5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid and its derivatives inhibit ionotropic γ-aminobutyric acid receptors by binding to the 4'-ethynyl-4-n-propylbicycloorthobenzoate site

Article abstract of DOI:10.1016/S0968-0896(00)00009-2

Acyclic noncompetitive antagonists of ionotropic γ-aminobutyric acid (GABA) receptors, bearing an ester or ether linkage, were designed, synthesized, and assayed for their inhibition of the specific binding of [³H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and housefly head membranes. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of [³H]EBOB in rat brain membranes, with an IC₅₀ of 88 nM. The potency conferred by the p-substituent decreased in the order C=C(CH₂)₂COOH>C=C(CH₂)₂COOCH₃>C=CH>Br. Pentyl phenyl ethers were equally potent compared with butyl benzoates, while phenyl pentanoates and benzyl butyl ethers were less potent. These compounds were generally less active in housefly head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in housefly GABA receptors, whereas this modification at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat receptors, this modification resulted in a decrease in potency except for a phenyl pentanoate. To confirm that DBCPP interferes with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC₅₀ of 0.54 μM and a Hill coefficient of 0.7. These findings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by binding to the EBOB site and that there might be structural difference in the noncompetitive antagonist-binding site between rat and housefly GABA receptors. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00009-2

Bioorganic & Medicinal Chemistry 8 (2000) 665±674
5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic Acid and
Its Derivatives Inhibit Ionotropic ꢀ-Aminobutyric Acid Receptors
by Binding to the 40-Ethynyl-4-n-propylbicycloorthobenzoate Site
Hiroshi Hamano, a Keiichi Nagata, b,y Nobuo Fukada, a Hiroshi Shimotahira, a
Xiu-Lian Ju a and Yoshihisa Ozoe a,*
aDepartment of Life Science and Biotechnology, Shimane University, Matsue 690-8504, Japan
bInstitute of Agriculture and Forestry, University of Tsukuba, Tsukuba 305-8572, Japan
Received 12 October 1999; accepted 16 December 1999
AbstractÐAcyclic noncompetitive antagonists of ionotropic g-aminobutyric acid (GABA) receptors, bearing an ester or ether
linkage, were designed, synthesized, and assayed for their inhibition of the speci®c binding of [3H]40-ethynyl-4-n-propylbicyclo-
orthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and house¯y head membranes. 5-[4-(3,3-Dimethyl-
butoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of
[3H]EBOB in rat brain membranes, with an IC50 of 88 nM. The potency conferred by the p-substituent decreased in the order
CꢀC(CH2)2COOH>CꢀC(CH2)2COOCH3>CꢀCH>Br. Pentyl phenyl ethers were equally potent compared with butyl benzo-
ates, while phenyl pentanoates and benzyl butyl ethers were less potent. These compounds were generally less active in house¯y
head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl
group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in house¯y GABA receptors, whereas this
modi®cation at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat
receptors, this modi®cation resulted in a decrease in potency except for a phenyl pentanoate. To con®rm that DBCPP interferes
with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the
primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC50 of
0.54 mM and a Hill coecient of 0.7. These ®ndings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by
binding to the EBOB site and that there might be structural di€erence in the noncompetitive antagonist-binding site between rat
and house¯y GABA receptors. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
GABA binding site. Similar but pharmacologically dif-
ferent ionotropic GABA receptors are present in the
central and peripheral nervous systems of invertebrate
species.2,3
The g-aminobutyric acid (GABA)A receptor is a hetero-
oligomeric ion channel protein that mediates inhibitory
neurotransmission in the vertebrate central nervous
system.1 GABA inhibits neuronal ®ring by increasing
the passage of chloride ions into the cell through the
channel. The GABAA receptor has binding sites for
several classes of medicinal or toxic compounds and for
ions, in addition to the binding site for GABA. These
chemicals allosterically modulate the action of GABA
on the receptor by binding to sites distinct from the
Noncompetitive antagonists inhibit the function of
ionotropic GABA receptors by binding to a site that is
labeled by a high-anity, selective radioligand, [3H]40-
ethynyl-4-n-propylbicycloorthobenzoate (EBOB).4 The
noncompetitive antagonists include diverse classes of
compounds such as plant terpenoid toxins (picrotox-
inin,5 picrodendrins,6 etc.), insecticides (lindane,7,8
dieldrin,9 etc.), and convulsants (bicyclophosphates,10
bicycloorthocarboxylates,11 dithianes,12 etc.). On the
basis of the antagonists' structure±activity relationships,
we previously constructed a model of the binding site
that can accommodate structurally diverse antagonists.
The generated model predicted that four important
*Corresponding author. Tel.: +81-852-32-6573; fax: +81-852-32-
6092; e-mail: ozoe-y@life.shimane-u.ac.jp
yPresent address: Laboratory of Memory and Learning, Brain Science
Institute, The Institute of Physical and Chemical Research (RIKEN),
Hirosawa 2-1, Wako 351-0198, Japan.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00009-2

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H. Hamano et al. / Bioorg. Med. Chem. 8 (2000) 665±674
interacting subsites are related to the antagonist activ-
ity;13 however, no antagonists supposed to interact with
all four sites have been found, probably because the
conformational rigidity of known antagonists precludes
the interaction of any one of them with all four subsites.
[3H]EBOB binding to rat brain membranes, respec-
tively. DBCPP increased an apparent dissociation con-
stant (Kd) of EBOB from 2.45 to 4.74 nM without the
signi®cant alteration of the maximum number of bind-
ing sites (Bmax) (2.38 and 2.36 pmol/mg protein in the
absence and presence of DBCPP, respectively), indicat-
ing that DBCPP competes with EBOB for a common
binding site in rat brain GABAA receptors. Scatchard
analysis was also performed for the inhibition of
[3H]EBOB binding by 3 mM 8 in house¯y head mem-
branes, suggesting that this type of benzoate acts as a
noncompetitive antagonist in house¯y GABA receptors
(data not shown).
In the present study, we have designed and synthesized
four classes of ¯exible acyclic antagonists (Fig. 1)
as molecular probes to verify the existence of the
four important subsites, and we here report that 5-[4-
(3,3-dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid
(DBCPP) and its derivatives are noncompetitive
antagonists interacting with the EBOB site. We also
discuss the structure±activity relationships of the acyclic
antagonists in relation to our binding-site model.
Phenyl pentanoates. Phenyl pentanoate 9, bearing
CꢀC(CH2)2COOCH3 at the p-position, poorly inhibited
[3H]EBOB binding in rat brain membranes (Table 1).
The introduction of an isopropyl group into the 2-position
of 9 led to a moderately active inhibitor. In house¯y head
membranes, both compounds were weakly active or
inactive. Analogues with a Br and CꢀCH substituent
were devoid of inhibitory activity in both rat brain and
house¯y head membranes (data not shown).
Results
E€ects of acyclic esters and ethers on [3H]EBOB binding
Butyl benzoates. 3,3-Dimethylbutyl benzoates (1, 3, 5
and 7) inhibited speci®c [3H]EBOB binding to rat brain
membranes, with IC50s of 0.088±3.77 mM (Table 1). 5-[4-
(3,3-Dimethylbutylbutoxycarbonyl)phenyl]-4-pentynoic
acid (DBCPP, 7) was the most potent inhibitor (Table 1
and Fig. 2A). The change of the p-substituent caused a
decrease in potency in the order CꢀC(CH2)2COOH
>C ꢀC(CH2)2COOCH3>C ꢀCH>Br. The introduction
of an isopropyl group into the 1-position of the 3,3-
dimethylbutyl group resulted in a reduction in potency.
