N-substituted 4-amino-3,3-dipropyl-2(3H)-furanones: New positive allosteric modulators of the GABAA receptor sharing electrophysiological properties with the anticonvulsant loreclezole

Article abstract of DOI:10.1021/jm011082k

1,4-Addition of benzylamine to 2(5H)-furanone followed by dialkylation of the 3-position with allylbromide gave (±)-4-benzyl-3,3-diallyl-2(3H)-furanone (8), which served as the intermediate for the synthesis of various N-substituted 4-amino-3,3-dipropyl-2

Full text of DOI:10.1021/jm011082k

2824
J . Med. Chem. 2002, 45, 2824-2831
N-Su bstitu ted 4-Am in o-3,3-d ip r op yl-2(3H)-fu r a n on es: New P ositive Alloster ic
Mod u la tor s of th e GABAA Recep tor Sh a r in g Electr op h ysiologica l P r op er ties
w ith th e An ticon vu lsa n t Lor eclezole
Ahmed El Hadri,† Ahmed Abouabdellah,† Urs Thomet,‡ Roland Baur,‡ Roman Furtmu¨ller,§ Erwin Sigel,‡
Werner Sieghart,§ and Robert H. Dodd*,†
Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique,
91198 Gif-sur-Yvette Cedex, France, Department of Pharmacology, University of Bern, Friedbu¨hlstrasse 49,
CH-3010 Bern, Switzerland, and Section of Biochemical Psychiatry, Department of Psychiatry and Brain Research Institute,
Section of Biochemistry and Molecular Biology, University of Vienna, A-1090, Vienna, Austria
Received October 29, 2001
1,4-Addition of benzylamine to 2(5H)-furanone followed by dialkylation of the 3-position with
allylbromide gave (()-4-benzyl-3,3-diallyl-2(3H)-furanone (8), which served as the intermediate
for the synthesis of various N-substituted 4-amino-3,3-dipropyl-2(3H)-furanones (()-9a -l. The
compounds were evaluated for their capacity to potentiate or inhibit GABA-evoked currents
in Xenopus laevis oocytes expressing recombinant R1â2γ2 GABAA receptors. The benzyl, ethyl,
and allyl carbamates ((R)-9a (100 µM), (()-9b (100 µM), (()-9c (200 µM)) stimulated GABA
currents by 279 ( 47%, 426 ( 8%. and 765 ( 61%, respectively, while the phenylcarboxamide
(()-9f (200 µM) stimulated currents by 420 ( 33%. Concentration-response studies showed
that compound 9c was approximately twice as potent in stimulating GABA currents as
R-EMTBL (2), the most potent 3,3-dialkylbutyrolactone known to date. On the other hand, the
N-sulfonyl analogues were much less active or even inhibited GABA-evoked currents. In vitro
radioligand displacement studies on rat brain membranes showed that these compounds did
not bind to the benzodiazepine or GABA recognition sites of the GABAA receptor. However,
these compounds generally weakly displaced [35S]-TBPS (approximately 50% displacement at
100 µM), though potencies did not correlate with GABA current potentiation. Results obtained
with R1â1 and mutant R1â2N265S receptors, which compared to R1â2 receptors are both much
less sensitive to current stimulation produced by the anticonvulsant loreclezole, suggest that
at least some of these aminobutyrolactones, (e.g., 9a , 9c), and interestingly also R-EMTBL,
share stimulatory properties with loreclezole.
In tr od u ction
recognition site (for which specific agonists and antago-
nists are also known, for example, muscimol and (+)-
bicuculline, respectively) but also by compounds inter-
acting at allosteric modulatory sites situated elsewhere
on the receptor complex.9 A number of such sites have
been described including that for benzodiazepines,
barbiturates, picrotoxin, neurosteroids, and ROD com-
pounds.10-13 An added feature of some of these binding
sites is that, depending on the chemical structure of the
ligand, either positive or negative allosteric modulation
of GABA-evoked currents (corresponding to stimulation
or inhibition of chloride ion flux) can result. Thus, while
the clinically useful 1,4-benzodiazepines are positive
allosteric modulators of the GABAA receptor,14 many
compounds belonging to the â-carboline family (e.g.,
methyl â-carboline-3-carboxylate or â-CCM) negatively
modulate the GABAA receptor by binding to the same
benzodiazepine recognition site.14,15
γ-Aminobutyric acid (GABA) is the major inhibitory
neurotransmitter of the central nervous system. GABA
interacts with three major receptor classes, the GABAA,
GABAB, and GABAC receptors. Of these three, the first
has by far received the most attention, and many
compounds interacting with this receptor (e.g., benzo-
diazepines such as Valium) have found great clinical
use. The GABAA receptor is a pentameric supramolecu-
lar complex forming a ligand-gated ion channel control-
ling neuronal chloride ion flux. The protein subunits
comprising the receptor have been classified as R, â, γ,
δ, ꢀ, π, and θ on the basis of sequence homology.1-6 Some
of these subunits also exist in the form of several
isoforms (e.g., R1-R6, â1-â3, etc.), consequently leading
to a potentially very large number of GABAA receptor
subtypes. In the mammalian brain, the major subtype
has the composition 2R12â2γ2.7,8
The 3,3- and 4,4-dialkylbutyrolactones, thiobutyro-
lactones, and related compounds, largely developed by
Covey and Ferrendelli over the past 2 decades, are
another class of compounds that appears to interact
with distinct allosteric binding sites on the GABAA
receptor.16-21 Thus, while the 4,4-dialkyl derivatives
(e.g., 1, Chart 1) were found to generally block GABA-
induced chloride currents in neurons and were conse-
Opening and closing of the chloride ion channel, and
thus the level of neuroinhibition, can be controlled not
only by the interaction of GABA with its particular
* To whom correspondence should be addressed. Phone: 33-1-
69824594. Fax: 33-1-69077247. E-mail: Robert.Dodd@icsn.cnrs-gif.fr.
† Institut de Chimie des Substances Naturelles.
‡ University of Bern.
§ University of Vienna.
10.1021/jm011082k CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/18/2002

N-Substituted Furanones
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2825
Ch a r t 1
N-substituted 4-amino-3,3-propyl-substituted butyro-
lactones are potent positive allosteric modulators of the
GABAA receptor that appear to exhibit an action par-
tially similar to that of loreclezole.
