Synthesis of novel GABA uptake inhibitors. Part 6: Preparation and evaluation of N-Ω asymmetrically substituted nipecotic acid derivatives

Article abstract of DOI:10.1016/S0968-0896(01)00148-1

In a previous series of potent GABA uptake inhibitors published from this laboratory, we noticed that asymmetry in the substitution pattern of the bis-aromatic moiety in known GABA uptake inhibitors such as 4 [1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid] and 5 [(R)-1-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid] was beneficial for high affinity. This led us to investigate asymmetric analogues of known symmetric GABA uptake inhibitors in which one of the aryl groups has been exchanged with an alkyl, alkylene or cycloalkylene moiety as well as other modifications in the lipophilic part. The in vitro values for inhibition of [³H]-GABA uptake in rat synaptosomes was determined for each compound, and it was found that several of the novel compounds inhibit GABA uptake as potently as their known symmetrical reference analogues. Several of the novel compounds were also evaluated for their ability to inhibit clonic seizures induced by a 15 mg/kg (ip) dose of methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) in vivo. Some of the compounds, for example 18 [(R)-1-(2-(((1,2-bis(2-fluorophenyl)ethylidene)amino)oxy)ethyl)-3- piperidinecarboxylic acid], show a high in vivo potency and protective index comparable with that of our recently launched anticonvulsant product, 5 [(R)-1-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid], and may therefore serve as second-generation drug candidates.

Full text of DOI:10.1016/S0968-0896(01)00148-1

Bioorganic& Medicinal Chemistry 9 (2001) 2773–2785
Synthesis of Novel GABA Uptake Inhibitors.
Part 6y: Preparation and Evaluation of N-X Asymmetrically
Substituted Nipecotic Acid Derivatives
Knud Erik Andersen,* Jesper Lau, Behrend F. Lundt, Hans Petersen, Per O. Huusfeldt,
Peter D. Suzdak and Michael D.B. Swedberg
˚
Health Care Discovery, Novo Nordisk A/S, Novo Nordisk Park, DK 2760 Maløv, Denmark
Received 26 March 2001; accepted 4 April 2001
Abstract—In a previous series of potent GABA uptake inhibitors published from this laboratory, we noticed that asymmetry in the
substitution pattern of the bis-aromaticmoiety in known GABA uptake inhibitors such as 4 [1-(4,4-diphenyl-3-butenyl)-3-piper-
idinecarboxylic acid] and 5 [(R)-1-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid] was beneficial for high affi-
nity. This led us to investigate asymmetricanalogues of known symmetricGABA uptake inhibitors in which one of the aryl groups
has been exchanged with an alkyl, alkylene or cycloalkylene moiety as well as other modifications in the lipophilic part. The in vitro
values for inhibition of [3H]-GABA uptake in rat synaptosomes was determined for each compound, and it was found that several
of the novel compounds inhibit GABA uptake as potently as their known symmetrical reference analogues. Several of the novel
compounds were also evaluated for their ability to inhibit clonic seizures induced by a 15 mg/kg (ip) dose of methyl 6,7-dimethoxy-
4-ethyl-b-carboline-3-carboxylate (DMCM) in vivo. Some of the compounds, for example 18 [(R)-1-(2-(((1,2-bis(2-fluoro-
phenyl)ethylidene)amino)oxy)ethyl)-3-piperidinecarboxylic acid], show a high in vivo potency and protective index comparable
with that of our recently launched anticonvulsant product, 5 [(R)-1-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic
acid], and may therefore serve as second-generation drug candidates. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
of GABA’ergicneurotransmission would only take
place under conditions where GABA is already being
released physiologically. GABA can be removed from
the synapse by either a high-affinity sodium-dependent
GABA uptake carrier into neuronal or glial cells or by
diffusion from the synapse. The GABA uptake system
has traditionally been classified as either neuronal or
glial on the basis of pharmacological selectivity of cyclic
amino acid GABA uptake inhibitors.5 However, several
investigators have recently cloned and sequenced sub-
types of the GABA uptake carrier and the selectivity of
novel and hitherto known GABA uptake inhibitors on
these subtypes has been investigated.17,18 It was found
that the type of GABA uptake inhibitors described in
this paper is binding to the GAT-1 subtype.
g-Aminobutyricaicd (GABA) is a major inhibitory
neurotransmitter in the mammalian central nervous
system (CNS).2À6 GABA has been estimated to be pre-
sent in 60–70% of all synapses in the CNS.7 A decrease
in GABA’ergicneurotransmission appears to be
involved in the etiology of several neurological dis-
orders, including anxiety, pain and epilepsy.4,5,8À10
Thus, numerous investigations have focused on finding
novel approaches to modulate GABA’ergic function in
man. These approaches include direct agonism of the
GABA receptors,11,12 the inhibition of enzymatic
breakdown of GABA,13,14 or by the inhibition of the
uptake of GABA into neuronal and glial cell bodies.5,15
It is well documented that GABA agonists are respon-
sible for a number of unacceptable side effects in man.16
However, in principle, GABA uptake inhibitors should
exert a more therapeutically useful influence than
GABA agonists. This is because a major enhancement
Nipecotic acid 1, guvacine 2 and homo-b-proline 319,20
(Fig. 1) which can be considered as conformationally-
restricted GABA analogues21 display in vitro activity as
inhibitors of [3H]-GABA uptake. However, compounds
1–3 do not readily cross the blood–brain barrier.19,22,23
Recently, novel series of lipophilic GABA uptake inhi-
bitors that possess potent activity in vitro and in vivo
were described.24À27 These compounds differ from
*Corresponding author. Tel.: +45-4443-4898; fax: +45-4466-3450;
e-mail: kea@novonordisk.com
ySee ref 1.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00148-1

