Synthesis and pharmacology of the baclofen homologues 5-amino-4-(4- chlorophenyl)pentanoic acid and the R- and S-enantiomers of 5-amino-3-(4- chlorophenyl)pentanoic acid

Article abstract of DOI:10.1021/jm990076+

(RS)-5-Amino-4-(4-chlorophenyl)pentanoic acid (10) and the R-form (11) and S-form (12) of (RS)-5-amino-3-(4-chlorophenyl)pentanoic acid, which are homologues of the 4-aminobutanoic acid(B) (GABA(B)) receptor agonist (RS)-4- amino-3-(4-chlorophenyl)butanoi

Full text of DOI:10.1021/jm990076+

J . Med. Chem. 1999, 42, 2053-2059
2053
Syn th esis a n d P h a r m a cology of th e Ba clofen Hom ologu es
5-Am in o-4-(4-ch lor op h en yl)p en ta n oic Acid a n d th e R- a n d S-En a n tiom er s of
5-Am in o-3-(4-ch lor op h en yl)p en ta n oic Acid
Rolf Karla,† Bjarke Ebert,‡ Christian Thorkildsen,‡ Claus Herdeis,† Tommy N. J ohansen,§ Birgitte Nielsen,§ and
Povl Krogsgaard-Larsen*,§
NeuroScience PharmaBiotec Research Centre, Departments of Pharmacology and Medicinal Chemistry,
The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark, and
Institut fu¨r Pharmazie und Lebensmittelchemie, Universita¨t Wu¨rzburg, Wu¨rzburg, Germany
Received February 16, 1999
(RS)-5-Amino-4-(4-chlorophenyl)pentanoic acid (10) and the R-form (11) and S-form (12) of
(RS)-5-amino-3-(4-chlorophenyl)pentanoic acid, which are homologues of the 4-aminobutanoic
acidB (GABAB) receptor agonist (RS)-4-amino-3-(4-chlorophenyl)butanoic acid (baclofen), were
synthesized. Compound 10 was synthesized by homologation at the carboxyl end of baclofen
using a seven-step reaction sequence. N-Boc-protected (4R,5R)-4-(4-chlorophenyl)-5-hydroxy-
2-piperidone (18) was deoxygenated via a modified Barton-McCombie reaction to give N-Boc-
protected (R)-4-(4-chlorophenyl)-2-piperidone (20), which was ring opened and deprotected to
give 11‚HCl. The corresponding S-enantiomer, 12‚HCl, was synthesized analogously from the
4S,5S-enantiomer of 18, compound 21. The enantiomeric purities of 11‚HCl (ee ) 99.8%) and
12‚HCl (ee ) 99.3%) were determined by chiral HPLC. Compound 10 did not show detectable
affinity for GABAA or GABAB receptor sites and was inactive as an agonist or an antagonist at
GABAB receptors in the guinea pig ileum. Like the enantiomers of baclofen, neither 11 nor 12
showed detectable affinity for GABAA receptor sites, and in agreement with the findings for
(S)-baclofen, 12 did not interact significantly with GABAB receptor sites. Compound 11 (IC50
) 7.4 ( 0.6 µM), a homologue of (R)-baclofen (2), was shown to be some 50 times weaker than
2 (IC50 ) 0.14 ( 0.01 µM) as an inhibitor of GABAB binding. Accordingly, 11 (EC50 ) 150 ( 23
µM) was shown to be weaker than 2 (EC50 ) 11 ( 1 µM) as an inhibitor of electrically induced
contractions of the guinea pig ileum. However, whereas this effect of 2 was sensitive to the
GABAB antagonist, CGP35348 (4), the inhibition by 11 was not significantly affected.
Furthermore, 12 (EC50 ) 310 ( 16 µM) was shown to be one-half as potent as 11 in this test
system, and this effect of 12 also was insensitive to 4. The dissimilarities of the pharmacological
effects of 2 and compounds 11 and 12 were emphasized by the observation that whereas 2
only inhibits the ileum contraction by 59 ( 5%, 11 as well as 12 were shown to inhibit this
response by approximately 94%. Neither 11 nor 12 appeared to affect significantly cholinergic
mechanisms in the ileum, and their mechanism(s) of action remain enigmatic.
