Affinity of 3-acyl substituted 4-quinolones at the benzodiazepine site of GABAA receptors

Article abstract of DOI:10.1016/j.bmc.2008.05.049

The finding that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate and N-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxamide derivatives may be high-affinity ligands at the benzodiazepine binding site of the GABA_A receptor, prompted a study of 3-acyl-1,4-dihydro-4-oxoquinoline (3-acyl-4-quinolones). In general, the affinity of the 3-acyl derivatives was found to be comparable with the 3-carboxylate and the 3-carboxamide derivatives, and certain substituents (e.g., benzyl) in position 6 were again shown to be important. As it is believed that the benzodiazepine binding site is situated between an α- and a γ-subunit in the GABA_A receptor, selected compounds were tested on the α₁β₂γ_2s, α₂β₂γ_2s and α₃β₂γ_2s GABA_A receptor subtypes. The 3-acyl-4-quinolones display various degrees of selectivity for α₁- versus α₂- and α₃-containing receptors, and high-affinity ligands essentially selective for α₁ over α₃ were developed.

Full text of DOI:10.1016/j.bmc.2008.05.049

Bioorganic & Medicinal Chemistry 16 (2008) 6936–6948
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Affinity of 3-acyl substituted 4-quinolones at the benzodiazepine
site of GABAA receptors
Erik Lager a, Jakob Nilsson a, Elsebet Østergaard Nielsen b, Mogens Nielsen c, Tommy Liljefors c,
Olov Sterner a,
*
a Division of Organic Chemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden
b NeuroSearch A/S, DK-2750 Ballerup, Denmark
c University of Copenhagen, Faculty of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
The finding that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate and N-alkyl-1,4-dihydro-4-oxoquino-
line-3-carboxamide derivatives may be high-affinity ligands at the benzodiazepine binding site of the
GABAA receptor, prompted a study of 3-acyl-1,4-dihydro-4-oxoquinoline (3-acyl-4-quinolones). In gen-
eral, the affinity of the 3-acyl derivatives was found to be comparable with the 3-carboxylate and the
3-carboxamide derivatives, and certain substituents (e.g., benzyl) in position 6 were again shown to be
Received 9 January 2008
Revised 12 May 2008
Accepted 22 May 2008
Available online 27 May 2008
important. As it is believed that the benzodiazepine binding site is situated between an
unit in the GABAA receptor, selected compounds were tested on the 1b2c2s 2b2c2s and
receptor subtypes. The 3-acyl-4-quinolones display various degrees of selectivity for 1- versus
3-containing receptors, and high-affinity ligands essentially selective for a1 over a3 were developed.
a
a
- and a
3b2c2s GABAA
2- and
c-sub-
Keywords:
a
,
a
3-Acyl-1,4-dihydro-4-oxoquinolines
Benzodiazepine binding site
GABAA receptor
GABAA receptor subtypes
Pharmacophore model
a
a
a
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
pine binding site is believed to be situated in the interface
between an
a- and a c-subunit, and a pharmacophore model of
c
-Aminobutyric acid, GABA, is a major neurotransmitter in the
the binding site has been developed.8 We have developed and re-
fined this model with additional pharmacophore elements follow-
ing a SAR study based on synthetic flavone derivatives.9,10 The
model has successfully guided us in identifying and optimizing no-
vel 4-quinolone derivatives of types A and B (see Fig. 1).11,12 Not
only was it possible to prepare very potent 4-quinolones, showing
Ki values down to 0.05 nM, but a preliminary study also indicated
that some of these compounds show significant subtype selectiv-
ity.12 However, 4-quinolones of types A and B can be expected to
be hydrolyzed rapidly by ester/amide hydrolysis in vivo, and are
mainly useful for the development of the pharmacophore model.
In the present study, we have therefore focused on three issues:
mammalian brain.1 GABA binds to three different receptor types:
the ligand gated chloride ion channels, GABAA and GABAC as well
as the G-protein coupled receptor, GABAB.2 GABAA receptors are
pentameric transmembrane proteins assembled by various combi-
nations of seven different subunits, which are mostly available as
multiple variants (
mon composition is two
a
1–6, b1–4
,
c
1–4, d,
e
, h, and
q
1–3).3 The most com-
c subunit. It is believed
a, two b and one
that receptors with different subtype compositions are responsible
for different physiologic responses. Many ligands, such as benzodi-
azepines, barbiturates, ethanol and certain steroids, are known to
allosterically alter the effect of GABA on the GABAA receptors.1
The pharmacological properties of benzodiazepines (anxiolytic,
anticonvulsant, muscle relaxant and sedative-hypnotic) have made
them the most common GABAA receptors modulating drugs in clin-
ical use,4 and the benzodiazepine binding site is consequently an
interesting target for development of novel drugs. Several other
types of compound are known to bind to the benzodiazepine bind-
ing site, such as the pyrimidin-5(6H)-ones, the b-carbolines, the
triazolopyridazines, the pyrazolo-quinolinones, the cyclopyrrol-
ones, the pyridodiindoles and the quinolines.5–7 The benzodiaze-
ꢀ To investigate the effect of an acyl substituent in position 3,
instead of a carboxylate or carboxamide, by preparing metabol-
ically more stable ketone analogues (i.e., type C) of the esters
and amides in Figure 1.
ꢀ To further explore the pharmacophore region called ‘interface’
(see Fig. 2) that has been proposed to be a channel-like cavity
at the interface between an a- and a c
-subunit.10
ꢀ To further evaluate the subtype selectivity of 4-quinolones.
Differences between subtypes has been evaluated as it is
believed that different subtypes mediate different physiological
* Corresponding author. Tel.: +46 462228213; fax: +46 462228209.
E-mail address: Olov.Sterner@organic.lu.se (O. Sterner).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.049

E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
6937
O
NH
O
NH
O
O
NH
R1 NH
R1
R1
O
O
O
R2
R2
R2
A
B
C
Figure 1. 4-Quinolone derivatives with affinity for the benzodiazepine site.
p-phenylaniline, p-benzylaniline, p-bromoaniline). The nitro pre-
cursors 28–30 were synthesized by various routes, including a
Heck coupling,17 a Wittig reaction18 and a Suzuki coupling.19 The
nitrobenzyl-pyridine derivative 31 was purchased, 33 was ob-
tained by reduction of the keto functionality of 3-benzoylpyri-
dine20 to yield 32, followed by nitration,21 while 34 was
prepared by nitration of 2-benzylpyridine.22 The nitro compounds
were reduced to the corresponding anilines 35–40 with hydrogen
in the presence of palladium on charcoal. A set of 3-acyl-4-quino-
lones were synthesized by reacting the 2-acyl-3-ethoxy acrylate
derivatives (see Scheme 3) with the anilines, to give the enamines
41–62.23,24 The enamines were isolated as Z/E mixtures, although
the E isomer was the major component (>90%) in all cases as indi-
cated by NMR. The enamines were subsequently cyclized in reflux-
ing diphenyl ether, yielding the desired 3-acyl-4-quinolone
derivatives 1–10 and 12–23 (see Table 1 and Section 5 for defini-
tion of R1 and R2 in each compound). Compound 11 was obtained
by direct nitration of 10.25 The 5,6,7,8-tetrahydro-4-quinolone 24
was prepared by the procedure depicted in Scheme 426. The con-
densation of the b-keto ester 27a with triethyl orthoformate in
the presence of acetic anhydride followed by the treatment of
the resulting intermediate with ammonia, resulted in the forma-
tion of amino analog 63 which was converted to 64 by the conden-
sation with cyclohexanone. 24 was obtained by cyclization of 64 in
refluxing diphenyl ether.
H1
A2
S1
3
S5
L1
L3
L2
S4
6
S2
H2/A3
S3
interface
Figure 2. The proposed binding mode of 14 in the pharmacophore model of the
benzodiazepine site of the GABAA receptors. The conformation of the 6-benzyl
group is arbitrary.
effects:
a1-containing receptors are involved with anterograde
amnesia and sedation, whereas
a
2-, a3- and possibly a5-containing
receptors are involved with anxiolytic activity.13,14 Consequently, a
subtype-selective compound could discriminate between the
many behaviors mediated by GABA transmission.
3. Results and discussion
The affinities of the tested 3-acyl-4-quinolone derivatives at the
benzodiazepine binding site were determined by displacement of
3H-flumazenil in rat cortical tissue in vitro (see the Section 5 for
details). The results are presented in Table 1. As discussed in detail
below, the affinities of the 3-acyl-4-quinolones are comparable
with those of the corresponding 3-carboxylates and 3-carboxam-
ides, indicating that the proposed interaction of a keto carbonyl
group with the hydrogen bond donor (H1, see Fig. 2) is comparable
with that of the ester or amide.
Our previous investigation of the corresponding 3-carboxylates
and 3-carboxamides has indicated that a chain length of the 3-sub-
stituent of 5 (including the ester/amide functionality) is optimal,12
and the focus of this investigation was therefore on the 3-valeryl
derivatives. However, the length of the unbranched chain is not
that critical in the acyl series, as discussed below. Branched or cyc-
2. Chemistry
The 24 3-acyl-4-quinolone derivatives prepared and investi-
gated in this study are to our knowledge all new compounds.
Important building blocks for the synthesis of the 4-quinolones
are the b-keto esters 27a–j, which were prepared by two different
routes outlined in Scheme 1 or purchased (27j).15,16 The b-keto es-
ters 27a–j were converted to the corresponding 2-acyl-3-ethoxy
acrylate derivatives, by condensation with triethyl orthoformate
in the presence of acetic anhydride (see Scheme 3), which were
used in the next step without purification or characterization.
The different substituents in the 6-position were introduced by uti-
lizing the corresponding aniline derivatives, synthesized according
to Scheme 2 if not commercially available (aniline, p-ethylaniline,
a: R1 = propyl
b: R1 = butyl
O
O
c: R1 = pentyl
d: R1 = 1-methylpropyl
HO
O
O
O
e: R1 = 2-methylpropyl
f: R1 = 2,2-dimethylpropyl
g: R1 = cyclopentyl
h: R1 = cyclohexyl
i: R1 = phenyl
R1
O
O
O
27a-j
O
j: R1 = ethyl
Scheme 1. Reagents and conditions: (a) 1—2 equiv n-BuLi, ꢁ78 to ꢁ5 °C, 10 min; 2—R1CH2COCl, ꢁ78 °C–rt, 15 min; (b) 1—NaH, 0 °C; 2—n-BuLi, 0 °C; 3—R1-X, 0 °C–rt, 30 min.

6938
E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
NO2
NH2
NO2
g
a
b
c
+
28
35
I
NO2
NH2
O
g
+
H
PPh3Br
29
36
NO2
NO2
NH2
NO2
B(OH)2
g
+
30
37
Br
NO2
NH2
g
31
38
N
N
NO2
NH2
g
d
e
O
N
N
32
33
39
N
N
NO2
NH2
g
f
N
34
40
N
N
Scheme 2. Reagents and conditions: (a) K2CO3, tetrabutylammonium bromide, Pd(OAc)2, DMF, 100 °C, 12 h; (b) n-BuLi, THF, ꢁ78 °C–rt; (c) PdCl2, K2CO3, acetone/H2O (3:1),
0 °C–rt, 3 h; (d) 10% Pd/C, concd HCl, H2, rt, 24 h; (e) NH4NO3, (CF3CO)2O, CHCl3, ꢁ10 °C–rt, 43 h; (f) concd HNO3, concd H2SO4, 0–50 °C, 5 min; (g) H2 (500 psi or 1 atm), 10%
Pd/C, MeOH or EtOH or EtOH/EtOAC, rt, 3 h or over night.
lic groups in the ketone moiety (4–9) will result in higher Ki values
than corresponding straight chain derivatives, as previously was
shown for the esters.12 The comparison of 25 and 26, or the corre-
sponding propyl esters and amides with R2 either as an ethyl or as
a benzyl group, shows that a benzyl group instead of an ethyl
group in the 6-position increase the affinity by a factor 10–20.12
Consequently, the receptor–ligand interaction in the vicinity of this
position is interesting to explore further, keeping the rest of the
molecule as the 3-valeryl-4-quinolone. By exchanging the ethyl
group of 1 and 7 for a benzyl group (to 14 and 16) the same effect
can be seen for the 3-acyl-4-quinolones as for 25 and 26, although
less pronounced. A 6-phenyl (13) is obviously not good, resulting
in a 165-fold decrease in affinity compared to the benzyl derivative
14. The low affinity of 13 could be explained by the requirement
for ligands to be planar in this receptor region.9 Conformational
analysis using the MMFF94s force field and the GB/SA hydration
model, performed as described in Section 5, shows that the energy
cost for the phenyl group of 13 to adopt a co-planar conformation
with respect to the bicyclic system is 2.9 kcal compared to its glo-
bal minimum. Increasing the linker length between position 6 and
the benzene ring of 14 with one methylene group (17) has a small
effect. However, with a propylene linker (18) or a larger aromatic
group (19) the Ki values increase, indicating a steric repulsive inter-
action further out in the ‘interface’ and/or higher energy penalties
due to the longer linker of 18 when adopting the right bioactive
conformation. In spite of the significantly higher desolvation en-
ergy of pyridylmethylene compared to benzyl, compounds 20–22
display only a slightly lower affinity than corresponding benzyl
compound 14. An explanation may be that part of the 6-substitu-
ent is solvated, with the result that no extra desolvation energy

