, 50mL, 50mmol) in THF (500mL), was
added dropwise BH3–THF complex (1M in THF,
800mL, 800mmol) over a period of 30min at room temp-
erature. After the addition was completed, stirring was
continued for 2h at ambient temperature. Then the reac-
tion mixture was cooled in an ice-bath, saturated NH4Cl
solution (400mL) was slowly added. After evaporation
of THF under reduced pressure, the residual solution
was partitioned between i-Pr2O (500mL) and H2O
(500mL). The mixture was stirred for 30min, and the
insoluble was filtered off. The organic layer was sepa-
rated from the filtrate, and the remaining aqueous layer
was extracted twice with i-Pr2O. Combined organic
layers were washed with brine, dried over MgSO4,
and concentrated under vacuum. The residue was chro-
matographed on silica gel, eluting with hexane/
EtOAc = 4/1 to 2/1 to give crude 6 as a solid. This solid
was dissolved in i-Pr2O (160mL), and to the solution,
hexane (240mL) was added dropwise with stirring.
The formed crystals were collected by filtration and
dried under vacuum to afford 6 (78g, 77%). Optical pur-
ity of 6 was determined to be >99% ee by a chiral HPLC
(Daicel Chiralpack AD column, 0.5mL/min, hexane/
ethanol 1/1, 25ꢁC). The retention times were: 6 (R)-iso-
mer, (9.38min); corresponding (S)-isomer, (8.34min).
5.1.4. 3-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-1H-indole
(1). A mixture of 811 (2.6g, 15mmol), 4 (4.76g,
10mmol), i-Pr2NEt (2.2mL, 13mmol), KI (1.7g,
10mmol), and MeCN (100mL) was heated under reflux
for 48h. After the reaction mixture was concentrated to
dryness under reduced pressure, the residue was parti-
tioned between EtOAc and H2O. The organic layer
was separated, washed with brine, dried over MgSO4,
and concentrated under vacuum. The residue was chro-
matographed on silica gel, eluting with CHCl3/
MeOH = 100/1 to give desired coupling product (3.4g,
60% from 4).
The compound obtained above was dissolved in EtOH
(10mL), treated with 4M HCl/EtOAc (30mL) at room
temperature for 2h. After the reaction mixture was con-
centrated in vacuo, the residue was partitioned between
EtOAc and aqueous K2CO3. The organic layer was
separated, washed with brine, dried over MgSO4,
and concentrated under vacuum. The residue was chro-
matographed on silica gel, eluting with CHCl3/
MeOH = 10/1 to give the title compound 1 (2.2g, 82%)
1
Mp: 65–66ꢁC; H NMR (CDCl3): 2.81 (d, J = 3.7Hz,
1H), 3.56 (dd, J = 10.6, 8.3Hz, 1H), 3.70 (dd, J = 10.6,
3.5Hz, 1H), 5.06 (m, 1H), 7.57 (m, 1H), 7.75 (m, 1H),
8.20 (m, 1H), 8.29 (m, 1H); Anal. Calcd for
C8H8BrNO3, 1/20i-Pr2O: C, 39.69; H, 3.49; N, 5.58.
Found: C, 39.58; H, 3.32; N, 5.70.
1
as an amorphous solid. H NMR (DMSO-d6): 0.97 (d,
5.1.2. (R)-2-Bromo-1-(tert-butyldimethylsilyloxy)-1-(3-
nitrophenyl)ethane (7a). To a stirred suspension of 6
(49g, 199mmol) and KBr (119g, 1.0mol) in DMF
(100mL), was added a solution of TBDMSCl (45g,
300mmol) and imidazole (41g, 603mmol) in DMF
(600mL). After 18h of stirring, the reaction mixture
was poured into 1L of water and extracted with i-
J = 6.1Hz, 3H), 2.55–2.74 (m, 2H), 2.87 (dd, J = 3.9,
4.6Hz, 1H), 2.98 (m, 1H), 3.44 (m, 1H), 4.55 (dd,
J = 7.9, 4.0Hz, 1H), 6.93–7.20 (m, 8H), 7.33 (d,
J = 8.0Hz, 1H), 7.47–7.51 (m, 2H), 7.81 (d, J = 5.0Hz,
1H), 10.81 (s, 1H); MS m/z: 456 (MH+); Anal. Calcd
for C23H25N3O3S2: C, 60.63; H, 5.53; N, 9.22. Found:
C, 60.65; H, 5.70; N, 8.82.

862
K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
5.1.5. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-1H-
indole (9). To a stirred solution of 8 (1.74g, 10mmol)
in CHCl3 (20mL), was added dropwise a solution di-
tert-butyl dicarbonate (2.6g, 12mmol) in CHCl3
(20mL) at room temperature. After stirring overnight,
the reaction mixture was concentrated under reduced
pressure. The residue was chromatographed on silica
gel, eluting with hexane/EtOAc = 3/1 to give 9 (2.8g,
quant.) as a colorless solid. 1H NMR (CDCl3): 1.13
(d, J = 6.6Hz, 3H), 1.43 (s, 9H), 2.85 (dd, J = 14.3,
6.9Hz, 1H), 2.97 (dd, J = 14.3, 5.1Hz, 1H), 4.03 (br,
1H), 4.44 (br, 1H), 7.02 (d, J = 2.2Hz, 1H), 7.09–7.22
(m, 2H), 7.36 (d, J = 7.9Hz, 1H), 7.64 (d, J = 7.7Hz,
1H), 8.07 (s, 1H).
ica gel, eluting with CHCl3/MeOH = 10/1 to give the title
compound 3 (0.15g, quant.) as an amorphous solid. H
1
NMR (DMSO-d6): 0.98 (d, J = 6.2Hz, 3H), 1.31 (t,
J = 7.2Hz, 3H), 2.60 (dd, J = 13.9, 7.9Hz, 1H), 2.67–
2.76 (m, 2H), 2.87 (dd, J = 13.9, 5.1Hz, 1H), 2.97 (m,
1H), 4.12 (q, J = 7.2Hz, 2H), 4.58 (dd, J = 7.7, 4.6Hz,
1H), 6.96–7.20 (m, 8H), 7.40 (d, J = 8.3Hz, 1H), 7.48
(dd, J = 3.8, 1.3Hz, 1H), 7.52 (d, J = 7.7Hz, 1H), 7.80
(dd, J = 5.1, 1.5Hz, 1H); MS m/z: 484 (MH+); Anal.
Calcd for C25H29N3O3S2: C, 62.08; H, 6.04; N, 8.69.
Found: C, 61.84; H, 6.13; N, 8.45.
5.1.8. (R)-7-Benzyloxy-3-[2-(tert-butoxycarbonylamino)-
propyl]-1H-indole (12). To a stirred solution of 1112
(112g, 399mmol) in EtOAc (800mL), was added drop-
5.1.6. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-1-ethyl-
1H-indole (10). To a stirred suspension of NaH (60% dis-
persion in mineral oil, 48mg, 1.2mmol) in THF (5mL),
was added a solution of 9 (0.27g, 1.0mmol) in THF
(5mL). After stirring at room temperature for 10min,
ethyl iodide (0.16mL, 2.0mmol) was added, and stirring
was continued for 2h. H2O was then added, and the mix-
ture was extracted with EtOAc. The organic layer was
washed with brine, dried over MgSO4, and concentrated
under vacuum. The residue was chromatographed on sil-
ica gel, eluting with hexane/EtOAc = 10/1 to give 10
(0.23g, 76%) as an amorphous solid. 1H NMR (CDCl3):
1.13 (d, J = 6.6Hz, 3H), 1.44 (s, 9H), 1.44 (t, J = 7.3Hz,
3H), 2.84 (dd, J = 14.3, 6.8Hz, 1H), 2.95 (dd, J = 14.3,
5.1Hz, 1H), 4.01 (br, 1H), 4.13 (q, J = 7.3Hz, 2H), 4.43
(br, 1H), 6.94 (s, 1H), 7.09 (m, 1H), 7.20 (m, 1H), 7.31
(d, J = 8.1Hz, 1H), 7.62 (d, J = 8.1Hz, 1H).
wise
a
solution di-tert-butyl dicarbonate (105g,
481mmol) in EtOAc (200mL) at 0ꢁC. After being stir-
red at room temperature for 3h, the reaction mixture
was concentrated under reduced pressure. To the result-
ing syrupy oil, hexane (400mL) was added, and the solid
formed was collected by filtration, dried under vacuum
to give 12 (146g, 96%). Mp: 94–95ꢁC; 1H NMR
(CDCl3): 1.11 (d, J = 6.6Hz, 3H), 1.43 (s, 9H), 2.83
(dd, J = 14.5, 6.7Hz, 1H), 2.94 (dd, J = 14.5, 5.1Hz,
1H), 4.00 (m, 1H), 4.44 (m, 1H), 5.18 (s, 2H), 6.71 (d,
J = 7.5Hz, 1H), 6.97 (d, J = 2.2Hz, 1H), 7.02 (dd,
J = 7.9, 7.5Hz, 1H), 7.26 (d, J = 7.9Hz, 1H), 7.24–7.51
(m, 5H), 8.30 (s, 1H).
