Potent and orally active ETA selective antagonists with 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acid structures

Article abstract of DOI:10.1016/j.bmc.2004.02.033

The synthesis and structure-activity relationships of a series of 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids are described. Our efforts have been focused on modification of the aryl ring at the 5-position and the alkyl substituent at the 2-p

Full text of DOI:10.1016/j.bmc.2004.02.033

Bioorganic & Medicinal Chemistry 12 (2004) 2139–2150
Potent and orally active ETA selective antagonists with 5,7-
diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acid structures
Takashi Yoshizumi,* Hirobumi Takahashi, Norikazu Ohtake, Hideki Jona,
Yoshiyuki Sato, Hiroyuki Kishino, Toshihiro Sakamoto, Satoshi Ozaki,
Hiroyuki Takahashi, Yoshihiro Shibata, Yasuyuki Ishii, Michiyasu Saito,
Megumu Okada, Takashi Hayama and Masaru Nishikibe
Banyu Tsukuba Research Institute in collaboration with Merck Research Laboratories, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
Received 3 December 2003; accepted 23 February 2004
Abstract—The synthesis and structure–activity relationships of a series of 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids
are described. Our efforts have been focused on modification of the aryl ring at the 5-position and the alkyl substituent at the
2-position of the bottom 4-methoxyphenyl ring in an effort to develop orally available ETA selective antagonists with safer profiles in
terms of the P-450 enzyme inhibitory activity. Incorporation of a hydroxymethyl group as an alkyl substituent in methylene-
dioxyphenyl and 6-dihydrobenzofuran derivatives led to the identification of orally bioavailable ETA selective antagonists 1f and 7f.
These compounds also showed not only excellent binding affinity (IC50 < 0.10 nM, more than 800-fold selectivity for the ETA
receptor over the ETB receptor) but also sufficient oral bioavailability, 48% and 56%, respectively, in rats. Furthermore, these
compounds did not exhibit either competitive or mechanism-based inhibition of human cytochrome P450 enzymes.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
disclosed. We have recently reported, both an ETA/ETB
mixed antagonist7 and an ETA selective antagonist (1b)8
from the series of the 5,7-diarylcyclopenteno[1,2-b]pyr-
idine-6-carboxylic acids. However, our ETA selective
antagonist lead, 3-hydroxy-2-methylpropyl derivative
(1b), had poor oral bioavailability (7.6%) in rats, while
the oral bioavailability of the advanced lead, 3-methoxy-
2-methylpropyl group derivative (1d) was significantly
higher (70%).
Endothelin-1 (ET-1)1 and its closely related isopeptides
(ET-2, ET-3) were identified as potent vasoconstrictor
peptides consisting of 21 amino acids. The endothelins
exert their diverse biological actions through distinct cell
surface G-protein coupled receptors (GPCR), termed
ETA and ETB.2 The ETA receptor subtype mediates
vasoconstriction and vascular smooth muscle prolifera-
tion, whereas the ETB receptor subtype is thought to
mediate either vasodilation or vasoconstriction. The
diversity of physiological effects elicited by the endo-
thelins has been implicated in the pathogenesis of a
variety of disease states including renal failure, cerebral
vasospasm, pulmonary hypertension, and congestive
heart failure. Since elevated levels of endothelins have
been observed in a number of these disease states,
endothelin receptor antagonists are expected to possess
clinical potential against endothelin-mediated disorders.3
CYPs inhibition is known to be due to oxidation of a
methylenedioxyphenyl ring, forming reactive species
such as a catechol, formate, and carbon monoxide.9
Indeed, the orally bioavailable derivative (1d) with a
methylenedioxyphenyl moiety showed significant inhib-
itory activity against CYP3A4 isozyme (vide infra). The
mechanism of this inhibition assumes the methylenedi-
oxyphenyl moiety is metabolized. Therefore, the effects
of the SARs of the 5-aryl rings and the 2-substituent on
the 7-(4-methoxyphenyl) ring on CYP3A4 inhibition
were investigated in order to identify a safer ETA
selective antagonist.
Several ETA selective4, ETB selective,5 and ETA/ETB
mixed6 nonpeptide endothelin antagonists have been
Herein, we describe the SARs of the 5-aryl ring and
alkyl substituent of the 7-aryl ring on the binding affinity
for the ETA and ETB receptors, and the inhibitory
* Corresponding author. Tel.: +81-298-77-2222; fax: +81-298-77-2027;
e-mail: yoszmitk@banyu.co.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.033

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T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
sequent reduction of the resulting aldehyde by NaBH4.
O
O
O
O
The two diastereomers were separated by column
chromatography. Further modification of these inter-
mediates (21a–b) based on the similar methods described
above gave 1a and 1b, respectively (Scheme 2).
CO2H
CO2H
*
N
N
N
H
CO2H
OH
OMe
OMe
ETA selective
ETA/ETB mixed
3. Results and discussion
(1b: polar diastereomer)
The synthesized compounds were evaluated in binding
assays (inhibitory activity against 125I-labeled ET-1
binding to both human ETA and ETB receptors).15
Selected compounds were further evaluated in CYP3A4
inhibition assays (inhibitory activities on testosterone
6b-hydroxylation after 60-min preincubation with
20 lM of test compounds) and in pharmacokinetic
studies (oral bioavailability in rats).
IC50 ETA: 0.11 nM
ETB: 0.60 nM
ETA: 0.041 nM
ETB: 54 nM
ETB/ETA: 1300
ETB/ETA: 5.6
Figure 1.
activities on testosterone 6b-hydroxylation in human
CYP3A4 (Fig. 1).
The effects of the 5-aryl ring in the 7-[2-(3-hydroxy-2-
methylpropyl)-4-methoxyphenyl]cyclopenteno[1,2-b]pyr-
idine core on the binding affinity for the ETA and ETB
receptors and ETA selectivity over the ETB receptor were
first investigated (Table 1).
2. Chemistry
The general synthesis of 5,7-diarylcyclopenteno[1,2-
b]pyridine-6-carboxylic acids with a variety of 5-aryl
rings was highlighted by the 2,3-dihydrobenzo[b]furan
Replacement of methylenedioxyphenyl with a 3,4-
dimethoxyphenyl group (2a–b) resulted in a more than
20-fold reduction in the binding affinity for the ETA and
ETB receptors, suggesting that the bicyclic structure at
the 5-aryl ring is important for retaining ETA and ETB
binding affinity. The 5-indan group (3a–b) clearly
retained its binding affinity comparable with 1a–b.
Interestingly, these compounds possessed higher selec-
tivity for the ETA than the ETB receptor relative to 1a–b.
The 5- and 6-benzofurans (4a–b and 6a–b) and the
corresponding dihydrobenzofuran (5a–b and 7a–b) were
acceptable in terms of ETA binding affinity and ETA
selectivity over ETB receptors. It is interesting to note
that a difluoromethylenedioxyphenyl moiety (11a–b)
dramatically improved the ETA selectivity over ETB
(ETB/ETA ¼ >10,000 fold) without loss of the ETA
binding affinity (11a: IC50¼0.11 nM).
derivative (7f).8 The addition of
a
2,3-di-
hydrobenzo[b]furan moiety10 to the enone 1211 via a
Grignard reaction yielded the corresponding tert-alco-
hol 13 in an 89% yield. Acetylation of 13 and subsequent
stereoselective reduction by heterogeneous hydrogena-
tion (Pd(OH)2 in THF/EtOH) produced the corre-
sponding cis–cis isomer (14).11 Thermodynamic
epimerization of 14 (tert-BuOK in dioxane–tert-BuOH)
followed by trifluoromethanesulfonylation (Tf2O,
DMAP) afforded the trans–trans triflate (15). This tri-
flate 15 was transformed into the vinyl analog via Stille
coupling (cat. PdCl2[(PPh3)2] in DMF at 125 ꢁC). The
subsequent stepwise oxidative cleavage of the vinyl
moiety (OsO4–NMO and NaIO4 system in THF–H2O)
yielded an aldehyde, which was subsequently reduced
using NaBH4 in MeOH to hydroxymethyl analog 16
(75% overall yield from 15). Protection of the primary
alcohol as an acetate followed by oxidation with
m-CPBA produced the corresponding pyridine N-oxide,
which was treated with N-isopropylbenzimidoyl chlo-
ride in the presence of triethylamine to yield 17.12
Optical resolution of 17 was achieved by liquid chro-
matography using a Chiralpak AD column to yield 18.
The protecting groups (N-benzoyl, O-acetyl, and tert-
butyl ester) of 18 were removed in two steps: (1)
reduction with diisobutylaluminum hydride (DIBAL)13
and (2) treatment with trifluoroacetic acid (TFA) to give
optically pure 7f (Scheme 1).14
Selected compounds (1a–b, 3a–b, 4a–b, 5a–b, 6a–b, 7a–
b, 11a–b) were tested in human CYP3A4 inhibition
assays (Table 2). Prior to derivatization, the replacement
of the methylenedioxyphenyl ring or the blockade of the
methylene spacer was expected to decrease the inhibi-
tory activity on CYP3A4.
Comparison of the two diastereomers of the methylene-
dioxyphenyl derivative (1a–b) indicated that the less
polar diastereomer (1a) had weaker CYP3A4 inhibition
than that of the polar diastereomer (1b).16 Unexpect-
edly, replacement of the methylenedioxyphenyl ring
with indan (3b), 5-benzofuran (4a–b), 5-dihydro-
benzofuran (5a–b), 6-benzofuran (6a–b) or difluorome-
thylenedioxyphenyl ring (11a–b) resulted in increasing
CYP3A4 inhibitory activity, when compared with those
of 1a–b. In contrast, the 6-dihydrobenzofuran ring (7a–
b) retained or slightly decreased the CYP3A4 inhibition
(7a: 22%, 7b: 35%).