Ch em istr y
The starting material for this study was 4-benzy-
lamino-4,5-dihydro-2(3H)-furanone 5,35 prepared first in
racemic form by conjugate addition of benzylamine to
2(5H)-furanone 4 in methanol (Scheme 1).36,37 Com-
pound (()-5 was then transformed into the 3,3-diallyl
derivative (()-8 by treatment at -78 °C with lithium
hexamethyldisilazide in THF followed by addition of
excess allyl bromide in HMPA.37 Minor amounts of the
cis and trans monoallyl derivatives ((()-6 and (()-7,
respectively) were also isolated from the reaction mix-
ture. Hydrogenation of compound (()-8 in the presence
of palladium catalyst led to simultaneous reduction of
the double bonds of the allyl groups and removal of the
N-benzyl group. The resulting primary amine function
was then acylated or sulfonylated using the appropriate
acyl or sulfonyl chloride (or Boc anhydride) in the
presence of triethylamine and DMAP, affording com-
pounds 9a -l as racemic mixtures (Table 1). Similarly,
for comparative purposes, compound (()-5 was con-
verted into the N-Cbz derivative (()-10 using the same
hydrogenation-acylation (with benzyl chloroformate)
sequence.
quently convulsant in vivo, the 3,3-dialkyl analogues
(best represented by R-EMTBL, 2)18,22-24 potentiated
currents and were anticonvulsant. Because both classes
of compounds displaced [35S]-tert-butylbicyclophospho-
rothionate ([35S]-TBPS), a ligand specific for the picro-
toxin binding site, in in vitro binding studies, it was at
first assumed that the lactones acted via the picrotoxin
site.22 However, subsequent electrophysiological studies,
especially with mutant receptors unresponsive to picro-
toxin, led to the hypothesis that the positive allosteric
modulatory butyrolactones act mainly at a distinct
binding site termed the lactone site, while the negative
modulatory activity would be predominantly due to
interaction with the picrotoxin site.23,24 Voltage-depend-
ent calcium channels have also been implicated in the
convulsant and anticonvulsant properties of these com-
pounds.25
The R and S enantiomers of the N-Cbz derivative (()-
9a could be separated by HPLC on a chiral OD column.
To assign the absolute configuration to the two fractions
obtained, (R)-9a was prepared unambiguously starting
from (R)-4-N-Cbz-aminobutyrolactone (R)-10 (Scheme
2), itself prepared from D-aspartic acid by a published
procedure.38,39 Because C-3 dialkylation of compound
(R)-10 was found to be inefficient using the conditions
that were successful for the N-benzyl analogue (()-5,
the N-Cbz group of (R)-10 was replaced by an N-benzyl
group. Thus, hydrogenolytic cleavage of the Cbz group
of (R)-10 followed by reductive alkylation of the result-
ing amine with benzaldehyde provided (R)-5, which
could now be dialkylated at C-3 as before using LiH-
MDS/allyl bromide to give (R)-8. Finally, the latter was
transformed into the 3,3-dipropyl N-Cbz derivative (R)-
9a by hydrogenation and acylation as before. Compound
(R)-9a was found to have a retention time corresponding
to that of the faster-moving component isolated by chiral
phase HPLC of (()-9a . The slower-moving component
could thus be assigned the S configuration.
Another anticonvulsant agent acting at the GABAA
receptor is loreclezole (3) whose activity was also orig-
inally hypothesized to be due to an interaction with the
benzodiazepine binding site.26,27 However, the observa-
tion that the anticonvulsant action of loreclezole is not
inhibited by the benzodiazepine receptor antagonist
flumazenil,28,29 together with various binding and elec-
trophysiological evidence, has pointed to a distinct
loreclezole-sensitive allosteric modulatory site. Only â2
and â3, but not â1, containing receptors respond strongly
to loreclezole, and a point mutation in â2 to the amino
acid residue present in the homologous position in â1,
that is, â2N265S, makes the receptor much less respon-
sive to loreclezole.30-34
Both the butyrolactone and loreclezole binding sites
of the GABAA receptor thus represent relatively unex-
plored targets for the development of agents having
fewer of the undesired effects of the widely prescribed
benzodiazepines (e.g., somnolence, amnesia, depen-
dence, tolerance). In this paper, then, we report that
Sch em e 1

2826 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
El Hadri et al.
Ta ble 1. Effect of Compounds 5-10 on GABA-Elicited
Currents in Xenopus Oocytes Expressing R1â2γ2 GABAA
Receptors
GABA-induced currents. In the following, the structural
requirements for modulation are discussed. The first
important observation that can be made is that an
amide-type functionality at C-4 of a butyrolactone ring
is more effective than an amine functionality in stimu-
lating GABA currents. Thus, the N-benzyl derivative
(()-5 (200 µM) had no effects on GABA currents while
the N-Cbz analogue (()-10 (200 µM) was able to
stimulate currents, albeit weakly (47 ( 5%). In both
cases, addition of alkyl or dialkyl substituents at C-3
had a beneficial effect on activity. Thus, the cis-3-allyl-
4-N-benzyl derivative and its 3,3-diallyl analogue ((()-6
and (()-8, respectively) now had significant, though still
relatively weak, stimulatory activity (73 ( 18% and 115
( 17%, respectively, at 200 µM). Interestingly, the trans
isomer (()-7 was totally inactive. In the case of the
N-Cbz analogue, the presence of the dialkyl substituents
at C-3 also had a very strong effect on activity, com-
pound (()-9a (100 µM) now stimulating currents by
about 280%. Furthermore, this stimulatory activity
resides mainly in the R enantiomer ((R)-9a , 279%
stimulation at 100 µM), the S enantiomer being ap-
proximately 2.5 times less active. Other carbamate
derivatives prepared (except for the sterically bulky
N-Boc compound (()-9d ) were even more potent (i.e.,
ethyl carbamate (()-9b, 426 ( 8% stimulation at 200
µM), stimulations being at the same level as those of
R-EMTBL (352 ( 23% stimulation at 200 µM), the most
active butyrolactone-type compound reported so far. In
the case of the allyl carbamate (()-9c (765 ( 61%
stimulation at 200 µM), the activity of R-EMTBL was
largely surpassed, a result confirmed in the concentra-
tion-response study described below. Finally, a car-
boxamide functionality ((()-9f) was found to display
stimulatory activity (420 ( 33% at 200 µM) of the same
order as the carbamate analogues. In contrast, and
except for the p-chlorophenylsulfonamide (()-9g (which
showed +23 ( 8% stimulation at 5 µM but could not be
assayed at higher concentrations because of insolubil-
ity), none of the sulfonamide derivatives synthesized
((()-9h -l) showed any significant current stimulation.
In fact, in some of these compounds [(()-9h ,i,k ], a weak
inhibitory effect on currents was observed.
% stimulation
of GABA-induced
compd
R1
R2
R3
currentsa (µM)
2 (R-EMTBL)
(()-5
+352 ( 23 (200)
0 ( 1 (200)
+73 ( 18 (200)
-1 ( 3 (200)
+115 ( 17 (200)
Bn
Bn
Bn
Bn
H
allyl
H
H
H
allyl
allyl
(()-6
(()-7
(()-8
allyl
(()-9a
(R)-9a
(S)-9a
(()-9b
(()-9c
(()-9d
(()-9e
(()-9f
(()-9g
(()-9h
(()-9i
(()-9j
(()-9k
(()-9l
(()-10
-CO2Bn
-CO2Bn
-CO2Bn
-CO2Et
propyl propyl +279 ( 94 (100)
propyl propyl +279 ( 47 (100)
propyl propyl +104 ( 4 (100)
propyl propyl +426 ( 8 (200)
propyl propyl +765 ( 61 (200)
-CO2allyl
-CO2-t-Bu
-CO2Ph
-COPh
-SO2Ph-p-Cl
-SO2Ph-p-OMe
-SO2Ph-p-CF3
-SO2-2-naphthyl propyl propyl
-SO2-2-thiophene propyl propyl
-SO2NMe2
-CO2Bn
propyl propyl
+46 ( 5 (200)
propyl propyl +330 ( 35 (200)
propyl propyl +420 ( 33 (200)
propyl propyl
propyl propyl
propyl propyl
+23 ( 8 (5)
-22 ( 10 (200)
-44 ( 1 (200)
+4 ( 3 (50)
-16 ( 5 (200)
-1 ( 6 (200)
+47 ( 5 (200)
propyl propyl
H
H
a
Determined electrophysiologically in X. laevis oocytes express-
ing R1â2γ2 GABAA receptors as previously described.40,41 Average
of three assays ( SEM.