In tr od u ction
methyl)phosphinic acid (CGP35348, 4) (Figure 1), is a
moderately potent GABAB antagonist capable of pen-
etrating the blood-brain barrier.2,9,10 The homologous
analogue of baclofen, (RS)-5-amino-2-(4-chlorophenyl)-
pentanoic acid (5), does not interact detectably with
GABAB receptors.11
4-Aminobutanoic acid (GABA, 1) is the major inhibi-
tory neurotransmitter in the central nervous system
(CNS), and GABA operates through ionotropic (GABAA
and GABAC) as well as G protein-coupled (GABAB)
receptors.1,2 There is a pharmacological and therapeutic
interest in agonists, partial agonists, and antagonists
for these receptors and in compounds interacting with
modulatory sites associated with GABA receptors.1-5
(R)-4-Amino-3-(4-chlorophenyl)butanoic acid [(R)-ba-
clofen, 2], which interacts stereospecifically with GABAB
receptors, is the classical GABAB agonist.6 Whereas the
phosphono amino acid analogue, (R)-phaclofen (3), is a
weak competitive GABAB antagonist,7,8 the phosphinic
acid analogue of GABA, P-(3-aminopropyl)-P-(diethoxy-
Partial GABA receptor agonists of appropriate rela-
tive efficacies may have particular therapeutic interest
as drugs adapted to regulation of GABA receptor dys-
functions,5 and we have previously described a number
of partial GABAA receptor agonists, some of which are
homologues of GABA.12,13 Whereas (R)-4-amino-3-hy-
droxybutanoic acid [(R)-3-OH-GABA, 6] is a selective
GABAB agonist,14 the GABA homologue, 5-aminopen-
tanoic acid (DAVA), is a nonselective GABAB antago-
nist.15 Using these two amino acids as leads, a number
of structural hybrids were synthesized and pharmaco-
logically characterized, including (RS)-5-amino-3-hydroxy-
pentanoic acid (3-OH-DAVA, 7), (S)-5-amino-2-hydroxy-
pentanoic acid [(S)-2-OH-DAVA, 8], and (R)-5-amino-
4-hydroxypentanoic acid [(R)-4-OH-DAVA, 9]14,16 (Fig-
ure 1). Whereas 7 did not significantly interact with
* Address correspondence to Prof. Povl Krogsgaard-Larsen. Phone:
(+45) 35370850, ext. 511. Fax: (+45) 35372209. E-mail: nordly@
medchem.dfh.dk.
† Universita¨t Wu¨rzburg.
‡ Department of Pharmacology, The Royal Danish School of Phar-
macy.
§ Department of Medicinal Chemistry, The Royal Danish School of
Pharmacy.
10.1021/jm990076+ CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/15/1999

2054 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Brief Articles
Sch em e 1a
a
Reagents: (i) MeOH, HCl; (ii) (Boc)2O, Et3N; (iii) LiBEt3H,
THF; (iv) (C6H5)3P, CCl4; (v) NaCN, DMF; (vi) MeOH, HCl, NaOH;
(vii) 6 M HCl.
Sch em e 2a
F igu r e 1. Structures of GABA (1), (R)-baclofen (2), and a
number of analogues of GABA, baclofen, and 5-aminopentanoic
acid (DAVA).
GABAB receptors, 8 showed GABAB antagonism and 9
low-efficacy partial agonism at GABAB receptors.14
In continuation of this project, the synthesis and
pharmacological characterization of (RS)-5-amino-4-(4-
chlorophenyl)pentanoic acid (10) and the R-form (11)
and the S-form (12) of 5-amino-3-(4-chlorophenyl)-
pentanoic acid, which are structural hybrids of baclofen
and DAVA, are described (Figure 1).
Resu lts
Ch em istr y a n d Ster eoch em istr y. Compound 10‚
HCl was synthesized as outlined in Scheme 1. The
N-Boc-protected methyl ester 14 of (RS)-baclofen (13)
was converted into the primary alcohol 15 using lithium
triethylborohydride as the reducing agent, and in two
steps, 15 was further transformed into the correspond-
ing nitrile 16. Methanolysis of the cyano group of 16
and subsequent cyclization of the N-deprotected methyl
ester formed gave (RS)-5-(4-chlorophenyl)-2-piperidone
(17), which has been synthesized previously using a
different procedure.17 Prolonged treatment of 17 with
6 M hydrochloric acid under reflux gave the target
compound 10‚HCl.
The syntheses of 11 and 12 are outlined in Scheme
2. Compound 11 was synthesized in a four-step reaction
sequence, using (4R,5R)-4-(4-chlorophenyl)-5-hydroxy-
2-piperidone (18)18 as the starting material. Removal
of the hydroxy group of 18, which is the key step of the
sequence, was unsuccessfully attempted using a variety
of reductive substitution reactions, based on zinc as the
reducing agent,19,20 or using platinum-catalyzed hydro-
genation conditions.21 Subsequently, deoxygenation at
a
Reagents: (i) phenyl chlorothionoformate, CH2Cl2, DMAP; (ii)
SnBu3H, acetone, di-tert-butyl peroxyoxalate; (iii) 1 M LiOH, THF,
0.5 M HCl; (iv) 6 M HCl.
C-5 of 18 was attempted using Barton-McCombie
reaction conditions. Compound 18 was successfully

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 2055
Ta ble 1. Receptor Binding and Pharmacological Data (Values ( SEM, n g 5)
receptor binding IC50 (µM)
guinea pig ileum pharmacology
EC50 (µM) sensitivity to CGP35348
compound
GABAA [3H]muscimol
GABAB [3H]GABA
(R)-baclofen (2)
(S)-baclofen
10
11
12
>100
>100
>100
>100
>100
0.14 ( 0.1
>100
11 ( 1
yes
nda
3300 ( 1300
>100
>3000
7.4 ( 0.6
>100
150 ( 23
310 ( 16
no
no
a
nd, not determined.
converted into the thioxocarbonate 19, but treatment
of this intermediate with tributyltin hydride in the
presence of AIBN, using previously described condi-
tions,22 led to extensive decomposition of starting mate-
rial. Finally, the deoxygenation reaction was completed
by treatment of 19 with tributyltin hydride in the
presence of small amounts of di-tert-butyl peroxyox-
alate23 to give 20 in 94% yield. Ring opening of 20 was
accomplished using lithium hydroxide as a base, and
treatment of the Boc-protected amino acid formed with
hydrochloric acid gave the target compound 11‚HCl. The
enantiomer 12‚HCl was synthesized using the 4S,5S-
enantiomer of 18, namely compound 21,18 as the start-
ing material and the reaction conditions described for
the synthesis of 11‚HCl (Scheme 2).
