Design, synthesis, and BK channel-opening activity of hexahydrodibenzazepinone derivatives

Article abstract of DOI:10.1016/j.bmc.2006.07.042

In order to explore new scaffolds for large-conductance Ca²⁺-activated K⁺ channel (BK channel) openers, we carried out molecular design and synthesis on the basis of the following two concepts: (1) introduction of a heteroatom into t

Full text of DOI:10.1016/j.bmc.2006.07.042

Bioorganic & Medicinal Chemistry 14 (2006) 8014–8031
Design, synthesis, and BK channel-opening activity
of hexahydrodibenzazepinone derivatives
Toshihiko Tashima,a Yoshimi Toriumi,a Yumi Mochizuki,a Taro Nonomura,a
Satoru Nagaoka,a Katsuo Furukawa,b Hiromichi Tsuru,b
Satomi Adachi-Akahaneb and Tomohiko Ohwadaa,*
aGraduate School of Pharmaceutical Sciences, The University of Tokyo, Laboratory of Organic and Medicinal Chemistry,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bDepartment of Pharmacology, Faculty of Medicine, School of Medicine, Toho University, 5-21-16, Omori-Nishi, Ota-ku,
Tokyo 143-8540, Japan
Received 19 June 2006; revised 21 July 2006; accepted 22 July 2006
Available online 10 August 2006
Abstract—In order to explore new scaffolds for large-conductance Ca2+-activated K+ channel (BK channel) openers, we carried out
molecular design and synthesis on the basis of the following two concepts: (1) introduction of a heteroatom into the dehydroabietic
acid (BK channel opener) skeleton would allow easier introduction of substituents. (2) Because of the fourfold symmetrical structure
of BK channels, dimeric compounds in which two pharmacophores are linked through a tether are expected to have a greater
binding probability to the channels, resulting in increased channel-opening activity. Herein, we explore the usefulness of the hexa-
hydrodibenzazepinone structure as a new scaffold for BK channel openers. The synthesized monomer compounds of hexahydrodi-
benzazepinone derivatives, which can be derived from dehydroabietic acid, were subjected to electrophysiological patch–clamp
studies, followed by Magnus contraction–relaxation assay using rabbit urinary bladder smooth muscle strips to assess overall activ-
ities. Dimeric compounds were designed by linking the monomeric hexahydrodibenzazepinone derivatives through a diacetyleneben-
zene tether, and their channel-opening activities were evaluated by electrophysiological methods. Finally, we concluded that the
critical structure for BK channel-opening activity is the hexahydrodibenzazepinone monomer substituted with a phenyl-bearing
alkynyl substituent on the lactam amide.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
action potential repolarization, neuronal excitability,
neurotransmitter release, hormone secretion, tuning of
cochlear hair cells,3 innate immunity,4 and modulation
of the tone of vascular, airway, uterine, gastrointestinal,
and urinary bladder smooth muscle tissues. Since BK
channels are activated by both elevation of intracellular
Ca2+ concentration and membrane depolarization, the
K+ efflux via BK channels results in membrane repolar-
ization and the suppression of Ca2+ influx through
voltage-dependent Ca2+ channels. Accordingly, BK
channels serve as a negative-feedback mechanism to
relax excessive muscle contraction in smooth muscles5
and to prevent aberrant cellular excitability of neurons.
Ion-selective channels are membrane proteins, which
generate electrical ionic signals and regulate signal trans-
duction events in living systems. Among such channels,
K+ channels are widely expressed in various tissues, such
as smooth muscles and neurons, and play an important
role in modulating membrane potential. In particular,
large-conductance Ca2+-activated K+ channels (BK
channels) elicit such a large K+ conductance that depo-
larized membrane potential can be effectively quenched
by the opening of BK channels.1 The BK channels are
distributed in both excitable and non-excitable cells,
which are involved in many cellular functions,2 such as
Like other members of the voltage-dependent K+ chan-
nel superfamily, BK channels are tetrameric proteins
composed of pore-forming a-subunits and auxiliary b-
subunits.6 Thus, the K+ channels are fourfold symmetri-
cal. Each BK channel a-subunit contains seven trans-
membrane spanning segments S0–S6 at the N-terminus
Keywords: BK channel opener; Hexahydrodibenzazepinones; Dimeric
compound; K+ channel.
*
Corresponding author. Tel.: +81 3 5841 4730; fax: +81 3 5841
4735; e-mail: ohwada@mol.f.u-tokyo.ac.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.042

T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
8015
and the four hydrophobic segments S7–S10 at the large
intracellular C-terminus. Unlike other classes of Ca2+
ATP-sensitive potassium channel (KATP), because it is
mostly absent in cardiac myocytes, except for mitochon-
dria.13 The BK channel openers comprise a large series
of synthetic benzimidazolone derivatives (Chart 1), such
as NS00414 and NS1619,15 the biaryl amines, such as
mefenamic and flufenamic acids,16 the biarylureas, such
as NS1608,17 the aryloxindoles (BMS-204352),18 the
arylpyrroles (NS-8),19 indole-3-carboxylic acid esters
(CGS-7184, CGS-7181)20 and natural modulators,
including dihydrosoyasaponin-1 (dehydrosoyasaponin-
1, DHS-1),21 and terpenes such as maxikdiol (1, Chart
2).22 Most of these compounds activate BK channels
as a subsidiary action in addition to their primary ac-
tion.23 In this context, the available scaffolds for BK
channel openers are rather limited in structural diversi-
ty. Both of the pioneering drugs NS004 and NS1619
are a-subunit-selective BK openers. We have found that
-
activated K+ channels, SK (small-conductance) and IK
(intermediate conductance) channels, the a-subunit of
the BK channels has an extra hydrophobic transmem-
brane segment (S0) that leads to the extracellular N-ter-
minus. The N-terminus acts as a binding domain for
b-subunits.7 Each a-subunit has a S4 voltage sensor8 and
a pore-forming region formed by S5–S6 and the P-loop.
BK channel openers have potential therapeutic applica-
tions because of possible involvement of BK channels in
various pathophysiological conditions such as hyperten-
sion,9 coronary artery spasm, urinary incontinence,10
progressive deafness,11 and several neurological disor-
ders.12 The BK channel has an advantage as a therapeu-
tic target compared to other K+ channels, such as the
H
N
H
N
H
N
F3C
F3C
F3C
O
O
O
N
N
F
OH
OH
OCH3
Cl
F3C
Cl
BMS-204352 (MaxiPost)
NS004
NS1619
H
N
F3C
CO2H
F
O
H
N
CF3
H
N
HN
NH2
CN
OH
H3C
Cl
NS1608
Flufenamic acid
NS-8
O
CO2Et
OH
CO2Et
OH
CO2H
O
N
N
F3C
F3C
O
OH
OH
O
NH
O
NH
OH
HO
O
O
O
OH
OH
CH3
Cl
CGS7181
CGS7184
DHS-I
O
CH3
OH
OH
OH
Chart 1.
R1
12
R1
12
OH
R2
14
R2
14
H
H
H
NH
H
CO2H
H
CO2H
H
H
OH
O
CO2H
CO2H
Maxikdiol (1)
Pimaric acid (2)
Abietic acid (3) Dehydroabietic acid
5
4a: R1 = R2 = Cl
4b: R1 = R2 = H
Chart 2.

8016
T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
pimaric acid and dehydroabietic acid act as BK channel
openers via direct interaction with the a-subunit of BK
channels.24 Several possible mechanisms can explain
the effect of the activators on BK channels: (i) modula-
tion of the gating (=open/close) kinetics of the a subunit,
(ii) modulation of the Ca2+ binding site at the C-termi-
nal end of the a-subunit, (iii) strengthening of the inter-
action between the a- and b-subunits, and (iv)
mimicking the interaction of b- with the a-binding site.
Novel synthetic channel modulators would be useful
tools to reveal the mechanism of the channel gating at
the atomic level, in addition to having therapeutic
potential.
acid (4), to examine the value of the hexahydrodibenzaz-
epinone structure as a new scaffold for BK channel
openers.
The synthesized monomer compounds were evaluated
by means of electrophysiological patch–clamp studies
in HEK293 cells expressing BK channels, followed by
the Magnus contraction–relaxation assay with rabbit
urinary bladder smooth muscle strips to evaluate overall
actions on smooth muscle. Dimeric compounds were de-
signed on the basis of the monomeric scaffolds, and their
channel opening activities were evaluated by electro-
physiological methods. Our results demonstrate that
the hexahydrodibenzazepinone structure is a potential
new scaffold for novel BK channel openers.
We have already found that pimaric acid (2) and dehy-
droabietic acid (4) exhibit BK channel opening activi-
ties, while the structurally related abietic acid (3) has
weak activity (Chart 2).24 In order to explore new struc-
tural scaffolds for BK channel openers, we carried out
molecular design on the basis of two concepts: (1) intro-
duction of a heteroatom into the dehydroabietic acid
skeleton would allow easier introduction of substituents.
(2) Because of the fourfold symmetrical structure of the
BK channels, dimeric compounds in which two pharma-
cophores are linked through a tether may have a greater
binding probability to the channels, resulting in in-
creased channel opening activity. Herein we describe
our exploration of the hexahydrodibenzazepinone deriv-
atives (5), which can be generated from dehydroabietic
2. Chemistry
2.1. Hexahydrodibenzazepinone derivatives
The hexahydrodibenzazepinone derivatives (11a and b)
were synthesized as shown in Scheme 1. The benzylic
oxidation of the dehydroabietic acid derivatives (6a
and b) was carried out with CrO3 in Ac2O/AcOH to give
the corresponding ketone compounds (7a and b). The
ketones were converted to the oximes (8a and b), and
the hydroxyl group of the oximes was tosylated. These
tosylated oximes (9a and b) were subjected to the Beck-
R1
R1
R1
NH2OH•HCl
CrO3
R2
R2
R2
EtOH / pyridine
Ac2O / AcOH
O
N
OH
H
H
H
CO2Me
CO2Me
CO2Me
6a R1 = R2 = Cl
7a R1 = R2 = Cl
R1 = R2 = Cl
8a
6b
7b
R1 = R2 = H
R1 = R2 = H
8b R1 = R2 = H
R1
R1
R1
1) KOH
/ crown ether
/ MeOH
R2
R2
TFA
TsCl
R2
NH
NH
pyridine
2) HCl
N
H
H
O
H
O
CO2Me OTs
CO2Me
CO2H
11a
11b
R1 = R2 = Cl
R1 = R2 = H
R1 = R2 = Cl
R1 = R2 = H
R1 = R2 = Cl
R1 = R2 = H
9a
9b
10a
10b
Scheme 1.
R1
R1
R1
1)KOH
/ crown ether
1)NaH / DMF
2)MeI
/ MeOH
R2
R2
R2
2)HCl
NH
O
N Me
N Me
H
H
H
O
O
CO2Me
CO2Me
CO2H
R1 = R2 = Cl
R = R = H
R = R = Cl
R1 = R2 = H
R = Cl, R = H
1 2
R1 = H, R2 = Cl
10a
10b
12a
12b
13a
13b
1
2
1
2
Scheme 2.

T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
8017
mann rearrangement in the presence of TFA at rt to give
the hexahydrodibenzazepinone derivatives (10a and b).
Finally, the methyl ester was hydrolyzed with KOH
and crown ether in methanol to give the corresponding
carboxylic acids (11a and b). The N-methyl group was
introduced with methyl iodide after deprotonation of
TsCl / Et3N
OH
NaI
I
OTs
CH2Cl2
acetone
14
16
15
R 1
R 1
1) NaH / DMF
2)
R 2
R 2
N
NH
O
I
H
H
O
16
CO2Me
CO2Me
10a
10b
R1 = R2 = Cl
R1 = R2 = H
17a R1 = R2 = Cl
17b
R1 = R2 = H
R1
R1
I
R2
R2
I
17a
N
O
N
Pd(PPh3)4 / CuI /Et3N
reflux
19 R1 = R2 = Cl
H
CO2R
H
O
R = Me
RO 2
C
17a
19
R = R = Cl
27
2) HCl
Condition a 1) KOH / crown ether / MeOH
2
R1= H
Pd(PPh3)4 /
Et3N
CuI
+
R 1
R1
R1
23
R5
R4
R 2
R2
R2
N
N
N
+
R3
H
H
H
MeO 2
O
O
O
CO2Me
18
CO2 Me
C
R = R = Cl, R = I, R = H, R =H
23
4
24 R11 = R22 = Cl, R33 =H, R4 = I, R55 = H
25
26
R1 = R2 = Cl, R3 =H, R4 = H, R5 = I
R1 = R2 = H, R3 =H, R4 = I, R5 =H
Pd(PPh3)2Cl2
/ CuI Et3N
17a
R1
R1
I
I
+ 24
+ 26
20
22
R2
R2
17a
17b
N
N
Pd(PPh3)2Cl2 / CuI /Et3N
H
CO2R
H
O
O
RO 2
C
20 R1 = R2 = Cl
Pd(PPh3)2Cl2
22
R1 = R2 = H
R = Me
17b
/ CuI
Et3N
a
a
28 R1 = R2 = Cl
R = H
30 R1 = R2 = H
R = H
R1
Pd(PPh3)2Cl2
/ CuI Et3N
I
17a
21
R2
R1
I
N
+ 25
17a
H
C
O
Pd(PPh3)2Cl2 / CuI /Et3N
R2
RO 2
N
H
CO 2R
O
21 R1 = R2 = Cl
R = Me
a
29 R1 = R2 = Cl
R=H
Scheme 3.