5.1.9. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-hydr-
oxy-1H-indole (13). A mixture of 12 (60g, 158mmol),
5% Pd/C (3g), and MeOH (500mL) was hydrogenated
under H2 at room temperature for 3h. The catalyst
was then removed by filtration, and the filtrate was con-
centrated under reduced pressure. Hexane (250mL) was
added to the resulting syrupy oil, and the solid formed
was collected by filtration, dried under vacuum to give
5.1.7. 1-Ethyl-3-{(2R)-2-{{(2R)-2-hydroxy-2-{3-[(2-thio-
phenesulfonyl)amino]phenyl}ethyl}amino}propyl}-1H-
indole (3). Compound 10 (0.35g, 1.16mmol) was treated
with 4M HCl in EtOAc (10mL) at room temperature
for 2h, and the resulting mixture was concentrated un-
der vacuum. The residue was partitioned between
EtOAc and aqueous K2CO3, and the organic layer was
separated, washed with brine, dried over MgSO4, and
concentrated under vacuum to give (R)-3-(2-aminopro-
pyl)-1-ethyl-1H-indole (0.15g, 64%), which was used in
the next step without further purification.
1
13 (46g, quant.). Mp: 164–166ꢁC; H NMR (CD3OD):
1.08 (d, J = 6.6Hz, 3H), 1.40 (s, 9H), 2.73 (dd, J = 13.9,
7.5Hz, 1H), 2.88 (m, 1H), 3.89 (m, 1H), 6.49 (d,
J = 7.5Hz, 1H), 6.80 (t, J = 7.7Hz, 1H), 6.98 (s, 1H),
7.08 (d, J = 7.9Hz, 1H).
5.1.10. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-meth-
oxy-1H-indole (14). A mixture of 13 (1.1g, 3.5mmol),
methyl iodide (1.1mL, 17.7mmol), K2CO3 (0.59g,
4.3mmol), and acetone (40mL) was refluxed overnight.
The solvent was then removed under vacuum, and the
residue was treated with EtOAc, washed successively
with H2O and brine. The organic layer was dried over
MgSO4 and concentrated under vacuum. The residue
was chromatographed on silica gel, eluting with hex-
ane/EtOAc = 4/1 to give 14 (1.1g, 99%) as a syrupy oil.
1H NMR (CDCl3): 1.11 (d, J = 6.6Hz, 3H), 1.43 (s,
9H), 2.83 (dd, J = 14.3, 7.0Hz, 1H), 2.94 (dd, J = 14.3,
5.1Hz, 1H), 3.95 (s, 3H), 3.98 (m, 1H), 4.45 (br, 1H),
6.64 (d, J = 7.7Hz, 1H), 6.98 (d, J = 2.0Hz, 1H), 7.03
(t, J = 7.7Hz, 1H), 7.24 (d, J = 7.7Hz, 1H), 8.29 (s, 1H).
A mixture of the product obtained above, 4 (0.33g,
0.69mmol), i-Pr2NEt (0.18mL, 1.0mmol), KI (0.11g,
0.69mmol), and MeCN (8mL) was heated under reflux
for 48h. After the reaction mixture was concentrated
under reduced pressure, the residue was partitioned be-
tween EtOAc and H2O. The organic layer was sepa-
rated, washed with brine, dried over MgSO4, and
concentrated under vacuum. The residue was chromato-
graphed on silica gel, eluting with CHCl3/MeOH = 100/
1 to give desired coupling product (0.19g, 46% from 4).
The compound obtained above was dissolved in EtOH
(4mL), treated with 4M HCl in EtOAc (10mL) at room
temperature for 2h. After the solvent was evaporated to
dryness, the residue was partitioned between EtOAc
and aqueous K2CO3. The organic layer was separated,
washed with brine, dried over MgSO4, and concentrated
under vacuum. The residue was chromatographed on sil-
5.1.11. 3-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-7-methoxy-
1H-indole (2). This compound was prepared from 14
(5.36g, 17.6mmol) using the procedure described for

K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
863
the preparation of 3. Yield 2.9g (50% from 4). 1H NMR
(DMSO-d6): 0.95 (d, J = 6.0Hz, 3H), 2.52–2.74 (m, 3H),
2.83 (dd, J = 13.9, 5.0Hz, 1H), 2.94 (m, 1H), 3.89 (s,
3H), 4.54 (dd, J = 8.2, 4.0Hz, 1H), 6.62 (d, J = 7.5Hz,
1H), 6.89 (t, J = 7.8Hz, 1H), 6.96–7.19 (m, 8H), 7.47
(dd, J = 3.7, 1.3Hz, 1H), 7.80 (dd, J = 5.0, 1.3Hz,
1H), 10.89 (s, 1H); MS m/z: 486 (MH+); Anal. Calcd
for C24H27N3O4S2: C, 59.36; H, 5.60; N, 8.65. Found:
C, 59.07; H, 5.68; N, 8.28.
2H); MS m/z: 576 (MH+); Anal. Calcd for
C31H33N3O4S2: C, 64.67; H, 5.78; N, 7.30. Found: C,
64.36; H, 5.78; N, 7.23.
5.1.16. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-(2-
chloroethoxy)-1H-indole (17a). To a stirred solution of
13 (0.58g, 2.0mmol), 2-chloroethanol (0.27mL,
4.0mmol), and triphenylphosphine (1.05g, 4.0mmol)
in THF (15mL), was added dropwise DEAD
(0.63mL, 4.0mmol) at room temperature. After stirring
overnight, the reaction mixture was concentrated under
reduced pressure. The residue was chromatographed on
silica gel, eluting with hexane/EtOAc = 4/1 to give 17a
(0.59g, 84%) as a colorless oil. 1H NMR (CDCl3):
1.11 (d, J = 6.6Hz, 3H), 1.43 (s, 9H), 2.83 (dd,
J = 14.3, 7.0Hz, 1H), 2.95 (dd, J = 14.3, 5.3Hz, 1H),
3.88 (t, J = 5.7Hz, 2H), 4.00 (br, 1H), 4.40 (t,
J = 5.7Hz, 2H), 4.43 (br, 1H), 6.63 (d, J = 7.7Hz, 1H),
6.99–7.04 (m, 2H), 7.28 (d, J = 7.9Hz, 1H), 8.33 (s, 1H).
5.1.12. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-meth-
oxy-1-methyl-1H-indole (15a). To a stirred suspension of
NaH (60% dispersion in mineral oil, 88mg, 2.2mmol) in
DMF (5mL) was added a solution of 14 (0.56g,
1.84mmol) in DMF (5mL) at room temperature. After
stirring for 10min, methyl iodide (0.137mL, 2.2mmol)
was added, and stirring was continued for 2h. The reac-
tion was then quenched with water, and the mixture was
extracted with EtOAc. The organic layer was washed
with brine, dried MgSO4, and concentrated under vac-
uum. The residue was chromatographed on silica gel,
eluting with hexane/EtOAc = 10/1 to give 15a (0.52g,
89%) as a colorless oil. 1H NMR (CDCl3): 1.11 (d,
J = 6.6Hz, 3H), 1.44 (s, 9H), 2.78 (dd, J = 14.3,
6.8Hz, 1H), 2.90 (dd, J = 14.3, 5.3Hz, 1H), 3.91 (s,
3H), 3.97 (br, 1H), 4.00 (s, 3H), 4.42 (br, 1H), 6.60 (d,
J = 7.7Hz, 1H), 6.74 (s, 1H), 6.97 (m, 1H), 7.18 (dd,
J = 8.0, 0.8Hz, 1H).