The 2-alkyl substituents in compounds (1b–f) were
introduced using either a Heck reaction or Stille cou-
pling of the triflate (20) with an appropriate reagent such
as allyltri-n-butyltin or allyl alcohol.8 The 3-hydroxyl-2-
methylpropyl derivatives (1a–b) were prepared via the
Heck reaction using a methallyl alcohol and the sub-

T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
2141
O
O
O
O
HO
a
b, c
d, e
CO2tBu
OBn
CO2tBu
OBn
CO2tBu
OH
CO2tBu
OTf
N
N
N
N
OMe
OMe
OMe
OMe
12
13
14 (cis-cis)
15 (trans-trans)
O
O
O
chiral form
j, k, l
m
f, g, h, i
CO2tBu
OH
CO2tBu
OAc
CO2tBu
OAc
N
N
N
N
N
Bz
Bz
OMe
OMe
OMe
O
O
16
18
17
n
o
CO2tBu
OH
CO2H
OH
N
N
N
N
H
H
OMe
OMe
19
7f
Scheme 1. The representative synthetic route: synthesis of 7f. Reagents and conditions: (a) 5-bromo-2,3-dihydrobenzo[b]furan, Mg, THF, ꢀ78 ꢁC,
t
t
89%; (b) Ac2O, DMAP, CHC13; (c) H2, Pd(OH)2, NaHCO3, THF–EtOH, rt, 82% in two steps; (d) BuOK, BuOH–THF, 0 ꢁC; (e) Tf2O, DMAP,
CHCl3, 0 ꢁC, 72% in two steps; (f) vinyl(tributyl)tin, PdCl2(PPh3)2, DMF, 125 ꢁC, quant.; (g) OsO4, NMO, THF–H2O, rt; (h) NaIO4, THF–H2O, rt;
(i) NaBH4, THF–H2O, 75% in three steps; (j) Ac2O, DMAP, THF, rt; (k) m-CPBA, CHCl3 0 ꢁC; (l) iPrN¼C(Cl)Ph, Et3N, CHC13, 70 ꢁC; (m) optical
resolution using Chiralpak AD, hexane-2-propanol, 33% from 16; (n) DIBAL, THF, ꢀ78 ꢁC, 91%; (o) TFA, rt, 92%.
O
O
O
O
a, b
1a or 1b
CO2tBu
CO2tBu
OTf
N
N
OH
OMe
OMe
21a (less polar isomer)
21b (polar isomer)
20
Scheme 2. Introduction of 3-hydroxy-2-methylpropyl group. Reagents and conditions: (a) methallyl alcohol (10 equiv), PdCl2(PPh3)2 (10 mol %),
NaHCO3 (2 equiv), DMF, 130 ꢁC, 12 h; (b) NaBH4, MeOH, rt.
Since several alkyl side chains at the 2-position of the
7-aryl ring were acceptable in terms of the ETA binding
affinity and the selectivity for the ETA receptor over the
ETB receptor,8 the effects of these side chains on
CYP3A4 inhibition were also investigated (Table 2).
Interestingly, the more lipophilic side chains [2-ethyl-3-
hydroxypropyl (1c) and 2-methyl-3-methoxypropyl (1d)]
had stronger inhibitory activity (58% and 57%) for
human CYP3A4 than 1b, whereas the more hydrophilic
side chains [3-hydroxypropyl (1e) and hydroxymethyl
(1f)] reduced the CYP3A4 inhibition (1e: 27%, 1f: 19%).
These results suggested that the side chain at the 2-
position of the 7-aryl ring plays a key role in reducing
the CYP3A4 inhibitory activity. Among them, the hy-
droxy- methyl group was determined to be one of the
most effective functional groups in terms of CYP3A4
inhibition. Incorporation of the hydroxymethyl group in
6-dihydrobenzofuran and difluoromethylenedioxyphen-
yl derivatives (7f and 11f) was clearly effective in
reducing the CYP3A4 inhibition without causing a

2142
T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
Table 1. Structure–activity relationship and CYP3A4 inhibition of a
series of cyclopenteno [1,2-b]pyridine with a variety of aryl groups at
5-position
The oral bioavailability of the hydroxymethyl deriva-
tives (1f and 7f) was examined and compared with that
of the other alkyl derivatives (1b and 1d). In contrast to
the low oral bioavailability of 1b, the hydroxymethyl
derivatives (1f and 7f) showed excellent oral bioavail-
ability (1f: 48%,7f: 56%) in rats.
Ar
CO2H
N
N
H
OH
Finally, the inhibitory activity of 1f and 7f on the
marker activities for human CYPs were also evaluated
in an effort to clarify whether they showed competitive
and mechanism-based inhibition of human P450s
(CYP1A2, 2A6, 2C9, 2C19, 2D6, and 3A4). The data
shown in Table 3 clearly indicates that these compounds
were neither competitive inhibitors nor mechanism-
based inactivators of human P450s.
OMe
Com-
pounda
Ar
IC50 (nM)
B/A
ratiob
CYP3A4
inh. (%)c
ETA
ETB
O
O
1a
b
0.059
0.041
51
54
860
1300
26
45
MeO
NTd
NTd
OMe
2a
b
2.6
1.1
2300
1100
890
1000
4. Conclusion
In conclusion, we have derivatized the 5-aryl rings and
the alkyl side chain of the 7-aryl ring of the 5,7-diaryl-
cyclopenteno[1,2-b]pyridine-6-carboxylic acids in order
to identify orally bioavailable ETA selective antagonists
without significant CYPs inhibitory activity. As a result,
6-dihydrobenzofuran was determined to be a replace-
able 5-aryl ring of the cyclopenteno[1,2-b]pyridine core
in place of the methylenedioxyphenyl ring. Moreover,
the SARs of the alkyl side chains at the 7-aryl ring on
CYP3A4 inhibitory activity suggested that the hydroxy-
methyl group was effective in reducing the CYP3A4
inhibitory activity. Among the test compounds, the
hydroxymethyl derivatives (1f and 7f) showed excellent
ETA binding affinity with more than 800-fold selectivity
for the ETA receptor over the ETB receptor. In addition,
they had excellent oral bioavailability in rats. These
compounds were determined to be neither competitive
inhibitors of P450 marker activities (even at 100 lM) nor
mechanism-based inactivators of P450s (up to 10 lM).
3a
b
0.12
0.21
310
300
2600
1400
71
58
O
O
4a
b
0.067
0.041
77
210
1200
1900
79
75
5a
b
0.20
0.12
180
290
900
2400
52
54
O
O
6a
b
0.085
0.16
75
270
880
690
78
77
7a
b
0.094
0.11
240
56
2600
510
22
35
O
8a
b
2.4
4.4
950
2100
400
480
NTd
NTd
O
9a
b
0.70
3.8
1100
3400
1600
890
NTd
NTd
5. Experimental
5.1. General
O
>1900 NTd
>2300 NTd
O
10a
b
5.2
4.3
>10000
>10000
All reagents and solvents were of commercial quality
and were used without further purification unless
otherwise noted. Melting points were determined using a
Yanako MP micromelting point apparatus and were not
corrected. Optical rotations were determined on a
JASCO P-1020 polarimeter. 1H NMR spectra were
recorded on a Varian Gemini-300 instrument at
300 MHz. Chemical shifts were reported in parts per
million as d units relative to tetramethylsilane as an
internal standard. Mass spectra were recorded using fast
atom bombardment (FAB) ionization on a JEOL JMS-
SX 102A spectrometer. Thin layer chromatography was
performed using E-Merck Kieselgel 60 F254 plates
(0.25 mm) and visualized using either UV light or
phosphomolybdic acid. Column chromatography was
performed using Wako gel C-300.
F
F
O
11a
b
0.11
0.16
>1100
>1100
>10000 59
>6900 63
O
a The less polar diastereomer and the polar diastereomer were shown in
the symbols a and b.
b IC50(ETB)/IC50(ETA).
c Inhibitory activities on testosterone 6b-hydroxylation activities for
human CYP3A4.
d Not tested.
significant loss in the ETA potency and ETA selectivity
over the ETB receptor.

T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
2143
Table 2. Biological profiles of cyclopenteno[1,2-b]pyridine analogs with several kinds of alkyl substituents
Ar
CO2H
N
N
H
R
OMe
IC50 (nM)
Compound
Ar
R
B/A ratio
1300
CYP3A4 inh.
(%)a
B/A (%)b
ETA
0.041
ETB
O
1bc
54
16
45
58
7.6
O
*
OH
OH
*
*
1cc
0.045
340
NTd
1dc
1e
1f
0.025
0.022
0.062
48
20
62
1900
910
57
27
19
70
OMe
OH
NTd
48
1000
OH
OH
O
7f
0.10
0.23
80
800
18
25
56
F
F
O
O
11f
430
1900
NTd
OH
a The inhibitory activities on testosterone 6b-hydroxylation activity for human CYP3A4 after 60 min preincubation of compounds, N ¼ 2.
b Bioavailability in rats (3mpk, po and 1 mpk, iv).
c Polar diastereomer.
d Not tested.