8018
T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
R1
R1
R2
R2
1) KOH / crown ether
/ MeOH
N
N
H
H
CO2H
2) HCl
O
O
CO2Me
17a R1 = R2 = Cl
31 R1 = R2 = Cl
R1
R1
R2
R2
PhI / Pd(PPh3)2Cl2
CuI / Et3N
N
N
H
O
H
O
CO2Me
CO2Me
17a R1 = R2 = Cl
32a R1 = R2 = Cl
R1
17b
R1 = R2 = H
32b
R1 = R2 = H
1) KOH / crown ether / MeOH
2) HCl
R2
N
H
CO2H
O
33a R1 = R2 = Cl
33b
R1 = R2 = H
Scheme 4.
the amide NH group with NaH (Scheme 2). Hydrolysis
of the methyl ester was carried out in a similar manner
to that described for 10, to give the carboxylic acids
(13a and b). The lactam structure of hexahydrodiben-
zazepinone derived from the Beckmann rearrangement
was reported previously,25 and our 1H NOE analysis be-
tween the N–CH3 group and the aromatic protons of the
compound 12a confirmed the structure.
was used under reflux in Et3N, to give the desired ortho
dimeric compound (19). Hydrolysis of the ester provided
the final carboxylic acid (27). The monomeric com-
pounds (31 and 33a,b), which bear a alkynyl substituent
on the lactam amide, were also synthesized as shown in
Scheme 4.
2.2. Molecular design of dimers
The syntheses of the dimeric compounds (27–30) are
illustrated in Scheme 3. Sonogashira coupling was the
key reaction. With respect to the linker moiety, 4-pen-
tyn-1-ol (14) was tosylated and converted to the iodide
(16) with NaI. N-Alkylation was carried out with the
iodide 16 to afford the alkyne derivatives (17a,b), which
were coupled with m-diiodobenzene or p-diiodobenzene
in the presence of Pd(PPh3)2Cl2/CuI in Et3N at rt (the
Sonogashira coupling reaction). The acetylene disubsti-
tuted products (20–22) and/or the acetylene monosubsti-
tuted products (24–26) were obtained. In the case of the
monosubstituted products, a second Sonogashira cou-
pling reaction was carried out to obtain the acetylene
disubstituted products (20–22). Finally, the ester group
was hydrolyzed with KOH and crown ether in methanol
to afford the corresponding dimeric compounds (27–29).
The alkyne (17) and o-diiodobenzene did not readily
undergo the Sonogashira coupling reaction with
Pd(PPh3)2Cl2/CuI in Et3N at rt. Under these reaction
conditions, in addition to the acetylene monosubstituted
product (23), the acetylene homodimeric product (18)
was obtained, probably because of steric hindrance.
Thus, on coupling of the alkyne (17a) with o-diiodoben-
zene, Pd(PPh3)4 was used as a catalyst and purified CuI
The X-ray structures of several K+ channels revealed
that the diameters of the pores (the gate region) in the
˚
closed state are 6 A (for the KcsA channel) and 8 A
˚
˚
(KirBac channel), and those in open state are 25 A
˚
(for the MthK channel) and 19 A (KvAP channel). In
the cases of other ion channels, the binding sites of small
organic molecules, such as channel blockers, are located
on the inner side of the pore.26 Moreover, the dichlo-
rodehydroabietic acid (4a) was shown to activate the
BK channel through interaction with the a subunit.24c
It seems reasonable to assume that the hexahydrodiben-
zazepinone derivatives (13) also interact with the a sub-
units of the BK channels.
We thus designed several dimeric compounds, 27–29, in
which two units of the pharmacophore, dichlorohexahy-
drodibenzazepinone 13a, are linked through the rigid
diacetylenebenzene unit. Because we have little informa-
tion about the arrangement and topology of the binding
sites, we added adjustable molecular-dynamic flexibility
by using an n-propyl chain as a tether between the nitro-
gen atom of the hexahydrodibenzazepinone core and the
rigid diacetylenyl benzene core. Ortho, meta, and para
substitution can change the direction of extension of

T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
8019
Figure 1. Conformational spaces of isomeric dimer compounds energy-minimized structures of the dimers in an n-octanol environment.
the two pharmacophores, and this molecular design al-
lowed us to change the distance between the two phar-
macophores in the dimer. In fact, molecular dynamic
calculations on the basis of conformational search with
OPLS2001 force field parameters in an n-octanol envi-
ronment (Macromodel 8.5) generated a range of dimer
structures.27 We obtained various numbers of indepen-
dent structures as follows: ortho (27): 2852 structures;
meta (28): 2581 structures; para (29): 2838 structures,
when the number of generated trial structures was set
to 3000 in each case. The minimum-energy structures
in each case are shown in Figure 1. The structural space
was not significantly different in the gas-phase or in
water as a solvent (data not shown). Because the carbox-
ylic acid group of dehydroabietic acid 4 is known to be
essential for BK channel opening activity,24 this group
presumably plays an important role in the interaction
with the channel proteins. A similar role of the carbox-
ylic acid group was expected for the hexahydrodibenzaz-
epinone derivatives. The average (with standard
deviation) minimum and maximum distances between
the two carbonyl oxygen atoms of the individual carbox-
ylic acids in the calculated dimer structures are as fol-
and this should result in an increase of channel opening
activity.
3. Results and discussion
3.1. Electrophysiological studies of hexahydrodibenzaz-
epinones derivatives
The activity of the monomeric hexahydrodibenzazepi-
none derivatives as BK channel openers was evaluated
by the patch–clamp technique with inside-out configura-
tion. Human BK channel subunits, hSlo a and b1, were
transiently expressed in tsA201 cells, a derivative of the
human embryonic kidney (HEK) cell line 293. In the
patch–clamp measurement, when a compound has chan-
nel opening activity, the conductance–voltage curve
shifts towards the negative voltage direction (leftward),
that is, a larger BK channel current is activated by the
same voltage step under the voltage–clamp condition.
The BK channel opening activities of the tested com-
pounds at 10 lM are represented as the shift of V1/2 val-
ue (mV, n = 3–5), the voltage activating the half-
maximum current, in comparison with the negative con-
trol in the presence of 100 lM Ca2+ on the intracellular
side of the membrane patch (Fig. 2). In this work, we
used dichlorodehydroabietic acid 4a as a reference com-
pound which has a related structure.24
˚
˚
lows: ortho (27): 6.54 4.02 A (minimum: 3.01 A;
˚
maximum: 15.37 A); meta (28): 7.04 5.54 A (mini-
˚
˚
˚
mum: 2.99 A; maximum: 18.42 A); para (29):
˚
˚
˚
22.23 1.66 A (minimum: 4.40 A; maximum: 25.09 A).
The distances between the pharmacophores, particularly
those of the carboxylic acids of 27–29, accord well with
the size of the pores of the K+ channels, which are well
conserved among the K+ channel superfamily. The pres-
ent designed dimeric compounds should be able to bind
to the two putative binding sites of a single BK channel,
if the binding sites are located close to the pore regions,
The voltage shift of V1/2 of 4a was À5.3 3.7 mV, and
that of the N-methyl derivative of the hexahydrodiben-
zazepinone 13a was À9.2 3.7 mV. Compound 13a
can open the BK channel, while 13b, its counterpart
lacking the dichloro-substituent, did not show channel

8020
T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
control
33a
C
A
B
Compound
4a 13a 13b 27 28 29 30 31 33a 33b
10
5
50 ms
0
1.0
0.8
0.6
0.4
0.2
0.0
-5
control
+33a
-10
-15
-20
-25
-30
-50
0
50
100
150
200
test potential (mV)
Figure 2. Effects of test compounds on the BK channel current. (A) Effect of 33a on BK channel currents recorded in the inside-out macro patch-
clamp configuration. BK channel currents were activated by test pulses to 100 mV from a holding potential at À100 mV. Current zero level is
indicated by a dotted line. Outward BK channel currents are shown in upward direction. (B) Conductance–voltage relationship (G–V curve) of BK
channel currents (n = 4). Compound 33a shifted G–V curve toward hyperpolarized direction. (C) Shifts of V1/2 value (mV) of G–V curve by test
compounds (n = 3–5). A negative value indicates a shift to hyperpolarizing voltages.
opening activity. In this context, the results of the
smooth muscle relaxation assay are consistent with
those of the patch–clamp experiments.
The prototype N-methyl hexahydrodibenzazepinone
13b, lacking the aromatic dichloro-substituents, did
not show BK channel opening activity in patch–clamp
experiments (Fig. 2). However, the m-dimeric com-
pound 30 showed channel opening activity with a hyper-
polarizing shift of V1/2 by À11.9 0.9 mV. This activity
is also probably derived from the fragment structure of
the dimer. In fact, the monomeric compound 33b also
showed a strong opening activity, with a leftward shift
of V1/2 by À18.9 3.8 mV, which is less than the shift
caused by 33a, but comparable in magnitude. The most
potent compound 33a expanded the width of the tail
current in the patch-clamp measurements (data not
shown), which suggests that this compound stabilizes
the BK channel in the open state.
Thus, the results of the electrophysiological study sup-
ported the idea that the dichloro-substituted N-methyl-
hexahydrodibenzazepinone 13a can serve as
pharmacophore for BK channel openers.
a
3.2. Electrophysiological study of the dimers and
N-substituent effects
These dimeric compounds bearing dichloro substituents
on the aromatic ring (27–29) were studied by means of
an inside-out patch–clamp configuration. They showed
BK channel opening activities, the left-shift of V1/2 being
À7.7 1.2 mV, À6.3 3.2 mV, and À5.4 1.6 mV,
respectively. While the channel opening activity of the
ortho derivative 27 is likely to be stronger than those
of the other isomers (28 and 29), the differences are
not significant. In addition, the magnitude of the shift
of 27 was as large as that of the monomer 13a. These re-
sults imply that a part of the structures common to these
dimeric isomers could be responsible for the activity,
rather than the dual binding of the dimeric compounds
to multiple binding sites of the BK channel. Thus, we
synthesized monomeric fragments of the dimers, that
is, 31 and 33a, the former bearing a N-alkyl acetylene
substituent and the latter bearing an additional terminal
benzene moiety. We examined the BK channel opening
activity of these compounds in patch clamp experiments
(Fig. 2). Unexpectedly, we found that 33a showed signif-
icant channel opening activity (À23.9 1.4 mV left-shift
of V1/2), while 31 showed activity (À6.6 1.5 mV) com-
parable in magnitude with that of the N-methyl deriva-
tive 13a.
3.3. Isolated rabbit detrusor smooth muscle assay as
overall activity
We evaluated the overall activities of hexa-
hydrodibenzazepine derivatives on detrusor smooth
muscle. Figure 3 shows the results of the rabbit detrusor
smooth muscle assay. The K+ channel-dependent mus-
cle dilating effect was evaluated as follows.28 Prior to
the application of test compounds, detrusor smooth
muscle strips were precontracted by depolarization
caused by raising the extracellular K+ concentration to
either 30 mM or 120 mM. With the extracellular K+
concentration at 30 mM, the small depolarization allows
Ca2+ influx through Ca2+ channels to produce contrac-
tion. If a test compound activates the BK channel, the
K+ efflux will shift the membrane potential toward the
equilibrium potential for K+ (EK) of approximately
À40 mV where Ca2+ channels are closed, thus causing
relaxation of the muscle strip. In contrast, with the
extracellular K+ concentration at 120 mM, the opening

T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
8021
Figure 3. Detrusor smooth muscle relaxation assay The relative magnitude of relaxation was normalized to the precontraction caused by high K+
(30 mM or 120 mM) as 100%. The relaxant activities of the test compounds were compared with that of 0.1% DMSO as the vehicle (control).
of the BK channel will shift the membrane potential to-
ward EK of around 0 mV, where Ca2+ channels are
open, and the dilating activity of the BK channel opener
should be abolished. The relative magnitude of relaxa-
tion was normalized to the contraction caused by high
K+ precontraction as 100%. The concentration of
DMSO as a vehicle was set to be no more than 0.1%
(v/v), because DMSO at high concentration caused
relaxation by itself. Moreover, the concentration of the
test compounds was fixed at 10 lM because of their rel-
atively poor solubility in DMSO–water. The relaxant
activities of the test compounds were compared with
that of vehicle (0.1% DMSO) measured in the same
muscle strip. All values are expressed as means SEM.
Statistical significance of differences was determined by
paired Student’s t-test, and P values less than 0.05 were
considered to be significant.
previous24 and present electrophysiological studies.
The dichloro-substituted 4a showed a statistically signif-
icant relaxation activity, while the unsubstituted 4b
showed weak activity (data not shown). The smooth
muscle relaxation study showed that the N-methyl deriv-
ative 13a, bearing aromatic dichloro substituents,
showed a statistically significant relaxation effect, while
the prototype NH amide 11a showed less potent activity
(data not shown). Even in the absence of the aromatic
dichloro substituents, the NH derivative 11b showed dis-
tinct relaxation activity, while the N-methyl derivative
13b did not.
The monomeric compounds 31 and 33a showed dis-
tinct rabbit urinary bladder relaxation activities in
detrusor smooth muscle strips (54.3 2.3% and
48.68 5.8%, respectively, n = 5), the activity of 33a
being statistically significant (Fig. 3). Reasonably,
the N-alkylated monomeric compound 33b also
exhibited potent relaxation activities (48.68 5.8%,
n = 5).
The results of the isolated rabbit detrusor smooth mus-
cle relaxation study of the dehydroabietic acid deriva-
tives were consistent with what we had found in the