Resu lts a n d Discu ssion
No radioligands are available for the proposed buty-
rolactone binding site of the GABAA receptor, and
furthermore, previous studies of this class of molecules
have not convincingly demonstrated any consistent
correlation between [35S]-TBPS displacement and GABA-
evoked current stimulations by these compounds.19 For
these reasons, it was decided to first perform electro-
physiological screening of the 4-aminobutyrolactone
derivatives and subsequently to investigate in vitro
binding characteristics of the more active compounds.
Thus, compounds 5-10 (200 µM, 100 µM or less,
depending on solubility) were tested for their aptitude
to activate, stimulate, or inhibit GABA-evoked currents
in Xenopus laevis oocytes expressing rat brain recom-
binant R1â2γ2 GABAA receptors.40,41 Results are shown
in Table 1. None of the tested compounds acted as an
agonist of the channel at the concentrations given in
Table 1, but many of them acted as modulators of
The in vitro binding profile of the more active com-
pounds of Table 1 was investigated with respect to
selected binding sites of the GABAA receptor. Thus,
interaction with the GABA, benzodiazepine, and picro-
toxin binding sites was evaluated by determination of
Sch em e 2

N-Substituted Furanones
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2827
Ta ble 2. Displacement of Radioactive Ligands from Rat Brain
Membranes by Selected 4-Aminobutyrolactones
Ta ble 3. Concentration-Response Values of Compounds 2,
(()-9c, and (()-9f on GABA-Elicited Currents in Xenopus
Oocytes Expressing R1â2γ2 Receptors
displacement of radioactive ligand at 100 µMa
% stimulation of GABA induced currentsa
compd
[3H]-flunitrazepam
[3H]-muscimol
[
35S]-TBPS
concn, µM
2 (R-EMTBL)
(()-9c
(()-9f
2 (R-EMTBL) 0%
0%
0%
0%
0%
0%
0%
25%
60%
60%
70%
30%
50%
(()-6
0%
2
20
200
+10 ( 2
+39 ( 11
+386 ( 25
+26 ( 5
+229 ( 14
+765 ( 61
+17 ( 3
+207 ( 17
+420 ( 33
(()-8
14% stimulation
0%
0%
0% (ce)
15% stimulation (fb)
25% stimulation
(()-9a
(()-9b
(()-9c
a
Average of three assays ( SEM.
(()-9e
(()-9f
25% stimulation (ce) 75%
0% (fb)
10% stimulation (ce) 65%
0% (fb)
Ta ble 4. Comparison of Current Stimulation of Compounds 2
(R-EMTBL), (R)-9a , (()-9c, and (()-9f in R1â2 and R1â2N265S
Receptors
15% stimulation
(()-9g
(()-9h
(()-9i
25% stimulation
0% stimulation
20% stimulation
0%
0%
0%
85%
80%
40% (ce)
70% (fb)
75%
% stimulation of GABA-induced currents
compda
(R)-9a
2 (R-EMTBL)
(()-9c
(()-9f
R1â2
R1â2N265S
324 ( 9
192 ( 36
2132 ( 569
557 ( 9
123 ( 20
83 ( 6
627 ( 95
648 ( 81
(()-9j
0%
0%
a
Determined in rat brain tissue as previously described.42,43
Values are given as percent displacement by each substance.
Experiments were performed at least three times in triplicate; “ce”
refers to rat cerebellar tissue; “fb” refers to rat forebrain tissue
(whole brain minus cerebellum). When tissue is not specified,
values were identical for fb and ce.
a
Compound 2 was assayed at 200 µM. The other compounds
were assayed at 100 µM.
problems that made analysis at higher concentrations
impossible. However (()-9c and (()-9f were more potent
than R-EMTBL at the same concentration. No evidence
for a biphasic effect was observed for the substances
investigated.
the percent displacement of the appropriate selective
radioligand (respectively, [3H]-muscimol, [3H]-fluni-
trazepam, and [35S]-tert-butylbicyclophosphorothionate
(([35S]-TBPS)) by 100 µM of the substance.42,43 As shown
in Table 2, none of the compounds tested showed any
binding affinity for the GABA recognition site. In fact,
a slight increase (10-25%) in [3H]-muscimol binding
could be observed in some cases in cerebellar tissue
(compounds (()-9e,f). Similarly, displacement of [3H]-
flunitrazepam was either negligible ((()-9b) or produced
a 15-25% stimulation of radioactive ligand binding ((()-
9e,f). This latter effect did not correlate with effects on
GABA current, since, for example, compound (()-9i,
which also stimulated [3H]-flunitrazepam binding by
20%, actually inhibited GABA currents by 44 ( 1%.
The 4-amino-3,3-dipropylbutyrolactones were some-
what more active in displacing [35S]-TBPS from its
binding site. Thus, compounds (()-9a -c,e,f displaced
from 30% to 75% of radioligand binding at 100 µM.
Though these values remain relatively modest, they are
superior to the displacement by R-EMTBL (25% at 100
µM). Again, there is little correlation between [35S]-
TBPS displacement potencies by these compounds and
effects on GABA-evoked currents. Thus, compound (()-
9j, which had practically no effect on currents, still
displaced 75% of [35S]-TBPS binding at 100 µM while
compound (()-9h , which actually inhibited currents
(-22 ( 10% at 100 µM) displaced 90% of [35S]-TBPS
binding at the same concentration. Studies by Covey
and co-workers using mutant GABA receptors insensi-
tive to picrotoxin (the postulated site of action of TBPS),
showed that R-EMBTL still potentiated GABA currents,
leading to the hypothesis that the dialkylbutyrolactones
are acting at two different sites, i.e., the picrotoxin/TBPS
site (which would account for inhibition of GABA
currents by low concentrations of R-EMTBL) and a
putative “butyrolactone” site (interaction that produces
current stimulations observed at higher concentrations
of R-EMTBL).24 Concentration-response values for
compounds (()-9c and (()-9f were also determined and
compared to those of R-EMTBL (Table 3). The EC50
values could not be determined because of solubility
Compound (R)-9a was also tested on R1â1 and R1â2
recombinant receptors. At 100 µM, the compound was
3- to 4-fold more active in R1â2 receptors than in R1â1
receptors (not shown). Interestingly, this profile re-
sembles that of the anticonvulsant loreclezole, which
similarly enhances the activity in â2 (as well as â3) but
not in â1 subunit containing GABAA receptors.33 To test
this hypothesis, the effect of (R)-9a on GABA currents
in the presence of the point mutant R1â2N265S receptor,
which shows a diminished response to loreclezole,44,45
was studied. As shown in Table 4, current stimulations
by (R)-9a were also greatly decreased by about 65% in
this case, again suggesting similarity in action with
loreclezole. Interestingly, the current stimulation by
R-EMTBL was also reduced in mutant R1â2N265S
receptors compared to wild-type R1â2 receptors by more
than 50% (Table 4).