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K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
compounds 1–3 in that they readily cross the blood–
brain barrier due to the attachment of a lipophilic
anchor to the nitrogen in compounds 1.
anti-epilepticagent has now been launched for add-on
therapy in the treatment of epilepsy.
With this new drug as a benchmark we have con-
tinuously searched for novel GABA uptake inhibitors
with improved properties as second-generation com-
pounds. This has now resulted in several series of highly
potent GABA uptake inhibitors of which 7 (IC50=137
nM48), 8 (IC50=55 nM49), and 9 (IC50=184 nM1) are
examples (Fig. 3). In this type of compound, the elec-
tronegative principle was applied in the chain by intro-
duction of heteroatoms bearing lone pairs and thereby
considerably improved GABA uptake inhibition com-
pared to compounds such as 4, 5 and 6. Among these
new series of compounds, we have previously reported
on the selection of compound 9 as a potential second-
generation compound to 5.1 In order to identify even
better compounds, the search for novel potent GABA
uptake inhibitors with improved biological properties
was continued. In the series of potent GABA uptake
inhibitors published earlier from this laboratory,1,42,48,49
we have noticed that asymmetry in the substitution
pattern of the bis-aromaticsystems represented in the
reference compounds 4–9 was beneficial for high affinity
for the GABA uptake site. This allowed asymmetry in
the bis-aromaticpart of the moleucle has led us to
investigate asymmetric analogues of the reference com-
pounds 4. Such analogues include (a) monoaryl com-
pounds in which one of the aryl groups has been
exchanged with an alkyl, alkylene or cycloalkylene
moiety, (b) biphenyl or aralkylaryl analogues or (c) the
two aryl groups have been placed in an asymmetric
pattern to each other either directly on the linker or in
the more constrained 2-phenyl-1,2,3,4-tetrahydro-
naphalene or 3-phenyl-1,2-dihydroindene ring systems.
The synthesis50À53 and biological activity of these series
of novel and selective GABA uptake inhibitors will now
be reported (Tables 1 and 2).
In the early 1980s, such lipophilic derivatives of amino
acids 1–3 were described for the first time. These com-
pounds, an example of which is 4 (SKF 89976A,
IC50=328 nM,48 Fig. 2), exhibited promising seizure
protection24,28,29 in some animal models predictive of
anticonvulsant activity.30 The compounds also dis-
played reduced CNS depressant effects compared with
some commonly used anticonvulsant drugs, such as
diazepam.31 These observations have also stimulated
others to investigate this field,32À40 leading to the dis-
covery of a highly lipophilic GABA uptake inhibitor
with CNS activity, 6 (CI-966, IC50=440 nM,48 Fig. 2).
This compound was discovered at Parke–Davis/War-
ner–Lambert, and has been investigated in a phase I
clinical trial.41 From this laboratory, we have previously
reported on the structure–activity studies leading to the
choice of 5 (tiagabine, NO-328, NNC 05-0328, IC50=67
nM,48 Fig. 2) as an anticonvulsant drug
candidate.25À27,42,43 Extensive pharmacological44,45 and
clinical investigations have been completed on 5 with
proven anticonvulsant efficacy in man.46,47 This novel
Figure 1. Three cyclic amino acids which act as GABA uptake inhi-
bitors, nipecotic acid 1, guvacine 2 and homo-b-proline 3.
Figure 3. Potent GABA uptake inhibitors with an electronegative
moiety introduced in the chain.
Figure 2. Lipophilicreference GABA uptake inhibitors.

K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
2775
Table 1. Chemical and biological data of oxime derivatives 14–28
No.
Method
Mp. (ꢀC)
Formula
Microanalysesa
GABA uptake IC50 (nM)b
137
7
c
14
15
16
17
18
19
20
21
22
23
24
A1C
A2B1C
A2B1C
A2B1C
A2B1C
A1C
130d
C18H24N2O3,HCl,0.25H2O
C19H28N2O3,HCl
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
1241
284
135
923
80
142–145d
Amorph.
Amorph.
125–130d
Amorph.
Amorph.
Amorph.
Amorph.
150–151e
43–45d
C22H26N2O3,HCl
C22H24F2N2O3,HCl,0.5H2O
C22H24F2N2O3,HCl
C23H28N2O3,HCl,0.5H2O
C19H26N2O3,HCl,1.25H2O
C20H28N2O3,HCl,1.25H2O
C21H30N2O3,HCl,1.25H2O
C20H28N2O3,1.5HCl,0.5H2O
C20H28N2O3,HCl,0.25H2O
1027
751
268
290
462
324
A1C
A1C
A1C
A1C
A1C
25
26
A1C
A1C
171–172e
116 dece
C20H26N2O3,HCl,0.25H2O
C,H,N
C,H,N
349
C21H30N2O3,HCl
9700
(continued)

2776
K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
Table 1 (continued)
No.
Method
A1C
Mp. (ꢀC)
160–162e
Formula
Microanalysesa
C,H,N
GABA uptake IC50 (nM)b
27
28
C24H28N2O3,HCl,0.25H2O
874
A1C
204–205e
C23H26N2O3,HCl
C,H,N
1405
aAll new compounds were analysed for C, H, and N and are within Æ 0.4% of the theoretical values. Microanalysis on the following compounds
showed deviations of N larger than Æ0.4% calcd (found): 19: 6.58 (6.03); 22: 6.71 (7.18).
bInhibition of GABA uptake in synaptosomes: IC50 (nM). Mean of two determinations.
cRef 48.
dCompounds were crystallised from CH2Cl2.
eCompounds were crystallised from acetone.
Table 2. Chemical and biological data of ether derivatives 29–45
No.
Method
Mp.ꢀC
Formula
Microanalysesa
GABA uptake IC50 (nM)b
e
55
8
c,d
29
30
31
32
33
34
35
36
1370
258
e
e
90
B2C
A3C
169–170f
171–172f
165–167f
148–149f
153–155f
C14H19NO3,HCl
C20H23NO3,HCl
C21H25NO3,HCl
C21H25NO3,HCl
C25H27NO3,HCl
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
>9000
5823
6946
334
A3C
A4B3C
A4B3C
7977
(continued)