8022
T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
4. Conclusion
a sealed tube. Then to this mixture was added a solution
of 2.8 M Cl2 in 10 mL CCl4 (28.0 mmol, 4 equiv) at rt,
and the whole was stirred for 4 days. The insoluble
materials were filtered off and the filtrate was then evap-
orated in vacuum. The residue was recrystallized from
n-hexane to afford 4a (574 mg, 1.554 mmol, yield
23%). Compound 4a: colorless fine needles. Mp 218–
In this work, we first demonstrated that the hexahydro-
dibenzazepinone structure represents a new scaffold for
BK channel openers. Second, we carried out molecular
design based on our hypothesis that dual binding of
linked pharmacophores might provide strong activation
even if the single pharmacophore is a rather weak open-
er. We found that the dimerization retained (in the case
of 27) or increased (in the case of 30) the channel open-
ing activity as compared with the corresponding mono-
meric pharmacophores (13a and 13b, respectively). In
the present design, ortho, meta, and para isomers (27–
29) of the dimeric compounds showed similar, not in-
creased, magnitudes of activity as compared with the
minimal monomer 13a, probably because the two phar-
macophores cannot bind two putative binding sites at
the same time. However, a portion of the molecules
clearly can gain access to the binding site. Because of
the highly hydrophobic nature of 33 and the dimers
27–30, and retention of the opening activities of the di-
mers 27–30, it is possible that there are hydrophobic
binding sites in the transmembrane region of the chan-
nels which allow these hydrophobic molecules to gain
access in a well-ordered conformation (at least partially)
via a membrane bilayer pathway, rather than a direct
aqueous pathway.29 Finally, we found that the phenyl-
bearing alkynyl substituent on the lactam amide nitro-
gen atom is crucial for BK channel opening activity.
Our findings provide a new basis for development of
novel BK channel openers.
1
222 ꢁC (n-hexane/AcOEt). H NMR (CDCl3) 1.20 (3H,
s, CH3), 1.28 (3H, s, CH3), 1.39 (6H, d, J = 7.3 Hz,
2CH3CH), 1.61–1.66 (2H, m, 2CH), 1.72–1.86 (5H, m,
5CH), 2.13 (1H, dd, J = 12.6 Hz, 2.2 Hz, CH), 2.24
(1H, d, J = 11.9 Hz, CH), 2.70–2.79 (1H, m, CH), 2.94
(1H, dd, J = 18.4 Hz, 6.0 Hz, CH), 3.91 (1H, br s,
CH3CH), 7.15 (1H, s, ArH). MS (FAB+) m/z 369
([M(35Cl2)+H]+),
371
([M(35Cl37Cl)+H]+),
373
([M(37Cl2)+H]+). Anal. Calcd for C20H26Cl2O2: C,
65.04; H, 7.10. Found: C, 64.95; H, 7.16.
12,14-Dichlorodehydroabietic acid methyl ester (6a). To
a solution of 12,14-dichlorodehydroabietic acid 4a
(442 mg, 1.197 mmol) in MeOH (2 mL) and PhMe
(4 mL) was dropwise added 2.0 M TMSCHN2 in
Et2O (0.78 mL, 1.557 mmol, 1.3 equiv) at rt over
5 min, and the whole was stirred at rt for 30 min. Ex-
cess TMSCHN2 was quenched with Et2O, then the
reaction mixture was evaporated in vacuum, and the
residue was purified by flash chromatography (n-hex-
ane only to n-hexane/AcOEt = 10:1) to afford 6a
(453 mg, 1.181 mol, yield 99%) as a colorless solid.
6a: colorless needles. Mp 126–129 ꢁC. 1H NMR
(CDCl3) 1.19 (3H, s, CH3), 1.26 (3H, s, CH3), 1.39
(6H, d, J = 7.1 Hz, 2CH3CH), 1.47–1.53 (2H, m,
2CH), 1.65–1.75 (5H, m, 5CH), 2.12 (1H, d,
J = 10.6 Hz, CH), 2.23 (1H, d, J = 11.7 Hz, CH),
2.71–2.73 (1H, m, CH), 2.92 (1H, did, J = 18.3 Hz,
6.2 Hz, CH), 3.67 (3H, s, CO2CH3), 3.91 (1H, br s,
CH3CH), 7.15 (1H, s). LRMS (FAB+) m/z 383
5. Experimental
5.1. Chemistry
Commercially available reagents were purchased and
were used without further purification unless otherwise
stated. 1H NMR spectra were recorded on a Bruker Ad-
vance (400 MHz) spectrometer. The chemical shifts
(ppm) were shown by using tetramethylsilane (TMS)
as an internal standard in deuterated chloroform
(CDCl3), and the peak of the solvent was used as an
internal standard in the case of deuterated methanol
(CD3OD). Mass spectra were recorded on a JEOL
MStation JMS-700 spectrometer. Elemental analyses
were recorded on a Yanaco CHN CORDER spectrom-
eter and were carried out at the Analysis Center of the
Graduate School of Pharmaceutical Sciences, the Uni-
versity of Tokyo. Flash column chromatography was
carried out with silica gel (Kanto Chemical 60 N, parti-
cle size 40–50 mm). Melting points were measured on a
Yanaco hot-stage microscope and are uncorrected.
Yields are shown in terms of those of isolated pure
materials.
([M(35Cl2)+H]+),
385
([M(35Cl37Cl)+H]+),
387
([M(35Cl2)+H]+). Anal. Calcd for C21H28Cl2O2: C,
65.79; H, 7.36. Found; C, 65.86; H, 7.14.
Dehydroabietic acid methyl ester (6b). A mixture of
dehydroabietic acid 4b (5.1 g, 0.017 mol) and concd
H2SO4 (6 mL) in MeOH (200 mL) was stirred at 85 ꢁC
for 19 h. Then concd H2SO4 (2 mL) was carefully added
to the reaction mixture. Stirring was continued for 2
days, then the mixture was cooled, the solvent was evap-
orated in vacuum, and the residue was diluted with
water (300 mL). The whole was extracted with CHCl3
(1· 200 mL, 2· 100 mL). The combined organic layer
was washed with brine (1· 100 mL), dried over Na2SO4,
filtered, and then the solvent was evaporated in vacuum.
The residue was purified by flash chromatography (n-
hexane/AcOEt = 10:1) to afford 6b (4.4 g, 0.014 mol,
yield 83%) as a colorless solid. Compound 6b: colorless
needles. Mp 45–49 ꢁC (n-hexane). 1H NMR (CDCl3)
1.21–1.23 (9H, m, 2CH3CH and CH3), 1.27 (3H, s,
CH3), 1.42 (1H, m, CH), 1.50 (1H, m, CH), 1.61–1.70
(5H, m, 5CH2), 2.24 (1H, dd, J = 12.5 Hz, 2.1 Hz,
CH), 2.30 (1H, d, J = 12.3 Hz, CH), 2.80–2.84 (1H, m,
CH3CH), 2.86–2.90 (1H, m, CH), 3.66 (3H, s, COCH3),
6.88 (1H, s, ArH), 6.99 (1H, d, J = 6.4 Hz, ArH), 7.16
(1H, d, J = 8.1 Hz, ArH). LRMS (FAB+) m/z 315
12,14-Dichlorodehydroabietic acid (4a). To a suspension
of dehydroabietic acid 4b (2.0 g, 6.66 mmol), 2% w/w
FeCl3 on SiO2 (1 g), and 1% w/w DDQ on SiO2 (0.1 g)
in 25 mL CCl4 was added a solution of 2.8 M Cl2 in
35.7 mL CCl4 (99.9 mmol, 15 equiv) at 0 ꢁC. The reac-
tion mixture was vigorously stirred at rt for one day in

T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
8023
([M+H]+). Anal. Calcd for C21H30O2: C, 80.21; H, 9.62.
Found: C, 80.07; H, 9.82.
The residue was purified by flash chromatography (n-hex-
ane/AcOEt = 1:1) toafford10a (166 mg, 0.403 mmol, yield
81%) as a colorless amorphous solid. Compound 10a: 1H
NMR (CDCl3): 1.42 (6H, d, J = 7.1 Hz, 2CH3CH), 1.45
(3H, s, CH3), 1.58 (3H, s, CH3), 1.67–1.76 (2H, m, 2CH),
1.81–1.83 (2H, m, 2CH), 1.90–1.93 (2H, m, 2CH), 2.29
(1H, d, J = 8.6 Hz, CH), 2.92 (1H, dd, J = 15.9 Hz,
6.8 Hz, CH), 3.47–3.55 (1H, m, CH), 3.64 (3H, s,
CO2CH3), 3.96 (1H, br s, CH3CH), 7.26 (1H, br s, ArH),
7.85 (1H, br s, NH). LRMS (FAB+) m/z 412
([M(35Cl2)+H]+), 414 ([M(35Cl37Cl)+H]+), 416 ([M(37Cl2)
+H]+).
13-Isopropyl-12,14-dichloro-7-oxopodocarpe-8,11,13-tri-
ene-15-carboxylic acid methyl ester (7a). To a solution of
CrO3 (287 mg, 2.867 mmol, 1.1 equiv) in Ac2O (9 mL)
and AcOH (4 mL) was dropwise added a suspension of
the methyl ester 6a (999 mg, 2.606 mmol) in AcOH
(15 mL) at 0 ꢁC over 10 min. The reaction mixture was
stirred at 50 ꢁC for 9 h, the whole was cooled, and poured
into ice-water (40 mL) and then the whole was extracted
with CHCl3 (3· 30 mL). The combined organic layer
was washed with water (1· 30 mL), satd NaHCO3 (2·
30 mL), and brine (1· 30 mL), dried over Na2SO4, fil-
tered, and then the solvent was evaporated in vacuum.
The residue was purified by flash chromatography (n-hex-
ane/AcOEt = 5:1) to afford 7a (847 mg, 2.130 mmol, yield
82%) as a pale yellow amorphous solid. Compound 7c: 1H
NMR (CDCl3) 1.17 (3H, s, CH3), 1.34 (3H, s, CH3), 1.41
(6H, d, J = 7.3 Hz, 2CH3CH), 1.75–1.79 (5H, m, 5CH),
2.20 (1H, d, J = 12.5 Hz, CH), 2.51 (1H, dd,
J = 16.9 Hz, 5.2 Hz, CH), 2.64 (2H, m, 2CH), 3.66 (3H,
s, CO2CH3), 3.94 (1H, br s, CH3CH), 7.19 (1H, br s, ArH).
12,14-Dichloro-13-isopropyl-8,11a-dimethyl-6-oxo-6,7,7a,
8,9,10,11,11a-octahydro-5H-dibenzo[b,d]azepine-8-carbox-
ylic acid (11a). A mixture of the methyl ester 10a (80 mg,
0.194 mmol), KOH (109 mg, 3.068 mmol, 10.0 equiv),
and 18-crown ether-6 (128 mg, 0.485 mmol, 2.5 equiv) in
MeOH (2 mL) was stirred at 80 ꢁC for 7.5 h, the whole
was cooled, and then the solvent was evaporated in vacu-
um. The residue was diluted with water (10 mL), acidified
with 2 N HCl (2 mL), and the whole was extracted with
CHCl3 (3· 20 mL). The combined organic layer was
washed with brine (1· 20 mL), dried over Na2SO4, filtered,
and then the solvent was evaporated in vacuum. The resi-
due was purified by flash chromatography (AcOEt only
to AcOEt/MeOH = 10:1) to afford 11a (35 mg,
0.087 mmol, yield 45%) as a colorless solid. Compound
11a: colorless powder. Mp 192–197 ꢁC (n-hexane/AcOEt/
12,14-Dichloro-13-isopropyl-7-hydroxyiminepodocarpe-
8,11,13-triene-15-carboxylic acid methyl ester (8a). A
mixture of the methyl ester 7a (198 mg, 0.498 mmol),
pyridine (0.06 mL), and NH2OHÆHCl (54 mg,
0.772 mmol, 1.6 equiv) in EtOH (2 mL) was stirred at
100 ꢁC for 3.5 h, the whole was cooled, and the solvent
was evaporated in vacuum. The residue was purified by
flash chromatography (n-hexane/AcOEt = 5:1) to afford
8a (207 mg, quantitative yield) as a colorless amorphous
1
MeOH). H NMR (CD3OD): 1.32 (6H, d, J = 7.1 Hz,
2CH3CH), 1.36 (3H, s, CH3), 1.47 (3H, s, CH3), 1.60
(2H, m, 2CH), 1.70 (2H, m, 2CH), 1.70 (2H, m, 2CH),
1.85 (2H, m, 2CH), 2.10 (1H, d, J = 8.4 Hz, CH), 2.86
(1H, dd, J = 15.9 Hz, CH), 3.27 (1H, m, CH), 3.90 (1H,
br s, CH3CH), 7.23 (1H, br s, ArH). LRMS (FAB+) m/z
398 ([M(35Cl2)+H]+), 400 ([M(35Cl37Cl)+H]+), 402
([M(37Cl2)+H]+). Anal. Calcd for C20H25Cl2NO3Æ3/
4H2O: C, 58.33; H, 6.30; N, 3.40. Found: C, 58.29; H,
6.39; N, 3.39.
1
solid. Compound 8a: H NMR (CDCl3) 1.06 (3H, s,
CH3), 1.22 (6H, d, J = 8.4 Hz, 2CH3CH), 1.26 (1H, dd,
J = 18.7 Hz, 6.4 Hz, CH), 1.72–1.77 (5H, m, 5CH),
2.15 (2H, dd, J = 12.1 Hz, 6.1 Hz, CH), 2.38 (1H, dd,
J = 18.7 Hz, 6.4 Hz, CH), 3.09 (1H, dd, J = 18.7 Hz,
13.0 Hz, CH), 3.66 (3H, s, CO2CH3), 3.69 (1H, br s,
CH3CH), 7.15 (1H, s, ArH).
13-Isopropyl-7-oxopodocarpe-8,11,13-triene-15-carboxyl-
ic acid methyl ester (7b). To a solution of CrO3 (790 mg,
7.886 mmol, 1.1 equiv) in Ac2O (28 mL) and AcOH
(14 mL) was dropwise added a suspension of dehydroa-
bietic acid methyl ester 6b (2.35 g, 7.456 mmol) in AcOH
(6 mL) at rt for 11 min. The reaction mixture was stirred
for 15 min at rt, then stirred at 50 ꢁC for 8.5 h. The mix-
ture was cooled, and the whole was poured into icewater
(100 mL) and then extracted with CHCl3 (1· 200 mL, 2·
100 mL). The combined organic layer was washed with
brine (1· 100 mL), dried over Na2SO4, filtered, and then
the solvent was evaporated in vacuum. The residue was
12,14-Dichloro-13-isopropyl-7-tosyloxyiminepodocarpe-8,
11,13-triene-15-carboxylic acid methyl ester (9a). To a solu-
tion of the methyl ester 8a (207 mg, 0.503 mmol) in pyri-
dine (1 mL) was added TsCl (144 mg, 0.754 mmol,
1.5 equiv) at rt. The reaction mixture was stirred for 17 h
at rt, and then the solvent was evaporated in vacuum.
The residue was purified by flash chromatography (n-hex-
ane/AcOEt = 5:1) to afford 9a (282 mg, 0.497 mmol, yield
99%) as a colorless oil. Compound 9a: 1H NMR (CDCl3)
1.03 (3H, s, CH3), 1.25 (3H, d, J = 7.1 Hz, CH3CH), 1.27
(3H, d, J = 7.1 Hz, CH3CH), 1.37 (3H, s, CH3), 1.74–
1.77 (5H, m, 5CH), 2.06–2.15 (2H, m, 2CH), 2.46 (3H, s,
CH3), 2.51 (1H, dd, J = 13.8 Hz, 5.0 Hz, CH), 3.02 (1H,
dd, J = 19.0 Hz, 12.9 Hz, CH), 3.67 (3H, s, CO2CH3),
3.94 (1H, br s, CH3CH), 7.12 (1H, br s, ArH), 7.35 (2H,
d, J = 8.4 Hz, ArH), 7.94 (2H, d, J = 8.2 Hz, ArH).
purified
by
flash
chromatography
(n-hexane/
AcOEt = 5:1) to afford the desired compound 7b
(570 mg, 1.746 mmol, yield 23%) as a pale yellow oil
and the undesired overreacted compound (550 mg,
1.431 mmol, yield 19%) as a yellow oil. Compound 7b:
1H NMR (CDCl3) 1.24–1.26 (9H, m, 2CH3CH and
CH3), 1.34 (3H, s, CH3), 1.61–1.80 (5H, m, 5CH),
2.32–2.38 (2H, m, 2CH), 2.71–2.73 (2H, m, 2CH),
2.91–2.95 (1H, m, CH3CH), 3.65 (3H, s, CO2CH3),
7.29 (1H, d, J = 8.1 Hz, ArH), 7.40 (1H, dd,
J = 8.2 Hz, 2.1 Hz, ArH), 7.87 (1H, d, J = 2.0 Hz,
12,14-Dichloro-13-isopropyl-8,11a-dimethyl-6-oxo-6,7,7a,
8,9,10,11,11a-octahydro-5H-dibenzo[b,d]azepine-8-carbox-
ylic acid methyl ester (10a). The mixture of the methyl ester
9a (282 mg, 0.497 mmol) in TFA (2 mL) was stirred at rt
for 1 h, and then the solvent was evaporated in vacuum.

