Enaminone amides as novel orally active GABAA receptor modulators

Article abstract of DOI:10.1021/jm070083v

A series of enaminone esters and amides have been developed as potent allosteric modulators of γ-aminobutyric acid_A (GABA _A) receptors. The compounds bind to a novel modulatory site that is independent of the benzodiazepine (BZ), isosteric GABA, and neuroactive steroid binding sites. Structure-activity relationship (SAR) studies resulted in the synthesis of the c-Bu amide 16h with an in vitro potency of 7 nM based on inhibition of [³⁵S]TBPS binding. The activity of the enaminones as positive allosteric modulators was confirmed with electrophysiological measurements in oocytes expressing α₁β₂γ _2L GABA_A receptors. The i-Pr, s-Bu, c-Pr, and c-Bu amides (16e-h) were orally active in mice with profound central nervous system depressant effects. The i-Pr amide 16e was an orally active anxiolytic in the mouse light-dark paradigm.

Full text of DOI:10.1021/jm070083v

J. Med. Chem. 2007, 50, 3369-3379
3369
Enaminone Amides as Novel Orally Active GABAA Receptor Modulators
Derk J. Hogenkamp,* Timothy B. C. Johnstone, Jin-Cheng Huang, Wen-Yen Li, Minhtam Tran, Edward R. Whittemore,
Rudy E. Bagnera, and Kelvin W. Gee
Department of Pharmacology, School of Medicine, Med Surge 2, UniVersity of CaliforniasIrVine, IrVine, California 92697
ReceiVed January 22, 2007
A series of enaminone esters and amides have been developed as potent allosteric modulators of
γ-aminobutyric acidA (GABAA) receptors. The compounds bind to a novel modulatory site that is independent
of the benzodiazepine (BZ), isosteric GABA, and neuroactive steroid binding sites. Structure-activity
relationship (SAR) studies resulted in the synthesis of the c-Bu amide 16h with an in vitro potency of 7 nM
based on inhibition of [35S]TBPS binding. The activity of the enaminones as positive allosteric modulators
was confirmed with electrophysiological measurements in oocytes expressing R1â2γ2L GABAA receptors.
The i-Pr, s-Bu, c-Pr, and c-Bu amides (16e-h) were orally active in mice with profound central nervous
system depressant effects. The i-Pr amide 16e was an orally active anxiolytic in the mouse light-dark
paradigm.
γ-Aminobutyric acid (GABAa) is the major inhibitory neu-
Chart 1. Structures of 1 and 2a
rotransmitter in the mammalian brain. There are three types of
GABA receptors, termed GABAA, GABAB, and GABAC
receptors.1 GABAA and GABAC receptors are ligand gated ion
channels that conduct chloride ions, whereas GABAB receptors
are G-protein coupled receptors. GABAA receptors are hetero-
pentameric in structure and belong to the cys-loop receptor
family that includes R7 nicotinic acetylcholine, glycine, and
serotonin-3A receptors. Allosteric modulators of GABAA recep-
tors2 such as benzodiazepines (BZ),3 neuroactive steroids,4 and
Chart 2. Z- and E-Isomers of 2a
barbiturates5 have been identified that are useful as anxiolytics,
anticonvulsants, anesthetics, and sedative-hypnotics. Numerous
non-BZ ligands that bind to the BZ-site on the GABAA receptor
have also been described, including imidazopyridines, pyrazol-
opyrimidines, and triazolopyrimidines.6 Side-effects associated
with BZ-site ligands include dependence, tolerance, strong
interactions with alcohol, and amnesic and myorelaxant effects.7
Novel allosteric modulators8 that bind to the GABAA receptor
at sites other than the BZ-site may offer an opportunity to
discover central nervous system (CNS) agents with improved
side effect profiles.
As part of a program to develop novel quinolones as
the dimethylenaminone (4a), which underwent substitution with
4-fluoroaniline at room temperature in EtOH to afford 2a.12 The
1H NMR of 2a in CDCl3 indicates that the compound is a 1:1
mixture of double bond isomers. The chemical shifts are very
different for the aniline NH in the two isomers. In one isomer
the aniline NH is a doublet at 12.7 ppm (coupled to the vinyl
proton) and in the second isomer the aniline NH is a doublet at
11.3 ppm. The coupling constant between the NH and the vinyl
proton in both isomers is 13 Hz, strongly suggesting an
antiperiplanar relationship for the two protons.13 The isomer
with the aniline NH at 12.7 ppm incorporates a hydrogen bond
(H-bond) between the aniline NH and the ester carbonyl (2aZ,
Chart 2), and the second isomer has an H-bond between the
aniline NH and the ketone carbonyl (2aE). These structure-
NMR correlations are supported by assignments in related
systems.14
modulators of GABAA receptors, 7-chloro-1-ethyl-6-[(1,2,3,4-
tetrahydro-1-naphthylenyl)amino]-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid (1, Chart 1) was identified as an allosteric
modulator of the GABAA receptor with an anxioselective profile
in vivo.9 An intermediate in the synthesis of N-1 aryl substituted
analogs of 1, ethyl 2,4-dichloro-5-fluoro-R-[[(4-fluorophenyl)-
amino]methylene]-â-oxobenzenepropanoate (2a), was found to
have GABAA receptor agonist activity based on its ability to
allosterically inhibit the binding of [35S]-tert-butylbicyclophos-
phorothionate ([35S]TBPS)10 to rat brain cortical homogenates
with an IC50 ) 150 nM (Imax 80%). The synthesis of 2a and its
analogs is shown in Scheme 1.11 Reaction of 2,4-dichloro-5-
fluorobenzoyl chloride (3a) with ethyl 2,2-dimethylaminoacryl-
ate in the presence of diisopropylethyl- or triethylamine afforded
* Corresponding author. Phone: (949) 824-5670. Fax: (949) 824-4855.
E-mail: dhogenka@uci.edu.
Analogs of 2a were synthesized with modifications to the
substituents on the aryl group of the ketone (Table 1). For
comparison, data for the endogenous neurosteroid 5R-pregnan-
3R-ol-20-one (3R,5R-P) are included.15 The 2- and 3-chloroaryl
ketones (2b and 2c) show 2- and 7-fold losses, respectively, in
a Abbreviations: GABA, γ-aminobutyric acid; TBPS, tert-butylbicyclo-
phosphorothionate; LRR, loss-of-righting reflex; 5R-pregnan-3R-ol-20-one,
3R,5R-P; N,N-dimethylformamide dimethyl acetal, DMFDMA; hydrogen
bond, H-bond; MED, minimum effective dose; TI, therapeutic index; PTZ,
pentylenetetrazole; 3R,20R-diol, 5R-pregnane-3R,20R-diol.
10.1021/jm070083v CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/16/2007

3370 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Scheme 1. Synthesis of Enaminones 2a-d
Hogenkamp et al.
Table 1. In Vitro Potency of Ethyl R-[[(4-Fluorophenyl)amino]methyl-
ene]-â-oxophenylpropanoatesa
Scheme 2. Synthesis of Acid 11
IC50 for inhibn
of [35S]TBPS
compd
R1
binding (nM)b
Imax (%)
3R,5R-Pc
-
50 ( 5
95 ( 1
87 ( 6
2a
2b
2c
2d
2,4-diCl, 5-F
2-Cl
3-Cl
130 ( 27
300 ( 55
900 ( 370
490 ( 84
93 ( 7
90 ( 16
100 ( 8
with 10 showing full displacement of [35S]TBPS binding.
Overall, the SAR indicates that adding steric bulk at the
4-position of the aniline does not improve in vitro potency in
the series where R2 ) F, Me, and i-Pr. The increased activity
for the 4-chloroaniline 5 is thus not due to its size but potentially
is related to its ability to act as a resonance donor to the aniline
ring. The influence of an H-bonding interaction with the halogen
at the 4-position of the aniline is unlikely because of the known
decrease in the strength of H-bonds when fluorine is replaced
with chlorine.16 Potency is reduced when the chlorine is shifted
to the 2- or 3-position of the aniline (6 and 7) presumably
because of unfavorable steric interactions with the receptor. The
increase in potency observed with 4-ethynyl substitution is likely
related to a specific interaction of the triple bond π-system with
the receptor.17
4-Cl
a Values are means and SEMs of three indpendent experiments. b The
concentration of test compound inhibiting 50% specific binding (IC50) and
the maximal extent of inhibition (Imax) were calculated by fitting the data
to the sigmodial function. c 5R-Pregnan-3R-ol-20-one (3R,5R-P) data are
taken from ref 15.
Table 2. In Vitro Potency of Ethyl 2-Chloro-â-oxo-R-[(phenylamino)-
methylene]benzenepropanoates
For comparison with the ethyl ester 5, the corresponding acid
11 was prepared as shown in Scheme 2. Meldrum’s acid was
acylated with 2-chlorobenzoyl chloride to give the expected
adduct (12), which was conveniently purified and stored by
conversion to the isopropylamine salt (12a).18 Partitioning the
salt between EtOAc and a 1 M aqueous HCl solution re-formed
the acid 12. The isolated acid was then heated with excess tert-
butyl alcohol to form the desired â-keto ester (13).19 Addition
of N,N-dimethylformamide dimethyl acetal (DMFDMA) then
gave the corresponding enaminone 14. Reaction of 14 with
4-chloroaniline afforded the tert-butyl ester 15, and subsequent
cleavage of the ester with trifluoroacetic acid gave the desired
acid 11.
IC50 for inhibn
of [35S]TBPS
binding (nM)
compd
R2
4-F
4-Cl
2-Cl
Imax (%)
2b
5
6
7
8
300 ( 55
90 ( 10
700 ( 110
600 ( 130
450 ( 150
360 ( 75
8 ( 1
93 ( 7
95 ( 10
100 ( 7
100 ( 8
93 ( 12
94 ( 8
3-Cl
4-Me
4-iPr
4-CCH
9
10
100 ( 7
potency compared to 2a, while the 4-chloroaryl ketone (2d)
shows almost a 4-fold loss in activity. All of the esters were
mixtures of isomers by H NMR.
1
Substitutions to the aniline ring were targeted because the
initial SAR on the aryl ketone did not lead to increases in
potency. For ease of synthesis, the SAR around the 2-chloride
(2b) was investigated further (Table 2). Within the halogen
series, the in vitro potency was found to increase to 90 nM for
the 4-chloroaniline 5. The 2- and 3-chloroanilines, 6 and 7, were
less potent than the 4-isomer, with IC50 values of 700 and 600
nM, respectively. Small alkyl groups at the 4-position resulted
in a loss in activity compared to 2b with the 4-methyl and
4-isopropyl compounds (8 and 9) having IC50 values of 450
and 360 nM, respectively. The 4-ethynyl compound 10 was the
most potent compound in this series, with an IC50 of 8 nM.