K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
2777
Table 2 (continued)
No.
Method
A3C
Mp.ꢀC
Formula
Microanalysesa
C,H,N
GABA uptake IC50 (nM)b
322
37
38
39
40
41
42
43
44
45
Amorph.
C22H27NO3,HCl
A4B3C
A5B3C
A5B3C
A4B3C
A4B3C
A3C
135–137f
122–124f
144–145f
214–215f
Amorph.
115–117g
Amorph.
112–114f
C22H27NO3,HCl
C22H27NO3,HCl
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
>3000
111
C23H29NO3,HCl
101
C23H27NO3,HCl
505
C23H27NO3,HCl,0.25H2O
C17H25NO3,HCl,0.25H2O
C20H32N2O3,2HCl,3H2O
C20H30ClFN2O3,HCl
>3000
4500
282
DB3C
DB3C
149
aAll new compounds were analysed for C, H, and N and are within Æ0.4% of the theoretical values. Microanalysis on the following compound
showed deviations of C larger than Æ0.4% calcd (found): 44: 50.52 (51.15).
bInhibition of GABA uptake in synaptosomes: IC50 (nM) Mean of two determinations.
cRef 42.
dRef 37.
eRef 49.
fCompounds were crystallised from acetone.
gCompounds were crystallised from EtOAc/acetone.
Only a few examples of this type of N-ꢀ asymmetrically
substituted or N-ꢀ monosubstituted derivatives of
nipecotic acid or guvacine are known from literature,
and these examples (10,54 11,34 12,32 and 1355) are listed
in Figure 4. Except for compound 12 (IC50=1.1 mM32)
these compounds show no significant activity (IC50>10
mM) for inhibiting synaptosomal GABA uptake.
method B3). The second strategy was via O-alkylation
of an oxime (Scheme 1; method A1) or an alcohol
(Scheme 2; method A3) with (R)-1-(2-bromoethyl)-3-
piperidinecarboxylic acid ethyl ester.56
The parent cyclic amino acids were protected as their
ester derivatives for this reaction. The separate enantio-
mers of 1 could be prepared by the published procedure
involving resolution with either l-(+)- or with d-(À)-
tartaricacid giving ( R)- or (S)-ethyl nipecotate, respec-
tively.57,58
Results
Two general strategies were used for preparation of the
new compounds presented in Tables 1 and 2. One
strategy was via N-alkylation of the parent cyclic amino
acid 1, with the appropriate halogenide (Schemes 1 and
2; method B1 or B2) or mesylate (Schemes 2 and 3;
The N-alkylated amino acid ester derivatives were
saponified under basic conditions (Schemes 1–3; method
C), to provide the free N-alkylated amino acids featured
in Tables 1 and 2, isolated generally as their crystalline
hydrochloride salts.

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K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
Starting from an oxime two methods were employed to
produce the O-alkylated oxime derivatives 14–28
(Scheme 1). When asymmetric ketones were used the
final oxime derivatives were generally obtained as a
mixture of geometricisomers that were diffiuclt to
separate. O-Alkylation of the appropriate oximes with
(R)-1-(2-bromoethyl)-3-piperidinecarboxylic acid ethyl
ester56 in acetone/K2CO3 at rt furnished the corre-
sponding amino acid ester derivatives directly (method
A1). In this alkylation reaction it is crucial to avoid
heating in order to reduce the quaternisation of (R)-1-
(2-bromoethyl)-3-piperidinecarboxylic acid ethyl ester
or the products by this very reactive bromide. Qua-
ternised by-products could easily be removed by column
chromatography and the purified ethyl esters were then
converted into the acids 14, 19–28 (Table 1) by the
general method C described above.
Alternatively, O-alkylation of the appropriate oximes
with 1,2-dibromoethane in acetone/K2CO3 gave the
corresponding 2-bromoethoxyimino derivatives (method
A2). These bromo derivatives were converted via N-
alkylation of (R)-3-piperidinecarboxylic acid ethyl ester
into the acids 15–18 (Table 1) by the general methods B1
and C described above.
The ether derivatives 32–43 (Table 2) were prepared by
several different routes as outlined in Scheme 2. Direct
N-alkylation of (R)-3-piperidinecarboxylic acid ethyl
ester with 2-phenoxyethylbromide in MIBK/K2CO3
(method B2) furnished the ester derivative 54 which was
then converted into the acid 32 (Table 2) by the general
method C described above. Another direct method is
the O-alkylation of the appropriate phenol or alcohol
with (R)-1-(2-bromoethyl)-3-piperidinecarboxylic acid
ethyl ester in the presence of a strong base like NaH
which produced the corresponding amino acid ester
derivatives, that is 49 (method A3). As described above
for the oxime derivatives heating must be avoided in
order to reduce quaternisation by-products. The amino
acid ester derivatives were then converted into the acids
33, 34, 37 and 43 (Table 2) by the general method C.
Alternative routes to the ether derivatives were via
elongation of the chain (method A4 or A5). In method
A4, the appropriate benzyl- or allylbromide derivative
was elongated by the Lindy method. Addition of n-Buli
at 10 ꢀC to ethylene glycol afforded the ethylene glyco-
late which was O-alkylated with the appropriate ben-
zylbromide. This afforded the corresponding 2-
hydroxyethylether derivatives, that is 50. In method A5,
the appropriate alcohol was O-alkylated with 2-bro-
moethyltetrahydro-2-pyranyl ether in KOH/DMSO
followed by hydrolysis in dilute acid of the THP pro-
tection group to give the corresponding 2-hydroxy-
ethylether derivatives, that is 52. The 2-
hydroxyethylether intermediates, prepared by methods
A4 and A5, were then converted via their mesylates by
N-alkylation of (R)-3-piperidinecarboxylic acid ethyl
ester into the acids 35, 36, 38–42 (Table 2) by the gen-
eral methods B3 and C described above.
The general synthesis of the aniline derivatives 44 and
45 is outlined in Scheme 3. Starting from the appro-
priate substituted N-(2-methyl-1-propyl)aniline59 the
Figure 4. Various known derivatives of nipecotic acid (1) and guva-
cine (2) which does not possess any significant affinity for the GABA
uptake site.
Scheme 1. Synthesis of oxime-ether derivatives 14–28. For definition of X, see Table 1. Method A1: (1) K2CO3, acetone, rt; method A2: K2CO3,
acetone, BrCH2CH2Br, reflux; method B1: K2CO3, acetone, reflux; method C: (1) NaOH, EtOH, rt; (2) HCl.

K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
2779
chain was built in a three step reaction (method D).
Acylation of the aniline with chloroacetic anhydride
produced the 2-chloroacetamide derivatives. Chain
elongation of these chlorides were then accomplished by
O-alkylation of ethylene glycol/DMF in the presence of
KtOBu to give the corresponding 2-(2-hydroxy-
ethoxy)acetamide derivatives. Reduction of these
amides with LiAlH4 furnished the corresponding 2-(2-
hydroxyethoxy)ethylamino derivatives which were then
converted via their mesylates by N-alkylation of (R)-3-
Scheme 2. Synthesis of ether derivatives 32–43. For definition of X, see Table 1. Method A3: NaH, toluene or THF, rt; method A4: n-BuLi, ethyl-
eneglycol, rt. Method A5: DMSO, KOH, 100 ꢀC; method B2: K2CO3, MIBK, reflux; method B3: (1) TEA, Et2O, MsCl, reflux or n-BuLi, THF,
MsCl, reflux; method C: (1) NaOH, EtOH, rt; (2) HCl.
Scheme 3. Synthesis of anilinoalkylethers 44–45. R1 and R2, see Table 1. Method D: (1) (ClCH2CO)2O, H2O, 70 ꢀC; (2) ethyleneglycol, DMF,
KOtBu, rt; (3) LiAlH4, toluene, THF, rt; method B3: (1) TEA, CH2Cl2, MsCl, À10 to 0 ꢀC; (2) Li2CO3, isopropyl acetate, reflux, method C: (1)
NaOH, EtOH, rt; (2) HCl.

