Selective inhibitors of GABA uptake: Synthesis and molecular pharmacology of 4-N-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol analogues

Article abstract of DOI:10.1016/j.bmc.2004.10.029

A series of lipophilic diaromatic derivatives of the glia-selective GABA uptake inhibitor (R)-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol [(R)-exo-THPO, 4] were synthesized via reductive amination of 3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazol-4-one (9) or via N-alkylation of O-alkylatedracemic 4. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation or primary cultures of mouse cortical neurons and glia cells (astrocytes), as well as HEK cells transfected with cloned mouse GABA transporter subtypes (GAT1-4). The activity against isoniazid-induced convulsions in mice after subcutaneous administration of the compounds was determined. All of the compounds were potent inhibitors of synaptosomal uptake the most potent compound being (RS)-4-[N-(1,1-diphenylbut-1-en-4-yl)amino]-4,5,6,7-tetrahydrobenzo[d] isoxazol-3-ol (17a, IC₅₀ = 0.14 μM). The majority of the compounds showed a weak preference for glial, as compared to neuronal, GABA uptake. The highest degree of selectivity was 10-fold corresponding to the glia selectivity of (R)-N-methyl-exo-THPO (5). All derivatives showed a preference for the GAT1 transporter, as compared with GAT2-4, with the exception of (RS)-4-[N-[1,1- bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d] isoxazol-3-ol (28d), which quite surprisingly turned out to be more potent than GABA at both GAT1 and GAT2 subtypes. The GAT1 activity was shown to reside in (R)-28d whereas (R)-28d and (S)-28d contributed equally to GAT2 activity. This makes (S)-28d a GAT2 selective compound, and (R)-28d equally effective in inhibition of GAT1 and GAT2 mediated GABA transport. All compounds tested were effective as anticonvulsant reflecting that these compounds have blood-brain barrier permeating ability.

Full text of DOI:10.1016/j.bmc.2004.10.029

Bioorganic & Medicinal Chemistry 13 (2005) 895–908
Selective inhibitors of GABA uptake: synthesis and
molecular pharmacology of 4-N-methylamino-
4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol analogues
Rasmus P. Clausen,a Ejner K. Moltzen,c Jens Perregaard,c Sibylle M. Lenz,c
Connie Sanchez,d Erik Falch,a Bente Frølund,a Tina Bolvig,b Alan Sarup,b
Orla M. Larsson,b Arne Schousboeb and Povl Krogsgaard-Larsena,*
aDepartment of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken,
DK-2100 Copenhagen, Denmark
bDepartment of Pharmacology, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken,
DK-2100 Copenhagen, Denmark
cDepartment of Medicinal Chemistry, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Copenhagen-Valby, Denmark
dDepartment of Neuropharmacological Research, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Copenhagen-Valby, Denmark
Received 4 June 2004; accepted 12 October 2004
Available online 2 November 2004
Abstract—A series of lipophilic diaromatic derivatives of the glia-selective GABA uptake inhibitor (R)-4-amino-4,5,6,7-tetra-
hydrobenzo[d]isoxazol-3-ol [(R)-exo-THPO, 4] were synthesized via reductive amination of 3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isox-
azol-4-one (9) or via N-alkylation of O-alkylatedracemic 4. The effects of the target compounds on GABA uptake mechanisms in
vitro were measured using a rat brain synaptosomal preparation or primary cultures of mouse cortical neurons and glia cells (ast-
rocytes), as well as HEK cells transfected with cloned mouse GABA transporter subtypes (GAT1-4). The activity against isoniazid-
induced convulsions in mice after subcutaneous administration of the compounds was determined. All of the compounds were
potent inhibitors of synaptosomal uptake the most potent compound being (RS)-4-[N-(1,1-diphenylbut-1-en-4-yl)amino]-4,5,6,7-
tetrahydrobenzo[d]isoxazol-3-ol (17a, IC50 = 0.14lM). The majority of the compounds showed a weak preference for glial, as com-
pared to neuronal, GABA uptake. The highest degree of selectivity was 10-fold corresponding to the glia selectivity of (R)-N-methyl-
exo-THPO (5). All derivatives showed a preference for the GAT1 transporter, as compared with GAT2-4, with the exception of
(RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (28d), which quite sur-
prisingly turned out to be more potent than GABA at both GAT1 and GAT2 subtypes. The GAT1 activity was shown to reside in
(R)-28d whereas (R)-28d and (S)-28d contributed equally to GAT2 activity. This makes (S)-28d a GAT2 selective compound, and
(R)-28d equally effective in inhibition of GAT1 and GAT2 mediated GABA transport. All compounds tested were effective as anti-
convulsant reflecting that these compounds have blood–brain barrier permeating ability.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
phenomena, a large number of in vitro and in vivo phar-
macological data are consistent with a major role of
GABA in this group of disorders.6 Thus, obstruction
of GABA biosynthesis or administration of GABA
antagonists in animal studies can induce convulsions
resembling those of epilepsy.7 Furthermore, enhance-
ment of GABA neurotransmission leads to protection
against seizures.8,9
4-Aminobutyric acid (GABA) is the major inhibitory
neurotransmitter in the central nervous system (CNS)
and has been implicated in a number of CNS disorders,
notably epilepsy.1–7 Although there is no direct proof of
impairment of GABA neurotransmission in epileptic
Enhancement of GABA transmission by inhibition
of GABA uptake has gained much attention as a
therapeutic strategy,10,11 since it functionally increases
the effect of GABA in a use-dependent manner, and
Keywords: GABA uptake inhibitor; Anticonvulsant; Gamma-amino-
butyric acid; Subtype selective.
*
Corresponding author. Tel.: +45 35 30 65 11; fax: +45 35 30 60 40;
e-mail: ano@carlsbergfondet.dk
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.029

896
R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
OH
OH
GABA uptake inhibitors have proved effective as anti-
convulsants in a variety of experimental models of
epilepsy and in epileptic patients.12–15 Pharmacological
intervention along these lines may be beneficial in other
CNS disorders and malfunctions, including chronic
pain, anxiety, and sleep disorders.
OH
H2N
HN
O
HN
O
O
(R)-Nipecotic acid (1)
Guvacine (2)
GABA
Me
NH2
OH
HN
OH
OH
N
At present, four different subtypes of mammalian
GABA transporters have been cloned and given spe-
cies-dependent nomenclature: GAT1–GAT4 in mouse
correspond to GAT-1, BGT-1, GAT-2, GAT-3, respec-
tively, in rat and human.16–24 The regional and cellular
distribution of these subtypes are highly heterogene-
ous.24 Early studies disclosed a pharmacological differ-
ence between GABA uptake in glial and neuronal cell
cultures suggesting that separate transporters on each
cell type could account for this difference.10 It was later
shown that there is no simple distinction between the
types of GABA transporter expressed at neurons and
astrocytes and the reasons for the pharmacological dif-
ferences observed using these cellular assay systems re-
main unclear,24 but may reflect different functional or
regulatory mechanisms in these cells. The role of the
transporter subtypes and their therapeutic potential as
targets will rely on development of potent and subtype
selective ligands toward GAT2–GAT4 penetrating the
blood–brain barrier (BBB).
HN
N
O
N
O
O
THPO (3)
(R)-exo-THPO (4) (R)-N-Me-exo-THPO (5)
OH
OH
S
N
O
N
O
S
SKF-89976A (6)
Tiagabine (7)
F3C
OH
O
O
N
CF3
CI-966 (8)
Three decades ago the discovery of 4,5,6,7-tetrahydrois-
oxazolo[4,5-c]pyridine-3-ol (THPO, 3) as a selective up-
take inhibitor without GABA receptor affinity led to the
discovery of (R)-nipecotic acid (1) and guvacine (2) as
potent and highly selective inhibitors of GABA trans-
port (Fig. 1).25,26 This discovery subsequently led to
the development of more potent derivatives of 1 and 2
as GABA uptake inhibitors, including 6–8 with in-
creased potency and showing ability to penetrate the
BBB.12,27–30 Tiagabine (7) is now used clinically as an
antiepileptic drug.15
Figure 1. Structure of GABA, the GABA uptake inhibitors
(R)-nipecotic acid (1), guvacine (2), (R)-4-amino-4,5,6,7-tetra-
hydrobenzo[d]isoxazol-3-ol [(R)-exo-THPO, 4], (R)-4-methylamino-
4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol [(R)-N-Me-exo-THPO, 5],
and lipophilic diaromatic analogues of 1 (6–8).
tives of 4 were synthesized of which the most potent
and glia-selective compound turned out to be the N-
methyl derivative 5. It was recently demonstrated that
4 and 5 effectively protect against seizures when injected
directly into the brain, the protective effect being corre-
lated with the inhibition of glial transport rather than
neuronal uptake inhibition.40 These results further sub-
stantiate the strategy of glia-selective intervention, but
the therapeutic potential of this strategy can only be elu-
cidated by the design of markedly more potent inhibi-
tors, selective for glial GABA transport, and capable
of penetrating the BBB.
Like GABA, the cyclic amino acids 1 and 2 are transport
substrates with similar affinity for the neuronal and glial
GABA transport systems.25,30–35 The bioisosteric ana-
logue of 1 and 2, THPO (3), however is not a substrate
for the GABA transporters and has a 2-fold higher affin-
ity for glial over neuronal transport.31,33 Although 3 is at
least an order of magnitude weaker than 1 as an inhibitor
of GABA uptake in vitro, it is capable of increasing
extracellular GABA levels more effectively than 1, and
3 is a more effective anticonvulsant than 1.31,36,37 Selec-
tive inhibition of glial GABA uptake might improve
the therapeutic potential of transport inhibition since
the glial transport leads to degradation of GABA,
whereas the re-uptake of GABA into neurons, which is
coupled to GABA release mechanisms, would be
strengthened.38 The observation that inhibitors selective
for neuronal uptake are proconvulsant is supportive evi-
dence of this pharmacological approach.37
These aspects have prompted us to synthesize deriva-
tives of exo-THPO containing lipophilic diaromatic side
chains with the aim of developing potent and glia-selec-
tive GABA uptake inhibitors, sufficiently lipophilic to
penetrate the BBB. We here report the results of these
efforts.
2. Results
2.1. Chemistry
Some years ago we redesigned the structure of THPO (3)
to give (R)-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-
3-ol [(R)-exo-THPO, 4], which showed increased selec-
tivity for glial uptake.39 A series of N-alkylated deriva-
Two principal routes were employed to obtain the deriv-
atives of (RS)-4, via reductive amination of ketone 939
(Scheme 1) or via N-alkylation of amines 15, 20, 23,