8024
T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
ArH). LRMS (FAB+) m/z 329 ([M+H]+). By-product:
(13-(1-Acetoxy-1-methylethyl)-7-oxopodocarpe-8,11,13-
triene-15-carboxylic acid methyl ester): 1H NMR
(CDCl3) 1.26 (3H, s, CH3), 1.34 (3H, s, CH3), 1.72–
1.79 (1H, m, 5CH and 2CH3CPh), 2.10 (3H, s, CH3CO),
2.32–2.36 (2H, m, 2CH), 2.70–2.74 (2H, m, 2· CH), 3.66
(3H, s, CO2CH3), 7.33 (1H, d, J = 8.2 Hz, ArH), 7.52
(1H, dd, J = 8.3 Hz, 2.3 Hz, ArH), 7.97 (1H, d,
J = 2.4 Hz, ArH).
LRMS (FAB+) m/z 343 ([M+H]+). Anal. Calcd for
C21H29NO3: C, 73.44; H, 8.51; N, 4.08. Found: C,
73.21; H, 8.29; N, 4.10.
13-Isopropyl-8,11a-dimethyl-6-oxo-6,7,7a,8,9,10,11,11a-
octahydro-5H-dibenzo[b,d] azepine-8-carboxylic acid (11b).
A mixture of the methyl ester 10b (100 mg, 0.291 mmol),
KOH (163 mg, 2.911 mmol, 10.0 equiv) and 18-crown
ether-6 (192 mg, 0.728 mmol, 2.5 eq.) in MeOH (2 mL)
was stirred at 80 ꢁC for 15 h, the whole was cooled,
and the solvent was evaporated in vacuum. The residue
was diluted with water (10 mL), acidified with 2 N HCl
(2 mL), and then the mixture was extracted with CHCl3
(3· 20 mL). The combined organic layer was washed
with brine (1· 20 mL), dried over Na2SO4, filtered,
and then the solvent was evaporated in vacuum. The res-
idue was purified by flash chromatography (n-hexane/
AcOEt = 1:2) to afford 11b (75 mg, 0.228 mmol, yield
78%) as a colorless oil. Compound 11b: colorless powder
13-Isopropyl-7-hydroxyiminepodocarpe-8,11,13-triene-15-
carboxylic acid methyl ester (8b). A mixture of the methyl
ester 7b (598 mg, 1.667 mmol), pyridine (0.2 mL), and
NH2OHÆHCl (180 mg, 2.585 mmol, 1.6 equiv) in EtOH
(4 mL) was stirred at 100 ꢁC for 3 h, the mixture was
cooled, and the solvent was evaporated in vacuum. The
residue was purified by flash chromatography (n-hexane/
AcOEt = 5:1) to afford 8b (390 mg, 1.137 mmol, yield
1
68%) as a colorless amorphous solid. Compound 8b: H
1
NMR (CDCl3) 1.12 (3H, s, CH3), 1.25 (6H, d,
J = 7.0 Hz, 2CH3CH), 1.38 (3H, s, CH3), 1.42–1.76 (5H,
m, 5CH), 2.28–2.35 (2H, m, 2CH), 2.64–2.67 (2H, m,
2CH), 2.88–2.91 (1H, m, CH3CH), 3.65 (3H, s, CO2CH3),
7.21 (2H, m, ArH), 7.70 (1H, s, ArH). LRMS (FAB+) m/z
344 ([M+H]+).
(n-hexane/AcOEt); Mp 135–140 ꢁC; H NMR (CDCl3)
1.22 (6H, d, J = 6.8 Hz, 2CH3CH), 1.42 (3H, s, CH3),
1.45 (3H, s, CH3), 1.74–1.88 (5H, m, 5CH), 1.95 (1H,
d, J = 11.3 Hz, CH), 2.28 (1H, dd, J = 14.9 Hz, 3.4 Hz,
CH), 2.67 (1H, dd, J = 14.9 Hz, 7.8 Hz, CH), 2.80
(1H, dd, J = 7.9 Hz, 3.7 Hz, CH), 2.83–2.86 (1H, m,
CH3CH), 6.70 (1H, d, J = 1.8 Hz, ArH), 7.00 (1H, dd,
J = 8.0 Hz, 1.8 Hz, ArH), 7.31 (1H, d, J = 8.4 Hz,
ArH), 7.99 (1H, s, ArH). LRMS (FAB+) m/z 330
([M+H]+). Anal. Calcd for C20H27NO3Æ1/6H2O: C,
72.26; H, 8.28; N, 4.21. Found: C, 72.21; H, 8.15; N, 4.16.
13-Isopropyl-7-tosyloxyiminepodocarpe-8,11,13-triene-
15-carboxylic acid methyl ester (9b). To a solution of the
methyl ester 8b (390 mg, 1.137 mmol) in pyridine (1 mL)
was added TsCl (325 mg, 1.705 mmol, 1.5 equiv) at rt.
The reaction mixture was stirred for 14 h at rt, and then
the solvent was evaporated in vacuum. The residue was
12,14-Dichloro-3-isopropyl-5,8,11a-trimethyl-6-oxo-6,7,7a,
8,9,10,11,11a-octahydro-5H-dibenzo[b,d]azepine-8-carbox-
ylic acid methyl ester (12a). To a suspension of NaH (60%
in mineral oil, washed twice with n-hexane) (10 mg,
0.244 mmol, 1.1 equiv) in DMF (1 mL) was dropwise
added a solution of the methyl ester 10a (91 mg,
0.222 mmol) in DMF (1 mL) at 0 ꢁC for 2 min in a flask
fitted with a CaCl2 tube. After stirring at 0 ꢁC for
15 min, MeI (0.07 mL, 1.108 mmol, 5.0 equiv) was added
to the reaction mixture at 0 ꢁC. Stirring was continued at
rt for 7 h, the solvent was evaporated in vacuum, and the
residue was diluted with 1NHCl (10 mL) andthen extract-
ed with CHCl3 (3· 20 mL). The combined organic layer
was washed with brine (1· 20 mL), dried over Na2SO4, fil-
tered, and then the solvent was evaporated in vacuum.
The residue was purified by flash chromatography (n-hex-
ane/AcOEt = 2:1) to afford 12a (73 mg, 0.171 mmol, yield
77%) as a colorless oil. Compound 12a: 1H NMR (CDCl3)
1.36 (3H, s, CH3), 1.42 (6H, d, J = 7.1 Hz, 2CH3CH), 1.46
(3H, s, CH3), 1.72–1.73 (2H, m, CH2), 1.82–1.91 (4H, m,
4CH), 2.21 (1H, d, J = 8.6 Hz, CH), 2.74 (1H, d,
J = 16.3 Hz, CH), 3.19 (3H, s, NCH3), 3.64 (3H, s,
CO2CH3), 3.81 (1H, dd, J = 16.2 Hz, 8.8 Hz, CH), 4.09
(1H, br s, CH3CH), 7.20 (1H, br s, ArH).
purified
by
flash
chromatography
(n-hexane/
AcOEt = 5:1) to afford 9b (545 mg, 1.095 mmol, yield
96%) as a colorless solid. Compound 9b: colorless pow-
der. Mp 158–160 ꢁC (n-hexane/AcOEt). 1H NMR
(CDCl3) 1.04 (3H, s, CH3), 1.20–1.28 (6H, m, 2CH3CH),
1.34 (3H, s, CH3), 1.68–1.74 (5H, m, 5CH), 2.26 (2H, t,
J = 9.3 Hz, CH2), 2.45 (3H, s, PhCH3), 2.66 (2H, d,
J = 8.6 Hz, CH2), 2.85–2.88 (1H, m, CH3CH), 3.66
(3H, s, CO2CH3), 7.18 (1H, d, J = 8.2 Hz, ArH), 7.25
(1H, dd, J = 8.2 Hz, 2.0 Hz, ArH), 7.35 (2H, d,
J = 8.6 Hz, ArH), 7.58 (1H, d, J = 1.8 Hz, ArH), 7.95
(2H, d, J = 8.4 Hz, ArH). LRMS (FAB+): m/z 498
([M+H]+). Anal. Calcd for C28H35NO5S: C, 67.58; H,
7.09; N, 2.81. Found: C, 69.39; H, 7.21; N, 2.91.
13-Isopropyl-8,11a-dimethyl-6-oxo-6,7,7a,8,9,10,11,11a-
octahydro-5H-dibenzo[b,d]azepine-8-carboxylic acid meth-
yl ester (10b). A mixture of the methyl ester 9b (505 mg,
1.015 mmol) in TFA (3 mL) was stirred at rt for 40 min,
and then the solvent was evaporated in vacuum. The res-
idue was purified by flash chromatography (n-hexane/
AcOEt = 1:1) to afford 10b (392 mg, quantitative yield)
as a colorless amorphous solid. Compound 10b: colorless
1
needles. Mp 148–149 ꢁC (n-hexane/AcOEt). H NMR
(CDCl3) 1.24 (6H, d, J = 7.0 Hz, 2CH3CH), 1.42 (3H, s,
CH3), 1.45 (3H, s, CH3), 1.69–1.93 (6H, m, 6CH), 2.18
(1H, d, J = 14.8 Hz, CH), 2.57 (1H, dd, J = 14.7 Hz,
8.2 Hz, CH), 2.78–2.81 (1H, dd, J = 8.2 Hz, 3.6 Hz,
CH), 2.85–2.88 (1H, m, CH3CH), 3.65 (3H, s, CO2CH3),
6.76 (1H, d, J = 2.0 Hz, ArH), 7.04 (1H, d, J = 8.2 Hz,
ArH), 7.33 (1H, d, J = 8.4 Hz, ArH), 8.62 (1H, s, NH).
12,14-Dichloro-13-isopropyl-5,8,11a-trimethyl-6-oxo-6,7,
7a,8,9,10,11,11a-octahydro-5H-dibenzo[b,d]-azepine-8-car-
boxylic acid (13a). A mixture of the methyl ester 12a
(73 mg, 0.171 mmol), KOH (96 mg, 1.705 mmol,
10.0 equiv) and 18-crown ether-6 (113 mg, 0.426 mmol,
2.5 equiv) in MeOH (1 mL) was stirred at 80 ꢁC for
13 h, the whole was cooled, and the solvent was evaporat-