2780
K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
piperidinecarboxylic acid ethyl ester into the acids 44
and 45 (Table 2) by the general methods B3 and C
described above.
example 43 compared to example 8. Therefore, some
residue must be left when one of the two aryl groups is
modified. Comparison of example 14 with example 15
and example 20 with examples 21–24 further indicate
that the non-aromaticresidue should represent some
bulkiness.
Discussion
In our continued effort to identify novel GABA uptake
inhibitors with improved potency and selectivity, a
range of such new inhibitors has been prepared.50À53
Some representative examples of these structures are
included in Tables 1 and 2. Their IC50 values for in vitro
inhibition of [3H]-GABA uptake determined essentially
by Fjalland’s method60 are included (mean of two
determinations is given).
Distortion of the attachment point of the two aryl
groups in the known and symmetrical GABA uptake
inhibitors is also allowed to some extent. Comparison of
example 7 with example 16 and example 30 with exam-
ple 41 show that affinity for the GABA uptake site is
not reduced significantly by such a distortion. However,
introduction of this asymmetry results in geometric iso-
mers. Generally, in the oxime series (Table 1) such geo-
metricisomers could not readily be separated, but in the
allyl ether derivatives 41 and 42 this was accomplished
through column chromatography. In these two exam-
ples the most conformationally extended E-isomer 42
showed the lowest potency. This tendency of low or
weak potency of compounds with two possible extended
aryl groups are further illustrated by the examples 19
and 38. As we have reported previously,42 ortho sub-
stitution in one or both aryl groups increase GABA
uptake inhibition. This is further illustrated by the
examples 18 and 45 when compared to the examples 16
and 44, respectively.
In these examples, mainly stericproperties of the lipo-
philicmoiety as well as the chain linking the lipophilic
moiety and the amino acid part of the molecules have
been varied in order to probe the requirements for inhi-
biting GABA transport at the site61À64 involved in the
uptake of GABA from the synapticcleft into neuronal
and glial cell bodies.
In general, inspection of the data presented in Tables 1
and 2 show that two aryl groups, as in the hitherto
known GABA uptake inhibitors (see Figs 2 and 3), are
not crucial in order to obtain high potency as exempli-
fied by compounds 15, 21–25 and 44. However, mod-
ifying one of the two aryl groups in the known GABA
uptake inhibitors is very delicate. Complete removal of
one aryl group seems not to be allowed as shown by
From the data in Table 2, it can be seen that the phe-
nolether examples 32–34 are very poor GABA uptake
inhibitors. This may be due to the short length of the
chain, and even the more flexible example 34 in which
the benzyl group can adapt several more extended
orientations is a poor GABA uptake inhibitor. How-
ever, extending the chain length with one carbon atom
as in the examples 35 and 37 improves potency by a
factor of 20 compared to examples 33 and 34, respec-
tively. The potency of examples 35 and 37 has even been
significantly improved compared to their closest sym-
metrical reference compound 29. Additional extension
of the chain length with one carbon atom as in the
examples 39 and 40 gives a further improvement in
potency, although only 3-fold. This very significant
improvement in potency when the chain is extended can
also be observed from example 29 to example 8.
Table 3. Anticonvulsant properties of selected compounds
No.
DMCM
ED50 (mg/kg)a
Rotarod
ED50 (mg/kg)b
Protective
indexc
4
5
7
8
3.1
1.2
3.2
1.7
10
15.7
1.8
4.1
1.3
5.5
7.4
1.4
7.4
3.0
35
—
5.5
—
9.0
16
80
—
21
6.8
24
>30
5.4
15
17.5
—
60
>60
>150
>60
10
—
4.6
—
5.3
1.6
5.1
—
5.1
5.2
4.4
>4.1
3.9
2.0
5.8
—
31
14
15
16
18
20
22
23
24
25
26
27
28
35
36
37
40
41
42
44
45
In Table 3, the in vivo anticonvulsant effect in mice of
representative compounds is illustrated, expressed as
ED50 values in milligram per kilogram. The convulsion
model used is based on observing the inhibition of clo-
nicseizures induced by a 15 mg/kg intraperitoneal (ip)
dose of the chemoconvulsant methyl 6,7-dimethoxy-4-
ethyl-b-carboline-3-carboxylate (DMCM), an inverse
benzodiazepine agonist. The experimental procedure
has been described previously.27 In Table 3, we have
also listed the ED50 values found in the rotarod perfor-
mance test and calculated the protective index as the
ratio between ED50 values for inhibition of rotarod and
inhibition of DMCM induced convulsions in mice. As
can be seen from Table 3 there is no correlation between
GABA uptake inhibition and in vivo potency which is
probably due to the diversity and difference in lipophi-
licity of the compounds in this investigation. As the in
23
>60
60
2.6
—
>2.5
>1.3
2.0
1.0
4.4
2.2
3.5
1.8
47
4.9
7.9
5.4
38
6.2
1.7
7.9
24
85
22
3.0
aInhibition of DMCM-induced seizures in mice: ED50 (mg/kg) after ip
administration.
bRotarod in mice: ED50 (mg/kg) after ip administration.
cRatio for ED50 values for inhibition of rotarod and inhibition of
DMCM induced convulsions in mice.