In oocytes expressing rat recombinant R1â2N265S
receptors, stimulation by (()-9c (100 µM) was also
reduced to about 30% of the current potentiation
observed in the wild-type receptor (Table 4). However,
stimulation by (()-9f (100 µM) was not significantly
affected by the point mutation â2N265S. Thus, at least
some (9a , 9c) but not all (9f) of the butyrolactones
analyzed may share similarity in their action to the
anticonvulsant loreclezole.
In conclusion, the addition of an N-carboxamide or
carbamate (but not N-sulfonyl) group at C-4 of a 3,3-
dialkylbutyrolactone has the effect of significantly in-
creasing the GABA-evoked current potentiation pro-
duced by this class of compounds possibly by directing
them to the loreclezole allosteric recognition site of the
GABAA receptor. While the latter point will obviously
require further study and the availability of more
selective compounds, it is clear that the addition of a
substituted amine functionality to the butyrolactone
nucleus allows for a great deal of structural variation

2828 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
El Hadri et al.
Continued elution of the chromatography column afforded
and considerable leeway for the optimization of activity.
These studies are currently underway.
compound (()-7 as an oil (100 mg, 18%): IR (film) 1773 cm-1
;
1
CIMS m/z 232 (MH)+; H NMR (300 MHz, CDCl3) δ 1.69 (s,
1H, NH), 2.37 (m, 1H, H-3), 2.52 (m, 2H, CH2CHdCH2), 3.39
(q, 1H, J ) 6.6 Hz, H-4), 3.75 (s, 2H, CH2N), 3.91 (dd, 1H,
J 4,5a ) 6.6 Hz, J gem ) 9.1 Hz, H-5a), 4.33 (dd, 1H, J 4,5b ) 6.6
Hz, J gem ) 9.1 Hz, H-5b), 5.12 (m, 2H, CHdCH2), 5.77 (m, 1H,
CHdCH2), 7.24-7.32 (m, 5H, ArH); 13C NMR (75 MHz, CDCl3)
δ 33.2, 46.1, 51.8, 58.5, 72.1, 118.5, 127.5, 128.1, 128.6, 134.1,
139.4, 177.2. Anal. (C14H17NO2) C, H, N.
Exp er im en ta l Section
Gen er a l. Melting points were determined on a Bu¨chi
apparatus and are uncorrected. IR spectra of samples were
obtained as KBr or as films with
a Nicolet 205 FT-IR
spectrometer. 1H NMR and 13C NMR were determined on a
Bruker 200, 250, or 300 MHz instrument. Chemical shifts are
given as δ values with reference to Me4Si as internal standard.
Electron impact and chemical ionization mass spectra were
recorded on AEI MS-50 and AEI MS-9 spectrometers, respec-
tively. Optical rotations were determined with a Perkin-Elmer
241 polarimeter. Thin-layer chromatography was performed
on Merck silica gel plates with fluorescent indicator. The plates
were visualized with UV light (254 nm) or with a 3.5% solution
of phosphomolybdic acid in ethanol. All column chromatogra-
phy was conducted on a Waters 990 apparatus. All reagents
were purchased from the Aldrich Chemical Co. and were used
without further purification. R-EMTBL was prepared as
described.46 Elemental analyses were performed at the ICSN,
CNRS, Gif-sur-Yvette.
(R,S)-4-Ben zyloxyca r b on yla m in o-4,5-d ih yd r o-2(3H )-
fu r a n on e ((()-10). A solution of compound (()-5 (306 mg, 1.6
mmol) in ethanol (10 mL) was hydrogenated at 40 psi for 16
h in the presence of 10% palladium on carbon (100 mg). The
reaction mixture was filtered through Celite, the filter pad was
washed with ethanol, and the combined filtrates were evapo-
rated under reduced pressure. The residue was dissolved in
dichloromethane (3 mL), and to the solution was added at 0
°C triethylamine (0.4 mL, 2.8 mmol), DMAP (45 mg, 0.37
mmol), and benzyl chloroformate (325 mg, 1.9 mmol). The
reaction mixture was stirred at 0 °C for 30 min and then at
room temperature for 12 h. The solvent was removed under
reduced pressure, and the residue was purified by flash column
chromatography on silica gel (ethyl acetate-hexane 1:1),
affording compound (()-10 as a white solid (230 mg, 61%): mp
101-102 °C (lit. mp (S isomer) 103-104 °C)47; CIMS m/z 236
(R,S)-4-Ben zyla m in o-4,5-d ih yd r o-2(3H)-fu r a n on e ((()-
5).35 A solution of 2(5H)-furanone (2.5 g, 29.7 mmol) in
methanol (3 mL) was treated at 0 °C with benzylamine (3.82
g, 35.7 mmol). The reaction mixture was stirred at 0 °C for 24
h, and the solvent was removed under reduced pressure. The
residue was purified by flash chromatography on silica gel
(ethyl acetate-hexane 1:1), affording compound (()-5 as a
yellow oil (3.5 g, 60%): CIMS m/z 191 (M+); 1H NMR (200 MHz,
1
(MH)+; H NMR (300 MHz, CDCl3) δ 2.47 (dd, 1H, J 3a,4 ) 3.0
Hz, J gem ) 17.7 Hz, H-3a), 2.85 (dd, 1H, J 3b,4 ) 7.6 Hz, J gem
) 17.7 Hz, H-3b), 4.23 (m, 1H, H-4), 4.50 (m, 2H, H-5),
5.12 (s, 2H, CH2O), 7.36 (s, 5H, ArH); 13C NMR (75 MHz,
CDCl3) δ 34.8, 48.1, 67.2, 73.6, 128.2, 128.4, 128.6, 135.9, 155.7,
175.1.
CDCl3) δ 1.62 (s, 1H, NH), 2.40 (dd, 1H, J 3a,4 ) 5.3 Hz, J gem
)
(R,S)-4-Ben zyloxycar bon ylam in o-4,5-dih ydr o-3,3-dipr o-
p yl-2(3H)-fu r a n on e ((()-9a ). Following the same procedure
as for the preparation of compound (()-10, compound (()-8
(100 mg, 0.37 mmol) was hydrogenated and then treated with
benzyl chloroformate (86 mg, 0.5 mmol) to afford compound
(()-9a as a white powder (61 mg, 52%): mp 91-92 °C; IR (KBr)
16.0 Hz, H-3a), 2.70 (dd, 1H, J 3b,4 ) 8.0 Hz, J gem ) 16.0 Hz,
H-3b), 3.68 (m, 1H, H-4), 3.80 (s, 2H, CH2Ph), 4.13 (dd, 1H,
J
5a,4 ) 1.6 Hz, J gem ) 10.7 Hz, H-5a), 4.48 (dd, 1H, J 5b,4 ) 5.3
Hz, J gem ) 10.7 Hz, H-5b), 7.20-7.50 (m, 5H, ArH); 13C NMR
(75 MHz, CDCl3) δ 35.9, 51.9, 54.0, 73.6, 127.7, 128.4, 128.9,
139.6, 176.5. Anal. (C11H13NO2) C, H, N.