5.1.17. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-(3-
chloropropoxy)-1H-indole (17b). This compound was
prepared from 13 (0.58g, 2.0mmol) and 3-chloro-1-pro-
panol (0.33mL, 4.0mmol) in a similar manner to that
described for 17a. Yield 0.60g (82%). 1H NMR
(CDCl3): 1.11 (d, J = 6.6Hz, 3H), 1.43 (s, 9H), 2.31
(m, 2H), 2.83 (dd, J = 14.1, 6.9Hz, 1H), 2.95 (dd,
J = 14.1, 5.1Hz, 1H), 3.78 (t, J = 6.4Hz, 2H), 4.00 (m,
1H), 4.29 (t, J = 5.9Hz, 2H), 4.44 (br, 1H), 6.66 (d,
J = 7.7Hz, 1H), 7.00 (s, 1H), 7.02 (t, J = 7.7Hz, 1H),
7.25 (d, J = 7.7Hz, 1H), 8.24 (s, 1H).
5.1.13. (R)-1-Benzyl-3-[2-(tert-butoxycarbonylamino)prop-
yl]-7-methoxy-1H-indole (15b). This compound was pre-
pared from 14 (0.51g, 1.68mmol) and benzyl bromide
(0.22mL, 1.85mmol) in a similar manner to that de-
scribed for 15a. Yield 0.68g (quant.). 1H NMR (CDCl3):
1.09 (d, J = 6.6Hz, 3H), 1.43 (s, 9H), 2.79 (dd, J = 14.3,
7.0Hz, 1H), 2.92 (dd, J = 14.3, 5.3Hz, 1H), 3.82 (s, 3H),
3.97 (m, 1H), 4.42 (br, 1H), 5.58 (s, 2H), 6.61(d,
J = 7.5Hz, 1H), 6.84 (s, 1H), 7.00 (t, J = 7.9Hz, 1H),
7.07–7.09 (m, 2H), 7.20–7.29 (m, 4H).
5.1.18. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-(4-
chlorobutoxy)-1H-indole (17c). This compound was pre-
pared from 13 (2.9g, 10mmol) and 4-chloro-1-butanol
(1.5mL, 15mmol) in a similar manner to that described
1
for 17a. Yield 1.45g (38%). H NMR (CDCl3): 1.12 (d,
J = 6.8Hz, 3H), 1.44 (s, 9H), 2.00–2.04 (m, 4H), 2.83
(dd, J = 14.2, 6.6Hz, 1H), 2.95 (dd, J = 14.2, 5.1Hz,
1H), 3.65 (m, 2H), 4.01 (m, 1H), 4.17 (m, 2H), 4.43
(br, 1H), 6.62 (d, J = 7.7Hz, 1H), 6.98–7.01 (m, 2H),
7.25 (m, 1H), 8.24 (s, 1H).
5.1.14. 3-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-7-methoxy-1-
methyl-1H-indole (16a). This compound was prepared
from 15a (0.83g, 2.6mmol) using the procedure de-
scribed for the preparation of 3. Yield 0.27g (49% from
4). 1H NMR (CDCl3): 1.11 (d, J = 6.2Hz, 3H), 2.57 (dd,
J = 12.1, 8.6Hz, 1H), 2.67–2.84 (m, 3H), 3.00 (m, 1H),
3.93 (s, 3H), 4.00 (s, 3H), 4.50 (dd, J = 8.6, 3.7Hz,
1H), 6.63 (d, J = 7.3Hz, 1H), 6.72 (s, 1H), 6.93–7.25
(m, 7H), 7.43–7.47 (m, 2H); MS m/z: 500 (MH+); Anal.
Calcd for C25H29N3O4S2: C, 60.10; H, 5.85; N, 8.41.
Found: C, 59.91; H, 5.91; N, 8.23.
5.1.19. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-(5-
chloropentyloxy)-1H-indole (17d). This compound was
prepared from 13 (1.74g, 6.0mmol) and 5-chloro-1-
pentanol (1.05mL, 9.0mmol) in a similar manner to that
described for 17a. Yield 2.0g (85%). 1H NMR (CDCl3):
1.11 (d, J = 6.6Hz, 3H), 1.44 (s, 9H), 1.68 (m, 2H), 1.81–
1.93 (m, 4H), 2.83 (dd, J = 14.3, 7.0Hz, 1H), 2.95 (dd,
J = 14.3, 5.1Hz, 1H), 3.58 (t, J = 6.6Hz, 2H), 4.00 (br,
1H), 4.14 (t, J = 6.2Hz, 2H), 4.44 (br, 1H), 6.62 (d,
J = 7.5Hz, 1H), 6.98–7.03 (m, 2H), 7.23 (d, J = 8.0Hz,
1H), 8.27 (s, 1H).
5.1.15. 1-Benzyl-3-{(2R)-2-{{(2R)-2-hydroxy-2-{3-[(2-thio-
phenesulfonyl)amino]phenyl}ethyl}amino}propyl}-7-meth-
oxy-1H-indole (16b). This compound was prepared from
15b (0.91g, 2.3mmol) using the procedure described for
the preparation of 3. Yield 0.27g (46% from 4). 1H
NMR (CDCl3): 1.10 (d, J = 6.2Hz, 3H), 2.55 (dd,
J = 12.1, 8.8Hz, 1H), 2.74–2.83 (m, 3H), 3.00 (m, 1H),
3.85 (s, 3H), 4.47 (dd, J = 8.3, 4.0Hz, 1H), 5.57 (d,
J = 15.7Hz, 1H), 5.61 (d, J = 15.7Hz, 1H), 6.65 (d,
J = 7.7Hz, 1H), 6.83–7.30 (m, 13H), 7.41–7.47 (m,
5.1.20. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-(3-
chloro-2,2-dimethylpropoxy)-1H-indole (17e). This com-
pound was prepared from 13 (1.16g, 4.0mmol) and
3-chloro-2,2-dimethyl-1-propanol (0.74g, 6.0mmol) by
the method described for 17a except that the reaction
was conducted under reflux temperature. Yield 1.26g
(80%). 1H NMR (CDCl3): 1.11 (d, J = 6.6Hz, 3H),
1.17 (s, 6H), 1.43 (s, 9H), 2.83 (dd, J = 14.3, 7.0Hz,

864
K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
1H), 2.95 (dd, J = 14.3, 5.1Hz, 1H), 3.62 (s, 2H), 3.93 (s,
2H), 4.00 (br, 1H), 4.45 (br, 1H), 6.66 (d, J = 7.7Hz,
1H), 6.99–7.04 (m, 2H), 7.25 (d, J = 8.0Hz, 1H), 8.26
(s, 1H).
(m, 4H), 2.80 (dd, J = 14.2, 6.6Hz, 1H), 2.91 (dd,
J = 14.2, 5.1Hz, 1H), 3.99 (m, 1H), 4.24 (t, J = 5.1Hz,
2H), 4.43 (br, 1H), 4.56 (m, 2H), 6.79–6.82 (m, 2H),
6.97 (t, J = 7.8Hz, 1H), 7.29 (dd, J = 7.8, 0.9Hz, 1H).
5.1.21. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-[2-
(2-chloroethoxy)ethoxy]-1H-indole (17f). This compound
was prepared from 13 (1.74g, 6.0mmol) and 2-(2-chlo-
roethoxy)ethanol (0.95mL, 9.0mmol) in a similar man-
ner to that described for 17a. Yield 1.5g (62%). 1H
NMR (CDCl3): 1.11 (d, J = 6.6Hz, 3H), 1.44 (s, 9H),
2.83 (dd, J = 14.3, 7.0Hz, 1H), 2.95 (dd, J = 14.3,
5.3Hz, 1H), 3.69 (t, J = 5.7Hz, 2H), 3.85 (t,
J = 5.7Hz, 2H), 3.92 (m, 2H), 4.00 (m, 1H), 4.31 (m,
2H), 4.44 (br, 1H), 6.67 (d, J = 7.5Hz, 1H), 6.98–7.03
(m, 2H), 7.27 (m, 1H), 8.58 (s, 1H).
5.1.26. (R)-7-[2-(tert-Butoxycarbonylamino)propyl]-3,4-
dihydro-3,3-dimethyl-2H-pyrrolo[1,2,3-ef]-1,5-benzoxaze-
pine (18e). This compound was prepared from 17e
(1.26g, 3.2mmol) by the method described for 18a ex-
cept that the reaction was conducted at 70ꢁC. Yield
1
0.52g (45%). H NMR (CDCl3): 1.14 (s, 6H), 1.14 (d,
J = 6.6Hz, 3H), 1.43 (s, 9H), 2.79 (dd, J = 14.5,
7.0Hz, 1H), 2.90 (dd, J = 14.5, 5.5Hz, 1H), 3.78 (s,
2H), 3.97 (s, 2H), 4.00 (m, 1H), 4.45 (br, 1H), 6.77 (d,
J = 7.5Hz, 1H), 6.82 (s, 1H), 6.99 (t, J = 7.7Hz, 1H),
7.24 (d, J = 7.9Hz, 1H).