K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
2781
vivo potency of these novel compounds in general is
Synaptosomal [3H]-GABA uptake
high these results are encouraging for the selection of an
efficacious compound with good kinetic properties.
Further, several examples show an in vivo potency and
protective index which are comparable with that of
potent reference compounds such as 5 and 8. Even more
examples show a comparable protective index with these
reference compounds, although they are less potent
in vivo.
Uptake of [3H]-GABA into synaptosomal preparations
was assayed by a filtration assay.60 Rat forebrain was
rapidly excised and homogenised in 20 mL of ice cold
0.32 M sucrose with a hand-driven Teflon/glass Potter-
Elvehjem homogeniser. The homogenate was cen-
trifuged for 10 min at 600g at 4 ꢀC. The pellet was re-
suspended in 50 volumes ice-cold buffer (120 mM NaCl,
0.18 mM KCl, 2.30 mM CaCl2, 4.0 mM MgSO4, 12.66
mM Na2HPO4, 2.97 mM NaH2PO4 and 10.0 mM glu-
cose, pH 7.4) at 4 ꢀC. Fifty microlitres of this synapto-
somal suspension (0.1 mg protein), diluted into 300 mL
of phosphate buffer and 100 mL of test substance solu-
tions in water, were pre-incubated for 8 min at 30 ꢀC.
Then 50 mL of [3H]-GABA (final concentration 0.9 nM)
and unlabelled GABA (final concentration 0.9 nM)
were added before continuing incubation for another 8
min. Synaptosomes were then recovered by rapid filtra-
tion through Whatman GF/F glass fibre filters under
vacuum. Filters were washed twice, each time with 10
mL of ice-cold isotonic saline, and the tritium trapped
on the filters was assessed by conventional scintillation
counting in 4 mL of Filter-Count (Packard). Non-car-
rier-mediated uptake was determined in the presence of
nipecotic acid (500 mM) and was subtracted from total
binding to give carrier-mediated [3H]-GABA uptake. The
IC50 value obtained for each example is shown in Table 1.
Conclusion
Previously,42 we have focussed on the selection of com-
pound 5 as an anticonvulsant drug candidate. Follow-
ing this selection we continued the search for a second-
generation compound with a longer duration of action
and broader therapeuticwindow. In this publication, we
have described a new series of GABA uptake inhibitors
in which the presence of two aryl groups, as in the
hitherto known GABA uptake inhibitors, are not cru-
cial in order to obtain high potency. However, modify-
ing one of the two aryl groups in the known GABA
uptake inhibitors is very delicate. Complete removal of
one of the aryl groups seems not to be allowed, and
some bulky residue must be left when one of the two
aryl groups is modified. Distortion of the attachment
point of the two aryl groups in the known and symme-
trical GABA uptake inhibitors is also allowed to some
extent. Ortho substitution in one or both aryl groups
increase GABA uptake inhibition as reported
previously42 for other series of GABA uptake inhibitors.
Antagonismof seizures induced by methyl 6,7-dime-
thoxy-4-ethyl-ꢀ-carboline-3-carboxylate (DMCM) in
mice.27 Female NMRI mice were used. The test drug
was injected ip 30 min prior to the seizure test. In this
test the animals were injected ip with 15 mg/kg of
DMCM and were observed for the next 30 min for the
presence of clonic seizures and death (N=5–10/dose).
The ED50 value obtained for each example is shown in
Table 3.
In this investigation, no correlation between GABA
uptake inhibition and in vivo potency could be found,
which is probably due to the diversity and difference in
lipophilicity of the compounds. However, several
examples, that is 18, 23 and 25, show an in vivo potency
and protective index that are comparable with that of
potent reference compounds such as 5 and 8.
Rotarod test in mice.27 The animals were pretrained in
the rotarod apparatus (Ugo-Basile, Italy) for 2 min
before to testing (speed: 6 rpm). The rod diameter was 3
cm. In the test procedure, the animals were placed on
the rotating rod. If the animal fell from the rod, the
animal was immediately picked up by the tail and again
placed on the rod. Testing was stopped when a total of
10 failures were obtained or 2 min had elapsed.
Experimental
General
Melting points were determined in open capillary tubes
on a Buchi 535 melting point apparatus and are uncor-
rected. The structures of all compounds are consistent
with spectroscopic data and satisfactory elemental ana-
lyses (for C, H, N with a Perkin–Elmer Model 240 ele-
mental analyser) were obtained within Æ0.4% of
Chemistry. Each of the methods A–D are illustrated by
the preparation of the following derivatives. Although
the methods are illustrated for specific compounds, the
methods have been found to be general for the examples
in Tables 1 and 2.
1
theoretical values where given. H NMR spectra were
recorded on a Bruker WM400 spectrometer with TMS
as internal standard, with illustrative chemical shifts
quoted in ppm (d) in the solvents indicated. Compounds
used as starting materials are either known compounds
or compounds which can be prepared by methods
known per se.59,65À73 Column chromatography was car-
ried out using the technique described by W.C. Still et
al.,74 on Merck silica gel 60 (Art 9385) using thick-wal-
led glass columns columns and TLCs on Merck silica
gel 60, 5Â20 cm plates (Art 5714).
(Method A1): (R)-1-(2-(((2-(Benzylidene)cyclohexylide-
ne)amino)oxy)ethyl)-3-piperidinecarboxylic acid ethyl es-
ter (46). A mixture of 2-benzylidenecyclohexanone
oxime69 (3.7 g, 18.4 mmol), (R)-1-(2-bromoethyl)-3-
piperidinecarboxylic acid ethyl ester hydrobromide56
(7.0 g, 20.2 mmol), K2CO3 (10.2 g, 73.5 mmol) and
acetone (150 mL) was stirred at rt for 11 days. The
mixture was filtered and the solvent evaporated. The
residue was purified by column chromatography on