In house¯y head membranes, benzoates with a Br and
CꢀCH substituent were weakly active or inactive, and
those with CꢀC(CH2)2COOCH3 and CꢀC(CH2)2
COOH were moderately active. The introduction of an
isopropyl group into the 1-position of the 3,3-dimethyl-
butyl group led to an increased (3 versus 4), unchanged
(7 versus 8), or decreased (5 versus 6) potency.
Benzyl butyl ethers. Alkyl benzyl ethers 13, 15 and 17
were moderately e€ective in inhibiting [3H]EBOB bind-
ing to rat brain membranes. Replacement of CꢀCH
with CꢀC(CH2)2COOCH3 and CꢀC(CH2)2COOH
resulted in a slight increase in potency. Compounds 15
and 17 were less potent in house¯y head membranes
than in rat brain membranes, while 13 was equally
active in both membranes. The introduction of an iso-
propyl group into the 1-position of the 3,3-dimethyl-
butyl group of these compounds caused a decrease in
potency in rat brain membranes. In contrast, the intro-
duction of an isopropyl group in 15 and 17 led to inhi-
bitors with increased potencies in house¯y head
membranes.
Figure 2B and C show the e€ects of 88 nM DBCPP on
the saturation isotherm and the Scatchard plot of
Pentyl phenyl ethers. The dependence of potency on the
p-substituent was similar to that of other classes of
compounds. Compounds 23 and 25 exhibited a high
level of potency, similar to the corresponding butyl
benzoates. These compounds were more potent in rat
brain membranes than in house¯y head membranes.
The e€ects of the isopropyl group were negative in both
rat brain and house¯y head membranes.
E€ects of DBCPP on GABA-induced currents
When the membrane potential was held at 60 mV in
the normal external solution, the bath application of
GABA produced an inward current in rat dorsal root
ganglion neurons. Responses to low concentrations of
GABA were maintained at a stable level over a period
of up to 60 min after the rupture of the membrane
(data not shown). The currents induced by 30 mM
GABA were suppressed when 0.1 or 1 mM DBCPP was
co-applied (Fig. 3A). During DBCPP suppression, the
desensitization of GABA-induced currents was acceler-
ated. Currents were completely recovered after the
membranes were washed with a DBCPP-free solution
(data not shown).
Figure 1. Structures of acyclic esters and ethers synthesized in the
present study.

H. Hamano et al. / Bioorg. Med. Chem. 8 (2000) 665±674
667
Table 1. Potency of DBCPP and its derivatives in inhibiting [3H]EBOB binding to rat brain and house¯y head membranes
No.
R1
L
R2
IC50 (mM)a
Rat
House¯y
Butyl benzoate
1
2
3
4
H
CH(CH3)2
H
CH(CH3)2
H
CH(CH3)2
H
CH(CH3)2
-O±CO-
-O±CO-
-O±CO-
-O±CO-
-O±CO-
-O±CO-
-O±CO-
-O±CO-
Br
Br
3.77 (2.69±5.27b)
>10 (15.4c)
>10 (22.4c)
>10 (15.9c)
>10 (27.3c)
CꢀCH
0.67 (0.53±0.84b)
>10 (39.1c)
CꢀCH
7.84 (5.06±12.16b)
4.15 (2.76±6.23b)
>10 (49.9c)
5
CꢀC(CH2)2COOCH3
CꢀC(CH2)2COOCH3
CꢀC(CH2)2COOH
CꢀC(CH2)2COOH
0.22 (0.18±0.29b)
4.83 (3.43±6.80b)
0.088 (0.068±0.110b)
1.41 (1.10±1.81b)
6
7 (DBCPP)
8
3.41 (2.26±5.15b)
2.90 (1.88±4.47b)
Phenyl pentanoate
9
10
H
CH(CH3)2
-CO±O-
-CO±O-
CꢀC(CH2)2COOCH3
CꢀC(CH2)2COOCH3
>10 (48.4c)
4.67 (3.31±6.59b)
>10 (17.6c)
>10 (35.1c)
Benzyl butyl ether
11
12
13
14
15
16
17
18
H
CH(CH3)2
H
CH(CH3)2
H
CH(CH3)2
H
CH(CH3)2
-O±CH2-
-O±CH2-
-O±CH2-
-O±CH2-
-O±CH2-
-O±CH2-
-O±CH2-
-O±CH2-
Br
Br
>10 (32.5c)
>10 (17.5c)
>10 (10.6c)
>10 (11.2c)
CꢀCH
3.41 (2.68±4.34b)
>10 (13.8c)
5.45 (3.79±7.84b)
6.02 (4.05±8.95b)
>10 (29.9c)
CꢀCH
CꢀC(CH2)2COOCH3
CꢀC(CH2)2COOCH3
CꢀC(CH2)2COOH
CꢀC(CH2)2COOH
1.99 (1.50±2.64b)
>10 (49.2c)
7.96 (5.01±12.67b)
>10 (37.2c)
1.68 (1.30±2.17b)
5.51 (4.09±7.41b)
2.72 (2.06±3.61b)
Pentyl phenyl ether
19
20
21
22
23
24
25
26
H
CH(CH3)2
H
CH(CH3)2
H
CH(CH3)2
H
CH(CH3)2
-CH2±O-
-CH2±O-
-CH2±O-
-CH2±O-
-CH2±O-
-CH2±O-
-CH2±O-
-CH2±O-
Br
Br
>10 (41.0c)
>10 (16.8c)
>10 (9.3c)
>10 ( 0.4c)
CꢀCH
5.16 (3.67±7.27b)
>10 (26.6c)
0.13 (0.10±0.18b)
9.05 (5.12±16.00b)
0.10 (0.080±0.13b)
2.90 (2.21±3.82b)
5.62 (3.74±8.45b)
>10 (37.5c)
CꢀCH
CꢀC(CH2)2COOCH3
CꢀC(CH2)2COOCH3
CꢀC(CH2)2COOH
CꢀC(CH2)2COOH
4.94 (3.13±7.79b)
>10 (11.1c)
5.21 (3.58±7.59b)
3.78 (3.23±4.41b)
aDetermined using 0.5 nM [3H]EBOB.
b95% Con®dence limit.
cInhibition percentage at 10 mM.
Figure 3B shows the concentration±response relation-
ship for the suppression of GABA-induced currents by
co-application with DBCPP. The currents induced by
10-s co-applications of 30 mM GABA and several
concentrations of DBCPP were measured when they
reached a steady-state level after repeated co-applica-
tions. DBCPP suppressed GABA-induced currents in
a concentration-dependent manner with an IC50 of
0.54 mM and a Hill coecient of 0.7.
ethers. Butyl benzoates are regarded as ring-opened
analogues of bicycloorthocarboxylate-type antagonists
such as EBOB. Phenyl pentanoates are the compounds
in which the acid and alcohol sides are reversed com-
pared with those of butyl benzoates. Benzyl butyl ethers
and pentyl phenyl ethers are the compounds that have a
methylene unit in place of the carbonyl group of butyl
benzoates and phenyl pentanoates, respectively. These
compounds were more potent in rat GABAA receptors
than in house¯y GABA receptors, or equipotent in both
receptors, except for four compounds (4, 14, 16 and 18).
The structural requirements for high potency are an
appropriately p-substituted phenyl group and a suitable
alkyl group on each side of their structures as well as a
central electronegative link (L of the structure in Table 1).
In rat brain membranes, butyl benzoates and pentyl
phenyl ethers were more potent than were phenyl
pentanoates and benzyl butyl ethers. This ®nding
might indicate that the presence of oxygen atom(s) in a
symmetrical position with respect to a line passing
through the p-substituents of the phenyl group is
Discussion
Structure±activity relationships
The present study demonstrates that four classes of
acyclic compounds are inhibitors of the speci®c binding
of [3H]EBOB, a radiolabeled noncompetitive GABA
antagonist, to rat brain and house¯y head membranes.