R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
897
racemic 13.39 Boc-protection of 13 was followed by
benzylation of the isoxazolol-ring to give 14. The Boc-
group was then removed with HCl to give 15 and alky-
lation with 29 and 30 yielded 16a and 16b. The benzyl
group was removed with hydrochloric acid furnishing
17a and 17b. The synthesis of 22 starts from the Boc-pro-
tected racemic 18, which is O-benzylated giving 19, fol-
lowed by removal of the Boc group with HCl yielding
20. N-Alkylation of O-benzyl protected 20 with bromide
29 gave 21 that upon deprotection in hydrochloric acid
furnished 22. Similarly tertiary amine derivatives 25a–d
were obtained by N-alkylation of O-ethyl-protected race-
mic N-methyl-exo-THPO (23)39 with halides 31–34, but
stronger acidic conditions were necessary in the depro-
tection of O-ethyl-protected 3-isoxazolols 24a–d to pro-
vide the desired compounds 25a–d. A series of tertiary
amine derivatives with acid-labile side chains 28a–d
was obtained starting from the pivaloyloxymethyl-pro-
tected derivative 26.39 N-Alkylation with 35–37 and 30
gave 27a–d and subsequent basic alcoholysis furnished
28a–d. In the case of (R)-28d and (S)-28d the starting
materials were (R)- and (S)-26,39 respectively.
R
R
O
NH
NH
OEt
N
OEt
N
OH
N
i
ii
O
O
O
9
11a-i
12a-i
R
R
R
S
a: (p-FPh)2CH(CH2)3
b: (o-MePh)2CH(CH2)3
c: (o-MePh)2C=CH(CH2)2
d:
e: Ph2CH(CH2)3
g:
N
S
f:
(CH2)3
h: Ph2CH(CH2)2
i: Ph(CH2)4
(CH2
)
3
(CH2)2
Scheme 1. Reagents and conditions: (i) R–NH2 (10a–i), NaCNBH3;
(ii) 33% HBr in HOAc (a–e, g–i) or 6M HCl (f).
and 26 (Scheme 2). A series of secondary amine deriva-
tives of racemic 4 were synthesized by the reductive ami-
nation of 9 with the appropriate amines 10a–i and
NaCNBH3 giving intermediates 11a–i (Scheme 1). These
O-ethyl-protected compounds were deprotected with
33% HBr in glacial acetic acid to give the free 3-isoxazol-
ols 12a–i. Two secondary amine derivatives 17a,b
(Scheme 2) were prepared via alkylation of O-benzyl
protected exo-THPO (15) obtained in three steps from
2.2. In vitro pharmacology
The inhibitory effect on GABA uptake in a crude prep-
aration of synaptosomes from rat brain were determined
Boc
R
R
R
R
NH2
NH
NH2
NH
NH
OH
N
OBn
N
OBn
N
OBn
N
OH
N
i,ii
iii
iv
iv
v
O
O
O
O
O
13
14
15
16a,b
17a,b
Boc
Me
Boc
Me
Me
Me
Me
N
N
HN
N
N
OH
N
OBn
N
OBn
N
OBn
N
OH
N
ii
iii
v
O
O
O
O
O
18
19
20
21
22
Product
R-X
Me
R
R
Me
R
R
Me
HN
HN
N
N
OEt
N
OEt
N
OH
N
17a
17b
22
25a
25b
25c
25d
Ph2C=CH(CH2)2-Br (29)
iv
v
(3-Me-2-thienyl)2C=CH(CH2)2-OMs (30)
Ph2C=CH(CH2)2-Br (29)
O
O
O
(p-FPh)2CH(CH2)3-Cl (31)
23
24a-d
25a-d
(o-MePh)2CH(CH2)3-I (32)
(o-MePh)2C=CH(CH2)2-I (33)
Me
Me
Me
OCH2O2CtBu
iv or vi
OCH2O2CtBu
vii
OH
N
N
N
(34)
(CH2)2-Br
N
N
O
O
O
28a
28b
28c
28d
Ph2CHO(CH2)2-OMs (35)
(p-FPh)2CHO(CH2)2-OMs (36)
(p-ClPh)2CHO(CH2)2-Cl (37)
(3-Me-2-thienyl)2C=CH(CH2)2-OMs (30)
26
(R)-26
(S)-26
27a-d
(R)-27d
(S)-27d
28a-d
(R)-28d
(S)-28d
Scheme 2. Reagents and conditions: (i) (Boc)2O, NaOH; (ii) BnBr, K2CO3; (iii) HCl in EtOAc; (iv) R–X (29–37), K2CO3; (v) HCl in water/EtOH;
(vi) R–X (30), Li2CO3 (28d); (vii) NaOH in EtOH.

898
R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
for all compounds.41 The compounds were equipotent
or more potent inhibitors than the classical inhibitors
1 and 2 in this assay with IC50 values below 6lM (Table
1). Several of the compounds were markedly more po-
tent with IC50 values below 1lM (12a,c,e,h, 17b), the
most potent compound being 17a (IC50 = 0.14lM).
Compounds with R2 = H showed a slightly higher affin-
ity than analogues R2 = Me.
the individual subtypes of the mouse GABA transporter
of the compounds were determined, a high preference
for GAT1 was evident as observed for the lipophilic
derivatives of 1, compounds 6 and 7.15 However, in
some cases significant GAT2 activity appeared, as exem-
plified by 12d, 17a,b, and 25e.
Surprisingly, in the case of 28d the GAT2 potency was
higher than GABA itself and at the level of GAT1 po-
tency relative to GABA. This interesting observation
prompted us to investigate whether this dual activity
was associated with a single enantiomer. We therefore
synthesized the enantiomers of 28d separately proving
that while inhibitory effects at GAT1 mainly resides in
(R)-28d, both enantiomers in the racemic mixture con-
tributed to inhibition of GAT2. The affinity in synapto-
somal uptake was also determined and found to be at
least 200-fold higher for (R)-28d as compared to (S)-
28d reflecting that GAT1 is the most abundant trans-
The compounds were subsequently subjected to further
pharmacological characterization using cultured cortical
neurons and astrocytes33 as well as monoclonal cell
cultures expressing GABA transporters from mouse
(GAT1-4)40 to probe the glial selectivity and the subtype
profile, respectively (Table 1). The majority of the com-
pounds maintained a preference for glial over neuronal
GABA uptake, the most selective compound showing
10-fold difference (12d), corresponding to the glia selec-
tivity of N-methyl-exo-THPO. When the affinities for
Table 1. [3H]GABA uptake inhibition into synaptosomes, cultured cortex astrocytes and neurons, and cells transfected with cloned GABA
transporters (GAT1-4) and anticonvulsant effects of GABA uptake inhibitors
Compd
GABA uptake inhibition IC50 (lM)
Seizure inhibition
ED50 (lmol/kg)
Synaptosomes Astrocytes Neurons GAT1
GAT2 GAT3 GAT4 Isoniazida
GABA
(R)-Nipecotic acid (Nip)
Guvacine
nd
5c
8b
16c
32b
12c
17b
24d
51b
2350d
1420d
15b
113d
228d
17b nd
1
2
159d nd
378d nd
12c
29c
32c
39d
(RS)-4 exo-THPO
(RS)-5 N-Me-exo-THPO
181c
63c
208c
28c
60c
883c
423c
510c
1000e
450e
nd
3000e >3000e >3000e nd
>3000e >3000e >3000e nd
5
7
(R)-N-Me-exo-THPO
Tiagabine (Nip-C14H15S2)
39c
nd
nd
>300e
nd
nd
2.7
0.067e
0.18e
0.36e
0.8e
300e
800e
R1
R2
12a
12b
12c
12d
12e
12f
12g
12h
12i
(p-FPh)2CH(CH2)3
(o-MePh)2CH(CH2)3
(o-MePh)2C@CH(CH2)2
H
H
H
H
H
H
H
H
H
H
H
0.24
0.84
0.3
1
20
2
2
7
4
1.5
>100
>200
>300
125
300
>200
>300
>100
>100
>200
>125
>300
>300 110
>200
>300
225
44
30
68
56
45
71
12
80
3
10
7
C
18H17
Ph2CH(CH2)3
C15H13
0.76
0.22
nd
7
0.5
4
>100
160
>100
>200
>125
S
2.5
3
6.5
5.5
6
C14H12NS
Ph2CH(CH2)2
Ph(CH2)4
0.96
0.2
14
2
>125
>300
1
15
1
30
>300 310
6.2
100
>300 >1000 >1000 150
17a
17b
22
Ph2C@CH(CH2)2
0.14
0.17
0.6
2
1.4
6
3
50
130
60
>100
>100
>300
>500
>500
>500
>300
>500
90 nd
>100 nd
C14H15S2
4
5
Ph2C@CH(CH2)2
(p-FPh)2CH(CH2)3
(o-MePh)2CH(CH2)3
(o-MePh)2C@CH(CH2)2
C18H17
Me 0.37
Me 0.73
Me 1.5
Me 0.7
Me 1.1
Me 0.36
Me 4.5
Me 4.9
Me 0.87
Me 0.48
Me >100
2
20
2
200
300
>100
>300
>500
>500
97
25a
25b
25c
25d
28a
28b
28c
28d
16
18
5
11
11
3
67
27
50
9
8
260
200
3
7.5
12
5
125
>300
>500
>500 160f
>300 160
Ph2CHO(CH2)2
(p-FPh)2CHO(CH2)2
(p-ClPh)2CHO(CH2)2
C14H15S2
1
4
16
17
2
15
13
2
12
17
7
>500
nd
55
81
63
24
>30 nd
26
22
34
>300
>150
>150
>300
>150
(R)-28d C14H15S2
(S)-28d C14H15S2
0.65
>100
1.5
>100
4
120
>150 >40
nd, not determined.
a Anticonvulsant effects in mice after subcutaneous treatment with isoniazid.
b Km value, Ref. 38.
c Ref. 39.
d Determined at rat GAT-1, human BGT-1, rat GAT-2, and GAT-3 corresponding to mouse GAT1-4, respectively; Ref. 24.
e Ref. 40.
f Biphasic curve, determined at maximum effect (60%).