1
1557, 1684, 1774 cm-1; CIMS m/z 320 (MH)+; H NMR (300
cis- an d tr a n s-3-Allyl-4-ben zylam in o-4,5-dih ydr o-2(3H)-
fu r a n on e ((()-6 a n d (()-7, Resp ectively) a n d (R,S)-4-
Ben zyl-3,3-d ia llyl-4,5-d ih yd r o-2(3H)-fu r a n on e ((()-8). To
a solution of lithium hexamethyldisilazide (5.4 mmol, 2.2
equiv) in anhydrous THF (50 mL) was added at -78 °C under
argon a solution of compound (()-5 (0.46 g, 2.44 mmol). The
solution was stirred at -78 °C for 30 min, and a solution of
allyl bromide (1.16 mL, 12.2 mmol) in HMPA (5 mL) was added
by cannula. The reaction mixture was stirred for 2 h at -78
°C, saturated aqueous ammonium chloride was added, and the
mixture was extracted with dichloromethane (3 × 20 mL). The
organic extracts were combined, the solvent was removed
under reduced presure, and the residue was purified by
chromatography on silica gel (ethyl acetate-hexane 1:1). The
first compound to be eluted was the diallyl derivative (()-8,
obtained as an oil (340 mg, 52%): IR (film) 1639, 1770, 3338
cm-1; CIMS m/z 272 (MH)+; 1H NMR (300 MHz, CDCl3) δ 1.52
(s, 1H, NH), 2.33 (m, 2H, CH2CHdCH2), 2.45 (m, 2H, CH2-
CHdCH2), 3.54 (dd, 1H, J 4,5a ) 8.4 Hz, J 4,5b ) 7.7 Hz, H-4),
3.80 (s, 2H, CH2N), 3.81 (dd, 1H, J 5a,4 ) 8.4 Hz, J gem ) 17.6
Hz, H-5a), 4.28 (dd, 1H, J 5b,4 ) 7.7 Hz, J gem ) 17.6 Hz, H-5b),
5.00-5.22 (m, 4H, 2 × CHdCH2), 5.68 (m, 1H, CHdCH2), 5.88
(m, 1H, CHdCH2), 7.31 (m, 5H, ArH); 13C NMR (75 MHz,
CDCl3) δ 36.0, 39.4, 49.1, 52.4, 59.1, 70.5, 119.3, 119.7, 127.5,
128.0, 128.6, 132.8, 133.1, 139.8, 178.7. Anal. (C17H21NO2) C,
H, N.
MHz, CDCl3) δ 0.90 (t, 3H, J ) 7.2 Hz, CH2CH3), 0.92 (t, 3H,
J ) 7.0 Hz, CH2CH3), 1.23-1.64 (m, 8H, 2 × CH2CH2), 3.84
(dd, 1H, J 4,5a ) 8.6 Hz, J 4,5b ) 7.4 Hz, H-4), 4.49 (m, 1H, H-5a),
4.57 (m, 1H, H-5b), 5.12 (s, 2H, CH2O), 7.36 (s, 5H, ArH); 13
C
NMR (75 MHz, CDCl3) δ 14.4, 14.5, 17.2 (2C), 32.8, 36.2, 48.2,
53.9, 67.5, 69.2, 127.8 (2C), 128.3 (2C), 128.7, 136.0, 155.8,
178.5. Anal. (C18H25NO4) C, H, N.
(R,S)-4,5-Dih ydr o-3,3-dipr opyl-4-eth yloxycar bon ylam i-
n o-2(3H)-fu r a n on e ((()-9b). Following the same procedure
as for the preparation of compound (()-10, compound (()-8
(100 mg, 0.37 mmol) was hydrogenated and the product was
treated with ethyl chloroformate (55 mg, 0.5 mmol) to afford
compound (()-9b as a white solid (73 mg, 77%): mp 111-112
°C; IR (KBr) 1548, 1690, 1788, 3326 cm-1; CIMS m/z 258
1
(MH)+; H NMR (250 MHz, CDCl3) δ 0.93 (t, 6H, J ) 7.0 Hz,
2 × CH2CH3), 1.27 (t, 3H, J ) 7.0 Hz, OCH2CH3), 1.33-1.70
(m, 8H, 2 × CH2CH2CH3), 3.84 (dt, 1H, J 4,5a ) J 4,5b ) 7.1 Hz,
J 4,NH ) 1.2 Hz, H-4), 4.15 (q, 2H, J ) 7.0 Hz, OCH2CH3), 4.45-
4.60 (m, 2H, H-5), 4.82 (br s, 1H, NH); 13C NMR (300 MHz,
CDCl3) δ 14.4, 14.6 (2C), 17.2 (2C), 32.7, 36.1, 48.2, 53.6, 61.6,
69.2, 156.2, 178.8. Anal. (C13H23NO4) C, H, N.
(R,S)-4-Allyloxyca r bon yla m in o-4,5-d ih yd r o-3,3-d ip r o-
p yl-2(3H)-fu r a n on e ((()-9c). Following the same procedure
as for the preparation of compound (()-10, compound (()-8
(100 mg, 0.37 mmol) was hydrogenated and the product was
treated with allyl chloroformate (60 mg, 0.5 mmol) to afford
compound (()-9c as a white solid (81 mg, 81%): mp 80-81
°C; IR (KBr) 1557, 1686, 1777, 3316 cm-1; CIMS m/z 270
Further elution of the chromatography column afforded
compound (()-6 as an oil (50 mg, 9%): IR (film) 1772 cm-1; 1H
NMR (300 MHz, CDCl3) δ 1.62 (s, 1H, NH), 2.39 (m, 2H, CH2-
CHdCH2), 2.65 (m, 1H, H-3), 3.59 (m, 1H, H-4), 3.80 (m, 2H,
CH2N), 4.15 (dd, 1H, J 4,5a ) 4.3 Hz, J gem ) 9.7 Hz, H-5a), 4.27
(dd, 1H, J 4,5b ) 1.6 Hz, J gem ) 9.7 Hz, J gem ) 9.7 Hz, H-5b),
5.14 (m, 2H, CHdCH2), 5.86 (m, 1H, CH-CH2), 7.26-7.36 (m,
5H, ArH); 13C NMR (75 MHz, CDCl3) δ 35.3, 45.9, 51.7, 58.4,
72.2, 118.4, 127.5, 128.1, 128.6, 134.2, 139.5, 179.1. HRCIMS
calcd for C14H18NO2 m/z 232.1337, found m/z 232.1339.
1
(MH)+; H NMR (200 MHz, CDCl3) δ 0.93 (t, 3H, J ) 6.8 Hz,
CH2CH3), 0.95 (t, 3H, J ) 6.6 Hz, CH2CH3), 1.22-1.68 (m, 8H,
2 × CH2CH2CH3), 3.87 (m, 1H, H-4), 4.44-4.61 (m, 4H, OCH2,
H-5), 4.95 (br s, 1H, NH), 5.23-5.39 (m, 2H, CH2dCH), 5.85-
5.99 (m, 1H, CH2dCH); 13C NMR (75 MHz, CDCl3) δ 14.4, 14.6,
17.1, 17.2, 32.6, 36.1, 48.2, 53.8, 66.2, 69.2, 118.4, 132.3, 155.6,
178.4. Anal. (C14H23NO4) C, H, N.

N-Substituted Furanones
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2829
(R,S)-4-t-Bu tyloxycar bon ylam in o-4,5-dih ydr o-3,3-dipr o-
p yl-2(3H)-fu r a n on e ((()-9d ). Following the same procedure
as for the preparation of compound (()-10, compound (()-8
(100 mg, 0.37 mmol) was hydrogenated and the product was
treated with BOC anhydride (110 mg, 0.5 mmol) to afford
compound (()-9d as a white solid (73 mg, 70%): mp 134-135
°C; IR (KBr) 1540, 1683, 1768, 3340 cm-1; CIMS m/z 286
) 8.9 Hz, ArH), 7.83 (d, 2H, J ) 8.9 Hz, ArH). Anal. (C17H25
NO5S‚0.1C6H14) C, H, N, S.