The compounds synthesized are butyl benzoates, phenyl
pentanoates, benzyl butyl ethers, and pentyl phenyl

668
H. Hamano et al. / Bioorg. Med. Chem. 8 (2000) 665±674
Figure 2. Inhibition of [3H]EBOB binding by DBCPP in rat brain membranes: (A) concentration±inhibition curves; (B) saturation isotherms of
[3H]EBOB binding in the absence (&) and presence (*) of 88 nM DBCPP; (C) Scatchard plots in the absence (&) and presence (*) of 88 nM
DBCPP.
CꢀC(CH2)2COOH into the p-position of the phenyl
group markedly increased the potency compared with
the Br analogues. This substitution has been reported to
confer high potency to dithiane-type antagonists.14 The
signi®cance of the special interaction of these functional
groups with the binding site should be pursued further
in future work. The isopropyl group (R1 of the structure
in Table 1) in the alkyl moiety was introduced because
a similar modi®cation rendered bicyclophosphoro-
thionate-type antagonists capable of exhibiting selectiv-
ity for house¯y GABA receptors versus rat GABAA
receptors.15 However, the isopropyl group's e€ect was
not prominent in the ¯exible, acyclic compounds exam-
ined in the present study, although the enhancement of
potency for house¯y receptors was observed in 4 (versus
3), 16 (versus 15) and 18 (versus 17).
Site of action
Scatchard analysis showed that the inhibition of
[3H]EBOB binding by DBCPP and 8 is competitive,
suggesting their noncompetitive antagonism toward
ionotropic GABA receptors (Fig. 2). To con®rm this
®nding by a functional assay, whole-cell patch clamp
experiments were performed with rat dorsal root gang-
lion neurons in the primary culture. When DBCPP was
co-applied with GABA for 10 s, the current was sup-
pressed in a dose-dependent manner (Fig. 3). During
DBCPP suppression, the desensitization of GABA-
induced currents was accelerated. These ®ndings indi-
cate that the DBCPP suppression of the GABA-induced
currents was use-dependent, being accelerated by fre-
quent openings of GABA-gated channels. Both bio-
chemical and electrophysiological data thus lead to the
conclusion that DBCPP and its derivatives are non-
competitive antagonists that share a common binding
site with EBOB.
Figure 3. Suppression of GABA-induced currents by co-application of
DBCPP and GABA in rat dorsal root ganglion neurons: (A) current
records in response to 10-s application of 30 mM GABA with and
without 0.1 (a) or 1 mM (b) DBCPP; (B) dose±response relationship
for the suppression of GABA-induced peak currents by co-application
of DBCPP.
Interaction with the binding site
advantageous for high potency (Fig. 4). In house¯y
head membranes, however, butyl benzoates and pentyl
phenyl ethers were not necessarily more potent than
were benzyl butyl ethers. As for the aromatic moiety,
the introduction of CꢀCH, CꢀC(CH2)2COOCH3 and
On the basis of the structure±activity relationships of
diverse classes of noncompetitive antagonists, we pre-
viously postulated that there are four major interacting
subsites in the noncompetitive antagonist-binding site

H. Hamano et al. / Bioorg. Med. Chem. 8 (2000) 665±674
669
Figure 4. Partial structures of acyclic GABA antagonists and position(s) of their oxygen atom(s).
within ionotropic GABA receptors (Fig. 5).6 Com-
pounds capable of interacting with two of the four sub-
sites were supposed to act as antagonists, although the
molecular mechanisms by which the antagonists exert
their e€ects remain to be elucidated. Subsite A probably
accepts an electronegative part of ligands. In both sides
of subsite A, there might be spaces (subsites C and D)
that accommodate hydrophobic and hydrophobic/
electronegative parts of ligands. In addition, we infer
the existence of one more subsite (B), which is located
near subsite C. With regard to subsite B, we have
reported that bicyclophosphorothionate GABA
antagonists, bearing an isopropyl group at the 3-posi-
tion, exhibit selectivity for house¯y GABA receptors
versus rat GABAA receptors, while the reversed selec-
tivity is the case for unsubstituted analogues.15 This
®nding allows us to speculate that the e€ect of the iso-
propyl group on the receptor selectivity is due to a
structural di€erence around subsite B between insect
and mammalian receptors. This hypothesis has been
substantiated by three-dimensional quantitative struc-
ture±activity relationship analyses.6,15,16
As mentioned above, the compounds synthesized in the
present study are open-ring analogues of bicycloortho-
benzoates. It thus seems reasonable to assume that they
act at the noncompetitive antagonist site in such a way
that their benzene rings lie in the same position, i.e.
subsite D (Fig. 5). We expected that esters and ethers
with R1=i-C3H7 would interact with all four subsites in
insect receptors, and that they would exhibit high
potency in inhibiting [3H]EBOB binding to house¯y
GABA receptors. Actually, although the compounds
showed anity for the EBOB binding site, they are less
potent than expected. Stable conformers of the con-
formationally ¯exible compounds might be incapable of
®tting to the binding site in terms of free energy,17
whereas compact, rigid compounds, such as 3-isopropyl
substituted bicyclophosphorothionates, are suitable for
binding. However, the isopropyl group's e€ect, i.e.
decreasing anity for rat receptors and increasing a-
nity for house¯y receptors, was observed, at least in 4,
16 and 18, being suggestive of the presence of subsite B
in the house¯y antagonist site and structural di€erence
between the rat and house¯y antagonist sites.
In conclusion, we have reported here, for the ®rst time,
a series of acyclic noncompetitive antagonists of iono-
tropic GABA receptors. In particular, 5, DBCPP (7), 23
and 25 were found to be potent antagonists, probably
interacting with subsites A, C, and D in our rat binding-
site model. While the data presented here also suggests
the existence of four subsites in the noncompetitive
antagonist site, further modi®cation will be necessary to
prepare highly active noncompetitive antagonists that
interact with all four subsites.
Experimental
Chemistry
General. Compounds were checked for their purity by
thin layer chromatography on silica gel G, mass spec-
trometry, and 1H NMR spectrometry. Mass spectra
were obtained on a Hitachi M-80B mass spectrometer.
1H NMR spectra were recorded in CDCl3 at 400 MHz
on a JEOL JNM-A-400 spectrometer, unless otherwise
noted. Spin multiplicities are described as: s (singlet), br
s (broad singlet), d (doublet), dd (doublet of doublets),
ddd (doublet of doublets of doublets), t (triplet), sp
(septet), dsp (doublet of septets), or m (multiplet).
Melting points were determined on a Yanako MP-500D
apparatus and are uncorrected. Distillation was per-
formed using a Shibata GTO-350D glass tube oven.
Figure 5. Possible interaction of noncompetitive antagonists with
their binding site. A, B, C and D refer to subsites A, B, C and D.
L=-O±CO-, -CO±O-, -O±CH2-, -CH2±O-; R1=H, i-C3H7; R2=Br,
CꢀCH, CꢀC(CH2)2COOCH3, CꢀC(CH2)2COOH.

670
H. Hamano et al. / Bioorg. Med. Chem. 8 (2000) 665±674
3,3-Dimethylbutyl 4-bromobenzoate (1). A solution of
3,3-dimethyl-1-butanol (2.04 g, 20 mmol) and Et3N
(2.02 g, 20 mmol) in dry Et2O (20 mL) was added
dropwise to an ice-cold, stirred solution of 4-bromo-
benzoyl chloride (4.37 g, 20 mmol) in dry Et2O (30 mL).