R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
899
porter in this assay. In contrast, Tiagabine (7) is only 3-
fold more potent than the corresponding (S)-form, as an
inhibitor of synaptosomal GABA uptake.28 Also in the
cortical glial and neuronal cell assay GABA uptake
could only be blocked by (R)-28d.
exo-THPO or N-Me-exo-THPO tested showed a high
degree of selectivity for GAT1. However, a comparison
of the secondary amines 17a and 17b with the corre-
sponding N-methylated derivatives 22 and 28d reveals
a surprising difference. Like 17a and 17b, compound
22 and 28d showed comparable inhibitory effects in all
GABA uptake assay systems but the GAT2 expressing
cell assay. Thus, compound 22 is not very potent as
inhibitor of GAT2 mediated transport whereas 28d is
more potent than GABA itself at both GAT1 and
GAT2. This makes 28d the first example of a GABA up-
take inhibitor showing greater potency than GABA at
GAT1 and GAT2 without significant effects at GAT3
and GAT4.
2.3. In vivo pharmacology
Most of the compounds were tested as anticonvulsants.
The compounds were given subcutaneously to mice trea-
ted with isoniazid and all tested compounds turned out
to be anticonvulsant, a majority of compounds showing
ED50 < 100lmol/kg (Table 1). The most active com-
pound in this assay was 25b (ED50 = 27lmol/kg). This
shows that generally the compounds have BBB permeat-
ing ability.
This unique profile of the racemic compound unexpect-
edly turned out to emerge from very different profiles of
the enantiomers. GAT1 activity could be assigned pri-
marily to (R)-28d, whereas (S)-28d and (R)-28d contrib-
utes equally to the potency of the racemate at GAT2.
Therefore, (S)-28d displays significant GAT2 selectivity
compared to GAT1 and both compounds carry a highly
unique pharmacological profile among GABA uptake
inhibitors.
3. Discussion
The classical GABA uptake inhibitors, nipecotic acid (1)
and guvacine (2), are the central components of the
derivatives containing lipophilic diaromatic side chains
on the amino group of these cyclic amino acids (Fig.
1). This substitution has proved to increase potency
and to disable these derivatives as substrates for the
GABA transporters.12,30 Numerous derivatives of 1
and 2 have now been synthesized and pharmacologically
characterized.27–29,42–47 Many of these derivatives are
highly potent and have ultimately led to a therapeutic
agent, Tiagabine (7), applicable to the treatment of epi-
lepsy. However, none of the derivatives tested so far
have shown significant selectivity for the glial versus
the neuronal GABA transport system. Our recent
discovery that (R)-exo-THPO (4) and, in particular, (R)-
N-Me-exo-THPO (5) show selectivity for the glial trans-
port system39,40 has prompted the synthesis of a series of
derivatives of racemic exo-THPO, mono- or disubsti-
tuted at the amino group. The substituted side chains
at the amino group were selected on the basis of those
previously employed for the synthesis of nipecotic acid
derivatives, such as 6–8. Furthermore some modification
was applied to these side chains in order to expand the
variation of these substituents.
Although Tiagabine (7), which is a derivative of (R)-
nipecotic acid (1) (Fig. 1), and (R)-28d are both tertiary
amines and contain identical lipophilic side chains, these
two compounds show very different relative potencies at
GAT1 and GAT2. Both compounds are now key exper-
imental tools in a comprehensive in vivo pharmacologi-
cal project with the aim of elucidating the physiological
role and pharmacological susceptibility of GAT1 and
GAT2 in the CNS.
Thus, although our modifications have not increased
glia selectivity of (R)-5, a potent and glia selective com-
pound 12d penetrating the BBB was obtained and, more
importantly, (R)-28d and (S)-28d with unique pharma-
cological profiles was discovered. All three compounds
will be relevant in future studies on the therapeutic
potential of GABA carriers in CNS disorders. Further-
more these compounds are the starting point for further
development of selective GABA uptake inhibitors.
The new compounds 12a–i, 17a,b, 22, 25a–d, and 28a–e
displayed a broad spectrum of pharmacological profiles.
Most of the compounds showed similar potencies at the
glial and neuronal transport carriers (Table 1). A nota-
ble exception is 12d which was an order of magnitude
more potent as inhibitor of glial GABA uptake and thus
comparable in selectivity to (R)-5. However no correl-
ation between the extent of glia selectivity of the
compounds and the anticonvulsant properties in the
isoniazid assay could be established. The intraperitonal
administration may obscure such correlation due to dif-
ferences in penetration. Nevertheless, all compounds
were anticonvulsant, thus demonstrating BBB
permeability.
4. Conclusion
A series of lipophilic diaromatic derivatives of the race-
mic forms of the glia-selective inhibitors exo-THPO and
N-methyl-exo-THPO was synthesized and evaluated
pharmacologically in vitro and in vivo. In general, the
compounds were effective as GABA uptake inhibitors
and anticonvulsants. A number of potent compounds
were discovered, including the glia-selective derivative
12d. Compound 28d appeared to be potent at GAT1
and GAT2, and (S)-28d turned out to be GAT2 selec-
tive. These compounds may be useful experimental tools
for investigating the relative pharmacological signifi-
cance of GABA transporters. In this regard exo-THPO
and N-methyl-exo-THPO continue to be useful lead
structures for the development of selective inhibitors
of GABA uptake.
The inhibitory action of the compounds at the cloned
GABA transporter subtypes was evaluated and, like
Tiagabine (7), most of the lipophilic derivatives of

900
R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
5. Experimental
additionally 1.5h. The mixture was cooled to rt and ex-
cess magnesium was filtered off in an inert atmosphere.
A solution of methyl 4-chlorobutyrate (40g, 0.29mol)
in dry THF (160mL) was added dropwise at 15–25ꢁC.
After further stirring for 30min, the mixture was poured
into an aqueous solution of NH4Cl and ice. Et2O
(500mL) was added. The organic phase was worked-
up according to the standard work-up procedure yield-
ing 65g of crude 4-chloro-1,1-diphenylbutan-1-ol. The
crude alcohol (30g) was dissolved in a mixture of glacial
HOAc (60mL) and 57% aqueous iodic acid (60mL).
Red phosphorus (5g) was added and the mixture was
refluxed for 6h. After slowly cooling to rt, the mixture
was poured into water and Et2O. The organic phase
was worked-up following the standard procedure above
yielding 39g of the title butyl iodide as a crude oil, which
was used without further purification. 1H NMR
(CDCl3): d 7.35–7.15 (m, 10H), 3.95 (t, 1H), 3.20 (t,
2H), 2.15 (p, 2H), 1.70 (p, 2H).
5.1. Chemistry
Melting points were determined on a Buchi SMP-20
¨
apparatus and are uncorrected. Mass spectra were ob-
tained on a Quattro MS–MS system from VG Biotech,
Fisons Instruments. The MS–MS system was connected
to an HP1050 modular HPLC system. A volume of 20–
50lL of the sample (10mg/mL) dissolved in a mixture of
1% HOAc in acetonitrile/water 1:1 was introduced via
the autosampler at a flow of 30lL/min into the electro-
spray source. Spectra were obtained at two standard sets
of operating conditions: one set to obtain molecular
weight information (MH+) (21eV) and the other set to
induce fragmentation patterns (70eV). The background
was subtracted. The relative intensities of the ions are
obtained from the fragmentation pattern. When no
intensity is indicated for the molecular ion (MH+), this
ion was only present under the first set of operating con-
ditions. 1H NMR spectra were recorded on a Bruker AC
200 F (200MHz), a Bruker Avance DRX 500, a Bruker
AC 250F (250MHz), or a Varian 360L (60MHz) spec-
trometer. Deuterated chloroform (99.8% D) dimethyl-
sulfoxide (99.9% D) or D2O were used as solvents.
TMS was used as internal reference standard. Chemical
shift values are expressed in ppm values. The following
abbreviations are used for multiplicity of NMR signals:
s = singlet, d = doublet, t = triplet, q = quartet, p = pen-
tet, dd = double doublet, dt = double triplet, m = multi-
plet, b = broad signal. NMR signals corresponding to
exchangeable protons are generally omitted. Content
of water in crystalline compounds was determined by
Karl Fischer titration. For column chromatography
(CC) silica gel of type Kieselgel 60, 230–400 mesh
ASTM was used. Optical rotations were measured in
thermostated cuvettes on a Perkin Elmer 241 polarime-
ter. Stereochemical purity were determined on an AGP
column (100 · 4.0mm) eluting with 50mM AcOH/
50mM ammonium acetate, pH4.0, H2O–MeOH
(70:30) with a flow of 0.5mL/min. Standard work-up
procedures refer to extraction with the indicated organic
solvent from proper aqueous solutions, drying of
combined organic extracts over anhydrous MgSO4
or Na2SO4, filtering, and evaporation of the solvent
in vacuo.
5.1.3. 2,2-Di(2-toluyl)tetrahydrofuran. To a suspension
of magnesium turnings (33g, 1.38mol) in dry THF
(150mL) was added 2-bromotoluene (4mL). The reac-
tion mixture was heated to reflux and an exothermic
reaction started. The heating mantle was removed and
2-bromotoluene (137mL) in dry THF (500mL) was
added dropwise over 1h at reflux temperature (exother-
mic reaction). The resulting reaction mixture was heated
under reflux for additionally 1.5h. The mixture was
cooled to rt and excess magnesium was filtered off in
an inert atmosphere. A solution of methyl 4-chlorobuty-
rate (56.4g, 0.41mol) in dry THF (200mL) was added
dropwise at 20ꢁC. The reaction mixture was stirred at
rt for additionally 1h and was then poured into an aque-
ous solution of NH4Cl and ice. The organic phase was
worked-up according to the standard work-up proce-
dures. After evaporation of the organic solvent the res-
idue was suspended in a mixture of n-heptane/EtOAc
(4:1). Filtration of the resulting crystals afforded 2,2-
di(2-toluyl)tetrahydrofuran (32.5g, 31%). 1H NMR
(CDCl3): d 7.65–7.57 (m, 2H), 7.23–7.07 (m, 4H),
7.07–7.00 (m, 2H), 4.02 (t, 2H), 2.57 (t, 2H), 2.10–1.96
(m, 2H), 1.96 (s, 6H).
5.1.4. 4,4-Di(2-toluyl)-1-butylamine (10b). Prepared like
4,4-diphenyl-1-butylamine (10e) without final HCl treat-
ment from 4,4-di(2-toluyl)-1-butyl iodide (32, 18.7g,
1
5.1.1. Synthesis of starting materials. 3-Ethoxy-4,5,6,7-
tetrahydrobenzo[d]isoxazol-4-one (9), (RS)-4-amino-
4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (13), (RS)-3-eth-
oxy-4-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazole
51mmol) and isolated as an oil. Yield: 11g (84%). H
NMR (CDCl3): d 7.16–7.05 (m, 8H), 4.23 (t, 1H), 2.70
(t, 2H), 2.27 (s, 6H), 2.03–1.87 (m, 2H), 1.58–1.42 (m,
2H), 1.20 (broad s, 2H).
(23),
(RS)-4-methylamino-3-pivaloyloxymethyloxy-
4,5,6,7-tetrahydrobenzo[d]isoxazole (26), and the corre-
sponding (R)- and (S)-forms of 26, were described
previously.39
5.1.5. 4,4-Di(2-toluyl)-3-butenylamine (10c). Prepared
like 4,4-diphenyl-1-butylamine (10e) without final HCl
treatment from 4,4-di(2-toluyl)-3-butenyl iodide (33,
34g, 94mmol) and isolated as an oil. Yield: 9.9g
(42%). H NMR (CDCl3): d 7.18–7.02 (m, 8H), 5.75
(t, 1H), 2.77 (t, 2H), 2.26 (s, 3H), 2.18 (q, 2H), 2.11 (s,
3H), 1.20 (broad s, 2H).
1
5.1.2. 4,4-Diphenyl-1-butyl iodide. To a suspension of
magnesium turnings (15g, 0.63mol) in dry THF
(75mL) was added bromobenzene (0.5g, 0.003mol).
After an initial exothermic reaction had started, the mix-
ture was heated to reflux and a solution of bromobenz-
ene (90g, 0.57mol) in dry THF (200mL) was added
dropwise during 30min. The mixture was heated for
5.1.6. 4,4-Diphenyl-1-butylamine hydrochloride (10e).48
To a solution of 4,4-diphenyl-1-butyl iodide (20g) in
dry DMF (150mL) was added sodium azide (10g). After