Table 3. Competitive inhibition (IC50s) of P450 marker activities and mechanism-based inactivation (%inh.) of P450 by 1f and 7f in human liver
microsomes
P450 marker metabolism (P450)
Competitive inhibition
Mechanism-based inactivation
1f
7f
1f
7f
IC50 (lM)
% inhibition @ 10lM
7-Ethoxyresorufin O-deethylation (CYP1A2)
Coumarin 7-hydroxylation (CYP2A6)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
None
None
None
None
None
None
None
None
None
None
None
None
Tolbutamide methylhydroxylation (CYP2C9)
S-Mephenytoin 40-hydroxylation (CYP2C19)
Bufurolol 10-hydroxylation (CYP2D6)
Testosterone 6b-hydroxylation(CYP3A4)
5.1.1. 7-(2-Benzyloxy-4-methoxyphenyl)-6-tert-butoxy-
carbonyl-5-(2,3-dihydrobenzo[b]furan-6-yl)-5-hydroxy-cyc-
lopenta-1,3-dieno[2,1-b]pyridine (13). A suspension of Mg
(28.7 g, 1.18 mol) in a 30 mL aliquot of a solution of 5-
bromo-2,3-dihydrobenzo[b]furan (224 g, 1.13 mol) in dry
THF (1.1 L) was heated under a N2 atmosphere. Upon
initiation of the exothermic reaction, the remainder of the
solution was added at a rate in which the reflux was
maintained. Following the addition, the mixture was
stirred at ambient temperature for 30 min. The resulting
Grignard reagent mixture was added to a solution of
enone 1211 (250 g, 564 mmol) in dry THF (2.5 L) at
ꢀ78 ꢁC under N2 atmosphere over 40 min, and subse-
quently maintained at the same temperature with stirring

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T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
for 20 min. The reaction mixture was quenched with
NH4Cl (saturated, 2 L), then extracted with EtOAc (4 L).
The organic layer was washed with water (4 L), brine
(1 L), and concentrated. The residue was washed with
diisopropyl ether and dried in vacuo to yield 13 as a pale
solved in dry CHCl3 (1.5 L) was added DMAP (197 g,
1.76 mol) and trifluoromethanesulfonic anhydride
(Tf2O, 102 mL, 606 mmol) at 0 ꢁC under a N2 atmo-
sphere. After maintaining the mixture at 0 ꢁC with stir-
ring for 30 min, the reaction mixture was diluted with
hexane (1.5 L) and EtOAc (4.5 L), washed with 0.5 M
HCl (2 L), 10% KHSO4 (2 L), NaHCO3 (saturated, 2 L),
and brine (2 L). The organic layer was dried over
MgSO4, concentrated, and subsequently treated with
diisopropyl ether (400 mL). The resulting crystalline
material was collected, washed with diisopropyl ether,
and dried in vacuo to give 15 as a white solid (172 g, 72%
in two steps). mp: 145–146 ꢁC; 1H NMR (CDCl3) d 1.38
(s, 9H), 3.22 (t, J ¼ 8:8 Hz, 2H), 3.25 (dd, J ¼ 9:9,
10.5 Hz, 1H), 3.83 (s, 3H), 4.60 (t, J ¼ 8:8 Hz, 2H), 4.65
(d, J ¼ 9:9 Hz, 1H), 4.91 (d, J ¼ 10:5 Hz, 1H), 6.68 (s,
1H), 6.78 (d, J ¼ 7:6 Hz, 1H), 6.91 (s, 1H), 6.93 (d,
J ¼ 7:6 Hz, 1H), 7.13 (dd, J ¼ 4:9, 7.7 Hz), 7.15–7.20
(m, 2H), 7.32 (d, J ¼ 7:7 Hz), 8.45 (d, J ¼ 4:9 Hz, 1H);
HRMS calcd for C29H28F3NO7S (MþH)þ: 592.1617.
Found 592.1619.
1
yellow crystal (282 g, 89% yield). mp: 183–184 ꢁC; H
NMR (CDCl3) d 1.18 (s, 9H), 3.15 (t, J ¼ 8:7 Hz, 2H),
3.87 (s, 3H), 4.41 (s, 1H), 4.54 (t, J ¼ 8:7 Hz, 2H), 5.10 (s,
2H), 6.67–6.70 (m, 2H), 6.80–6.96 (br, 2H), 7.08 (dd,
J ¼ 5:0, 7.5 Hz), 7.11–7.16 (m, 1H), 7.22–7.47 (m, 6H),
7.53 (dd, J ¼ 1:5, 7.5 Hz, 1H), 8.48 (dd, J ¼ 1:5, 5.0 Hz,
1H); HRMS calcd for C35H33NO6 (MþH)þ: 564.2386.
Found 564.2387.
5.1.2. (5RS,6RS,7SR)-7-(2-Hydroxy-4-methoxyphenyl)-
6-tert-butoxycarbonyl-5-(2,3-dihydrobenzo[b]furan-6-yl)-
cyclopenteno[1,2-b]pyridine (14). To a solution of 13
(282 g, 500 mmol) and DMAP (183 g, 1.50 mol) in
chloroform (2.0 L) was added acetic anhydride (189 mL,
2.00 mol), and the mixture was stirred at ambient tem-
perature for 1 h. The reaction mixture was diluted with
EtOAc (8 L) and hexane (1.5 L), and then washed with
0.5 M HCl (3 L), 10% KHSO4 (2 L), NaHCO3 (satu-
rated, 2 L), and brine (1 L). The organic layer was dried
over MgSO4 and concentrated, and the resulting crude
product was used for the next step without further
purification.
5.1.4. (5RS,6SR,7SR)-7-(2-Hydroxymethyl-4-methoxy-
phenyl)-6-tert-butoxycarbonyl-5-(2,3-dihydrobenzo[b]-
furan-6-yl)cyclopenteno[1,2-b]pyridine (16). A mixture
of 15 (140 g, 237 mmol), vinyl(tributyl)tin (90.2 g,
284 mmol), dichloro[bis(triphenylphosphine)]palladium
(16.6 g, 23.7 mmol), and LiCl (30.1 g, 710 mmol) in
DMF (1.1 L) was stirred at 125 ꢁC under a N2 atmo-
sphere for 4 h. After cooling to ambient temperature,
EtOAc (1.0 L) and 20% aqueous KF (1.5 L) was added,
and the resulting mixture was vigorously stirred for
30 min. The mixture was filtered through a pad of Celite,
and the organic and aqueous layers of the filtrate were
separated. The organic layer was washed with 20%
aqueous KF (0.5 L), and the combined aqueous layers
were extracted with EtOAc (0.5 L). The combined
organic layers were washed with water (2 L) and brine
(1.5 L), dried over MgSO4, and concentrated. The resi-
due was purified by silica gel column chromatography
eluted with hexane/EtOAc to give the vinyl analog as a
The crude product was dissolved in THF (1.6 L) and
EtOH (1.2 L), followed by the addition of 20% palla-
dium hydroxide on carbon (160 g) and NaHCO3 (50.4 g,
600 mmol). After stirring the mixture under an H2
atmosphere (1 atm) at room temperature for 45 h, the
reaction mixture was filtered through a pad of Celite and
the cake was washed with THF (2 L). The combined
filtrates were concentrated, and the resulting solid was
washed with diisopropyl ether (1.5 L), and dried in
vacuo to give 14 as a white powder (189 g, 82% in two
steps). mp: 178–181 ꢁC; 1H NMR (CDCl3) d 0.93 (s,
9H), 3.21 (t, J ¼ 8:6 Hz, 2H), 3.77 (s, 3H), 3.94 (dd,
J ¼ 5:9, 7.3 Hz, 1H), 4.58 (t, J ¼ 8:6 Hz, 2H), 4.73 (d,
J ¼ 7:3 Hz, 1H), 4.97 (d, J ¼ 5:9 Hz, 1H), 6.37 (dd,
J ¼ 2:4, 8.5 Hz, 1H), 6.52 (d, J ¼ 2:4 Hz, 1H), 6.73 (s,
1H), 6.84 (d, J ¼ 7:0 Hz, 1H), 7.08 (d, J ¼ 8:5 Hz, 1H),
7.18 (dd, J ¼ 5:0, 7.8 Hz, 1H), 7.21 (d, J ¼ 7:0 Hz, 1H),
7.58 (d, J ¼ 7:8 Hz, 1H), 8.36 (d, J ¼ 5:0 Hz, 1H);
HRMS calcd for C28H29NO5 (MþH)þ: 460.2124. Found
460.2122.
1
pale green oil (112 g): H NMR (CDCl3) d 1.34 (s, 9H),
3.23 (t, J ¼ 8:6 Hz, 2H), 3.28 (dd, J ¼ 9:7, 10.8 Hz, 1H),
3.83 (s, 3H), 4.59 (t, J ¼ 8:6 Hz, 2H), 4.60 (1H, d,
J ¼ 4:6 Hz, 1H), 4.99 (d, J ¼ 9:7 Hz, 1H), 5.28 (d,
J ¼ 10:8 Hz, 1H), 5.63 (d, J ¼ 17:3 Hz, 1H), 6.68 (s,
1H), 6.77 (d, J ¼ 7:5 Hz, 1H), 6.83 (dd, J ¼ 2:7, 8.6 Hz,
1H), 6.91–7.04 (br s, 1H), 6.98 (d, J ¼ 8:6 Hz, 1H), 7.05
(d, J ¼ 2:7 Hz, 1H), 7.09 (dd, J ¼ 4:8, 7.6 Hz, 1H), 7.17
(d, J ¼ 7:5 Hz, 1H), 7.31 (dd, J ¼ 1:2, 7.6 Hz, 1H), 8.45
(dd, J ¼ 1:2, 4.8 Hz, 1H); HRMS calcd for C30H31NO4
(MþH)þ: 470.2331. Found 470.2325.