After 6-h stirring at room temperature, the solution was
extracted with CHCl3. The CHCl3 extract was washed
with water, dried, and concentrated. The residue was
puri®ed by column chromatography on silica gel with
benzene to give 1 as a colorless liquid (3.51 g, 61.8%
Methyl 5-[4-(3,3-dimethylbutoxycarbonyl)phenyl]-4-pen-
tynoate (5). A solution of 4-pentynoic acid (2.5 g, 25.5
mmol) and H2SO4 (1.5 g) in dry MeOH (50 mL, 1.2 mol)
was heated under re¯ux for 12 h. The solution was
extracted with CH2Cl2, and the CH2Cl2 extract was
washed with a saturated NaHCO3 solution and con-
centrated to give crude methyl 4-pentynoate (2.3 g,
79.2% yield). A solution of 1 (1.4 g, 5 mmol), methyl
4-pentynoate (1.3 g, 12 mmol), bis(triphenylphos-
phine)palladium(II) chloride (87.5 mg, 0.13 mmol), and
copper iodide(I) (25 mg, 0.13 mmol) in Et3N (50 mL)
was re¯uxed under a nitrogen atmosphere for 24 h. The
solution was extracted with Et2O, and the Et2O extract
was dried and concentrated. The residue was puri®ed by
column chromatography on silica gel with n-hexane:
CH2Cl2 (9:1) to give 5 as a white solid (1.25 g, 79.3%
1
yield): H NMR d 0.99 (9H, s, (CH3)3C), 1.70 (2H, t,
J=7.3 Hz, (CH3)3CCH2), 4.37 (2H, t, J=7.3 Hz,
CH2O), 7.57, 7.89 (4H, AA0XX0, J=8.8, 2.1 Hz, Ar);
CIMS m/z 285 (M+1, 99.5%), 286 (M+2, 66.7%), 287
(M+3, 100%), 288 (M+4, 61.3%).
yield): mp 32.2 ꢁC; H NMR d 0.99 (9H, s (CH3)3C),
1
1-Isopropyl-3,3-dimethylbutyl 4-bromobenzoate (2). A
solution of 2,5,5-trimethyl-3-hexanol (1.44 g, 10 mmol),
4-bromobenzoic acid (2.01 g, 10 mmol), dicyclohexyl-
carbodiimide (3.50 g, 17 mmol), and 4-(dimethylamino)-
pyridine (0.37 g, 3 mmol) in toluene (200 mL) was stir-
red at room temperature for 4 days. The solution was
extracted with CHCl3, and the CHCl3 extract was
washed with water, dried, and concentrated. The residue
was puri®ed by column chromatography on silica gel
with benzene to give 2 as a colorless liquid (1.34 g,
1.70 (2H, t, J=7.3 Hz, (CH3)3CCH2), 2.65, 2.76 (4H, 2t,
CꢀC(CH2)2), 3.73 (3H, s, COOCH3), 4.37 (2H, t,
J=7.3 Hz, CH2O), 7.43, 7.94 (4H, 2d, J=8.1 Hz, Ar);
CIMS m/z 317 (M+1, 62.0%).
Methyl 5-[4-(1-isopropyl-3,3-dimethylbutoxycarbonyl)-
phenyl]-4-pentynoate (6). This compound was obtained
as a yellow liquid (53.4% yield), using the procedure
described for 5: 1H NMR d 0.90 (9H, s, (CH3)3C), 0.92,
0.94 (6H, 2d, J=6.8 Hz, CH(CH3)2), 1.45 (1H, dd,
J=14.9, 1.5 Hz, CH2), 1.65 (1H, dd, J=14.9, 9.0 Hz,
CH2), 1.92 (1H, dsp, J=6.8, 4.4 Hz, CH(CH3)2), 2.65,
2.76 (4H, ddd, CꢀC(CH2)2), 3.72 (3H, s, COOCH3),
5.18 (1H, ddd, J=9.0, 4.4, 1.5 Hz, CHO), 7.43, 7.96
(4H, AA0XX0, J=8.7, 1.8 Hz, Ar); CIMS m/z 359
(M+1, 20.0%).
1
41.2% yield): H NMR d 0.90 (9H, s, (CH3)3C), 0.92,
0.94 (6H, 2d, J=6.8 Hz, CH(CH3)2), 1.46 (1H, dd,
J=14.9, 1.5 Hz, CH2), 1.65 (1H, dd, J=14.9, 9.1 Hz,
CH2), 1.92 (1H, dsp, J=6.8, 4.5 Hz, CH(CH3)2), 5.17
(1H, ddd, J=9.1, 4.5, 1.5 Hz, CHO), 7.58, 7.91 (4H,
AA0XX0, J=8.5, 2.3 Hz, Ar); CIMS m/z 327 (M+1,
30.0%), 329 (M+3, 24.3%).
3,3-Dimethylbutyl 4-ethynylbenzoate (3). A solution of 1
(0.57 g, 2 mmol), (trimethylsilyl)acetylene (0.39 g, 4
mmol), triphenylphosphine (24 mg, 0.09 mmol), and
palladium(II) acetate (12 mg, 0.05 mmol) in Et3N (7 mL)
was re¯uxed under a nitrogen atmosphere for 24 h.
After the solution was concentrated, a mixture of dry
tetrahydrofuran (THF) (12 mL) and a THF solution of
1 M tetrabutylammonium ¯uoride (2.4 mL) were added
to the residue at 0 ꢁC. After 2-h stirring at room tem-
perature, the solution was concentrated and extracted
with CH2Cl2. The CH2Cl2 extract was washed with
water, dried, and concentrated. The residue was puri®ed
by column chromatography on silica gel with n-hexane:
CH2Cl2 (9:1) toꢁgive 3 as a yellow solid (264 mg, 57.3%
5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic
acid (7). A mixture of 5 (80 mg, 0.25 mmol), LiOH (42
mg, 1 mmol), THF (5 mL), and H2O (3 mL) was stirred
in an ice bath for 30 min. The solution was washed with
n-hexane, and the aqueous layer was acidi®ed with
dilute HCl and extracted with EtOAc. The EtOAc
extract was dried and concentrated. The residue was
recrystallized from Et2O to give 7 as a white solid (18
mg, 23.8%): mp 116.0 ꢁC; H NMR d (DMSO-d6) 0.96
1
(9H, s, (CH3)3C), 1.65 (2H, t, J=7.1 Hz, (CH3)3CCH2),
2.54, 2.67 (4H, 2t, J=6.8 Hz, CꢀC(CH2)2), 4.33 (2H, t,
J=7.1 Hz, CH2O), 7.51, 7.91 (4H, 2d, J=8.2 Hz, Ar),
12.36 (1H, br s, COOH); CIMS m/z 303 (M+1, 15.0%).
1
yield): mp 50.7 C; H NMR d 1.00 (9H, s, (CH3)3C),
5-[4-(1-Isopropyl-3,3-dimethylbutoxycarbonyl)phenyl]-4-
pentynoic acid (8). This compound was obtained as a
colorless viscous liquid (1.2% yield), using the proce-
1.71 (2H, t, J=7.3 Hz, (CH3)3CCH2), 3.22 (1H, s,
CꢀCH), 4.38 (2H, t, J=7.3 Hz, CH2O), 7.55, 7.89 (4H,
2d, J=8.3 Hz, Ar); CIMS m/z 231 (M+1, 100%).
1
dure described for 7: H NMR d (DMSO-d6) 0.86 (9H,
s, (CH3)3C), 0.87, 0.89 (6H, 2d, J=7.3 Hz, CH(CH3)2),
1.50 (1H, d, J=14.8 Hz, (CH3)3CCH2), 1.63 (1H,
dd, J=14.8, 9.0 Hz, (CH3)3CCH2), 1.87 (1H, m,
CH(CH3)2), 2.55, 2.68 (4H, 2t, J=7.1 Hz, CꢀC(CH2)2),
5.07 (1H, dd, J=9.0, 4.4 Hz, CHO), 7.52, 7.93 (4H, 2d,
J=8.3 Hz, Ar), 12.37 (1H, br s, COOH); CIMS m/z 345
(M+1, 30.0%).