E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
6939
carboxylates and 3-carboxamides series. The butyl ketones 1
(0.9 nM) and 14 (0.17 nM) are comparable with the corresponding
propyl esters, 1.8 nM and 0.17 nM, respectively.12 However, it is
obvious that the 3-acyl series is less sensitive to the length of the
acyl carbon chain compared to the esters. A 3-butyryl instead of
a 3-valeryl does practically not affect the affinity, 15 versus 14,
but the corresponding carbon chain decrease in the ester series
gave rise to a compound with 8 times lower affinity (ethyl ester
26 vs its corresponding propyl ester).12 Also, increasing the chain
length from C5 (1) to C6 (2) and C7 (3) has a minor effect in the acyl
series while the affinity decreases 20 times for the esters.12 The
most potent 4-quinolone prepared so far is the amide (type B,
Fig. 1) with R1 = propyl and R2 = benzyl, with the Ki value
0.048 nM. This is considerably more potent compared to the corre-
sponding ester and ketone (both 0.17 nM), and a possible explana-
tion is that the NH of the amide supports the binding by giving a
(weak) hydrogen bond to H2/A3 (see Fig. 2).
O
O
O
R1
O
O
O
a
R1
O
27a-j
O
O
R1
O
O
NH
R1
HN
b
c
O
R2
1-10 and 12-23
R2
41-62
Compound 24 was considered to be potentially interesting, as
partly saturated derivatives in other types of compounds have dis-
played high-affinity at the benzodiazepine binding site.5 However,
the affinity was 8 times lower compared to its unsaturated parent
compound (10), indicating that the electronic properties of 24,
and/or the small deviation from being planar, affect the affinity
compared to the planar compound 10 in a negative way.
Scheme 3. Reagents and conditions: (a) triethyl orthoformate, acetic anhydride,
100 °C, over night; (b) para-substituted aniline derivatives, 100 °C, 1 h; (c) diphenyl
ether, reflux, 30 min R1 according to Scheme 1 and Table 1, R2 according to Table 1.
Table 1
Ki values of 3-acyl-4-quinolones 1–23 and 3-pentanoyl-5,6,7,8-tetrahydro-4-quino-
lone 24 tested on 3H-flumazenil binding in vitro to rat cortical membranes
In order to assess the subtype specificity of the 3-acyl-4-quino-
lones, selected compounds were tested on recombinant
2b2c2s and 3b2c2s receptor subtypes (results are shown in Table
2). We have previously shown that selectivity for 1- over 3-con-
a1b2c2s,
O
O
NH
NH
a
a
O
NH
R1
a
a
O
taining receptors canbe obtained by including substituentsinteract-
ing in the receptor ‘interface’ region as well as the L1 and L2 regions
of the pharmacophore model (see Fig. 2).12 One aim of the present
O
O
O
R2
R2
study was therefore to reveal possible differences between
and 3-containing receptor subtypes focusing on the ‘interface’ re-
gion. All compounds investigated in this part of the study display a
preference for 1b2c2 over 2b2c2 and 3b2c2 receptor subtypes,
respectively. The a2 a1 Ki ratio remained essentially the same for
the compounds that were assayed, whereas the a3 1 Ki ratio obvi-
a1-, a2-,
1-23
25-26
24
R2
a
a
Compound
R1
Ki value (nM)
a
a
a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
CH3CH2CH2–
CH3CH2CH2CH2–
CH3CH2CH2CH2CH2–
CH3CH2CH(CH3)–
(CH3)2CHCH2–
(CH3)3CCH2–
Cyclopentyl–
Cyclohexyl–
Phenyl–
CH3CH2CH2–
CH3CH2CH2–
CH3CH2CH2–
CH3CH2CH2–
CH3CH2CH2–
CH3CH2–
Cyclopentyl–
CH3CH2CH2–
CH3CH2CH2–
CH3CH2CH2–
CH3CH2CH2–
CH3CH2CH2–
CH3CH2CH2–
Cyclopentyl–
CH3CH2–
CH3CH2–
CH3CH2–
CH3CH2–
CH3CH2–
CH3CH2–
CH3CH2–
CH3CH2–
CH3CH2–
H–
NO2–
Br–
Phenyl–
Benzyl–
Benzyl–
Benzyl–
Benzyl–CH2–
Benzyl–CH2CH2–
2-Naphthyl–CH2–
4-Pyridyl–CH2–
3-Pyridyl–CH2–
2-Pyridyl–CH2–
3-Pyridyl–CH2–
0.9 0.2
0.98 0.19
2.1 0.3
18 4.4
/
/a
ously depends on the substituent in position 6 of the 4-quinolone
derivatives. The following discussion will therefore focus on differ-
2.5 1.7
19
3
ences between
In the ester series, the substitution of an ethyl to a benzyl group
in the 6-position (25 vs 26) resulted in 3 times higher a3 1Ki ratio
as well as an affinity increase. The a3 a1 Ki ratio remained essen-
a3- versus a1-containing receptors.
7.9 1.1
30 11
69 21
2.2 0.4
0.87 0.09
0.54 0.23
/a
/
tially unaffected for a similar ethyl to benzyl group substitution
in the ketone series (1 vs 14). No significant subtype difference
in the ‘interface’ region could be traced by bulkier 6-position sub-
stituents (17–19). This indicates that the three different subtypes
all accept bulky substituents to the same extent in the ‘interface’
region, at least when it comes to 3-valeryl-4-quinolones. The pyr-
28
9
0.17 0.01
0.22 0.05
1.0 0.3
0.42 0.11
2.5 0.3
idyl derivative 20 displays the same a3
zyl compound 14. However, and interestingly, the other two
pyridyl isomers 21 and 22 show a 3-fold enhanced a3 a1 Ki ratio
/a1 Ki ratio as its parent ben-
1.3 0.2
0.53 0.11
0.27 0.09
0.23 0.07
1.5 0.2
/
compared to 20. This indicates that there are differences between
the two receptor subtypes in the ‘interface’ region, which could
be included in a future subtype specific pharmacophore model.
The fused benzene ring of the 4-quinolones interact, according to
pharmacophore model, with the same receptor region as the satu-
rated ring structures in imidazopyridopyrimidinone- and pyrazolo-
3-pyridinone derivatives that display functional selectivity at
receptor subtypes.5,26 Therefore, we were interested in investigat-
ing the affinity of a partly saturated 4-quinolone derivative (10 vs
17.1 2.1
CH3CH2–
Benzyl–
20 5b
1.4 0.2b
a
Each Ki value is the mean SD of three determinations.
Previously published data.11
b
would be lost for the pyridyl derivatives when binding to the
receptor. This is in accordance with the channel-like cavity that
has been proposed to be situated in this area.10
24). The affinity decreased for 24 at both
a1- and a3-containing
receptors, and the a3 a1 Ki ratio decreased indicating additional
/
A detailed comparison of the impact of the keto function on cor-
tical tissue affinity can be made in those cases where affinity data
of the corresponding 4-quinolones are available for the 3-acyl, 3-
differences between the receptor subtypes. Previous research has
shown that bulky substituents in the L2 region result in an increase
of the a3/a
1 Ki ratio.6,12 The less bulky propyl ketone 15 displayed,

6940
E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
O
O
O
O
O
O
a
b
O
27a
O
O
O
O
O
O
NH
d
c
O
HN
O
H2N
64
63
24
Scheme 4. Reagents and conditions: (a) triethyl orthoformate, acetic anhydride, 100 °C, over night; (b) NH3, EtOH, rt, 5 h; (c) cyclohexanon, p-TsOH, toluene, Dean-Stark
conditions, 24 h; (d) diphenyl ether, reflux, 30 min.
Table 2
The affinity of selected 4-quinolones tested on 3H-flumazenil binding to
a
1b2c2s
,
a
2b2c2s and a3b2c2s GABAA receptor subtypes
O
NH
O
O
O
NH
NH
NH
O
NH
O
O
O
O
O
O
R1
R1
R1
1, 10, 14, 17-22
7, 16, 23
15
24
25, 26
Substance
R1
Kia1 (nM)
Kia2 (nM)
Kia3 (nM)
Ki ratio a2
/
a1
Ki ratio a3/a1
1
7
CH3CH2–
CH3CH2–
H–
0.38 0.05a
5.1 0.8a
1.6 0.4a
2.8 0.1a
74 24a
9.2 2.3a
4
6
—
3
7
—
3
5
8
7
—
—
—
—
—
—
7
15
9
31 10a
10
14
15
16
17
18
19
20
21
22
23
24
25
26
0.97 0.15a
0.12 0.04a
0.12 0.05a
0.51 0.05a
0.53 0.31a
3.2 1.5a
Not tested
0.40 0.15a
0.81 0.13a
Not tested
1.7 0.7a
16 8a
Benzyl–
0.61 0.11a
1.56 0.96a
13 2a
5
13
25
2
4
3
Benzyl–
Benzyl-CH2–
1.1 0.1a
14.3 9.6a
8.8 5.5a
2.1 0.8a
2.2 0.2a
2.2 0.6a
23 2a
Benzyl-CH2CH2–
(2-Naphthyl)CH2–
(4-Pyridyl)CH2–
(3-Pyridyl)CH2–
(2-Pyridyl)CH2–
(3-Pyridyl)CH2–
2.7 1.0a
22 13a
0.39 0.20a
0.12 0.02a
0.14 0.02a
0.88 0.27a
11 1a
2.7 1.3a
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
5
18
16
26
5
9
27
51 9a
CH3CH2–
Benzyl–
5.9 0.4a
52 8a
0.27 0.06a,b
7.2 0.03a,b
a
Each Ki value is the mean SD of three determinations.
b
Previously published data.11
however, a higher a3
tone 14. Also the cyclopentylketone 7 showed a higher
ity than corresponding butylketone 1.
In order to investigate if substances with a 3-cyclopentyl meth-
ylene ketone and a bulky substituent in position 6 could display
synergetic effects, for example, higher subtype selectivity than 7,
compounds 16 and 23 were synthesized and tested. This was
apparently a successful approach, resulting in with two high-affin-
/
a1 Ki ratio than the corresponding butyl ke-
in spite of the differences in the electronic properties of the car-
bonyl function in the three compound types. Nevertheless, set of
compounds prepared and assayed in this investigation have per-
mitted additional characterization of the ‘interface’, as defined in
the pharmacophore model. The decreased affinity that is displayed
by 18 and 19, with bulky 6-substituents, indicate the presence of
receptor essential volumes (steric repulsive interactions) further
out in the ‘interface’. Several substances have been tested on the
a1 selectiv-
ity ligands that display a a3
and this path will be further investigated.
/
a1 Ki ratio of 25 and 26, respectively,
a
1b2c2s
these results, 16 and 23 were designed to display selectivity for
1- over 3-containing receptors, and were shown to have strong
affinity at the 1b2c2s subtype (Ki < 1 nM) and a3 a1 Ki ratios of
, a2b2c2s and a3b2c2s GABAA receptor subtypes. Based on
a
a
4. Conclusion
a
/
25 and 26, respectively. As the ketones can be expected to be met-
abolically more stable than the corresponding esters or amides, se-
lected 4-quinolones from present study are currently undergoing
in vivo studies.
The affinity of the 3-acyl-4-quinolones for the benzodiazepine
binding site of the GABAA receptor is to a large extent similar to
that of the corresponding esters and amides studied previously,