Increases in potency did not come at the expense of efficacy,
The tert-butyl ester 15 was active in vitro ([35S]TBPS IC50
) 320 nM), while the acid 11 was essentially inactive ([35S]-
TBPS IC50 ) 7000 nM). Because of the poor activity of the
acid, it is very unlikely that the ethyl esters are simply prodrugs
that are converted to the acid under the [35S]TBPS assay
conditions. Compound 5 was selected for initial profiling in vivo
because of its good in vitro potency when compared to known
modulators of the GABAA receptor (e.g., 3R,5R-P) and the hope
that the chloride substitution on the aryl ketone and aniline
would retard metabolism of these two rings. The ethyl ester 5
caused all animals tested to lose their righting reflex (LRR)9
for 40 min when dosed intraperitoneally (ip) at 50 mg/kg in
mice. No mortality was observed at this dose after 24 h. Not

Enaminone Amides as GABAA Receptor Modulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3371
Table 3. In Vitro Potency of N-Alkyl-2-chloro-R-[[(4-chlorophenyl)-
amino]methylene]-â-oxobenzenepropanamidesa
Scheme 3. Synthesis of Enaminone Amide 16a
IC50 for inhibn
of [35S]TBPS
compd
R3
binding (nM)
Imax (%)
16b
16c
16a
16d
16e
16f
16g
16h
16i
H
Me
Et
1700 ( 770
160 ( 27
50 ( 5
45 ( 7
40 ( 13
20 ( 2
30 ( 4
7 ( 2
40 ( 18
74 ( 8
82 ( 4
90 ( 7
80 ( 10
86 ( 4
92 ( 5
88 ( 6
88 ( 5
86 ( 6
100
Chart 3. Comparison of Hydrogen Bonding within Enaminone
Amides and Esters
Pr
i-Pr
s-Bu
c-Pr
c-Bu
c-Pentyl
1-ethylpropyl
t-Bu
30 ( 4
80 ( 9
3000
16j
16k
16l
1,2-diMePr
400 ( 42
94 ( 7
a Values are means and SEMs of three independent experiments, except
for 16k (n ) 1).
Scheme 4. Synthesis of Amides 16b,c,f, and i-l from Acid 11
surprisingly, however, the ethyl ester 5 was not active in
inducing LRR when tested orally (po) at 50 mg/kg in mice. 5
was inactive at this dose when evaluated for its ability to
suppress locomotor activity. It was likely that the ester was
rapidly being converted to the inactive acid 11 when dosed
orally.
On the basis of these results, the ethyl ester in 5 was replaced
with an ethyl amide as a potential metabolically stable bioiso-
stere. The ethyl amide 16a was prepared as shown in Scheme
3. Reaction of 2-chlorobenzoyl Meldrum’s acid (12) with
ethylamine afforded the expected â-ketoamide 17a.20 The
corresponding enaminone 18a was prepared as in Scheme 2,
and addition of 4-chloroaniline at reflux in toluene gave the
desired enaminone amide 16a.
Scheme 5. Alternative Synthesis of â-Ketoamides 17d,e,g,h,
and k
The amide 16a was found to be more potent in vitro than
the corresponding ester 5 ([35S]TBPS IC50 ) 50 and 90 nM,
1
respectively). The H NMR of 16a, unlike the corresponding
4). This method worked well for the preparation of most amides.
However, when the crude acid chloride derived from 11 was
treated with isopropyl-, cyclopropyl-, or cyclobutylamine, only
very low yields of the desired amides were isolated. These
compounds (16e, 16g, and 16h) and the propyl amide 16d were
prepared by using the route in Scheme 3 but using an alternative
route to the intermediate â-ketoamide 17 given in Scheme 5.
The tert-butyl ester 13 was prepared as in Scheme 2 and then
cleaved to the â-keto acid 19. Amide bond formation with
dicyclohexyl carbodiimide (DCC) or via the corresponding acid
chloride gave the â-ketoamide 17. Conversion to the desired
ester, did not show a mixture of isomers. Only a single aniline
NH and vinyl proton were apparent. This difference between
the esters and amides can be understood if the amide exists as
the Z-isomer (16aZ, Chart 3), where two H-bonds can form,
one between the aniline NH and the carbonyl of the amide and
a second between the amide NH and the ketone carbonyl. The
corresponding E-isomer (16aE) can benefit from only one
H-bond, between the aniline NH and the ketone carbonyl,
making it less favored energetically than the Z-isomer. In the
esters, both the Z- and E-isomers have a single H-bond (5Z
and 5E, Chart 3), and the two isomers are closer in energy than
in the enaminone amides. Enaminones amides related to 16a
have been reported to exist as single isomers.21
The ethyl amide 16a was dosed in mice as described for the
corresponding ester. While all mice tested showed LRR at 50
mg/kg ip, no LRR or rotarod9 deficit was apparent orally. The
amide side chain (R3, Table 3) was systematically varied in order
to better understand this result. A more convergent method for
the synthesis of analogs of amide 16a was employed where the
acid 11 was converted to the corresponding acid chloride with
thionyl chloride and then treated with the desired amine (Scheme
1
product was accomplished as in Scheme 3. In each case, H
NMR showed the amides to exist as single isomers.
The unsubstituted amide 16b was a weak inhibitor of [35S]-
TBPS binding, exhibiting an 34-fold loss in potency compared
to the ethyl amide 16a. The methyl amide (16c) was 3-fold less
potent in vitro than the ethyl amide. The lack of oral activity
for the ethyl amide may be due to dealkylation to the weakly
active unsubstituted amide 16b. The corresponding tert-butyl
amide 16k was prepared to eliminate this possibility. Surpris-
ingly, given the activity of the corresponding tert-butyl ester
(15, [35S]TBPS IC50 ) 320 nM), the tert-butyl amide was only

3372 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Hogenkamp et al.
Figure 1. Dose-dependent effect of 2-chloro-R-[[(4-chlorophenyl)-
amino]methylene]-N-isopropyl-â-oxobenzenepropanamide (16e, 1 and
3 mg/kg po, N ) 10) and 3R,21-dihydroxy-3â-trifluoromethyl-5â-19-
norpregnan-20-one (Co 2-6749; 10 mg/kg po, N ) 59) compared to
vehicle (3:1 PEG 400/5% dextrose in water, N ) 19) on time spent in
the dark chamber in the mouse light-dark paradigm. *Standard
deviation from vehicle P < 0.001 Anova, <0.5 Dunnett’s multiple
comparison test.
Figure 2. Dose response of ethyl 2-chloro-R-[[(4-ethynylphenyl)amino]-
methylene]-â-oxobenzenepropanoate (10), 2-chloro-R-[[(4-ethynylphe-
nyl)amino]methylene]-â-oxo-N-propylbenzenepropanamide (20), 3R,5R-
P, and clonazepam on 0.2 nM [3H]flunitrazepam binding in rat cortex.
Chart 4. Structures of 10 and 2-Chloro-R-[[(4-ethynylphenyl)-
amino]methylene]-â-oxo-N-propylbenzenepropanamide (20)
weakly active in vitro ([35S]TBPS IC50 ) 3000 nM). The propyl
amide (16d) was prepared with the goal of adding bulk to the
ethyl amide without compromising in vitro activity. We found
16d to be equipotent to the ethyl amide 16a. Amide 16d
exhibited an in vivo profile identical to that of the ethyl amide:
mice showed LRR when dosed ip at 50 mg/kg but not orally at
50 mg/kg. To further increase the steric bulk of R3, the isopropyl,
sec-butyl, and cyclopentyl amides 16e, 16f, and 16i were
prepared. All three compounds were similar in potency to 16a.
The 1-ethylpropyl amide 16j was also targeted and found to be
1.5-fold less potent than 16a. The 1,2-dimethylpropyl homolog
of 16f (16l) resulted in a 8-fold loss in activity. The cyclopropyl
amide 16g inhibited [35S]TBPS binding with an IC50 of 30 nM.
The cyclobutyl amide 16h was the most potent enaminone amide
tested with an IC50 of 7 nM. The in vitro data indicate a positive
role for lipophilicity in the series R3 ) H, Me, and Et. The
propyl amide 16d does not have improved potency. Within the
branched amides, the isopropyl amide 16e is similar in potency
to the ethyl amide. Adding bulk to give the sec-butylamide 16f
doubles potency, while the addition of an additional methyl
group in the 1-ethylpropyl amide 16j results in a 4-fold loss
relative to 16f. The activity of the cyclic amides shows a
preference for the cyclobutyl amide 16h. Ring contraction or
expansion results in a 4-fold loss in activity. Taken together,
these data support the presence of a lipophilic pocket in the
receptor of limited size that accommodates bulk only in specific
directions.
The isopropyl, sec-butyl, cyclopropyl, and cyclobutylamides
(16e, 16f, 16g, and 16h) were found to cause LRR orally in
mice at 50 mg/kg. The anxiolytic activity of the isopropylamide
16e was determined in the mouse light-dark paradigm.23 When
dosed orally at 3 mg/kg, 16e significantly reduced the time mice
spent in the dark (Figure 1) compared to vehicle alone. No effect
was observed for 16e orally at 1 mg/kg. The neurosteroid 3R,-
21-dihydroxy-3â-trifluoromethyl-5â-19-norpregnan-20-one (Co
2-6749) was used as a positive control and was active orally at
10 mg/kg.24 The CNS depressant potential of 16e was evaluated
in the rotarod test. No rotarod deficit was observed orally at 3
mg/kg, and only one of seven animals was found to fail the
test at 30 mg/kg. On the basis of these data, 16e has a therapeutic
index (TI) of >10 (TI ) light-dark MED/rotarod TD50).
The oral activity observed with the amides tested with
branching at the carbon atom bonded to the amide nitrogen
(16e-h) is likely due to reduced metabolism to the inactive
unsubstituted amide 16b or acid 11 when dosed orally.
None of the amides were found to be active as anticonvulsants
when tested ip at 50 mg/kg prior to sc dosing of 85 mg/kg of
the chemical convulsant pentylenetetrazole (PTZ).
In order to confirm that the enaminones are positive allosteric
modulators of GABAA receptors in a functional assay, 16e was
evaluated for its ability to potentiate GABA currents in human
R1â2γ2L GABAA receptors expressed in Xenopus oocytes.25
Amide 16e showed robust activity as a modulator electrophysi-
ologically, with an EC50 of 34 ( 2 nM and modulation of the
GABA EC10 to 88% of the maximum GABA response. In the
absence of GABA, 16e did not elicit a response up to a
concentration of 10 µM (limit of solubility), confirming its mode
of action as an allosteric modulator.