1-Isopropyl-3,3-dimethylbutyl 4-ethynylbenzoate (4).
This compound was obtained as a yellow liquid (8.8%
yield) from 2, using the procedure described for 3: H
NMR d 0.91 (9H, s, (CH3)3C), 0.92, 0.94 (6H, 2d,
J=6.8 Hz, CH(CH3)2), 1.46 (1H, dd, J=14.9, 1.5 Hz,
CH2), 1.66 (1H, dd, J=14.9, 9.0 Hz, CH2), 1.93 (1H,
dsp, J=6.8, 4.4 Hz, CH(CH3)2), 3.22 (1H, s, CꢀCH),
5.18 (1H, ddd, J=9.0, 4.4, 1.5 Hz, CHO), 7.55, 8.00
(4H, AA0XX0, J=8.5, 1.6 Hz, Ar); CIMS m/z 273
(M+1, 3.3%).
1
Methyl 5-[4-(4,4-dimethylpentanoyloxy)phenyl]-4-pen-
tynoate (9). A solution of 3,3-dimethyl-1-butanol (20.7
g, 202.9 mmol) in pyridine (2 g, 25 mmol) was added

H. Hamano et al. / Bioorg. Med. Chem. 8 (2000) 665±674
671
dropwise to an ice-cold, stirred solution of PBr3 (23 g,
85.8 mmol) containing a few drops of pyridine. After
12-h re¯ux, the solution was distilled to give 1-bromo-
3,3-dimethylbutane (33.0 g, 99.2% yield): bp 136±
138 ꢁC. A solution of 1-bromo-3,3-dimethylbutane (5.6
g, 34 mmol) in dry Et2O (50 mL) was added dropwise to
an ice-cold, stirred mixture of magnesium turnings (0.82
g, 34 mg-atom) in dry Et2O (50 mL). After the solution
was stirred for 2 h at room temperature, dry CO2 gas
was passed into the solution for 1 h. The reaction mix-
ture was poured dropwise into a 25% NH4Cl solution
and extracted with EtOAc. The EtOAc extract was
washed with water and concentrated. The residue was
washed with an NaHCO3 solution and washed with n-
hexane. The aqueous layer was acidi®ed with 1 N HCl
and extracted with EtOAc. The EtOAc extract was
dried and concentrated to give crude 4,4-dimethylpen-
tanoic acid (1.95 g, 43.9% yield). The reaction of 4,4-
dimethylpentanoic acid with 4-bromophenol in a man-
ner similar to that described for 2 gave 4-bromophenyl
4,4-dimethylpentanoate as a white solid (27.5% yield):
mp 54.8 ꢁC. 9 was obtained as a white solid (77.3%
yield) from 4-bromophenyl 4,4-dimethylpentanoate and
methyl 4-pentynoate, using the procedure described for
2.42, 2.44 (1H, ddd, J=10.4, 6.8, 1.2 Hz, CHCO), 2.63,
2.72 (4H, 2ddd, CꢀC(CH2)2), 3.72 (3H, s, COOCH3),
6.98, 7.38 (4H, AA0XX0, J=8.5, 2.3 Hz, Ar); CIMS m/z
359 (M+1, 100%).
4-Bromobenzyl 3,3-dimethylbutyl ether (11). 4-Bromo-
benzyl bromide (4.96 g, 20 mmol) was added to sodium
3,3-dimethyl butoxide prepared from 3,3-dimethyl-1-
butanol (3.06 g, 30 mmol) and sodium (0.46 g, 20 mg-
atom) in dry n-butyl ether (20 mL). The mixture was
re¯uxed for 2 h and partitioned between Et2O and H2O.
The Et2O layer was concentrated, puri®ed by column
chromatography on silica gel with benzene, and distilled
to give 11 as a colorless liquid (2.29 g, 42.5% yield): bp
115±118 ꢁC/4 mmHg; 1H NMR d 0.92 (9H, s, (CH3)3C),
1.56 (2H, t, J=7.5 Hz, (CH3)3CCH2), 3.52 (2H, t,
J=7.5 Hz, CH2CH2O), 4.44 (2H, s, OCH2Ph), 7.21,
7.46 (4H, AA0XX0, J=8.5, 2.0 Hz, Ar); CIMS m/z 269
(M-1, 100%), 270 (M, 99.3%), 271 (M+1, 99.5%), 272
(M+2, 99.3%), 273 (M+3, 59.3%).
4-Bromobenzyl 1-isopropyl-3,3-dimethylbutyl ether (12).
This compound was obtained as a colorless liquid
(10.8% yield) by the reaction of 2,5,5-trimethyl-3-hex-
anol and 4-benzyl bromide in 1,4-dioxane as described
5: mp 41.2 ꢁC; H NMR d 0.95 (9H, s, (CH3)3C), 1.67,
1
ꢁ
1
2.52 (4H, 2m, (CH3)3C(CH2)2), 2.63, 2.72 (4H, 2ddd,
CꢀC(CH2)2), 3.72 (3H, s, COOCH3), 7.00, 7.38 (4H,
AA0XX0, J=8.8, 2.3 Hz, Ar); CIMS m/z 317 (M+1,
45.3%).
for 11: bp 120±124 C/4 mmHg; H NMR d 0.87, 0.89
(6H, 2d, J=6.8 Hz, CH(CH3)2), 0.92 (9H, s, (CH3)3C),
1.28 (1H, dd, J=14.6, 2.4 Hz, (CH3)3CCH2), 1.36 (1H,
dd, J=14.6, 7.3 Hz, (CH3)3CCH2), 2.05 (1H, dsp,
J=6.8, 3.4 Hz, CH(CH3)2), 3.31 (1H, ddd, J=7.3, 3.4,
2.4 Hz, CHO), 4.34 , 4.50 (2H, 2d, J=11.5 Hz,
OCH2Ph), 7.21, 7.44 (1H, AA0XX0, J=8.5, 2.0 Hz, Ar);
CIMS m/z 311 (M-1, 39.3%), 313 (M+1, 57.3%), 314
(M+2, 10.7%), 315 (M+3, 22.0%).
Methyl 5-[4-(2-isopropyl-4,4-dimethylpentanoyloxy)-
phenyl]-4-pentynoate (10). A solution of diisopropyl-
amine (2.3 g, 23 mmol) in dry THF (15 mL) was stirred
at 20 ꢁC under a nitrogen atmosphere. To this solution
were successively added an n-hexane solution of 1.6 M
n-BuLi (15.5 mL) and 4,4-dimethylpentanoic acid (1.5 g,
12 mmol) at such a rate that the temperature does not
exceed 0 ꢁC. After hexamethylphosphoramide (2.5 g, 13
mmol) was added to the solution, the mixture was stir-
red at room temperature for 30 min. To the mixture
maintained at 0 ꢁC was added isopropyl iodide (3.0 g, 17
mmol), and the mixture was stirred at room tempera-
ture for 3 h. After the reaction mixture was acidi®ed
with 3 N HCl, it was extracted with petroleum ether.