E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
6941
(MeOD) d 0.93 (6H, m), 1.28 (4H, m), 1.44 (1H, m), 2.02 (1H, m),
2.80 (2H, q, J = 7.5), 2.96 (1H, m), 3.23 (1H, m), 7.51 (1H, d,
J = 8.4), 7.62 (1H, d, J = 8.4), 8.16 (1H, s), 8.53 (1H, s); 13C NMR
(MeOD) d 11.8, 16.0, 19.9, 29.6, 30.9, 32.4, 51.2, 119.5, 119.8,
125.3, 129.2, 134.6, 138.8, 143.4, 145.4, 178.1, 202.5. HRMS (ESI):
for C17H22NO2 calcd: 272.1651 [MH]+; found: 272.1646.
5. Experimental
Reagents and solvents (except THF) were used from commercial
sources without purification. THF was distilled from sodium/ben-
zophenone prior to use. 1H and 13C NMR were recorded at room
temperature with a Bruker ARX300 or a Bruker DRX400 spectrom-
eter. The spectra were recorded in CDCl3, DMSO-d6, and CD3OD,
and the solvent signals (7.27 and 77.0, 2.50 and 39.5 or 3.31 and
49.0 ppm, respectively) were used as reference. The raw data were
transformed and the spectra were evaluated with the standard
Bruker UXNMR software (rev. 941001). Analytical thin-layer chro-
matography (TLC) was performed on Kiselgel 60 F254 plates
(Merck). Column chromatography was performed on SiO2 (Matrex
LC-gel: 60A, 35–70 MY, Grace). Melting points (uncorrected) were
determined with a Reichert microscope. EI mass spectra were re-
corded at 70 eV with a Jeol SX102 spectrometer and ESI spectra
were recorded with Micromass Q-TOF Micro. The purity of the as-
sayed compounds was verified with 1H NMR and HPLC, and only
used if more than 98% pure.
5.5. 6-Ethyl-3-(4-methylpentanoyl)-4-quinolone (5)
The title compound was prepared and purified according to the
procedure described for 1, starting from 45 (vide infra). The reac-
tion yielded 5 (35%), a white solid (mp: 196–198 °C). 1H NMR
(MeOD) d 0.95 (6H, d, J = 6.5), 1.30 (3H, t, J = 7.6), 1.55 (2H, m),
1.64 (1H, m), 2.80 (2H, q, J = 7.6), 3.20 (2H, t, J = 7.6), 7.50 (1H, d,
J = 8.4), 7.61 (1H, d, J = 8.4), 8.16 (1H, s), 8.53 (1H, s); 13C NMR
(MeOD) d 16.0, 22.9, 22.9, 29.2, 29.6, 34.5, 42.3, 119.1, 119.8,
125.2, 129.2, 134.6, 138.8, 143.4, 145.3, 178.2, 202.8. HRMS (ESI):
for C17H22NO2 calcd: 272.1651 [MH]+; found: 272.1657.
5.6. 3-(4,4-Dimethylpentanoyl)-6-ethyl-4-quinolone (6)
5.1. 6-Ethyl-3-pentanoyl-4-quinolone (1)
The title compound was prepared and purified according to the
procedure described for 1, starting from 46 (vide infra). The reac-
tion yielded 6 (34%), a white solid (mp: 217–219 °C). 1H NMR
(DMSO-d6) d 0.91 (9H, s), 1.23 (3H, t, J = 7.5), 1.48 (2H, m), 2.74
(2H, q, J = 7.5), 3.09 (2H, m), 7.55 (1H, d, J = 8.4), 7.59 (1H, d,
J = 8.4), 8.06 (1H, s), 8.46 (1H, s), 12.44 (1H, s); 13C NMR (DMSO-
d6) d 15.5, 27.9, 29.2, 29.2, 29.2, 29.9, 37.7, 38.2, 117.3, 118.8,
123.9, 127.8, 132.7, 137.2, 140.6, 143.7, 175.0, 199.8. HRMS (ESI):
for C18H24NO2 calcd: 286.1807 [MH]+; found: 286.1808.
41 (vide infra) (0.134 g, 0.442 mmol) was dissolved in diphenyl
ether (2 ml). The mixture was refluxed for 1 h and then allowed to
cool to rt. Petroleum ether was added and the resulting crystals
were collected and washed with a large amount of petroleum
ether. The crystals were purified by trituration with diethyl ether
yielding 1 (0.068 g, 0.264 mmol, 60%), a white solid (mp: 206–
208 °C). 1H NMR (MeOD) d 0.96 (3H, t, J = 7.3), 1.30 (3H, t,
J = 7.6), 1.42 (2H, m), 1.65 (2H, m), 2.80 (2H, q, J = 7.6), 3.19 (2H,
t, J = 7.3), 7.51 (1H, d, J = 8.4), 7.63 (1H, dd, J = 8.5, 2.0), 8.16 (1H,
m), 8.54 (1H, s); 13C NMR (DMSO-d6) d 13.9, 15.5, 22.0, 26.1,
27.9, 42.2, 117.3, 118.8, 123.8, 127.8, 132.7, 137.2, 140.6, 143.7,
5.7. 3-(Cyclopentylacetyl)-6-ethyl-4-quinolone (7)
175.0, 199.2. HRMS (ESI): for
C16H19NO2Na calcd: 280.1313
The title compound was prepared and purified according to the
procedure described for 1, starting from 47 (vide infra). The reac-
tion yielded 7 (50%), a white solid (mp: 209–211 °C). 1H NMR
(MeOD) d 1.21 (2H, m), 1.30 (3H, t, J = 7.6), 1.60 (4H, m), 1.84
(2H, m), 2.35 (1H, m), 2.80 (2H, q, J = 7.6), 3.22 (2H, d, J = 7.1),
7.51 (1H, d, J = 8.4), 7.62 (1H, dd, J = 8.4, 1.9), 8.16 (1H, d, J = 1.9),
8.53 (1H, s); 13C NMR (MeOD) d 16.0, 26.0, 26.0, 29.6, 33.7, 33.7,
37.2, 50.4, 119.3, 119.8, 125.2, 129.3, 134.6, 138.8, 143.4, 145.3,
178.2, 202.3. HRMS (ESI): for C18H22NO2 calcd: 284.1651 [MH]+;
found: 284.1649.
[MNa]+; found: 280.1320.
5.2. 6-Ethyl-3-hexanoyl-4-quinolone (2)
The title compound was prepared and purified according to the
procedure described for 1, starting from 42 (vide infra). The reaction
yielded 2 (57%), a white solid (mp: 209–211 °C). 1H NMR (MeOD) d
0.92 (3H, t, 7.1), 1.30 (3H, t, J = 7.6), 1.37 (4H, m), 1.67 (2H, m), 2.80
(2H, q, J = 7.6), 3.17 (2H, t, J = 7.4), 7.50 (1H, d, J = 8.4), 7.62 (1H, dd,
J = 8.4, 1.9), 8.15 (1H, d, J = 1.9), 8.54 (1H, s); 13C NMR (MeOD) d
14.4, 16.0, 23.7, 25.3, 29.6, 32.9, 44.2, 119.1, 119.8, 125.2, 129.2,
134.6, 138.8, 143.4, 145.3, 178.2, 202.6. HRMS (ESI): for C17H22NO2
calcd: 272.1651 [MH]+; found: 272.1662.
5.8. 3-(Cyclohexylacetyl)-6-ethyl-4-quinolone (8)
The title compound was prepared and purified according to the
procedure described for 1, starting from 48 (vide infra). The reac-
tion yielded 8 (68%), a white solid (mp: 229-231 °C). 1H NMR
(DMSO-d6) d 0.95 (2H, m), 1.17 (6H, m), 1.65 (5H, m), 1.82, (1H,
s), 2.73 (2H, q, J = 7.6), 2.99 (2H, d, J = 6.8), 7.56 (2H, m), 8.04
(1H, s), 8.46 (1H, s); 13C NMR (DMSO-d6) d 15.5, 25.8, 25.8, 25.9,
27.9, 32.9, 32.9, 33.7, 49.9, 117.6, 118.9, 123.9, 127.8, 132.7,
137.3, 140.6, 143.8, 174.9, 198.7. HRMS (ESI): for C19H24NO2 calcd:
298.1807 [MH]+; found: 298.1796.
5.3. 6-Ethyl-3-heptanoyl-4-quinolone (3)
The title compound was prepared and purified according to the
procedure described for 1, starting from 43 (vide infra). The reac-
tion yielded 3 (46%), a white solid (mp: 208–210 °C). 1H NMR
(MeOD) d 0.90 (3H, t, J = 6.8), 1.33 (9H, m), 1.66 (2H, m), 2.80
(2H, q, J = 7.6), 3.18 (2H, t, J = 7.4), 7.50 (1H, d, J = 8.4), 7.62 (1H,
dd, J = 8.4, 1.9), 8.15 (1H, d, J = 1.9), 8.54 (1H, s); 13C NMR (MeOD)
d 14.4, 16.0, 23.7, 25.5, 29.6, 30.3, 33.0, 44.2, 119.1, 119.8, 125.2,
129.2, 134.6, 138.8, 143.4, 145.3, 178.2, 202.5. HRMS (ESI): for
5.9. 6-Ethyl-3-(phenylacetyl)-4-quinolone (9)
C
18H23NO2Na calcd: 308.1626 [MNa]+; found: 308.1631.
The title compound was prepared and purified according to the
procedure described for 1, starting from 49 (vide infra). The reac-
tion yielded 9 (59%), a white solid (mp: 227–229 °C). 1H NMR
(MeOD) d 1.31 (3H, t, J = 7.6), 2.80 (2H, q, J = 7.6), 4.54 (2H, s),
7.22 (5H, m), 7.49 (1H, d, J = 8.5), 7.61 (1H, dd, J = 8.4, 1.6) 8.18
(1H, d, J = 1.6), 8.51 (1H, s); 13C NMR (MeOD) d 16.0, 29.6, 49.9,
118.8, 119.9, 125.3, 127.5, 129.2, 129.3, 129.3, 130.9, 130.9,
5.4. 6-Ethyl-3-(3-methylpentanoyl)-4-quinolone (4)
The title compound was prepared and purified according to the
procedure described for 1, starting from 44 (vide infra). The reac-
tion yielded 4 (47%), a white solid (mp: 183–184 °C). 1H NMR