5.1.3. (5RS,6SR,7SR)-7-(2-Trifluoromethanesulfonyloxy-
4-methoxyphenyl)-6-tert-butoxycarbonyl-5-(2,3-dihydro-
benzo[b]furan-6-yl)cyclopenteno [1,2-b]pyridine (15). A
solution of 14 (185 g, 403 mmol) and potassium tert-
butoxide (90.3 g, 805 mmol) in dry THF (1.5 L) and tert-
butyl alcohol (1.0 L) was stirred at 0 ꢁC under N2
atmosphere for 20 min. The reaction mixture was
quenched using AcOH (69.2 mL, 1.21 mol), then
extracted with EtOAc/hexane (1/1, 4 L). The organic
layer was washed with NaHCO3 (saturated) and brine,
then dried over MgSO4 and concentrated to afford the
crude trans–trans isomer. To the crude product dis-
To a solution of this vinyl compound (112 g, 237 mmol)
in THF (800 mL) was added 4-methylmorpholine-N-
oxide (41.9 g, 358 mmol) and 0.05 M OsO4 in water
(158 mL, 7.9 mmol). After stirring the mixture at ambi-
ent temperature for 12 h, the reaction mixture was
quenched using 10% Na2S2O3 (1.5 L). The mixture was
extracted with EtOAc (1.6 L), then the organic layer was
washed with brine, dried over MgSO4, and concen-
trated. To the crude residue dissolved in THF (300 mL)

T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
2145
was added a solution of NaIO4 (34.1 g, 159 mmol) in
water (300 mL) over 10 min at 0 ꢁC. After stirring for an
additional 10 min, NaBH4 (9.04 g, 239 mmol) was added
in small portions, and the mixture was stirred at ambient
temperature for 20 min. To the mixture was added water
(0.6 L) and EtOAc (0.9 L). The organic layer was sepa-
rated, washed with brine, dried over MgSO4, and con-
centrated. The residue was purified using silica gel
column chromatography eluted with hexane/EtOAc to
give hydroxymethyl analog 16 as an oil (84.2 g, 75% in
1H), 6.63–6.99 (m, 4H), 7.03 (dd, J ¼ 1:2, 7.9 Hz, 1H),
7.12–7.25 (m, 6H); HRMS calcd for C41H44N2O7
(MþH)þ: 677.3227. Found 677.3228.
5.1.6. Optically pure compound 18. Racemic 17 (72.9 g)
was optically resolved using chiral HPLC (Chiralpak
AD, 50 · 500 mm, hexane/2-propanol (70:30), flow
rate¼100 mL/min) to give chiral compound 18 (32.2 g,
44%). Chiral analytical HPLC indicated that enantio-
27
1
four steps). H NMR (CDCl3) d 1.36 (s, 9H), 3.26 (t,
meric excess (ee) of 18 was >99.9%; ½aꢁ þ22.8 (c 1.00,
D
J ¼ 8:7 Hz, 2H), 3.62 (dd, J ¼ 9:6, 11.8 Hz, 1H), 3.84 (s,
3H), 4.59–4.61 (m, 2H), 4.63 (t, J ¼ 8:7 Hz, 2H), 4.95 (d,
J ¼ 11:8 Hz, 1H), 5.14 (d, J ¼ 9:6 Hz, 1H), 6.73 (d,
J ¼ 1:5 Hz, 1H), 6.80 (dd, J ¼ 1:5, 7.5 Hz, 1H), 6.85 (dd,
J ¼ 2:8, 8.6 Hz, 1H), 7.06 (d, J ¼ 2:8 Hz, 1H), 7.08 (dd,
J ¼ 4:8, 7.7 Hz, 1H), 7.12 (d, J ¼ 8:6 Hz, 1H), 7.22 (d,
J ¼ 7:5 Hz, 1H), 7.35 (dd, J ¼ 1:3, 7.7 Hz, 1H), 8.34 (dd,
J ¼ 1:3, 4.8 Hz, 1H); HRMS calcd for C29H31NO5
(MþH)þ: 474.2280. Found 474.2274.
EtOH).
5.2. tert-Butyl
5.2.1. (5S,6R,7R)-(+)-5-(2,3-dihydrobenzo[b]furan-6-yl)-
7-(2-hydroxylmethyl-4-methoxyphenyl)-2-(N-isopropyl-
amino)cyclopenteno[1,2-b]pyridine-6-carboxylate (19). To
a solution of 18 (30.0 g, 44.3 mmol) in THF (300 mL)
was added 1.01 M diisobutylaluminum hydride in tolu-
ene (175 mL, 177 mmol) over 30 min at ꢀ78 ꢁC. The
mixture was maintained at the same temperature with
stirring for 1.5 h. To the reaction mixture was added
NH4Cl (saturated, 1.0 L), EtOAc (0.90 L), water
(0.50 L), and 10% KHSO4 (0.10 L). The layers were
separated and the organic layer was washed with 10%
KHSO4 (0.60 L) and brine (0.60 L), dried over MgSO4,
and concentrated. The residue was purified by silica gel
column chromatography with hexane/EtOAc as the
mobile phase to give 19 as an off-white solid (21.5 g,
5.1.5. (5RS,6SR,7SR)-7-(2-Acetoxymethyl-4-methoxy-
phenyl)-6-tert-butoxycarbonyl-5-(2,3-dihydrobenzo[b]-fur-
an-6-yl)-2-(N-benzoyl-isopropylamino)cyclopenteno-[1,2-
b]pyridine (17). To a solution of 16 (84.1 g, 178 mmol) in
THF (420 mL) were added DMAP (65.2 g, 534 mmol)
and acetic anhydride (67.0 mL, 710 mmol). After stirring
the mixture at ambient temperature for 20 min, the
reaction mixture was diluted with EtOAc (1.2 L), wa-
shed with 10% KHSO4 (1.5 L), NaHCO3 (saturated,
1 L), and brine (1 L), then dried over MgSO4, and con-
centrated. The residue was passed through a short col-
umn using a silica gel, and the resulting crude product
was used in the next step without further purification.
1
91%). mp: 89–92 ꢁC; H NMR (DMSO-d6) d 0.97 (d,
J ¼ 6:4 Hz, 3H), 1.04 (d, J ¼ 6:4 Hz, 3H), 3.00 (dd,
J ¼ 8:3, 8.9 Hz, 1H), 3.15 (t, J ¼ 8:9 Hz, 2H), 3.39–3.47
(m, 1H), 3.60–3.71 (m, 1H), 3.73 (s, 3H), 4.32 (d,
J ¼ 8:3 Hz, 1H), 4.46–4.57 (m, 1H), 4.51 (t, J ¼ 8:9 Hz,
2H), 4.62 (d, J ¼ 8:9 Hz, 1H), 4.59–4.64 (m, 1H), 6.25
(d, J ¼ 8:5 Hz, 1H), 6.59 (d, J ¼ 1:4 Hz, 1H), 6.70 (dd,
J ¼ 1:4, 7.6 Hz, 1H), 6.74 (dd, J ¼ 2:8, 8.5 Hz, 1H), 6.89
To a solution of this acetoxy analog (74.2 g, 144 mmol)
in CHCl3 (700 mL) was added m-CPBA (74.5 g,
432 mmol) in small portions at 0 ꢁC. After stirring the
mixture at 0 ꢁC for 15 h, the reaction mixture was
quenched with 15% Na2S2O3 (1.5 L), and extracted with
hexane (1.4 L)-EtOAc (0.7 L). The organic layer was
washed with NaHCO3 (saturated, 1.5 L) and brine, dried
over MgSO4, and concentrated to give the correspond-
ing pyridine N-oxide, which was used in the next step
without further purification.
(d, J ¼ 7:9 Hz, 1H), 6.92 (d, J ¼ 7:9 Hz, 1H), 7.00 (d,
27
J ¼ 2:8 Hz, 1H), 7.17 (d, J ¼ 7:6 Hz, 1H); ½aꢁ þ20.9 (c
D
1.00, EtOH); HRMS calcd for C32H38N2O5 (MþH)þ:
531.2859. Found 531.2858.
5.2.2. (5S,6R,7R)-(+)-5-(2,3-Dihydrobenzo[b]furan-6-yl)-
7-(2-hydroxylmethyl-4-methoxyphenyl)-2-(N-isopropyl-
amino)cyclopenteno[1,2-b]pyridine-6-carboxylic acid (7f)14.
A solution of 19 (21.0 g, 39.6 mmol) in TFA (63 mL) was
stirred at room temperature for 1 h, then concentrated.