The enantiomeric purities of 11 and 12 were deter-
mined by chiral HPLC using a Crownpak CR(-) column.
The enantiomeric excess (ee) of the more slowly eluting
enantiomer, compound 12, was determined to be 99.8%.
Under the analysis conditions used, compound 11 did
not contain detectable amounts of 12, and based on the
analysis of a spiked sample of 11, ee was found to be
>99.3%.
Compound 10 was devoid of affinity for GABAB receptor
sites and was inactive in this functional assay.
The sensitivity of the inhibitory effects of (RS)-
baclofen, 11, and 12 to the GABAB receptor antagonist
CGP3534810 was studied, and the results are illustrated
in Figure 2C,D. Whereas the addition of this antagonist
caused a parallel shift of the dose-response curve for
(RS)-baclofen corresponding to a Ki value of approxi-
mately 10 µM, the effects of 11 (Figure 2D) or 12 (not
illustrated) were not significantly affected by the pres-
ence of CGP35348.
Prompted by these latter, quite surprising, observa-
tions, the effects of 11 and 12 on acetylcholine-mediated
contractions of the guinea pig ileum muscle preparation
were studied. At a concentration of 3 µM, 11 was shown
to inhibit the acetylcholine-mediated muscle contraction
as shown by a shift of the dose-response curve to acet-
ylcholine to the right by a factor of 2 (not illustrated).
This inhibitory effect of 11 was, however, concentration-
independent, and 12 as well as (RS)-baclofen turned out
to be inactive at concentrations up to 100 µM.
Discu ssion
Recep tor Bin d in g. The affinities of 10-12 for
GABAA receptor sites were measured using [3H]musci-
mol as radioligand,24 whereas GABAB receptor affinity
was studied as inhibition of baclofen-sensitive and
isoguvacine-insensitive [3H]GABA binding.14 None of
the compounds studied showed detectable affinity for
GABAA receptor sites (Table 1). Like (S)-baclofen, the
S-enantiomer, 12, did not significantly affect the binding
of [3H]GABA to GABAB receptor sites. The R-enanti-
omer, 11, on the other hand, was shown to be an
inhibitor of GABAB receptor binding, though some 50
times weaker than 2. Compound 10 was inactive in the
GABAB receptor binding assay.
We have previously described the design of partial
GABAB agonists and GABAB antagonists14,25,26 as struc-
tural hybrids between the GABAB agonist (R)-3-OH-
GABA (6)14,27 and the GABAB antagonist DAVA.15
Within this series of hydroxylated GABA homologues
(Figure 1), 3-OH-DAVA (7) turned out to be inactive,
whereas (S)-2-OH-DAVA (8) and (R)-4-OH-DAVA (9)
showed GABAB antagonist and low-efficacy partial
GABAB agonist effects, respectively.14 On the basis of
computer modeling studies, low-energy conformations
of 6, 8, and 9 could be superimposed in a flexible four-
point fit involving all oxygen and nitrogen atoms.14
Although this approach had provided GABAB receptor
ligands showing the desired pharmacological profiles,
the key compounds 8 and 9 were not sufficiently potent
to be useful as pharmacological tools or potential drugs.
This conclusion provoked an analogous approach involv-
ing the use of baclofen as a lead. Although both 2 and
6 have the R-configuration, the stereochemical orienta-
tion of the 4-chlorophenyl group of 2 is “opposite” to that
of the hydroxy group of 6 (Figure 1) suggesting that
these groups interact with different structural element
of the GABAB receptor recognition site.25 Furthermore,
2 is somewhat more potent than 6 as a GABAB agonist,
and these aspects prompted us to synthesize structural
hybrids between baclofen and DAVA as potential new
GABAB receptor ligands. Soon after the start of this
project, the synthesis of (RS)-5-amino-2-(4-chlorophen-
yl)pentanoic acid (5) and its inactivity as a GABAB
receptor ligand were reported,11 and we decided to
synthesize the isomeric C-4-substituted DAVA analogue
Gu in ea P ig Ileu m P h a r m a cology. The isolated
longitudinal guinea pig ileum muscle, which previously
has been used to characterize GABAB ligands,7 was used
as a functional test system for comparative studies of
(RS)-baclofen, baclofen enantiomers, compound 10, and
11 and 12. Following published procedures,7,14 the
compounds were characterized as inhibitors of electri-
cally evoked muscle contractions. As shown in Figure
2A,B, 11 and 12 were weak inhibitors of muscle con-
tractions showing EC50 values of 150 ( 23 and 310 (
16 µM, respectively (Table 1). The maximal percentage
inhibitions by 11 or 12 were, however, significantly
higher (more than 94%) than those produced by (R)-
baclofen (2) or (RS)-baclofen (59 ( 5%; t-test: p > 99.5%)
(Figure 2B). In this functional test system, 2 (EC50
)
11 ( 1 µM) was typically some 15 times more potent
than 11, whereas (S)-baclofen was essentially inactive
(EC50 ) 3300 ( 1300 µM) (Figure 2A and Table 1).

