Refinement and evaluation of a pharmacophore model for flavone derivatives binding to the benzodiazepine site of the GABAA receptor

Article abstract of DOI:10.1021/jm020839k

To further develop and evaluate a pharmacophore model previously proposed by Cook and co-workers (Drug Des. Discovery 1995, 12, 193-248) for ligands binding to the benzodiazepine site of the GABA_A receptor, 40 new flavone derivatives have been synthesized and their affinities for the benzodiazepine site have been determined. Two new regions of steric repulsive interactions between ligand and receptor have been characterized, and the receptor region in the vicinity of 6- and 3′-substituents has been mapped out. 2′-Hydroxy substitution is shown to give a significant increase in affinity, which is interpreted in terms of a novel hydrogen bond interaction with the previously proposed hydrogen bond-accepting site A2. On the basis of the results of these studies and the refined pharmacophore model, 5′-bromo-2′-hydroxy-6-methylflavone, the highest affinity flavone derivative reported so far (Ki = 0.9 nM), was successfully designed. A comparison of the pharmacophore model with a recently proposed alternative model (Marder; et al. Bioorg. Med. Chem., 2001, 9, 323-335) has been made.

Full text of DOI:10.1021/jm020839k

4188
J . Med. Chem. 2002, 45, 4188-4201
Refin em en t a n d Eva lu a tion of a P h a r m a cop h or e Mod el for F la von e Der iva tives
Bin d in g to th e Ben zod ia zep in e Site of th e GABAA Recep tor
Pia Kahnberg,† Erik Lager,† Celia Rosenberg,† J ette Schougaard,‡ Linda Camet,† Olov Sterner,†
Elsebet Østergaard Nielsen,§ Mogens Nielsen,‡ and Tommy Liljefors*,‡
Department of Organic and Bioorganic Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, Sweden, Department of
Medicinal Chemistry, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark, and
NeuroSearch A/ S, Ballerup, Denmark
Received J anuary 31, 2002
To further develop and evaluate a pharmacophore model previously proposed by Cook and
co-workers (Drug Des. Discovery 1995, 12, 193-248) for ligands binding to the benzodiazepine
site of the GABAA receptor, 40 new flavone derivatives have been synthesized and their affinities
for the benzodiazepine site have been determined. Two new regions of steric repulsive
interactions between ligand and receptor have been characterized, and the receptor region in
the vicinity of 6- and 3′-substituents has been mapped out. 2′-Hydroxy substitution is shown
to give a significant increase in affinity, which is interpreted in terms of a novel hydrogen
bond interaction with the previously proposed hydrogen bond-accepting site A2. On the basis
of the results of these studies and the refined pharmacophore model, 5′-bromo-2′-hydroxy-6-
methylflavone, the highest affinity flavone derivative reported so far (Ki ) 0.9 nM), was
successfully designed. A comparison of the pharmacophore model with a recently proposed
alternative model (Marder; et al. Bioorg. Med. Chem., 2001, 9, 323-335) has been made.
In tr od u ction
GABA, such as benzodiazepines, â-carbolines, barbitu-
rates, ethanol, and certain steroids1. The pharmacologi-
cal effects of the benzodiazepines (anxiolytic, anticon-
vulsant, muscle relaxant, and sedative-hypnotic) make
them the most important GABAA receptor-modulating
drugs in clinical use.8 It has been shown that many
classes of compounds bind to the benzodiazepine recep-
tor with high affinity, e.g., â-carbolines, triazolopy-
ridazines, pyrazoloquinolinones, cyclopyrrolones, pyr-
idodiindoles, and quinolines.9,10 In addition, it has been
found that flavones, naturally occurring and synthetic
derivatives, bind with high affinity to the benzodiaz-
epine site of the GABAA receptor.11,12 Unified pharma-
cophore models (including agonists, antagonists, and
inverse agonists) have been developed for the benzodi-
azepine site,13,5 and the model by Cook and co-workers5
has been selected for fitting flavonoids into a pharma-
cophore model and for further development by us14 and
others.15 A quantitative structure-activity relationship
(QSAR) model that confirms the interactions proposed
by us14 has been developed by Huang et al.16 In the
present investigation, 40 new flavone derivatives have
been synthesized and tested and used to further refine
the previously proposed pharmacophore model. A com-
parison of this model with a recently suggested alterna-
tive is made.