-
(R,S)-4,5-Dih ydr o-3,3-dipr opyl-4-(p-tr iflu or om eth ylph e-
n ylsu lfon a m id o)-2(3H)-fu r a n on e ((()-9i). Following the
same procedure as for the preparation of compound (()-10,
compound (()-8 (100 mg, 0.5 mmol) was hydrogenated and
the product was treated with p-trifluoromethylphenylsulfonyl
chloride (127 mg, 0.5 mmol), affording compound (()-9i as a
white solid (81 mg, 57%): mp 106-107 °C; IR(KBr) 1169, 1323,
1753 cm-1; CIMS m/z 394 (MH)+; 1H NMR (250 MHz, CDCl3)
δ 0.78 (t, 3H, J ) 7.1 Hz, CH2CH3), 0.86 (t, 3H, J ) 7.0 Hz,
CH2CH3), 0.93-1.62 (m, 8H, 2 × CH2CH2CH3), 3.82 (dd, 1H,
J gem ) 9.2 Hz, J 4,5a ) 7.7 Hz, H-5a), 4.03 (dt, 1H, J 4,NH ) 9.4
1
(MH)+; H NMR (250 MHz, CDCl3) δ 0.93 (t, 3H, J ) 7.0 Hz,
CH2CH3), 0.94 (t, 3H, J ) 7.1 Hz, CH2CH3), 1.25-1.44 (m, 4H,
2 × CH2CH3), 1.45 (s, 9H, (CH3)3C), 1.52-1.65 (m, 4H, 2 ×
CH2CH2), 3.83 (m, 1H, H-4), 4.47 (m, 2H, H-5), 4.66 (br s, 1H,
NH); 13C NMR (50 MHz, CDCl3) δ 14.5, 14.7, 17.2 (2C), 28.4
(3C), 32.6, 36.2, 48.4, 53.6, 69.5, 69.6, 157.2, 178.5. Anal.
(C15H27NO4) C, H, N.
Hz, J 4,5a ) 7.7 Hz, J 4,5b ) 7.5 Hz, H-4), 4.27 (dd, 1H, J gem
)
9.2 Hz, J 4,5b ) 7.5 Hz, H-5b), 5.60 (d, 1H, J 4,NH ) 9.4 Hz, NH),
7.83 (d, 2H, J ) 8.4 Hz, ArH), 8.05 (d, 2H, J ) 8.4 Hz, ArH);
13C NMR (75 MHz, CDCl3) δ 14.1, 14.4, 16.9, 17.2, 32.8, 35.5,
48.1, 55.4, 69.0, 126.6, 127.4, 127.6, 135.1, 143.7, 177.4. Anal.
(C17H22NO4SF3‚0.1H2O) C, H, N, S.
(R,S)-4,5-Dih yd r o-3,3-d ip r op yl-4-p h en yloxyca r b on y-
la m in o-2(3H)-fu r a n on e ((()-9e). Following the same pro-
cedure as for the preparation of compound (()-10, compound
(()-8 (100 mg, 0.36 mmol) was hydrogenated and the product
was treated with phenyl chloroformate (79 mg, 0.5 mmol) to
afford compound (()-9e as an off-white powder (73 mg, 67%):
(R,S)-4,5-Dih ydr o-3,3-dipr opyl-4-(1-n aph th alen esu lfon a-
m id o)-2(3H)-fu r a n on e ((()-9j). Following the same proce-
dure as for the preparation of compound (()-10, compound
(()-8 (100 mg, 0.5 mmol) was hydrogenated and the product
was treated with 1-naphthalenesulfonyl chloride (114 mg, 0.5
mmol), affording compound (()-9j as an off-white solid (67 mg,
50%): mp 210-211 °C; IR(KBr) 1165, 1332, 1755, 2962, 3240
mp 138-139 °C; IR(KBr) 1542, 1705, 1768, 2960, 3312 cm-1
;
1
CIMS m/z 306 (MH)+; H NMR (250 MHz, CDCl3) δ 0.93 (t,
3H, J ) 7.1 Hz, CH2CH3), 0.97 (t, 3H, J ) 7.0 Hz, CH2CH3),
1.29-1.70 (m, 8H, 2 × CH2CH2CH3), 3.93 (m, 1H, H-4), 4.57
(m, 2H, H-5), 5.24 (br s, 1H, NH), 7.05-7.13 (m, 2H, ArH),
7.23-7.26 (m, 1H, ArH), 7.35-7.41 (m, 2H, ArH); 13C NMR
(62.5 MHz, CDCl3) δ 14.5, 14.7, 17.3 (2C), 32.9, 36.2, 48.5, 54.1,
69.2, 121.5, 122.9, 125.9, 129.3, 129.6, 150.7, 154.2, 178.4.
Anal. (C17H23NO4‚0.2H2O) C, H, N.
1
cm-1; CIMS m/z 376 (MH)+; H NMR (250 MHz, DMSO-d6) δ
0.69 (t, 3H, J ) 7.0 Hz, CH2CH3), 0.72 (t, 3H, J ) 6.6 Hz,
CH2CH3), 0.84-1.48 (m, 8H, 2 × CH2CH2CH3), 3.85 (dd, 1H,
J gem ) 8.6 Hz, J 4,5a) 7.6 Hz, H-5a), 4.06 (m, 1H, H-4), 4.17 (s,
1H, NH), 4.26 (dd, 1H, J gem ) 8.6 Hz, J 4,5b ) 8.4 Hz, H-5b),
7.75-7.90 (m, 3H, ArH), 8.25 (dd, 1H, J ) 1.3 and 7.7 Hz,
ArH), 8.31 (d, 1H, J ) 7.2 Hz, ArH), 8.39 (d, 1H, J ) 7.9 Hz,
ArH), 8.79 (d, 1H, J ) 8.3 Hz, ArH); 13C NMR (75 MHz,
DMSO-d6) δ 13.6, 13.8, 15.6, 16.2, 31.9, 34.0, 47.2, 54.3, 67.5,
124.2, 126.6, 126.9, 127.7 (2C), 128.6, 128.7, 133.5, 134.0,
135.1, 177.1. Anal. (C20H25NO4S), C, H, N.