2056 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Brief Articles
F igu r e 2. A: Dose-dependent inhibition of contractions of the longitudinal guinea pig ileum. Percent inhibition is expressed
relative to maximum inhibition observed for each of the four compounds tested. Data were fitted to the following equation: %
inhibition ) 100 × (1 - [Inh]n/([Inh]n + EC50n)), where [Inh] is the concentration of inhibitor and n is the Hill slope of the curve.
EC50 values are shown in Table 1. B: Inhibition, expressed relative to the maximum muscle contraction obtainable, for each of
the three compounds tested. Data were fitted to the following equation: % inhibition ) max × (1 - [Inh]n/([Inh]n + EC50n)) +
baseline, where max is the maximum muscle contraction, [Inh] is the concentration of inhibitor, n is the Hill slope of the curve,
and baseline is the muscle contraction insensitive to the test compound. C: Effects of the competitive GABAB antagonist CGP353489
on the inhibition of muscle contraction by (RS)-baclofen, the inhibition being expressed relative to the maximum inhibition obtained.
Data were fitted to the following equation: % inhibition ) 100 × (1 - [Inh]n/([Inh]n + EC50n)), where [Inh] is the concentration
of inhibitor and n is the Hill slope of the curve. The dose ratio, calculated as the ratio between the concentration of agonist in the
presence or absence of 100 µM CGP35348, was transformed into a Ki value by the following equation: log(dose ratio - 1) ) -log
Ki - log [antagonist]. D: Effects of the competitive GABAB antagonist CGP35348 on the inhibition of muscle contraction by 11,
as described above for (RS)-baclofen (C). For all of the dose-response data illustrated in panels A-D, values are mean values (
SEM of at least five experiments.
10 and the R-form (11) and S-form (12) of the analogue
substituted at C-3 of the DAVA backbone (Figure 1).
On the basis of receptor binding studies, the structure-
activity relationship (SAR) of 5 and 10-12 is comple-
mentary to that of the hydroxylated analogues of DAVA,
7-9. Thus, whereas 3-OH-DAVA (7) is the only member
of the latter series of compounds that is devoid of
GABAB receptor affinity,14,25 only a C-3-substituted
analogue, compound 11, in the former series of com-
pounds shows significant GABAB receptor affinity (Table
1). Notably, this receptor affinity, like that of (R)-
baclofen (2), resides in the R-enantiomer, 11, which also
shows agonist properties.
Thus, compounds 11 and 12 show atypical “GABAB-
like” pharmacological effects. These homologues of ba-
clofen appear to interact with GABAB receptors in a
manner different from that of (R)-baclofen (2), or
alternatively, 11 and 12 may show a GABAB receptor
subtype selectivity different from that of 2. At present,
we are not in a position to study the effects of these
compounds on cloned subtypes of GABAB receptors, but
such future studies may shed light on the mechanism-
(s) of action of 11 and 12 and may indicate new
directions of GABAB receptor pharmacology.
Exp er im en ta l Section
Pharmacological data, obtained using the guinea pig
ileum GABAB receptor functional assay, are, however,
not fully consistent with the SAR based on receptor
binding data (Table 1). Thus, although 12 is devoid of
GABAB receptor affinity, 12 turned out to be ap-
proximately one-half as potent as 11 in this ileum assay.
Furthermore, both 11 and 12 showed markedly higher
efficacy than baclofen in this assay, but in contrast to
the effects of baclofen, those of 11 and 12 were not
significantly attenuated by the standard GABAB an-
tagonist, CGP35348 (4) (Figure 2).
Ch em istr y. Gen er a l P r oced u r es. Column chromatogra-
phy (CC) was performed on Merck silica gel 60. Thin-layer
chromatography (TLC) was performed on Merck precoated
silica gel 60 F254 plates. Compounds were visualized on TLC
plates using UV light (254 nm) and/or by iodine vapor. Melting
points were determined on a Bu¨chi 510 apparatus and are
uncorrected. Evaporations were performed in a vacuum on a
rotary evaporator at approximately 15 mmHg. IR spectra were
recorded on a Perkin-Elmer spectrophotometer 681. NMR
spectra were measured on a Bruker AC 200 instrument (200
MHz for 1H NMR and 50 MHz for 13C NMR). TMS or
1,4-dioxane (δ 3.70) was used as internal standard for spectra

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 2057
evaporated and the residue subjected to CC (light petroleum-
recorded in organic solvents or D2O, respectively. Electron
impact mass spectra (MS) were determined by the Mass
Spectrometry Laboratory of the Department of Organic Chem-
istry at the University of Wu¨rzburg on a Finnigan MAT 8200
instrument at 70 eV. Optical rotations were determined with
a Perkin-Elmer polarimeter 241 at 589 nm. Elemental analy-
ses were carried out at the Microanalytical Laboratory at the
Department of Inorganic Chemistry at the University of
Wu¨rzburg.