5.1.22. (R)-6-[2-(tert-Butoxycarbonylamino)propyl]-2,3-di-
hydropyrrolo[1,2,3-de]-1,4-benzoxazine (18a). To a stir-
red solution of 17a (0.59g, 1.67mmol) in DMF (8mL),
was added NaH (60% dispersion in mineral oil, 0.13g,
3.36mmol) at 0ꢁC. After stirring at room temperature
for 2h, the reaction mixture was quenched with 1M
aqueous HCl, extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and concen-
trated under vacuum. The residue was chromatographed
on silica gel, eluting with hexane/EtOAc = 4/1 to give
5.1.27. (R)-9-[2-(tert-Butoxycarbonylamino)propyl]-2,3,5,
6-tetrahydropyrrolo[1,2,3-gh]-1,4,7-benzodioxazonine
(18f). A mixture of 17f (0.97g, 2.4mmol), KOH (0.26g,
4.0mmol), and DMSO (8mL) was heated at 80ꢁC. After
stirring for 66h, the reaction mixture was poured into
H2O, and extracted with EtOAc. The extract was
washed with brine, dried over MgSO4, and concentrated
under vacuum. The residue was chromatographed on
silica gel, eluting with hexane/EtOAc = 2/1 to give 18f
(0.28g, 32%) as an amorphous solid. 1H NMR
(CD3OD): 1.08 (d, J = 6.6Hz, 3H), 1.39 (s, 9H), 2.72
(dd, J = 14.3, 7.3Hz, 1H), 2.87 (m, 1H), 3.75 (m, 2H),
3.82 (m, 2H), 3.80-4.50 (m, 5H), 6.75 (dd, J = 7.7,
0.9Hz, 1H), 6.89 (s, 1H), 6.91 (t, J = 7.7Hz, 1H), 7.30
(d, J = 7.7Hz, 1H).
1
18a (0.43g, 81%) as a colorless oil. H NMR (CDCl3):
1.13 (d, J = 6.6Hz, 3H), 1.43 (s, 9H), 2.84 (dd,
J = 14.3, 6.8Hz, 1H), 2.92 (dd, J = 14.3, 5.1Hz, 1H),
4.00 (m, 1H), 4.22 (t, J = 4.7Hz, 2H), 4.46 (br, 1H),
4.51 (t, J = 4.7Hz, 2H), 6.64 (d, J = 7.5Hz, 1H), 6.89
(s, 1H), 6.97 (t, J = 7.8Hz, 1H), 7.18 (d, J = 8.0Hz, 1H).
5.1.28. 6-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-2,3-dihydro-
pyrrolo[1,2,3-de]-1,4-benzoxazine (19a). This compound
was prepared from 18a (0.8g, 2.5mmol) using the proce-
dure described for the preparation of 3. Yield 0.30g
(50% from 4). 1H NMR (DMSO-d6): 0.98 (d,
J = 6.2Hz, 3H), 2.59 (dd, J = 13.7, 7.5Hz, 1H), 2.68
(d, J = 6.2Hz, 2H), 2.82 (dd, J = 13.7, 5.2Hz, 1H),
2.94 (m, 1H), 4.20 (t, J = 4.6Hz, 2H), 4.44 (t,
J = 4.6Hz, 2H), 4.54 (t, J = 6.2Hz, 1H), 6.50 (d,
J = 7.5Hz, 1H), 6.84 (t, J = 7.8Hz, 1H), 6.98–7.08 (m,
5H), 7.13–7.21 (m, 2H), 7.47 (dd, J = 3.7, 1.3Hz, 1H),
7.80 (dd, J = 4.9, 1.3Hz, 1H); MS m/z: 498 (MH+);
Anal. Calcd for C25H27N3O4S2: C, 60.34; H, 5.47; N,
8.44. Found: C, 59.97; H, 5.60; N, 8.16.
5.1.23. (R)-7-[2-(tert-Butoxycarbonylamino)propyl]-3,4-di-
hydro-2H-pyrrolo[1,2,3-ef]-1,5-benzoxazepine (18b). This
compound was prepared from 17b (0.6g, 1.64mmol)
using the procedure described for the preparation of
18a. Yield 0.31g (57%). 1H NMR (CDCl3): 1.14 (d,
J = 6.6Hz, 3H), 1.43 (s, 9H), 2.35 (m, 2H), 2.81 (dd,
J = 14.3, 6.8Hz, 1H), 2.92 (dd, J = 14.3, 5.7Hz, 1H),
4.00 (m, 1H), 4.12 (m, 2H), 4.29 (t, J = 4.7Hz, 2H),
4.43 (br, 1H), 6.81 (d, J = 7.7Hz, 1H), 6.90 (s, 1H),
7.00 (t, J = 7.7Hz, 1H), 7.25 (d, J = 7.7Hz, 1H).
5.1.24. (R)-8-[2-(tert-Butoxycarbonylamino)propyl]-2,3,4,5-
tetrahydropyrrolo[1,2,3-fg]-1,6-benzoxazocine (18c).
This compound was prepared from 17c (1.45g,
3.8mmol) using the procedure described for the prepara-
tion of 18a. Yield 1.28g (99%). H NMR (CDCl3): 1.12
1
5.1.29. 7-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-3,4-dihydro-
2H-pyrrolo[1,2,3-ef ]-1,5-benzoxazepine (19b). This com-
pound was prepared from 18b (0.73g, 2.2mmol) using
the procedure described for the preparation of 3. Yield
0.30g (51% from 4). 1H NMR (DMSO-d6): 0.97 (d,
J = 6.2Hz, 3H), 2.24 (m, 2H), 2.56 (dd, J = 14.0,
7.3Hz, 1H), 2.67 (d, J = 6.2Hz, 2H), 2.79 (dd,
J = 14.0, 5.3Hz, 1H), 2.91 (m, 1H), 4.08 (t, J = 5.6Hz,
2H), 4.22 (dd, J = 5.7, 3.7Hz, 2H), 4.53 (t, J = 6.2Hz,
1H), 6.64 (d, J = 7.7Hz, 1H), 6.89 (t, J = 7.7Hz, 1H),
6.96–7.06 (m, 4H), 7.09–7.20 (m, 3H), 7.46 (dd,
J = 3.7, 1.3Hz, 1H), 7.79 (dd, J = 4.9, 1.3Hz, 1H); MS
m/z: 512 (MH+); Anal. Calcd for C26H29N3O4S2: C,
(d, J = 6.6Hz, 3H), 1.43 (s, 9H), 1.60 (m, 2H), 2.01 (m,
2H), 2.81 (dd, J = 14.3, 6.8Hz, 1H), 2.92 (dd, J = 14.3,
5.5Hz, 1H), 3.99 (m, 1H), 4.24 (t, J = 5.4Hz, 2H),
4.43 (br, 1H), 4.47 (t, J = 6.2Hz, 2H), 6.77 (s, 1H),
6.81 (d, J = 7.1Hz, 1H), 6.98 (t, J = 7.7Hz, 1H), 7.35
(d, J = 8.1Hz, 1H).
5.1.25. (R)-9-[2-(tert-Butoxycarbonylamino)propyl]-3,4,5,
6-tetrahydro-2H-pyrrolo[1,2,3-gh]-1,7-benzoxazonine
(18d). This compound was prepared from 17d (1.09g,
2.9mmol) using the procedure described for the prepara-
1
tion of 18a. Yield 0.19g (18%). H NMR (CDCl3): 1.12
(d, J = 6.6Hz, 3H), 1.43 (s, 9H), 1.49 (m, 2H), 1.74–1.83

K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
865
61.03; H, 5.71; N, 8.21. Found: C, 60.75; H, 5.89; N,
7.90.
2H), 3.84 (m, 2H), 3.92–4.42 (m, 4H), 4.60 (dd,
J = 7.9, 5.0Hz, 1H), 6.77 (d, J = 7.0Hz, 1H), 6.83 (s,
1H), 6.89–6.98 (m, 3H), 7.03 (m, 1H), 7.07–7.16 (m,
2H), 7.43 (dd, J = 3.8, 1.3Hz, 1H), 7.57 (dd, J = 5.1,
1.3Hz, 1H); MS m/z: 542 (MH+); Anal. Calcd for
C27H31N3O5S2, 1/2H2O: C, 58.89; H, 5.86; N, 7.63.
Found: C, 58.81; H, 5.85; N, 7.26.
5.1.30. 8-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-2,3,4,5-tetra-
hydropyrrolo[1,2,3-fg]-1,6-benzoxazocine (19c). This
compound was prepared from 18c (1.34g, 3.9mmol)
using the procedure described for the preparation of 3.