To more fully define the in vitro pharmacological profile of
the enaminones, the ethyl ester 10 ([35S]TBPS IC50 ) 8 nM)
and the corresponding propyl amide, 2-chloro-R-[[(4-
ethynylphenyl)amino]methylene]-â-oxo-N-propylbenzenepro-
panamide (20, Chart 4), were tested for their ability to interact
with the BZ binding site on the GABAA receptor. The amide
20 was prepared by using the general method in Scheme 3 and
was a potent modulator of [35S]TBPS binding with IC50 ) 54
( 1 nM (Imax ) 91 ( 3%). Both 10 and 20 dose-dependently
enhance the binding of [3H]flunitrazepam (EC50 ) 6 ( 2 and
60 ( 2 nM, respectively), indicating that the enaminones do
not bind directly to the BZ-site on the GABAA receptor (Figure
2). As expected, the neuroactive steroid 3R,5R-P also dose-
dependently enhanced the binding of [3H]flunitrazepam, while
the BZ-site ligand clonazepam dose-dependently inhibited the
binding of [3H]flunitrazepam in the same assay. Both 10 and
20 enhance the binding of [3H]muscimol, ruling out activity
for the enaminones at the isosteric GABA site on the GABAA
receptor (Figure 3).

Enaminone Amides as GABAA Receptor Modulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3373
their pharamacological profile and to elucidate the basis for their,
thus far, atypical profile as GABAA receptor modulators.
Experimental Section
Chemistry. 1H and 13C NMR spectra were recorded on a Varian
Unity Inova spectrometer at 400 and 100 MHz, respectively, in
CDCl3 referenced to CHCl3 (7.26 ppm) or to tetramethylsilane (0.00
1
ppm) and to CDCl3 at 77.00 ppm. H and 13C NMR spectra in
DMSO-d6 were referenced to DMSO-d5 (2.50 ppm) and to DMSO-
d6 (39.51 ppm), respectively. Melting points were determined on
an Electrothermal MEL-TEMP 3.0 apparatus (Barnstead Interna-
tional, Dubuque, IA) and are not corrected. Elemental analyses were
performed by Robertson Microlit Laboratories, Madison, NJ. Flash
chromatography was carried out by using the method of Still et
al.,26 on 230-400 mesh silica gel (silica gel 60, Geduran) obtained
from EMD. Reverse phase high performance liquid chromatography
(RPHPLC) was performed on a Shimatzu system with CH3CN/
water mixtures. Solvents were HPLC grade and were obtained from
EMD. DMSO, GABA, and pentylenetetrazole (PTZ) were obtained
from Sigma Chemical Co. Anhydrous solvents were obtained from
Aldrich Chemical Co. (Milwaukee, WI). Other synthetic reagents
were obtained from Aldrich or Lancaster Synthesis (Windham, NH)
and were used as received unless otherwise indicated. 4-Fluoro-
aniline (Lancaster) was purified by bulb-to-bulb distillation before
use. [3H]Flunitrazepam, [35S]TBPS, [3H]muscimol, and unlabeled
TBPS were obtained from Perkin-Elmer Bioscience (Boston, MA).
The neuroactive steroid 3R,21-dihydroxy-3â-trifluoromethyl-5â-
19-norpregnan-20-one (Co 2-6749) was prepared as described in
the literature.24b
Figure 3. Dose response of ethyl 2-chloro-R-[[(4-ethynylphenyl)amino]-
methylene]-â-oxobenzenepropanoate (10), 2-chloro-R-[[(4-ethynylphe-
nyl)amino]methylene]-â-oxo-N-propylbenzenepropanamide (20), and
GABA on 5 nM [3H]muscimol binding in rat cortex.
Ethyl 2,4-Dichloro-r-[(dimethylamino)methylene]-5-fluoro-
â-oxobenzenepropanoate (4a). A mixture of ethyl 3,3-dimethyl-
aminoacrylate (3.10 g, 21.6 mmol) and N,N-diisopropylethylamine
(8.0 mL, 5.94 g, 45.9 mmol) was stirred at room temperature and
a solution of 2,4-dichloro-5-fluorobenzoyl chloride (Lancaster; 4.92
g, 21.6 mmol) was added dropwise via addition funnel over 20
min. The cloudy yellow solution that formed was placed in an oil
bath at 85-90 °C. After 3 h, the mixture that formed was filtered
and the solid was washed with benzene. The dark filtrate was
concentrated and the residue was triturated with hexanes (50 mL).
The solid that formed was collected and washed with hexanes (20
mL) and partitioned between water and EtOAc. The EtOAc layer
was washed with water (3 × 25 mL) and brine, dried (Na2SO4),
filtered, and concentrated to 5.0 g (69%) of 4a. Recrystallization
from 4:1 hexanes/EtOAc gave the product as an off-white solid.
Figure 4. Dose response of 3R,20R-diol in the absence and presence
of 30 nM ethyl 2-chloro-R-[[(4-ethynylphenyl)amino]methylene]-â-
oxobenzenepropanoate (10) or 100 nM 2-chloro-R-[[(4-ethynylphenyl)-
amino]methylene]-â-oxo-N-propylbenzenepropanamide (20) on 2 nM
[35S]TBPS binding in rat cortex.
The [35S]TBPS dose response for the partial agonist neuroactive
steroid 5R-pregnane-3R,20R-diol (3R,20R-diol) in the presence
of 10 or 20 (Figure 4) shows that the effects of the enaminones
and the steroid are additive. Thus, the enaminones do not interact
directly with the neuroactive steroid site of the GABAA receptor.
1
Mp: 94.5-95.5 °C (lit27 mp: 94-95 °C). H NMR (400 MHz,
CDCl3): δ 7.87 (s, 1H), 7.39 (d, 1H, JH,F ) 6.4 Hz), 7.20 (d, 1H,
JH,F ) 8.7 Hz), 3.97 (q, 2H, J ) 7.1 Hz), 3.37 (s, 3H), 2.97 (s,
3H), 0.96 (t, 3H, J ) 7.1 Hz). 13C NMR (100 MHz, CDCl3) 187.59,
167.00, 159.27, 156.54 (d, JC,F ) 250 Hz), 142.39 (d, JC,F ) 5.3
Hz), 130.72, 126.06 (d, JC,F ) 3.8 Hz), 121.78 (d, JC,F ) 19.1 Hz),
116.51 (d, JC,F ) 23.7 Hz), 101.73, 59.91, 48.03, 42.79, 13.72 ppm.
Anal. (C14H14Cl2FNO3) C, H, N.
In conclusion, a series of enaminone esters and amides has
been identified as novel allosteric modulators of the GABAA
receptor. In vitro binding studies indicate that this class of
modulators likely interacts with a novel site on the GABAA
receptor distinct from the BZ, GABA, and neuroactive steroid
binding sites. The cyclobutylamide (16h) was identified from
SAR studies with low nanomolar in vitro potency as a modulator
of [35S]TBPS binding. Electrophysiological studies in oocytes
confirmed that the enaminone esters and amides are positive
allosteric modulators of human GABAA receptors. The isopro-
pyl, sec-butyl, cyclopropyl, and cyclobutyl amides (16e-h) may
have use as sedative-hypnotics because they were found to cause
LRR orally in mice. Compound 16e was orally active as an
anxiolytic in the mouse light-dark paradigm with a MED of 3
mg/kg and TI of >10. These modulators have no apparent
anticonvulsant activity, despite their robust modulation of
GABAA receptors. The in vivo profile of 16e (inactive against
PTZ-induced seizures and active as an anxiolytic) is similar to
that reported for the quinolone 1.9 Additional studies are
underway to more fully probe the interactions of this novel class
of compounds with GABAA receptors in order to characterize
Ethyl 2-Chloro-r-[(dimethylamino)methylene]-â-oxoben-
zenepropanoate (4b). A mixture of ethyl 3,3-dimethylaminoacryl-
ate (Acros; 4.68 g, 32.7 mmol) and N,N-diisopropylethylamine (12
mL, 8.9 g, 69 mmol) was stirred at room temperature and a solution
of 2-chlorobenzoyl chloride (5.72 g, 32.7 mmol) in 30 mL of
toluene was added over 5 min. The yellow solution that formed
was placed in an oil bath at 85-90 °C. After 3 h, the mixture that
formed was filtered and the solid was washed with toluene (4 ×
25 mL). The pooled toluene washes were extracted with water (3
× 50 mL) and brine (1 × 30 mL), dried (Na2SO4), filtered, and
concentrated. The dark filtrate was concentrated and the oily residue
was triturated with hexanes (100 mL). The solid that formed was
isolated by filtration and washed with hexanes (25 mL). The crude
product was dissolved in a minimum volume of EtOAc and added
to 16.5 cm of flash silica gel in a 5 cm diameter column. Elution
with 100% EtOAc afforded an oil that solidified after trituration
with hexanes to give 4b (5.68 g, 62%). Mp: 70-71.5 °C. 1H NMR
(400 MHz, CDCl3): δ 7.81 (br s, 1H), 7.34-7.23 (m, 4H), 3.89

3374 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Hogenkamp et al.
(q, 2H, J ) 7.2 Hz), 3.32 (br s, 3H), 2.96 (br s, 3H), 0.83 (t, 3H,
J ) 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 190.33, 167.45,
158.23, 142.29, 130.80, 129.69, 129.34, 128.61, 126.26, 102.92,
59.72, 47.76 (br), 42.31 (br), 13.46 ppm. Anal. (C14H16ClNO3) C,
H, N.
Ethyl 2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-â-oxo-
benzenepropanoate (5). A precipitate formed as the reaction was
stirred in EtOH. The product was isolated by filtration and washed
with EtOH, affording a 47% yield of the dichloride as a solid. Mp:
1
121.5-122.5 °C. H NMR (400 MHz, CDCl3): δ (major isomer)
12.71 (d, 1H, J ) 12.8 Hz), 8.58 (d, 1H, J ) 13.4 Hz), 7.40 (d,
2H, J ) 8.9 Hz), 7.37-7.28 (m, 4H), 7.22 (d, 2H, J ) 8.9 Hz),
4.01 (q, 2H, J ) 7.1 Hz), 0.93 (t, 3H, J ) 7.0 Hz). Anal. (C18H15-
Cl2NO3) C, H, N.
Ethyl 3-Chloro-r-[(dimethylamino)methylene]-â-oxoben-
zenepropanoate (4c). A mixture of 3,3-dimethylaminoacrylate
(1.44 g, 10.0 mmol) and triethylamine (3.0 mL, 2.2 g, 22 mmol)
was treated with a solution of 3-chlorobenzoyl chloride (1.78 g,
10.2 mmol) in 10 mL of toluene. The resulting mixture was heated
at 95 °C for 4.5 h. Workup as described for the 2-isomer gave the
crude product as a dark orange oil. Column chromatography (1:1
EtOAc/hexanes) and trituration of the resulting light yellow oil with
hexanes gave 902 mg (32%) of 4c as a light yellow solid. Mp:
Ethyl 2-Chloro-r-[[(2-chlorophenyl)amino]methylene]-â-oxo-
benzenepropanoate (6) was isolated as a viscous oil after chro-
matography on silica gel eluting with 6:1 hexanes/EtOAc. The oil
1
solidified on standing to give a white solid. Mp: 92-95 °C. H
NMR (400 MHz, CDCl3): δ (major isomer) 12.99 (d, 1H, J )
13.1 Hz), 8.66 (d, 1H, J ) 13.4 Hz), 7.48 (d, 1H, J ) 8.5 Hz),
7.45-7.31 (m, 6H), 7.19-7.12 (m, 1H), 4.03 (q, 2H, J ) 7.1 Hz),
0.94 (t, 3H, J ) 7.2 Hz). Anal. (C18H15Cl2NO3) C, H, N.