Water (100 mL) adjusted to pH 6.5 using NaHCO3
(saturated) was added to the residue dissolved in EtOAc
(200 mL). The layers were separated and the aqueous
layer was extracted with EtOAc (2 · 100 mL). The
combined organic layers were concentrated, and the
residue was washed with EtOAc (200 mL) then dried in
vacuo to give 7f as a colorless crystal (15.7 g). The
washings were concentrated and purified by silica gel
column chromatography to afford a second crop (1.6 g)
for a combined total yield of 17.3 g (92%). mp: 220–
222 ꢁC; 1H NMR (DMSO-d6) d 0.98 (d, J ¼ 6:4 Hz, 3H),
1.04 (d, J ¼ 6:4 Hz, 3H), 3.01 (dd, J ¼ 8:6, 8.8 Hz, 1H),
3.15 (t, J ¼ 8:8 Hz, 2H), 3.60–3.71 (m, 1H), 3.73 (s, 3H),
A mixture of this pyridine N-oxide, N-isopropylbenz-
imidoyl chloride (78.5 g, 432 mmol), and triethylamine
(141 mL, 1.01 mol) in dry CHCl3 (600 mL) was refluxed
under a N2 atmosphere for 17 h. After cooling to
ambient temperature, NaHCO3 (saturated, 1.5 L) was
added and the layers were separated. The organic layer
was washed with brine, dried over MgSO4, and con-
centrated. The residue was purified by silica gel column
chromatography with hexane/EtOAc as an eluent to
1
give 17 as a pale green oil (72.9 g, 61% from 16). H
NMR (CDCl3) d 1.12 (d, J ¼ 7:7 Hz, 3H), 1.17 (d,
J ¼ 7:7 Hz, 3H), 1.35 (s, 9H), 2.10 (s, 3H), 3.21 (t,
J ¼ 8:7 Hz, 2H), 3.29 (dd, J ¼ 9:6, 9.9 Hz, 1H), 3.85 (s,
3H), 4.48 (d, J ¼ 9:6 Hz, 1H), 4.59 (t, J ¼ 8:7 Hz, 2H),
4.94–5.02 (m, 1H), 4.96 (d, J ¼ 9:9 Hz, 1H), 5.21–5.32
(m, 2H), 6.52 (d, J ¼ 1:2 Hz, 1H), 6.57 (d, J ¼ 7:9 Hz,

2146
T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
4.32 (d, J ¼ 8:6 Hz, 1H), 4.44–4.56 (m, 3H), 4.60–4.72
(m, 2H), 6.25 (d, J ¼ 8:5 Hz, 1H), 6.60 (s, 1H), 6.70 (d,
J ¼ 7:7 Hz, 1H), 6.75 (dd, J ¼ 2:9, 8.6 Hz, 1H), 6.89 (d,
3.20–3.35 (m, 2H), 3.58–3.65 (m, 1H), 3.70 (s, 3H), 3.77
(s, 3H), 3.83 (s, 3H), 4.25 (d, J ¼ 6:5 Hz, 1H), 4.99 (d,
J ¼ 6:6 Hz, 1H), 6.34 (d, J ¼ 8:8 Hz, 1H), 6.52 (br s,
1H), 6.62–6.83 (m, 5H), 7.24–7.26 (m, 1H); HRMS calcd
for C31H38N2O6 (MþH)þ: 535.2808. Found 535.2803.
J ¼ 7:7 Hz, 1H), 6.92 (d, J ¼ 7:6 Hz, 1H), 7.02 (d,
20
J ¼ 2:9 Hz, 1H), 7.18 (d, J ¼ 7:6 Hz, 1H); ½aꢁ þ71.9 (c
D
1.00, 1 M NaOH); HRMS calcd for C28H30N2O5
(MþH)þ: 475.2233. Found 475.2239.
2b; White solid; mp 114–116 ꢁC; H NMR (CDCl3) d
1
1.07 (d, J ¼ 6:5 Hz, 3H), 1.12 (d, J ¼ 6:3 Hz, 3H), 1.18
(d, J ¼ 6:3 Hz, 3H), 2.06–2.09 (m, 1H), 2.49–2.57 (m,
1H), 2.95–3.04 (m, 1H), 3.23 (dd, J ¼ 7:7, 8.1 Hz, 1H),
3.35–3.45 (m, 2H), 3.61–3.66 (m, 1H), 3.76 (s, 3H), 3.85
(s, 3H), 3.88 (s, 3H), 4.49 (d, J ¼ 7:7 Hz, 1H), 5.07 (d,
5.2.3. (5RS,6SR,7SR)-7-[2-(3-Hydroxy-2-methylpropyl)-
4-methoxyphenyl]-6-tert-butoxycarbonyl-5-(3,4-methylene-
dioxyphenyl)cyclopenteno[1,2-b]pyridine (21a,b). The
mixture of PdCl2 (PPh3)2 (7.70 g, 0.011 mol), NaHCO3
(18.4 g, 0.22 mol), methallyl alcohol (93.1 mL, 1.10 mol)
and 20 (64.9 g, 0.11 mol) in DMF (650 mL) was heated
with stirring at 130 ꢁC for 12 h under N2. After cooling
to room temperature, the reaction mixture was diluted
with EtOAc and H2O, and the organic layer was washed
with water and brine, dried over Na2SO4 and then
concentrated. The residue was dissolved in MeOH and
NaBH4 (8.32 g, 0.22 mol) was added to the solution in
small portions at 0 ꢁC. After an additional stirring for
15 min, the mixture was quenched using saturated
NH4Cl and diluted with CHCl3. The organic layer was
washed with brine, dried over Na2SO4, and concen-
trated. The residue was purified by silica gel column
chromatography eluted with hexane/EtOAc to give less
polar diastereomer 21a as a colorless solid (9.91 g, 18%
yield) and polar diastereomer 21b as an oil (8.62 g, 15%
J ¼ 8:1 Hz, 1H), 6.22 (d, J ¼ 8:9 Hz, 1H), 6.67–6.91 (m,
27
6H), 7.12 (d, J ¼ 8:2 Hz, 1H); ½aꢁ þ21.0 (c 1.00,
D
EtOH); HRMS calcd for C31H38N2O6 (MþH)þ:
535.2808. Found 535.2816.
5.2.5.
(5S,6R,7R)-(+)-5-(Indan-5-yl)-7-[2-(3-hydroxy-2-
methylpropyl)-4-methoxyphenyl]-2-(N-isopropylamino)-cyc-
lopenteno[1,2-b]pyridine-6-carboxylic acid (3a,b). 3a; White
solid; mp 122–125 ꢁC; 1H NMR (CDCl3) d 1.06 (d,
J ¼ 7:6 Hz, 3H), 1.09 (d, J ¼ 6:4 Hz, 3H), 1.14 (d,
J ¼ 6:4 Hz, 3H), 2.03–2.13 (m, 3H), 2.52–2.57 (m, 1H),
2.87–3.02 (m, 5H), 3.25 (dd, J ¼ 8:6, 8.9 Hz, 1H), 3.33–
3.51 (m, 2H), 3.54–3.68 (m, 1H), 3.75 (s, 3H), 4.49 (d,
J ¼ 8:6 Hz, 1H), 5.05 (d, J ¼ 8:9 Hz, 1H), 6.19 (d, J ¼
29
D
8.2 Hz, 1H), 6.63–6.74 (m, 2H), 6.85–7.21 (m, 5H); ½aꢁ
(MþH)þ: 515.2910. Found 515.2914.
þ23.3 (c ¼ 1:00, EtOH); HRMS calcd for C32H38N2O4
1
yield). 21a; H NMR (CDCl3) d 8.37 (d, J ¼ 5:0 Hz,
1H), 7.27 (d, J ¼ 7:3 Hz 1H), 7.07 (dd, J ¼ 7:3 and
5.0 Hz, 1H), 6.87 (d, J ¼ 7:7 Hz, 1H), 6.62–6.83 (m, 5H),
5.97 (s, 2H), 5.04 (d, J ¼ 9:9 Hz, 1H), 4.51 (d,
J ¼ 9:9 Hz, 1H), 3.78 (s, 3H), 3.40–3.63 (m, 2H), 3.27 (t,
J ¼ 9:9 Hz, 1H), 2.66–2.98 (m, 2H), 1.88–2.07 (m, 1H),
1.32 (s, 9H), 1.00 (d, J ¼ 6:6 Hz, 3H). HRMS calcd for
C31H36NO6 (MþH)þ 518.2543. Found 518.2540.
1
3b; White solid; mp 117–121 ꢁC; H NMR (CDCl3) d
0.93 (d, J ¼ 6:4 Hz, 3H), 1.12 (d, J ¼ 6:3 Hz, 3H), 1.14
(d, J ¼ 6:3 Hz, 3H), 1.92–2.10 (m, 3H), 2.60–2.72 (m,
2H), 2.84–2.93 (m, 4H), 3.20 (dd, J ¼ 8:7, 8.9 Hz, 1H),
3.30–3.49 (m, 2H), 3.58–3.67 (m, 1H), 3.73 (s, 3H), 4.47
(d, J ¼ 8:7 Hz, 1H), 4.87 (d, J ¼ 8:9 Hz, 1H), 6.18 (d,
J ¼ 8:8 Hz, 1H), 6.64–6.73 (m, 2H), 6.90–7.20 (m, 5H);
29
D
1
21b; H NMR (CDCl3) d 8.35 (d, J ¼ 5:0 Hz, 1H), 7.33
½aꢁ
þ40.3 (c ¼ 1:00, EtOH); HRMS calcd for
(d, J ¼ 7:7 Hz, 1H), 7.10 (dd, J ¼ 7:7 and 5.0 Hz, 1H),
6.86 (d, J ¼ 8:4 Hz, 1H), 6.69–6.83 (m, 5H), 5.98 (s, 2H),
5.13 (d, J ¼ 9:7 Hz, 1H), 4.57 (d, J ¼ 9:7 Hz, 1H), 3.78
(s, 3H), 3.53 (dd, J ¼ 11:4 and 3.1 Hz, 1H), 3.43 (dd,
J ¼ 11:4 and 4.4 Hz, 1H), 3.26 (t, J ¼ 9:7 Hz, 1H), 3.00–
3.17 (m, 1H), 2.46–2.61 (m, 1H), 2.04–2.19 (m, 1H), 1.34
(s, 9H), 1.09 (d, J ¼ 6:6 Hz, 3H). HRMS calcd for
C31H36NO6 (MþH)þ 518.2543, found 518.2543.
C32H38N2O4 (MþH)þ: 515.2910. Found 515.2915.