1
Yield 0.33g (59% from 4). H NMR (DMSO-d6): 0.97
5.1.34. (R)-3-[2-(tert-Butoxycarbonylamino)propyl]-7-(3-
chloro-2,2-difluoropropoxy)-1H-indole (20). A solution
of 13 (1.16g, 4.0mmol), 3-chloro-2,2-difluoro-1-propa-
nol (0.73mL, 8.0mmol), and CMBP (1.93g, 8.0mmol)
in toluene (30mL) was refluxed for 18h under Ar
atmosphere. The reaction mixture was concentrated
under reduced pressure, and the residue was chromato-
graphed on silica gel, eluting with hexane/EtOAc =
4/1 to give 20 (1.61g, quant.) as a colorless oil. 1H
NMR (CDCl3): 1.12 (d, J = 6.6Hz, 3H), 1.43 (s, 9H),
2.83 (dd, J = 14.3, 7.0Hz, 1H), 2.95 (dd, J = 14.3,
5.1Hz, 1H), 3.95 (t, J = 12.5Hz, 2H), 4.00 (m, 1H),
4.45 (br, 1H), 4.47 (t, J = 11.3Hz, 2H), 6.67 (d,
J = 7.5Hz, 1H), 7.01-7.06 (m, 2H), 7.33 (d, J = 8.0Hz,
1H), 8.31 (s, 1H).
(d, J = 6.2Hz, 3H), 1.43 (m, 2H), 1.99 (m, 2H), 2.56
(dd, J = 14.0, 7.5Hz, 1H), 2.65–2.68 (m, 2H), 2.80 (dd,
J = 14.0, 5.3Hz, 1H), 2.93 (m, 1H), 4.15 (t, J = 5.2
Hz, 2H), 4.39 (t, J = 6.1Hz, 2H), 4.53 (t, J = 6.2Hz,
1H), 6.70 (d, J = 7.6Hz, 1H), 6.89 (t, J = 7.7Hz, 1H),
6.95–7.01 (m, 3H), 7.05 (dd, J = 4.9, 3.9Hz, 1H), 7.13–
7.20 (m, 2H), 7.26 (d, J = 7.9Hz, 1H), 7.47 (dd,
J = 3.9, 1.3Hz, 1H), 7.80 (dd, J = 4.9, 1.3Hz, 1H); MS
m/z: 526 (MH+); Anal. Calcd for C27H31N3O4S2: C,
61.69; H, 5.94; N, 7.99. Found: C, 61.44; H, 6.07; N,
7.65.
5.1.31. 9-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-3,4,5,6-tetra-
hydro-2H-pyrrolo[1,2,3-gh]-1,7-benzoxazonine
(19d).
This compound was prepared from 18d (0.6g,
1.68mmol) using the procedure described for the prepa-
ration of 3. Yield 0.26g (42% from 4). 1H NMR
(DMSO-d6): 0.98 (d, J = 6.2Hz, 3H), 1.34 (m, 2H),
1.62–1.74 (m, 4H), 2.59 (dd, J = 13.8, 7.7Hz, 1H),
2.63–2.78 (m, 2H), 2.85 (dd, J = 13.8, 5.1Hz, 1H), 2.99
(m, 1H), 4.15 (m, 2H), 4.47 (m, 2H), 4.58 (m, 1H),
6.74 (d, J = 7.6Hz, 1H), 6.90 (t, J = 7.7Hz, 1H), 6.95–
7.08 (m, 4H), 7.14–7.23 (m, 3H), 7.50 (dd, J =
3.9, 1.3Hz, 1H), 7.82 (dd, J = 4.9, 1.3Hz, 1H); MS m/
z: 540 (MH+); Anal. Calcd for C28H33N3O4S2: C,
62.31; H, 6.16; N, 7.79. Found: C, 62.02; H, 6.27; N,
7.68.
5.1.35. (R)-7-[2-(tert-Butoxycarbonylamino)propyl]-3,3-di-
fluoro-3,4-dihydro-2H-pyrrolo[1,2,3-ef]-1,5-benzoxazepine
(21). This compound was prepared from 20 (0.8g,
2.0mmol) using the procedure described for the prepara-
1
tion of 18f. Yield 0.38g (52%). H NMR (CDCl3): 1.13
(d, J = 6.6Hz, 3H), 1.43 (s, 9H), 2.78 (dd, J = 14.5,
6.8Hz, 1H), 2.88 (m, 1H), 4.00 (m, 1H), 4.43 (br, 1H),
4.52 (t, J = 12.7Hz, 2H), 4.53 (t, J = 12.6Hz, 2H),
6.80–6.84 (m, 2H), 7.00 (t, J = 7.9Hz, 1H), 7.33 (d,
J = 7.9Hz, 1H).
5.1.36. 7-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-3,3-difluoro-
3,4-dihydro-2H-pyrrolo[1,2,3-ef]-1,5-benzoxazepine (22).
This compound was prepared from 21 (0.66g, 1.8mmol)
using the procedure described for the preparation of 3.
5.1.32. 7-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-3,4-dihydro-
3,3-dimethyl-2H-pyrrolo[1,2,3-ef]-1,5-benzoxazepine (19e).
This compound was prepared from 18e (0.52g,
1.45mmol) using the procedure described for the prepa-
ration of 3. Yield 0.19g (37% from 4). 1H NMR
(DMSO-d6): 0.99 (d, J = 6.0Hz, 3H), 1.05 (s, 6H), 2.57
(dd, J = 14.2, 7.6Hz, 1H), 2.70 (d, J = 6.2Hz, 2H),
2.81 (dd, J = 14.2, 5.3Hz, 1H), 2.98 (m, 1H), 3.80 (s,
2H), 3.94 (s, 2H), 4.57 (t, J = 6.2Hz, 1H), 6.63 (d,
J = 6.9Hz, 1H), 6.88 (t, J = 7.8Hz, 1H), 6.99–7.03 (m,
3H), 7.05 (dd, J = 4.9, 3.9Hz, 1H), 7.12–7.21 (m, 3H),
7.48 (dd, J = 3.7, 1.4Hz, 1H), 7.81 (dd, J = 5.0, 1.4Hz,
1H); MS m/z: 540 (MH+); Anal. Calcd for
C28H33N3O4S2: C, 62.31; H, 6.16; N, 7.79. Found: C,
62.01; H, 6.30; N, 7.49.
1
Yield 0.11g (20% from 4). H NMR (DMSO-d6): 0.97
(d, J = 6.2Hz, 3H), 2.55 (dd, J = 13.9, 7.7Hz, 1H),
2.61–2.73 (m, 2H), 2.80 (dd, J = 13.9, 5.1Hz, 1H), 2.94
(m, 1H), 4.54 (m, 1H), 4.58 (t, J = 12.6Hz, 2H), 4.70
(t, J = 13.1Hz, 2H), 6.76 (d, J = 7.6Hz, 1H), 6.92–
7.02 (m, 3H), 7.06 (dd, J = 5.0, 3.7Hz, 1H), 7.11–7.21
(m, 3H), 7.26 (d, J = 7.7Hz, 1H), 7.48 (dd, J = 3.9,
1.4Hz, 1H), 7.82 (dd, J = 5.0, 1.3Hz, 1H); MS m/z:
548 (MH+); Anal. Calcd for C26H27F2N3O4S2: C,
57.02; H, 4.97; N, 7.67. Found: C, 57.20; H, 5.34; N,
7.37.
5.1.37. (R)-7-Allyloxy-3-[2-(tert-butoxycarbonylamino)-
propyl-1H-]indole (23). This compound was prepared
from 13 (1.45g, 5.0mmol) and allyl iodide (0.91mL,
10mmol) in a similar manner to that described for 14.
Yield 1.36g (82%). 1H NMR (CDCl3): 1.12 (d,
J = 6.6Hz, 3H), 1.43 (s, 9H), 2.83 (dd, J = 14.3,
6.9Hz, 1H), 2.95 (dd, J = 14.3, 5.1Hz, 1H), 4.01 (br,
1H), 4.44 (br, 1H), 4.69 (dt, J = 5.3, 1.5Hz, 2H), 5.31
(m, 1H), 5.45 (m, 1H), 6.12 (m, 1H), 6.64 (d,
J = 7.5Hz, 1H), 6.98–7.03 (m, 2H), 7.24 (d, J = 8.2Hz,
1H), 8.30 (s, 1H).
5.1.33. 9-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-2,3,5,6-tetra-
hydropyrrolo[1,2,3-gh]-1,4,7-benzodioxazonine
(19f).