T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
8025
1
ed in vacuum. The residue was diluted with water
(10 mL), acidified with 2 N HCl (2 mL), and the whole
was extracted with CHCl3 (3· 20 mL). The combined
organic layer was washed with brine (1· 20 mL), dried
over Na2SO4, filtered, and the solvent was evaporated in
vacuum. The residue was purified by flash chromatogra-
phy (AcOEt only) to afford 13a (65 mg, 0.156 mmol, yield
92%) as a colorless solid. Compound 13a: colorless pow-
der. Mp 155–159 ꢁC (n-hexane). 1H NMR (CDCl3) 1.34
(3H, s, CH3), 1.39 (6H, d, J = 7.1 Hz, 2CH3CH), 1.43
(3H, s, CH3), 1.54 (2H, m, 2CH), 1.73–1.88 (4H, m,
4CH), 2.18 (1H, d, J = 8.6 Hz, CH), 2.83 (1H, d,
J = 16.3 Hz, CH), 3.19 (3H, s, NCH3), 3.79–3.85 (1H,
m, CH), 3.94 (1H, br s, CH3CH), 7.19 (1H, br s, ArH),
10.03 (1H, br s, CO2H). LRMS (FAB+) m/z 412
127 ꢁC (n-hexane/AcOEt). H NMR (CDCl3) 1.24 (6H,
d, J = 7.0 Hz, 2CH3CH), 1.31 (3H, s, CH3), 1.50 (3H, s,
CH3), 1.67–1.78 (4H, m, 2CH), 1.98–2.04 (3H, m, 3CH),
2.50–2.62 (2H, m, 2CH), 2.86–2.90 (1H, m, CH3CH),
3.28 (3H, s, NCH3), 6.97 (1H, d, J = 1.8 Hz, ArH), 7.06
(1H, dd, J = 8.2 Hz, 1.9 Hz, ArH), 7.26 (1H, d,
J = 8.2 Hz,ArH). LRMS(FAB+)m/z344([M+H]+).Anal.
CalcdforC21H29NO3:C, 73.44;H,8.51;N,4.08. Found:C,
73.28; H, 8.31; N, 4.03.
Toluene-4-sulfonic acid pent-4-ynyl ester (15). To a solu-
tion of 4-pentyn-1-ol 14 (1.10 mL, 0.012 mol) and Et3N
(1.81 mL, 0.013 mol, 1.1 equiv) in CH2Cl2 (8 mL) was
dropwise added a solution of TsCl (2.49 g, 0.0113 mol,
1.1 equiv) in CH2Cl2 (20 mL) at 0 ꢁC for 20 min, in a
flask fitted with a CaCl2 tube. Stirring was continued
at rt for 20 h, and the whole was poured into icewater
(50 mL). The separated aqueous layer was extracted
with CHCl3 (3· 30 mL). Then the combined organic
layer was washed with water (1· 30 mL) and brine (1·
30 mL), dried over Na2SO4, filtered, and then the sol-
vent was evaporated in vacuum. The residue was puri-
fied by flash chromatography (n-hexane/AcOEt = 4:1)
to afford 15 (2.37 g, 0.010 mol, yield 84%) as a colorless
liquid. Compound 15: 1H NMR (CDCl3) 1.85–1.89 (2H,
m, CH2CH2CH2), 1.89 (1H, t, J = 2.7 Hz, HCC), 2.26
(2H, dt, J = 6.9 Hz, 2.7 Hz, CCH2), 2.45 (3H, s, CH3),
4.15 (2H, t, J = 6.1 Hz, CH2O), 7.35 (2H, d,
J = 8.2 Hz, ArH), 7.80 (2H, d, J = 8.4 Hz, ArH).
([M(35Cl2)+H]+),
414
([M(35Cl37Cl)+H]+),
416
([M(37Cl2)+H]+). Anal. Calcd for C21H27Cl2NO2Æ1/4H2O:
C, 60.51; H, 6.64; N, 3.36. Found: C, 60.72; H, 6.82; N, 3.26.
13-Isopropyl-5,8,11a-trimethyl-6-oxo-6,7,7a,8,9,10,11,11a-
octahydro-5H-dibenzo[b,d]azepine-8-carboxylic acid methyl
ester (12b). To a suspension of NaH (60% in mineral oil,
washed twice with n-hexane) (13 mg, 0.032 mmol,
1.1 equiv) in DMF (1 mL) was dropwise added a solution
of the carboxylic acid 10b (100 mg, 0.291 mmol) in DMF
(1 mL) at 0 ꢁC for 1 min, in a flask fitted with a CaCl2 tube.
After stirring at 0 ꢁC for 15 min, MeI (0.091 mL,
1.456 mmol, 5.0 equiv) was added to the reaction mixture
at 0 ꢁC. Stirring was continued at rt for 14 h, the solvent
was evaporated in vacuum, and the residue was diluted
with 1NHCl and then extracted with CHCl3 (3· 20 mL).
The combined organic layer was washed with brine (1·
20 mL), dried over Na2SO4, filtered, and then the solvent
was evaporated in vacuum. The residue was purified by
flash chromatography (n-hexane/AcOEt = 2:1) to afford
12b (90 mg, 0.252 mmol, yield 87%) as a pale orange oil.
Compound 12b: colorless plates. Mp 149–150 ꢁC (n-hex-
5-Iodopent-1-yne (16). A mixture of toluene-4-sulfonic
acid pent-4-ynyl ester 15 (817 mg, 3.430 mmol) and
NaI (1.234 g, 8.232 mmol, 2.4 equiv) in acetone (8 mL)
was stirred at 70 ꢁC for 1 h, the mixture was cooled,
and poured into ice-water (20 mL). The whole was
extracted with Et2O (3· 20 mL). The combined organic
layer was washed with satd NaHCO3 (1· 20 mL) and
brine (1· 20 mL), dried over Na2SO4, filtered, and then
the solvent was evaporated in vacuum to afford 16
(600 mg, 3.093 mmol, yield 90%) as a yellow liquid.
Compound 16: 1H NMR (CDCl3) 2.00 (1H, t,
J = 2.6 Hz, HCC), 2.01 (2H, tt, J = 6.7 Hz, 6.7 Hz,
CH2CH2I), 2.35 (2H, dt, J = 6.7 Hz, 2.6 Hz, CCH2),
3.32 (2H, t, J = 6.7 Hz, CH2I).
1
ane/AcOEt). H NMR (CDCl3) 1.26 (6H, d J = 7.0 Hz,
2CH3CH), 1.31 (3H, s, CH3), 1.48 (3H, s, CH3), 1.65–
1.68 (2H, m, 2CH), 1.74–1.77 (2H, m, 2CH), 1.90 (1H, d,
J = 13.6 Hz, CH), 1.89–1.98 (3H, m, 3CH), 2.50–2.63
(2H, m, 2CH), 2.88–2.92 (1H, m, CH3CH), 3.27 (3H, s,
NCH3), 3.60 (3H, s, CO2CH3), 7.01 (1H, d, J = 1.8 Hz,
ArH), 7.07 (1H, dd, J = 8.2 Hz, 1.9 Hz, ArH), 7.28 (1H,
d, J = 8.2 Hz, ArH). LRMS (FAB+) m/z 358 ([M+H]+).
Anal. Calcd for C22H31NO3: C, 73.91; H, 8.74; N, 3.92.
Found: C, 73.78; H, 8.89; N, 3.97.
12,14-Dichloro-13-isopropyl-8,11a-dimethyl-6-oxo-5-pent-
4-ynyl-6,7,7a,8,9,10,11,11a-octahydro-5H-dibenzo[b,d]aze-
pine-8-carboxylic acid methyl ester (17a). To a suspension
of NaH (60% in mineral oil, washed twice with n-hexane)
(6 mg, 0.138 mmol, 1.1 equiv) in DMF (1 mL) was drop-
wise added a solution of the methyl ester 10a (52 mg,
0.125 mmol) in DMF (1.5 mL) at 0 ꢁC for 2 min, in a flask
fitted with a CaCl2 tube. After stirring at 0 ꢁC for 30 min, a
solution of 5-iodopent-1-yne 16 (122 mg, 0.627 mmol,
5.0 equiv)inDMF (1.5 mL) wasaddedtothe reaction mix-
ture at 0 ꢁC. Stirring was continued at rt for 17 h, the sol-
vent was evaporated in vacuum, and the residue was
diluted with water (20 mL) and the whole was extracted
with CHCl3 (3· 20 mL). The combined organic layer was
washed with brine (1· 20 mL), dried over Na2SO4, filtered,
and then evaporated in vacuum. The residue was purified
by flash chromatography (n-hexane/AcOEt = 4:1) to af-
ford 17a (44 mg, 0.093 mmol, yield 74%) as a colorless
13-Isopropyl-5,8,11a-trimethyl-6-oxo-6,7,7a,8,9,10,11,11a-
octahydro-5H-dibenzo[b,d]azepine-8-carboxylic acid (13b).
A mixture of the methyl ester 12b (50 mg, 0.141 mmol),
KOH (79 mg, 1.410 mmol, 10.0 equiv) and 18-crown
ether-6 (93 mg, 0.352 mmol, 2.5 equiv) in MeOH (1 mL)
was stirred at 80 ꢁC for 18 h, the mixture was cooled, and
the solvent was evaporated in vacuum. The residue was
diluted with water (10 mL), acidified with 2 N HCl
(2 mL), and then extracted with CHCl3 (3· 20 mL). The
combined organic layer was washed with brine (1·
20 mL), dried over Na2SO4, filtered, and then the solvent
was evaporated in vacuum. The residue was purified by
flash chromatography (n-hexane/AcOEt = 1:2) to afford
13b (42 mg, 0.121 mmol, yield 86%) as a colorless amor-
phous solid. Compound 13b: colorless flakes. Mp 123–