Meth yl (RS)-4-[N-(ter t-Bu tyloxyca r bon yl)a m in o]-3-(4-
ch lor op h en yl)bu ta n oa te (14). A suspension of (RS)-baclofen
(13) (3.44 g, 16.2 mmol) in MeOH (50 mL) was saturated with
HClg to give a clear solution. The addition of HCl was
continued for a further 10 min, and then the solution was
evaporated to give methyl (RS)-4-amino-3-(4-chlorophenyl)-
butanoate‚HCl (3.82 g, 89%) as colorless crystals: mp 182-
184 °C; 1H NMR (DMSO-d6) δ 8.23 (3H, s), 7.50-7.40 (4H,
m), 3.56-3.40 (4H, m), 3.07-2.90 (3H, m), 2.90-2.63 (1H, dd,
J ) 9.5 and 16.4 Hz); 13C NMR (CDCl3) δ 171.4, 139.2, 132.2,
129.9, 128.7, 51.5, 43.2, 39.3, 37.5. Anal. (C11H14ClNO2‚HCl)
H, N; C: calcd, 50.02; found, 49.43.
1
EtOAc) to give 16 (0.91 g, 79%): mp 71.0-72.0 °C; H NMR
(CDCl3) δ 7.27-7.03 (4H, m), 4.58 (1H, s), 3.49-2.73 (3H, m),
2.31-1.56 (4H, m), 1.33 (9H, s); 13C NMR (CDCl3) δ 155.7,
138.5, 133.0, 129.1, 129.0, 118.9, 79.3, 45.4, 44.6, 28.5, 28.1,
15.0. Anal. (C16H21ClN2O2) C, H, N.
(RS)-5-(4-Ch lor op h en yl)-2-p ip er id on e (17). A solution
of 16 (0.60 g, 1.94 mmol) in MeOH (50 mL) was saturated with
HClg, and with continuous addition of HClg, this solution was
refluxed for 2 h. The solvent was evaporated and the crystal-
line residue taken up in 2 M NaOH (50 mL). This mixture
was extracted with CH2Cl2 (3 × 50 mL). The combined organic
phases were washed with brine, dried (Na2SO4), and evapo-
rated. The crystalline residue was subjected to CC (CH2Cl2-
MeOH) to give 17 (0.35 g, 86%): mp 174 °C (lit. mp 171.5-
172.5 °C17); 1H NMR (CDCl3) δ 7.34-7.27 (4H, m), 3.59-3.33
(1H, m), 3.34 (1H, dd, J ) 10.5 Hz), 3.15-2.92 (1H, ddd, J )
5.0 and 10.3 Hz), 2.63-2.38 (2H, m), 2.16-1.98 (2H, m); 13C
NMR (CDCl3) δ 172.1, 140.1, 132.6, 128.7, 128.2, 48.1, 38.7,
30.9, 27.5. Anal. (C11H12ClNO) H, N; C: calcd, 63.01; found,
62.42.
Without further purification, this compound (2.19 g, 8.8
mmol) was dissolved in EtOH (100 mL), and to this solution
was added at 0 °C a solution of Et3N (3.61 mL, 26.3 mmol)
and di-tert-butyl dicarbonate (4.70 g, 21.7 mmol) in C2H5OH
(20 mL). This solution was left at 0 °C for 1.5 h and then at
25 °C for 30 min, and after evaporation of most of the content
of EtOH, the remaining suspension was dissolved in a mixture
of H2O (50 mL) and EtOAc (100 mL). After acidification of the
suspension with HOAc, the phases were separated and the
aqueous phase was extracted with EtOAc (2 × 50 mL). The
combined and dried (Na2SO4) organic phases were evaporated,
and the residue was subjected to CC (light petroleum-EtOAc)
(RS)-5-Am in o-4-(4-ch lor op h en yl)p en ta n oic Acid Hy-
d r och lor id e (10‚HCl). A solution of 17 (0.21 g, 1.00 mmol)
in 6 M HCl (10 mL) was refluxed for 18 h. After cooling to 25
°C and addition of charcoal (50 mg), the mixture was heated
to reflux for 30 min. After cooling to 25 °C, filtration, and
evaporation of the solvent, the oily residue was dissolved in
EtOH. After addition of acetone and CH2Cl2, 10‚HCl (90 mg,
34%) crystallized: mp 163-165 °C; 1H NMR (DMSO-d6) δ
8.41-7.67 (1H, s), 7.43-7.24 (4H, m), 3.42 (3H, s), 3.35-2.91
(4H, m), 2.21-1.93 (3H, m); 13C NMR (DMSO-d6) δ 173.9,
139.5, 131.9, 130.1, 128.8, 43.6, 42.2, 31.4, 28.4. Anal. (C11H14
-
ClNO2‚HCl‚0.5H2O) C, H, N.
1
to give 14 (2.40 g, 76%): mp 84.0-85.0 °C; H NMR (CDCl3)
(4R,5R)-1-(ter t-Bu tyloxyca r bon yl)-4-(4-ch lor op h en yl)-
5-[O-(ph en yloxyth iocar bon yl)h ydr oxy]-2-piper idon e (19).