This compound was prepared from 18f (0.27g,
0.75mmol) using the procedure described for the prepa-
ration of 3. Yield 0.08g (25% from 4). 1H NMR
(CD3OD): 1.11 (d, J = 6.0Hz, 3H), 2.68 (dd, J = 12.1,
4.9Hz, 1H), 2.74–2.86 (m, 3H), 3.02 (m, 1H), 3.74 (m,

866
K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
5.1.38. (R)-1-Allyl-7-allyoxy-3-[2-(tert-butoxycarbonyl-
amino)propyl]indole (24). This compound was prepared
from 23 (6.1g, 18.4mmol) and allyl bromide (1.88mL,
22mmol) in a similar manner to that described for
15a. Yield 6.6g (97%). 1H NMR (CDCl3): 1.11 (d,
J = 6.6Hz, 3H), 1.43 (s, 9H), 2.79 (dd, J = 14.5,
7.0Hz, 1H), 2.91 (dd, J = 14.5, 5.3Hz, 1H), 3.98 (m,
1H), 4.41 (m, 1H), 4.64 (dd, J = 3.8, 1.4Hz, 2H), 4.91–
5.09 (m, 4H), 5.29 (dd, J = 10.4, 1.5Hz, 1H), 5.43 (dd,
J = 17.2, 1.7Hz, 1H), 5.97–6.17 (m, 2H), 6.62 (d,
J = 7.7Hz, 1H), 6.82 (s, 1H), 6.96 (t, J = 7.7Hz, 1H),
7.21 (d, J = 7.9Hz, 1H).
5.1.42. 6-[(2R)-2-(tert-Butoxycarbonylamino)propyl]-2,3-
dihydro-(2RS)-2-methylpyrrolo[1,2,3-de]-1,4-benzoxazine
(28). A suspension of 27 (1.5g, 3.4mmol) and 20%
Pd(OH)2 (0.3g) in EtOH (20mL) was hydrogenated un-
der H2 for 2h. The catalyst was then removed by filtra-
tion, and the filtrate was evaporated under reduced
pressure. The residue was chromatographed on silica
gel, eluting with hexane/EtOAc = 2/1 to give the corre-
sponding 7-hydroxyindole derivative (0.99g, 83%).
To a stirred solution of the 7-hydroxyindole derivative
obtained above and triphenylphosphine (0.97g,
3.7mmol) in THF (20mL), was added dropwise DEAD
(0.58mL, 3.7mmol) at room temperature. After stirring
overnight, the reaction mixture was concentrated under
reduced pressure. The residue was chromatographed on
silica gel, eluting with hexane/EtOAc = 3/1 to give the
5.1.39. (R)-8-[2-(tert-Butoxycarbonylamino)propyl]-2,5-di-
hydropyrrolo[1,2,3-fg]-1,6-benzoxazocine (25). To a stir-
red solution of 24 (6.6g, 17.8mmol) in toluene (1L),
was added Cl2(PCy3)2Ru@CHPh (2.2g, 2.67mmol),
and the mixture was heated under reflux for 4h. The
reaction mixture was concentrated under vacuum, and
the residue was chromatographed on silica gel, eluting
with hexane/EtOAc = 4/1 to give 25 (3.9g, 64%) as an
amorphous solid. 1H NMR (CDCl3): 1.12 (d,
J = 6.6Hz, 3H), 1.43 (s, 9H), 2.80 (dd, J = 14.1,
6.6Hz, 1H), 2.90 (dd, J = 14.1, 5.3Hz, 1H), 3.97 (m,
1H), 4.42 (br, 1H), 4.85 (m, 2H), 4.90 (d, J = 8.3Hz,
2H), 5.64 (m, 1H), 6.03 (m, 1H), 6.83 (s, 1H), 6.88
(dd, J = 7.5, 0.8Hz, 1H), 7.00 (t, J = 7.8Hz, 1H), 7.38
(dd, J = 8.0, 0.8Hz, 1H).
1
title compound 28 (0.77g, 82%) as a colorless oil. H
NMR (CDCl3): 1.13 (d, J = 6.6Hz, 3H), 1.43 (s, 9H),
1.55 (d, J = 6.4Hz, 3H), 2.80–2.96 (m, 2H), 3.88 (dd,
J = 12.1, 9.2Hz, 1H), 4.00 (m, 1H), 4.18 (dd, J = 12.1,
3.0Hz, 1H), 4.35–4.56 (m, 2H), 6.63 (d, J = 7.5Hz,
1H), 6.86 (s, 1H), 6.96 (dd, J = 8.1, 7.5Hz, 1H), 7.17
(d, J = 8.1Hz, 1H).
5.1.43. 6-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}2,3-dihydro-
(2RS)-2-methylpyrrolo[1,2,3-de]-1,4-benzoxazine
(29).
This compound was prepared from 28 (0.77g, 2.3mmol)
using the procedure described for the preparation of 3.
Yield 0.29g (55% from 4). H NMR (DMSO-d6): 0.97
5.1.40. 8-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-2,5-dihydro-
pyrrolo[1,2,3-fg]-1,6-benzoxazocine (26). This compound
was prepared from 25 (0.6g, 1.75mmol) using the proce-
dure described for the preparation of 3. Yield 0.16g
(23% from 4). 1H NMR (DMSO-d6): 0.97 (d,
J = 6.2Hz, 3H), 2.56 (dd, J = 13.9, 7.8Hz, 1H), 2.64–
2.76 (m, 2H), 2.82 (dd, J = 13.9, 4.6Hz, 1H), 2.96 (m,
1H), 4.57 (dd, J = 7.3, 5.0Hz, 1H), 4.78 (s, 2H), 4.85
(d, J = 8.0Hz, 2H), 5.67 (m, 1H), 5.99 (m, 1H), 6.80
(d, J = 7.3Hz, 1H), 6.93 (t, J = 7.7Hz, 1H), 6.99–7.07
(m, 4H), 7.14–7.21 (m, 2H), 7.29 (d, J = 7.7Hz, 1H),
7.49 (dd, J = 3.7, 1.3Hz, 1H), 7.82 (dd, J = 5.0, 1.3Hz,
1H); MS m/z: 524 (MH+); Anal. Calcd for
C27H29N3O4S2, 1/4H2O: C, 61.40; H, 5.63; N, 7.96.
Found: C, 61.39; H, 5.65; N, 7.73.
1
(d, J = 6.2Hz, 3H), 1.45 (d, J = 6.2Hz, 3H), 2.58 (m,
1H), 2.68 (d, J = 6.4Hz, 2H), 2.82 (m, 1H), 2.95 (m,
1H), 3.80 (m, 1H), 4.32 (m, 1H), 4.43 (m, 1H), 4.54
(m, 1H), 6.50 (m, 1H), 6.84 (m, 1H), 6.97–7.07 (m,
5H), 7.13–7.20 (m, 2H), 7.47 (m, 1H), 7.81 (m, 1H);
MS m/z: 512 (MH+); Anal. Calcd for C26H29N3O4S2:
C, 61.03; H, 5.71; N, 8.21. Found: C, 60.70; H, 5.91;
N, 7.87.
5.1.44. 7-Benzyloxy-3-[(2R)-2-(tert-butoxycarbonylamino)-
propyl]-1-[(2RS)-2-methoxycarbonylethyl]-1H-indole
(30). To a stirred mixture of 12 (1.52g, 4.0mmol), NaH
(60% dispersion in mineral oil, 0.19g, 4.8mmol), and
DMF (10mL), was added dropwise methyl 2-bromo-
propionate (0.54mL, 4.8mmol) at room temperature.
After stirring for 30min, the reaction mixture was
quenched with aqueous 1M HCl, extracted with EtOAc.
The organic layer was washed with brine, dried over
MgSO4, and concentrated under vacuum. The residue
was chromatographed on silica gel, eluting with hex-
ane/EtOAc = 4/1 to give 30 (1.68g, 90%) as a colorless
5.1.41. 7-Benzyloxy-3-[(2R)-2-(tert-butoxycarbonylamino)-
propyl]-1-[(2RS)-2-hydroxypropyl]-1H-indole (27).