R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
901
reflux for 1.5h, the mixture was cooled to rt and
subsequently poured into Et2O and water. The organic
phase was worked-up following the standard procedure
above. Yield of 4,4-diphenyl-1-butyl azide: 14g. The
crude azide (10g) was dissolved in EtOH (150mL),
water (10mL), and glacial HOAc (10mL). 2% Palla-
dium on activated charcoal was added and the mixture
was hydrogenated in a Parr apparatus at 3atm for
1.5h. The catalyst was filtered off and the solvents
evaporated in vacuo. The remaining viscous oil was
dissolved in water and CH2Cl2. Aqueous NaOH
solution was added to adjust the pH to >11. The organic
phase was separated and worked according to the
standard procedure above. The hydrochloric salt was
prepared by addition of HCl to a solution of the
free amino compound in Et2O. Yield 3.4g. Mp 172–
175ꢁC.
5.1.10. (RS)-4-N-[1,1-Di(2-toluyl)but-1-en-4-yl]amino-3-
ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazole (11c). Pre-
pared according to the procedure for 11b from 939
(2.0g, 11mmol) and 4,4-di(2-toluyl)-3-butenylamine
(10c), and isolated as an oil. Yield: 1.9g (41%). 1H
NMR (250MHz, CDCl3): d 7.17–7.03 (m, 8H), 5.80 (t,
1H), 4.27 (q, 2H), 3.67 (t, 1H), 2.76 (t, 2H), 2.70–2.40
(m, 2H), 2.30–2.17 (m, 5H), 2.10 (s, 3H), 2.05–1.40
(m,5H), 1.33 (t, 3H).
5.1.11.
(RS)-4-N-[3-(10,11-Dihydrodibenzo[a,d]cyclo-
hept-5-ylidene)prop-1-yl]amino-3-ethoxy-4,5,6,7-tetra-
hydrobenzo[d]isoxazole (11d). Prepared according to the
procedure for 11e from 939 (4.0g, 22mmol) and 3-
(10,11-dihydrodibenzo[a,d]cyclohept-5-ylidene)propyl-
amine, hydrochloride,52 and isolated as an oil. Yield:
1
6.7g (74%). H NMR (250MHz, CDCl3): d 7.33–7.21
(m, 1H), 7.21–7.07 (m, 6H), 7.07–7.00 (m, 1H), 5.90 (t,
1H), 4.25 (q, 2H), 3.65 (t, 1H), 3.50–2.85 (m, 3H),
2.85–2.65 (m, 2H), 2.65–2.40 (m, 2H), 2.35 (q, 2H),
2.05–1.50 (m, 6H), 1.40 (t, 3H).
5.1.7. 3-(Thioxanthen-9-yl)-1-propylamine (10f). A mix-
ture of 3-(thioxanthen-9-yl)-1-propyl iodide49 (6.9g,
18.8mmol) and sodium azide (6.1g, 94.2mmol) in dry
DMF (100mL) was heated to reflux for 2h followed
by stirring at rt for 16h. Water (300mL) was added fol-
lowed by extraction with Et2O (2 · 200mL). The com-
bined organic phase was washed with water
(2 · 250mL) and brine (2 · 250mL). Drying of the or-
ganic phase over Na2SO4, filtration, and removal of sol-
vent in vacuo gave a dark oil of crude 3-(thioxanthen-9-
yl)-1-propyl azide (5.0g, 94%).
5.1.12. (RS)-4-N-(4,4-Diphenylbut-1-yl)amino-3-ethoxy-
4,5,6,7-tetrahydrobenzo[d]isoxazole (11e). Molecular
˚
sieve powder (3A) (4g) was added to a solution of 4,4-
diphenylbutylamine
hydrochloride
(10e)
(3.0g,
11.4mmol) and 939 (1.0g, 5.52mmol) in MeOH. NaC-
NBH3 (1.0g, 15.9mmol) was added and the mixture
was stirred for 16h at rt. After filtering off the sieves,
water (200mL) and Et2O (200mL) were added and pH
adjusted to >11 by addition of aqueous NaOH. The or-
ganic phase was worked-up according to the general
procedure above leaving an oil, which was purified by
CC on silica gel (EtOAc/heptane/NEt3 50:50:4) and iso-
The oil was dissolved in EtOH (100mL) and HOAc
(7mL), water (7mL), and 2% palladium on activated
charcoal (0.55g) were added. The mixture was hydro-
genated in a Parr apparatus at 3atm for 2h. Filtration
and removal of solvent in vacuo gave an oil, which
was subjected to CC (eluent: (1) n-heptane/EtOAc 1:1,
(2) EtOAc + 2% NEt3). Evaporation gave the title com-
pound as an oil (1.5g, 32%). 1H NMR (250MHz,
CDCl3): d 7.45–7.35 (m, 2H), 7.30–7.10 (m, 6H), 4.00
(t, 1H), 2.60 (broad t, 2H), 1.75 (t, 2H), 1.50–1.25 (m,
4H).
1
lated as an oil. Yield: 0.8g (37%). H NMR (250MHz,
CDCl3): d 7.30–7.10 (m, 10H), 4.30 (q, 2H), 3.85 (t,
1H), 3.65 (t, 1H), 2.65 (t, 2H), 2.60–2.45 (m, 2H),
2.20–2.00 (m, 2H), 2.00–1.85 (m, 1H), 1.85–1.65 (m,
3H), 1.40–1.55 (m, 2H), 1.35 (t, 3H).
5.1.13. (RS)-4-N-[3-(Thioxanthen-9-yl)prop-1-yl]amino-
3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazole (11f). Pre-
pared according to the procedure for 11b from 939
(1.0g, 5.5mmol) and 3-(thioxanthen-9-yl)-1-propyla-
mine (10f). Yield: 0.58g (25%) as an oil. 1H NMR
(250MHz, CDCl3): d 7.45–7.35 (m, 2H), 7.30–7.15 (m,
6H), 4.25 (q, 2H), 3.95 (t, 1H), 3.60 (t, 1H), 2.60–2.50
(m, 4H), 2.05–1.85 (m, 1H), 1.80–1.65 (m, 3H), 1.65–
1.50 (m, 3H), 1.50–1.35 (m, 1H), 1.30 (t, 3H).
5.1.8. (RS)-4-N-[4,4-Bis(4-fluorophenyl)but-1-yl]amino-3-
ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazole (11a). Pre-
pared according to the procedure for 11e from 939
(0.40g, 2.2mmol) and 4,4-bis(4-fluorophenyl)butylam-
ine,50 and isolated as an oil. Yield: 0.7g (75%). 1H
NMR (250MHz, CDCl3): d 7.15 (dd, 4H), 6.95 (t,
4H), 4.30 (q, 2H), 3.85 (t, 1H), 3.70 (t, 1H), 2.65 (t,
2H), 2.60–2.45 (m, 2H), 2.10–1.90 (m, 3H), 1.85–1.60
(m, 3H), 1.55–1.40 (m, 3H), 1.35 (t, 3H).
5.1.14. (RS)-4-N-[3-(Phenothiazin-10-yl)prop-1-yl]amino-
3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazole (11g).
A
5.1.9.
(RS)-4-N-[4,4-Di(2-toluyl)but-1-yl]amino-3-eth-
solution of 939 (654mg, 3.61mmol) and 3-(phenothia-
zin-10-yl)-propylamine53 (1.02g, 3.98mmol) in toluene
(130mL) was heated under reflux for 6h. p-Toluenesulf-
onic acid monohydrate (10mg) was added to the reflux-
ing solution, which was heated under reflux for
additionally 16h. This solution was cooled to 5ꢁC and
was then added to a solution of NaCNBH3 (635mg,
10.1mmol) in MeOH (50mL) at 10ꢁC. The resulting
reaction mixture was stirred for 20min, before addition
of further NaCNBH3 (500mg, 7.96mmol). The reaction
mixture was stirred for additional 10min at 10ꢁC. The
oxy-4,5,6,7-tetrahydrobenzo[d]isoxazole (11b). The title
compound was prepared according to a literature proce-
dure51 from 4,4-di(2-toluyl)-1-butylamine (10b) (1.7g,
6.7mmol), 939 (1.0g, 5.5mmol), titanium(IV)isopropyl-
ate (4.3mL), NaCNBH3 (0.6g, 9.5mmol), and EtOH
(20mL). Yield of the title compound as an oil was
1
1.0g (43%). H NMR (250MHz, CDCl3): d 7.15–7.05
(m, 8H), 4.27 (q, 2H), 3.68 (t, 1H), 2.75–2.45 (m, 4H),
2.27 (s, 6H), 2.05–1.85 (m, 3H), 1.85–1.40 (m, 7H),
1.32 (t, 3H).