1
65-66 °C. H NMR (400 MHz, CDCl3): δ 7.76 (br s, 1H), 7.63
(br s, 1H), 7.44-7.29 (m, 4H), 3.97 (q, 2H, J ) 7.0 Hz), 3.05 (2
br s, 6H), 0.92 (t, 3H, J ) 7.0 Hz). 13C NMR (100 MHz, CDCl3):
δ 192.08, 168.19, 156.24, 143.06, 134.09, 131.06, 129.15, 128.55,
126.60, 99.89 (br), 59.73, 44.41 (br), 13.80 ppm.28 Anal. (C14H16-
ClNO3) C, H, N.
Ethyl 2-Chloro-r-[[(3-chlorophenyl)amino]methylene]-â-oxo-
1
benzenepropanoate (7). Mp: 107-108 °C. H NMR (400 MHz,
CDCl3): δ (major isomer) 12.65 (d, 1H, J ) 13.4 Hz), 8.59 (d,
1H, J ) 13.1 Hz), 7.38-7.21 (m, 7H), 7.17 (dt, 1H, J ) 7.8, 2.0
Hz), 4.02 (q, 2H, J ) 7.0 Hz), 0.94 (t, 3H, J ) 7.0 Hz). Anal.
(C18H15Cl2NO3) C, H, N.
Ethyl 4-Chloro-r-[(dimethylamino)methylene]-â-oxobenzene-
propanoate (4d) was prepared as described above for 4c as a light
yellow solid. Mp: 78.5-79.5 °C. 1H NMR (400 MHz, CDCl3): δ
7.70 (br m, 3H), 7.35 (d, 2H, J ) 8.5 Hz), 3.96 (q, 2H, J ) 7.1
Hz), 3.00 (br s, 6H), 0.93 (t, 3H, J ) 7.2 Hz). 13C NMR (100
MHz, CDCl3): δ 192.40, 168.30, 156.15, 139.48, 137.45, 129.95,
128.09, 99.89, 46.50 (br), 42.20 (br), 59.72, 13.85 ppm.28 Anal.
(C14H16ClNO3) C, H, N.
Ethyl 2,4-Dichloro-5-fluoro-r-[[(4-fluorophenyl)amino]meth-
ylene]-â-oxobenzenepropanoate (2a). A suspension of 4a (512
mg, 1.53 mmol) in 5 mL of EtOH was treated with neat
4-fluoroaniline (172 µL, 1.55 mmol). After stirring overnight, the
precipitate that formed was isolated and washed with 1.5 mL of
EtOH, affording 311 mg (51%) of 2a as a white solid. Mp: 119-
120 °C, as a 55:45 mixture of isomers (lit.11a mp: 111-112 °C).
1H NMR (400 MHz, CDCl3): δ (major isomer) 12.70 (d, 1H, J )
13.1 Hz), 8.59 (d, 1H, J ) 14.0 Hz), 7.41 (dd, 1H, J ) 6.3, 2.3
Hz), 7.28-7.20 (m, 4H), 7.06 (d, 1H, J ) 8.5 Hz), 4.07 (q, 2H, J
) 7.0 Hz), 1.06 (t, 3H, J ) 7.0 Hz). Anal. (C18H13Cl2FNO3) C, H,
N.
Ethyl 2-Chloro-r-[[(4-methylphenyl)amino]methylene]-â-oxo-
1
benzenepropanoate (8). Mp: 106-108 °C. H NMR (400 MHz,
CDCl3): δ 12.74 (d, 1H, J ) 13.7 Hz), 8.62 (d, 1H, J ) 13.4 Hz),
2.37 (s, 3H), 7.37-7.25 (m, 4H), 7.23 (d, 2H, J ) 8.8 Hz), 7.17
(d, 2H, J ) 8.5 Hz), 4.00 (q, 2H, J ) 7.1 Hz), 0.93 (t, 3H, J ) 7.0
Hz). Anal. (C19H18ClNO3) C, H, N.
Ethyl 2-Chloro-r-[[(4-isopropylphenyl)amino]methylene]-â-
oxobenzenepropanoate (9). Mp: 87-88 °C. 1H NMR (400 MHz,
CDCl3): δ (major isomer) 12.74 (d, 1H, J ) 13.4 Hz), 8.63 (d,
1H, J ) 13.4 Hz), 7.37-7.25 (m, 6H), 7.21 (d, 2H, J ) 8.5 Hz),
4.00 (q, 2H, J ) 7.2 Hz), 2.93 (heptet, 1H, J ) 7.0 Hz), 1.26 (d,
6H, J ) 7.0 Hz), 0.93 (t, 3H, J ) 7.0 Hz). Anal. (C21H22ClNO3)
C, H, N.
Ethyl 2-Chloro-r-[[(4-ethynylphenyl)amino]methylene]-â-
oxobenzenepropanoate (10). Mp: 131-132.5 °C. 1H NMR (400
MHz, CDCl3): δ (major isomer) 12.70 (d, 1H, J ) 13.4 Hz), 8.62
(d, 1H, J ) 13.4 Hz), 7.55 (d, 2H, J ) 8.9 Hz), 7.36-7.27 (m,
4H), 7.24 (d, 2H, J ) 8.9 Hz), 4.02 (q, 2H, J ) 7.1 Hz), 3.14 (s,
1H), 0.94 (t, 3H, J ) 7.2 Hz). Anal. (C20H16ClNO3) C, H, N.
5-(2-Chlorobenzoyl)-2,2-dimethyl-1,3-dioxane-4,6-dione (12).
A solution of 2,2-dimethyl-1,3-dioxane-4,6-dione (24.0 g, 166.5
mmol) and 4-(dimethylamino)pyridine (DMAP, 47.47 g, 209 mmol)
in CH2Cl2 (500 mL) was cooled in an ice-salt bath to -10 °C,
and a solution of 2-chlorobenzoyl chloride (32.44 g, 185 mmol) in
CH2Cl2 (125 mL) was added dropwise via addition funnel over 2
h. The resulting yellow solution was kept in the cooling bath for 4
h and allowed to stir at room temperature overnight. The orange-
yellow solution was then cooled in an ice-water bath and washed
with 3 × 100 mL of a 1 M aqueous HCl solution and water. The
organic layer was dried over MgSO4 and filtered, and the filtrate
was evaporated to dryness. The residue was triturated with hexanes
(500 mL), and the resulting yellow solid was collected by filtration,
washed with hexanes (100 mL), and dried under vacuum to give
45.08 (96%) of 12 as a yellow powder. The organic layer was
extracted with EtOAc (3 × 50 mL), and the pooled organic layers
were washed with water and brine, dried (MgSO4), filtered, and
concentrated to afford an additional 2.19 g of crude product. A
suspension of 23 g (81 mmol) of crude 12 in 800 mL of toluene
was stirred at room temperature for 45 min and then filtered to
remove insoluble material. The toluene solution was then treated
with neat isopropylamine (6.0 mL, 4.2 g, 70 mmol) added dropwise
via syringe. The resulting precipitate was isolated by filtration and
washed with toluene (3 × 50 mL) and hexanes (2 × 50 mL) to
Ethyl 2-Chloro-r-[[(4-fluorophenyl)amino]methylene]-â-oxo-
benzenepropanoate (2b) was prepared as described above for 2a.
Mp: 111-112 °C. 1H NMR (400 MHz, CDCl3): δ (major isomer)
12.75 (d, 1H, J ) 13.7 Hz), 8.56 (d, 1H, J ) 13.4 Hz), 7.37-7.24
(m, 6H), 7.14 (t, 2H, J ) 8.4 Hz), 4.01 (q, 2H, J ) 7.1 Hz), 0.93
(t, 3H, J ) 7.0 Hz). Anal. (C18H15ClFNO3) C, H, N.
Ethyl 3-Chloro-r-[[(4-fluorophenyl)amino]methylene]-â-oxo-
benzenepropanoate (2c). A mixture of ethyl 3-chloro-R-[(di-
methylamino)methylene]-â-oxobenzenepropanoate (4c, 204 mg,
0.724 mmol) and 4-fluoroaniline (80 mg, 0.72 mmol) in 4 mL of
EtOH was allowed to stand at room temperature for 5 d. The
resulting yellow solution was concentrated in vacuo. The residue
was dissolved in 10 mL of EtOAc and extracted with a 1 M HCl
solution (3 × 10 mL), a saturated NaHCO3 solution, and brine (10
mL of each). The EtOAc layer was dried (Na2SO4), filtered, and
concentrated. The oil that remained was subjected to flash chro-
matography (4:1 hexanes/EtOAc), affording 162 mg (65%) of 2c
as a light yellow oil. Trituration with hexanes gave a white solid.
Mp: 70-80 °C. By 1H NMR, 2c is a 1.4:1 mixture of isomers. 1H
NMR (major isomer, 400 MHz, CDCl3): δ 12.23 (d, 1H, J ) 13.1
Hz), 8.49 (d, 1H, J ) 13.4 Hz), 7.52-7.30 (m, 4H), 7.24-7.08
(m, 4H), 4.06 (q, 2H, J ) 7.1 Hz), 1.02 (t, 3H, J ) 7.2 Hz). Anal.
(C18H15ClFNO3) C, H, N.
Ethyl 4-Chloro-r-[[(4-fluorophenyl)amino]methylene]-â-oxo-
benzenepropanoate (2d) was isolated as a white solid in 28% yield.
Mp: 110-111 °C. 1H NMR (400 MHz, CDCl3): δ (major isomer)
12.70 (d, 1H, J ) Hz), 8.47 (d, 1H, J ) 13.1 Hz), 7.59 (d, 1H, J
) 8.2 Hz), 7.46-7.07 (m, 7H), 4.07 (q, 2H, J ) 7.0 Hz), 1.05 (t,
3H, J ) 7.2 Hz). Anal. (C18H15ClFNO3) C, H, N.
1
afford the salt 12a as a yellow solid. Mp: 147-149 °C (dec). H
NMR (400 MHz, DMSO-d6): δ 7.75 (br s, 3H), 7.24-7.15 (m,
3H), 7.02 (dd, 1H, J ) 7.0, 2.1 Hz), 3.26 (heptet, 1H, J ) 6.4 Hz),
1.53 (s, 6H), 1.16 (d, 6H, J ) 6.4 Hz). 13C NMR (100 MHz,
DMSO-d6) 187.51, 163.59, 145.71, 128.28, 128.19, 127.33, 126.94,
Compounds 5-10 were prepared by using the method used above
for the synthesis of 2a-d.

Enaminone Amides as GABAA Receptor Modulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3375
126.05, 99.50, 88.32, 42.85, 26.14, 20.20 ppm. Anal. (C16H20ClNO5)
C, H, N. The salt was then suspended in CH2Cl2 and washed a 1
M aqueous HCl solution. The organic layer was separated, dried
(MgSO4), filtered, and concentrated to give 12 as a yellow solid.