One of the quantitatively most important neurotrans-
mitters in the central nervous system (CNS) is γ-ami-
nobutyric acid (GABA).1 It is estimated that depending
on the brain region, 20-50% of all CNS synapses use
GABA as their transmitter.2,3 GABA exerts its physi-
ological effects by binding to three major classes of
receptors: two types of ligand-operated chloride chan-
nels, GABAA and GABAC receptors, and GABAB recep-
tors, which are coupled to G-proteins. When the GABA
receptors are activated, the communication between the
different cells in the CNS decreases. GABAA receptors
are transmembrane proteins that are assembled from
subunits to form a pentameric structure. The various
subunits that belong to eight classes with multiple
isoforms have been identified by their sequence homol-
ogy4 (R1-R6, â1-â4, γ1-γ4, δ, ꢀ, π, θ, and F1-F3). Most
GABAA receptors are composed of R-, â-, and γ-subunits.
The so far most common combination is two R-, two â-,
and one γ-subunit. Experiments with site-directed mu-
tagenesis have shown that changes in the R- and
γ-subunits alter the activity of the benzodiazepine
receptor-mediated response,5 and the benzodiazepine
site is believed to be situated between an R- and a
γ-subunit. It is also believed that different subunits
mediate different physiological effects, that sedation and
anterograde amnesia are mediated by R1-containing
receptors, and that anxiolytic activity is mediated by
R2-, R3-, or possibly R5-containing receptors.6,7 The
GABAA receptor possesses binding sites for compounds
that allosterically modify the chloride channel-gating of
Resu lts a n d Discu ssion
Ch em istr y. The structures of the flavones investi-
gated in this study are shown in Chart 1. They were
synthesized according to procedures that previously
have been described for other flavones.17 The synthesis
involves an acetylation, a Fries rearrangement, a modi-
fied Schotten-Baumann reaction, and a Baker Venka-
taraman rearrangement followed by a cyclization cata-
lyzed by acid (see Scheme 1). Aminoflavones were
* To whom correspondence should be addressed. Tel. +45-35 30 65
05. Fax: +45-35 30 60 40. E-mail:tl@dfh.dk.
† Lund University.
‡ Royal Danish School of Pharmacy.
§ NeuroSearch A/S.
10.1021/jm020839k CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/09/2002

Pharmacophore Model for Flavone Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4189
Ta ble 1. KI Values of Flavones Tested on 3H-Ro 15-1788
Binding In Vitro to Rat Cortical Membranes
Ch a r t 1
Ki value
no.
name
(nM)
1
2
3
4
5
6
7
8
9
flavone
4200 ( 30
180 ( 40
180 ( 40
480 ( 80
720 ( 140
>1700
6-methylflavone
6-ethylflavone
propylflavone
6-(1-methylethyl)flavone
8-methylflavone
4′,6-dimethylflavone
3′,4′,6-trimethylflavone
3′,5′,6-trimethylflavone
4400
>1700
>1500
700 ( 46
1200
10 2′-amino-6-methylflavone
11 3′-amino-6-methylflavone
12 4′-amino-6-methylflavone
2600
13 2′-methoxy-6-methylflavone
14 2′-hydroxy-6-methylflavone
15 3′-methoxy-6-methylflavone
16 3′-hydroxy-6-methylflavone
17 3′-butoxy-6-methylflavone
18 3′-(2-ethylbutyloxy)-6-methylflavone
19 2′-methoxy-5′,6-dimethylflavone
20 2′-hydroxy-5′,6-dimethylflavone
21 methyl 3′-(6-methylflavone)carboxylate
22 3′-(6-methylflavone)carboxylic acid
23 isopropyl 3′-(6-methylflavone)carboxylate
24 2-ethylbutyl 3′-(6-methylflavone)carboxylate
25 methyl 3′-(5′-methoxy-6-methylflavone)-
carboxylate
820 ( 110
6.2 ( 0.2
99 ( 15
390 ( 130
710 ( 150
1000 ( 210
>1500
7.8 ( 0.9