5.2.6. (5S,6R,7R)-(+)-5-(Benzofuran-5-yl)-7-[2-(3-hydr-
oxy-2-methylpropyl)-4-methoxyphenyl]-2-(N-isopropyl-
amino)cyclopenteno[1,2-b]pyridine-6-carboxylic acid (4a,b).
1
4a; White solid; mp 116–119 ꢁC; H NMR (CDCl3) d
1.00 (d, J ¼ 6:5 Hz, 3H), 1.08 (d, J ¼ 6:2 Hz, 3H), 1.13
(d, J ¼ 6:2 Hz, 3H), 1.95–2.10 (m, 1H), 2.49 (dd,
J ¼ 4:9, 13.8 Hz, 1H), 2.93 (d, J ¼ 13:8 Hz, 1H), 3.22
(dd, J ¼ 8:2, 8.3 Hz, 1H), 3.30–3.50 (m, 2H), 3.52–3.65
(m, 1H), 3.70 (s, 3H), 4.60 (d, J ¼ 8:2 Hz, 1H), 5.04 (d,
J ¼ 8:3 Hz, 1H), 6.19 (d, J ¼ 8:6 Hz, 1H), 6.63–6.75 (m,
3H), 6.90 (d, J ¼ 9:2 Hz, 1H), 7.07 (d, J ¼ 8:6 Hz, 1H),
The preparation of methylenedioxyphenyl derivatives
(1a–f) was previously reported.8 Compounds 2a–7b and
8a–11b were prepared from intermediate 12 in a similar
manner using the corresponding arylbromide. The syn-
thesis of 11f was analogous to the method used for the
synthesis of 7f.
7.20 (d, J ¼ 8:5 Hz, 1H), 7.44 (d, J ¼ 8:5 Hz, 1H), 7.56
30
(s, 1H), 7.60 (s, 1H); ½aꢁ þ32.4 (c 1.00, EtOH); HRMS
D
calcd for C31H34N2O5 (MþH)þ: 515.2546. Found
5.2.4. (5S,6R,7R)-5-(2,3-Dimethoxyphenyl)-7-[2-(3-hy-
droxy-2-methylpropyl)-4-methoxyphenyl]-2-(N-isopropyl-
amino)cyclopenteno[1,2-b]pyridine-6-carboxylic acid (2a,b).
515.2549.
4b; White crystals; mp 219–221 ꢁC; 1H NMR (CDCl3) d
0.90 (d, J ¼ 6:3 Hz, 3H), 1.12 (d, J ¼ 6:5 Hz, 3H), 1.14
(d, J ¼ 6:5 Hz, 3H), 1.90–2.05 (m, 1H), 2.50–2.65 (m,
1H), 2.65–2.75 (m, 1H), 3.21 (dd, J ¼ 8:3, 8.4 Hz, 1H),
3.30–3.50 (m, 2H), 3.55–3.65 (m, 1H), 3.69 (s, 3H), 4.58
1
2a; White solid; mp 124–127 ꢁC; H NMR (CDCl3) d
0.75 (d, J ¼ 6:4 Hz, 3H), 1.14 (d, J ¼ 6:3 Hz, 3H), 1.18
(d, J ¼ 6:3 Hz, 3H), 1.79–1.91 (m, 1H), 2.00–2.14 (m,
1H), 2.99 (dd, J ¼ 6:5, 6.6 Hz, 1H), 3.08–3.12 (m, 1H),

T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
2147
(d, J ¼ 8:3 Hz, 1H), 4.89 (d, J ¼ 8:4 Hz, 1H), 6.20 (d,
J ¼ 8:2 Hz, 1H), 6.60–6.75 (m, 3H), 6.94 (d, J ¼ 8:2 Hz,
30
HRMS calcd for C31H34N2O5 (MþH)þ: 515.2546.
Found 515.2539.
1H), 7.07 (d, J ¼ 8:8 Hz, 1H), 7.19 (d, J ¼ 8:5 Hz, 1H),
7.44 (d, J ¼ 8:5 Hz, 1H), 7.49 (s, 1H), 7.61 (s, 1H); ½aꢁ
D
þ48.5 (c 0.30, EtOH); HRMS calcd for C32H38N2O4
(MþH)þ: 515.2546. Found 515.2526.
5.2.9. (5S,6R,7R)-5-(2,3-Dihydrobenzo[b]furan-6-yl)-7-
[2-(3-hydroxy-2-methylpropyl)-4-methoxyphenyl]-2-(N-
isopropylamino)cyclopenteno[1,2-b]pyridine-6-carboxylic
acid (7a,b). 7a; white solid; 1H NMR (CDCl3) d 1.06 (d,
J ¼ 6:9 Hz, 3H), 1.10 (d, J ¼ 6:3 Hz, 3H), 1.14 (d,
J ¼ 6:3 Hz, 3H), 2.10–2.12 (m, 1H), 2.49–2.62 (m, 1H),
3.01 (dd, J ¼ 8:9, 9.0 Hz, 1H), 3.15–3.25 (m, 1H), 3.21
(t, J ¼ 8:7 Hz, 2H), 3.37–3.42 (m, 2H), 3.56–3.67 (m,
1H), 3.75 (s, 3H), 4.49 (d, J ¼ 9:0 Hz, 1H), 4,58 (t,
J ¼ 8:7 Hz, 2H), 5.05 (d, J ¼ 8:9 Hz, 1H), 6.19 (d,
J ¼ 8:5 Hz, 1H), 6.66–6.77 (m, 3H), 6.81 (d, J ¼ 7:3 Hz,
5.2.7. (5S,6R,7R)-(+)-5-(2,3-Dihydrobenzo[b]furan-5-yl)-
7-[2-(3-hydroxy-2-methylpropyl)-4-methoxyphenyl]-2-(N-
isopropylamino)cyclopenteno[1,2-b]pyridine-6-carboxylic
1
acid (5a,b). 5a; White solid; mp 199–201 ꢁC; H NMR
(CDCl3) d 1.05 (d, J ¼ 6:2 Hz, 3H), 1.09 (d, J ¼ 6:2 Hz,
3H), 1.14 (d, J ¼ 6:2 Hz, 3H), 1.98–2.12 (m, 1H), 2.48–
2.57 (m, 1H), 2.91–3.02 (m, 1H), 3.15–3.25 (m, 3H),
3.32–3.50 (m, 2H), 3.56–3.65 (m, 1H), 3.74 (s, 3H), 4.45
(d, J ¼ 8:5 Hz, 1H), 4,56 (t, J ¼ 8:6 Hz, 2H), 5.02 (d,
J ¼ 8:7 Hz, 1H), 6.18 (d, J ¼ 8:4 Hz, 1H), 6.65–6.78 (m,
1H), 6.91 (d, J ¼ 8:5 Hz, 1H), 7.09 (d, J ¼ 8:5 Hz, 1H),
26
7.15 (d, J ¼ 7:3 Hz, 1H); ½aꢁ þ14.1 (c 1.00, EtOH);
D
HRMS calcd for C31H36N2O5 (MþH)þ: 517.2702.
Found 517.2682.
3H), 6.89 (d, J ¼ 8:2 Hz, 1H), 7.02 (d, J ¼ 7:7 Hz, 1H),
31
7.07 (d, J ¼ 8:4 Hz, 1H), 7.12 (s, 1H); ½aꢁ þ18.6 (c 0.44,
7b; White solid; 1H NMR (CDCl3) d 0.92 (d, J ¼ 6:6 Hz,
3H), 1.12 (d, J ¼ 6:3 Hz, 3H), 1.14 (d, J ¼ 6:3 Hz, 3H),
1.90–2.03 (m, 1H), 2.53–2.75 (m, 2H), 3.12–3.23 (m,
3H), 3.32–3.46 (m, 2H), 3.56–3.66 (m, 1H), 3.72 (s, 3H),
4.45 (d, J ¼ 8:4 Hz, 1H), 4,56 (t, J ¼ 8:4 Hz, 2H), 4.87
(d, J ¼ 8:4 Hz, 1H), 6.20 (d, J ¼ 8:6 Hz, 1H), 6.62–6.72
(m, 3H), 6.76 (d, J ¼ 7:2 Hz, 1H), 6.90 (d, J ¼ 8:6 Hz,
1H), 7.07–7.18 (m, 2H); HRMS calcd for C31H36N2O5
(MþH)þ: 517.2702. Found 517.2706.