Mp: 88.5-91 °C (dec). 1H NMR (400 MHz, CDCl3): δ 15.10 (br
s, 1H), 7.54-7.38 (m, 4H), 1.81 (s, 6H).
2H), 3.43-3.31 (m, 2H), 1.21 (enol CH3; t, 3H, J ) 7.3 Hz), 1.17
(keto CH3; t, 3H, J ) 7.3 Hz). Anal. (C11H12ClNO2) C, H, N.
2-Chloro-r-[(dimethylamino)methylene]-N-ethyl-â-oxoben-
zenepropanamide (18a). A solution of 17a (5.63 g, 25.0 mmol)
in 60 mL of CH2Cl2 was treated with DMFDMA (4.0 mL, 3.6 g,
30 mmol) and stirred at room temperature overnight. The reaction
was concentrated to dryness and then subjected to column chro-
matography. Elution with 3% and 4% MeOH/CH2Cl2 afforded 3.37
g of the crude product as an oil. Trituration with hexanes afforded
2.97 g (42%) of 18a as a yellow solid. Mp: 97.5-100 °C. 1H NMR
(400 MHz, CDCl3): δ 8.23 (br s, 1H), 7.41-7.39 (m, 1H), 7.34-
7.27 (m, 3H), 7.15 (br s, 1H), 3.37 (pentet, 1H, J ) 6.7 Hz), 3.08
(br s, 6H), 1.18 (t, 3H, J ) 6.7 Hz). 13C NMR (100 MHz, CDCl3):
δ 190.18, 165.55, 161.18, 140.60, 131.03, 129.86, 129.80, 128.45,
126.71, 105.96, 45.00 (br), 34.16, 14.72 ppm. Anal. (C14H17ClN2O2)
C, H, N.
1,1-Dimethylethyl 2-Chloro-â-oxobenzenepropionate (13). A
mixture of 12 (11.5 g, 41.0 mmol) and t-BuOH (15 mL) was
refluxed for 5 h and then concentrated to dryness. The residue was
purified by silica gel chromatography. Elution with 1:9 EtOAc/
hexanes gave 7.23 g (70%) of ester 13 as a viscous yellow oil that
1
was carried on without further purification. H NMR (400 MHz,
CDCl3): δ (mixture of keto and enol forms, ratio 1.9:1) 12.62 (s,
1H, enol OH), 7.61-7.30 (m, 4H), 5.45 (s, 1H, enol dCH), 3.94
(s, 2H, keto CH2), 1.54 (s, 9H, enol tBu), 1.39 (s, 9H, keto tBu).
Anal. (C13H15ClO3) C, H, N.
2-Chloro-â-oxo-N-propylbenzenepropanamide (17d) was iso-
1,1-Dimethylethyl 2-Chloro-r-[(dimethylamino)methylene]-
â-oxobenzenepropanoate (14). A solution of the 1,1-dimethylethyl
ester 13 (2.00 g, 7.86 mmol) and 1.20 g (10 mmol) of DMFDMA
in toluene (25 mL) was stirred at 80 °C for 16 h. The reaction was
concentrated in vacuo and the residue was subjected to column
chromatography. Elution with 3:2 hexanes/EtOAc afforded 1.96 g
(80%) of 14 as an oil that solidified on standing. Mp: 97-99 °C.
1H NMR (400 MHz, CDCl3): δ 7.77 (s, 1H), 7.40 (br s, 1H), 7.35-
7.23 (m, 4H), 3.31 (br s, 3H), 2.94 (br s, 3H), 1.10 (s, 9H). Anal.
(C16H20ClNO3) C, H, N.
lated as a colorless oil after chromatography with 1% MeOH/CH2-
1
Cl2. H NMR (400 MHz, CDCl3; 2.3:1 mixture of keto and enol
forms): δ 14.21 (enol OH; s, 1H), 7.56 (keto ArH; d, 1H, J ) 7.9
Hz), 7.44-7.29 (m, 3H), 6.96 (keto NH; br s, 1H), 5.47 (enol NH;
br s, 1H), 5.29 (enol dCH; s, 1H), (3.93 (keto CH2; s, 2H), 3.31
(enol CH2CH3; q, 3H, J ) 6.7 Hz), 3.27 (keto CH2CH3; q, 3H, J
) 7.0 Hz), 1.59 (enol CH2CH3, hextet, 2H, J ) 7.3 Hz), 1.56 (keto
CH2CH3, hextet, 2H, J ) 7.3 Hz), 0.96 (enol CH3; t, 3H, J ) 7.3
Hz), 0.94 (keto CH3; t, 3H, J ) 7.3 Hz). Anal. (C12H14ClNO2) C,
H, N.
1,1-Dimethylethyl 2-Chloro-r-[[(4-chlorophenyl)amino]meth-
ylene]-â-oxobenzenepropanoate (15). A solution 14 (3.86 g, 12.5
mmol) and 4-chloroaniline (1.59 g, 12.5 mmol) in EtOH (25 mL)
was stirred at room temperature for 45 h. The resulting precipitate
was isolated and washed with EtOH, giving 2.91 g of 15 as a white
solid. Mp: 129-132 °C. The EtOH mother liquor was concentrated
in vacuo and the residue was purified by flash chromatography,
2-Chloro-r-[(dimethylamino)methylene]-â-oxo-N-propylben-
zenepropanamide (18d) was isolated as a light yellow solid after
chromatography with 2.5% MeOH/CH2Cl2 and trituration with
1
hexanes. Mp: 85-90 °C. H NMR (400 MHz, CDCl3): δ 8.29
(br s, 1H), 7.39 (d, 1H, J ) 7.0 Hz), 7.34-7.27 (m, 3H), 7.14 (br
s, 1H), 3.31 (q, 2H, J ) 6.6 Hz), 3.07 (br s, 6H), 1.58 (hextet, 2H,
J ) 7.0 Hz), 0.96 (t, 3H, J ) 7.3 Hz). 13C NMR (100 MHz,
CDCl3): δ 191.17, 165.55, 161.23, 140.52, 130.95, 129.83, 129.76,
128.42, 126.66, 105.88, 45.00 (br), 41.11, 22.75, 11.43 ppm. Anal.
(C15H19ClN2O2) C, H, N.
1
affording an additional 1.55 g of 15 (91% total yield). H NMR
(400 MHz, CDCl3): δ 12.56 (d, 1H, J ) 13.4 Hz), 8.54 (d, 1H, J
) 13.4 Hz), 7.38 (d, 2H, J ) 8.5 Hz), 7.36-7.24 (m, 4H), 7.20 (d,
2H, J ) 8.9 Hz), 1.16 (s, 9H). Anal. (C20H19Cl2NO3) C, H, N.
2-Chloro-N-methyl-â-oxobenzenepropanamide (17c) was iso-
lated as a light yellow oil in 82% yield after chromatography with
1:1 hexanes/EtOAc. 1H NMR (400 MHz, CDCl3; 2.6:1 mixture of
keto and enol forms): δ 14.20 (enol OH; br s, 1H), 7.59-7.29 (m,
4H), 7.00 (keto NH; br s, 1H), 5.50 (enol NH; br s, 1H), 5.38 (enol
dCH; s, 1H), 3.95 (keto CH2, s, 2H), 2.91 (enol Me; d, 3H, J )
4.6 Hz), 2.86 (keto Me; d, 3H, J ) 4.9 Hz). Anal. (C10H10ClNO2)
C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-â-oxoben-
zenepropanoic Acid (11). To a solution of tert-butyl ester 15 (1.30
g, 3.30 mmol) in CH2Cl2 (20 mL) was added trifluoroacetic acid
at room temperature, and the solution was stirred at room
temperature for 16 h. The resulting yellow reaction was evaporated
to dryness and the residue was dissolved in toluene and concentrated
in vacuo. The residual solid was triturated with hexanes, filtered,
washed with hexanes, and dried under vacuum to give 830 mg
(75%) of the carboxylic acid 11 as a light yellow solid. Mp: 179-
184 °C (dec). 1H NMR (400 MHz, CDCl3): δ 14.11 (s, 1H), 12.22
(d, 1H, J ) 12.8 Hz), 7.77 (d, 1H, J ) 13.4 Hz), 7.53-7.38 (m,
4H), 7.34 (d, 2H, J ) 8.7 Hz), 6.97 (d, 2H, J ) 8.7 Hz). 13C NMR
(100 MHz, CDCl3): δ 195.47, 170.14, 156.62, 136.45, 136.42,
132.47, 131.56, 130.72, 130.28, 130.15, 129.01, 127.29, 119.56,
101.54 ppm. Anal. (C16H11Cl2NO3) C, H, N.
2-Chloro-r-[(dimethylamino)methylene]-N-methyl-â-oxoben-
zenepropanamide (18c) was isolated as a light yellow oil that
solidified on standing. Mp: 84-88 °C, after chromatography with
1
5% MeOH/CH2Cl2. H NMR (400 MHz, CDCl3): δ 8.34 (br s,
1H), 7.11 (br s, 1H), 3.07 (s, 6H), 2.90 (d, 3H, J ) 4.6 Hz). 13C
NMR (100 MHz, CDCl3) 191.21, 166.12, 161.50, 140.37, 130.84,
129.79, 129.72, 128.34, 126.64, 105.57, 47.30 (br), 42.30 (br), 25.84
ppm. Anal. (C13H15ClN2O2) C, H, N.
2-Chloro-r-[(dimethylamino)methylene]-N-ethyl-â-oxoben-
zenepropanamide (18a). General procedure for the direct conver-
sion of acyl Meldrum’s acid 12 to â-ketoamides 17 and conversion
to enaminones 18.
2-Chloro-N-isopropyl-â-oxobenzenepropanamide (17e) was
isolated as a white solid (65% yield) after chromatography with
3:1 hexanes/EtOAc. Mp: 85-86 °C. 1H NMR (400 MHz, CDCl3;
mixture of keto and enol forms, keto form given): δ 7.56 (d, 1H,
J ) 7.3 Hz), 7.44-7.30 (m, 3H), 6.72 (br s, 1H), 3.91 (s, 2H),
1.18 (d, 6H, J ) 6.4 Hz). Anal. (C12H14ClNO2) C, H, N.
2-Chloro-r-[(dimethylamino)methylene]-N-isopropyl-â-oxo-
benzenepropanamide (18e). The crude reaction was concentrated
to dryness and partitioned between EtOAc and water. The EtOAc
layer was washed with a dilute aqueous NaHCO3 solution and brine.