To a solution of 1818 (0.31 g, 0.93 mmol) and phenyl chlo-
rothionoformate (0.20 g, 1.14 mmol) in CH2Cl2 (15 mL) was
added a solution of 4-(dimethylamino)pyridine (DMAP) (2.17
g, 17.8 mmol) in CH2Cl2 (15 mL) under N2 and within a period
of 15 min. After stirring for 18 h at 25 °C, the reaction mixture
was quenched with 1 M HCl, and then CH2Cl2 (40 mL) was
added. The organic phase was washed twice with 1 M HCl
and subsequently with a saturated aqueous solution of NaH-
CO3 and brine. The dried (Na2SO4) organic phase was evapo-
rated and the residue subjected to CC (light petroleum-
EtOAc) to give 19 (0.37 g, 85%): mp 153-154 °C; [R]20D ) +31°
(c ) 1.2, CH2Cl2); 1H NMR (CDCl3) δ 7.45-7.02 (9Η, m), 5.80-
5.58 (1Η, m), 4.30-4.07 (1Η, dd, J ) 4.0 and 14.5 Ηz), 3.87-
3.65 (1Η, dd, J ) 3.8 and 14.5 Ηz), 3.78-3.53 (1Η, m), 3.12-
2.90 (1Η, dd, J ) 6.1 and 17.0 Hz), 2.90-2.68 (1H, dd, J ) 7.4
and 17.0 Hz), 1.54 (9H, s); 13C NMR (CDCl3) δ 193.8, 168.9,
153.1, 151.7, 137.4, 133.7, 129.6, 129.3, 128.5, 126.8, 121.6,
83.8, 80.3, 46.2, 41.3, 37.3, 27.9; MS m/e (rel intensities) 307
(2), 251 (78), 172 (56), 165 (47), 115 (17), 94 (47), 77 (17), 57
(100), 41 (44). Anal. (C23H24ClNO5S) C, H, N, S.
δ 7.27-7.08 (4H, m), 4.64 (1H, s), 3.54 (3H, s), 3.39-3.17 (3H,
m), 2.77-2.55 (1H, dd, J ) 5.9 and 15.8 Hz), 2.67-2.42 (1H,
dd, J ) 7.9 and 15.7 Hz), 1.36 (9H, s); 13C NMR (CDCl3) δ
172.0, 155.6, 139.7, 132.6, 128.8, 128.7, 79.2, 51.5, 45.4, 41.7,
37.8, 28.2. Anal. (C16H22ClNO4) C, H, N.
(R S )-4-[N -(t er t -Bu t yloxyca r b on yl)a m in o]-3-(4-ch lo-
r op h en yl)bu ta n ol (15). To a solution of 14 (1.30 g, 3.9 mmol)
in CH2Cl2 (100 mL) was added at -30 °C lithium triethyl-
borohydride28 (11.7 mL, 11.7 mmol, 1 M in THF). The solution
was left at -30 °C for 0.5 h and then at 0 °C for 1 h. The
reaction was quenched with a saturated aqueous solution of
NH4Cl, and the aqueous phase was extracted with CH2Cl2 (2
× 50 mL). The combined organic phases were dried (Na2SO4)
and evaporated, and the residue was subjected to CC (light
petroleum-EtOAc) to give 15 as a colorless oil (0.92 g, 78%):
1H NMR (CDCl3) δ 7.22-7.01 (4H, m), 4.61 (1H, s), 3.54-3.28
(3H, m), 3.21-2.91 (2H, m), 2.91-2.74 (1H, m), 1.95-1.61 (2H,
m), 1.30 (9H, s); 13C NMR (CDCl3) δ 156.0, 140.9, 132.3, 129.1,
128.6, 79.5, 59.9, 45.9, 42.0, 35.9, 28.2. Anal. (C15H22ClNO3)
C, H, N.
(R S )-4-[N -(t er t -Bu t yloxyca r b on yl)a m in o]-3-(4-ch lo-
r op h en yl)bu ta n en itr ile (16). A solution of 15 (2.58 g, 8.6
(R)-1-(ter t-Bu tyloxyca r bon yl)-4-(4-ch lor op h en yl)-2-p i-
p er id on e (20). To a solution of 19 (0.35 g, 0.75 mmol) in
acetone (50 mL) were added tributyltin hydride (0.30 g, 1.39
mmol) and subsequently di-tert-butyl peroxyoxalate (10 mg).
The solution was left at 25 °C for 18 h. The opalescent solution
was evaporated, and the oily residue was subjected to CC (light
petroleum-EtOAc) to give 20 (0.22 g, 94%): mp 95.0-96.0 °C;
29
mmol) and triphenylphosphine (2.37 g, 9.0 mmol) in CCl4
(100 mL) was heated to reflux for 5 days. After cooling,
triphenylphosphine oxide was filtered. The filtrate was evapo-
rated and the residue subjected to CC (light petroleum-
EtOAc) to give (RS)-4-[N-(tert-butyloxycarbonyl)amino]-1-
chloro-3-(4-chlorophenyl)butane (3.14 g, 97%): mp 61.0-61.5
°C; 1H NMR (CDCl3) δ 7.24-7.02 (4H, m), 4.58 (1H, t, J ) 5.1
Hz), 3.43-3.24 (2H, m), 3.24-3.07 (2H, m), 3.07-2.85 (1H,
m), 2.38-1.53 (2H, m), 1.31 (9H, s); 13C NMR (CDCl3) δ 155.6,
139.3, 132.6, 129.1, 128.8, 79.1, 45.5, 42.6, 42.2, 35.8, 28.1.
Anal. (C15H21Cl2NO2) H, N; C: calcd, 56.61; found, 57.27.