8026
T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
oil. Compound 17a: 1H NMR (CDCl3) 1.42 (3H, s, CH3),
1.42 (6H, d, J = 7.0 Hz, 2CH3CH), 1.47 (3H, s, CH3), 1.72
(2H, m, 2CH), 1.83–1.91 (5H, m, 5CH), 2.01 (1H, t,
J = 2.3 Hz, HCC), 2.21 (1H, d, J = 8.5 Hz, CH), 2.30
(2H, t, J = 7.0 Hz, CCH2), 2.90 (1H, d, J = 16.2 Hz, CH),
3.05–3.12 (1H, m, CH), 3.66 (3H, s, CO2CH3), 3.70 (1H,
dd, J = 16.4 Hz, 8.9 Hz, CH), 3.96 (1H, br s, CH3CH),
4.11–4.28 (1H, m, CH), 7.21 (1H, br s, ArH). LRMS
(FAB+) m/z 478 ([M(35Cl2)+H]+), 480 ([M(35Cl37Cl)+H]+),
482 ([M(37Cl2)+H]+).
was filtered, the solvent was evaporated in vacuum, and
the resultant residue was purified by flash chromatogra-
phy (n-hexane/AcOEt = 2:1) to afford 21 (32 mg,
0.031 mmol, yield 84%) as a colorless oil.
1,4-Bis[[5-(12,14-dichloro-13-isopropyl-8,11a-dimethyl-8-
hydroxycarbonyl-6-oxo-6,7,7a,8,9,10,11,11a-octahydro-
5H-dibenzo[b,d]azepin-5-yl)]pent-1-ynyl]benzene (29). A
mixture of the methyl ester 21 (46 mg, 0.045 mmol),
KOH (25 mg, 0.445 mmol, 10.0 equiv) and 18-crown
ether-6 (59 mg, 0.223 mmol, 5.0 equiv) in MeOH
(2 mL) was stirred at 80 ꢁC for 19 h, the mixture was
cooled, and the solvent was evaporated in vacuum.
The residue was diluted with water (20 mL), acidified
with 2 N HCl (2 mL) and then extracted with CHCl3
(3· 20 mL). The combined organic layer was washed
with brine (1· 20 mL), dried over Na2SO4, filtered,
and then the solvent was evaporated in vacuum. The
residue was purified by flash chromatography (AcOEt/
MeOH = 10:1) to afford 29 (29 mg, 0.029 mmol, yield
65%) as a colorless solid. Compound 29: colorless pow-
der. Mp 211–214 ꢁC (n-hexane/AcOEt). 1H NMR
(CD3OD) 1.31 (6H, s, 2CH3), 1.43–1.49 (12H, m,
2CH3CH and 2CH3), 1.76–2.18 (14H, m, 14CH), 2.27
(2H, d, J = 8.6 Hz, 2CH), 2.54–2.55 (4H, m, 4CH),
3.15 (2H, d, J = 16.5 Hz, 2CH), 3.65–3.72 (2H,
m, 2CH), 4.02 (2H, br s, 2CH3CH), 4.30 (2H, m,
2CH), 7.32 (4H, s, ArH), 7.35 (2H, br s, ArH). LRMS
12,14-Dichloro-13-isopropyl-8,11a-dimethyl-6-oxo-5-[5-(4-
iodophenyl)pent-4-ynyl]-6,7,7a,8,9,10,11,11a-octahydro-5H-
dibenzo[b,d]azepine-8-carboxylic acid methyl ester (23) and
1,4-bis[[5-(2,4-dichloro-3-isopropyl-8,11a-dimethyl-8-meth-
oxycarbonyl-6-oxo-6,7,7a,8,9,10,11,11a-octahydro-5H-dib-
enzo[b,d]azepin-5-yl)]pent-1-ynyl]benzene (21). A mixture
of the methyl ester 17a (44 mg, 0.093 mmol), p-diiodoben-
zene (34 mg, 0.102 mmol, 1.1 equiv), Pd(PPh3)2Cl2 (1 mg,
0.001 mmol, 0.02 equiv), and CuI (1 mg, 0.005 mmol,
0.06 equiv) in Et3N (2 mL) was stirred at rt for 39 h under
Ar atmosphere. The reaction mixture was filtered, the sol-
vent was evaporated in vacuum, and the resultant residue
was purified by flash chromatography (n-hexane/
AcOEt = 2:1) to afford the mixture of the monosubstitut-
ed compound 25 and p-diiodobenzene (55 mg) as pale yel-
low oil and the disubstituted compound 21 (14 mg,
0.013 mmol, yield 28%) as pale yellow amorphous solid.
Then the former mixture was purified by flash chromatog-
raphy (n-hexane/AcOEt = 4:1) again to afford the mono-
substituted compound 25 (32 mg, 0.046 mmol, yield 50%)
(FAB+)
m/z
1001
([M(35Cl4)+H]+),
1003
([M(35Cl337Cl)+H]+), 1005 ([M(35Cl237Cl2)+H]+), 1007
([M(35Cl37Cl3)+H]+), 1009 ([M(37Cl4)+H]+). HRMS
(FAB+) found: 1001.3432. Calcd for C56H6435Cl4N2O6:
1001.3449. Anal. Calcd for C56H64Cl4N2O6Æ3/2H2O: C,
65.30; H, 6.56; N, 2.72. Found: C, 65.34; H, 6.59; N, 2.60.
1
as a pale orange amorphous solid. Compound 25: H
NMR (CDCl3) 1.41 (6H, d, J = 5.3 Hz, 2CH3CH), 1.42
(3H, s, CH3), 1.46 (3H, s, CH3), 1.70 (2H, m, 2CH),
1.81–1.97 (5H, m, 5CH), 2.02–2.09 (1H, m, CH), 2.21
(1H, d, J = 8.5 Hz, CH), 2.50 (2H, t, J = 6.9 Hz, CH2),
2.86 (1H, d, J = 16.2 Hz, CH), 3.06–3.50 (1H, m, CH),
3.51 (3H, s, CO2CH3), 3.72 (1H, dd, J = 16.1 Hz,
8.6 Hz, CH), 3.95 (1H, br s, CH3CH), 4.33–4.39 (1H, m,
CH), 7.14 (2H, d, J = 8.1 Hz, ArH), 7.20 (1H, br s,
ArH), 7.62 (2H, d, J = 8.1 Hz, ArH). LRMS (FAB+) m/
z 680 ([M(35Cl2)+H]+), 682 ([M(35Cl37Cl)+H]+), 684
([M(37Cl2)+H]+). Compound 21: 1H NMR (CDCl3)
1.43 (12H, d, J = 6.0 Hz, 4CH3CH), 1.44 (6H, s, 2CH3),
1.48 (6H, s, 2CH3), 1.71 (4H, m, 4CH), 1.83–1.99 (10H,
m, 10CH), 2.04–2.09 (2H, m, 2CH), 2.22 (2H, d,
J = 8.5 Hz, 2CH), 2.53 (4H, d, J = 6.9 Hz, 4CH), 2.89
(2H, d, J = 16.3 Hz, 2CH), 3.09–3.16 (2H, m, 2CH),
3.53 (6H, s, 2CO2CH3), 3.74 (2H, dd, J = 16.1 Hz,
8.6 Hz, 2CH), 3.97 (2H, br s, 2CH3CH), 4.35–4.41 (2H,
m, 2CH), 7.22 (2H, br s, ArH), 7.35 (4H, s, ArH). LRMS
13-Isopropyl-8,11a-dimethyl-6-oxo-5-pent-4-ynyl-6,7,7a,8,9,
10,11,11a-octahydro-5H-dibenzo[b,d]-azepine-8-carbox-
ylic acid methyl ester (17d). To a suspension of NaH (60%
in mineral oil, washed twice with n-hexane) (12 mg,
0.292 mmol, 1.1 equiv) in DMF (1 mL) was dropwise
added a solution of the methyl ester 10b (91 mg,
0.265 mmol) in DMF (1.5 mL) at 0 ꢁC over 1 min, in a
flask fitted with a CaCl2 tube. After stirring at 0 ꢁC for
30 min, a solution of 5-iodopent-1-yne 16 (257 mg,
1.326 mmol, 5.0 equiv) in DMF (1.5 mL) was added to
the reaction mixture at 0 ꢁC. Stirring was continued at
rt for 18 h, and the solvent was evaporated in vacuum,
and the residue was diluted with water (20 mL) and then
extracted with CHCl3 (3· 20 mL). The combined organ-
ic layer was washed with brine (1· 20 mL), dried over
Na2SO4, filtered and the solvent was evaporated in vac-
uum. The residue was purified by flash chromatography
(n-hexane/AcOEt = 4:1) to afford 17b (76 mg,
0.85 mmol, yield 70%) as a colorless oil. Compound
17b: 1H NMR (CDCl3) 1.27 (6H, d, J = 6.9 Hz,
2CH3CH), 1.32 (3H, s, CH3), 1.48 (3H, s, CH3), 1.66–
1.68 (2H, m, 2CH), 1.75–1.78 (2H, m, 2CH), 1.90 (1H,
d, J = 13.3 Hz, CH), 1.95–2.04 (2H, m, 2CH), 1.96
(1H, t, J = 2.6 Hz, HCC), 2.10–2.17 (1H, m, CH),
2.19–2.37 (2H, m, 2CH), 2.50–2.59 (2H, m, 2CH),
2.88–2.96 (1H, m, CH3CH), 3.48–3.56 (1H, m, CH),
3.60 (3H, s, CO2CH3), 3.90–3.97 (1H, m, CH), 7.08
(1H, dd, J = 8.2 Hz, 1.9 Hz, ArH), 7.11 (1H, d,
(FAB+)
m/z
1029
([M(35Cl4)+H]+),
1031
([M(35Cl337Cl)+H]+), 1033 ([M(35Cl237Cl2)+H]+), 1035
([M(35Cl37Cl3)+H]+).
1,4-Bis[[5-(12,14-dichloro-13-isopropyl-8,11a-dimethyl-8-
methoxycarbonyl-6-oxo-6,7,7a,8,9,10,11,11a-octahydro-
5H-dibenzo[b,d]azepin-5-yl)]pent-1-ynyl]benzene
(21).
The mixture of the alkyne 17a (17 mg, 0.036 mmol), the
iodide 25 (32 mg, 0.047 mmol, 1.3 equiv), Pd(PPh3)2Cl2
(1 mg, 0.001 mmol, 0.04 equiv), and CuI (1 mg,
0.005 mmol, 0.14 equiv) in Et3N (2 mL) was stirred at rt
for 72 h under Ar atmosphere. The reaction mixture

T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
8027
J = 1.9 Hz, ArH), 7.29 (1H, d, J = 8.2 Hz, ArH). LRMS
(FAB+) m/z 410 ([M+H]+).
KOH (38 mg, 0.672 mmol, 10.0 equiv), and 18-crown
ether-6 (89 mg, 0.336 mmol, 5.0 equiv) in MeOH
(2 mL) was stirred at 80 ꢁC for 18 h, the whole was
cooled, and the solvent was evaporated in vacuum.
The resultant residue was diluted with water (20 mL),
acidified with 2 N HCl (2 mL), and then extracted with
CHCl3 (3· 20 mL). The combined organic layer was
washed with brine (1· 20 mL), dried over Na2SO4, fil-
tered, and then the solvent was evaporated in vacuum.
The residue was purified by flash chromatography
(AcOEt/MeOH = 10:1) to afford the desired compound
28 (50 mg, 0.050 mmol, yield 75%) as a colorless solid.
Compound 28: colorless powder. Mp 207–213 ꢁC (n-
12,14-Dichloro-13-isopropyl-8,11a-dimethyl-6-oxo-5-[5-(3-
iodophenyl)pent-4-ylnyl]-6,7,7a,8,9,10,11,11a-octahydro-
5H-dibenzo[b,d]azepine-8-carboxylic acid methyl ester
(24) and 1,3-bis[[5-(12,14-dichloro-13-isopropyl-8,11a-di-
methyl-8-methoxycarbonyl-6-oxo-6,7,7a,8,9,10,11,11a-
octahydro-5H-dibenzo[b,d]azepin-5-yl)]pent-1-ynyl]ben-
zene (20). A mixture of the alkyne 17b (62 mg,
0.129 mmol), m-diiodobenzene (21 mg, 0064 mmol,
0.5 equiv), Pd(PPh3)2Cl2 (1 mg, 0.001 mmol, 0.01 equiv),
and CuI (1 mg, 0.005 mmol, 0.04 equiv) in Et3N (3 mL)
was stirred at rt for 49 h under Ar atmosphere. The reac-
tion mixture was filtered, the solvent was evaporated in
vacuum. The resultant residue was purified by flash chro-
matography (n-hexane/AcOEt = 4:1) to afford the mono-
substituted compound 24 (32 mg, 0.046 mmol, yield 36%)
as an orange oil and the disubstituted compound 20
(31 mg, 0.030 mmol, yield 47%) as an orange oil. Com-
1
hexane/CHCl3). H NMR (CDCl3) 1.29 (6H, s, 2CH3),
1.32 (12H, d, J = 7.1 Hz, 4CH3CH), 1.33 (6H, s,
2CH3), 1.61 (4H, m, 4CH), 1.75–1.89 (10H, m, 10CH),
2.13 (2H, d, J = 8.6 Hz, 2CH), 2.21 (4H, m, 4CH),
2.92–3.00 (2H,, m, 2CH), 2.98 (2H, d, J = 16.7 Hz,
2CH), 3.50–3.57 (2H, m, 2CH), 3.91 (2H, br s,
2CH3CH), 4.13–4.20 (2H, m, 2CH), 7.09 (3H, m,
ArH), 7.24 (2H, br s, ArH), 7.30 (1H, s, ArH). LRMS
1
pound 24: H NMR (CDCl3) 1.41 (6H, d, J = 5.9 Hz,
2CH3CH), 1.42 (3H, s, CH3), 1.47 (3H, s, CH3), 1.70–
1.71 (2H, m, 2CH), 1.82–1.93 (5H, m, 5CH), 2.03–2.10
(1H, m, CH), 2.20 (1H, d, J = 8.5 Hz, CH), 2.51 (2H, t,
J = 7.0 Hz, CH2), 2.87 (1H, d, J = 16.2 Hz, CH), 3.07–
3.14 (1H, m, CH), 3.52 (3H, s, CO2CH3), 3.72 (1H, dd,
J = 16.1 Hz, 8.6 Hz, CH), 3.96 (1H, br s, CH3CH),
4.32–4.39 (1H, m, CH), 7.02 (1H, t, J = 7.9 Hz, ArH),
7.20 (1H, br s, ArH), 7.37 (1H, td, J = 7.8 Hz, 1.2 Hz,
ArH), 7.61 (1H, td, J = 8.5 Hz, 1.4 Hz, ArH), 7.77
(1H, t, J = 1.6 Hz, ArH). LRMS (FAB+) m/z
680 ([M(35Cl2)+H]+), 682 ([M(35Cl37Cl)+H]+), 684
([M(37Cl2)+H]+). Compound 20: 1H NMR (CDCl3)
1.41 (6H, s, 2CH3), 1.41 (12H, d, J = 6.3 Hz, 4CH3CH),
1.47 (6H, s, 2CH3), 1.69–1.70 (4H, m, 4CH), 1.81–1.93
(10H, m, 10CH), 2.03–2.08 (2H, m, 2CH), 2.20 (2H, d,
J = 8.5 Hz, 2CH), 2.50 (4H, d, J = 7.0 Hz, 4CH), 2.88
(2H, d, J = 16.2 Hz, 2CH), 3.07–3.14 (2H, m, 2CH),
3.51 (6H, s, 2CO2CH3), 3.73 (2H, dd, J = 16.2 Hz,
8.7 Hz, 2CH), 3.96 (2H, br s, 2CH3CH), 4.32–4.39 (2H,
m, 2CH), 7.20 (2H, br s, ArH), 7.21 (2H, t, J = 7.7 Hz,
ArH), 7.32 (2H, d, J = 7.8 Hz, ArH), 7.44 (1H, s, ArH).
LRMS (FAB+) m/z 1029 ([M(35Cl4)+H]+), 1031
([M(35Cl337Cl)+H]+), 1033 ([M(35Cl237Cl2)+H]+), 1035
(M(35Cl37Cl3)+H)+).
(FAB+)
m/z
1001
([M(35Cl4)+H]+),
1003
([M(35Cl337Cl)+H]+), 1005 ([M(35Cl237Cl2)+H]+), 1007
([M(35Cl37Cl3)+H]+), 1009 ([M(37Cl4)+H]+). HRMS
(FAB+) found: 1001.4380. Calcd for C56H6535Cl4N2O6:
1001.4302. Anal. Calcd for C56H64Cl4N2O6Æ3/2H2O: C,
65.30; H, 6.56; N, 2.72. Found: C, 65.03; H, 6.78; N, 2.62.
13-Isopropyl-8,11a-dimethyl-6-oxo-5-[5-(3-iodophenyl)pent-
4-ylnyl]-6,7,7a,8,9,10,11,11a-octahydro-5H-dibenzo[b,d]aze-
pine-8-carboxylicacidmethyl ester(26) and1,3-bis[[5-(13-iso-
propyl-8,11a-dimethyl-8-methoxycarbonyl-6-oxo-6,7,7a,8,9,
10,11,11a-octahydro-5H-dibenzo[b,d]azepin-5-yl)]pent-1-ynyl]-
benzene (22). A mixture of the alkyne 17b (66 mg,
0.161 mmol), m-diiodobenzene (53 mg, 0.161 mmol),
Pd(PPh3)2Cl2 (1 mg, 0.001 mmol, 0.01 equiv), and CuI
(1 mg, 0.005 mmol, 0.03 equiv) in Et3N (2 mL) was stirred
at rt for 19 h under Ar atmosphere. The reaction mixture
was filtered, the solvent was evaporated in vacuum, and the
resultant residue was purified by flash chromatography (n-
hexane/AcOEt = 4:1–2:1)toaffordthemonosubstitutedcom-
pound 26 (63 mg, 0.103 mmol, yield 64%) as yellow oil and
the disubstituted compound 22 (26 mg, 0.029 mmol, yield
1
36%) as a yellow oil. Compound 26: H NMR (CDCl3)
1.23 (6H, d, J = 6.9 Hz, 2CH3CH), 1.34 (3H, s, CH3), 1.50
(3H, s, CH3), 1.68–1.69 (2H, m, 2CH), 1.77–1.79 (2H, m,
2CH), 1.92 (1H, d, J = 8.3 Hz, CH), 1.97–2.00 (2H, m,
2CH), 2.09 (1H, m, CH), 2.22–2.23 (1H, m, CH), 2.46–2.64
(4H, m, 4CH), 2.83–2.90 (1H, m, CH3CH), 3.55–3.61 (1H,
m, CH), 3.62 (3H, s, CO2CH3), 3.95–4.02 (1H, m, CH), 7.00
(1H, t, J = 7.9 Hz, ArH), 7.08 (1H, dd, J = 8.2 Hz, 1.8 Hz,
ArH), 7.13 (1H, d, J = 1.8 Hz, ArH), 7.30 (1H, t,
J = 8.3 Hz, ArH), 7.32 (1H, dt, J = 8.0 Hz, 1.4 Hz, ArH),
7.60 (1H, td, J = 7.9 Hz, 1.2 Hz, ArH), 7.73 (1H, t,
J = 1.6 Hz, ArH). LRMS (FAB+) m/z 612 ([M+H]+). Com-
1,3-Bis[[5-(12,14-dichloro-13-isopropyl-8,11a-dimethyl-8-
methoxycarbonyl-6-oxo-6,7,7a,8,9,10,11,11a-octahydro-
5H-dibenzo[b,d]azepin-5-yl)]pent-1-ynyl]benzene (20). A
mixture of the alkyne 17a (22 mg, 0.046 mmol), the iodide
24 (32 mg, 0.046 mmol), Pd(PPh3)2Cl2 (1 mg,
0.001 mmol, 0.03 equiv), and CuI (1 mg, 0.005 mmol,
0.1 equiv) in Et3N (2 mL) was stirred at rt for 48 h under
Ar atmosphere. The reaction mixture was filtered, and the
solvent was evaporated in vacuum, and the residue was
1
purified
AcOEt = 2:1) to afford 20 (33 mg, 0.032 mmol, yield
by
flash
chromatography
(n-hexane/
pound 22: H NMR (CDCl3) 1.19 (12H, d, J = 6.9 Hz,
4CH3CH), 1.33 (6H, s, 2CH3), 1.48 (6H, s, 2CH3), 1.67–
1.68 (4H, m, 4CH), 1.75–1.77 (4H, m, 4CH), 1.90 (2H, d,
J = 7.3 Hz, 2CH), 2.01–2.11 (2H, m, 2CH), 2.17–2.24 (2H,
m, 2CH), 2.40–2.62 (8H, m, 8CH), 2.80–2.90 (2H, m,
2CH3CH), 3.42–3.60 (2H, m, 2CH), 3.61 (6H, s, 2CO2CH3),
3.92–4.00 (2H, m, 2CH), 7.05 (2H, dd, J = 8.2 Hz, 1.9 Hz,
ArH), 7.11 (2H, d, J = 1.9 Hz, ArH), 7.16 (1H, d,
70%) as an orange oil.
1,3-Bis[[5-(12,14-dichloro-13-isopropyl-8,11a-dimethyl-8-
hydroxycarbonyl-6-oxo-6,7,7a,8,9,10,11,11a-octahydro-
5H-dibenzo[b,d]azepin-5-yl)]pent-1-ynyl]benzene (28). A
mixture of the methyl ester 20 (69 mg, 0.067 mmol),