After drying (Na2SO4), the mixture was filtered and concentrated
to a light yellow foam. Trituration with hexanes gave a light yellow
solid which was further purified by RPHPLC, affording the product
as a white solid. Mp: 96-97 °C. 1H NMR (400 MHz, CDCl3): δ
7.37 (dd, 1H, J ) 7.6, 0.9 Hz), (7.31-7.24 (m, 3H), 7.18 (br s,
1H), 4.07 (m, 1H), 3.05 (br s, 6H), 1.14 (d, 6H, J ) 5.2 Hz). 13C
NMR (100 MHz, CDCl3) 190.90, 164.87, 160.65, 140.42, 130.84,
2-Chloro-N-ethyl-â-oxobenzenepropanamide (17a). A suspen-
sion of 12 (23.5 g, 83.2 mmol) in 800 mL of toluene was stirred at
room temperature for 30 min. The mixture was filtered and the
toluene solution was treated with a 2 M solution of ethylamine in
THF (24 mL, 48 mmol) added dropwise via addition funnel. The
resulting precipitate was collected and washed with toluene (50
mL) and hexanes (2 × 50 mL). A suspension of the salt (18.54 g,
56.58 mmol) in 250 mL of benzene was heated at reflux for 5 h.
Once at room temperature, the reaction was concentrated in vacuo
to give the crude â-ketoamide as an oil. Chromatography with 1:1
1
hexanes/EtOAc afforded 17a as a light yellow oil. H NMR (400
MHz, CDCl3; mixture of keto and enol forms): δ 14.19 (enol OH;
s, 1H), 7.56 (d, 1H, J ) 7.6 Hz), 7.44-7.29 (m, 3H), 6.91 (br s,
1H), 5.42 (br s, 1H), 5.36 (enol dCH; s, 1H), 3.93 (keto CH2; s,

3376 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Hogenkamp et al.
129.83, 129.71, 128.23, 126.66, 105.89, 47.79 (br), 41.93 (br),
41.07, 22.56 ppm. Anal. (C15H19ClN2O2) C, H, N.
1H, J ) 7.6 Hz), 1.62 (m, 4H), 0.99 (t, 6H, J ) 7.5 Hz). 13C NMR
(100 MHz, CDCl3): δ 193.13, 168.33, 155.84, 139.19, 137.60,
130.72, 130.62, 130.52, 130.04, 129.89, 128.75, 126.95, 118.58,
104.27, 51.58, 27.25, 10.37 ppm. Anal. (C21H22Cl2N2O2) C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-(1,2-di-
methylpropyl)-â-oxobenzenepropanamide (16l). Mp: 110 °C. 1H
NMR (400 MHz, CDCl3): δ 12.99 (d, 1H, J ) 12.5 Hz), 9.57 (d,
1H, J ) 8.5 Hz), 7.65 (d, 1H, J ) 12.2 Hz), 7.36 (t, 1H, J ) 7.9
Hz), 7.26 (d, 2H, J ) 7.6 Hz), 6.83 (d, 2H, J ) 8.5 Hz), 4.01
(hextet, 1H, J ) 7.0 Hz), 1.86 (octet, 1H, J ) 6.7 Hz), 1.22 (d,
3H, J ) 6.7 Hz). 13C NMR (100 MHz, CDCl3): δ 193.12, 167.91,
155.83, 139.14, 137.57, 130.69, 130.62, 130.51, 130.02, 129.88,
128.75, 126.94, 118.55, 104.28, 49.61, 32.88, 18.60, 18.55, 17.51
ppm. Anal. (C21H22Cl2N2O2) C, H, N.
2-Chloro-N-cyclopropyl-â-oxobenzenepropanamide (17g) was
isolated as an off-white solid after chromatography with 2:1
1
hexanes/EtOAc. Mp: 54.5-59 °C. H NMR (400 MHz, CDCl3;
4:1 mixture of keto and enol forms, keto form given): δ 7.44-
7.29 (m, 4H), 7.03 (br s, 1H), 3.91 (s, 2H), 2.81-2.73 (m, 1H),
0.86-0.77 (m, 2H), 0.61-0.53 (m, 2H). Anal. (C12H12ClNO2) C,
H, N.
2-Chloro-N-cyclopropyl-r-[(dimethylamino)methylene]-â-oxo-
benzenepropanamide (18g). A solution of 17g (234 mg, 0.984
mmol) and DMFDMA (0.5 mL, 3.75 mmol) in 3 mL of CH2Cl2
was stirred at room temperature for 4 d. The resulting yellow
solution was then concentrated in vacuo and the residue was purified
by RPHPLC and recrystallized from CH2Cl2/hexanes to give a white
solid. Mp: 121-122 °C. 1H NMR (400 MHz, CDCl3): δ 8.43 (br
s, 1H), 7.38 (d, 1H, J ) 7.6 Hz), 7.33-7.24 (m, 3H), 7.15 (br s,
1H), 3.08 (br s, 6H), 2.80 (m, 1H), 0.75 (br s, 2H), 0.50 (br s, 2H).
13C NMR (100 MHz, CDCl3) 191.05, 166.91, 161.25, 140.34,
130.75, 129.80, 129.66, 128.22, 126.62, 47.67 (br), 42.53 (br),
22.26, 6.31 ppm. Anal. (C15H17ClN2O2) C, H, N.
2-Chloro-N-(1,1-dimethylethyl)-â-oxobenzenepropanamide
(17k) was isolated as a white solid. Mp: 109-110 °C after
chromatography (100% CH2Cl2). 1H NMR (400 MHz, CDCl3;
mixture of keto and enol forms, NMR of keto form given): δ 7.56
(d, 1H, J ) 7.3 Hz), 7.43-7.30 (m, 3H), 6.66 (br s, 1H), 3.85 (s,
2H), 1.36 (s, 9H). Anal. (C13H16ClNO2) C, H, N.
2-Chloro-â-oxobenzenepropionic Acid (19). TFA (10 mL) was
added slowly to a solution of 1,1-dimethylethyl 2-chloro-â-
oxobenzenepropionate (13, 7.23 g, 28.4 mmol) in CH2Cl2 (70 mL)
at room temperature with stirring. The resulting purple solution
was stirred at room temperature for 5 h. The yellow solution that
formed was evaporated to dryness, the residual solid was triturated
with hexanes (50 mL) and filtered, and the solid was washed with
hexanes (10 mL) and dried under vacuum to give 4.5 g (80%) of
the carboxylic acid as an off-white powder. Mp: 101 °C (dec).
1
The H NMR (CDCl3) indicated that it was a mixture of keto and
enol forms. It was used for the next reaction without further
purification. 1H NMR (400 MHz, CDCl3): δ (1.1:1 mixture of keto
and enol forms) 12.14 (s, 1H, enol OH), 10.18 (br s, 1H), 7.66-
7.31 (m, 4H), 5.61 (s, 1H, enol dCH), 4.13 (s, 2H, keto CH2).
Anal. (C9H7ClO3) C, H.
2-Chloro-r-[(dimethylamino)methylene]-N-(1,1-dimethylethyl)-
â-oxobenzenepropanamide (18k). A solution of 17k (879 mg, 3.46
mmol) in 5 mL of CH2Cl2 was treated with 1.0 mL (7.5 mmol) of
DMFDMA. After stirring at room temperature for 3 d, the reaction
was concentrated to dryness and the residue was partitioned between
EtOAc and water. The EtOAc layer was separated, washed with a
brine solution, dried (MgSO4), filtered, and concentrated. The oil
that formed was purified by flash chromatography (silica gel; 3
and 5% MeOH/CH2Cl2) and RPHPLC. The resulting oil (200 mg;
2-Chloro-N-cyclobutyl-â-oxobenzenepropanamide (17h). A
solution of the carboxylic acid 19 (1.99 g, 10.0 mmol) obtained
above and DCC (2.1 g, 11 mmol) in CH2Cl2 (30 mL) was stirred
at room temperature for 1.5 h and a solution of cyclobutylamine
(710 mg, 10.0 mmol) in CH2Cl2 (10 mL) was added. After stirring
for 5 h, the resulting mixture was filtered to remove a white solid
and the filtrate was evaporated to dryness. Purification by chro-
matography over silica gel (7/3 hexanes/EtOAc) gave 1.99 g (80%)
of the cyclobutyl amide as a white sold. It was used for the next
reaction without further purification. Mp: 82-83 °C. 1H NMR (400
MHz, CDCl3): δ (1:1 mixture of keto and enol forms) 14.17 (br s,
1H, enol OH), 7.59-7.28 (m, 4H), 7.07 (br s, 1H), 5.58 (br s, 1H),
5.34 (s, 1H, enol dCH), 4.49 (hextet, 1H, J ) 8.1 Hz), 4.42 (hextet,
1H, J ) 8.1 Hz), 3.90 (s, 2H, keto CH2), 2.42-2.31 (m, 2H), 1.98-
1.88 (m, 2H), 1.78-1.69 (m, 2H). Anal. (C13H14ClNO2) C, H, N.
2-Chloro-N-cyclobutyl-r-[(dimethylamino)methylene]-â-oxo-
benzenepropanamide (18h). A solution of 2-chloro-N-cyclobutyl-
â-oxobenzenepropanamide (1.88 g, 7.52 mmol) obtained above and
DMFDMA (1.0 g, 8.3 mmol) in toluene (25 mL) was stirred at
80-85 °C for 15 h and evaporated to dryness. The residue was
purified by chromatography over silica gel (20/1 CH2Cl2/MeOH)
to give 1.75 g (95%) of the enaminone as an off-white foam that
1
20% yield) was carried on without further purification. H NMR
(400 MHz, CDCl3): δ 7.38 (d, 1H, J ) 7.3 Hz), 7.31-7.26 (m,
3H), 3.06 (br s, 6H), 1.31 (s, 9H). 13C NMR (100 MHz, CDCl3)
190.62, 165.25, 159.97 (br), 140.72, 130.84, 129.78, 129.63, 128.25,
126.69, 50.70, 47.71 (br), 41.63 (br), 28.58 ppm. Anal. (C16H21-
ClN2O2‚1/3H2O) C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-(1-meth-
ylpropyl)-â-oxobenzenepropanamide (16f). The following is a
general method for the conversion of acid 11 to amides 16. To a
solution of the carboxylic acid 11 (250 mg, 0.74 mmol) in CH2Cl2
(15 mL) was added SOCl2 (0.5 mL). The resulting solution was
stirred at room temperature for 16 h. The reaction was evaporated
to dryness, dissolved in toluene, and then reconcentrated to give
the crude acid chloride. A solution of the acid chloride in CH2Cl2
(15 mL) was added dropwise into a solution of sec-butylamine (100
mg, 1.37 mmol) in CH2Cl2 (3 mL) and stirred at room temperature
for 5 h. The reaction was then evaporated to dryness and the residue
was purified by chromatography over silica gel, eluting with 7/3
hexanes/EtOAc to give 240 mg (83%) of the amide 16f as an off-
1
gave a solid after trituration with hexanes. Mp: 112-113 °C. H
NMR (400 MHz, CDCl3): δ 8.60 (br s, 1H), 7.39 (d, 1H, J ) 7.6
Hz), 7.34-7.26 (m, 3H), 7.21 (br s, 1H), 4.43 (m, 1H), 3.06 (br s,
6H), 2.34 (m, 2H), 1.94 (m, 2H), 1.72 (m, 2H). 13C NMR (100
MHz, CDCl3): δ 191.10, 164.68, 161.19, 140.66, 131.02, 129.85,
129.78, 128.44, 126.69, 105.70, 44.85, 44.80 (br), 31.06, 15.37 ppm.