D
EtOH); HRMS calcd for C31H36N2O5 (MþH)þ:
517.2702. Found 517.2698.
1
5b: White solid; mp 212–215 ꢁC; H NMR (CDCl3) d
0.88 (d, J ¼ 6:4 Hz, 3H), 1.12 (d, J ¼ 5:9 Hz, 3H), 1.17
(d, J ¼ 5:9 Hz, 3H), 1.84–1.98 (m, 1H), 2.40–2.52 (m,
1H), 2.64–2.78 (m, 1H), 3.08 (dd, J ¼ 8:2, 8.3 Hz, 1H),
3.16 (t, J ¼ 8:6 Hz, 2H), 3.25–3.40 (m, 2H), 3.52–3.65
(m, 1H), 3.68 (s, 3H), 4.39 (d, J ¼ 8:2 Hz, 1H), 4,54 (t,
J ¼ 8:6 Hz, 2H), 4.83 (d, J ¼ 8:3 Hz, 1H), 6.23 (d,
J ¼ 8:6 Hz, 1H), 6.65 (s, 1H), 6.67 (d, J ¼ 8:8 Hz, 1H),
6.71 (d, J ¼ 8:3 Hz, 1H), 6.88 (d, J ¼ 8:8 Hz, 1H), 6.97
5.2.10. (5S,6R,7R)-5-(Benzofuran-4-yl)-7-[2-(3-hydroxy-
2-methylpropyl)-4-methoxyphenyl]-2-(N-isopropylamino)-
cyclopenteno[1,2-b]pyridine-6-carboxylic acid (8a,b). 8a;
White solid; mp 124–126 ꢁC; 1H NMR (CDCl3) d 0.98 (d,
J ¼ 6:7 Hz, 3H), 1.09 (d, J ¼ 6:3 Hz, 3H), 1.15 (d,
J ¼ 6:3 Hz, 3H), 1.98–2.08 (m, 1H), 2.41–2.52 (m, 1H),
2.88–3.01 (m, 1H), 3.26 (dd, J ¼ 7:3; 8:4 Hz, 1H), 3.32–
3.48 (m, 2H), 3.58–3.66 (m, 1H), 3.69 (s, 3H), 4.87 (d,
J ¼ 7:3 Hz, 1H), 5.07 (d, J ¼ 8:4 Hz, 1H), 6.22 (d,
(d, J ¼ 8:3 Hz, 1H), 7.06 (s, 1H), 7.10 (d, J ¼ 8:6 Hz,
20
1H); ½aꢁ þ35.9 (c 0.92, EtOH); HRMS calcd for
D
C31H36N2O5 (MþH)þ: 517.2702. Found 517.2693.
5.2.8. (5S,6R,7R)-5-(Benzofuran-6-yl)-7-[2-(3-hydroxy-2-
methylpropyl)-4-methoxyphenyl]-2-(N-isopropylamino)-
cyclopenteno[1,2-b]pyridine-6-carboxylic acid (6a,b).
J ¼ 8:5 Hz, 1H), 6.60–6.72 (m, 2H), 6.89 (d, J ¼ 8:5 Hz,
31
6a; White solid; 1H NMR (CDCl3)
d
1.00 (d,
1H), 7.07–7.53 (m, 4H); ½aꢁ ꢀ0.75 (c 1.00, EtOH);
D
J ¼ 6:6 Hz, 3H), 1.09 (d, J ¼ 6:3 Hz, 3H), 1.14 (d,
J ¼ 6:3 Hz, 3H), 1.95–2.07 (m, 1H), 2.43–2.55 (m, 1H),
2.88–2.99 (m, 1H), 3.22 (dd, J ¼ 8:1, 8.4 Hz, 1H), 3.32–
3.48 (m, 2H), 3.55–3.66 (m, 1H), 3.70 (s, 3H), 4.62 (d,
J ¼ 8:4 Hz, 1H), 5.04 (d, J ¼ 8:1 Hz, 1H), 6.20 (d,
J ¼ 8:4 Hz, 1H), 6.62–6.69 (m, 2H), 6.74 (d, J ¼ 1:5 Hz,
1H), 6.89 (d, J ¼ 8:4 Hz, 1H), 7.08 (d, J ¼ 8:4 Hz, 1H),
7.17 (d, J ¼ 8:4 Hz, 1H), 7.43 (s, 1H), 7.53 (d,
J ¼ 8:4 Hz, 1H), 7.60 (d, J ¼ 1:5 Hz, 1H); HRMS calcd
for C31H34N2O5 (MþH)þ: 515.2546. Found 515.2523.
6b; White solid; 1H NMR (CDCl3) d 0.86 (d, J ¼ 6:9 Hz,
3H), 1.13 (d, J ¼ 6:9 Hz, 3H), 1.15 (d, J ¼ 6:9 Hz, 3H),
1.83–1.96 (m, 1H), 2.40–2.52 (m, 1H), 2.65–2.77 (m,
1H), 3.18 (dd, J ¼ 8:3, 8.2 Hz, 1H), 3.28–3.42 (m, 2H),
3.55–3.65 (m, 1H), 3.67 (s, 3H), 4.58 (d, J ¼ 8:3 Hz, 1H),
4.89 (d, J ¼ 8:2 Hz, 1H), 6.22 (d, J ¼ 8:4 Hz, 1H), 6.60
(d, J ¼ 2:4 Hz, 1H), 6.66 (dd, J ¼ 2:4, 8.4 Hz, 1H), 6.73
(d, J ¼ 2:4 Hz, 1H), 6.90 (d, J ¼ 8:4 Hz, 1H), 7.10 (d,
J ¼ 8:4 Hz, 1H), 7.15 (d, J ¼ 8:4 Hz, 1H), 7.40 (s, 1H),
7.52 (d, J ¼ 8:4 Hz, 1H), 7.59 (d, J ¼ 2:4 Hz, 1H);
HRMS calcd for C31H34N2O5 (MþH)þ: 515.2546.
Found 515.2517.
1
8b; White solid; mp 126–127 ꢁC; H NMR (CDCl3) d
0.85 (d, J ¼ 6:5 Hz, 3H), 1.12 (d, J ¼ 6:5 Hz, 3H), 1.15
(d, J ¼ 6:5 Hz, 3H), 1.85–1.95 (m, 1H), 2.37–2.49 (m,
1H), 2.67–2.81 (m, 1H), 3.23 (dd, J ¼ 8:2, 8.8 Hz, 1H),
3.29–3.40 (m, 2H), 3.52–3.65 (m, 1H), 3.65 (s, 3H), 4.83
(d, J ¼ 8:8 Hz, 1H), 4.90 (d, J ¼ 8:2 Hz, 1H), 6.23 (d,
J ¼ 8:5 Hz, 1H), 6.55–6.70 (m, 3H), 6.89 (d, J ¼ 8:5 Hz,
1H), 7.06 (d, J ¼ 7:5 Hz, 1H), 7.03–7.14 (m, 2H), 7.40
31
(d, J ¼ 8:2 Hz, 1H), 7.51 (d, J ¼ 2:1 Hz, 1H); ½aꢁ þ15.9
D
(c 0.94, EtOH); HRMS calcd for C31H34N2O5 (MþH)þ:
515.2546. Found 515.2533.
5.2.11. (5S,6R,7R)-5-(2,3-Dihydrobenzo[b] furan-4-yl)-7-
[2-(3-hydroxy-2-methylpropyl)-4-methoxyphenyl]-2-(N-iso-
propylamino)cyclopenteno[1,2-b]pyridine-6-carboxylic
1
acid (9a,b). 9a; White solid; mp 128–130 ꢁC. H NMR
(CDCl3) d 1.06 (d, J ¼ 6:9 Hz, 3H), 1.10 (d, J ¼ 6:3 Hz,

2148
T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
1
3H), 1.14 (d, J ¼ 6:3 Hz, 3H), 2.10–2.12 (m, 1H), 2.49–
2.62 (m, 1H), 3.01 (dd, J ¼ 8:9, 9.0 Hz, 1H), 3.15–3.25
(m, 1H), 3.21 (t, J ¼ 8:7 Hz, 2H), 3.37–3.42 (m, 2H),
3.56–3.67 (m, 1H), 3.75 (s, 3H), 4.49 (d, J ¼ 9:0 Hz, 1H),
4,58 (t, J ¼ 8:7 Hz, 2H), 5.05 (d, J ¼ 8:9 Hz, 1H), 6.19
(d, J ¼ 8:5 Hz, 1H), 6.66–6.77 (m, 3H), 6.81 (d,
11b; White form; mp 119–120 ꢁC. H NMR (CDCl3) d
0.90 (d, J ¼ 6:8 Hz, 3H), 1.13 (d, J ¼ 6:9 Hz, 3H), 1.15
(d, J ¼ 6:9 Hz, 3H), 1.86–2.01 (m, 1H), 2.47–2.72 (m,
2H), 3.06 (dd, J ¼ 8:8,9.2 Hz, 1H), 3.28–3.47 (m, 2H),
3.59–3.70 (m, 1H), 3.71 (s, 3H), 4.46 (d, J ¼ 8:8 Hz, 1H),
4.87 (d, J ¼ 9:2 Hz, 1H), 6.24 (d, J ¼ 8:6 Hz, 1H), 6.61–
J ¼ 7:3 Hz, 1H), 6.91 (d, J ¼ 8:5 Hz, 1H), 7.09 (d,
6.70 (m, 2H), 6.84 (d, J ¼ 8:6 Hz, 1H), 6.93–7.02 (m,
32
32
J ¼ 8:5 Hz, 1H), 7.15 (d, J ¼ 7:3 Hz, 1H); ½aꢁ ꢀ8.0 (c
3H), 7.08 (d, J ¼ 8:7 Hz, 1H); ½aꢁ þ27.7 (c 1.00,
D
D
1.00, EtOH); HRMS calcd for C31H36N2O5 (MþH)þ:
EtOH). HRMS calcd for C30H32F2N2O6 (MþH)þ:
517.2702. Found 517.2690.
555.2307. Found 555.2280.
1
9b; White solid; mp 127–129 ꢁC. H NMR (CDCl3) d
0.87 (d, J ¼ 6:5 Hz, 3H), 1.12 (d, J ¼ 6:6 Hz, 3H), 1.15
(d,J ¼ 6:6 Hz, 3H), 1.82–1.95 (m, 1H), 2.40–250 (m,
1H), 2.83–2.95 (m, 1H), 3.05–3.17 (m, 2H), 3.18 (dd,
J ¼ 8:2; 8:8 Hz), 3.25–3.40 (m, 2H), 3.53–3.66 (m, 1H),
3.68 (s, 3H), 4.40–4.53 (m, 2H), 4.53 (d, J ¼ 8:2 Hz, 1H),
4.83 (d, J ¼ 8:8 Hz, 1H), 6.22 (d, J ¼ 8:5 Hz, 1H), 6.55–
5.2.14. (5S,6R,7R)-(+)-5-(2,2-Difluorobenzo[1,3]dioxol-
5-yl)-7-(2-hydroxymethyl-4-methoxyphenyl)-2-(N-isopro-
pylamino)cyclopenteno[1,2-b]pyridine-6-carboxylic acid
(11f). 11f; Pale yellow solid; mp 139–142 ꢁC. 1H NMR
(CDCl3) d 1.24–1.28 (m, 6H), 3.86 (dd, J ¼ 8:9, 9.2 Hz,
1H), 3.59–3.70 (m, 1H), 3.76 (s, 3H), 4.60 (d, J ¼ 9:2 Hz,
1H), 4.62 (d, J ¼ 12:8 Hz, 1H), 4.98 (d, J ¼ 12:8 Hz,
6.70 (m, 4H), 6.87 (d, J ¼ 8:5 Hz, 1H), 7.03–7.18 (m,
32
2H); ½aꢁ þ6.3 (c 1.00, EtOH); HRMS calcd for
1H), 5.50 (d, J ¼ 8:9 Hz, 1H), 6.56 (d, J ¼ 9:0 Hz, 1H),
D
20
C31H36N2O5 (MþH)þ: 517.2702. Found 517.2689.