6942
E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
134.6, 137.0, 138.8, 143.5, 145.6, 178.2, 199.9. HRMS (ESI): for
19H18NO2 calcd: 292.1338 [MH]+; found: 292.1338.
26.1, 40.6, 42.2, 117.4, 119.0, 125.1, 126.1, 127.8, 128.5, 128.5,
128.8, 128.8, 133.4, 137.4, 138.3, 140.9, 143.8, 174.9, 199.1. HRMS
(ESI): for C21H22NO2 calcd: 320.1651 [MH]+; found: 320.1655.
C
5.10. 3-Pentanoyl-4-quinolone (10)
5.15. 6-Benzyl-3-butyryl-4-quinolone (15)
The title compound was prepared and purified according to the
procedure described for 1, starting from 50 (vide infra). The reac-
tion yielded 10 (63%), a white solid (mp: 225–227 °C). 1H NMR
(MeOD) d 0.95 (3H, t, J = 7.4), 1.42 (2H, m), 1.65 (2H, m), 3.18
(2H, t, J = 7.4), 7,48 (1H, ddd, J = 8.2, 7.1, 1.1), 7.57 (1H, d, J = 8.2),
7.74 (1H, ddd, J = 8.2, 7.1, 1.4), 8.33 (1H, dd, J = 8.2, 1.4), 8.57 (1H,
s); 13C NMR (MeOD) d 14.4, 23.7, 27.7, 43.9, 119.4, 119.8, 126.6,
127.2, 129.3, 134.1, 140.6, 145.8, 178.2, 202.4.HRMS (ESI): for
The title compound was prepared and purified according to the
procedure described for 1, starting from 54 (vide infra). The reac-
tion yielded 15 (71%), a white solid (mp: 230–232 °C). 1H NMR
(DMSO-d6) d 0.87 (3H, t, J = 7.3), 1.54 (2H, sex, J = 7.3), 3.02 (2H,
t, J = 7.3), 4.05 (2H, s), 7.17 (1H, m), 7.25 (4H, m), 7.52 (1H, d,
J = 8.4), 7.57 (1H, dd, J = 8.4, 1.8), 8.01 (1H, d, J = 1.8), 8.44 (1H, s),
12.44 (1H, br s); 13C NMR (DMSO-d6) d 13.9, 17.3, 40.6, 44.5,
117.4, 119.0, 125.1, 126.1, 127.8, 128.5, 128.5, 128.8, 128.8,
133.4, 137.4, 138.3, 140.9, 143.8, 174.9, 199.0. HRMS (ESI): for
C
14H16NO2 calcd: 230.1181 [MH]+; found: 230.1189.
5.11. 6-Nitro-3-pentanoyl-4-quinolone (11)
A solution of 68% HNO3 (52 l, 0.785 mmol) in 1.5 ml concd
C
20H20NO2 calcd: 306.1494 [MH]+; found: 306.1493.
l
5.16. 6-Benzyl-3-(cyclopentylacetyl)-4-quinolone (16)
H2SO4 was added to a solution of 10 (150 mg, 0.654 mmol) in
1.5 ml H2SO4 at ꢁ10 °C. The mixture was stirred at ꢁ10 °C for 4 h
whereupon it was poured on ice. The mixture was extracted with
diethyl ether, and the extract was dried with MgSO4, and then con-
centrated. The crude product was purified by chromatography
(CHCl3/EtOH, 50:1) followed by recrystallization from ethanol to
give 11 (76 mg, 0.277 mmol, 42%), a white solid (mp: 278–
280 °C). 1H NMR (DMSO-d6) d 0.89 (3H, t, J = 7.4), 1.33 (2H, sex,
J = 7.4), 1.55 (2H, quint, J = 7.4), 3.08 (2H, t, J = 7.4), 7.80 (1H, d,
J = 9.1), 8.46 (1H, dd, J = 9.1, 2.7), 8.6 (1H, s), 8.90 (1H, d, J = 2.7);
13C NMR (DMSO-d6) d 13.9, 22.0, 25.9, 42.2, 118.7, 120.8, 122.0,
126.7, 127.3, 143.0, 143.8, 145.6, 174.4, 198.7. HRMS (ESI): for
The title compound was prepared and purified according to the
procedure described for 1, starting from 55 (vide infra). The reac-
tion yielded 16 (88%), a white solid (mp: 215–217 °C). 1H NMR
(DMSO-d6) d 1.12 (2H, m), 1.46 (2H, m), 1.57 (2H, m), 1.74 (2H,
m), 2.24 (1H, m), 3.11 (2H, d, J = 7.1), 4.08 (2H, s), 7.21 (1H, m),
7.28 (4H, m), 7.55 (1H, d, J = 8.4), 7.61 (1H, dd, J = 8.4, 2.0), 8.03
(1H, d, J = 2.0), 8.46 (1H, s), 12.5 (1H, br s); 13C NMR (DMSO-d6)
d 24.5, 24.5, 32.2, 32.2, 35.4, 40.6, 48.6, 117.5, 119.0, 125.0,
126.1, 127.8, 128.5, 128.5, 128.8, 128.8, 133.4, 137.4, 138.2,
140.9, 143.8, 174.9, 198.9. HRMS (ESI): for C23H24NO2 calcd:
346.1807 [MH]+; found: 346.1795.
C
14H15N2O4 calcd: 275.1032 [MH]+; found: 275.1020.
5.17. 3-Pentanoyl-6-(2-phenylethyl)-4-quinolone (17)
5.12. 6-Bromo-3-pentanoyl-4-quinolone (12)
The title compound was prepared and purified according to the
procedure described for 1, starting from 56 (vide infra). The reac-
tion yielded 17 (67%), a white solid (mp: 223–225 °C). 1H NMR
(DMSO-d6) d 0.89 (3H, t, J = 7.4), 1.33 (2H, sex, J = 7.4), 1.55 (2H,
quint, J = 7.4), 2.93 (2H, m), 3.01 (2H, m), 3.09 (2H, t, J = 7.4), 7.17
(1H, m), 7.25 (4H, m), 7.53 (1H, d, J = 8.4), 7.59 (1H, dd, J = 8.4,
1.8), 8.06 (1H, d, J = 1.8), 8.46 (1H, d, J = 5.9), 12.44 (1H, d,
J = 5.9); 13C NMR (DMSO-d6) d 13.9, 22.1, 26.1, 36.7, 36.9, 42.2,
117.3, 118.7, 124.8, 125.9, 127.7, 128.2, 128.2, 128.4, 128.4,
133.2, 137.3, 138.4, 141.2, 143.7, 175.0, 199.2. HRMS (ESI): for
The title compound was prepared and purified according to the
procedure described for 1, starting from 51 (vide infra). The reac-
tion yielded 12 (80%), a white solid (mp: 295–297 °C). 1H NMR
(DMSO-d6) d 0.89 (3H, t, J = 7.3), 1.32 (2H, m), 1.55 (2H, m), 3.08
(2H, t, J = 7.3), 7.60 (1H, d, J = 8.8), 7.87 (1H, dd, J = 8.8, 2.3), 8.29
(1H, d, J = 2.3), 8.54 (1H, s); 13C NMR (DMSO-d6) d 13.9, 22.0,
25.9, 42.2, 117.6, 117.8, 121.5, 127.9, 129.3, 135.2, 138.1, 144.6,
173.7, 198.9. HRMS (ESI): for
C14H151NO2Br calcd: 308.0286
[MH]+; found: 308.0296.
C
22H24NO2 calcd: 334.1807 [MH]+; found: 334.1819.
5.13. 3-Pentanoyl-6-phenyl-4-quinolone (13)
5.18. 3-Pentanoyl-6-(3-phenylpropyl)-4-quinolone (18)
The title compound was prepared and purified according to the
procedure described for 1, starting from 52 (vide infra). The reac-
tion yielded 13 (82%), a white solid (mp: 267–269 °C). 1H NMR
(DMSO-d6) d 0.90 (3H, t, J = 7.3), 1.34 (2H, m), 1.57 (2H, m), 3.13
(2H, t, J = 7.4), 7.41 (1H, m), 7.52 (2H, m), 7.74 (3H, m), 8.06 (1H,
dd, J = 8.5, 2.1), 8.46 (1H, d, J = 2.1), 8.54 (1H, s); 13C NMR
(DMSO-d6) d 14.0, 22.1, 26.1, 42.3, 117.7, 119.8, 123.2, 123.2,
126.8, 127.8, 128.1, 129.2, 129.2, 131.2, 136.7, 138.5, 139.2,
144.2, 175.1, 199.2. HRMS (ESI): for C20H20NO2 calcd: 306.1494
[MH]+; found: 306.1508.
The title compound was prepared and purified according to the
procedure described for 1, starting from 57 (vide infra). The reac-
tion yielded 18 (41%), a white solid (mp: 171-173 °C). 1H NMR
(DMSO-d6) d 0.88 (3H, t, J = 7.3), 1.32 (2H, sex, J = 7.3), 1.54 (2H,
quint, J = 7.3), 1.92 (2H, quint, J = 7.5), 2.60 (2H, t, J = 7.5), 2.73
(2H, t, J = 7.5), 3.09 (2H, t, J = 7.3), 7.23 (5H, m), 7.54 (1H, d,
J = 8.4), 7.58 (1H, dd, J = 8.4, 1.7), 8.03 (1H, br s), 8.46 (1H, d,
J = 6.5), 12.48 (1H, d, J = 6.5); 13C NMR (DMSO-d6) d 14.1, 22.2,
26.2, 32.7, 34.5, 34.7, 42.3, 117.5, 119.0, 124.7, 125.9, 127.9,
128.4, 128.4, 128.4, 128.4, 133.3, 137.4, 139.0, 142.0, 143.9,
5.14. 6-Benzyl-3-pentanoyl-4-quinolone (14)
175.2, 199.5. HRMS (ESI): for
C23H25NO2Na calcd: 370.1783
[MNa]+; found: 370.1792.
The title compound was prepared and purified according to the
procedure described for 1, starting from 53 (vide infra). The reaction
yielded 14 (72%), a white solid (mp: 209–211 °C). 1H NMR (DMSO-
d6) d 0.88 (3H, t, J = 7.4), 1.32 (2H, sex, J = 7.4), 1.54 (2H, quint,
J = 7.4), 3.08 (2H, t, J = 7.4), 4.08 (2H, s), 7.26 (5H, m), 7.55 (1H, d,
J = 8.4), 7.61 (1H, dd, J = 8.4, 2.0), 8.04 (1H, d, J = 2.0), 8.46 (1H, d,
J = 6.7), 12.46 (1H, d, J = 6.7); 13C NMR (DMSO-d6) d 13.9, 22.0,
5.19. 6-(2-Naphthylmethyl)-3-pentanoyl-4-quinolone (19)
The title compound was prepared and purified according to the
procedure described for 1, starting from 58 (vide infra). The reac-
tion yielded 19 (65%), a white solid (mp: 222–224 °C). 1H NMR
(DMSO-d6) d 0.86 (3H, t, J = 7.4), 1.30 (2H, sex, J = 7.4), 1.52 (2H,