220 ( 30
>1700
180 ( 10
610 ( 170
3000
26 methyl 3′-(5′-hydroxy-6-methylflavone)-
carboxylate
>1600
27 3′-(5′-methoxy-6-methylflavone)carboxylic acid >1600
28 3′-(5′-hydroxy-6-methylflavone)carboxylic acid
29 5′-hydroxymethyl-3′-methoxy-6-methylflavone
30 dimethyl 3′,5′-(6-methylflavone)dicarboxylate
31 3′-5′-(6-methylflavone)dicarboxylic acid
>1600
>1600
>1600
>1600
Sch em e 1a
32 methyl 3′-(5′-hydroxymethyl-6-methylflavone)- >1500
carboxylate
33 3′-5′-bis(hydroxymethyl)-6-methylflavone
34 methyl 3′-(6-methyl-5′-nitroflavone)carboxylate >2100
35 3′-(6-methyl-5′-nitroflavone)carboxylic acid
36 methyl 3′-(5′-amino-6-methylflavone)-
carboxylate
>1500
>1700
>1900
37 ethyl 3′-(5′-amino-6-methylflavone)carboxylate 3600
38 3′-(trifluoromethyl)-6-methylflavone
9.8 ( 2.7
39 3′-bromo-6-methylflavone
12 ( 2
40 5′-bromo-2′-methoxy-6-methylflavone
41 5′-bromo-2′-hydroxy-6-methylflavone
>1500
0.9 ( 0.2
42 6-methyl-2-(3-pyridyl)-4H-1-benzopyran-4-one 130 ( 12
(3′-aza-6-methylflavone)
micromolar concentrations. Compound 41 displays the
highest affinity in the present series with a Ki value of
0.9 nM, and it is the highest affinity flavone derivative
reported so far.
a
Reagents: (a) Ac2O, pyridine. (b) AlCl3. (c) Substituted or
nonsubstituted benzoyl chloride, pyridine. (d) KOH, pyridine. (e)
H2SO4.
prepared by reduction of the corresponding nitro com-
pound.18 Esters were hydrolyzed with LiOH in tetrahy-
drofuran (THF) and water 1:1. Esters of benzoic acids
were prepared by an acid-catalyzed reaction with the
alcohol in THF or by the reaction of the alkyl halide in
dimethyl formamide (DMF)/K2CO3. The latter reaction
was also used for preparing ethers. 2-Methoxy-5-meth-
ylbenzoic acid was prepared from the corresponding
aldehyde,19 instead of from 2-bromo-4-methylanisol.20
Ethers were cleaved by exposing them to BBr3 in CH2-
Cl2 at -78 °C. Esters were reduced to alcohols with
LiAlH4 in anhydrous THF at 0 °C. The ether 18 and
the ester 24 were made from 16 and 22, respectively,
under Mitsonobu conditions.21
P h a r m a cop h or e Mod el. The pharmacophore model
developed by Cook and co-workers5 and previously
employed by us for structure-activity analysis of a
series of flavonoids14 is shown in Figure 1. The proposed
binding mode of compound 2 is shown. H1 and A2 are
hydrogen bond donor and acceptor sites, respectively,
whereas H2/A3 is a bifunctional hydrogen bond donor/
acceptor site. L1-L3 are three lipophilic pockets and
S1-S3 denote regions of steric repulsive ligand-recep-
tor interactions (receptor-essential volumes).
The main conclusions in our previous structure-
activity analysis on flavonoids were that in order to bind
to the benzodiazepine site, the flavone skeleton should
be planar or close to planar. Small substituents such
as methyl and bromine in the 6-position significantly
increase the affinity, whereas a 4′-NO2 group signifi-
cantly decreases the affinity. A 3′-NO2 or 3′-methyl
Recep tor Bin d in g. As shown in Table 1, the tested
3
flavonoids inhibit H-Ro 15-1788 binding with a range
of affinities from low nanomolar (14, 20, and 41) to

4190 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Kahnberg et al.
display a conformational energy difference of 1.7 kcal/
mol and the 2′-hydroxy compounds 14, 20, and 41
display an energy difference of 1.3 kcal/mol. All com-
pounds without a 2′-substituent display conformational
energies for adopting a planar conformation of less than
0.2 kcal/mol.