Without further purification, a suspension of this compound
(1.19 g, 3.6 mmol) and NaCN (0.40 g, 8.2 mmol) in DMF (30
mL) was heated at 90 °C for 4 h. After evaporation of the
solvent, the residue was taken up in a mixture of H2O (50 mL)
and EtOAc (100 mL). The organic phase was washed with a
saturated aqueous solution of NaHCO3 and then with 1 M HCl
and brine, and after drying (Na2SO4) this solution was
[R]20 ) +22° (c ) 2.1, CH2Cl2); 1H NMR (CDCl3) δ 7.26-7.06
D
(4H, m), 3.68-3.44 (1H, dd, J ) 4.4 and 10.8 Hz), 3.60-3.36
(1H, dd, J ) 4.3 and 10.1 Hz), 3.12-2.93 (1H, m), 2.86-2.63
(1H, ddd, J ) 2.0, 5.4, and 17.0 Hz), 2.61-2.38 (1H, dd, J )
11.1 and 17.0 Hz), 2.26-2.03 (1H, ddd, J ) 2.0, 4.1, and 13.5
Hz), 1.97-1.80 (1H, m), 1.47 (9H, s); 13C NMR (CDCl3) δ 169.9,
152.6, 141.5, 132.7, 128.9, 127.8, 85.1, 46.7, 41.9, 37.8, 30.2,
28.0; M/S m/e (rel intensities) 311 (M+, 2), 254 (18), 209 (25),
181 (6), 138 (22), 97 (41), 57 (100).
(R)-5-Am in o-3-(4-ch lor op h en yl)p en ta n oic Acid Hyd r o-
ch lor id e (11‚HCl). To a solution of 20 (0.31 g, 1.0 mmol) in
THF (15 mL) was added an aqueous solution of LiOH (1 M,

2058 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Brief Articles
1.5 mL), and the mixture was stirred for 2 h. After evaporation
of the THF, the pH of the aqueous solution was adjusted to
1.5 by addition of 0.5 M HCl. The aqueous layer was extracted
with Et2O (3 × 30 mL), and the combined and dried (Na2SO4)
organic phases were evaporated. The remaining colorless oil
was purified by CC (EtOAc-CH2Cl2-AcOH) to give (R)-5-[N-
(tert-butyloxycarbonyl)amino]-3-(4-chlorophenyl)pentanoic acid
curves were obtained by adding cumulative amounts of test
compound to the organ bath. When an antagonist was used,
it was added to the organ bath 5-7 min prior to the first
application of agonist. Between each recorded dose-response
curve, the preparations were allowed to recover for 20-30 min
until the contractions had returned to baseline level. Each
experiment was performed on at least four preparations
obtained from two animals. The effect of compounds on
contractions elicited by acetylcholine in the absence of electri-
cal stimulation was characterized using the same setup and
allowing the compound to equilibrate with the tissue for at
least 5 min prior to the first application of agonist.
(0.22 g, 64%) as a colorless oil; [R]20 ) +4° (c ) 0.6, CH2Cl2);
D
1H NMR (DMSO-d6) δ 10.88 (1H, s), 7.31-7.14 (4H, m), 4.83
(1H, s), 3.40-3.25 (1H, m), 3.15-3.02 (2H, m), 2.99-2.77 (1H,
dd, J ) 6.6 and 15.5 Hz), 2.87-2.60 (1H, dd, J ) 8.5 and 15.5
Hz), 2.10-1.86 (2H, m), 1.44 (9H, s); 13C NMR (CDCl3) δ 176.0,
156.0, 141.5, 132.2, 128.7, 128.6, 40.9, 38.7, 38.6, 35.8.
Without further purification, this compound (0.19 g, 0.58
mmol) was mixed with 6 M HCl (10 mL) and the solution
heated to reflux for 2 h. After evaporation, the remaining
crystalline product was recrystallized (EtOH-acetone-CH2-
Recep tor Bin d in g. GABAA and GABAB receptor binding
assays were performed using rat brain synaptic membranes
from male Sprague-Dawley rats, tissue preparations being
prepared as earlier described.14 On the day of the assay, the
membrane preparation was thawed at room temperature for
45 min, suspended in 75 volumes (w/v) of 5 mM Tris-HCl buffer
(pH 7.1) using an Ultra-Turrax homogenizer, and centrifuged
at 48.000g for 20 min at 4 °C (Sorvall rotor SM34). This step
was repeated four times. The final pellet was resuspended in
the incubation buffer used in the particular binding assay.
GABAA binding was studied using [3H]muscimol as radio-
ligand.24 Membranes were resuspended in 50 mM Tris-HCl
buffer (pH 7.4) at 1 mg/mL, and aliquots were incubated with
[3H]muscimol (10 nM) for 1 h at 20 °C in a total volume of 1.0
mL in the presence of various GABAA agonists or antagonists
at concentrations ranging from 10-10 to 10-3 M. Nonspecific
binding was defined with 1 mM GABA. Membranes were
filtered through Whatman GF/B filters, which were washed
three times with ice-cold buffer, and radioactivity was counted
by liquid scintillation spectroscopy.