A
mixture of 12 (1.52g, 4.0mmol), t-BuOK (0.54g,
4.8mmol), propylene oxide (10mL), and THF (10mL)
was stirred at room temperature overnight. The reaction
mixture was then concentrated under reduced pressure,
and the residue was partitioned between EtOAc and
aqueous 1M HCl. The organic layer was separated,
washed with brine, dried over MgSO4, and concentrated
under vacuum. The residue was chromatographed on
silica gel, eluting with hexane/EtOAc = 2/1 to give 27
(0.77g, 44%) as an amorphous solid. 1H NMR (CDCl3):
0.95 (d, J = 6.2Hz, 3H), 1.10–1.16 (m, 3H), 1.33–
1.44 (m, 9H), 2.76–2.84 (m, 2H), 3.85–4.12 (m, 3H),
4.38–4.48 (m, 2H), 5.09–5.17 (m, 2H), 6.72 (m, 1H),
6.86 (m, 1H), 7.00 (m, 1H), 7.23 (m, 1H), 7.35–7.47
(m, 5H).
1
oil. H NMR (CDCl3): 1.11 (d, J = 6.6Hz, 3H), 1.43
(s, 9H), 1.72 (d, J = 7.3Hz, 3H), 2.76–2.96 (m, 2H),
3.57 (s, 3H), 4.00 (br, 1H), 4.49 (br, 1H), 5.17 (s, 2H),
5.93 (q, J = 7.3Hz, 1H), 6.69 (d, J = 7.7Hz, 1H), 6.95–
7.01 (m, 2H), 7.23 (d, J = 8.1Hz, 1H), 7.34–7.44 (m,
5H).
5.1.45. 7-Benzyloxy-3-[(2R)-2-(tert-butoxycarbonylamino)-
propyl]-1-{[(1RS)-1-hydroxymethyl]ethyl}-1H-indole
(31). A mixture of 30 (1.68g, 3.6mmol), aqueous 1M
NaOH (15mL), and MeOH (15mL) was stirred at room

K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
867
temperature for 18h. After evaporation of MeOH under
5.2. Pharmacological studies
reduced pressure, the residue was partitioned between
EtOAc and aqueous 1M HCl. The organic layer was
separated, washed with brine, dried over MgSO4, and
concentrated under vacuum. The residue was chromato-
graphed on silica gel, eluting with hexane/EtOAc = 4/1
to 2/1 to give the corresponding carboxylic acid (1.5g,
92%) as an amorphous solid.
5.2.1. cAMP accumulation studies. Each subtype of hu-
man b1-, b2-, and b3-ARs was expressed on Chinese
hamster ovary (CHO) cells. These cells were cultured
with DulbeccoÕs MEM containing 10% fetal bovine ser-
um, 100lM nonessential amino acids, and 200lg/mL
G-418. On the day of assay, cells were harvested and
resuspended in HankÕs balanced salt solution containing
20mM HEPES, 0.5mM 3-isobutyl-methylxanthine, and
1mM L(+)-ascorbic acid. The cell suspension was mixed
with test compounds and allowed to stand at 37ꢁC.
After 30min of incubation, the reaction was terminated
by boiling for 5min. After centrifugation of the reaction
mixture at 900g for 5min at room temperature, cAMP
concentrations in the supernatant were determined with
a cAMP enzyme immunoassay (EIA) system (Amer-
sham Bioscience).
To a stirred solution of the carboxylic acid obtained
above in THF (10mL), was added dropwise BH3–THF
complex (1M in THF, 10mL, 10mmol) at room temper-
ature. After 2h stirring, the reaction mixture was
quenched with saturated NH4Cl solution, and extracted
with EtOAc. The organic layer was washed with brine,
dried over MgSO4, and concentrated under vacuum.
The residue was chromatographed on silica gel, eluting
with hexane/EtOAc = 4/1 to give 31 (0.74g, 51%) as a
1
syrupy oil. H NMR (CDCl3): 1.11–1.17 (m, 3H), 1.28–
1.47 (m, 9H), 1.46–1.49 (m, 3H), 2.78–2.90 (m, 2H),
3.74 (m, 2H), 3.97 (m, 1H), 4.37 (br, 1H), 5.19 (s, 2H),
5.41 (m, 1H), 6.69–6.74 (m, 1H), 6.95–7.08 (m, 2H),
7.21 (d, J = 8.0Hz, 1H), 7.35–7.48 (m, 5H).
5.2.2. Radioligand binding studies. Three CHO cell lines,
expressing each subtype of human b1-, b2-, and b3-ARs,
were cultured with the above-mentioned medium. Pre-
confluent cells were washed with ice-cold PBS, and har-
vested with the ice-cold lysis buffer (10mM Tris, 2mM
EDTA, 5lg/mL leupeptin, 5lg/mL benzamidine, and
10lg/mL soybean trypsin inhibitor, pH7.4 at 4ꢁC).
Harvested membranes were washed twice in lysis buffer
by centrifugation at 57,000g for 20min and pellets were
resuspended in the assay buffer (50mM Tris, 2mM
EDTA, and 12.5mM MgCl2, pH7.4 at 37ꢁC). Cell
membranes were incubated for 30min at 37ꢁC with vari-
ous concentrations of test compounds in the presence of
5.1.46. 6-[(2R)-2-(tert-Butoxycarbonylamino)propyl]-2,3-
dihydro-(3RS)-3-methylpyrrolo[1,2,3-de]-1,4-benzoxazine
(32). A suspension of 31 (0.74g, 1.7mmol) and 20%
Pd(OH)2 (0.2g) in EtOH (15mL) was hydrogenated
under H2 for 3h. The catalyst was then removed by
filtration, and the filtrate was concentrated under
reduced pressure. The residue was chromatographed
on silica gel, eluting with hexane/EtOAc = 2/1 to give
the corresponding 7-hydroxyindole derivative (0.53g,
89%) as an amorphous solid.
[
125I]-iodocyanopindolol (PerkinElmer) (1nM for b3-,
0.1nM for b1-, and 0.04nM for b2-ARs). Nonspecific
binding was determined in the presence of 10lM (ꢀ)-
propranolol. The reaction was terminated by rapid fil-
tration through GF/C filters (Whatman). The radioacti-
vity of the ligand trapped on the filters was measured
using a gamma counter (ALOKA). Ki values were calcu-
lated from IC50 values according to the method of
Cheng and Prusoff.19
To a stirred solution of the 7-hydroxyindole derivative
obtained above and triphenylphosphine (0.52g,
2.0mmol) in THF (20mL) was added dropwise DEAD
(0.31mL, 2.0mmol) at room temperature. After stirring
overnight, the reaction mixture was concentrated under
reduced pressure. The residue was chromatographed on
silica gel, eluting with hexane/EtOAc = 3/1 to give the
1
title compound 32 (0.42g, 84%) as a colorless solid. H
5.3. Caco-2 monolayer permeability studies
NMR (CDCl3): 1.13–1.15 (m, 3H), 1.43 (s, 9H), 1.51
(d, J = 6.4Hz, 3H), 2.80–2.98 (m, 2H), 4.01 (m, 1H),
4.09 (dd, J = 11.0, 7.1Hz, 1H), 4.30–4.49 (m, 3H), 6.64
(d, J = 7.3Hz, 1H), 6.94–7.00 (m, 2H), 7.18 (d,
J = 8.0Hz, 1H).
5.3.1. Materials. The Caco-2 cell line was obtained from
the American Type Culture Collection at passage 38.
DulbeccoÕs modified Eagle medium (DMEM) was pur-
chased from Sigma. Nonessential amino acid (NEAA),
fetal bovine serum (FBS), trypsin, EDTA, and penicil-
lin–streptomycin (5000IU/mL and 5000lg/mL) were
purchased from ICN Biomedical, Inc. HankÕs balanced
salt solution (HBSS) was purchased from Gibco Labo-
ratories. HEPES was purchased from Nakarai Tesque.
D(+)-glucose, bovine serum albumin (BSA), and 2-Mor-
pholinoethanesrufonic acid monohydrate (MES) were
purchased from Wako.
5.1.47. 6-{(2R)-2-{{(2R)-2-Hydroxy-2-{3-[(2-thiophene-
sulfonyl)amino]phenyl}ethyl}amino}propyl}-2,3-dihydro-
(3RS)-3-methylpyrrolo[1,2,3-de]-1,4-benzoxazine
(33).
This compound was prepared from 32 (0.61g, 1.8mmol)
using the procedure described for the preparation of 3.
Yield 0.36g (66% from 4). H NMR (DMSO-d6): 0.98
1
(d, J = 5.9Hz, 3H), 1.41 (d, J = 6.4Hz, 3H), 2.59 (m,
1H), 2.68 (t, J = 7.1Hz, 2H), 2.83 (m, 1H), 2.96 (m,
1H), 4.05 (m, 1H), 4.35 (m, 1H), 4.46 (dd, J = 11.2,
3.1Hz, 1H), 4.54 (m, 1H), 6.51 (d, J = 7.5Hz, 1H),
6.84 (t, J = 7.8Hz, 1H), 6.98–7.08 (m, 4H), 7.14–7.20
(m, 3H), 7.47 (m, 1H), 7.81 (m, 1H); MS m/z: 512
(MH+); Anal. Calcd for C26H29N3O4S2: C, 61.03; H,
5.71; N, 8.21. Found: C, 60.66; H, 5.91; N, 7.89.