6.81–7.10 (m, 6H), 7.39 (d, J ¼ 9:0 Hz, 1H); ½aꢁ þ37.8
D
(c 1.00, EtOH); HRMS calcd for C27H27F2N2O6
(MþH)þ: 513.1837. Found 513.1812.
5.2.12. (5S,6R,7R)-(+)-5-(2,2-Dimethylbenzo[1,3]dioxol-
5-yl)-7-[2-(3-hydroxy-2-methylpropyl)]-4-methoxyphenyl-
2-(N-isopropylamino)cyclopenteno[1,2-b]pyridine-6-car-
boxylic acid (10a,b). 10a; White solid; mp 126–127 ꢁC.
1H NMR (CDCl3) d 0.98 (d, J ¼ 6:7 Hz, 3H), 1.12 (d,
J ¼ 6:7 Hz, 3H), 1.15 (d, J ¼ 6:7 Hz, 3H), 1.66 (s, 6H),
1.98–2.10 (m, 1H), 2.40–2.52 (m, 1H), 2.90–3.00 (m,
1H), 3.15 (t, J ¼ 9:0 Hz, 1H), 3.30–3.50 (m, 2H), 3.55–
3.66 (m, 1H), 3.73 (s, 3H), 4.43 (d, J ¼ 9:0 Hz, 1H), 5.02
(d, J ¼ 9:0 Hz, 1H), 6.20 (d, J ¼ 8:2 Hz, 1H), 6.52–6.72
5.3. Endothelin receptor binding assay
The binding affinities were determined according to the
reported method15 by inhibition of specific binding of
[
125I]ET-1 using membranes prepared from human
neuroblastoma-derived SK-N-MC cells and Girardi
heart cells that were reported to possess only ETA and
ETB receptors, respectively.17
(m, 4H), 6.86 (d, J ¼ 8:2 Hz, 1H), 7.08–7.17 (m, 2H);
26
½aꢁ
þ22.7 (c 2.16, EtOH); HRMS calcd for
D
5.4. Screening assay for CYP3A4 inhibition
C32H38N2O6 (MþH)þ: 547.2808. Found 547.2813.
Human liver microsome (1.0 mg/mL) in buffer A [con-
sisting of 0.1 M phosphate buffer (pH 7.4), 3.0 mM
MgCl2, 0.4 l/mL glucose-6-phosphate dehydrogenase,
3.3 mM glucose-6-phosphate and 1.3 mM NADP] was
incubated with tested compound (20 lM) at 37 ꢁC for
60 min. Testosterone (0.2 mM) in buffer A at was
subsequently incubated for 5 min at 37 ꢁC. After the
addition of a 2-fold volume of methanol, the amount of
6b-hydroxytestosterone was measured by HPLC (col-
umn: Capcelpak AG 120 C18, Mobile Phase: MeOH/
10 mM AcONH4 (3:2), flow rate: 1.0 mL/min).
1
10b; White solid; mp 122–124 ꢁC. H NMR (CDCl3) d
0.93 (d, J ¼ 6:3 Hz, 3H), 1.14 (d, J ¼ 6:6 Hz, 3H), 1.16
(d, J ¼ 6:6 Hz, 3H), 1.66 (s, 6H), 1.91–2.06 (m, 1H),
2.55–2.83 (m, 2H), 3.17 (t, J ¼ 8:4 Hz, 1H), 3.32–3.52
(m, 2H), 3.56–3.67 (m, 1H), 3.72 (s, 3H), 4.45 (d,
J ¼ 8:4 Hz, 1H), 4.90 (d, J ¼ 8:4 Hz, 1H), 6.22 (d,
J ¼ 8:7 Hz, 1H), 6.60–6.75 (m, 4H), 6.90 (d, J ¼ 8:7 Hz,
26
1H), 7.08–7.17 (m, 2H). ½aꢁ þ38.3 (c 2.46, EtOH);
D
HRMS calcd for C32H38N2O6 (MþH)þ: 547.2808.
Found 547.2805.
5.2.13. (5S,6R,7R)-(+)-5-(2,2-Difluorobenzo[1,3]dioxol-
5-yl)-7-[2-(3-hydroxy-2-methylpropyl)-4-methoxyphenyl]-
2-(N-isopropylamino)cyclopenteno[1,2-b]pyridine-6-car-
boxylic acid (11a,b). 11a; White form; mp 121–124 ꢁC.
1H NMR (CDCl3) d 1.05 (d, J ¼ 6:0 Hz, 3H), 1.10 (d,
J ¼ 6:3 Hz, 3H), 1.15 (d, J ¼ 6:3 Hz, 3H), 1.98–2.10 (m,
1H), 2.48–2.56 (m, 1H), 2.99 (dd, J ¼ 8:2, 10.2 Hz, 1H),
3.10–3.19 (m, 1H), 3.30–3.50 (m, 2H), 3.54–3.70 (m,
1H), 3.76 (s, 3H), 4.50 (d, J ¼ 8:2 Hz, 1H), 5.04 (d,
J ¼ 10:2 Hz, 1H), 6.20 (d, J ¼ 8:5 Hz, 1H), 6.68–6.73
5.5. Competitive inhibition of human CYPs marker
activities
Human liver microsomes were preincubated for 5 min at
37 ꢁC in the presence of a NADPH-generating system
(10 mM glucose-6-phosphate, 1 unit/mL glucose-6-
phosphate dehydrogenase, 3 mM MgCl2, 1 mM
b-NADPþ in 100 mM potassium phosphate buffer
pH 7.4). The reaction was initiated by adding test com-
pound (1f or 7f up to 100 lM) and a CYP marker
substrate at a concentration similar to its Km value.18
The reaction was terminated by adding 2 vol of an
actonitrile–methanol mixture (2:1, v/v) except that
(m, 2H), 6.83 (d, J ¼ 8:5 Hz, 1H), 7.01–7.09 (m, 4H).
20
½aꢁ
þ17.8 (c 1.0, EtOH); HRMS calcd for
D
C30H32F2N2O6 (MþH)þ: 555.2307. Found 555.2291.

T. Yoshizumi et al. / Bioorg. Med. Chem. 12 (2004) 2139–2150
2149
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N.; Barrish, J. C. J. Med. Chem. 2003, 46, 125.
0.1 vol of 60% HClO4aq was used for bufuralol. The
amount of metabolite formation specific for the CYP
activity was measured by either HPLC-UV or HPLC-
fluorescence method. The percent inhibition of the CYP
activity was calculated from the ratio of metabolite
formation with or without the test compound.
5. (a) Balwierczak, J. L.; Bruseo, C. W.; Del Grande, D.;
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A.; Kumar, C.; Lee, J. A.; Bean, J. W.; Debrosse, C. W.;
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5.6. Mechanism-based inactivation of human CYPs
Mechanism-based inactivation was examined by prein-
cubating with human liver microsomes and 10 lM of
either 1f or 7f in the presence of a NADPH-generating
system. After the 30-min preincubation, a CYP marker
substrate at a concentration similar to its Km value18 was
added to the mixture. The reaction was terminated by
adding 2 vol of an actonitrile–methanol mixture (2:1, v/
v) except that 0.1 vol of 60% HClO4aq was used for
bufuralol. The amount of metabolite formation specific
for the CYP activity was measured by either HPLC-UV
or HPLC-fluorescence method. The percent inhibition
of the CYP activity was calculated from the ratio of
metabolite formation with or without the test com-
pound.
Acknowledgements
We are grateful to Mr. Shinnosuke Abe and Mr. Koichi
Osawa for analytical support (Mass spectra).
References and notes
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18. Concentration of marker substrates, incubation time and
microsomal protein concentration used in this study are as
follows: 7-Ethoxyresorufin (CYP1A2): 0.2 lM, 10 min
(0.25 mg/mL microsomal protein concentration), Couma-
rin (CYP2A6): 0.3 lM, 25 min (0.005 mg/mL), tolbutamide
(CYP2C9): 100 lM, 60 min (0.25 mg/mL), S-Mephenytoin
(CYP2C19): 100 lM, 60 min (1.0 mg/mL), Bufurolol
(CYP2D6): 20 lM, 20 min (0.25 mg/mL), Testosterone
(CYP3A4): 100 lM, 10 min (0.25 mg/mL).