The petroleum ether extract was washed with H2O,
concentrated, and extracted with a saturated NaHCO3
solution. The aqueous solution was washed with n-hexane,
acidi®ed with 3 N HCl, and extracted with EtOAc. The
EtOAc extract was dried, concentrated, and puri®ed by
column chromatography on silica gel with petroleum
ether:EtOAc (3:1) to give 2-isopropyl-4,4-dimethyl-
pentanoic acid (1.2 g, 58.4%) as a colorless liquid. The
reaction of 2-isopropyl-4,4-dimethylpentanoic acid and
4-bromophenol in a manner similar to that described
for 2 gave 4-bromophenyl 2-isopropyl-4,4-dimethyl-
pentanoate as a colorless liquid (45.3% yield). Compound
10 was obtained as a colorless liquid (25.4% yield)
from 4-bromophenyl 2-isopropyl-4,4-dimethylpentanoate
and methyl 4-pentynoate, using the procedure described
for 5: 1H NMR d 0.95 (9H, s, (CH3)3C), 1.02, 1.03 (6H,
2d, J=6.8 Hz, CH(CH3)2), 1.36 (1H, dd, J=14.2, 1.2
Hz, (CH3)3CCH2), 1.90 (1H, dd, J=14.5, 10.4 Hz,
(CH3)3 CCH2), 1.93 (1H, sp, J=6.8 Hz, CH(CH3)2),
4-Ethynylbenzyl 3,3-dimethylbutyl ether (13). This com-
pound was prepared as a yellow liquid (6.3% yield)
from 11 and trimethylsilylacetylene, using the procedure
1
described for 3: H NMR d 0.95 (9H, s, (CH3)3C), 1.57
(2H, t, J=7.4 Hz, (CH3)3CCH2), 3.06 (1H, s, CꢀCH),
3.53 (2H, t, J=7.4 Hz, CH2CH2O), 4.49 (2H, s,
OCH2Ph), 7.29, 7.47 (4H, 2d, J=8.3 Hz, Ar); CIMS m/z
217 (M+1, 100%).
4-Ethynylbenzyl 1-isopropyl-3,3-dimethylbutyl ether (14).
This compound was prepared as a black liquid (17.9%
yield) from 12 and trimethylsilylacetylene, using the
1
procedure described for 3: H NMR d 0.88, 0.89 (6H,
2d, J=6.8 Hz, CH(CH3)2), 0.93 (9H, s, (CH3)3C), 1.27
(1H, dd, J=14.6, 2.3 Hz, (CH3)3CCH2), 1.37 (1H, dd,
J=14.6, 7.5 Hz, (CH3)3CCH2), 2.06 (1H, dsp, J=6.8,
3.6 Hz, CH(CH3)2), 3.05 (1H, s, CꢀCH), 3.31 (1H, ddd,
J=7.5, 3.6, 2.3 Hz, CHO), 4.40, 4.55 (2H, 2d, J=11.6
Hz, OCH2Ph), 7.30, 7.46 (4H, 2d, J=8.2 Hz, Ar);
CIMS m/z 259 (M+1, 29.8%).
Methyl 5-[4-(3,3-dimethylbutoxymethyl)phenyl]-4-pen-
tynoate (15). This compound was obtained as a yellow
liquid (37.9% yield) from 11 and methyl 4-pentynoate,
1
using the procedure described for 5: H NMR d 0.91
(9H, s, (CH3)3C), 1.56 (2H, t, J=7.4 Hz, (CH3)3CCH2),
2.64, 2.73 (4H, 2ddd, CꢀC(CH2)2), 3.51 (2H, t, J=7.4
Hz, CH2CH2O), 3.72 (3H, s, COOCH3), 4.47 (2H, s,

672
H. Hamano et al. / Bioorg. Med. Chem. 8 (2000) 665±674
OCH2Ph), 7.25, 7.37 (4H, 2d, J=8.2 Hz, Ar); CIMS m/z
303 (M+1, 83.0%).
in dry THF (10 mL) and dry N,N-dimethylformamide
(DMF) (4 mL) under a nitrogen atmosphere. The sus-
pension was stirred in an ice bath for 1 h and at room
temperature for 1 h. To the suspension cooled in an ice-
bath was added 4-bromophenol (1.77 g, 10.3 mmol).
After stirring in an ice bath for 1 h and at room tem-
perature for 24 h, a small amount of MeOH was added,
and the solution was extracted with Et2O. The Et2O
extract was washed with water, dried, and concentrated.
The residue was puri®ed by column chromatography on
silica gel with benzene: n-hexane (1:1) to give 19 as a
Methyl 5-[4-(1-isopropyl-3,3-dimethylbutoxymethyl)-
phenyl]-4-pentynoate (16). This compound was obtained
as a black liquid (52.4% yield) from 12 and methyl
4-pentynoate, using the procedure described for 5:
1H NMR d 0.87, 0.88 (6H, 2d, J=6.8 Hz, CH(CH3)2),
0.92 (9H, s, (CH3)3C), 1.28 (1H, dd, J=14.6, 2.2 Hz,
(CH3)3CCH2), 1.36 (1H, dd, J=14.6, 7.6 Hz, (CH3)3
CCH2), 2.05 (1H, dsp, J=6.8, 3.4 Hz, CH(CH3)2), 2.63,
2.73 (4H, 2ddd, CꢀC(CH2)2), 3.30 (1H, ddd, J=7.6,
3.5, 2.2 Hz, CHO), 3.72 (3H, s, COOCH3), 4.37, 4.53
(2H, 2d, J=11.6 Hz, OCH2Ph), 7.26, 7.34 (4H, 2d,
J=8.3 Hz, Ar); CIMS m/z 345 (M+1, 62.3%).
1
colorless liquid (650 mg, 23.4% yield): H NMR d 0.92
(9H, s, (CH3)3C), 1.31 (2H, m, (CH3)3CCH2), 1.71±1.78
(2H, m, CH2CH2CH2), 3.89 (2H, t, J=6.7 Hz, CH2O),
6.77, 7.36 (4H, AA0XX0, J=9.0, 2.8 Hz, Ar); CIMS m/z
270 (M, 45.0%), 271 (M+1, 18.3%), 272 (M+2,
44.3%), 273 (M+3, 16.7%).
5-[4-(3,3-Dimethylbutoxymethyl)phenyl]-4-pentynoic acid
(17). A mixture of 15 (100 mg, 0.33 mmol), KOH (20
mg, 0.36 mmol), H2O (10 mg, 0.56 mmol), MeOH (0.5
mL) and CH2Cl2 (20 mL) was stirred at room tempera-
ture for 3 h. The aqueous layer was acidi®ed with 1 N
HCl and extracted with CH2Cl2. The CH2Cl2 solution
was concentrated and extracted with an NaHCO3 solu-
tion. The aqueous layer was washed with n-hexane,
acidi®ed with 1 N HCl, and extracted with CH2Cl2. The
CH2Cl2 layer was dried and concentrated. The residue
was recrystallized to yield 17 as a white solid (15.3 mg,
16.3% yield): mp 125.1 ꢁC; 1H NMR d 0.91 (9H, s,
(CH3)3C), 1.56 (2H, t, J=7.4 Hz, (CH3)3CCH2), 2.71
(4H, m, CꢀC(CH2)2), 3.51 (2H, t, J=7.4 Hz, CH2
CH2O), 4.47 (2H, s, OCH2Ph), 7.25, 7.36 (4H, 2d,
J=8.3 Hz, Ar); CIMS m/z 289 (M+1, 21.1%).
4-Bromophenyl 2-isopropyl-4,4-dimethylpentyl ether (20).
A mixture of 2-isopropyl-4,4-dimethylpentanoic acid
(370 mg, 2.2 mmol), dry methanol (50 mL), and H2SO4
(3 g) was re¯uxed for 12 h. The solution was extracted
with CH2Cl2, and the CH2Cl2 extract was washed with
an NaHCO2 solution, dried, and concentrated to give
methyl 2-isopropyl-4,4-dimethylpentanoate as a yellow
liquid (216 mg, 54.0% yield). Methyl 2-isopropyl-4,4-
dimethylpentanoate (3.2 g, 17 mmol) in dry THF (15
mL) was added dropwise to an ice-cold, stirred suspen-
sion of LiAlH4 (3.2 g, 85 mmol) in dry THF (100 mL),
and the suspension was re¯uxed for 10 h. The suspen-
sion was poured into a 3 N H2SO4 solution and extrac-
ted with EtOAc. The EtOAc extract was dried and
concentrated. The residue was puri®ed by column
chromatography on silica gel with n-hexane:EtOAc (9:1)
to give 4,4-dimethyl-2-isopropyl-1-pentanol as a color-
less liquid (1.9 g, 72.1% yield). A solution of PBr3 (1.5 g,
5.5 mmol) containing a few drops of pyridine was
slowly added to a stirred solution of 2-isopropyl-4,4-
dimethyl-1-pentanol (1.7 g, 11 mmol) and pyridine
(0.2 g, 2.5 mmol) at 20 ꢁC. The solution was stirred at
20 ꢁC for 1 h and was heated at 80±90 ꢁC for 12 h.