2782
K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
silica gel (200 g, heptane/EtOAc=3:2) to give 2.5 g
(35%) of 46 as an oil. TLC: Rf : 0.27 (SiO2; heptane/
EtOAc=2:3). H NMR (CDCl3, 400 MHz) d 7.20–7.35
(m, 5H, ArH); 6.91 (s, 1H, C¼CH); 4.28 (t, 2H, J=5
Hz, OCH2CH2N); 4.12 (q, 2H, J=7 Hz, CH2CH3);
3.06–3.12 (m, 1H, NCH2CH); 2.80–2.87 (m, 1H,
NCH2CH); 2.75 (t, 2H, J=5 Hz, OCH2CH2N); 2.55–
2.70 (m, 5H, CHCO2Et and ¼CCH2CH2CH2CH2C¼);
2.25–2.33 (m, 1H, NCH2CH2CH2CH); 2.10–2.17 (m,
acidic extract was washed with a small portion of
toluene and the organicphases were disacrded. The
acidic aqueous solution was neutralised with 4 N NaOH
and the mixture was extracted with EtOAc (150 mL).
The organicextract was dried (Na 2SO4) and the solvent
1
1
was evaporated to give 1.7 g (46%) of 49 as an oil. H
NMR (CDCl3, 400 MHz) d 6.80–7.25 (m, 9H, ArH);
4.10 (q, 2H, J=7 Hz, CH2CH3); 4.06 (t, 2H, J=5 Hz,
OCH2CH2N); 3.96 (s, 2H, ArCH2Ar); 3.03–3.08 (m,
1H, NCH2CH); 2.75–2.85 (m, 3H, OCH2CH2N and
NCH2CH); 2.50.60 (m, 1H, CHCO2Et); 2.30–2.40 (m,
1H, NCH2CH2CH2CH); 2.10–2.18 (m, 1H, NCH2CH2-
CH2CH); 1.88.95 (m, 1H, NCH2CH2CH2CH); 1.42–
1.68 (m, 3H, NCH2CH2CH2CH); 1.25 (t, 3H, J=7 Hz,
CH2CH3).
1H,
NCH2CH2CH2CH);
1.90–2.00
(m,
1H,
NCH2CH2CH2CH); 1.45–1.75 (m, 7H, NCH2CH2-
CH2CH and ¼CCH2CH2CH2CH2C¼); 1.25 (t, 3H,
J=7 Hz, CH2CH3).
(Method A2B1): (R)-1-(2-(((1,2-Diphenylethylidene)ami-
no)oxy)ethyl)-3-piperidine-carboxylic acid ethyl ester
(47). A suspension of 1,2-diphenylethanone oxime66,67
(5.0 g, 24 mmol), 1,2-dibromoethane (25 mL) and
K2CO3 (10.0 g, 72 mmol) in acetone (25 mL) was heated
at reflux for 1 h, then stirred at rt for 72 h and finally
heated at reflux for 4.5 h. KI (0.5 g) was added and
refluxing was continued for an additional 16 h. H2O
(100 mL) was added and the resulting mixture was
extracted with CH2Cl2 (2Â100 mL). The combined
organicextracts were washed with H 2O (100 mL), brine
(10 mL) and dried (MgSO4). The solvent was evapo-
rated and the residue was purified by column chroma-
tography on silica gel (60 g, n-heptane/EtOAc=10:1) to
give 3.6 g of 1,2-diphenylethanone O-(2-bromoethy-
(Method A4B3): 2-((3-Benzylbenzyloxy)ethanol (50). A
2.5 M solution of n-BuLi in hexanes (36 mL) was added
drop wise to dry ethylene glycol (120 mL) placed on an
ice-bath under N2. When addition was complete the
mixture was stirred for 0.5 h and 3-benzylbenzyl-
bromide73 was added in one portion and the reaction
mixture was stirred at rt for 72 h. H2O (200 mL) was
added and the resulting mixture was extracted with
Et2O (2Â150 mL). The combined organic extracts were
dried (MgSO4) and the solvent was evaporated to give a
residue which was purified by column chromatography
on silica gel (600 mL, heptane/EtOAc=3:2). This affor-
ded 8.0 g (55%) of 50. TLC: Rf 0.30 (SiO2; heptane/
1
1
l)oxime (48) as an oil. H NMR (CDCl3, 200 MHz) d
EtOAc=1:1). H NMR (CDCl3, 400 MHz) d 7.15–7.30
7.20–7.70 (m, 10H, ArH); 4.45 (t, 2H, J=5 Hz,
OCH2CH2Br); 4.21 (s, 2H, CH2Ar); 3.60 (t, 2H, J=5
Hz, OCH2CH2Br).
(m, 9H, ArH); 4.62 (t, 1H, J=5 Hz, HOCH2CH2O);
4.45 (s, 2H, OCH2Ar); 3.94 (s, 2H, ArCH2Ar); 3.50 (q,
2H, J=5 Hz, HOCH2CH2O); 3.44 (t, 2H, J=5 Hz,
HOCH2CH2O).
A mixture of 48 (3.5 g, 11 mmol), (R)-3-piper-
idinecarboxylic acid ethyl ester (3.1 g, 20 mmol), K2CO3
(4.1 g, 30 mmol), NaI (0.15 g, 1 mmol) and acetone (100
mL) was heated at reflux for 16 h. H2O (100 mL) was
added and the resulting mixture was extracted with
CH2Cl2 (2Â100 mL). The combined organic extracts
were washed with H2O (100 mL), brine (10 mL) and
dried (MgSO4). The solvent was evaporated to give 5.0
g of 47 as an oil. 1H NMR (CDCl3, 200 MHz) d 7.15.65
(m, 10H, ArH); 4.37 (t, 2H, J=5 Hz, OCH2CH2N);
4.13 (s, 2H, CH2Ar); 4.08 (q, 2H, J=7 Hz, CH2CH3);
3.00–3.09 (m, 1H, NCH2CH); 2.82.–9090 (m, 1H,
NCH2CH); 2.75 (t, 2H, J=5 Hz, OCH2CH2N); 2.55–
2.60 (m, 1H, CHCO2Et); 2.23–2.33 (m, 1H,
NCH2CH2CH2CH); 1.85.–215 (m, 2H, NCH2CH2-
CH2CH); 1.35.–175 (m, 3H, NCH2CH2CH2CH); 1.22
(t, 3H, J=7 Hz, CH2CH3).
(R)-1-(2-(3-Benzylbenzyloxy)ethyl)-3-piperidinecarboxylic
acid ethyl ester (51). To a solution of 50 (5.0 g, 21
mmol) in dry Et2O (100 mL), TEA (7.3 mL) was added.
A solution of MsCl (3.5 g, 31 mmol) in dry Et2O (20
mL) was added dropwise at rt. When addition was
complete, the reaction mixture was left overnight. H2O
(50 mL) was added and the mixture was stirred for 10
min. The phases were separated, the organicphase was
dried (MgSO4) and the solvent was evaporated to give a
residue which was dissolved in acetone (150 mL). To
this solution (R)-3-piperidinecarboxylic acid ethyl ester
tartrate (9.5 g, 31 mmol) and K2CO3 (7.3 g, 53 mmol)
were added and the mixture was heated at reflux for 96
h. The mixture was allowed to cool, diluted with ace-
tone (150 mL) and filtered. The solvent was evaporated
to give a residue which was purified by column chro-
matography on silica gel (500 mL, heptane/
EtOAc=1:1). This afforded 4.3 g (54%) of 51 as an oil.
TLC: Rf 0.19 (SiO2; heptane/EtOAc=1:1). 1H NMR
(CDCl3, 400 MHz) d 7.10–7.30 (m, 9H, ArH); 4.42 (s,
2H, OCH2Ar); 4.03 (q, 2H, J=7 Hz, CH2CH3); 3.94 (s,
2H, ArCH2Ar); 3.48 (t, 2H, J=5 Hz, OCH2CH2N);
2.82–2.90 (m, 1H, NCH2CH); 2.60–2.68 (m, 1H,
NCH2CH); 2.45–2.55 (m, 3H, CHCO2Et and
OCH2CH2N); 2.15–2.20 (m, 1H, NCH2CH2CH2CH);
1.98.–207 (m, 1H, NCH2CH2CH2CH); 1.72–1.80 (m, 1H,
NCH2CH2CH2CH); 1.30–1.60 (m, 3H, NCH2CH2-
CH2CH); 1.15 (t, 3H, J=7 Hz, CH2CH3).
(Method A3): (R)-1-(2-(2-Benzylphenoxy)ethyl)-3-piperi-
dinecarboxylic acid ethyl ester (49). NaH (0.8 g, 20
mmol, 60% oil dispersion) was added portionwise to a
stirred solution of 2-benzylphenol (1.8 g, 10 mmol) in
toluene (30 mL) placed under N2. The mixture was
stirred for 0.5 h and (R)-1-(2-bromoethyl)-3-piper-
idinecarboxylic acid ethyl ester hydrobromide (3.5 g, 10
mmol) was added portionwise. The reaction mixture
was stirred for 2 h at rt and H2O (50 mL) was added.
The phases were separated and the organicphase was
extracted with a 10% citric acid solution (200 mL). This