(R,S)-4-Ben zam ido-4,5-dih ydr o-3,3-dipr opyl-2(3H)-fu r a-
n on e ((()-9f). Following the same procedure as for the
preparation of compound (()-10, compound (()-8 (100 mg, 0.36
mmol) was hydrogenated and the product was treated with
benzoyl chloride (70 mg, 0.5 mmol) to afford compound (()-9f
as a white solid (59 mg, 57%): mp 130-131 °C; IR(KBr) 1547,
1
1637, 1767, 3306 cm-1; CIMS m/z 290 (MH)+; H NMR (300
MHz, CDCl3) δ 0.91 (t, 3H, J ) 7.9 Hz, CH2CH3), 0.95 (t, 3H,
J ) 7.2 Hz, CH2CH3), 1.26-1.74 (m, 8H, 2 × CH2CH2CH3),
4.03 (dd, 1H, J 4,5a ) 9.7 Hz, J 4,5b ) 5.5 Hz, H-4), 4.61 (dd, 1H,
J 4,5a ) 9.7 Hz, J gem ) 7.1 Hz, H-5a), 4.98 (m, 1H, H-5b), 6.65
(br s, 1H, NH), 7.39-7.57 (m, 3H, ArH), 7.78 (dd, 2H, J ) 7.1
and 1.4 Hz, ArH); 13C NMR (75 MHz, CDCl3) δ 14.4, 14.7, 17.1,
17.2, 32.3, 36.1, 48.8, 52.8, 70.2, 127.0, 128.8, 132.1, 133.7,
167.3, 178.9. Anal. (C17H23NO3) C, H, N.
(R,S)-4,5-Dih yd r o-3,3-d ip r op yl-4-(2-th iop h en esu lfon a -
m id o)-2(3H)-fu r a n on e ((()-9k ). Following the same proce-
dure as for the preparation of compound (()-10, compound
(()-8 (100 mg, 0.36 mmol) was hydrogenated and the product
was treated with 2-thiophenesulfonyl chloride (92 mg, 0.5
mmol), affording compound (()-9k as a white powder (94 mg,
79%): mp 92-93 °C; IR(KBr) 1154, 1350, 1774, 2962, 3258
cm-1; CIMS m/z 332 (MH)+; 1H NMR (250 MHz, CDCl3) δ 0.83
(t, 3H, J ) 7.0 Hz, CH2CH3), 0.90 (t, 3H, J ) 6.8 Hz, CH2CH3),
1.09-1.58 (m, 8H, 2 × CH2CH2CH3), 3.82 (dd, 1H, J 4,5a ) 8.2
Hz, J gem ) 9.2 Hz, H-5a), 4.07-4.16 (m, 1H, H-4), 4.29 (dd,
(R,S)-4-(p-Ch lor op h en ylsu lfon a m id o)-4,5-d ih yd r o-3,3-
d ip r op yl-2(3H)fu r a n on e ((()-9g). Following the same pro-
cedure as for the preparation of compound (()-10, compound
(()-8 (100 mg, 0.36 mmol) was hydrogenated and the product
was treated with p-chlorophenylsulfonyl chloride (106 mg, 0.5
mmol), affording compound (()-9g as a white solid (100 mg,
84%): mp 96-97 °C; IR(KBr) 1174, 1478, 1758 cm-1; CIMS
1H, J gem ) 9.2 Hz, J 4,5b ) 7.8 Hz, H-5b), 5.17 (d, 1H, J 4,NH
)
8.6 Hz, NH), 7.14 (t, 1H, J ) 4.6 Hz, ArH), 7.68 (d, 2H, J )
4.6 Hz, ArH); 13C NMR (75 MHz, CDCl3) δ 14.4, 14.6, 17.3,
17.4, 33.4, 35.9, 48.2, 55.5, 69.1, 127.9, 133.1 (2C), 140.6, 177.2.
Anal. (C14H21NO4S2) C, H, N, S.
1
m/z 360 (MH+ with 35Cl), 362 (MH+ with 37Cl); H NMR (250
MHz, CDCl3) δ 0.80 (t, 3H, J ) 7.1 Hz, CH2CH3), 0.88 (t, 3H,
J ) 6.9 Hz, CH2CH3), 1.01-1.63 (m, 8H, 2 × CH2CH2CH3),
3.79 (dd, 1H, J gem ) 9.1 Hz, J 4,5a ) 8.0 Hz, H-5a), 4.01 (dt, 1H,
J 4,NH ) 9.1 Hz, J 4,5 ) 8.0 Hz, H-4), 4.25 (dd, 1H, J gem ) 9.1
Hz, J 4,5b ) 8.0 Hz, H-5b), 5.28 (d, 1H, J 4,NH ) 9.1 Hz, NH),
7.54 (d, 2H, J ) 9.1 Hz, ArH), 7.84 (d, 2H, J ) 9.1 Hz, ArH);
13C NMR (75 MHz, CDCl3) δ 14.1, 14.4, 17.0, 17.2, 33.0, 35.6,
(R,S)-4,5-Dih yd r o-4-(d im eth yla m in osu lfon a m id o)-3,3-
d ip r op yl-2(3H)-fu r a n on e ((()-9l). Following the same pro-
cedure as for the preparation of compound (()-10, compound
(()-8 (100 mg, 0.36 mmol) was hydrogenated and the product
was treated with dimethylsulfamoyl chloride (72 mg, 0.5
mmol), affording compound (()-9l as an off-white powder (58
mg, 55%): mp 62-63 °C; IR(KBr) 1149, 1349, 1778, 2960, 3277
cm-1; CIMS m/z 293 (MH)+; 1H NMR (200 MHz, CDCl3) δ 0.94
(t, 6H, J ) 6.9 Hz, 2 × CH2CH3), 1.18-1.73 (m, 8H, 2 ×
CH2CH2CH3), 2.83 (s, 6H, N(CH3)2), 3.99 (m, 1H, H-5a), 4.13
(m, 1H, H-4), 4.48-4.62 (m, 2H, H-5b, NH); 13C NMR (75 MHz,
CDCl3) δ 14.5, 14.6, 17.4, 17.6, 33.2, 35.9, 38.2, 48.2, 55.6, 69.5,
177.7. Anal. (C12H24N2O4S) C, H, N.
(R)-4-Ben zyla m in o-4,5-d ih yd r o-2(3H)-fu r a n on e ((R)-5).
A solution of (R)-4-benzyloxycarbonylamino-4,5-dihydro-2(3H)-
furanone ((R)-10, 150 mg, 0.64 mmol) (prepared from D-
aspartic acid)38,39 in ethyl acetate (10 mL) was stirred under
hydrogen (30 psi) at room temperature for 4 h in the presence
of 10% palladium on carbon (100 mg). The reaction mixture
was filtered through Celite, the filter pad was washed with
ethyl acetate, and the combined filtrate and washings were
48.0, 55.2, 68.9, 128.5, 129.8, 138.5, 140.1, 177.4. Anal. (C16H22
-
NO4SCl‚0.1C6H14) C, H, N, S.