After cooling in an ice bath, H2O was added to destroy
excess PBr3, and the mixture was extracted with
CH2Cl2. The CH2Cl2 extract was dried, concentrated,
and distilled to give 1-bromo-2-isopropy-4,4-dimethyl-
pentane as a colorless liquid (1.0 g, 42.9% yield): bp 60±
65 ꢁC/20 mmHg. To an ice-cold, stirred suspension of
NaH (260 mg, 11 mmol), dry THF (18 mL), and dry
DMF (7 mL) was slowly added a solution of 4-bromo-
phenol (470 mg, 2.7 mmol) in dry THF (5 mL) under a
nitrogen atmosphere. After the mixture was stirred in an
ice bath for 1 h and at room temperature for 1 h, a
solution of 1-bromo-2-isopropyl-4,4-dimethylpentane
(600 mg, 2.7 mmol) in dry THF (5 mL) was slowly
added to the ice-cold, stirred mixture. The mixture was
stirred in an ice bath for 1 h and at room temperature
for 24 h. A small amount of MeOH was added to the
mixture to destroy excess NaH, and the solution was
extracted with Et2O. The Et2O extract was washed with
H2O, dried, and concentrated. The residue was puri®ed
by column chromatography on silica gel with n-hexane
to give 20 as a colorless liquid (435 mg, 51.1% yield):
1H NMR d 0.88, 0.91 (6H, 2d, J=6.8 Hz, CH(CH3)2),
5-[4-(1-Isopropyl-3,3-dimethylbutoxymethyl)phenyl]-4-
pentynoic acid (18). This compound was obtained as a
yellow solid (35.5% yield) from 16, using the procedure
described for 17: mp 65.2 ꢁC; 1H NMR d 0.87, 0.88 (6H,
2d, J=6.8 Hz, CH(CH3)2), 0.92 (9H, s, (CH3)3C), 1.28
(1H, dd, J=14.6, 2.2 Hz, (CH3)3CCH2), 1.36 (1H, dd,
J=14.6, 7.4 Hz, (CH3)3CCH2), 2.05 (1H, dsp, J=6.8,
3.7 Hz, CH(CH3)2), 2.73 (4H, m, CꢀC(CH2)2), 3.30
(1H, ddd, J=7.4, 3.7, 2.2 Hz, CHO), 4.38, 4.53 (2H, 2d,
J=11.7 Hz, OCH2Ph), 7.26, 7.35 (4H, 2d, J=8.3 Hz,
Ar); CIMS m/z 331 (M+1, 3.3%).
4-Bromophenyl 4,4-dimethylpentyl ether (19). A solution
of 4,4-dimethylpentanoic acid (5.5 g, 42.3 mmol) in dry
Et2O (50 mL) was added dropwise to a suspension of
LiAlH4 (1.6 g, 42.3 mmol) in dry Et2O (100 mL). After
5-h re¯ux, the suspension was slowly poured into an
ice-cold 15% H2SO4 solution (100 mL). The solution
was partitioned between EtOAc and H2O, and the
EtOAc layer was dried and concentrated to give 4,4-
dimethyl-1-pentanol as a colorless liquid (2.1 g, 44.8%
yield). A solution of 4,4-dimethyl-1-pentanol (2.2 g,
18.9 mmol) in pyridine (0.5 g, 6.3 mmol) was added to
an ice-cold, stirred solution of PBr3 (2.2 g, 8.2 mmol)
containing a few drops of pyridine. After 12-h re¯ux,
the solution was distilled to give 1-bromo-4,4-dimethyl-
pentane as a colorless liquid (1.8 g, 54.4% yield): bp
148±149 ꢁC. A solution of 1-bromo-4,4-dimethylpentane
(1.8 g, 10.3 mmol) in dry THF (6 mL) was added to an
ice-cold, stirred suspension of NaH (0.3 g, 12.5 mmol)

H. Hamano et al. / Bioorg. Med. Chem. 8 (2000) 665±674
673
0.92 (9H, s, (CH3)3C), 1.05 (1H, dd, J=14.3, 6.3 Hz,
(CH3)3CH2), 1.26 (1H, dd, J=14.3, 2.7 Hz, (CH3)3
CH2), 1.74 (1H, m, CHCH2O), 1.96 (1H, dsp, J=6.8,
3.8 Hz, CH(CH3)2), 3.76 (1H, dd, J=9.2, 7.6 Hz,
CH2O), 3.80 (1H, dd, J=9.2, 5.1 Hz, CH2O), 6.77, 7.35
(4H, AA0XX0, J=9.0, 2.8 Hz, Ar); EIMS m/z 312 (M,
20.0%), 314 (M+2, 19.0%).
5-[4-(2-Isopropyl-4,4-dimethylpentoxy)phenyl]-4-pentynoic
acid (26). This compound was obtained as white needles
(36.4% yield) from 24, using the procedure described
for 7: mp 96.4±97.6 ꢁC; H NMR d 0.88, 0.90 (6H, 2d,
1
J=6.8 Hz, CH(CH3)2), 0.91 (9H, s, (CH3)3C), 1.05 (1H,
dd, J=14.4, 6.6 Hz, (CH3)3CH2), 1.25 (1H, dd, J=14.4,
2.7 Hz, (CH3)3CH2), 1.74 (1H, m, CHCH2O), 1.96 (1H,
dsp, J=6.8, 3.7 Hz, CH(CH3)2), 2.66±2.75 (4H, m,
CꢀC(CH2)2), 3.78 (1H, dd, J=9.3, 7.6 Hz, CH2O), 3.82
(1H, dd, J=9.3, 5.4 Hz, CH2O), 6.79, 7.30 (4H,
AA0XX0, J=8.9, 2.5 Hz, Ar); CIMS m/z 331 (M+1,
65.3%).
4-Ethynylphenyl 4,4-dimethylpentyl ether (21). This
compound was obtained as a colorless liquid (15.0%
yield) from 19 and trimethylsilylacetylene, using the
procedure described for 3: 115-120 ꢁC/2 mmHg; 1H
NMR d 0.92 (9H, s, (CH3)3C), 1.32 (2H, m, (CH3)3
CCH2), 1.72±1.79 (2H, m, CH2CH2CH2), 2.99 (1H, s,
CꢀCH), 3.93 (2H, t, J=6.7 Hz, CH2O), 6.83, 7.41 (4H,
AA0XX0, J=8.8, 2.4 Hz, Ar); CIMS m/z 217 (M+1,
83.0%).
Equilibrium binding studies
Rat brain P2 membranes were prepared from 5-week-
old male Wistar rats by a modi®cation of the method
of Squires et al.18 Forebrains stored at 80 ꢁC were
thawed and homogenized in ice-cold 1 mM EDTA
using a Te¯on-glass homogenizer. The homogenate was
centrifuged at 1000Âg for 10 min. The supernatant was
then centrifuged at 25,000Âg for 30 min. The resulting
pellets were suspended in 1 mM EDTA and dialyzed
three times (2 h each) in cellophane tubing against 2 L
of distilled, deionized water at 4 ꢁC. After dialysis, the
inner solution was recentrifuged at 25,000Âg for 30 min,
and the pellets were stored at 80 ꢁC until use. The
frozen pellet was thawed and suspended in 10 mM
sodium phosphate bu€er containing 300 mM NaCl (pH
7.5) (bu€er A) for the binding assays.