E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
6943
quint, J = 7.4), 3.05 (2H, t, J = 7.4), 4.25 (2H, s), 7.39 (1H, dd, J = 8.4,
5.24. 3-Pentanoyl-5,6,7,8-tetrahydro-4-quinolone (24)
1.5), 7.47 (2H, m), 7.56 (1H, d, J = 8.4), 7.66 (1H, dd, J = 8.4, 1.9),
7.78 (1H, br s), 7.85 (3H, m), 8.07 (1H, d, J = 1.5), 8.45 (1H, s); 13C
NMR (DMSO-d6) d 14.0, 22.2, 26.2, 40.9, 42.3, 117.5, 119.3, 125.3,
125.7, 126.3, 126.9, 127.5, 127.6, 127.7, 127.9, 128.2, 131.8,
133.3, 133.7, 137.6, 138.3, 138.6, 144.0, 175.1, 199.4. HRMS (ESI):
for C25H23NO2Na calcd: 392.1626 [MNa]+; found: 392.1638.
64 (vide infra) (94 mg, 0.337 mmol) was refluxed in 1 ml of di-
phenyl ether for 30 min. The mixture was concentrated under re-
duced pressure, whereupon the crude product was purified by
chromatography (petroleum ether/EtOAc, 30:1–1:1) yielding 24
(25 mg, 0.107 mmol, 32%), a white solid (mp: 174–176 °C). 1H
NMR (CDCl3) d 0.96 (3H, t, J = 7.4), 1.42 (2H, m), 1.73 (2H, m),
1.83 (4H, m), 2.67 (2H, t, J = 6.1), 2.87 (2H, t, J = 6.1), 3.01 (2H, t,
J = 7.4), 8.70 (1H, s); 13C NMR (CDCl3) d 13.8, 21.5, 21.7, 22.2,
22.4, 26.6, 32.2, 38.4, 115.1, 122.4, 147.6, 161.3, 167.5, 206.1.
5.20. 3-Pentanoyl-6-(pyridin-4-yl-methyl)-4-quinolone (20)
The title compound was prepared and purified according to the
procedure described for 1, starting from 59 (vide infra). The reac-
tion yielded 20 (47%), a white solid (mp: 152–154 °C). 1H NMR
(MeOD) d 0.95 (3H, t, J = 7.4), 1.40 (2H, sex, J = 7.4), 1.64 (2H, quint,
J = 7.4), 3.16 (2H, t, J = 7.4), 4.19 (2H, s), 7.32 (2H, d, J = 5.8), 7.55
(1H, d, J = 8.5), 7.62 (1H, dd, J = 8.5, 1.6), 8.20 (1H, d, J = 1.6), 8.43
(2H, d, J = 5.8), 8.55 (1H, s); 13C NMR (MeOD) d 14.5, 23.8, 27.8,
41.7, 44.1, 119.5, 120.6, 126.1, 126.1, 127.1, 129.5, 135.3, 138.4,
139.6, 145.9, 150.3, 150.3, 152.8, 178.0, 202.5. HRMS (ESI): for
HRMS (ESI): for
234.1487.
C
14H20NO2 calcd: 234.1494 [MH]+; found:
5.25. Ethyl 3-oxoheptanoate (27a)
n-Butyllithium (12.29 ml 2.5 M in hexane, 30.73 mmol) was
added slowly to
a
ꢁ78 °C solution of monoethyl malonate
(2.030 g, 15.37 mmol) in 40 ml anhydrous THF, while allowing
the temperature to rise to ꢁ10 °C near the end of the addition.
After 10 min at ꢁ10 °C, the mixture was cooled to ꢁ78 °C and
valeryl chloride was added dropwise. Cooling was removed and
after 10 min the reaction was quenched with 1 M HCl (aq)
(30 ml). The mixture was extracted with diethyl ether (60 ml).
The organic extract was washed with satd NaHCO3 (aq) (2ꢂ
20 ml), H2O (20 ml) and brine (20 ml), then dried with MgSO4.
Evaporation of the solvent yielded 27a (1.479 g, 8.59 mmol,
95%), as a colourless oil. Characterization data agreed with previ-
ous publication.15
C
20H21N2O2 calcd: 321.1603 [MH]+; found: 321.1589.
5.21. 3-Pentanoyl-6-(pyridin-3-yl-methyl)-4-quinolone (21)
The title compound was prepared and purified according to the
procedure described for 1, starting from 60 (vide infra). The reac-
tion yielded 21 (72%), a white solid (mp: 197–199 °C). 1H NMR
(DMSO-d6) d 0.88 (3H, t, J = 7.4), 1.32 (2H, sex, J = 7.4), 1.54 (2H,
quint, J = 7.4), 3.08 (2H, t, J = 7.4), 4.12 (2H, s), 7.33 (1H, dd,
J = 7.8, 4.8), 7.57 (1H, d, J = 8.4), 7.64 (2H, m), 8.05 (1H, d, J = 1.7),
8.43 (1H, d, J = 4.8), 8.47 (1H, s), 8.55 (1H, s), 12.48 (1H, br s); 13C
NMR (DMSO-d6) d 13.9, 22.1, 26.1, 37.6, 42.2, 117.5, 119.2, 123.7,
125.1, 127.9, 133.3, 136.3, 136.5, 137.5, 137.5, 143.9, 147.5,
149.9, 174.9, 199.2. HRMS (ESI): for C20H21N2O2 calcd: 321.1603
[MH]+; found: 321.1592.
5.26. Ethyl 3-oxooctanoate (27b)
Ethyl acetoacetate (0.204 g, 1.57 mmol) was added dropwise to
a 0 °C solution of NaH (69 mg with 60% mineral oil, 1.72 mmol) in
5 ml anhydrous THF. After 20 min, tert-butyllithium (1.05 ml 1.5 M
soln in pentane, 1.57 mmol) was added dropwise and the resulting
mixture was stirred at 0 °C for 30 min. 1-Iodobutane in 5 ml of
anhydrous THF was added dropwise to the dianion solution where-
upon cooling was removed. After approximately 15 min, the reac-
tion was quenched by 10 ml of satd NH4Cl (aq), whereupon the
mixture was extracted with diethyl ether (3ꢂ 15 ml). The com-
bined organic extract was washed with brine (15 ml) and dried
with MgSO4. The crude product was purified by chromatography
(petroleum ether/EtOAc, 30:1–20–1), yielding 27b (0123 g,
0.660 mmol, 42%) as a colourless oil. Characterization data agreed
with previous publication.27
5.22. 3-Pentanoyl-6-(pyridin-2-yl-methyl)-4-quinolone (22)
The title compound was prepared and purified according to the
procedure described for 1, starting from 61 (vide infra). The reac-
tion yielded 22 (55%), a white solid (mp: 191-193 °C). 1H NMR
(DMSO-d6) d 0.88 (3H, t, J = 7.4), 1.32 (2H, sex, J = 7.4), 1.54 (2H,
quint, J = 7.4), 3.08 (2H, t, J = 7.4), 4.22 (2H, s), 7.22 (1H, dd,
J = 7.4, 4.9), 7.33 (1H, d, J = 7.8), 7.56 (1H, d, J = 8.4), 7.65 (1H, dd,
J = 8.4, 1.8), 7.72 (1H, dt, J = 7.7, 1.7), 8.08 (1H, d, J = 1.8), 8.46
(1H, d, J = 6.6), 8.49 (1H, m), 12.47 (1H, d, J = 6.6); 13C NMR
(DMSO-d6) d 13.9, 22.0, 26.1, 42.2, 43.3, 117.4, 119.0, 121.6,
123.2, 125.4, 127.8, 133.6, 136.8, 136.8, 137.5, 143.8, 149.2,
160.2, 174.9, 199.2. HRMS (ESI): for C20H21N2O2 calcd: 321.1603
[MH]+; found: 321.1587.
5.27. Ethyl 3-oxononanoate (27c)
The title compound was prepared and purified according to
the procedure described for 27b, using 1-bromopentane in-
stead of 1-iodobutane. The reaction yielded 27c (30%), a col-
ourless oil. Characterization data agreed with previous
publication.28
5.23. 3-(Cyclopentylacetyl)-6-(pyridin-3-yl-methyl)-4-
quinolone (23)
The title compound was prepared and purified according to the
procedure described for 1, starting from 62 (vide infra). The reaction
yielded 23 (49%), a white solid (mp: 184–186 °C). 1H NMR (DMSO-
d6) d 1.11 (2H, m), 1.46 (2H, m), 1.57 (2H, m), 1.74 (2H, m), 2.24
(1H, m), 3.11 (2H, d, J = 7.1), 4.12 (2H, s), 7.34 (1H, dd, J = 7.8, 4.8),
7.57 (1H, d, J = 8.4), 7.63 (1H, dd, J = 8.4, 1.9), 7.67 (1H, dt, J = 7.8,
1.3), 8.04 (1H, d, J = 1.9), 8.44 (1H, dd, J = 4.8, 1.3), 8.46 (1H, d,
J = 6.6), 8.55 (1H, d, J = 1.3), 12.49 (1H, d, J = 6.6); 13C NMR (DMSO-
d6) d 24.5, 24.5, 32.2, 32.2, 35.4, 37.6, 48.6, 117.6, 119.2, 123.7,
125.1, 127.9, 133.3, 136.5, 136.5, 137.4, 137.5, 143.9, 147.4, 149.8,
174.9, 198.9. HRMS (ESI): for C22H23N2O2 calcd: 347.1760 [MH]+;
found: 347.1751.
5.28. Ethyl 5-methyl-3-oxoheptanoate (27d)
The title compound was prepared and purified according to the
procedure described for 27b, using 2-iodobutane instead of 1-iodo-
butane. The reaction yielded 27d (24%), a colourless oil. 1H NMR
(CDCl3) d 0.79 (6H, m), 1.16 (5H, m), 1.84 (1H, m), 2.23 (1H, dd,
J = 16.6, 8.0), 2.43 (1H, dd, J = 16.6, 5.7), 3.31 (2H, s), 4.08 (2H, q,
J = 7.1); 13C NMR (CDCl3) d 11.0, 18.9, 29.1, 29.4, 30.2, 49.4, 49.8,
61.0, 166.9, 202.3. HRMS (ESI): for C10H18O3Na calcd: 209.1154
[MNa]+; found: 209.1157.

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E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
5.29. Ethyl 6-methyl-3-oxoheptanoate (27e)
5.35. 2-(4-Nitrobenzyl)naphthalene (30)
The title compound was prepared and purified according to the
procedure described for 27b, using 1-iodo-2-methylpropane in-
stead of 1-iodobutane. The reaction yielded 27e (51%), a colourless
oil. Characterization data agreed with previous publication.29
PdCl2 (6 mg, 0.0334 mmol) was added to a 0 °C suspension of 2-
naphthaleneboronic acid (0.344 g, 2.00 mmol), 4-nitrobenzyl bro-
mide (0.432 g, 2.00 mmol) and K2CO3 (0.692 g, 5.00 mmol) in ace-
tone/H2O (30:10 ml). The mixture was then allowed to reach rt and
stirred for 3 h. The mixture was concentrated under reduced pres-
sure, whereupon 25 ml H2O was added. The mixture was extracted
with diethyl ether (2ꢂ 40 ml), and the combined organic extract
was washed with brine (40 ml) and dried with MgSO4. The crude
product was purified by chromatography (petroleum ether), yield-
ing 30 (0.253 g, 9.61 mmol, 48%) as a brown solid (mp: 76–78 °C).
1H NMR (CDCl3) d 4.25 (2H, s), 7.28 (1H, d, J = 6.8), 7.40 (2H, d,
J = 8.5), 7.49 (2H, m), 7.65 (1H, s), 7.82 (3H, m), 8.17 (2H, d,
J = 8.5); 13C NMR (CDCl3) d 41.9, 123.8, 123.8, 124.0, 125.8, 126.3,
127.2, 127.4, 127.5, 127.7, 128.6, 129.7, 129.7, 132.3, 133.6,
136.6, 148.7. HRMS (EI): for C17H13NO2 calcd: 263.0946 [M]+;
found: 263.0944.
5.30. Ethyl 6,6-dimethyl-3-oxoheptanoate (27f)
Oxalyl chloride (0.305 ml, 3.49 mmol) was added dropwise to a
0 °C solution of 4,4-dimethylpentanoic acid30 (0.303 g, 2.33 mmol)
and a catalytic amount of DMF in 5 ml CH2Cl2. The mixture was al-
lowed to reach rt over 1 h and was then stirred for 2 h. The mixture
was concentrated under reduced pressure and the resulting acid
chloride was used, without further purification, as a reactant in-
stead of valeroyl chloride according to the procedure described
for 27a. The crude product was purified by chromatography (petro-
leum ether/EtOAc, 30:1), yielding 27f (0.208 g, 1.04 mmol, 45%), a
colourless oil. 1H NMR (CDCl3) d 0.89 (9H, s), 1.29 (3H, t, J = 7.1),
1.51 (2H, m), 2.52 (2H, m), 1.73 (2H, s), 4.21 (2H, q, J = 7.1); 13C
NMR (CDCl3) d 13.9, 28.9, 28.9, 28.9, 29.7, 36.7, 38.6, 49.2, 61.1,
167.1, 203.3. HRMS (ESI): for C11H21O3 calcd: 201.1491 [MH]+;
found: 201.1483.
5.36. 3-Benzylpyridine (32)
3-Benzoylpyridine (2.081 g, 11.36 mmol) and 10% Pd/C (0.8 g)
was stirred in 100 ml of ethanol and 4 ml of concd HCl (aq) under
H2 at atmospheric pressure. After 24 h, the mixture was filtered
and the filtrate was basified by 50 ml of 1 M NaOH (aq) followed
by concentration under reduced pressure. The resulting mixture
was extracted with diethyl ether (3ꢂ 50 ml). The combined organic
extract was washed with 50 ml of brine and dried with MgSO4, to
give 32 (1.803 g, 10.65 mmol, 94%), a colourless oil. 1H NMR
(CDCl3) d 3.99 (2H, s), 7.26 (6H, m), 7.48 (1H, m), 8.47 (1H, d,
J = 4.8), 8.53 (1H. m); 13C NMR (CDCl3) d 38.4, 122.9, 125.9,
128.1, 128.1, 128.3, 128.3, 135.7, 136.0, 139.3, 147.0, 149.6. HRMS
(ESI): for C12H12N calcd: 170.0970 [MH]+; found: 170.0970.
5.31. Ethyl 4-cyclopentyl-3-oxobutanoate (27g)
The title compound was prepared according to the procedure
described for 27a, using cyclopentylacetyl chloride instead of vale-
royl chloride. The crude product was purified by chromatography
(petroleum ether/EtOAc), yielding 27g (64%), a colourless oil. Char-
acterization data agreed with previous publication.31
5.32. Ethyl 4-cyclohexyl-3-oxobutanoate (27h)
The title compound was prepared and purified according to
the same procedure described for 27f, using phenylacetic acid in-
stead of 4,4-dimethylpentanoic acid. The reaction yielded 27h
(78%), a colourless oil. 1H NMR (CDCl3) d 0.94 (2H, m), 1.16 (2H,
m), 1.28 (5H, m), 1.70 (4H, m), 1.86 (1H, m), 2.41 (2H, d,
J = 6.8), 3.42 (2H, s), 4.21 (2H, q, J = 7.1); 13C NMR (CDCl3) d
14.0, 25.9, 26.0, 26.0, 32.9, 32.9, 33.5, 49.7, 50.5, 61.1, 167.1,
5.37. 3-(4-Nitrobenzyl)pyridine (33)
NH4NO3 (1.677 g, 20.954 mmol) was added in portions to a
solution of 32 (1.773 g, 10.477 mmol), 10 ml trifluoroacetic anhy-
dride and 15 ml CHCl3 at ꢁ10 °C. The mixture was allowed to reach
rt slowly while stirring under N2. After 43 h, 150 ml of satd NaH-
CO3 (aq) was added and the mixture was extracted with CH2Cl2
(3ꢂ 50 ml). The combined organic extract was washed with satd
NaHCO3 (aq) and dried with MgSO4. The crudeproduct was purified
by chromatography (heptane/ethyl acetate, 2:3), yielding 33
(1.233 g, 5.756 mmol, 55%) as a yellow solid (mp 87–88 °C). 1H
NMR (CDCl3) d 4.06 (2H, s), 7.22 (1H, dd, J = 7.8, 4.8), 7.31 (2H, d,
J = 8.7), 7.46 (1H, m), 8.10 (2H, d, J = 8.7), 8.47 (2H, m); 13C NMR
(CDCl3) d 38.5, 123.6, 123.7, 123.7, 129.5, 129.5, 134.7, 136.4,
146.5, 147.2, 147.9, 149.7. HRMS (ESI): for C12H11N2O2 calcd:
215.0821 [MH]+; found: 215.0810.
202.3. HRMS (ESI): for
found: 235.1317.
C
12H20O3Na calcd: 235.1310 [MNa]+;
5.33. Ethyl 3-oxo-4-phenylbutanoate (27i)
The title compound was prepared and purified according to
the procedure described for 27a, using phenylacetyl chloride
instead of valeroyl chloride. The reaction yielded 27i (quantita-
tive), a colourless oil. Characterization data agreed with previous
publication.15
5.38. 4-(2-Phenylethyl)aniline (35)
5.34. 1-Nitro-4-[(E)-2-phenylvinyl]benzene (28)
28 (0.398 g, 1.77 mmol) and a catalytic amount of 10% Pd/C,
in 25 ml MeOH was stirred over night under H2, 500 psi. The
mixture was filtered through Celite, yielding 35 (300 mg,
1.52 mmol, 86%), characterization data agreed with previous
publication.33
Styrene (0.698 ml, 6.02 mmol), 1-iodo-4-nitrobenzene (0.500 g,
2.01 mmol), K2CO3 (0.833 g, 6.02 mmol), tetrabutylammonium
bromide (0.647 g, 2.01 mmol) and Pd(OAc)2 (22 mg, 0.100 mmol)
were mixed in 20 ml of DMF for 10 min, while flushing with N2.
The mixture was heated at 100 °C for 12 h, whereupon addition-
ally styrene (0.698 ml, 6.02 mmol) was added and the mixture
was stirred over night. Heating was removed and 25 ml of H2O
was added. The mixture was extracted with ether (3ꢂ 40 ml).
The combined organic extract was washed with H2O (40 ml)
and dried with MgSO4, yielding 4-nitrostilbene 28 (0.416 g,
1.85 mmol, 92%), characterization data agreed with previous
publication.32
5.39. 4-(3-Phenylpropyl)aniline (36)
Triphenyl(2-phenylethyl)phosphonium bromide was prepared as
previously reported.18 Butyllithium (1 ml 2.5 M soln in hexanes,
2.51 mmol) was added to a suspension of the phosphonium salt
(1.07 g, 2.39 mmol) in 10 ml of THF at ꢁ78 °C, whereupon cooling
was removed. After 20 min the mixture was cooled to ꢁ78 °C and 4-