Anal. (C16H19ClN2O2) C, H, N.
1
white solid. Mp: 104-105 °C. H NMR (400 MHz, CDCl3): δ
12.99 (d, 1H, J ) 11.9 Hz), 9.46 (d, 1H, J ) 7.9 Hz), 7.64 (d, 1H,
J ) 12.5 Hz), 7.47 (dd, 1H, J ) 7.6, 1.4 Hz), 7.40 (dt, 1H, J )
7.0, 2.0 Hz), 7.36 (dt, 1H, J ) 7.0, 1.4 Hz), 7.32 (dd, 1H, J ) 7.0,
2.0 Hz), 7.26 (d, 2H, J ) 8.8 Hz), 6.83 (d, 2H, J ) 8.8 Hz), 4.04
(heptet, 1H, J ) 6.7 Hz), 1.60 (m, 2H), 1.27 (d, 3H, J ) 6.7 Hz),
1.61 (m, 2H), 1.00 (t, 3H, J ) 7.5 Hz). 13C NMR (100 MHz,
CDCl3): δ 192.98, 167.96, 155.83, 139.34, 137.74, 130.80, 130.66,
130.44, 130.00, 129.88, 128.79, 126.88, 118.58, 104.47, 46.17,
29.57, 20.27, 10.30 ppm. Anal. (C20H20Cl2N2O2) C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-cyclobu-
tyl-â-oxobenzenepropanamide (16h). A solution of 2-chloro-N-
cyclobutyl-R-[(dimethylamino)methylene]-â-oxobenzenepropana-
mide (175 mg, 0.57 mmol) and 4-chloroaniline (73 mg, 0.57 mmol)
in toluene (6 mL) was stirred at 75-80 °C for 5 h and evaporated
to dryness. The residue was purified by chromatography over silica
gel (7/3 hexanes/EtOAc) to give 175 mg (79%) of the product as
a white powder. Mp: 161-162 °C. 1H NMR (400 MHz, CDCl3):
δ 12.95 (d, 1H, J ) 12.3 Hz), 9.72 (d, 1H, J ) 7.0 Hz), 7.64 (d,
1H, J ) 12.5 Hz), 7.47 (d, 1H, J ) 7.5 Hz), 7.41 (dt, 1H, J ) 7.3,
1.8 Hz), 7.37 (t, 1H, J ) 7.2 Hz), 7.31 (dd, 1H, J ) 7.5, 1.8 Hz),
6.82 (d, 2H, J ) 8.6 Hz), 4.49 (h, 1H, J ) 8.1 Hz), 2.41 (m, 2H),
2.09 (m, 2H), 1.80 (m, 2H). 13C NMR (100 MHz, CDCl3): δ
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-(1-ethyl-
propyl)-â-oxobenzenepropanamide (16j). Mp: 111-112 °C. 1H
NMR (400 MHz, CDCl3): δ 12.99 (d, 1H, J ) 12.2 Hz), 9.43 (d,
1H, J ) 8.5 Hz), 7.65 (d, 1H, J ) 12.5 Hz), 7.43 (d, 1H, J ) 7.3
Hz), 7.41 (dt, 1H, J ) 7.0, 1.8 Hz), 7.36 (t, 1H, J ) 7.9 Hz), 7.33
(dd, 1H, J ) 7.3, 2.3 Hz), 6.83 (d, 2H, J ) 8.8 Hz), 3.93 (hextet,

Enaminone Amides as GABAA Receptor Modulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3377
192.90, 167.62, 155.88, 139.21, 137.60, 130.75, 130.72, 130.46,
129.99, 129.86, 128.73, 126.90, 118.58, 104.18, 44.34, 30.99, 15.54
ppm. Anal. (C20H18Cl2N2O2) C, H, N.
4H). 13C NMR (100 MHz, CDCl3): δ 192.92, 168.10, 155.76,
139.28, 137.70, 130.77, 130.66, 130.44, 129.99, 129.87, 128.77,
126.88, 118.56, 104.44, 50.67, 33.17, 23.84 ppm. Anal. (C21H20-
Cl2N2O2) C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-ethyl-â-
oxobenzenepropanamide (16a). Mp: 150.5-151.5 °C. 1H NMR
(400 MHz, CDCl3): δ 12.99 (d, 1H, J ) 12.5 Hz), 9.54 (br t, 1H),
7.65 (d, 1H, J ) 12.5 Hz), 7.47 (dd, 1H, J ) 7.8, 1.5 Hz), 7.41 (dt,
1H, J ) 7.3, 1.8 Hz), 7.36 (dt, 1H, J ) 7.3, 1.5 Hz), 7.31 (dd, 1H,
J ) 7.3, 2.1 Hz), 7.26 (d, 2H, J ) 8.9 Hz), 6.83 (d, 2H, J ) 8.8
Hz), 3.45 (br m, 2H), 1.29 (t, 3H, J ) 7.3 Hz). 13C NMR (100
MHz, CDCl3): δ 193.01, 168.49, 155.85, 139.24, 137.66, 130.79,
130.75, 130.48, 130.04, 129.92, 128.76, 129.93, 118.61, 104.34,
33.68, 14.73 ppm. Anal. (C18H16Cl2N2O2) C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-(1,1-di-
methylethyl)-â-oxobenzenepropanamide (16k). Mp: 177-177.5
1
°C. H NMR (400 MHz, CDCl3): δ 12.98 (d, 1H, J ) 12.2 Hz),
9.56 (s, 1H), 7.62 (d, 1H, J ) 12.2 Hz), 7.46 (d, 1H, J ) 7.6 Hz),
7.40 (dd, 1H, J ) 1.5 Hz), 7.35 (t, 1H, J ) 6.7 Hz), (dd, 1H, J )
7.0, 2.0 Hz), 7.25 (d, 2H, J ) 8.5 Hz), 6.84 (d, 2H, J ) 8.8 Hz),
1.49 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 193.11, 168.04,
156.11, 139.49, 137.87, 130.81, 130.65, 130.42, 130.02, 129.88,
128.80, 126.91, 118.72, 105.22, 50.96, 29.03 ppm. Anal. (C20H20-
Cl2N2O2) C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-â-oxoben-
1
zenepropanamide (16b). Mp: 186.5-187.5 °C. H NMR (400
2-Chloro-r-[[(4-ethynylphenyl)amino]methylene]-â-oxo-N-
propylbenzenepropanamide (20). A solution of 2-chloro-R-[(di-
methylamino)methylene]-â-oxo-N-propylbenzenepropanamide (104
mg, 0.35 mmol) and 41 mg (0.35 mmol) of 4-ethynylaniline in 2
mL of toluene was stirred at room temperature for 4 d. The reaction
was diluted with 10 mL of EtOAc; extracted with a 1 M aqueous
HCl solution (3 × 10 mL), saturated aqueous NaHCO3, and NaCl
solutions; dried (Na2SO4); filtered; and concentrated. The residue
was triturated with 3 mL of MeOH to give 74 mg (58%) of the
product as a light yellow solid. Mp: 175-176.5 °C. 1H NMR (400
MHz, CDCl3): δ 13.03 (d, 1H, J ) 12.5 Hz), 9.59 (s, 1H), 7.70
(d, 1H, J ) 12.5 Hz), 7.48 (dd, 1H, J ) 7.9, 1.2 Hz), 7.47-7.40
(m, 1H), 7.37 (dt, 1H, J ) 7.3, 1.2 Hz), 7.41 (d, 2H, J ) 8.5 Hz),
7.32 (dd, 1H, J ) 7.3, 1.8 Hz), 6.84 (d, 2H, J ) 8.5 Hz), 3.39 (br
s, 2H), 3.08 (s, 1H), 1.68 (hextet, 2H, J ) 7.3 Hz), 1.03 (t, 3H, J
) 7.5 Hz). 13C NMR (100 MHz, CDCl3): δ 193.19, 168.58, 155.40,
139.31, 139.18, 133.76, 130.89, 130.55, 130.11, 128.82, 126.97,
119.09, 117.08, 104.74, 82.75, 40.68, 22.79, 11.59 ppm. Anal.
(C21H19ClN2O2) C, H, N.
MHz, CDCl3): δ 12.75 (d, 1H, J ) 12.8 Hz), 9.28 (br s, 1H), 7.61
(d, 1H, J ) 12.8 Hz), 7.48 (dd, 1H, J ) 7.6, 1.2 Hz), 7.42 (dt, 1H,
J ) 7.0, 1.8 Hz), 7.38 (dt, 1H, J ) 7.2, 1.4 Hz), 7.33 (dd, 1H, J )
7.2, 2.0 Hz), 7.28 (d, 2H, J ) 8.9 Hz), 6.79 (d, 2H, J ) 8.8 Hz),
5.59 (br s, 1H). 13C NMR (100 MHz, CDCl3): δ 192.67, 170.61,
156.28, 139.17, 137.51, 131.02, 130.71, 130.56, 130.03, 129.90,
128.74, 126.98, 118.86, 104.02 ppm. Anal. (C16H12Cl2N2O2) C, H,
N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-methyl-â-
1
oxobenzenepropanamide (16c). Mp: 169.5-170.5 °C. H NMR
(400 MHz, CDCl3): δ 12.98 (d, 1H, J ) 12.2 Hz), 9.49 (br s, 1H),
7.65 (d, 1H, J ) 12.5 Hz), 7.47 (dd, 1H, J ) 7.9, 1.2 Hz), 7.41 (dt,
1H, J ) 7.3, 1.8 Hz), 7.37 (dt, 1H, J ) 7.6, 1.2 Hz), 7.31 (dd, 1H,
J ) 7.3, 1.8 Hz), 7.26 (d, 2H, J ) 8.8 Hz), 6.83 (d, 2H, J ) 8.8
Hz), 2.98 (d, 3H, J ) 4.6 Hz). 13C NMR (100 MHz, CDCl3): δ
193.00, 169.26, 155.78, 139.22, 137.66, 130.81, 130.52, 130.06,
129.95, 128.75, 126.96, 118.63, 104.43, 25.34 ppm. Anal. (C17H14-
Cl2N2O2) C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-â-oxo-N-
[35S]TBPS Binding Assay. The cortex from male Sprague-
Dawley rats (weighing 160-200 g) was removed immediately after
euthanization and dissected over ice. A P2 homogenate was prepared
for binding assay as previously described.10 The tissue was
homogenized in 0.32 M sucrose (J.T. Baker Chemical Co.,
Phillipsburg, NJ) with a Teflon-coated pestle, followed by cen-
trifugation at 1000g for 10 min. The supernatant was collected and
centrifuged at 9000g for 20 min. The resultant P2 pellet was
resuspended in ice-cold 50 mM sodium potassium phosphate (J.T.