Dim en sion s of th e L2 Region . Methyl and ethyl
substitution in the 6-position give essentially identical
affinities (Table 1). However, on increasing the size of
the 6-substituent from ethyl (3) to propyl (4) and
isopropyl (5), the affinity decreases although the lipo-
philicity of the substituent increases. This indicates the
presence of steric repulsions with the receptor for
substituents of the size of a propyl group or larger.
Compound 6, in which the methyl group in 2 is moved
to the 8-position, displays a decreased affinity as
compared to 2. The decreased affinity of the 8-methyl
compound 6 may be due to a steric conflict with the H2/
A3 site (Figure 1).
F igu r e 1. Proposed binding mode of compound 2 in the
4′-Su bstitu ted F la von es. In our previous study, we
observed that a 4′-NO2 group decreased the affinity by
a factor of about 60.14 However, it was not clear from
the available data if the strong decrease in affinity is
due to steric repulsive interactions or electrostatic
repulsions between the 4′-NO2 group and the receptor.
Compounds 7 and 12 were prepared and tested in order
to obtain more information on this interaction. The 4′-
methyl-substituted compound 7 displays an affinity
decrease as compared to 2 by a factor of 24, similar to
that of the 4′-NH2 compound 12 and slightly lower than
the corresponding factor displayed by the 4′-NO2-
substituted compound. The 3′,4′-dimethyl-substituted
compound 8 displays a reduced affinity by a factor of
>59 as compared to the corresponding 3′-methyl-
substituted compound (Ki ) 29 nM).14 Thus, the major
part of the affinity decrease caused by 4′-substitution
is most likely due to steric repulsions with the receptor
although additional electrostatic or hydrogen-bonding
interactions for the 4′-NO2- and the 4′-NH2-substituted
compounds cannot be excluded.
P r op er ties of th e Recep tor Region in th e Vicin -
ity of th e 3′-Su bstitu en t. This region is of particular
interest as we14 and others23-25 have shown that 3′-
substituents as methyl, nitro, and bromo significantly
increase the affinity. In the context of the pharmaco-
phore model shown in Figure 1, we have previously
concluded that a 3′-substituent should be directed
“downwards”.14 The low affinity of the 3′,5′-dimethyl
compound 9 (Table 1) clearly shows that a methyl group
in the alternative equivalent position (5′) suffers strong
steric repulsive interactions. Furthermore, all other
3′,5′-substituted compounds in Table 1 (compounds 25-
37) display Ki values higher than 1500 nM. Thus, it is
concluded that the sterically repulsive region in the
vicinity of a 4′-substituent discussed above extends to
the region about the 5′-position.
pharmacophore model developed by Cook and co-workers.
group (directed “downwards” in Figure 1) strongly
increases the affinity making this substituent position
of great interest for further investigations. None of the
previously studied flavonoids was able to interact with
the hydrogen bond accepting site A2, although this has
been found to be an important interaction site for
compounds that display potent inverse agonism.5 De-
spite this, 6-methyl-3′-nitroflavone was shown to be a
high affinity ligand with a Ki of 5.6 nM and displaying
inverse agonism.14
The present series of flavonoids was designed to
explore the dimensions of the lipophilic pocket L2
including the regions in the vicinity of L2 (compounds
3-6) and the nature of the previously observed repul-
sive interactions between a 4′-substituent and the
receptor (compounds 7, 8, and 12). Furthermore, a series
of 3′-substituted compounds were synthesized and
tested in order to map out the properties of the region
in the vicinity of a 3′-substituent (compounds 11, 15-
18, 22-24, 38, and 39). The 3′,5′-disubstituted com-
pounds 9 and 25-37 were studied in order to obtain
information on the effects of substitution in both of the
equivalent 3′- and 5′-positions. Finally, to probe the
possibility of obtaining favorable interactions with the
hydrogen bond acceptor site A2, the 2′(6′)-aminoflavone
10 and the 2′(6′)-hydroxyflavone 14 were prepared
together with the 2′,5′-disubstituted compounds 20 and
41. The corresponding O-methylated compounds 13, 19,
and 40 were prepared for comparisons.