E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
6945
nitrobenzaldehyde (0.361 g, 2.39 mmol) in 5 ml of THF was added
dropwise. Cooling was removed and the mixture was stirred at rt for
90 min. Ten milliliters of brine was added and the mixture was ex-
tractedwith ether(2ꢂ 25 ml).Thecombinedorganicextractwasdried
with MgSO4. The crude product was purified by chromatography to
give a mixture of the E and Z alkenes (29). The E/Z ratio was estimated
to 75:25 on the basis of NMR data (identical with previous published
data).34 29 and a catalytic amount of 10% Pd/C was stirred in 25 ml
MeOH over night under H2, 500 psi. The mixture was filtered through
Celite, yielding 36 (0.338 g, 1.60 mmol, 67%) as a brown oil. 1H NMR
(CDCl3) d 1.99 (2H, quint, J = 7.6), 2.62 (2H, t, J = 7.6), 2.71 (2H, t,
J = 7.6), 6.28 (2H, br s), 6.93 (2H, d, J = 7.8), 7.10 (2H, d, J = 7.8), 7.28
(3H, m), 7.36 (2H, d, J = 6.9); 13C NMR (CDCl3) d 33.0, 34.5, 35.2,
117.4, 117.4, 125.6, 128.2, 128.2, 128.3, 128.3, 129.2, 129.2, 135.2,
139.4, 142.2. HRMS (ESI): for C15H18N calcd: 212.1439 [MH]+; found:
212.1429.
crude product was purified by chromatography (petroleum ether/
ethyl acetate, 30:1) yielding 41 (1.47 g, 4.84 mmol, 61%), a yellow
oil. 1H NMR (CDCl3) d 0.94 (3H, t, J = 7.3), 1.22 (3H, t, J = 7.6), 1.36
(5H, m), 1.62 (2H, quint, J = 7.5), 2.62 (2H, q, J = 7.6), 2.95 (2H, t,
J = 7.5), 4.24 (2H, q, J = 7.1), 7.09 (2H, d, J = 8.3), 7.19 (2H, d, J = 8.3),
8.48 (1H, d, J = 13.2), 12.80 (1H, d, J = 13.2); 13C NMR (CDCl3) d 14.0,
14.4, 15.4, 22.5, 27.0, 28.2, 41.9, 59.7, 101.9, 117.6, 117.6, 129.1,
129.1, 136.8, 141.7, 152.1, 166.9, 202.7. HRMS (ESI): for C18H26NO3
calcd: 304.1913 [MH]+; found: 304.1902.
5.45. Ethyl (2E)-3-[(4-ethylphenyl)amino]-2-hexanoylacrylate
(42)
The title compound was prepared and purified according to the
procedure described for 41, starting from 27b as reacting b-keto
ester. The reaction yielded 42 (73%), a yellow oil. 1H NMR (CDCl3)
d 0.91 (3H, t, J = 6.7), 1.24 (3H, t, J = 7.6), 1.36 (7H, m), 1.66 (2H, m), 2.65
(2H, q, J = 7.6), 2.96 (2H, t, J = 7.6), 4.26 (2H, q, J = 7.1), 7.11 (2H, d,
J = 8.5), 7.22 (2H, d, J = 8.5), 8.49 (1H, d, J = 13.2), 12.81 (1H, d,
J = 13.2); 13C NMR (CDCl3) d 14.0, 14.5, 15.5, 22.6, 24.7, 28.2, 31.7,
42.3, 59.8, 102.0, 117.8, 117.8, 129.2, 129.2, 136.9, 141.8, 152.2, 167.0,
202.9. HRMS (ESI): for C19H28NO3 calcd: 318.2069 [MH]+; found:
318.2056.
5.40. 4-(2-Naphthylmethyl)aniline (37)
2-(4-Nitrobenzyl)naphthalene (30) (0.247 g, 0.938 mmol) and a
catalytic amount of 10% Pd/C in 20 ml EtOAc and 5 ml of EtOH was
stirred over night under H2, 500 psi. The mixture was filtered
through Celite, washing with additionally EtOAc yielding 37
(218 mg, 0.938 mmol, quantitative), a brown solid (mp: 62–
64 °C). 1H NMR (CDCl3) d 4.16 (2H, s), 4.56 (2H, br s), 6.73 (2H, d,
J = 8.3), 7.16 (2H, d, J = 8.1), 7.45 (1H, dd, J = 8.5, 1.6), 7.57 (2H,
m), 7.75 (1H, s), 7.90 (3H, m); 13C NMR (CDCl3) d 41.1, 115.4,
115.4, 125.1, 125.8, 126.7, 127.4, 127.5, 127.8, 129.7, 129.7,
131.0, 131.9, 133.5, 139.3, 144. HRMS (ESI): for C17H16N calcd:
234.1283 [MH]+; found: 234.1286.
5.46. Ethyl (2E)-3-[(4-ethylphenyl)amino]-2-heptanoylacrylate
(43)
The title compound was prepared and purified according to the
procedure described for 41, starting from 27c as reacting b-keto es-
ter. The reaction yielded 43 (55%), a yellow oil. 1H NMR (CDCl3) d
0.88 (3H, t, J = 6.9), 1.22 (3H, t, J = 7.6), 1.34 (9H, m), 1.63 (2H, m),
2.62 (2H, q, J = 7.6), 2.95 (2H, t, J = 7.5), 4.24 (2H, q, J = 7.1), 7.09 (2H,
d, J = 8.5), 7.19 (2H, d, J = 8.5), 8.48 (1H, d, J = 13.2), 12.80 (1H, d,
J = 13.2); 13C NMR (CDCl3) d 14.0, 14.4, 15.4, 22.5, 24.8, 28.1, 29.2,
31.7, 42.2, 59.7, 101.9, 117.6, 117.6, 129.1, 129.1, 136.8, 141.6, 152.1,
166.8, 202.7. HRMS (ESI): for C20H29NO3Na calcd: 354.2045 [MNa]+;
found: 354.2035.
5.41. 4-(Pyridin-4-ylmethyl)aniline (38)
4-(4-Nitrobenzyl)pyridine (31) (0.500 g, 2.33 mmol) and a cata-
lytic amount of 10% Pd/C in 50 ml EtOH was stirred for 3 h under
H2 at atmospheric pressure. The mixture was filtered through Cel-
ite, washing with additionally EtOH yielding 38 (417 mg,
2.26 mmol, 97%). Characterization data agreed with previous
publication.35
5.47. Ethyl (2E)-3-[(4-ethylphenyl)amino]-2-(3-methylpentanoyl)
acrylate (44)
5.42. 4-(Pyridin-3-ylmethyl)aniline (39)
The title compound was prepared and purified according to the
procedure described for 41, starting from 27d as reacting b-keto
ester. The reaction yielded 44 (78%), a yellow oil. 1H NMR (CDCl3)
d 0.91 (6H, m), 1.30 (8H, m), 1.99 (1H, m), 2.63 (2H, q, J = 7.6), 2.78
(1H, dd, J = 15.3, 7.9), 2.97 (1H, dd, J = 15.3, 5.8), 4.20 (2H, q, J = 7.1),
7.10 (2H, d, J = 8.4), 7.21 (2H, d, J = 8.4), 8.48 (1H, d, J = 13.2), 12.84
(1H, d, J = 13.2); 13C NMR (CDCl3) d 11.4, 14.5, 15.5, 19.4, 28.2, 29.7,
31.5, 49.1, 59.8, 102.4, 117.7, 117.7, 129.1, 129.1, 136.9, 141.7,
152.2, 167.0, 202.5. HRMS (ESI): for C19H28NO3 calcd: 318.2069
[MH]+; found: 318.2063.
The title compound was prepared according to the procedure
described for 38, using 33 as reacting nitro compound. The reaction
yielded 39 (133 mg, 0.722 mmol, 96%), a white solid (mp: 117–
119 °C). 1H NMR (CDCl3) d 3.84 (2H, s), 6.61 (2H, d, J = 8.1), 6.94
(2H, d, J = 8.1), 7.15 (1H, dd, J = 7.5, 4.7), 7.44 (1H, d, J = 7.5), 8.43
(1H, d, J = 4.7), 8.49 (1H, s); 13C NMR (CDCl3) d 37.8, 115.0, 115.0,
123.1, 129.2, 129.4, 129.4, 135.9, 137.0, 144.8, 147.0, 149.7. HRMS
(ESI): for C12H13N2 calcd: 185.1079 [MH]+; found: 185.1076.
5.43. 4-(Pyridin-2-ylmethyl)aniline (40)
5.48. Ethyl (2E)-3-[(4-ethylphenyl)amino]-2-(4-methylpenta-
2-(4-Nitrobenzyl)pyridine (34) was prepared from 2-benzylpyri-
dine and used in the synthesis of 40.22
noyl) acrylate (45)
The title compound was prepared and purified according to
the procedure described for 41, starting from 27e as reacting b-
keto ester. The reaction yielded 45 (81%), a yellow oil. 1H NMR
(CDCl3) d 0.94 (6H, d, J = 6.6), 1.22 (3H, t, J = 7.6), 1.34 (3H, t,
J = 7.1), 1.53 (2H, m), 1.62 (1H, m), 2.63 (2H, q, J = 7.6), 2.97
(2H, t, J = 7.8), 4.25 (2H, q, J = 7.1), 7.09 (2H, d, J = 8.4), 7.20 (2H,
d, J = 8.4), 8.48 (1H, d, J = 13.2), 12.81 (1H, d, J = 13.2); 13C NMR
(CDCl3) d 14.5, 15.4, 22.5, 22.5, 27.9, 28.2, 33.9, 40.3, 59.8,
102.0, 117.7, 117.7, 129.1, 129.1, 136.9, 141.7, 152.2, 166.9,
203.0. HRMS (ESI): for C19H28NO3 calcd: 318.2069 [MH]+; found:
318.2054.
5.44. Ethyl (2 E)-3-[(4-ethylphenyl)amino]-2-pentanoylacrylate
(41)
27a (1.367 g, 7.94 mmol), triethyl orthoformate (3.97 ml,
23.81 mmol) and acetic anhydride (1.13 ml, 11.91 mmol) were
mixed and heated at 100 °C, under nitrogen, over night. Low boiling
components were evaporated under reduced pressure. The resulting
crude product was mixed with 4-ethyl aniline (0.99ml, 7.94 mmol),
whereupon the mixture was heated at 100 °C for 1 h. Low boiling
components were evaporated under reduced pressure. The resulting