8028
T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
J = 8.4 Hz, ArH), 7.24 (2H, dd, J = 8.6 Hz, 1.5 Hz, ArH),
7.28 (2H, d, J = 8.3 Hz, ArH), 7.36 (1H, m, ArH). LRMS
(FAB+) m/z 893 ([M+H]+).
2.04 (2H, m, 2CH), 2.20 (2H, d, J = 8.5 Hz, 2CH),
2.36 (4H, d, J = 7.1 Hz, 4CH), 2.83 (2H, d, J = 16.2 Hz,
2CH), 2.98–3.05 (2H, m, 2CH), 3.67 (6H, s, 2CO2CH3),
3.69 (2H, dd, J = 16.0 Hz, 8.6 Hz, 2CH), 3.94
(2H, br s, 2CH3CH), 4.26–4.32 (2H, m, 2CH), 7.19–
7.23 (2H, br s, ArH). LRMS (FAB+) m/z 1029
([M(35Cl4)+H]+), 1031 ([M(35Cl337Cl)+H]+), 1033
([M(35Cl237Cl2)+H]+), 1035 ([M(35Cl37Cl3)+H]+), 1037
([M(37Cl4)+H]+).
1,3-Bis[[5-(13-isopropyl-8,11a-dimethyl-8-hydroxycarbon-
yl-6-oxo-6,7,7a,8,9,10,11,11a-octahydro-5H-dibenzo[b,d]aze-
pin-5-yl)]pent-1-ynyl]benzene (30). A mixture of the methyl
ester 22 (26 mg, 0.029 mmol), KOH (16 mg, 0.289 mmol,
10.0 equiv), and 18-crown ether-6 (38 mg, 0.145 mmol,
5.0 equiv) in MeOH (2 mL) was stirred at 80 ꢁC for
17 h, the whole was cooled, and the solvent was evaporat-
ed in vacuum. The residue was diluted with water
(20 mL), acidified with 2 N HCl (2 mL), and then extract-
ed with CHCl3 (3· 20 mL). The combined organic layer
was washed with brine (1· 20 mL), dried over Na2SO4, fil-
tered, and then the solvent was evaporated in vacuum.
The residue was purified by flash chromatography
(AcOEt/MeOH = 10:1) to afford 30 (9 mg, 0.010 mmol,
yield 36%) as a colorless amorphous solid. Compound
30: colorless powder. Mp 159–163 ꢁC (n-hexane/CHCl3).
1H NMR (CD3OD) 1.04 (12H, d, J = 6.9 Hz, 4CH3CH),
1.22 (6H, s, 2CH3), 1.33 (6H, s, 2CH3), 1.57–1.68 (8H, m,
8CH), 1.84–1.88 (8H, m, 8CH), 2.21–2.27 (2H, m, 2CH),
2.38–2.48 (8H, m, 8CH), 2.73–2.78 (2H, m, 2CH3CH),
3.30–3.38 (2H, m, 2CH), 3.80 (2H, m, 2CH), 7.01–7.08
(6H, m, ArH), 7.23–7.25 (4H, m, ArH). LRMS (FAB+)
m/z 866 ([M+H]+). Anal. Calcd for C56H68N2O6Æ2H2O:
C, 74.64; H, 8.05; N, 3.11. Found: C, 74.45; H, 8.31; N,
2.83.
1,2-Bis[[5-(12,14-dichloro-13-isopropyl-8,11a-dimethyl-8-
methoxycarbonyl-6-oxo-6,7,7a,8,9,10,11,11a-octahydro-
5H-dibenzo[b,d]azepin-5-yl)]pent-1-ynyl]benzene (19). A
mixture of the alkyne 17a (15 mg, 0.032 mmol), the io-
dide 23 (22 mg, 0.032 mmol), Pd(PPh3)4 (2 mg,
0.002 mmol, 0.05 equiv), and CuI (1 mg, 0.010 mmol,
0.3 equiv) in Et3N (1 mL) was stirred at rt for 19 h under
Ar atmosphere. The reaction mixture was filtered, the
solvent was evaporated in vacuum, and the resultant res-
idue was purified by flash chromatography (n-hexane/
AcOEt = 2:1) to afford 19 (11 mg, 0.011 mmol, yield
33%) as an orange oil. Compound 19: 1H NMR
(CDCl3) 1.41 (6H, s, 2CH3), 1.42 (12H, d, J = 6.6 Hz,
4CH3CH), 1.46 (6H, s, 2CH3), 1.68 (4H, m, 4CH),
1.81–1.92 (8H, m, 8CH), 1.95–2.00 (2H, m, 2CH),
2.07–2.14 (2H, m, 2CH), 2.20 (2H, d, J = 8.0 Hz,
2CH), 2.57 (4H, t, J = 6.9 Hz, 2CH2), 2.89 (2H, d,
J = 16.2 Hz, 2CH), 3.13–3.20 (2H, m, 2CH), 3.47 (6H,
s, 2CO2CH3), 3.74 (2H, dd, J = 16.2 Hz, 8.6 Hz, 2CH),
3.96 (2H, br s, 2CH3CH), 4.33–4.38 (2H, m, 2CH),
7.20 (2H, dd, J = 5.7 Hz, 3.3 Hz, ArH), 7.19–7.20 (2H,
m, ArH), 7.41 (2H, dd, J = 5.7 Hz, 3.4 Hz, ArH). LRMS
12,14-Dichloro-13-isopropyl-8,11a-dimethyl-6-oxo-5-[5-(2-
iodophenyl)pent-4-ynyl]-6,7,7a,8,9,10,11,11a-octahydro-
5H-dibenzo[b,d]azepine-8-carboxylic acid methyl ester
(23) and 1,10-bis[5-(12,14-dichloro-13-isopropyl-8,11a-di-
methyl-8-methoxycarbonyl-6-oxo-6,7,7a,8,9,10,11,11a-
octahydro-5H-dibenzo[b,d]azepin-5-yl)]decane-4,6-diyne (18).
A mixture of the alkyne 17a (77 mg, 0.161 mmol), o-di-
iodobenzene (0.010 mL, 0.080 mmol, 0.5 equiv),
Pd(PPh3)2Cl2 (1 mg, 0.001 mmol, 0.01 equiv), and CuI
(1 mg, 0.005 mmol, 0.03 equiv) in Et3N (2 mL)was stirred
at rt for 48 h under Ar atmosphere. The reaction mixture
was filtered, the solvent was evaporated in vacuum and
the resultant residue was purified by flash chromatogra-
phy (n-hexane/AcOEt = 2:1) to afford the monosubstitut-
ed compound 23 (17 mg, 0.025 mmol, yield 15%) as
orange oil and the homodimeric compound 18 (52 mg,
0.054 mmol, yield 67%) as an orange oil. Compound 23:
1H NMR (CDCl3) 1.43 (6H, d, J = 7.5 Hz, 2CH3CH),
1.44 (3H, s, CH3), 1.49 (3H, s, CH3), 1.70 (2H, m,
2CH), 1.85–1.92 (4H, m, 2CH), 2.02–2.03 (1H, m, CH),
2.12–2.17 (1H, m, CH), 2.22 (1H, d, J = 8.5 Hz, CH),
2.61 (2H, t, J = 6.9 Hz, CH2), 2.91 (1H, d, J = 16.3 Hz,
CH), 3.20–3.27 (1H, m, CH), 3.52 (3H, s, CO2CH3),
3.77 (1H, dd, J = 16.1 Hz, 8.6 Hz, CH), 3.98 (1H, br s,
CH3CH), 4.34–4.38 (1H, m, CH), 6.98 (1H, dt,
J = 7.7 Hz, 1.7 Hz, ArH), 7.22 (1H, br s, ArH), 7.29
(1H, dt, J = 7.6 Hz, 1.2 Hz, ArH), 7.44 (1H, dd,
J = 7.7 Hz, 1.6 Hz, ArH), 7.84 (1H, dd, J = 8.0 Hz,
1.6 Hz, ArH). LRMS (FAB+) m/z 680 ([M(35Cl2)+H]+),
682 ([M(35Cl37Cl)+H]+), 684 ([M(37Cl2)+H]+). Com-
pound 18: 1H NMR (CDCl3) 1.40 (6H, s, 2CH3),
1.41 (12H, d, J = 5.8 Hz, 4CH3CH), 1.46 (6H, s, 2CH3),
1.71 (4H, m, 4CH), 1.79–1.92 (10H, m, 10CH), 1.95–
(FAB+)
m/z
1029
([M(35Cl4)+H]+),
1031
([M(35Cl337Cl3)+H]+), 1033 ([M(35Cl237Cl2)+H]+), 1035
([M(35Cl37Cl3)+H]+).
1,2-Bis[[5-(12,14-dichloro-13-isopropyl-8,11a-dimethyl-8-
hydroxycarbonyl-6-oxo-6,7,7a,8,9,10,11,11a-octahydro-
5H-dibenzo[b,d]azepin-5-yl)]pent-1-ynyl]benzene (27). A
mixture of the methyl ester 19 (11 mg, 0.011 mmol),
KOH (60 mg, 0.108 mmol, 10.0 equiv), and 18-crown
ether-6 (14 mg, 0.054 mmol, 5.0 equiv) in MeOH
(2 mL) was stirred at 80 ꢁC for 16 h, the whole was
cooled, and the solvent was evaporated in vacuum,
and the resultant residue was diluted with water
(20 mL), acidified with 2 N HCl (2 mL) and then
extracted with CHCl3 (3· 20 mL). The combined organ-
ic layer was washed with brine (1· 20 mL), dried over
Na2SO4, filtered, and then the solvent was evaporated
in vacuum. The residue was purified by flash chromatog-
raphy (AcOEt/MeOH = 20:1) to afford 27 (4 mg,
0.004 mmol, yield 41%) as a colorless solid. Compound
27: pale yellow powder. Mp 181–184 ꢁC (n-hexane/
1
AcOEt). H NMR (CD3OD) 1.28 (6H, s, 2CH3), 1.34
(12H, m, 2CH3CH and 2CH3), 1.73 (14H, m, 14CH),
2.21 (6H, m, 6CH), 2.98 (4H, m, 4CH), 3.54 (2H, m,
2CH), 3.91 (2H, br s, 2CH3CH), 4.15 (2H, m, 2CH),
7.06 (2H, s, ArH), 7.27 (4H, m, ArH). LRMS (FAB+)
m/z 1001 ([M(35Cl4)+H]+), 1003 ([M(35Cl337Cl)+H]+),
1005 ([M(35Cl237Cl2)+H]+), 1007 ([M(35Cl37Cl3)+H]+),
1009 ([M(37Cl4)+H]+). Anal. Calcd for C56H64Cl4N2O6Æ
3/2H2O: C, 65.30; H, 6.56; N, 2.72. Found: C, 65.17; H,
6.79; N, 2.69.