Con for m a tion a l An a lyses. As mentioned above, a
coplanar or close to coplanar arrangement of the two
ring systems in the flavones is required for binding to
the receptor. As steric hindrance to coplanarity may
occur for the 2′-substituted compounds 10, 13, 14, 19,
20, 40, and 41, the energy difference between the lowest
energy minimum (with a twisted 2-phenyl ring) and the
coplanar conformation was calculated for each of these
compounds. The calculations were performed as previ-
ously described by using the MM3(92) force field.14,22
The 2′-amino-substituted compound 10 displays a con-
formational energy difference of 3.5 kcal/mol between
the coplanar conformation and the lowest energy one,
whereas the 2′-methoxy compounds 13, 19, and 40
For compound 2 and six compounds with small
3′-substituents NH2 (11), OMe (15), OH (16), Me,14
CF3 (38), and Br (39), an excellent correlation between
logKi values and Hansch’s π-values26 can be obtained
(logKi ) -0.960π + 1.984, R2 ) 0.97, data not shown)
indicating that the affinites of these compounds may
be understood in terms of a favorable transfer from the
aqueous phase to a receptor phase with properties

Pharmacophore Model for Flavone Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4191
F igu r e 3. Proposed binding mode of compound 41 in the
pharmacophore model displaying the proposed novel interac-
tion between a 2′ (6′)-OH-substituted flavone and the A2 site.
F igu r e 2. Steric repulsive interaction sites S4 and S5
identified in the present work and the proposed properties of
the receptor region in vicinity of the 3′-position in flavones.
substituents in the 2′(6′)-position, compounds 10 and
14 were synthesized and tested. As shown in Table 1, a
2′(6′)-OH group (14) increases the affinity by a factor
of 29, whereas a 2′(6′)-NH2 group (10) decreases the
affinity by a factor of 5 as compared to reference
compound 2. The lower affinity of compound 10 as
compared to 14 by a factor of ca. 100 is most probably
due to a combination of a higher energy required for 10
to adopt a coplanar arrangement of the ring systems
(see above) and to the significant difference between the
hydrogen-bonding energies involving a phenolic hydroxy
group and an aniline type amino group, respectively.
Halgren has calculated the phenol-water (with phenol
as the hydrogen bond donor) and the aniline-water
hydrogen bond interaction energies by high level ab
initio quantum chemical methodology (MP2/6-31+G**
basis set).28 The calculations show that the phenol-
water interaction is 3.5 kcal/mol stronger than the
aniline-water interaction.
Introducing a 6′-hydroxy substituent to the 3′-meth-
yl14 and 3′-bromo25 (39) compounds to give 20 and 41,
respectively, increases the affinity significantly as pre-
dicted on the basis of the high affinity of the 2′(6′)-
hydroxy compound 14 and the pharmacophore model
(Figure 1), which implies that the 6′-hydroxy group
interacts with the A2 site and the 3′-substituent is
directed “downwards” as shown in Figure 3. It should
be noted that 3′-bromo-6′-hydroxy-6-methyl flavone (41)
is the highest affinity flavone derivative reported so far
with respect to binding to the benzodiazepine site of the
GABAA receptor.
similar to 1-octanol. The 6-methyl,3′-nitro-subtituted
compound with a Ki of 5.6 nM14 is about 30 times more
active than predicted from the regression equation
indicating that additional favorable interactions, e.g.,
hydrogen bonding, are available for this strongly polar
substituent. The presence of a hydrogen-bonding envi-
ronment about the 3′-position is also supported by the
affinity of the pyridyl compound 42. Although the
desolvation energy of the pyridyl group is significantly
higher than that of a phenyl group, compounds 2 and
42 have virtually identical affinities (Table 1). The low
affinity of compound 22 is most probably due to the very
high desolvation energy of a carboxylate group, which
cannot be sufficiently compensated for by interactions
in the receptor region about the 3′-position.
The affinities of the methyl and isopropyl esters, ester
21, and 23 are slightly lower than that of the 3′-methoxy
compound 15. With larger alkyl ester groups and alkyl
ether groups as in compound 24 and compounds 17 and
18, the affinity slowly decreases with increasing size of
the alkyl substituent. In the alkyl ester series, all
affinites are within a factor of 3, whereas the corre-
sponding factor for the alkyl ether series is 10.