902
R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
reaction mixture was poured onto water and the phases
were separated. The aqueous phase was extracted with
EtOAc (3 · 250mL). The combined organic phases were
washed with brine, dried (Na2 SO4), and evaporated in
vacuo. The residue was subjected to CC (n-heptane/
EtOAc 1:1) to give 640mg (42%) of the title compound
5.1.20.
hept-5-ylidene)propan-1-yl]amino-4,5,6,7-tetrahydroben-
zo[d]isoxazol-3-ol hydrobromide (12d). Prepared
(RS)-4-N-[3-(10,11-Dihydrodibenzo[a,d]cyclo-
according to the procedure for 12e from 11d (4.0g,
1
9.7mmol). Yield 1.8g (40%). Mp 228–230ꢁC (dec). H
NMR (250MHz, DMSO-d6): d 7.28–7.00 (m, 8H),
5.80 (t, 1H), 3.87–3.77 (m, 1H), 3.40–3.10 (m, 2H),
3.00–2.65 (m, 4H), 2.65–2.15 (m, 4H), 2.00–1.55 (m,
4H). MS m/z (%): 387 (MH+, 5%), 233 (7%), 138
(41%), 43 (100%). Anal. Calcd for C25H27BrN2O2: C,
64.23; H, 5.83; N, 5.99. Found: C, 63.99; H, 5.82; N,
6.01.
1
as an oil. H NMR (250MHz, CDCl3): d 7.18–7.08 (m,
4H), 6.95–6.85 (m, 4H), 4.26 (q, 2H), 3.97 (t, 2H), 3.65
(t, 1H), 2.84–2.70 (m, 2H), 2.60–2.40 (m, 2H), 2.00–
1.50 (m,7H), 1.33 (t, 3H).
5.1.15. (RS)-4-N-(3,3-Diphenylprop-1-yl)amino-3-ethoxy-
4,5,6,7-tetrahydrobenzo[d]isoxazole (11h). Prepared
according to the procedure for 11e from 939 (3.0g,
16.6mmol) and 3,3-diphenylpropylamine.48 Yield: 5.5g
5.1.21.
(RS)-4-N-(4,4-Diphenylbut-1-yl)amino-4,5,6,7-
tetrahydrobenzo[d]isoxazol-3-ol hydrobromide (12e). A
solution of compound 11e (0.7g, 1.79mmol) in a 33%
HBr solution in glacial HOAc (40mL) was heated at
80–90ꢁC for 1h. Excess of solvents were cautiously
evaporated in vacuo and ethanol/Et2O (1:1) was added.
The hydrobromide salt of the title compound 12e was
filtered off and dried. Yield 0.6g (76%). Mp 221–
222ꢁC. 1H NMR (250MHz, DMSO-d6): d 7.40–7.15
(m, 10H), 4.20 (broad s, 1H), 3.95 (t, 1H), 3.05 (t,
2H), 2.75–2.55 (m, 2H), 2.10–1.70 (m, 6H), 1.65–1.45
(m, 2H). MS m/z (%): 363 (MH+, 100%), 138 (89%).
Anal. Calcd for C23H27BrN2O2: C, 62.30; H, 6.15; N,
6.32. Found: C, 62.27; H, 6.17; N, 6.37.
1
(89%) as an oil. H NMR (250MHz, CDCl3): d 7.35–
7.15 (m, 10H), 4.25 (q, 2H), 4.10 (t, 1H), 3.65 (t, 1H),
2.65–2.50 (m, 3H), 2.25 (q, 2H), 2.00–1.85 (m, 1H),
1.80–1.60 (m, 4H), 1.60 (s, 1H), 1.40 (t, 3H).
5.1.16. (RS)-4-N-(4-Phenylbut-1-yl)amino-3-ethoxy-4,5,6,7-
tetrahydrobenzo[d]isoxazole (11i). Prepared according to
the procedure for 11e from 939 (3.0g, 16.6mmol) and
1
4-phenylbutylamine.54 Yield: 3.5g (68%) as an oil. H
NMR (250MHz, CDCl3): d 7.35–7.15 (m, 5H), 4.30
(q, 2H), 3.70 (t, 1H), 2.70–2.50 (m, 6H), 2.00–1.50 (m,
8H), 1.60 (s, 1H), 1.40 (t, 3H).
5.1.17. (RS)-4-N-[4,4-Bis(4-fluorophenyl)but-1-yl]amino-
4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol hydrobromide (12a).
Prepared according to the procedure for 12e from 11a
(0.58g, 1.4mmol). Yield: 0.4g (61%). Mp 205–
206ꢁC. 1H NMR (250MHz, DMSO-d6): d 7.35 (dd,
4H), 7.15 (t, 4H), 4.20 (broad s, 1H), 4.00 (t, 1H), 3.05
(t, 2H), 2.70–2.55 (m, 2H), 2.10–1.70 (m, 6H), 1.60–
1.45 (m, 2H). MS m/z (%): 399 (MH+, 4%), 138
(100%), 67 (84%). Anal. Calcd for C23H25BrF2N2O2:
C, 57.62; H, 5.27; N, 5.84. Found: C, 57.16; H, 5.35;
N, 5.86.
5.1.22.
(RS)-4-N-[3-(Thioxanthen-9-yl)propan-1-yl]-
amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol hydrochlo-
ride (12f). The ethoxy derivative 11f (0.42g, 1.0mmol)
was heated under ref1ux in 6M HCl for 66h. After
work-up the hydrochloride salt was isolated by addition
and subsequent evaporation of hydrochloric acid and
crystallization from EtOH. Yield: 0.37g (88%). Mp
221–223ꢁC. 1H NMR (250MHz, DMSO-d6): d 7.45–
7.35 (m, 4H), 7.35–7.25 (m, 4H), 4.20–4.10 (m, 2H),
2.90 (t, 2H), 2.65–2.45 (m, 2H), 2.15–1.90 (m, 2H),
1.85–1.45 (m, 6H). MS m/z (%): 393 (MH+, 6%), 239
(37%), 197 (26%), 138 (100%). Anal. Calcd for
C23H25BrN2O2S, 3/4H2O: C, 62.42; H, 6.05; N, 6.33.
Found: C, 62.27; H, 5.75; N, 6.29.
5.1.18. (RS)-4-N-[4,4-Di(2-toluyl)but-1-yl]amino-4,5,6,7-
tetrahydrobenzo[d]isoxazol-3-ol hydrobromide (12b). Pre-
pared according to the procedure for 12e from 11b
(1.0g, 2.4mmol). Yield: 0.39g (33%). Mp 217–219ꢁC
5.1.23.
(RS)-4-N-[3-(Phenothiazin-10-yl)propan-1-yl]-
1
(dec). H NMR (250MHz, DMSO-d6): d 7.20–7.03 (m,
amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol hydrobro-
mide (12g). Prepared according to the procedure for
12e from 11g (0.9g, 2.1mmol). Yield 0.27g (27%). Mp
187–189ꢁC. 1H NMR (250MHz, DMSO-d6): d 7.26–
7.10 (m, 4H), 7.07 (d, 2H), 6.97 (dd, 2H), 4.20–4.09
(m, 1H), 3.95 (t, 2H), 3.16–3.00 (m, 2H), 2.71–2.50 (m,
2H), 2.12–1.65 (m, 6H). MS m/z (%): 394 (MH+, 3%),
256 (7%), 138 (18%), 43 (100%). Anal. Calcd for
C22H24BrN3O2S: C, 55.69; H, 5.11; N, 8.86. Found: C,
55.90; H, 5.14; N, 8.90.
8H), 4.27–4.13 (m, 2H), 3.06 (t, 2H), 2.75–2.52 (m,
2H), 2.25 (d, 6H), 2.18–1.55 (m, 8H). MS m/z (%): 391
(MH+, 7%), 195 (15%), 145 (80%), 138 (92%), 105
(100%). Anal. Calcd for C25H31BrN2O2: C, 63.68; H,
6.64; N, 5.94. Found: C, 63.33; H, 6.74; N, 6.12.
5.1.19. (RS)-4-N-[1,1-Di(2-toluyl)but-1-en-4-yl]amino-3-
hydroxy-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol hydro-
bromide (12c). Prepared according to the procedure
for 12e from 11c (1.9g, 4.6mmol). Yield 1.2g (57%).
Mp 209–211ꢁC (dec). 1H NMR (250MHz, DMSO-
5.1.24. (RS)-4-N-(3,3-Diphenylpropan-1-yl)amino-4,5,6,7-
tetrahydrobenzo[d]isoxazol-3-ol hydrobromide (12h). Pre-
pared according to the procedure for 12e from 11h
(1.9g, 5.0mmol). Yield: 0.7g (33%). Mp 218–220ꢁC
d6):
d 7.26–6.96 (m, 8H), 5.73 (t, 1H), 4.24–
4.16 (m, 1H), 3.11 (t, 2H), 2.73–2.54 (m, 2H), 2.45–
2.27 (m, 2H), 2.22 (s, 3H), 2.15–1.70 (m, 7H), MS m/z
(%): 389 (MH+, 5%), 143 (33%), 138 (100%), 105
(29%), 67 (44%). Anal. Calcd for C25H29BrN2O2: C,
63.96; H, 6.24; N, 5.97. Found: C, 63.62; H, 6.29; N,
6.18.
1
(EtOH). H NMR (DMSO-d6): d 7.40–7.15 (m, 10H),
4.25 (broad s, 1H), 4.05 (t, 1H), 2.95 (t, 2H), 2.70–2.55
(m, 2H), 2.40 (t, 2H), 2.10–1.70 (m, 4H). MS m/z (%):
349 (MH+, 50%), 138 (100%), 67 (30%). Anal. Calcd

R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
903
for C22H25BrN2O2: C, 61.54; H, 5.88; N, 6.53. Found:
C, 61.46; H, 5.88; N, 6.61.
24h. The solvent was evaporated and H2O (15mL)
was added followed by extraction with Et2O
(3 · 20mL). The combined organic phases were dried
and evaporated. The remaining oil was purified by
gradient CC on silica gel (starting with 1% HOAc in
toluene followed by 1% HOAc in MeOH/EtOAc
(1:9)). Yield of the title compound as an oil was
0.28g (41%). 1H NMR (60MHz, CDCl3): d 7.5–
7.1 (m, 15H), 5.87 (t, 1H), 5.22 (s, 2H), 3.95–3.75
(m, 1H), 3.0–2.7 (m, 2H), 2.55–2.2 (m, 4H), 1.9–
1.7 (m, 7H).
5.1.25. (RS)-4-N-(4-Phenylbut-1-yl)amino-4,5,6,7-tetra-
hydrobenzo[d]isoxazol-3-ol hydrobromide (12i). Prepared
according to the procedure for 12e from 11i (3.5g,
11mmol). Yield: 2.9g (71%). Mp 202–204ꢁC (EtOH).
1H NMR (DMSO-d6): d 7.35–7.15 (m, 5H), 4.25 (broad
s, 1H), 3.00 (broad t, 2H), 2.70–2.50 (m, 4H), 2.15–1.70
(m, 4H), 1.65–1.55 (m, 4H). MS m/z (%): 287 (MH+,
6%), 138 (100%), 91 (42%), 67 (63%). Anal. Calcd for
C17H23BrN2O2: C, 55.58; H, 6.32; N, 7.63. Found: C,
55.74; H, 6.32; N, 7.65.
5.1.29. (RS)-4-[N-[1,1-Bis(3-methyl-2-thienyl)but-1-en-4-
yl]-amino]-3-benzyloxy-4,5,6,7-tetrahydrobenzo[d]isoxaz-
ole (16b). A mixture of 17 (182mg, 0.65mmol), 1,1-
bis(3-methyl-2-thienyl)but-1-en-4-yl methanesulfonate
(30) (334mg, 0.98mmol), K2CO3 (269mg, 1.95mmol),
and NaI (30mg, 0.2mmol) in DMF was heated at
100ꢁC for 24h. 1,1-Bis(3-methyl-2-thienyl)but-1-en-4-yl
methanesulfonate (30) (334mg, 0.98mmol) was added
and stirring continued for 24h. To the solution was
added H2O (12mL) by extraction with Et2O. The com-
bined organic phases were dried and evaporated. The
residue was purified by CC (toluene) yielding 0.103g
(33%). 1H NMR (60MHz, CDCl3): d 7.45–7.30 (m,
5H), 7.18 (d, 1H), 7.04 (d, 1H), 6.82 (d, 1H), 6.73 (d,
1H), 6.00 (t, 1H), 5.28 (s, 2H), 3.75–3.65 (m, 1H),
2.80–2.15 (m, 6H), 1.98 (s, 3H), 1.92 (s, 3H), 2.05–1.65
(m, 7H).
5.1.26. (RS)-4-N-(tert-Butyloxycarbonyl)amino-3-benzyl-
oxy-4,5,6,7-tetrahydrobenzo[d]isoxazole (14). To a solu-
tion of 1339 (1.0g, 4.24mmol) in a mixture of H2O
(10mL) and THF (5mL) at 0ꢁC was added saturated
aqueuos NaOH until pH ꢀ 9. Di-tert-butyldicarbonate
(0.39g, 9.75mmol) in THF (5mL) was added and
cooling was removed. A vigorous stirring at rt was
continued for 5h. Et2O (20mL) and H2O (15mL) was
added. The aqueous phase was separated and cooled.
Then EtOAc (20mL) was added followed by KHSO4
(aq) until pH ꢀ 4. The aqueous phase was extracted
with EtOAc (2 · 20mL) and the combined organic
phases was dried and evaporated. Yield of (RS)-4-
N-(tert-butyloxycarbonyl)amino-4,5,6,7-tetrahydroben-
zo[d]isoxazol-3-ol 0.56g (52%). 1H NMR (60MHz,
CDCl3): d 5.2–4.9 (m, 1H), 4.5–4.2 (m, 1H), 2.70–2.40
(m, 2H), 1.95–1.65 (m, 4H), 1.50 (s, 9H). A solution
of (RS)-4-N-(tert-butyloxycarbonyl)amino-4,5,6,7-tetra-
hydrobenzo[d]isoxazol-3-ol (0.55g, 2.16mmol) in DMF
(12mL) was added K2CO3 (595mg, 4.3mmol) and
stirred for 45min at 40ꢁC. Benzyl bromide (0.77mL,
6.5mmol) was added and stirring continued at 40ꢁC
overnight. The solvent was evaporated and H2O was
added followed by extraction with EtOAc (3 · 25mL).
The combined organic phases was dried and evapo-
rated. The residue was subjected to CC (gradient: tol–
EtOAc) to yield 360mg (48%) of the title compound
as an oil. 1H NMR (60MHz, CDCl3): d 7.4–7.2 (m,
5H), 5.2 (s, 2H), 4.8–4.5 (m, 2H), 2.6–2.4 (m, 2H),
1.9–1.7 (m, 4H), 1.4 (s, 9H).
5.1.30.
4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol
(RS)-4-[N-(1,1-Diphenylbut-1-en-4-yl)amino]-
hydrochloride
(17a). A solution of 16a (280mg, 0.62mmol) in a mix-
ture of 4M HCl (5mL) and EtOH (10mL) was refluxed
for 72h followed by evaporation. EtOH was added and
the solution evaporated. The residue was dissolved in
MeCN/EtOH (6:1, 3.5mL) heated and filtered hot.
The filtrate was added Et2O and precipitated at 5ꢁC
for 3days. The precipitate was filtered off and dried.
Yield 77mg (31%). Mp 134–138ꢁC. 1H NMR
(200MHz, D2O/DMSO-d6 (3:2)): d 7.4–7.2 (m, 10 H),
6.12 (t, 1H), 4.2–4.1 (m, 1H), 3.2–3.1 (m, 2H), 2.6–
2.35 (m, 4H), 1.9–1.7 (m, 4H). Anal. Calcd for
C23H25ClN2O2, 5/4H2O: C, 65.86; H, 6.61; N, 6.68.
Found: C, 65.67; H, 6.17; N, 6.70.
5.1.27. (RS)-4-Amino-3-benzyloxy-4,5,6,7-tetrahydroben-
zo[d]isoxazole (15).
A
suspension of 13 (344mg,
5.1.31. (RS)-4-[N-[1,1-Bis(3-methyl-2-thienyl)but-1-en-4-
yl]amino]-3-hydroxy-4,5,6,7-tetrahydrobenzo[d]isoxazole
hydrochloride (17b). To a solution of 16b (103mg,
0.215mmol) in EtOH (6mL) was added 4M HCl
(3mL) and the solution was heated under reflux for
54h. Additional EtOH (10mL) was added followed by
activated charcoal treatment. After evaporation the res-
idue was dissolved in EtOH and evaporated. The prod-
uct was precipitated twice from a mixture MeCN (6mL)
and EtOH (2mL) by careful addition of Et2O. Yield
34mg (36%). Mp 188–191ꢁC. 1H NMR (200MHz,
D2O/DMSO-d6 (3:2)): d 7.42 (d, 1H), 7.27 (d, 1H),
6.99 (d, 1H), 6.92 (d, 1H), 6.11 (t, 1H), 4.3–4.2 (m,
1H), 3.30–3.15 (m, 2H), 2.75–2.45 (m, 4H), 2.08 (s,
6H), 2.05–1.85 (m, 4H). Anal. Calcd for
C21H25ClN2O2S2: C, 57.72; H, 5.77; N, 6.41. Found:
C, 57.56; H, 5.78; N, 6.67.
1.0mmol) in Et2O (10mL) was added 2.5M HCl in
EtOAc (10mL). A clear solution appeared and stirring
was continued for 24h. Et2O (15mL) was added and
the precipitate was filtered and washed with Et2O. Yield:
232mg (83%). Mp 179–182ꢁC. 1H NMR (60MHz,
D2O): d 7.50 (m, 5H), 5.32 (s, 2H), 4.5–4.2 (m, 1H),
2.8–2.5 (m, 2H), 2.1–1.8 (m, 4H).
5.1.28. (RS)-4-[N-(1,1-Diphenylbut-1-en-4-yl)amino]-3-
benzyloxy-4,5,6,7-tetrahydrobenzo[d]isoxazole
acetate
(16a). A mixture of 15 (375mg, 1.33mmol), 4,4-diphen-
yl-3-butenyl bromide27 (573mg, 2.0mmol), K2CO3
(553mg, 4.0mmol), and NaI (60mg, 0.4mmol) in
DMF was heated to 120ꢁC and stirred for 24h.
Additional 4,4-diphenyl-3-butenyl bromide (573mg,
2.0mmol) was added and stirring continued for another