(R,S)-4,5-Dih yd r o-3,3-d ip r op yl-4-(p -m et h oxyp h en yl-
su lfon a m id o)-2(3H)-fu r a n on e ((()-9h ). Following the same
procedure as for the preparation of compound (()-10, com-
pound (()-8 (100 mg, 0.36 mmol) was hydrogenatated and the
product was treated with p-methoxyphenylsulfonyl chloride
(104 mg, 0.5 mmol), affording compound (()-9h as a white
powder (107 mg, 84%): mp 103-104 °C; IR(KBr) 1168, 1323,
1754 cm-1; CIMS m/z 356 (MH)+; 1H NMR (300 MHz, CDCl3)
δ 0.78 (t, 3H, J ) 7.1 Hz, CH2CH3), 0.88 (t, 3H, J ) 6.9 Hz,
CH2CH3), 1.07-1.59 (m, 8H, 2 × CH2CH2CH3), 3.76 (dd, 1H,
J gem ) 9.1 Hz, J 4,5a ) 8.3 Hz, H-5a), 3.89 (s, 3H, OCH3), 3.98
(q, 1H, J ) 8.3 Hz, H-4), 4.21 (dd, 1H, J gem ) 9.1 Hz, J 4,5b
)
8.3 Hz, H-5b), 4.99 (d, 1H, J 4,NH ) 8.3 Hz, NH), 7.01 (d, 2H, J

2830 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
El Hadri et al.
evaporated under reduced pressure. The residue was taken
up in methanol (3 mL), benzaldehyde (0.065 mL, 0.64 mmol)
was added with cooling, and the reaction mixture was stirred
for 1 h. Methanol (17 mL) and platinum oxide catalyst (100
mg) were added, and the mixture was hydrogenated at 40 psi
for 3 h. After removal of the catalyst by filtration through
Celite, the filtrate was evaporated to dryness under reduced
pressure and the residue was purified by column chromatog-
raphy on silica gel (ethyl acetate-hexane 1:1), affording
compound (R)-5 as a colorless oil (70 mg, 57%) whose spectral
characteristics were identical to racemic 5 prepared above:
GABAA receptor channel or to allosterically modulate currents
elicited by GABA. For this purpose, recombinant GABAA
receptors were expressed in Xenopus oocytes. Briefly, Xenopus
laevis oocytes were prepared, injected, and defolliculated and
currents were recorded as described.40,41 Oocytes were injected
with 50 nL of cRNA dissolved in 5 mM K-Hepes (pH 6.8). This
solution contained the transcripts coding for the different
subunits at a concentration of 10 nM for R1, 10 nM for â2,
and 50 nM for γ2. For dual subunit combinations, a concentra-
tion of 100 nM was used for each transcript (R1, â2, and the
point mutant â2N265S). RNA transcripts were synthesized
from linearized plasmids encoding the desired protein using
the message machine kit (Ambion) according to the recom-
mendation of the manufacturers. A poly(A) tail of about 300
residues was added to the transcripts by using yeast poly(A)
polymerase (USB or Amersham). The cRNA combinations were
coprecipitated in ethanol and stored at -20 °C. Transcripts
were quantified on agarose gels after staining with Radiant
Red RNA stain (Bio-Rad) by comparing staining intensities
with various amounts of molecular weight markers (RNA-
Ladder, Gibco-BRL). Electrophysiological experiments were
performed by the two-electrode voltage clamp method at a
holding potential of -80 mV. The medium contained 90 mM
NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM Na-
Hepes (pH 7.4). GABA eliciting about 2-4% of the maximal
current amplitude was applied for 20 s, and a washout period
of 4 min was allowed to ensure full recovery from desensitiza-
tion. The perfusion system was cleaned between drug applica-
tions by washing with dimethyl sulfoxide to avoid contami-
nation. Drugs were applied at a concentration of 100 µM in
the absence of GABA to see whether the drugs could act as a
channel agonist. To study allosteric modulation, GABA was
first applied alone and subsequently in combination with
either 0.1 or 100 µM of the drug.
[R]25 +16.5° (c 1.0, CHCl3).
D
(R)-4-Ben zyla m in o-3,3-d ia llyl-4,5-d ih yd r o-2(3H)-fu r a -
n on e ((R)-8). Following the same procedure as for the
preparation of racemic 8, a solution of the lithium enolate of
compound (R)-5 (92 mg, 0.49 mmol) in THF was treated at
-78 °C with a solution of allyl bromide (0.23 mL, 2.44 mmol)
in HMPA (1 mL). The reaction mixture was stirred at -78 °C
and then worked up as before. Isolation of the least polar
product by chromatography on silica gel (ethyl acetate-hexane
1:1) afforded compound (R)-8 as an oil (68 mg, 52%) whose
spectroscopic characteristics were identical to racemic 8: [R]25
+1.4° (c 0.8, CHCl3).
D
(R)-4-Ben zyloxyca r bon yla m in o-4,5-d ih yd r o-3,3-d ip r o-
p yl-2(3H)-fu r a n on e ((R)-9a ). Following the same procedure
as for the preparation of compound (()-9a , compound (R)-8
(100 mg, 0.36 mmol) was hydrogenated and the product was
treated with benzyl chloroformate (86 mg, 0.5 mmol) to afford
compound (R)-9a as a colorless oil (65 mg, 57%). The spectro-
scopic characteristics of compound (R)-9a were identical to
those of racemic 9a : [R]25 -0.28° (c 1.44, CHCl3).
D
Sep a r a tion of th e En a n tiom er s of Com p ou n d 9a .
Racemic 9a (40 mg) was subjected to HPLC on a chiral OD
column using hexane-2-propanol (9:1) as the eluent. The
fraction having the shorter retention time had an optical
rotation ([R]25 -0.28° (c 1.0, CHCl3)) corresponding to R-9a ,
Ack n ow led gm en t. We thank the European Union
(Grants BI04-CT96-0585 and BBW96.0010) and the
Swiss National Science Foundation (Grant 3100-
053599.98/1) for financial support.
D
prepared from D-aspartic acid as described above. The fraction
having the longer retention time ([R]25D +0.24° (c 1.0, CHCl3))
thus corresponded to (S)-9a .
Recep tor Bin d in g Stu d ies. The synthesized compounds
were tested in vitro for their capacity to bind to different sites
on the GABAA receptor, including the GABA site itself (by
displacement studies of [3H]-muscimol), the benzodiazepine
site (by displacement of [3H]-flunitrazepam), and the picrotoxin
site (by displacement of [35S]-TBPS). Briefly, frozen mem-
branes from cerebellum or from brain without cerebellum were
thawed, centrifuged, and resuspended in 50 mM Tris-citrate
buffer, pH 7.4, at a protein concentration of about 1 mg/mL.
Membranes (0.5 mL) were then incubated in a total of 1 mL
of a solution containing 50 mM Tris-citrate buffer, pH 7.4, 150
mM NaCl, and 2 nM [3H]-flunitrazepam or 2 nM [3H]-
muscimol in the absence or presence of various concentrations
of the compounds to be investigated or of 10 µM diazepam or
10 µM GABA, for 90 min at 4 °C, respectively. For [35S]-TBPS
binding, membranes were incubated in a total of 1 mL of a
solution containing 50 mM Tris-citrate buffer, pH 7.4, 200 mM
NaBr, and 2 nM [35S]-TBPS in the absence or presence of
various concentrations of the compounds to be investigated
or of 10 µM TBPS or picrotoxinin for 180 min at room
temperature.42,43
Membranes were then filtered through Whatman GF/B
filters. When [3H]-flunitrazepam or [3H]-muscimol binding was
investigated, the filters were rinsed twice with 5 mL of ice-
cold 50 mM Tris-citrate buffer. When [35S]-TBPS binding was
investigated, the filters were rinsed three times with 3.5 mL
of this buffer. Filters were transferred to scintillation vials and
subjected to scintillation counting after addition of 3.5 mL of
scintillation fluid. Nonspecific binding determined in the
presence of 10 µM diazepam, 10 µM GABA, or 10 µM TBPS
was subtracted from total [3H]-flunitrazepam, [3H]-muscimol,
or [35S]-TBPS binding, respectively, to result in specific bind-
ing.
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