Our interpretation of the affinity data for the 3′-
substituted compounds is that the receptor region close
to the 3′-position is a partly lipophilic region with
possibilities for hydrogen bonding and that the exten-
sion of this region is channel-like in which the larger
substituents of compounds 17, 18, and 24 are only partly
desolvated. Such a channel may exist at the interface
between an R- and a γ-subunit in the GABAA receptor
where the benzodiazepine site most probably is lo-
cated.27
Methylation of the hydroxy group in 20, 14, and 41
to give 19, 13, and 40 significantly reduces the affinity
due to the disruption of the hydrogen bond to A2,
according to the pharmacophore model (Table 1, Figure
3). For the highest affinity 6′-hydroxy compound 41,
O-methylation decreases the affinity by more than a
factor of 1500. This decrease in the affinity is similar
to what is observed for compound 43 (Chart 2) as
reported by Cook and co-workers.5 The affinity de-
creases by more than a factor of 2500 on going from
RdH to Rdmethyl in 43. Compound 43 with RdH is a
close analogue of CGS-9896 (44), which was used by
Figure 2 summarizes our conclusions on the proper-
ties of the receptor regions about the 3′-, 4′-, and 5′-
positions.
In ter a ction s w ith th e Hyd r ogen Bon d -Accep tin g
A2 Site. As shown by the low affinities of the 3′,5′-
disubstituted compounds discussed above, favorable
interactions with the A2 site by substituents in the 5′-
position (“upwards” in Figure 2) are not possible due to
strong steric repulsions in this region. To investigate if
interactions with the A2 site may be obtained by

4192 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Kahnberg et al.
Ch a r t 2
the C(5)dN(4) bond in diazepam and the A-ring in the
flavones superimposes the 5-phenyl ring in diazepam
(Chart 3). With this superimposition, the CdO group
in the flavones is interacting with the H2/A3 site (Figure
1) and the 2-phenyl ring (B in Chart 3) is positioned
close to the lipophilic region L2. In this model, there
are no interactions between the flavones and the H1 and
no possibility for interactions with A2. The drawback
with the model suggested by Marder et al. is that it is
not able to rationalize a number of observed important
SAR for the flavones. The significantly increased affinity
due to the 2′-OH group in compounds 14, 20, and 41
cannot be understood by the model, and the strong
decrease in affinity due to the 4′-methyl group in 7
cannot be rationalized. This methyl group would in the
model proposed by Marder et al. be positioned in the
L2 lipophilic pocket with a predicted increase in affinity.
The explanation provided for the low affinity of 4′-
substituted flavones in terms of an electron rich site
located close to L2 is not convincing as the methyl group
in the 4′-position does not cause any significant changes
of the electron distribution of the flavone molecule and
the methyl group itself is certainly not a polar substitu-
ent.
Ch a r t 3
Cook and co-workers to establish the presence of a
hydrogen-bonding site A25 and which superimposes
extremely well with the flavones.14 In this superimposi-
tion, the N-H bonds in 43 (RdH) and 44 superimposes
with the OH bond in 41 and other 2′(6′)-hydroxy
flavones. This strongly supports the superimpositions
previously proposed by us and in particular the conclu-
sion that the 2′(6′)-hydroxy group in the flavone series
is directed “upwards” as in Figure 3 interacting with
the A2 site and that the 3′-position consequently is
directed “downwards”.14
Com p a r ison s w ith a P r op osed Alter n a tive P h a r -
m a cop h or e Mod el. Recently, Marder et al. proposed
an alternative pharmacophore model in which the
flavones are superimposed with diazepam.15 According
to the pharmacophore model developed by Cook and co-
workers5 and used as a starting point by us as well as
by Marder et al., 1,4-benzodiazepines such as diazepam
display interactions with the H1, L1, L2, and H2/A3
sites and, in addition, with the lipophilic region L3
(Figure 1).5 It should be noted that the 1,4-benzodiaz-
epines in this model do not interact with the A2 site
and, according to our analysis, not with the site occupied
by the 2-phenyl ring in the flavones.