904
R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
5.1.32.
(RS)-4-[N-(tert-Butyloxycarbonyl)-N-methyl-
5.1.36.
(RS)-4-[N-[4,4-Bis(4-fluorophenyl)but-1-yl]-N-
amino]-3-benzyloxy-4,5,6,7-tetrahydrobenzo[d]isoxazole
(19). A mixture of 1839 (1.20g, 4.47mmol), benzyl bro-
mide (1.59mL, 13.4mmol), and K2CO3 (1.24g,
8.94mmol) in DMF (25mL) was stirred overnight at
40ꢁC. The solvent was evaporated and H2O (25mL)
was added followed by extraction with CH2Cl2
(3 · 50mL). The combined organic phases was dried,
evaporated, and the residue purified by CC (gradient:
heptane/tol (1:1)–tol–EtOAc) yielding 0.62g (39%) as
methyl-amino]-3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isox-
azole (24a). To a solution of 2339 (1.0g, 5.10mmol) in
4-methylpentan-2-one (10mL) was added bis-4,4-(4-
fluorophenyl)-1-butyl chloride55 (2.0g, 7.12mmol),
K2CO3 (1.0g, 7.24mmol), and KI (0.5g, 3.01mmol).
The mixture was heated under reflux overnight. Inorganic
salts were filtered off and the solvent was evaporated. The
remaining oil was purified by CC on silica gel (heptane/
EtOAc 2:3). Yield of the title compound as an oil was
1
1
an oil. H NMR (60MHz, CDCl3): d 7.4–7.3 (m, 5H),
5.23 (s, 2H), 5.2–5.1 (m, 1H), 2.7–2.5 (m, 2H), 2.60 (s,
3H), 2.1–1.7 (m, 4H), 1.45 (s, 9H).
1.6g (71%). H NMR (250MHz, CDCl3): d 7.15 (dd,
4H), 6.95 (t, 4H), 4.25 (q, 2H), 3.85 (t, 1H), 3.60 (t, 1H),
2.60–2.35 (m, 4H), 2.20 (s, 3H), 2.05–1.90 (m, 3H),
1.75–1.60 (m, 3H), 1.50–1.35 (m, 2H), 1.35 (t, 3H).
5.1.33. (RS)-4-N-Methylamino-3-benzyloxy-4,5,6,7-tetra-
hydrobenzo[d]isoxazole hydrochloride (20). A solution of
19 (0.62g, 1.73mmol) in EtOH (20mL) was added 0.5M
HCl (aq, 20mL) and stirred at 45ꢁC for 2h followed by
evaporation. EtOH was added and evaporated. The
residue was dissolved in MeCN (6mL) and added
Et2O (20mL) and left at 5ꢁC for 24h. The precipitate
was filtered and dried, yielding 0.46g (90%). Mp 156–
5.1.37.
(RS)-4-[N-[4,4-Di(2-toluyl)but-1-yl]-N-methyl-
amino]-3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazole (24b).
Prepared by the procedure for 24a via alkylation of 2339
(1.5g, 7.6mmol) with 4,4-di(2-toluyl)-1-butyl iodide (32)
1
in acetone and isolated as an oil. Yield: 1.8g (55%). H
NMR (250MHz, CDCl3): d 7.17–7.00 (m, 8H), 4.23 (q,
2H), 3.60 (t, 1H), 2.60–2.35 (m, 4H), 2.26 (dd, 6H), 2.20
(s, 3H), 2.05–1.87 (m, 3H), 1.80–1.60 (m, 4H), 1.56–1.42
(m, 2H), 1.28 (t, 3H).
1
159ꢁC. H NMR (200MHz, D2O): d 7.5–7.35 (m, 5H),
5.33 (s, 2H), 4.3–4.2 (m, 1H), 2.73 (s, 3H), 2.7–2.6 (m,
2H), 2.05–1.85 (m, 4H). 13C NMR (50MHz, D2O): d
175.4, 135.9, 129.6, 129.5, 129.0, 99.7, 72.9, 50.8, 31.7,
25.9, 22.6, 17.6. Anal. Calcd for C15H19ClN2O2: C,
61.12; H, 6.50; N, 9.50. Found: C, 60.65; H, 6.52; N,
9.64.
5.1.38. (RS)-4-[N-[1,1-Di(2-toluyl)but-1-en-4-yl]-N-meth-
ylamino]-3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazole (24c).
Prepared by the procedure for 24a via alkylation of 2339
(1.9g, 9.7mmol) with 4,4-di(2-toluyl)-3-butenyl iodide
(33) in acetone and isolated as an oil. Yield: 2.5g
1
5.1.34. (RS)-4-[N-(1,1-Diphenylbut-1-en-4-yl)-N-methyl-
amino]-3-benzyloxy-4,5,6,7-tetrahydrobenzo[d]isoxazole
(21). A mixture of 20 (442mg, 1.50mmol), 4,4-diphenyl-
3-butenyl bromide27 (646mg, 2.25mmol), K2CO3
(622mg, 4.50mmol), and NaI (75mg, 0.50mmol) in
DMF (8mL) was stirred at 120ꢁC for 24h. 4,4-Diphen-
yl-3-butenyl bromide (500mg, 1.74mmol) was added
and stirring continued for another 24h. The solvent
was evaporated and H2O (20mL) added followed by
extraction with Et2O (3 · 25mL). The combined organic
phases was dried and evaporated. The residue was
purified by gradient CC (toluene to 10% MeOH in
EtOAc) yielding 0.59g (85%). 1H NMR (60MHz,
CDCl3): d 7.3–7.1 (m, 15H), 6.0 (t, 1H), 5.23 (s, 2H),
3.6–3.4 (m, 1H), 2.7–2.1 (m, 6H), 2.14 (s, 3H), 1.9–1.7
(m, 4H).
(60%). H NMR (250MHz, CDCl3): d 7.17–7.00 (m,
8H), 5.81 (t, 1H), 4.26 (q, 2H), 3.57 (t, 1H), 2.67–2.45
(m, 4H), 2.26 (s, 3H), 2.17 (dd, 6H), 2.07–1.90 (m,
2H), 1.80–1.55 (m, 4H), 1.33 (t, 3H).
5.1.39.
(RS)-4-[N-[3-(10,11-Dihydrodibenzo[a,d]cyclo-
hept-5-ylidene)propane-1-yl]-N-methylamino]-3-ethoxy-
4,5,6,7-tetrahydrobenzo[d]isoxazole (24d). Prepared by
the procedure for 24a via alkylation of 2339 (1.0g,
5.1mmol) with 3-(10,11-dihydrodibenzo[a,d]cyclohept-
5-ylidene)-1-propyl bromide52 in acetone and isolated
as an oil. Yield: 0.71g (32%). 1H NMR (250MHz,
CDCl3): d 7.30–6.98 (m, 8H), 5.88 (t, 1H), 4.24 (q,
2H), 3.63–3.50 (m, 1H), 3.50–2.70 (m, 4H), 2.70–2.45
(m, 4H), 2.35–2.20 (m, 2H), 2.20 (s, 3H), 2.06–1.87 (m,
1H), 1.74–1.60 (m, 3H), 1.30 (t, 3H).
5.1.35. (RS)-4-[N-(1,1-Diphenylbut-1-en-4-yl)-N-methyl-
amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol
5.1.40.
(RS)-4-[N-[4,4-Bis(4-fluorophenyl)but-1-yl]-N-
hydro-
methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol
hydrobromide (25a). Prepared according to the proce-
dure for 12e from 24a (5.5g, 13mmol). Yield: 3.7g
(69%). Mp 78–82ꢁC. H NMR (250MHz, DMSO-d6,
60ꢁC): d 7.35 (dd, 4H), 7.10 (t, 4H), 4.45 (t, 1H), 4.05 (t,
1H), 3.20 (broad t, 2H), 2.70 (broad s, 3H), 2.70–2.55
(m, 2H), 2.15–1.60 (m, 8H). MS m/z (%): 413 (MH+,
10%), 203 (13%), 138 (100%), 109 (12%), 67 (58%). Anal.
Calcd for C24H27BrF2N2O2, 1/3H2O: C, 57.72; H, 5.58;
N, 5.61. Found: C, 57.36; H, 5.67; N, 5.66.
chloride (22). To a solution of 21 (0.59g, 1.27mmol)
in EtOH (15mL) was added 4M HCl (7.5mL) and
refluxed for 3days. The solution was evaporated and
the residue was dissolved in EtOH followed by
evaporation. The residue was dissolved in acetone
(8mL) and EtOH (2mL), treated with activated
charcoal and precipitated by slow addition of Et2O at
ꢁ20ꢁC. Filtration gave 228mg (44%). Mp 140–
143ꢁC. 1H NMR (200MHz, D2O/DMSO-d6 (2:1)): d
7.4–7.0 (m, 10 H), 5.96 (t, 1H), 4.25–4.15 (m, 1H),
3.2–3.05 (m, 2H), 2.6 (s, 3H), 2.55–2.35 (m, 4H), 1.9–
1.7 (m, 4H). Anal. Calcd for C24H27ClN2O2, 3/4EtOH:
C, 68.75; H, 7.13; N, 6.29. Found: C, 69.13; H, 7.17; N,
6.25.
1
5.1.41.
(RS)-4-[N-[4,4-Di(2-toluyl)but-1-yl]-N-methyl-
amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol hydrobro-
mide (25b). Prepared according to the procedure for 12e
from 24b (1.8g, 4.2mmol). Yield: 1.2g (60%). Mp 193–