4-Ethynylphenyl 2-isopropyl-4,4-dimethylpentyl ether (22).
This compound was obtained as a yellow liquid (18.8%
yield) from 20 and trimethylsilylacetylene, using the
1
procedure described for 3: H NMR d 0.88, 0.91 (6H,
2d, J=6.8 Hz, CH(CH3)2), 0.92 (9H, s, (CH3)3C), 1.06
(1H, dd, J=14.4, 6.6 Hz, (CH3)3CH2), 1.26 (1H, dd,
J=14.4, 2.7 Hz, (CH3)3CH2), 1.75 (1H, m, CHCH2O),
1.96 (1H, dsp, J=6.8, 3.8 Hz, CH(CH3)2), 2.98 (1H, s,
CꢀCH), 3.80 (1H, dd, J=9.3, 7.6 Hz, CH2O), 3.84 (1H,
dd, J=9.3, 5.4 Hz, CH2O), 6.83, 7.41 (4H, AA0XX0,
J=8.9, 2.4 Hz, Ar); CIMS m/z 259 (M+1, 8.2%).
Methyl 5-[4-(4,4-dimethylpentoxy)phenyl]-4-pentynoate
(23). This compound was obtained as a yellow liquid
(72.2% yield) from 19 and methyl 4-pentynoate, using
House¯y head P2 membranes were prepared from 5- to
10-day-old adult house¯ies (Musca domestica L.) of
WHO strain by a modi®cation of the method of Deng
et al.19 The heads were homogenized in 10 mM Tris±
HCl bu€er containing 0.25 M sucrose (pH 7.5) with a
glass±Te¯on homogenizer, ®ltered through four layers
of 64-mm mesh nylon screen and centrifuged at 500Âg
for 5 min. The supernatant was ®ltered through four
layers of nylon screen and centrifuged at 25,000Âg for
30 min. The pellets were suspended in the bu€er and
allowed to stand in an ice bath for 30 min. The suspen-
sion was centrifuged at 25,000Âg for 30 min. The pellets
were resuspended in bu€er A and used immediately for
binding assays without freezing.
1
the procedure described for 5: H NMR d (DMSO-d6)
0.91 (9H, s, (CH3)3C), 1.31 (2H, m, (CH3)3CCH2), 1.71±
1.78 (2H, m, CH2CH2CH2), 2.60±2.66, 2.69±2.74 (4H,
2m, CꢀC(CH2)2), 3.72 (3H, s, COOCH3), 3.91 (2H, t,
J=6.7 Hz, CH2O), 6.79, 7.30 (4H, AA0XX0, J=9.0, 2.4
Hz, Ar); CIMS m/z 303 (M+1, 100%).
Methyl 5-[4-(2-isopropyl-4,4-dimethylpentoxy)phenyl]-4-
pentynoate (24). This compound was obtained as a col-
orless viscous liquid (12.3% yield) from 20 and methyl
4-pentynoate, using the procedure described for 5:
1H NMR d 0.88, 0.91 (6H, 2d, J=6.8 Hz, CH(CH3)2),
0.91 (9H, s, (CH3)3C), 1.05 (1H, dd, J=14.4, 6.3 Hz,
(CH3)3CH2), 1.25 (1H, dd, J=14.4, 2.7 Hz, (CH3)3
CH2), 1.74 (1H, m, CHCH2O), 1.96 (1H, dsp, J=6.8,
3.7 Hz, CH(CH3)2), 2.60±2.66, 2.69±2.76 (4H, 2m
CꢀC(CH2)2), 3.71 (3H, s, COOCH3), 3.78 (1H, dd,
J=9.3, 7.6 Hz, CH2O), 3.82 (1H, dd, J=9.3, 5.1 Hz,
CH2O), 6.79, 7.30 (4H, AA0XX0, J=9.0, 2.4 Hz, Ar);
CIMS m/z 245 (M+1, 28.4%).
[3H]EBOB binding assays were carried out according to
Cole and Casida4 and Deng et al.19 Brie¯y, DBCPP and
its derivatives were incubated with rat brain membranes
(125 mg protein) or house¯y head membranes (200 mg
protein) and 0.5 nM [3H]EBOB (38 Ci/mmol, NEN Life
Science Products, Inc.) in 1.0 mL of bu€er A at 37 ꢁC
for 90 min (rat) or at 22 ꢁC for 70 min (house¯y). Pro-
tein was measured by the method of Bradford.20 Test
compounds and unlabeled EBOB were added as DMSO
solutions (4 mL) to reaction mixtures so as to give the
desired ®nal concentrations. After incubation, the mix-
tures were ®ltered through GF/B ®lters (Whatman
International Ltd.) and were rapidly rinsed twice with
5 mL of ice-cold bu€er A using a Brandel M-24 cell
harvester (Biomedical Research and Development
Laboratories, Inc.). The radioactivity of [3H]EBOB that
speci®cally bound to membranes on the ®lters was
5-[4-(4,4-Dimethylpentoxy)phenyl]-4-pentynoic acid (25).
This compound was obtained as a colorless solid
(10.2% yield) from 23, using the procedure described
for 7: mp 135.5 ꢁC; H NMR d (DMSO-d6) 0.89 (9H, s,
1
(CH3)3C), 1.28 (2H, m, (CH3)3CCH2), 1.63±1.70 (2H,
m, CH2CH2CH2),2.50, 2.59 (4H, 2m, CꢀC(CH2)2), 3.94
(2H, t, J=6.5 Hz, CH2O), 6.88, 7.27 (4H, AA0XX0,
J=8.9, 2.4 Hz, Ar), 12.30 (1H, s, COOH); CIMS m/z
289 (M+1, 5.3%).

674
H. Hamano et al. / Bioorg. Med. Chem. 8 (2000) 665±674
measured with a Beckman LS 6000SE liquid scintilla-
tion spectrometer. Nonspeci®c binding was determined
in the presence of 5 mM unlabeled EBOB. Each experi-
ment was performed in triplicate and repeated twice.
IC50 values were obtained by the Probit method.
slope factor (Hill coecient) of DBCPP were calculated
from the equation:
I ˆ ImaxCn=ꢀCn ‡ IC5n0†
where I is the amplitude of GABA-induced current, Imax
the maximum current, C the DBCPP concentration, and
n the Hill coecient. The nonlinear regression analysis
was carried out using the least squares ®tting method
(Sigmaplot, Version 4.0) by a microcomputer. Whole-
cell current records were analyzed by the pClamp ver-
sion 6.0 software (Axon instruments).
Whole-cell current recording
The dorsal root ganglia were dissected from the lumbo-
dorsal region of newborn rats (1±5 days postnatal) and
were immediately placed into a Ca2+ and Mg2+-free
phosphate-bu€ered saline solution supplemented with
6 g/L of glucose. The ganglia were digested in a phos-
phate-bu€ered saline solution containing 2.5 mg/mL of
trypsin (Sigma Chemical Co.) for 20 min at 37 ꢁC. The
ganglia were then dissociated by repeated triturations
using a ®re-polished Pasteur pipette in Dulbecco's
Modi®ed Eagle Medium containing 0.1 mg/mL of fetal
bovine serum and 0.08 mg/mL of gentamicin. The dis-
sociated cells were placed on coverslips coated with
poly-l-lysine. Neurons were maintained in Dulbecco's
Modi®ed Eagle Medium containing serum and genta-
micin in a 90% air±10% CO2 atmosphere controlled at
37 ꢁC. Neurons cultured for 2 to 5 days were used for
experiments.
Acknowledgements
This work was supported in part by Grants-in-Aid for
Scienti®c Research from the Ministry of Education,
Science, Sports and Culture of Japan (to K.N. and
Y.O.).
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