Baker) buffer (pH 7.4) containing 200 mM NaCl (J.T. Baker) and
used immediately in binding assays. A 2 nM concentration of [35S]-
TBPS (86 Ci/mmol) was incubated with 100 µL of tissue homo-
genate (10% w/v) in the presence or absence of 5 µM GABA and
5 µL aliquots of test drug dissolved in dimethyl sulfoxide (e10
µL of solvent used in all assays). At the concentration (e1%) used,
dimethyl sulfoxide had no effect on specific [35S]TBPS binding.
All assays were brought to a final volume of 1 mL with 50 mM
sodium potassium phosphate buffer (pH 7.4) containing 200 mM
NaCl. Nonspecific binding was defined as binding in the presence
of 2 µM TBPS and accounted for ∼30% of the total binding. Assays
were terminated after a 90 min steady-state incubation at 25 °C by
rapid filtration through glass fiber filters (no. 32; Schleicher &
Schuell, Keene, NH). Filter-bound radioactivity was quantified by
liquid scintillation spectrophotometry (LSC). The data were evalu-
ated by nonlinear regression (GraphPad, Inc., San Diego, CA) to
obtain IC50 (concentration at which half-maximal inhibition of
radioligand occurs) values and Imax values (maximal inhibition).
[3H]Flunitrazepam Binding Assay. The [3H]flunitrazepam
binding assay was carried using the conditions and tissue preparation
described for the [35S]TBPS assay, with the exception that 1 µM
GABA was used instead of 5 µM GABA. [3H]Flunitrazepam (0.2
nM, 75 Ci/mmol) was used to label BZ-sites. Nonspecific binding
was defined as binding in the presence of 1 µM clonazepam. Values
are means and SEMs of three independent experiments.
1
propylbenzenepropanamide (16d). Mp: 119-120 °C. H NMR
(400 MHz, CDCl3): δ 12.99 (d, 1H, J ) 12.2 Hz), 9.60 (br s, 1H),
7.65 (d, 1H, J ) 12.5 Hz), 7.47 (dd, 1H, J ) 7.9, 1.2 Hz), 7.41 (dt,
1H, J ) 7.2, 2.0 Hz), 7.37 (dt, 1H, J ) 7.2, 1.4 Hz), 7.32 (dd, 1H,
J ) 7.3, 1.8 Hz), 7.26 (d, 2H, J ) 8.5 Hz), 6.83 (d, 2H, J ) 8.8
Hz), 3.38 (br s, 2H), 1.68 (hextet, 2H, J ) 7.3 Hz), 1.03 (t, 3H, J
) 7.5 Hz). 13C NMR (100 MHz, CDCl3): δ 193.04, 168.64, 155.86,
139.36, 137.76, 130.86, 130.78, 130.50, 130.07, 129.96, 118.64,
104.50, 40.66, 22.79, 11.57 ppm. Anal. (C19H18Cl2N2O2) C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-isopropyl-
â-oxobenzenepropanamide (16e). Mp: 130.5-131.5 °C. 1H NMR
(400 MHz, CDCl3): δ 13.01 (d, 1H, J ) 12.2 Hz), 9.46 (d, 1H, J
) 7.0 Hz), 7.47 (dd, 1H, J ) 7.6, 1.2 Hz), 7.41 (dt, 1H, J ) 7.3,
2.0 Hz), 7.36 (dt, 1H, J ) 7.3, 1.2 Hz), 7.31 (dd, 1H, J ) 7.3, 2.1
Hz), 7.25 (d, 2H, J ) 8.8 Hz), 7.64 (d, 1H, J ) 12.5 Hz), 6.83 (d,
2H, J ) 8.5 Hz), 4.20 (octet, 1H, J ) 6.8 Hz), 1.30 (d, 6H, J )
6.4 Hz). 13C NMR (100 MHz, CDCl3): δ 192.97, 167.77, 155.89,
139.32, 137.73, 130.80, 130.70, 130.47, 130.03, 129.91, 128.79,
126.92, 118.61, 104.43, 40.89, 22.74 ppm. Anal. (C19H18Cl2N2O2)
C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-cyclopro-
pyl-â-oxobenzenepropanamide (16g). Mp: 158-159 °C. 1H NMR
(400 MHz, CDCl3): δ 12.99 (d, 1H, J ) 12.5 Hz), 9.58 (br s, 1H),
7.64 (d, 1H, J ) 12.5 Hz), 7.47 (d, 1H, J ) 7.9 Hz), 7.42 (dt, 1H,
J ) 7.7, 1.4 Hz), 7.36 (t, 1H, J ) 7.3 Hz), 7.29 (d, 1H, J ) 7.2
Hz), 7.27 (d, 2H, J ) 8.8 Hz), 6.83 (d, 2H, J ) 8.8 Hz), 2.86
(hextet, 1H, J ) 3.7 Hz), 0.87 (m, 2H), 0.68 (m, 2H). 13C NMR
(100 MHz, CDCl3): δ 192.84, 170.14, 155.68, 139.05, 137.50,
130.75, 130.67, 130.47, 129.97, 129.87, 128.65, 126.88, 118.51,
104.11, 21.97, 6.28 ppm. Anal. (C19H16Cl2N2O2) C, H, N.
2-Chloro-r-[[(4-chlorophenyl)amino]methylene]-N-cyclopen-
tyl-â-oxobenzenepropanamide (16i). Mp: 165-166 °C. 1H NMR
(400 MHz, CDCl3): δ 13.00 (d, 1H, J ) 12.2 Hz), 9.58 (d, 1H, J
) 6.4 Hz), 7.63 (d, 1H, J ) 12.2 Hz), 7.47 (dd, 1H, J ) 7.9, 1.2
Hz), 7.41 (dt, 1H, J ) 7.0, 2.0 Hz), 7.36 (dt, 1H, J ) 7.2, 1.4 Hz),
7.30 (dd, 1H, J ) 7.2, 2.0 Hz), 6.83 (d, 2H, J ) 8.8 Hz), 4.32
(hextet, 1H, J ) 6.6 Hz), 2.06 (m, 2H), 1.78 (m, 2H), 1.64 (m,
[3H]Muscimol Binding Assay. The cortex from male Sprague-
Dawley rats (weighing 160-200 g) was removed immediately after
euthanization and dissected over ice. The tissue was homogenized
in 15 vol of 0.32 M sucrose followed by centrifugation at 1000g

3378 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Hogenkamp et al.
for 10 min. The supernatant was transferred to a 38 mL polycar-
bonate tube (Beckman Instruments, Palo Alto CA) and centrifuged
at 20000g for 20 min. The membrane pellet was resuspended in
10 vol of distilled water and centrifuged at 8000g for 20 min. The
resulting pellet was washed with distilled water once and with Na+-
free assay buffer (40 mM KH2PO4, 100 mM KCl, pH 7.4). The
pellet was resuspended in 35 mL of Na+-free assay buffer, incubated
at 37 °C for 30 min, and then centrifuged at 31000g for 20 min.
The final pellet was resuspended in 10 vol of Na+-free assay buffer.
The protein concentration was about 1 mg/mL by BCA reagent
protein assay. Aliquots of membrane suspension (100 µL) were
incubated in Na+-free assay buffer with 5 nM [3H]muscimol (25
Ci/mmol) and 5 µL of DMSO or drug dissolved in DMSO. The
final volume of the incubation medium was 1 mL. Nonspecific
binding was defined as binding in the presence of 1 mM GABA.
After the membranes were added, the tubes were briefly vortexed
and incubated at 4 °C in the dark. The incubation was terminated
after 60 min by rapid filtration through glass filters followed by
three washes with ice-cold assay buffer. Filter-bound radioactivity
was quantified by LSC after overnight extraction. The data were
evaluated as described for the [35S]TBPS assay.
Locomotor Activity Paradigm. Nonhabituated male non-Swiss
albino (NSA) mice were injected with test compounds dissolved
in 100% PEG 400 immediately before being placed in an E63-12
Tru Scan activity chamber (Colbourn Instruments, Allentown, PA).
Activity was monitored by horizontal beam breaks initiated by the
test animal and recorded every 5 min. Habituated male NSA mice
were exposed for 30 min to the activity chamber for three
consecutive days. On the fourth day, habituated male NSA mice
were injected with the test compound in PEG 400 or PEG 400 and
immediately placed into the activity chamber. Activity in habituated
mice was recorded every 5 min for 1 h. Activity for habituated
and nonhabituated animals was defined as the total distance traveled
(cm). All animals were tested between 9 a.m. and 2 p.m.
LRR Method. Male NSA mice were dosed with test compounds
at 50 mg/kg ip or po in PEG 400. After injection animals were
placed on their backs. Animals that righted themselves within 1
min were considered not to have lost their righting reflex. Those
that remained on their backs for more than 1 min were considered
to have lost their righting reflex.
Rotarod Test. The CNS depressant potential of compounds was
evaluated in the rotarod test as previously described.9 A dose that
causes behavioral toxicity in half the animals (toxic dose; TD50)
was calculated on the basis of each dose-response function by the
method of Litchfield and Wilcoxon.29
Light-Dark Paradigm. Male NSA mice (25-30 g) were used.
The apparatus consisted of an open-topped box divided into small
and large area by a partition that has a hole at floor level. The
small compartment was painted black and the large compartment
white. The white compartment was illuminated with light and the
black compartment with red light. The time spent in the light
compartment was recorded during a 3 min test session. Vehicle or
test compounds were administered 30 min prior to the test.
Anticonvulsant Assay. Adult male NSA mice (25-30 g) were
used. Time to peak anticonvulsant effect was determined against
pentylenetetrazole (PTZ) induced seizures. Mice were injected (ip)
with various doses of drug dissolved in PEG 400 or PEG 400 at
the time of peak effect before administration (sc) of a CD97 dose
of PTZ (85 mg/kg) or vehicle (0.9% saline 5 µL/g body weight).
Immediately after the injection mice are observed for a period of
45-60 min. Six animals were used per dose of test drug. The
number of animals with tonic/clonic convulsions was recorded.
Electrophysiology. Preparation, maintenance, and microinjection
of Xenopus oocytes were performed as reported previously.25
Individual oocytes were microinjected with ∼1-5 ng of cRNA
encoding human R1, â2, and γ2L GABAA receptor subunits.
Membrane current responses were measured at a holding potential
of -70 mV using conventional two-electrode voltage clamp
techniques, 3-11 days following injection. All drugs were dissolved
in DMSO and diluted with frog Ringer ((in mM) NaCl, 115; KCl,
2; CaCl2, 1.8; HEPES, 5; pH adjusted to 7.4 with NaOH; final
DMSO concentration <1%) and applied to oocytes via a linear
array rapid perfusion system.25 Modulatory effects of compounds
were assayed on control responses that were 10% of the maximum
GABA response (EC10) in an individual oocyte. Modulation was
measured as a percentage of Imax. Numerical data values listed refer
to value (SEM.
Acknowledgment. This work was funded by a University
of California Discovery Grant (bio04-10469 to K.W.G.).
Supporting Information Available: Table of elemental analysis
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
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