R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
905
1
195ꢁC (dec). H NMR (250MHz, DMSO-d6): d 7.21–
2-(bis(4-chlorophenyl)methoxy)ethyl chloride.42 Yield:
0.5g (21%) as an oil. H NMR (250MHz, CDCl3): d
7.35–7.15 (m, 8H), 5.90 (d, 1H), 5.90 (dd, 2H), 3.70
(dt, 1H), 3.50 (t, 2H), 2.85–2.65 (m, 2H), 2.55 (t, 2H),
2.30 (s, 3H), 2.10–1.95 (m, 1H), 1.85–1.60 (m, 3H),
1.20 (s, 9H).
1
7.03 (m, 8H), 4.47–4.36 (m, 1H), 4.25 (t, 1H), 3.35–
3.15 (m, 2H), 2.80–2.55 (m, 5H), 2.26 (s, 6H), 2.20–
1.65 (m, 8H). MS m/z (%): 405 (MH+, 4%), 268 (27%),
138 (30%), 43 (100%). Anal. Calcd for C26H33BrN2O2:
C, 64.32; H, 6.86; N, 5.77. Found: C, 64.17; H, 6.91;
N, 5.79.
5.1.47. (RS)-4-[N-[1,1-Bis(3-methyl-2-thienyl)but-1-en-4-
yl]-N-methylamino]-3-pivaloyloxymethyloxy-4,5,6,7-
tetrahydrobenzo[d]isoxazole (27d). To a solution of 2639
(8.46g, 30.0mmol) and 1,1-bis(3-methyl-2-thienyl)but-
1-en-4-yl methanesulfonate (30) (15g, 43.8mmol) in iso-
propyl acetate (40mL) was added Li2CO3 (2.5g,
33.8mmol). The mixture was heated under reflux for
24h while continuously adding additional Li2CO3 in
small amounts (4 · 0.5g, 27.1mmol). Et2O was added
and the solution filtered. The organic phase was washed
with H2O. The aqueous phase was extracted and the
combined organic phases were washed with brine,
dried (MgSO4), and evaporated. CC (Et3N/EtOAc/hep-
tane 5:10:85) gave 27d (9.0g, 56%). 1H NMR (500MHz,
CDCl3): d 7.20 (d, 1H), 7.04 (d, 1H), 6.84 (d, 1H), 6.75
(d, 1H), 6.10 (t, 1H), 5.88 (d, 1H), 5.86 (d, 1H), 3.62 (t,
1H), 2.65–2.50 (m, 4H), 2.30–2.24 (m, 2H), 2.20 (s, 3H),
2.04 (s, 3H), 1.99 (s, 3H), 1.75–1.60 (m, 4H), 1.21 (s,
9H). 13C NMR (125MHz, CDCl3): d 176.8, 171.4,
168.2, 139.6, 135.3, 135.1, 133.7, 133.3, 131.0, 129.4,
127.9, 124.0, 122.4, 104.8, 84.5, 55.3, 53.6, 38.6, 38.2,
28.7, 26.8, 25.9, 23.0, 19.7, 14.6, 14.2.
5.1.42. (RS)-4-[N-[4,4-Di(2-toluyl)but-3-en-1-yl]-N-methyl-
amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol hydrobro-
mide (25c). Prepared according to the procedure for 12e
from 24c (2.5g, 5.8mmol). Yield: 1.3g (46%). Mp 177–
179ꢁC. 1H NMR (250MHz, DMSO-d6): d 7.30–6.98
(m, 8H), 5.74 (t, 1H), 4.47–4.35 (m, 1H), 3.40–3.15 (m,
2H), 2.80–2.60 (m, 6H), 2.21 (s, 3H), 2.14–1.70 (m,
8H). MS m/z (%): 403 (MH+, 19%), 266 (40%), 143
(77%), 138 (100%), 105 (49%), 67 (20%). Anal. Calcd
for C26H31BrN2O2: C, 64.58; H, 6.48; N, 5.80. Found:
C, 64.31; H, 6.62; N, 6.02.
5.1.43.
(RS)-4-[N-[3-(10,11-Dihydrodibenzo[a,d]cyclo-
hept-5-ylidene)prop-1-yl]-N-methylamino]-4,5,6,7-tetra-
hydrobenzo[d]isoxazol-3-ol hydrobromide (25d). Prepared
according to the procedure for 12e from 24d (0.71g,
1.7mmol). Yield: 0.18g (22%). Mp 215–217ꢁC (dec).
1H NMR (250MHz, DMSO-d6): d 7.30–7.05 (m, 8H),
5.80 (t, 1H), 4.43–4.34 (m, 1H), 3.44–3.05 (m, 7H),
2.95–2.40 (m, 6H), 2.10–1.70 (m, 4H). MS m/z (%):
401 (MH+, 26%), 265 (66%), 233 (30%), 138 (84%), 43
(100%). Anal. Calcd for C26H29BrN2O2, 1/2H2O: C,
63.66; H, 6.18; N, 5.71. Found: C, 63.95; H, 6.14; N,
5.87.
5.1.48. (R)-4-[N-[1,1-Bis(3-methyl-2-thienyl)but-1-en-4-
yl]-N-methylamino]-3-pivaloyloxymethyloxy-4,5,6,
7-tetrahydrobenzo[d]isoxazole [(R)-27d]. Prepared accor-
ding to the procedure for 27d from the (R)-2639 (1.40g,
5.0mmol). Yield: 1.54g (59%). 1H NMR (500MHz,
CDCl3): d 7.20 (d, 1H), 7.04 (d, 1H), 6.84 (d, 1H),
6.75 (d, 1H), 6.10 (t, 1H), 5.88 (d, 1H), 5.86 (d, 1H),
3.62 (t, 1H), 2.65–2.50 (m, 4H), 2.30–2.24 (m, 2H),
2.20 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.75–1.60 (m,
4H), 1.21 (s, 9H). 13C NMR (125MHz, CDCl3): d
176.8, 171.4, 168.2, 139.6, 135.3, 135.1, 133.7, 133.3,
131.0, 129.4, 127.9, 124.0, 122.4, 104.8, 84.5, 55.3,
53.6, 38.6, 38.2, 28.7, 26.8, 25.9, 23.0, 19.7, 14.6, 14.2.
5.1.44. (RS)-4-[N-[2-(Diphenylmethoxy)ethyl]-N-methyl-
amino]-3-pivaloyloxymethyloxy-4,5,6,7-tetrahydrobenzo-
[d]isoxazole (27a). Prepared by the procedure below for
27b via alkylation of 2639 (1.3g, 4.6mmol) with 2-
(diphenylmethoxy)ethyl methanesulfonate (35). Yield:
1
1.2g (53%) as an oil. H NMR (250MHz, CDCl3): d
7.40–7.15 (m, 10H), 5.90 (dd, 2H), 5.35 (s, 1H), 3.65
(dt, 1H), 3.50 (dt, 2H), 2.80–2.60 (m, 2H), 2.55 (t,
2H), 2.30 (s, 3H), 2.05–1.95 (m, 1H), 1.80–1.60 (m,
3H), 1.20 (s, 9H).
5.1.45. (RS)-4-[N-[2-[Bis(4-fluorophenyl)methoxy]ethyl]-
N-methylamino]-3-pivaloyloxymethyloxy-4,5,6,7-tetra-
hydrobenzo[d]isoxazole (27b). To a solution of 2639
(1.1g, 3.90mmol) in 4-methylpentan-2-one (18mL) were
added K2CO3 (0.7g, 5.06mmol) and 2-(bis(4-fluorophen-
5.1.49. (S)-4-[N-[1,1-Bis(3-methyl-2-thienyl)but-1-en-4-
yl]-N-methylamino]-3-pivaloyloxymethyloxy-4,5,6,
7-tetrahydrobenzo[d]isoxazole
[(S)-27d].
Prepared
according to the procedure for 27d from the (S)-2639
(1.37g, 4.9mmol). Yield: 1.75g (68%). 1H NMR
(500MHz, CDCl3): d 7.20 (d, 1H), 7.04 (d, 1H), 6.84
(d, 1H), 6.75 (d, 1H), 6.10 (t, 1H), 5.88 (d, 1H), 5.86
(d, 1H), 3.62 (t, 1H), 2.65–2.50 (m, 4H), 2.30–2.24 (m,
2H), 2.20 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.75–1.60
(m, 4H), 1.21 (s, 9H). 13C NMR (125MHz, CDCl3): d
176.8, 171.4, 168.2, 139.6, 135.3, 135.1, 133.7, 133.3,
131.0, 129.4, 127.9, 124.0, 122.4, 104.8, 84.5, 55.3,
53.6, 38.6, 38.2, 28.7, 26.8, 25.9, 23.0, 19.7, 14.6, 14.2.
yl)methoxy)ethyl
methanesulfonate
(36)
(1.8g,
5.26mmol). The mixture was heated under reflux over-
night. Inorganic salts were filtered off and the solvent
was evaporated. CC afforded pure 27b. Yield: 1.4g
1
(68%) as an oil. H NMR (250MHz, CDCl3): d 7.55
(d, 2H), 7.30 (dd, 4H), 7.00 (t, 4H), 5.90 (dd, 2H),
5.35 (s, 1H), 3.65 (dt, 1H), 3.50 (t, 2H), 2.80–2.60 (m,
2H), 2.55 (broad t, 2H), 2.25 (s, 3H), 2.05–1.95 (m,
1H), 1.80–1.60 (m, 3H), 1.20 (s, 9H).
5.1.50. (RS)-4-[N-[2-(Diphenylmethoxy)ethyl]-N-methyl-
hydro-
chloride (28a). Prepared according to the procedure
below for 28b from 27a (1.2g, 2.4mmol). The obtained
sodium salt was converted to the title hydrochloride salt
5.1.46. (RS)-4-[N-[2-[Bis(4-chlorophenyl)methoxy]ethyl]-
N-methylamino]-3-pivaloyloxymethyloxy-4,5,6,7-tetra-
hydrobenzo[d]isoxazole (27c). Prepared by the procedure
for 27b via alkylation of 2639 (1.2g, 2.1mmol) with
amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol

906
R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
from CH2Cl2 by addition of concentrated hydrochloride
acid to pH = 1. Yield: 0.5g (55%). Mp: 108–113ꢁC
(amorphous) (Et2O). H NMR (250MHz, DMSO-d6):
d 7.45–7.20 (m, 10H), 5.55 (s, 1H), 4.55 (broad s, 1H),
3.80 (broad t, 2H), 3.60–3.40 (m, 2H), 2.80 (broad s,
3H), 2.75–2.60 (m, 2H), 2.30–2.10 (m, 2H), 2.10–1.70
(m, 2H). MS m/z (%): 379 (MH+, 4%), 167 (100%),
152 (74%), 138 (36%), 67 (32%). Anal. Calcd for
C23H27ClN2O3: C, 66.57; H, 6.57; N, 6.75. Found: C,
66.21; H, 6.73; N, 6.64.
5.1.54. (R)-(ꢁ)-4-[N-[1,1-Bis(3-methyl-2-thienyl)but-1-
en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isox-
azol-3-ol hydrochloride [(R)-28d]. Prepared according to
the procedure for 28d from (R)-27d (1.54g, 2.9
1
mmol). Yield: 0.88g (67%). Mp: 160–162ꢁC. Ee =
20
98.5% (HPLC), ½aꢂ ꢁ16.5 (c 1.12, CHCl3). Anal. Calcd
D
for C22H27ClN2O2S2: C, 58.58; H, 6.03; N, 6.21. Found:
C, 58.09; H, 6.14; N, 6.15.
5.1.55. (S)-(+)-4-[N-[1,1-Bis(3-methyl-2-thienyl)but-1-en-
4-yl]-N-methylamino]-3-hydroxy-4,5,6,7-tetrahydro-1,2-
benzo[d]isoxazole hydrochloride [(S)-28d]. Prepared
according to the procedure for 28d from (S)-27d
5.1.51. (RS)-4-[N-[2-[Bis(4-fluorophenyl)methoxy]ethyl]-
N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol
sodium salt (28b). To the pivaloyloxymethyl protected
derivative 27b (0.6g, 1.13mmol) in EtOH (7mL) was
added water (1.4mL) and NaOH powder (0.7g,
17.5mmol). The mixture was stirred overnight. EtOH
was evaporated in vacuo and water (25mL) was added.
The precipitated crystalline product was filtered off and
washed with water. After drying overnight at 70–80ꢁC
in vacuo 0.35g (71%) of pure title compound remained.
Mp: 178–181ꢁC. 1H NMR (250MHz, DMSO-d6): d 7.40
(d, 2H), 7.35 (d, 2H), 7.15 (t, 4H), 5.50 (s, 1H), 3.40 (t,
2H), 3.25 (t, 1H), 2.90–2.60 (m, 2H), 2.35–2.10 (m, 2H),
2.20 (s, 3H), 1.90–1.75 (m, 1H), 1.75–1.60 (m, 1H), 1.60–
1.45 (m, 1H), 1.45–1.30 (m, 1H). MS m/z (%): 415
(MH+, 4%), 203 (100%), 183 (63%), 138 (42%), 67
(30%). Anal. Calcd for C23H23F2N2NaO3, 1/2H2O: C,
62.01; H, 5.44; N, 6.29. Found: C, 61.55; H, 5.43; N,
5.93.
(1.75g, 3.3mmol). Yield: 0.94g (63%). Mp: 161–
20
D
163ꢁC. Ee = 99.1% (HPLC), ½aꢂ 16.7 (c 1.02, CHCl3).
Anal. Calcd for C22H27ClN2O2S2: C, 58.58; H, 6.03;
N, 6.21. Found: C, 58.34; H, 5.96; N, 6.07.
5.1.56. 1,1-Bis(3-methyl-2-thienyl)but-1-en-4-yl methane-
sulfonate (30). Magnesium (14.0g, 0.58mol) was sus-
pended in THF (10mL). 2-Bromo-3-methylthiophen
(100g, 0.57mol) in THF (250mL) was added slowly to
maintain gentle reflux. The reaction was heated under re-
flux for another 15min. The reaction was cooled to 0ꢁC
and 4-butyrolactone (21.5g, 0.25mol) in THF (50mL)
was added dropwise. The reaction was stirred another
30min at rt upon addition. The reaction was poured on
NH4Cl (100g) in H2O (600mL). The phases were sepa-
rated and the aqueous phase extracted with THF. The
combined organic phases were washed with brine, dried
(MgSO4), and evaporated. Recrystallization in THF/n-
heptane gave the title compound (50.2g, 63%). Mp:
128–130ꢁC. 1H NMR (500MHz, CDCl3): d 7.10 (d,
2H), 6.77 (d, 2H), 3.72 (broad s, 1H), 3.66 (t, 2H), 3.38
(broad s, 1H), 2.54 (t, 2H), 1.90 (s, 6H), 1.69 (p,
2H); 13C NMR (125MHz, CDCl3): d 142.7, 133.6,
131.7, 121.7, 75.2, 62.9, 39.1, 26.9, 14.4. To a solution
of 1,1-bis(3-methyl-2-thienyl)butane-1,4-diol (14.3g, 50.6
mmol) in MeOH was added 4M HCl (10mL) and
refluxed for 3h. Saturated aqueous K2CO3 was carefully
added. The solution was then extracted with Et2O. The
combined organic phases were washed with brine, dried
(MgSO4), and evaporated giving 4,4-bis(3-methyl-2-thie-
nyl)but-3-en-1-ol (13.1g, 98%) as an oil, which was used
without further purification. 1H NMR (500MHz,
CDCl3): d 7.20 (d, 1H), 7.04 (d, 1H), 6.84 (d, 1H), 6.75
(d, 1H), 6.10 (t, 1H), 3.68 (t, 2H), 2.39 (q, 2H), 2.04 (s,
3H), 1.99 (s, 3H); 13C NMR (125MHz, CDCl3): d 139.2,
135.2, 134.9, 133.5, 131.1, 131.0, 129.4, 124.2, 122.6,
62.0, 33.2, 14.6, 14.2. To a solution of 4,4-bis(3-methyl-
2-thienyl)but-3-en-1-ol (13.1g, 48.4mmol) in Et2O
(150mL) was added MeSO2Cl (5mL, 7.4g, 64.6mmol)
and Et3N (9mL, 6.55g, 64.7mmol). The reaction was stir-
red for 1h. The resulting suspension was filtered and the
precipitate was washed with Et2O and the filtrate was
evaporated giving 1,1-bis(3-methyl-2-thienyl)but-1-en-4-
yl methanesulfonate (17.0g, 100%) as an oil, which was
5.1.52. (RS)-4-[N-[2-[Bis(4-chlorophenyl)methoxy]ethyl]-
N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol
(28c). Prepared according to the procedure for 28b from
27c (0.50g, 0.89mmol). Yield: 0.38g (91%). Mp: 201–
1
203ꢁC (water/EtOH). H NMR (250MHz, DMSO-d6):
d 7.45 (s, 8H), 5.55 (s, 1H), 3.50–3.35 (m, 2H), 3.35 (t,
1H), 2.90–2.65 (m, 2H), 2.20 (s, 3H), 2.35–2.20 (m,
2H), 1.85–1.75 (m, 1H), 1.70–1.60 (m, 1H), 1.60–1.40
(m, 2H). MS m/z (%): 447 (MH+); 235 (78%), 165
(57%), 138 (100%), 67 (56%). Anal. Calcd for
C23H23Cl2N2NaO3: C, 58.85; H, 4.95; N, 5.97. Found:
C, 58.46; H, 4.99; N, 5.95.
5.1.53. (RS)-4-[N-[1,1-Bis(3-methyl-2-thienyl)but-1-en-4-
yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-
3-ol hydrochloride (28d). To a solution of 27d (9.0g,
17.0mmol) in dry EtOH (150mL) was added NaOH
(9g, 225mmol) on an ice bath. The solution was stirred
overnight followed by evaporation. Water (100mL) and
EtOAc (100mL) was added and adjusting to pH7 with
4M HCl (ꢀ40mL) enabled extraction with EtOAc.
The organic phase was washed with brine, dried
(MgSO4), and evaporated. The residue was dissolved
in HCl in EtOH then evaporated. Precipitation from
EtOH by adding Et2O gave 28d (6.3g, 82%). Mp: 154–
157ꢁC. 1H NMR (500MHz, DMSO-d6): d 7.49 (d,
1H), 7.32 (d, 1H), 6.94 (d, 1H), 6.83 (d, 1H), 6.03 (br,
1H), 4.44 (br, 1H), 3.45–3.25 (m, 2H), 2.80–2.2.55 (m,
4H), 2.78 (br, 1H), 2.52 (s, 3H), 2.15–2.05 (m, 2H),
2.03 (s, 3H), 1.98 (s, 3H), 2.0–1.9 (m, 1H), 1.8–1.7 (m,
1H). Anal. Calcd for C22H27ClN2O2S2: C, 58.58; H,
6.03; N, 6.21. Found: C, 58.16; H, 6.10; N, 6.08.
1
used without further purification. H NMR (500MHz,
CDCl3): d 7.22 (d, 1H), 7.06 (d, 1H), 6.86 (d, 1H), 6.77
(d, 1H), 6.03 (t, 1H), 4.28 (t, 2H), 2.97 (s, 3H), 2.58 (q,
2H), 2.04 (s, 3H), 2.00 (s, 3H); 13C NMR (125MHz,
CDCl3): d 138.6, 135.5, 134.2, 134.0, 131.1, 130.9, 129.6,
128.0, 124.6, 123.1, 68.5, 37.3, 29.6 14.6, 14.1.

R. P. Clausen et al. / Bioorg. Med. Chem. 13 (2005) 895–908
907
5.1.57. 4,4-Di(2-toluyl)-1-butyl iodide (32). 2,2-Di(2-tolu-
yl)tetrahydrofuran (28g, 0.11mol) was dissolved in
HOAc (250mL). 5% Palladium on activated charcoal
(3g) was added and the mixture was hydrogenated in a
Parr apparatus at 3atm at 55ꢁC for 5h. The catalyst
was filtered off and the solvent was evaporated in vacuo.
The remaining oil was subjected to CC (n-heptane/EtOAc
15:1) to give 4,4-di(2-toluyl)-1-butanol (19g). A solution
of 4,4-di(2-toluyl)-1-butanol (19g) in HOAc (400mL)
was heated under reflux for 3h. The cooled solution was
evaporated in vacuo to give 4,4-di(2-toluyl)-1-butyl ace-
tate (17g) as an oil. A solution of 4,4-di(2-toluyl)-1-butyl
acetate (9.2g) in 57% aqueous iodic acid (150mL) was
heated under reflux for 3h. The cooled solution was
poured into a mixture of ice and water and the aqueous
phase was extracted with Et2O. The combined organic
phases were washed with water and brine, dried
(Na2SO4), and evaporated in vacuo to give the crude title
compound (11.7g) as an oil, which was used without fur-
ther purification. 1H NMR (CDCl3): d 7.12 (s, 8H), 4.26
(t, 1H), 3.17 (t, 2H), 2.28 (s, 6H), 1.80–2.10 (m, 4H).
are means of 2–3 four-fold experiments and variations
are less than 15%.
5.3. Determination of anticonvulsant effects
The anticonvulsant effects of the compounds were deter-
mined by subcutaneous injection into isoniazid-treated
mice using the experimental conditions described
previously.39
Acknowledgements
This work was supported by grants from the Lundbeck
Foundation, The Danish Medical Research Council and
Apotekerfonden af 1991.
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