K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
2783
(Method A5B3): 2-(2-(2-Benzylphenyl)ethoxy)ethanol
(52). To a solution of 2-benzylphenylacetic acid72 (5.5
g, 24 mmol) in THF (100 mL) a solution of LiAlH4 in
THF (24 mL, 24 mmol, 1 M) was added dropwise on an
ice-bath. When addition was complete the mixture was
stirred for 1 h at rt. H2O (1 mL), 4 N NaOH (2 mL) and
H2O (4 mL) were added successively. The mixture was
filtered and the filtrate diluted with Et2O (250 mL). The
phases were separated and the organicphase was
washed with saturated NH4Cl (3Â100 mL) and dried
(MgSO4). The solvent was evaporated to give 4.8 g of
crude 2-(2-benzylphenyl)ethanol as an oil. To a mixture
of this alcohol (4.8 g, 23 mmol), 2-bromoethyltetrahy-
dro-2-pyranyl ether (9.6 g, 46 mmol) and DMSO (100
mL), KOH (2.6 g, 46 mmol) was added. The reaction
mixture was stirred at rt for 20 h and then heated at
100 ꢀC for 3 h. The reaction mixture was allowed to
cool, poured into ice-water (200 mL) and extracted with
Et2O (2Â150 mL). The combined organic extracts were
dried (MgSO4) and the solvent was evaporated to give
4.6 g of an oily residue. This oil was purified by column
chromatography on silica gel (3Â40 cm, heptane/
heated at reflux overnight. The reaction mixture was
allowed to cool and then filtered. The solvent was
evaporated and the residue was purified by column
chromatography on silica gel (800 g, heptane/
EtOAc=4:1) to give 17.1 g (62%) of 54 as an oil. TLC:
1
Rf 0.20 (SiO2; heptane/EtOAc=3:2). H NMR (CDCl3,
400 MHz) d 7.27 (t, 2H, J=8 Hz, m-ArH); 6.95 (t, 1H,
J=8 Hz, p-ArH); 6.90 (d, 2H, J=8 Hz, o-ArH); 4.14 (q,
2H, J=7 Hz, CH2CH3); 4.08 (t, 2H, J=7 Hz,
OCH2CH2N); 3.05–3.12 (m, 1H, NCH2CH); 2.80–2.90
(m, 3H, OCH2CH2N and NCH2CH); 2.55–2.63 (m, 1H,
CHCO2Et); 2.28–2.37 (m, 1H, NCH2CH2CH2CH);
2.11–2.20 (m, 1H, NCH2CH2CH2CH); 1.90–2.00 (m,
1H, NCH2CH2CH2CH); 1.45–1.75 (m, 3H, NCH2CH2-
CH2CH); 1.25 (t, 3H, J=7 Hz, CH2CH3).
(Method C): Hydrolysis of 3-piperidinecarboxylic acid
ester derivatives. General method. The ester under con-
sideration (1.0 mmol) was dissolved in ethanol (3 mL)
and 3 mmol of 4 or 12 N NaOH was added. The reac-
tion mixture was stirred at rt until TLC indicated com-
plete reaction (3–6 h). A concentrated aqueous HCl
solution was added with cooling on an ice-bath until pH
1. Then CH2Cl2 (100 mL) was added and the resulting
emulsion was dried (Na2SO4). The solvent was evapo-
rated to give a residue which was crystallized. Recrys-
tallization afforded the pure acid hydrochlorides for
which the data are shown in Tables 1 and 2.
1
EtOAc=1:4). This afforded 1.3 g of 52 as an oil. H
NMR (CDCl3, 400 MHz) d 7.10–7.30 (m, 9H, ArH);
4.05 (s, 2H, ArCH2Ar); 3.66 (q, 2H, J=5 Hz, HOCH2-
CH2O); 3.53 (t, 2H, J=5 Hz, OCH2CH2Ar); 3.45 (t,
2H, J=5 Hz, HOCH2CH2O); 2.90 (t, 2H, J=5 Hz,
OCH2CH2Ar); 1.92 (t, 1H, J=5 Hz, HOCH2CH2O).
(R)-1-(2-(2-(2-Benzylphenyl)ethoxy)ethyl)-3-piperidine-
carboxylic acid ethyl ester (53). TEA (10 mL) was
added to a solution of 52 (1.3 g, 6.1 mmol) in CH2Cl2
(50 mL) and then MsCl (0.77 g, 6.7 mmol) was added
dropwise at rt. When addition was complete the reac-
tion mixture was diluted with CH2Cl2 (100 mL), washed
with H2O (3Â50 mL) and dried (MgSO4). The solvent
was evaporated to give a residue which was dissolved in
acetone (100 mL). To this solution (R)-3-piper-
idinecarboxylic acid ethyl ester tartrate (2.8 g, 9.5
mmol) and K2CO3 (2.1 g, 15.3 mmol) were added and
the mixture was heated at reflux for 72 h. An additional
portion of (R)-3-piperidinecarboxylic acid ethyl ester
tartrate (1.4 g, 4.8 mmol) was added and heating was
continued for another 24 h. The mixture was allowed to
cool and then filtered. The solvent was evaporated to
give a residue which was purified by column chromato-
graphy on silica gel (3Â30 cm, heptane/
EtOAc=1:1!3:7). This afforded 0.9 g of 53 as an oil.
1H NMR (CDCl3, 400 MHz) d 7.10–7.30 (m, 9H, ArH);
4.12 (q, 2H, J=7 Hz, CH2CH3); 4.05 (s, 2H, ArCH2Ar);
3.50 (2Ât, 4H, J=5 Hz, CH2OCH2); 2.98–3.05 (m, 1H,
NCH2CH); 2.88 (t, 2H, J=5 Hz, OCH2CH2Ar); 2.74–
2.82 (m, 1H, NCH2CH); 2.50–2.60 (m, 3H, CHCO2Et
and OCH2CH2N); 2.12–2.20 (m, 1H, NCH2CH2-
CH2CH); 1.96–2.04 (m, 1H, NCH2CH2CH2CH); 1.92
(m, 1H, NCH2CH2CH2CH); 1.35–1.70 (m, 3H,
NCH2CH2CH2CH); 1.25 (t, 3H, J=7 Hz, CH2CH3).
(Method
DB3):
2-Chloro-N-(2-methyl-1-propyl)-N-
phenylacetamide (55). A mixture of N-(2-methyl-1-pro-
pyl)aniline59 (14.9 g, 100 mmol), chloroacetic anhydride
(19.0 g, 111 mmol) and H2O (100 mL) was heated at
70 ꢀC for 1 h. The reaction mixture was poured into ice-
water (300 mL) and concentrated HCl was added until
pH 2. The precipitate was collected and dried to give
11.3 g (50%) of 55. TLC: Rf 0.37 (SiO2; heptane/
EtOAc=4:1). H NMR (CDCl3, 200 MHz) d 7.20–7.50
(m, 5H, ArH); 3.80 (s, 2H, CH2Cl); 3.60 (d, 2H, NCH2);
1.78 (hept., 1H, CHMe2); 0.93 (d, 6H, 2ÂCH3).
1
2-(2-Hydroxyethoxy)-N-(2-methyl-1-propyl)-N-phenyl-
acetamide (56). To a solution of dry ethylene glycol
(12.4 g, 200 mmol) in DMF (50 mL) was carefully
added KOtBu (16.8 g, 150 mmol) while the temperature
was kept below 70 ꢀC. The resulting mixture was stirred
at rt for 0.5 h, then a solution of 55 (11.3 g, 50 mmol) in
DMF (5 mL) was added in one portion and the mixture
was stirred at rt for 1.5 h. The reaction mixture was
poured into ice-water (400 mL) and extracted with
CH2Cl2 (3Â50 mL). The combined organic extracts
were dried (MgSO4) and the solvent was evaporated to
give 12.5 g (99%) of 56 as an oil. TLC: Rf 0.47 (SiO2;
1
EtOAc). H NMR (CDCl3, 200 MHz) d 7.15–7.45 (m,
5H, ArH); 3.85 (s, 2H, OCH2C¼O); 3.50–3.70 (m, 6H,
OCH2CH2OH and NCH2); 1.75 (hept., 1H, CHMe2);
0.91 (d, 6H, 2ÂCH3).
(Method B2): (R)-1-(2-Phenoxyethyl)-3-piperidinecar-
boxylic acid ethyl ester (54). A mixture of 2-phenox-
2-(2-(N-(2-Methyl-1-propyl)-N-phenylamino)ethoxy)ethanol
(57). To a suspension of LiAlH4 (2.8 g, 75 mmol) in dry
toluene (16 mL), dry THF (20 mL) was added dropwise.
A solution of 56 (12.5 g, 49 mmol) in dry toluene (5 mL)
was added dropwise while the temperature was kept
yethylbromide
(20
g,
100
mmol),
(R)-3-
piperidinecarboxylic acid ethyl ester (34 g, 112 mmol),
K2CO3 (41 g, 300 mmol) and MIBK (200 mL) was