6946
E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
5.49. Ethyl (2E)-2-(4,4-dimethylpentanoyl)-3-[(4-ethylphenyl)
amino]acrylate (46)
quint, J = 7.5), 2.96 (2H, t, J = 7.5), 4.26 (2H, q, J = 7.1), 7.17 (3H,
m), 7.38 (2H, t, J = 7.8), 8.50 (1H, d, J = 13.1), 12.79 (1H, d,
J = 13.1); 13C NMR (CDCl3) d 14.0, 14.4, 22.5, 27.0, 42.0, 59.8,
102.4, 117.6, 117.6, 125.4, 129.8, 129.8, 139.1, 151.9, 166.8,
202.9. HRMS (ESI): for C16H22NO3 calcd: 276.1600 [MH]+; found:
276.1590.
The title compound was prepared according to the procedure
described for 41, starting from 27f as reacting b-keto ester. The fi-
nal crude product was purified by chromatography (petroleum
ether/ethyl acetate, 30:1) followed by recrystallization from petro-
leum ether at ꢁ18 °C, yielding 46 (21%), a colourless solid (mp: 71–
73 °C). 1H NMR (CDCl3) d 0.95 (9H, s), 1.23 (3H, t, J = 7.6), 1.35 (3H,
t, J = 7.1), 1.55 (2H, m), 2.64 (2H, q, J = 7.6), 2.95 (2H, m), 4.26 (2H,
q, J = 7.1), 7.09 (2H, d, J = 8.4), 7.21 (2H, d, J = 8.4), 8.49 (1H, d,
J = 1.2), 12.81 (1H, d, J = 13.2); 13C NMR (CDCl3) d 14.5, 15.5, 28.2,
29.3, 29.3, 29.3, 30.1, 37.9, 38.6, 59.8, 102.0, 117.7, 117.7, 129.1,
129.1, 136.9, 141.7, 152.2, 166.9, 203.3. HRMS (ESI): for C20H29NO3-
Na calcd: 354.2045 [MNa]+; found: 354.2031.
5.54. Ethyl (2E)-3-[(4-bromophenyl)amino]-2-
pentanoylacrylate (51)
The title compound was prepared and purified according to the
procedure described for 41, but using 4-bromoaniline instead of
4-ethylaniline. The reaction yielded 51 (59%), a yellow oil. 1H
NMR (CDCl3) d 0.90 (3H, t, J = 7.2), 1.31 (3H, t, J = 6.9), 1.35 (2H, m),
1.57 (2H, quint, J = 7.4), 2.91 (2H, t, J = 7.4), 4.22 (2H, q, J = 6.9),
7.00 (2H, d, J = 8.2), 7.43 (2H, d, J = 8.2), 8.38 (1H, d, J = 12.7), 12.74
(1H, d, J = 12.7); 13C NMR (CDCl3) d 13.9, 14.3, 22.4, 26.8, 42.0, 59.9,
102.8, 118.0, 118.9, 118.9, 132.7, 132.7, 138.1, 151.3, 166.5, 202.9.
HRMS (ESI): for C16H21NO3Br calcd: 354.0705 [MH]+; found:
354.0692.
5.50. Ethyl (2E)-2-(cyclopentylacetyl)-3-[(4-ethylphenyl)
amino]acrylate (47)
The title compound was prepared and purified according to the
procedure described for 41, starting from 27g as reacting b-keto es-
ter. The reaction yielded 47 (76%), a yellow oil. 1H NMR (CDCl3) d
1.19 (2H, m), 1.24 (3H, t, J = 7.6), 1.35 (3H, t, J = 7.1), 1.59 (4H,
m), 1.84 (2H, m), 2.32 (1H, m), 2.65 (2H, q, J = 7.6), 3.00 (2H, d,
J = 7.1), 4.26 (2H, q, J = 7.1), 7.11 (2H, d, J = 8.3), 7.22 (2H, d,
J = 8.3), 8.49 (1H, d, J = 13.2), 12.82 (1H, d, J = 13.2); 13C NMR
(CDCl3) d 14.5, 15.5, 25.0, 25.0, 28.2, 32.7, 32.7, 36.3, 48.3, 59.8,
102.2, 117.8, 117.8, 129.2, 129.2, 137.0, 141.8, 152.2, 167.0,
202.6. HRMS (ESI): for C20H28NO3 calcd: 330.2069 [MH]+; found:
330.2060.
5.55. Ethyl (2E)-3-(1,10-biphenyl-4-ylamino)-2-pentanoylacry-
late (52)
The title compound was prepared and purified according to the
procedure described for 41, but using 4-phenylaniline instead of 4-
ethylaniline. The reaction yielded 52 (65%), a yellow oil. 1H NMR
(CDCl3) d 0.96 (3H, t, J = 7.3), 1.37 (3H, t, J = 7.1), 1.40 (2H, m),
1.64 (2H, m), 2.99 (2H, t, J = 7.5), 4.29 (2H, q, J = 7.1), 7.27 (2H, d,
J = 8.7), 7.37 (1H, m), 7.46 (2H, m), 7.61 (4H, m), 8.56 (1H, d,
J = 13.1), 12.89 (1H, d, J = 13.1); 13C NMR (CDCl3) d 14.1, 14.5,
22.6, 27.1, 42.1, 60.0, 102.6, 118.0, 118.0, 126.8, 126.8, 127.5,
128.4, 128.4, 128.9, 128.9, 138.3, 138.4, 139.9, 151.7, 166.9,
203.1. HRMS (ESI): for C22H26NO3 calcd: 352.1913 [MH]+; found:
352.1899.
5.51. Ethyl (2E)-2-(cyclohexylacetyl)-3-[(4-ethylphenyl)amino]-
acrylate (48)
The title compound was prepared and purified according to the
procedure described for 41, starting from 27h as reacting b-keto
ester. The reaction yielded 48 (62%), a yellow oil. 1H NMR (CDCl3)
d 1.02 (2H, m), 1.17 (2H, m), 1.24 (3H, t, J = 7.5), 1.29 (2H, m), 1.35
(3H, t, J = 7.1), 1.70 (4H, m), 1.88 (1H, m), 2.65 (2H, q, J = 7.5), 2.85
(2H, d, J = 6.7), 4.26 (2H, q, J = 7.1), 7.11 (2H, d, J = 8.3), 7.22 (2H, d,
J = 8.3), 8.49 (1H, d, J = 13.2), 12.85 (1H, d, J = 13.2); 13C NMR
(CDCl3) d 15.0, 15.5, 26.3, 26.3, 26.4, 28.2, 33.4, 33.4, 34.9, 49.6,
59.9, 102.5, 117.8, 117.8, 129.2, 129.2, 136.9, 141.8, 152.3, 167.0,
202.3. HRMS (ESI): for C21H30NO3 calcd: 344.2226 [MH]+; found:
344.2222.
5.56. Ethyl (2E)-3-[(4-benzylphenyl)amino]-2-pentanoylacry-
late (53)
The title compound was prepared and purified according to the
procedure described for 41, but using 4-benzylaniline instead of 4-
ethylaniline. The reaction yielded 53 (68%), a yellow oil. 1H NMR
(CDCl3) d 0.97 (3H, t, J = 7.2), 1.36 (3H, t, J = 7.1), 1.40 (2H, m), 1.65
(2H, quint, J = 7.4), 2.99 (2H, t, J = 7.4), 3.99 (2H, s), 4.27 (2H, q,
J = 7.1), 7.12 (2H, d, J = 8.3), 7.22 (5H, m), 7.32 (2H, d, J = 7.4), 8.50
(1H, d, J = 13.2), 12.82 (1H, d, J = 13.2); 13C NMR (CDCl3) d 14.0,
14.4, 22.5, 27.0, 41.2, 42.0, 59.8, 102.1, 117.8, 117.8, 126.2, 128.5,
128.5, 128.8, 128.8, 130.2, 130.2, 137.3, 138.6, 140.5, 152.0, 166.8,
202.8. HRMS (ESI): for C23H28NO3 calcd: 366.2069 [MH]+; found:
366.2056.
5.52. Ethyl (2E)-3-[(4-ethylphenyl)amino]-2-(phenylacetyl)-
acrylate (49)
The title compound was prepared and purified according to the
procedure described for 41, starting from 27i as reacting b-keto es-
ter. The reaction yielded 49 (46%), a yellow oil. 1H NMR (CDCl3) d
1.26 (3H, t, J = 7.6), 1.39 (3H, t, J = 7.1), 2.67 (2H, q, J = 7.6), 4.32
(2H, q, J = 7.1), 4.38 (2H, s), 7.11 (2H, d, J = 8.5), 7.31 (7H, m),
8.56 (1H, d, J = 13.3), 12.77 (1H, d, J = 13.3); 13C NMR (CDCl3) d
14.5, 15.5, 28.2, 48.3, 60.0, 101.7, 117.8, 117.8, 126.4, 128.3,
128.3, 129.2, 129.2, 129.8, 129.8, 136.7, 142.0, 152.7, 166.9,
199.5. HRMS (ESI): for C21H23NO3Na calcd: 360.1576 [MNa]+;
found: 360.1586.
5.57. Ethyl (2E)-3-[(4-benzylphenyl)amino]-2-butyrylacrylate
(54)
The title compound was prepared and purified according to
the procedure described for 41, starting from ethyl 3-oxohexano-
ate as reacting b-keto ester and using 4-benzylaniline instead of
4-ethylaniline. The reaction yielded 54 (79%), a yellow oil. 1H
NMR (CDCl3) d 1.01 (3H, t, J = 7.4), 1.35 (3H, t, J = 7.1), 1.70 (2H,
sex, J = 7.4), 2.97 (2H, t, J = 7.4), 3.97 (2H, s), 4.27 (2H, q, J = 7.1),
7.11 (2H, d, J = 7.1), 7.21 (5H, m), 7.30 (2H. m), 8.49 (1H, d,
J = 13.2), 12.82 (1H, d, J = 13.2); 13C NMR (CDCl3) d 13.9, 14.4,
18.1, 41.1, 44.1, 59.7, 102.1, 117.7, 117.7, 126.1, 128.4, 128.4,
128.7, 128.7, 130.1, 130.1, 137.2, 138.5, 140.5, 151.9, 166.7,
202.6. HRMS (ESI): for C22H26NO3 calcd: 352.1913 [MH]+; found:
352.1899.
5.53. Ethyl (2E)-3-anilino-2-pentanoylacrylate (50)
The title compound was prepared and purified according to the
procedure described for 41, but using aniline instead of 4-ethylan-
iline. The reaction yielded 50 (68%), a yellow oil. 1H NMR (CDCl3) d
0.94 (3H, t, J = 7.3), 1.34 (3H, t, J = 7.1), 1.39 (2H, m), 1.63 (2H,