T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
8029
12,14-Dichloro-13-isopropyl-8,11a-dimethyl-6-oxo-5-pent-
4-ynyl-6,7,7a,8,9,10,11,11a-octahydro-5H-dibenzo[b,d]aze-
pine-8-carboxylic acid (31). A mixture of the methyl ester
17a (41 mg, 0.086 mmol), KOH (48 mg, 0.863 mmol,
10.0 equiv) and 18-crown ether-6 (57 mg, 0.216 mmol,
2.5 equiv) in MeOH (1 mL) was stirred at 80 ꢁC for 17 h,
the whole was cooled, and the solvent was evaporated in
vacuum. The residue was diluted with water (10 mL), acid-
ified with 2 N HCl (2 mL), and then extracted with CHCl3
(3· 20 mL). The combined organic layer was washed with
brine (1· 20 mL), dried over Na2SO4, filtered, and then the
solvent was evaporated in vacuum. The residue was puri-
fied by flash chromatography (AcOEt/MeOH = 10:1) to
afford 31 (29 mg, 0.0624 mmol, yield 72%) as a colorless
solid. Compound 31: colorless flakes. Mp 122–126 ꢁC (n-
residue was purified by flash chromatography (n-hex-
ane/AcOEt = 10:1) to afford 33a (31 mg, 0.058 mmol,
yield 87%) as a colorless oil. Compound 33a: colorless
powder. Mp 127–130 ꢁC (n-hexane/AcOEt). H NMR
(CDCl3) 1.37–1.44 (12H, m, 4CH3), 1.70 (2H, m, 2CH),
1.79–1.88 (5H, m, 5CH), 1.90–2.04 (1H, m, CH), 2.11
(1H, d, J = 8.5 Hz, CH), 2.47 (2H, t, J = 6.9 Hz, CH2),
2.89 (1H, d, J = 16.3 Hz, CH), 3.07–3.14 (1H, m, CH),
3.48 (1H, dd, J = 16.1 Hz, 8.6 Hz, CH), 3.94 (1H, br s,
CH3CH), 4.27–4.36 (1H, m, CH), 7.13–7.36 (6H, m,
ArH). LRMS (FAB+) m/z 540 ([M(35Cl2)+H]+), 542
([M(35Cl37Cl)+H]+), 544 ([M(37Cl2)+H]+). Anal. Calcd
for C22H29Cl2NO3: C, 68.88; H, 6.53; N, 2.59. Found:
C, 68.62; H, 6.65; N, 2.58.
1
1
hexane/AcOEt). H NMR (CDCl3) 1.39 (3H, s, CH3),
13-Isopropyl-8,11a-dimethyl-6-oxo-5-(5-phenylpent-4-ynyl)-
6,7,7a,8,9,10,11,11a-octahydro-5H-dibenzo[b,d]azepine-8-
carboxylic acid methyl ester (32b). A mixture of the alkyne
17b (88 mg, 0.215 mmol), iodobenzene (0.029 mL,
0.258 mmol, 1.2 equiv), Pd(PPh3)2Cl2 (2 mg, 0.002 mmol,
0.01 equiv), and CuI (1 mg, 0.005 mmol, 0.02 equiv) in
Et3N (1 mL) was stirred at rt for 21 h under Ar atmo-
sphere. The reaction mixture was filtered, the solvent
was evaporated in vacuum, and the resultant residue was
1.40 (6H, d, J = 5.5 Hz, 2CH3CH), 1.44 (3H, s, CH3),
1.69–1.74 (2H, m, 2CH), 1.73 (1H, t, J = 2.4 Hz, HCC),
1.81–1.89 (5H, m, 5CH), 2.16 (1H, d, J = 8.5 Hz, CH),
2.50–2.53 (2H, m, 2CH), 2.94–3.01 (1H, s, CH), 3.04
(1H, d, J = 16.4 Hz, CH), 3.74 (1H, dd, J = 15.9 Hz,
9.0 Hz, CH), 3.95 (1H, br s, CH3CH), 4.36–4.43 (1H, m,
CH), 7.19 (1H, br s, ArH). LRMS (FAB+) m/z 464
([M(35Cl2)+H]+),
466
([M(35Cl37Cl)+H]+),
468
([M(37Cl2)+H]+). Anal. Calcd for C25H31Cl2NO3: C,
64.65; H, 6.73; N, 3.02. Found: C, 64.63; H, 6.87; N, 2.94.
purified
by
flash
chromatography
(n-hexane/
AcOEt = 2:1) to afford 32b (81 mg, 0.167 mmol. yield
78%) as a pale orange amorphous solid. Compound 32b:
1H NMR (CDCl3) 1.20 (6H, d, J = 6.9 Hz, 2CH3CH),
1.33 (3H, s, CH3), 1.49 (3H, s, CH3), 1.66–1.68 (2H, m,
2CH), 1.74–1.77 (2H, m, 2CH), 1.91 (1H, d, J = 13.3 Hz,
CH), 1.92–1.98 (2H, m, 2CH), 2.05–2.12 (1H, m, CH),
2.16–2.27 (1H, m, CH), 2.41–2.62 (4H, m, 4CH), 2.79–
2.90 (1H, m, CH3CH), 3.56–3.63 (1H, m, CH), 3.60 (3H,
s, CO2CH3), 3.95–4.02 (1H, m, CH), 7.06 (1H, dd,
J = 8.2 Hz, 1.7 Hz, ArH), 7.13 (1H, d, J = 1.9 Hz, ArH),
7.24–7.26 (3H, m, ArH), 7.28 (1H, d, J = 8.3 Hz, ArH),
7.34–7.37 (2H, m, ArH). LRMS (FAB+) m/z 486
([M+H]+).
12,14-Dichloro-13-isopropyl-8,11a-dimethyl-6-oxo-5-(5-phe-
nylpent-4-ynyl)-6,7,7a,8,9,10,11,11a-octahydro-5H-diben-
zo[b,d]azepine-8-carboxylic acid methyl ester (32c). A
mixture of the alkyne 17a (20 mg, 0.063 mmol), iodoben-
zene (0.008 mL, 0.075 mmol, 1.2 equiv), Pd(PPh3)2Cl2
(1 mg, 0.001 mmol, 0.02 equiv), and CuI (1 mg,
0.005 mmol, 0.08 equiv) in Et3N (1 mL) was stirred at rt
for 14 h under Ar atmosphere. The reaction mixture was
filtered, the solvent was evaporated in vacuum, and the
resultant residue was purified by flash chromatography
(n-hexane/AcOEt = 5:1) to afford 32a (37 mg, quantita-
tive yield) as a pale yellow amorphous solid. Compound
32a: 1H NMR (CDCl3) 1.42 (6H, d, J = 6.7 Hz,
2CH3CH), 1.42 (3H, s, CH3), 1.47 (3H, s, CH3), 1.69
(2H, m, 2CH), 1.81–1.98 (5H, m, 5CH), 2.06–2.11 (1H,
m, CH), 2.20 (1H, d, J = 8.5 Hz, CH), 2.52 (2H, t,
J = 7.0 Hz, CH2), 2.89 (1H, d, J = 16.3 Hz, CH), 3.10–
3.17 (1H, m, CH), 3.48 (3H, s, CO2CH3), 3.73 (1H, dd,
J = 16.2 Hz, 8.5 Hz, CH), 3.96 (1H, br s, CH3CH), 4.32–
4.39 (1H, m, CH), 7.20 (1H, br s, ArH), 7.27–7.29 (3H,
m, ArH), 7.41 (2H, dd, J = 7.9 Hz, 1.9 Hz, ArH). LRMS
13-Isopropyl-8,11a-dimethyl-6-oxo-5-(5-phenylpent-4-ynyl)-
6,7,7a,8,9,10,11,11a-octahydro-5H-dibenzo[b,d]azepine-
8-carboxylic acid (33b). A mixture of the methyl ester
32b (67 mg, 0.139 mmol), KOH (93 mg, 0.167 mmol,
1.2 equiv), and 18-crown ether-6 (92 mg, 0.347 mmol,
2.5 equiv) in MeOH (1 mL) was stirred at 80 ꢁC for
15 h, the whole was cooled, and the solvent was evapo-
rated in vacuum. The residue was diluted with water
(20 mL), acidified with 2 N HCl (2 mL), and then
extracted with CHCl3 (3· 20 mL). The combined organ-
ic layer was washed with brine (1· 20 mL), dried over
Na2SO4, filtered, and then evaporated in vacuum. The
residue was purified by flash chromatography (n-hex-
ane/AcOEt = 1:1) to afford the unreacted compound
32b (24 mg, 0.049 mmol, yield 35% after recrystalliza-
tion) as a colorless amorphous solid and the desired
compound 33b (32 mg, 0.068 mmol, yield 49%) as a col-
orless amorphous solid. Compound 33b: pale yellow
powder. Mp 76–79 ꢁC (n-hexane). 1H NMR (CDCl3)
1.24 (6H, d, J = 7.0 Hz, 2CH3CH), 1.26 (3H, s, CH3),
1.46 (3H, s, CH3), 1.65–1.77 (4H, m, 4CH), 1.88–2.03
(2H, m, 2CH), 1.97 (1H, d, J = 13.3 Hz, CH), 2.05–
2.12 (1H, m, CH), 2.46–2.57 (4H, m, 4CH), 2.84–2.91
(1H, m, CH3CH), 3.23–3.31 (1H, m, CH), 3.74–3.80
(FAB+),
m/z
554
([M(35Cl2)
+H]+),
556
([M(35Cl37Cl)+H]+), 558 ([M(37Cl2)+H]+).
12,14-Dichloro-13-isopropyl-8,11a-dimethyl-6-oxo-5-(5-
phenylpent-4-ynyl)-6,7,7a,8,9,10,11,11a-octahydro-5H-
dibenzo[b,d]azepine-8-carboxylic acid (33a). A mixture of
the methyl ester 32a (37 mg, 0.067 mmol), KOH (38 mg,
0.669 mmol, 10.0 equiv), and 18-crown ether-6 (44 mg,
0.167 mmol, 2.5 equiv) in MeOH (1 mL) was stirred at
80 ꢁC for 15 h, the whole was cooled, and the solvent
was evaporated in vacuum. The residue was diluted with
water (10 mL), acidified with 2 N HCl (2 mL), and then
extracted with CHCl3 (3· 20 mL). The combined organic
layer was washed with brine (1· 20 mL), dried over
Na2SO4, filtered, and then evaporated in vacuum. The

8030
T. Tashima et al. / Bioorg. Med. Chem. 14 (2006) 8014–8031
(1H, m, CH), 7.05 (1H, dd, J = 8.2 Hz, 1.8 Hz, ArH),
7.08 (1H, d, J = 1.8 Hz, ArH), 7.18 (2H, d, J = 8.2 Hz,
ArH), 7.21–7.25 (2H, m, ArH), 7.29 (1H, d,
J = 7.4 Hz, ArH). LRMS (FAB+) m/z 472 ([M+H]+).
Anal. Calcd for C31H37Cl2NO3: C, 78.95; H, 7.91; N,
2.97. Found: C, 78.65; H, 8.07; N, 2.80.
Na+ in Krebs solution were replaced with K+ to main-
tain isotonicity. The relaxant activities of the test com-
pounds were compared with that of vehicle (0.1%
DMSO) measured in the same muscle strip. All values
are expressed as means SEM. Statistical significance
of differences was determined by paired Student’s t-test,
and P values less than 0.05 were considered to be
significant.
5.2. Biological assays
5.2.1. Electrophysiological measurements. Human BK
channel subunits (hslo a and hslo b1) were subcloned
into pcDNA3 (Invitrogen, CA, USA) and pTracer-
CMV2 (Invitrogen), respectively, and transiently co-ex-
pressed in TSA201 cells with FuGENE6 transfection re-
agent (Roche Diagnostics Corporation, IN, USA). BK
channel currents were measured with the inside-out
patch clamp technique (Patch/Whole Cell Clamp Ampli-
fier CEZ-2400, Nihon Koden). Experiments was per-
formed within 24–48 h after the transfection. All the
experiments were carried out at room temperature
(20–25 ꢁC). Test compounds were diluted in the intracel-
lular solution (in mM; 140 K-Mes (Alfa Aesar, MA,
USA), 10 HEPES (Sigma–Aldrich, Inc., MO, USA), 5
HEDTA (Nacalai Tesque, Inc., Kyoto, Japan), and
100 nM free-Ca2+, pH 7.4) at the final concentration
of 10 lM and applied to the intracellular side of the
patch membrane. The resistance of the patch pipette
was 2–5 MX when filled with the pipette solution. Test
voltages from À100 mV to 190 mV were applied at
10 mV steps. The BK channel conductance was deter-
mined by measuring the tail current at each test voltage,
and plotted as conductance–voltage curves. V1/2 (half-
maximal activation voltage) values were calculated
based on Boltzmann’s fit of the conductance–voltage
curves. The BK channel opening activities of tested com-
pounds were evaluated as the hyperpolarizing shift of
Acknowledgments
We are grateful to Prof. Ligia Toro (University of
California at Los Angeles, U.S.A.) for the gifts of
hsloa and hslob1. This work was supported by
the Health and Labour Sciences Research Grants,
Research on Risk of Chemical Substances, from
Ministry of Health, Labour and Welfare to T.O. and
S.A.-A.
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