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K. E. Andersen et al. / Bioorg. Med. Chem. 9 (2001) 2773–2785
below 75 ꢀC. When addition was complete, the mixture
was stirred at rt for 1 h. H2O (10 mL) was carefully
added and when the initial exotherm had subsided
K2CO3 (10 g) was added in one portion. The mixture
was stirred for 15 min and then filtered. The filterpad
was washed with CH2Cl2 (2Â100 mL) and the phases
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were separated. The organicphase was dried (MgSO )
4
and the solvent evaporated to give 10.6 g (91%) of 57 as
an oil. TLC: Rf 0.75 (SiO2; EtOAc).
(R)-1-(2-(2-(N-(2-Methyl-1-propyl)-N-phenylamino)ethoxy)-
ethyl)-3-piperidinecarboxylic acid ethyl ester (58). A
mixture of 57 (10.6 g, 45 mmol), TEA (7.3 mL, 53
mmol) and CH2Cl2 (100 mL) was cooled to À10 ꢀC and
MsCl (3.3 mL, 42 mmol) was added dropwise while the
temperature was kept below À5 ꢀC. When addition was
complete the mixture was stirred at 0 ꢀC for 1 h and
then at rt for 0.5 h. The reaction mixture was washed
with 0.1 N HCl (2Â50 mL), H2O (50 mL) and brine (10
mL). The organicphase was dried (MgSO 4) and the
solvent was evaporated. A mixture of the residue, (R)-3-
piperidinecarboxylic acid ethyl ester (10.0 g, 64 mmol),
Li2CO3 (3.1 g, 43 mmol) and isopropyl acetate (100 mL)
was stirred at rt for 16 h and then heated at reflux for 24
h. The reaction mixture was filtered and the filtrate was
concentrated. The residue was dissolved in CH2Cl2 (100
mL), dried (MgSO4) and the solvent evaporated. The
residue was purified by column chromatography on
silica gel (100 g, n-heptane/EtOAc=4:1) to give 8.0 g
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1
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