Cl2) to give 11‚HCl (0.11 g, 70%): mp 178 °C; [R]20 ) +12° (c
D
1
) 0.6, MeOH); H NMR (DMSO-d6) δ 8.12 (1H, s), 7.38 (4H,
m), 3.59 (3H, s), 3.30-3.20 (2H, m), 2.82-2.34 (3H, m), 1.99-
1.89 (2H, m); 13C NMR (MeOH-d4) δ 175.8, 142.2, 133.7, 130.2,
129.8, 41.9, 40.1, 38.9, 24.3. Anal. (C11H14ClNO2‚HCl‚H2O) C,
N; H: calcd, 6.07; found, 5.65.
(S)-5-Am in o-3-(4-ch lor op h en yl)p en ta n oic Acid Hyd r o-
ch lor id e (12‚HCl). Compound 12‚HCl was prepared from 2118
(0.12 g, 0.36 mmol) following a synthetic sequence and using
reaction conditions identical with those described above for
the synthesis of 11‚HCl starting with compound 18. The
following intermediates showed IR, 1H NMR, and 13C NMR
spectroscopic data essentially identical with those of the
corresponding enantiomeric compounds:
(4S,5S)-1-(ter t-Bu tyloxyca r bon yl)-4-(4-ch lor op h en yl)-
5-[O-(ph en yloxyth iocar bon yl)h ydr oxy]-2-piper idon e (0.15
g, 89%): mp 154-156 °C; [R]20D ) -34° (c ) 0.9, CH2Cl2). Anal.
(C23H24ClNO5S) C, H, N, S.
GABAB receptor binding assays were carried out as previ-
ously described.14 Membranes corresponding to 0.5 mg of
protein were incubated in 50 mM Tris-HCl buffer (pH 7.4)
containing 2.5 mM CaCl2, in the presence of 20 µM isoguvacine
and 50 nM of [3H]GABA. Nonspecific binding was determined
using 100 µM (RS)-baclofen. Following incubation at 24 °C for
45 min, the membranes were filtered through Whatman GF/B
filters, which were washed three times with ice-cold buffer,
and radioactivity was counted by liquid scintillation spectros-
copy.
(S)-1-(ter t-Bu tyloxyca r bon yl)-4-(4-ch lor op h en yl)-2-p i-
p er id on e (0.41 g, 90%): mp 95-96 °C; [R]20D ) -22° (c ) 2.0,
CH2Cl2); MS m/e (rel intensities) 311 (M+, 2), 254 (18), 209
(25), 181 (6), 138 (22), 97 (41), 57 (100).
(S)-5-[N-(ter t-Bu t yloxyca r b on yl)a m in o]-3-(4-ch lor o-
p h en yl)p en ta n oic a cid (0.15 g, 63%): colorless oil; [R]20
-6° (c ) 1.1, CH2Cl2).
)
D
12‚HCl (0.07 g, 61%): mp 179 °C; [R]20 ) -12° (c ) 0.7,
D
MeOH). Anal. (C11H14ClNO2‚HCl‚H2O) C, H, N.
Ack n ow led gm en t. This work was supported by
grants from the Danish State Biotechnology Program
and the Lundbeck Foundation. We wish to express our
gratitude to Dr. Th. Linker, Institut fu¨r Organische
Chemie der Universita¨t Wu¨rzburg, for fruitful discus-
sion in the field of radical chemistry, to Dr. W. Froestl,
Novartis, Basel, Switzerland, for the generous gift of
CGP35348, and to A. Nordly for skillful secretarial
assistance.
Deter m in a tion of th e En a n tiom er ic P u r ity. A 150- ×
4-mm Crownpak CR(-) column (Diacel) was thermostated at
38 °C and eluted at 0.8 mL/min with aqueous perchloric acid,
pH 2, using a Waters instrumentation consisting of a M510
pump connected to a UK6 injector and a M991 photodiode
array detector with a detection wavelength of 200 nm.
Gu in ea P ig Ileu m P h a r m a cology. The method used is
described in detail by Kristiansen et al.14 In short, male guinea
pigs were stunned and bled; 2-4-cm long segments of the
distal ileum, taken 10 to 30 cm from the ileocecal valve, were
quickly removed, cleared of intraluminal contents, and placed
in a modified Krebs-bicarbonate solution of the composition
(mM): NaCl (151.0), KCl (4.7), MgSO4 (0.6), CaCl2 (2.8),
NaHCO3 (16.3), NaH2PO4 (1.3), and glucose (7.7), which had
been equilibrated with a gas mixture of 5% CO2 and 95% O2,
resulting in a pH of 7.4 at 37 °C. Strips of longitudinal muscle
with myenteric plexus attached, obtained by the method of
Paton and Zar,30 were mounted vertically in a 10-mL organ
bath containing the modified Krebs-bicarbonate buffer con-
tinuously gassed with the mixture of 5% CO2 and 95% O2 and
maintained at 37 °C. After 60 min of equilibration, the muscle
segments were stimulated electrically (0.1 Hz; duration, 5 ms;
supramaximal voltage, 100 V) using an HSE stimulator.
Isometric muscle contractions were recorded using a force
transducer (Grass model FT-03) coupled to a BBC-Goerz
Metrawatt recorder. Effects of test compounds were measured
as inhibition of the electrically induced contractions and
expressed either relative to maximum inhibition induced by
the GABAB agonist (RS)-baclofen or relative to maximum
inhibition induced by the compound itself. The dose-response
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