5.3.2. Preparation of Caco-2 monolayers. Caco-2 cells
were grown in DMEM supplemented with 10% FBS
and 1% NEAA at 37ꢁC in dishes (IWAKI, Japan) in a
humidified air–5% CO2 atmosphere. The cells were
harvested with trypsin–EDTA, and suspended in culture
medium containing 100IU/mL penicillin and 100lg/mL

868
K. Mizuno et al. / Bioorg. Med. Chem. 13 (2005) 855–868
Nardone, N. A.; Tess, D. A.; DaSilva-Jardine, P. Bioorg.
Med. Chem. Lett. 2004, 14, 3235–3240.
4. Sher, P. M.; Mathur, A.; Fisher, L. G.; Wu, G.; Skwish,
S.; Michel, I. M.; Seiler, S. M.; Dickinson, K. E. J. Bioorg.
Med. Chem. Lett. 1997, 7, 1583–1588.
streptomycin. This suspension was seeded onto polycar-
bonate filters (3lm pores, 0.31cm2 growth area) inside
HTS multiwell insert well plate (Nippon Becton
Dikinson, Japan) at a density of 1 105 cells/cm2. The
*
culture medium (0.3mL in the insert and 40mL in the
feeder tray) was replaced every 48h from 7days to
12days after seeding and every 24h thereafter.
5. Shih, T. L.; Candelore, M. R.; Cascieri, M. A.; Chiu, S.
H., Jr.; Colwell, L. F., Jr.; Deng, L.; Feeney, W. P.;
Forrest, M. J.; Hom, G. J.; MacIntyre, D. E.; Miller, R.
R.; Stearns, R. A.; Strader, C. D.; Tota, L.; Wyvratt, M.
J.; Fisher, M. H.; Weber, A. E. Bioorg. Med. Chem. Lett.
1999, 9, 1251–1254.
6. Uehling, D. E.; Donaldson, K. H.; Deaton, D. N.;
Hyman, C. E.; Sugg, E. E.; Barrett, D. G.; Hughes, R.
G.; Reitter, B.; Adkison, K. K.; Lancaster, M. E.; Lee, F.;
Hart, R.; Paulik, M. A.; Sherman, B. W.; True, T.;
Cowan, C. J. Med. Chem. 2002, 45, 567–583.
7. van de Waterbeemd, H.; Smith, D. A.; Beaumont, K.;
Walker, D. K. J. Med. Chem. 2001, 44, 1313–1333.
8. Mizuno, K.; Sawa, M.; Harada, H.; Tateishi, H.; Oue, M.;
Tsujiuchi, H.; Furutani, Y.; Kato, S. Bioorg. Med. Chem.
Lett., see doi:10.1016/j.bmc.2004.10.032.
9. Corey, E. J.; Helal, C. J. Angew. Chem., Int. Ed. 1998, 37,
1986–2012. A review.
10. Hu, B.; Ellingboea, J.; Gunawana, I.; Hanb, S.; Largisb,
E.; Lia, Z.; Malamasa, M.; Mulveyb, R.; Oliphanta, A.;
Suma, F. W.; Tillettb, J.; Wong, V. Bioorg. Med. Chem.
Lett. 2001, 11, 757–760.
11. Repk, D. B.; Ferguson, W. J. J. Heterocycl. Chem. 1976,
13, 775–778.
12. Fujii, A.; Fujima, Y.; Harada, H.; Ikunaka, M.; Inoue, T.;
Kato, S.; Matsuyama, K. Tetrahedron: Asymmetry 2001,
12, 3235–3240.
5.3.3. Drug transport study. Apical to Basal permeability
of the drugs was measured using Caco-2 monolayers.
The transport medium used for the drug transport study
was HBSS supplemented with 25mM glucose after
adjusting the pH to 6.0 (Apical side) or 7.4 (Basal side)
with 25mM MES or 10mM HEPES, respectively. After
20min of incubation of both sides of the monolayers
with the drug-free transport medium, each side of the
medium was replaced with a drug-containing transport
medium (apical side) or fresh transport medium with
4.5% BSA or without 4.5% BSA (basal side). Thereafter,
the Caco-2 monolayers were incubated for 2h at 37ꢁC.
Drug concentration in the basal side medium was meas-
ured using a reversed-phase HPLC system (LC-2010C,
Shimadzu, Japan). Permeability (apparent permeability
coefficient, Papp (cm/s)) of each drug was calculated
according to the following equation:
Papp ¼ dM=dt ꢃ 1=AC0
where dM/dt is the rate of the drug in amount trans-
ported to the basal side, C0 is the initial drug concentra-
tion on the apical side, and A is the surface area of the
monolayer.
13. Heaney, H.; Ley, S. V. J. Chem. Soc., Perkin Trans. 1
1973, 499–500.
14. Guiot, J.; Ameduri, B.; Boutevin, B.; Fruchier, A. New J.
Chem. 2002, 26, 1768–1773.
15. Tsunoda, T.; Ozaki, F.; Ito, S. Tetrahedron Lett. 1994, 35,
5081–5082.
References and notes
16. Schwab, P.; Grubbs, R. H.; Ziller, J. W. J. Am. Chem. Soc.
1996, 118, 100–110.
1. (a) de Souza, C. J.; Burkey, B. F. Curr. Pharm. Des. 2001,
7, 1433–1449; (b) Dow, R. L. Exp. Opin. Invest. Drugs
1997, 6, 1811–1825.
2. (a) Igawa, Y.; Yamazaki, Y.; Takeda, H.; Hayakawa, K.;
Akahane, M.; Ajisawa, Y.; Yoneyama, T.; Nishizawa, O.;
Andersson, K. E. Br. J. Pharmacol. 1999, 126, 819–825;
(b) Igawa, Y.; Yamazaki, Y.; Takeda, H.; Akahane, M.;
Ajisawa, Y.; Yoneyama, T.; Nishizawa, O. Acta Physiol.
Scand. 1998, 164, 117–118.
3. (a) Washburn, W. N.; Sun, C. Q.; Bisacchi, G.; Wu, G.;
Cheng, P. T.; Sher, P. M.; Ryono, D.; Gavai, A. V.; Poss,
K.; Girotra, R. N.; McCann, P. J.; Mikkilineni, A. B.;
Dejneka, T. C.; Wang, T. C.; Merchant, Z.; Morella, M.;
Arbeeny, C. M.; Harper, T. W.; Slusarchyk, D. A.;
Skwish, S.; Russell, A. D.; Allen, G. T.; Tesfamariam, B.;
Frohlich, B. H.; Abboa-Offei, B. E.; Cap, M.; Waldron, T.
L.; George, R. J.; Young, D.; Dickinson, K. E.; Seymour,
A. A. Bioorg. Med. Chem. Lett. 2004, 14, 3525–3529; (b)
Dow, R. L.; Paight, E. S.; Schneider, S. R.; Hadcock, J.
R.; Hargrove, D. M.; Martin, K. A.; Maurer, T. S.;
17. In a previous report,20 we used CHO cells expressing a
high level of the b3-AR to measure compound activity,
that is, the receptor densities were 150,000 receptors/cell
(b3-AR), 12,000 receptors/cell (b1-AR), and 30,000 recep-
tors/cell (b2-AR). In this study, to better evaluate subtype
selectivity, we used CHO cells expressing a low density of
the b3-AR (13,000 receptors/cell) and high densities of the
b1- and b2-ARs (320,000 and 600,000 receptors/cell,
respectively).
18. Mitsuya, M.; Kobayashi, K.; Kawakami, K.; Satoh, A.;
Ogino, Y.; Kakikawa, T.; Ohtake, N.; Kimura, T.; Hirose,
H.; Sato, N.; Numazawa, T.; Hasegawa, T.; Noguchi, K.;
Mase, T. J. Med. Chem. 2000, 43, 5017–5029.
19. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099–3108.
20. Harada, H.; Hirokawa, Y.; Suzuki, K.; Hiyama, Y.;
Oue, M.; Kawashima, H.; Yoshida, N.; Furutani, Y.;
Kato, S. Bioorg. Med. Chem. Lett. 2003, 13, 1301–
1305.