E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
6947
5.58. Ethyl (2E)-3-[(4-benzylphenyl)amino]-2-(cyclopentylace-
tyl) acrylate (55)
The reaction yielded 59 (46%), a yellow oil. 1H NMR (CDCl3) d 0.94
(3H, t, J = 7.6), 1.34 (3H, t, J = 7.1), 1.38 (2H, m), 1.62 (2H, quint,
J = 7.5), 2.96 (2H, t, J = 7.5), 3.96 (2H, s), 4.25 (2H, q, J = 7.1), 7.15
(6H, m), 8.49 (3H, m), 12.80 (1H, d, J = 13.0); 13C NMR (CDCl3) d
14.0, 14.4, 25.6, 27.0, 40.5, 42.0, 59.9, 102.4, 117.6, 118.0, 118.0,
124.1, 124.1, 130.4, 130.4, 136.1, 137.9, 149.7, 149.7, 151.9,
166.8, 203.0. HRMS (ESI): for C22H27N2O3 calcd: 367.2022 [MH]+;
found: 367.2015.
The title compound was prepared and purified according to the
procedure described for 41, starting from 27g as reacting b-keto es-
ter and using 4-benzylaniline instead of 4-ethylaniline. The reac-
tion yielded 55 (72%), a yellow oil. 1H NMR (CDCl3) d 1.22 (2H,
m), 1.34 (3H, t, J = 7.1), 1.55 (2H, m), 1.63 (2H, m), 1.84 (2H, m),
2.33 (1H, m), 3.01 (2H, d, J = 7.1), 3.97 (2H, s), 4.26 (2H, q,
J = 7.1), 7.11 (2H, d, J = 8.4), 7.18 (3H, m), 7.22 (2H, d, J = 7.5),
7.30 (2H, t, J = 7.5), 8.48 (1H, d, J = 13.1), 12.83 (1H, d, J = 13.1);
13C NMR (CDCl3) d 14.4, 24.9, 24.9, 32.6, 32.6, 36.2, 41.1, 48.2,
59.7, 102.3, 117.7, 117.7, 126.2, 128.5, 128.5, 128.7, 128.7, 130.1,
130.1, 137.3, 138.5, 140.5, 151.9, 166.8, 202.5. HRMS (ESI): for
5.63. Ethyl (2E)-2-pentanoyl-3-{[4-(pyridin-3-ylmethyl)phenyl]-
amino} acrylate (60)
The title compound was prepared and purified according to the
procedure described for 41, but using 39 instead of 4-ethylaniline.
The reaction yielded 60 (48%), a yellow oil. 1H NMR (CDCl3) d 0.90
(3H, t, J = 7.3), 1.30 (3H, t, J = 7.1), 1.35 (2H, m), 1.59 (2H, quint,
J = 7.5), 2.93 (2H, t, J = 7.5), 3.93 (2H, s), 4.22 (2H, q, J = 7.1), 7.09
(2H, d, J = 8.5), 7.18 (3H, m), 7.43 (1H, m), 8.44 (3H, m), 12.77
(1H, d, J = 13.2); 13C NMR (CDCl3) d 13.9, 14.3, 22.5, 26.9, 38.2,
41.9, 59.8, 102.3, 117.8, 117.8, 123.4, 130.1, 130.1, 135.9, 136.1,
137.0, 137.6, 147.6, 149.9, 151.8, 166.7, 202.8. HRMS (ESI): for
C
25H30NO3 calcd: 392.2226 [MH]+; found: 392.2222.
5.59. Ethyl (2E)-2-pentanoyl-3-{[4-(2-phenylethyl)phenyl]-
amino}acrylate (56)
The title compound was prepared and purified according to the
procedure described for 41, but using 35 instead of 4-ethylaniline.
The reaction yielded 56 (46%), a yellow oil. 1H NMR (CDCl3) d 0.99
(3H, t, J = 7.3), 1.39 (3H, t, J = 7.1), 1.43 (2H, m), 1.67 (2H, quint,
J = 7.5), 2.96 (4H, s), 3.01 (2H, t, J = 7.5), 4.30 (2H, q, J = 7.1), 7.13
(2H, d, J = 8.5), 7.22 (5H, m), 7.32 (2H, d, J = 7.1), 8.53 (1H, d,
J = 13.2), 12.84 (1H, d, J = 13.2); 13C NMR (CDCl3) d 14.0, 14.4,
22.6, 27.0, 37.1, 37.7, 42.0, 59.8, 102.0, 117.6, 117.6, 125.9, 128.3,
128.3, 128.4, 128.4, 129.8, 129.8, 137.1, 139.1, 141.1, 152.0,
166.9, 202.8. HRMS (ESI): for C24H30NO3 calcd: 380.2226 [MH]+;
found: 380.2222.
C
22H27N2O3 calcd: 367.2022 [MH]+; found: 367.2017.
5.64. Ethyl (2E)-2-pentanoyl-3-{[4-(pyridin-2-ylmethyl)-
phenyl]amino} acrylate (61)
The title compound was prepared and purified according to the
procedure described for 41, but using 4022 instead of 4-ethylani-
line. The reaction yielded 61 (51%), a yellow oil. 1H NMR (CDCl3)
d 0.88 (3H, t, J = 7.3), 1.27 (3H, t, J = 7.1), 1.33 (2H, m), 1.57 (2H,
quint, J = 7.5), 2.90 (2H, t, J = 7.5), 4.07 (2H, s), 4.19 (2H, q,
J = 7.1), 7.05 (4H, m), 7.22 (2H, d, J = 8.3), 7.52 (1H, dt, J = 7.7,
1.5), 8.42 (1H, d, J = 13.1), 8.48 (1H, m), 12.74 (1H, d, J = 13.1);
13C NMR (CDCl3) d 13.8, 14.3, 22.4, 26.8, 41.8, 43.7, 59.6, 102.0,
117.7, 117.7, 121.2, 122.8, 130.2, 130.2, 136.4, 136.7, 137.3,
149.2, 151.8, 160.2, 166.6, 202.6. HRMS (ESI): for C22H27N2O3
calcd: 367.2022 [MH]+; found: 367.2015.
5.60. Ethyl (2E)-2-pentanoyl-3-{[4-(3-phenylpropyl)phenyl]-
amino} acrylate (57)
The title compound was prepared and purified according to the
procedure described for 41, but using 36 instead of 4-ethylaniline.
The reaction yielded 57 (60%), a yellow oil. 1H NMR (CDCl3) d 0.96
(3H, t, J = 7.3), 1.35 (3H, t, J = 7.1), 1.40 (2H, m), 1.64 (2H, m), 1.95
(2H, m), 2.65 (4H, m), 2.98 (2H, t, J = 7.5), 4.27 (2H, q, J = 7.1), 7.11
(2H, d, J = 8.5), 7.20 (5H, m), 7.30 (2H, m), 8.50 (1H, d, J = 13.2),
12.83 (1H, d, J = 13.2); 13C NMR (CDCl3) d 14.0, 14.5, 22.6, 27.1,
32.8, 34.7, 35.3, 42.0, 59.8, 102.0, 117.7, 117.7, 125.8, 128.3, 128.3,
128.3, 128.3, 129.7, 129.7, 137.0, 139.8, 141.9, 152.1, 166.9, 202.8.
HRMS (ESI): for C25H32NO3 calcd: 394.2382 [MH]+; found: 394.2384.
5.65. Ethyl (2E)-2-(cyclopentylacetyl)-3-{[4-(pyridin-3-ylmethyl)
phenyl]amino}acrylate (62)
The title compound was prepared and purified according to the
procedure described for 41, starting from 27g as reacting b-keto es-
ter and using 39 instead of 4-ethylaniline. The reaction yielded 62
(38%), a yellow oil. 1H NMR (CDCl3) d 1.14 (2H, m), 1.29 (3H, t,
J = 7.1), 1.53 (4H, m), 1.78 (2H, m), 2.26 (1H, m), 2.95 (2H, d,
J = 7.1), 3.92 (2H, s), 4.20 (2H, q, J = 7.1), 7.08 (2H. d. J = 8.5), 7.16
(3H, m), 7.41 (1H, d, J = 7.8), 8.43 (3H, m), 12.8 (1H, d, J = 13.2);
13C NMR (CDCl3) d 14.3, 24.8, 24.8, 32.5, 32.5, 36.1, 38.2, 48.1,
59.7, 102.4, 117.8, 117.8, 123.3, 130.0, 130.0, 135.9, 136.1, 136.9,
137.6, 147.6, 149.8, 151.7, 166.7, 202.5. HRMS (ESI): for
5.61. Ethyl (2E)-3-{[4-(2-naphthylmethyl)phenyl]amino}-2-
pentanoylacrylate (58)
The title compound was prepared and purified according to the
procedure described for 41, but using 37 instead of 4-ethylaniline.
The reaction yielded 58 (68%), a yellow oil. 1H NMR (CDCl3) d 0.99
(3H, t, J = 7.3), 1.36 (3H, t, J = 7.1), 1.43 (2H, m), 1.67 (2H, quint,
J = 7.5), 3.01 (2H, t, J = 7.5), 4.13 (2H, s), 4.28 (2H, q, J = 7.1),
7.12 (2H, d, J = 8.4), 7.25 (2H, d, J = 8.4), 7.31 (1H, dd, J = 8.4,
1.6), 7.47 (2H, m), 7.63 (1H, br s), 7.81 (3H, m), 8.51 (1H, d,
J = 13.2), 12.85 (1H, d, J = 13.2); 13C NMR (CDCl3) d 14.0, 14.4,
22.5, 27.0, 41.3, 42.0, 59.8, 102.1, 117.7, 117.7, 125.4, 126.0,
127.0, 127.3, 127.4, 127.5, 128.1, 130.2, 130.2, 132.0, 133.5,
137.3, 138.0, 138.4, 151.9, 166.8, 202.8. HRMS (ESI): for
C
24H29N2O3 calcd: 393.2178 [MH]+; found: 393.2166.
5.66. Ethyl (2E)-3-amino-2-pentanoylacrylate (63)
The acyl-ethoxy acrylate-intermediate was prepared as de-
scribed in the synthesis of 41. This crude intermediate was stirred
at rt in 5 ml of 2.0 M NH3 solution in EtOH for 5 h. Solvent was
evaporated under reduced pressure and the crude product was
purified by chromatography (petroleum ether/ethyl acetate, 6:1)
yielding 63 (42%), a yellow oil. 1H NMR (CDCl3) d 0.93 (3H, t,
J = 7.3), 1.30 (3H, t, J = 7.1), 1.37 (2H, m), 1.59 (2H, quint, J = 7.6),
2.89 (2H, t, J = 7.6), 4.20 (2H, q, J = 7.1), 5.91 (1H, br s), 8.11 (1H,
dd, J = 15.1, 8.7), 10.31 (1H, br s); 13C NMR (CDCl3) d 14.0, 14.4,
22.6, 27.1, 42.2, 59.6, 101.8, 158.1, 167.2, 203.2. HRMS (ESI): for
C
27H30NO3 calcd: 416.2226 [MH]+; found: 416.2225.
5.62. Ethyl (2E)-2-pentanoyl-3-{[4-(pyridin-4-ylmethyl)
phenyl]amino}acrylate (59)
The title compound was prepared and purified according to the
procedure described for 41, but using 38 instead of 4-ethylaniline.
C
10H18NO3 calcd: 200.1287 [MH]+; found: 200.1289.

6948
E. Lager et al. / Bioorg. Med. Chem. 16 (2008) 6936–6948
5.67. Ethyl (2E)-3-(cyclohex-1-en-1-ylamino)-2-
pentanoylacrylate (64)
tions were carried out using the Polak-Ribiere conjugate gradient
algorithm (PRCG) as implemented in MacroModel 7.0.
63 (145 mg, 0.728 mmol), cyclohexanone (107 mg, 1.09 mmol)
and a catalytic amount of p-TsOH were refluxed in 10 ml of toluene
under Dean-Stark conditions for 24 h. The mixture was neutralized
by a small amount of Na2CO3, followed by concentration under re-
duced pressure. The resulting crude product was purified by chro-
matography (petroleum ether/EtOAc, 9:1) yielding 64 (97 mg,
0.347 mmol, 48%), a yellow oil. 1H NMR (CDCl3) d 0.88 (3H, t,
J = 7.4), 1.27 (3H, t, J = 7.1), 1.33 (2H, pent, J = 7.4), 1.55 (4H, m),
1.73 (2H, m), 2.10 (2H, m), 2.17 (2H, m), 2.86 (2H, t, J = 7.6), 4.17
(2H, q, J = 7.1), 5.47 (1H, t, J = 4.1), 8.07 (1H, d, J = 13.5), 12.49
(1H, d, J = 13.5); 13C NMR (CDCl3) d 13.9, 14.4, 21.8, 21.8, 22.5,
24.0, 24.0, 27.2, 41.8, 59.4, 100.2, 113.0, 135.0, 150.4, 167.1,
202.1. HRMS (ESI): for C16H26NO3 calcd: 280.1913 [MH]+; found:
280.1899.
Acknowledgments
Financial support from the Swedish Board for Scientific Re-
search (VR), the Research School for Pharmaceutical Sciences at
Lund University, and the NeuroScience PharmaBiotec Research
Center, Denmark, is gratefully acknowledged.
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Binding of 3H-flumazenil (87 Ci/mmol) to rat cortical mem-
branes and to a membrane suspension of HEK 293 cells express-
ing human a1b2c2s a2b2c2s or a3b2c2s GABAA receptors was done
following methods previously described by Kahnberg et al.11In
brief: tissue is homogenized in 20 ml Tris, HCl (30 mM, pH 7.4)
using an Ultra-Turrax homogenizer. The suspensions are centri-
fuged at 27,000g for 15 min followed by three centrifugations
resuspensions cycles. The washed pellet is resuspended in
20 ml buffer, incubated at 37 °C for 30 min and then centrifuged
for 10 min (27,000g). The pellet is washed once and the final pel-
let is resuspended in 30 ml Tris–HCl buffer (50 mM, pH 7.1) and
stored at ꢁ20 °C until use. For binding studies, frozen membrane
suspensions were thawed and centrifuged (27,000g, 10 min). The
pellet was resuspended into Tris,citrate buffer (50 mM, pH 7.1) at
a tissue concentration: cortex preparation ca. 50
0.55 ml assay (1 mg original tissue/0.55 ml assay) and HEK cells
ca 25 g protein per 0.55 ml assay. Aliquots of 0.5 ml membrane
lg protein/
l
preparation are added to 25
final concentration) and 25
l
l
l of 3H-flumazenil solution (1 nM
l containing test substance and
incubated at an ice-bath (0–4 °C) for 40 min. The incubated sam-
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7.1), poured direcly onto Whatman GF/C glass fibre filters under
suction and immediately washed with 5 ml ice-cold buffer. Non-
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conventional protein assay method using Bovine serum albumin
as standard.
IC50 values were determined using 4–6 different concentrations
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(1 + (L)/KD), (L) is the concentration (nM) of 3H-flumazenil; KD is
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