As discussed above, it is well-established that a planar
or close to planar structure is required for active
flavones. To make the two aromatic rings in diazepam
(Chart 3) reasonably coplanar, the seven-membered ring
(B) in diazepam, which has a boat conformation in the
lowest energy minimum, was flattened and the 5′-
phenyl ring was reoriented with a total calculated
conformational energy cost of 7-9 kcal/mol.15 The
superimposition of flavones and diazepam as suggested
by Marder et al. is in our opinion highly problematic.
The contrast between the high conformational energy
calculated for the proposed bioactive conformation of
diazepam and the very low one calculated for the
flavones is hardly compatible with the high affinity of
diazepam (IC50 ) 8.1 nM).5
Marder et al. cast doubts on our superimpositions of
CGS-9896 (44, Chart 2) and the flavones14 and argue
for a superimposition of the flavones with diazepam on
the basis that these compounds do not bind to the
R6â3γ2-subtype, whereas CGS-9896 binds to this sub-
type. Affinity data for CGS-9896 on specific subtypes
are to our knowledge not available, but the bromo
analogue of 44 binds to the R6â3γ2-subtype with an
affinity that is 31-222 times lower than the affinities
for the R(1-5)â3γ2-subtypes.29 Very few affinity data for
flavones interacting with subtypes have been reported.
Preliminary data for 6,8-dibromochrysin (5,7-dihydroxy-
6,8-dibromoflavone) display a lower affinity for the
R6â3γ2-subtype by a factor of >3-19.15 Amentoflavone
has a significant selectivity for the R(1-5)â3γ2-subtypes,
but because this molecule is a biflavone, it must occupy
other receptor sites than the simple flavones do and can
therefore not be compared directly to the flavones
studied by us in this and previous works. Thus, it is in
our opinion premature to draw conclusions on the
subtype selectivity of flavones. However, CGS-9896 used
in our model is only one of several classes of compounds
to which the flavones may be superimposed and which
also superimposes with CGS-9896 exceedingly well as
shown by Cook and co-workers.5 For instance, dihydro-
pyrido[3,4-b:5,4-b′]diindoles could equally well be used
as a template as shown in our previous work.14 Accord-
ing to Huang et al.,29 these compounds show high
selectivity for the R(1-5)â3γ2-subtypes. Thus, compounds
that differ in their binding to a specific subtype do not
necessarily require different binding modes in terms of
the pharmacophore model developed by Cook and co-
workers and employed by us for structure-activity
analysis of flavones.
Con clu sion s
The pharmacophore model for the benzodiazepine site
of the GABAA receptor originally proposed by Cook and
co-workers5 has been further validated and refined by
the identification of two new regions of steric repulsive
In the model of Marder et al., the flavones are fitted
to diazepam so that the flavone CdO bond superimposes

Pharmacophore Model for Flavone Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4193
15.9, 28.1, 92.3, 118.6, 126.8, 126.8, 127.1, 128.7, 128.7, 128.8,
132.3, 133.7, 134.7, 135.8, 160.6, 177.4, 195.5. MS m/z (% rel
int): 268 (M+, 52%), 149 (24%), 105 (100%), 77 (42%).
6-Eth ylfla von e (3). Concentrated sulfuric acid (95-97%)
(0.22 g, 2.22 mmol) was added to a suspension of the diketone
II (0.56 g, 2.22 mmol) in 3 mL of glacial acetic acid and heated
to 80 °C for 2 h, whereafter the mixture was poured onto 14 g
of ice. After 30 min, the white crystals that formed were
collected by filtration, washed with 30 mL of water, and
recrystallized from acetone to give white needles (yield 54%);
mp 96-98 °C. 1H NMR (CDCl3): 1.31 (t, 3H, J ) 7.6), 2.78 (q,
2H, J ) 7.6), 6.83 (s, 1H), 7.52 (m, 5H), 7.93 (d, 2H, J ) 8.2),
8.06 (d, 1H, J ) 2.2). 13C NMR (CDCl3): 15.4, 28.3, 107.4,
117.9, 123.7, 123.8, 126.2, 126.2, 129.0, 129.0, 131.4, 131.9,
133.9, 141.5, 154.6, 163.2, 178.6. MS m/z (% rel int): 250.1002
(M+, 100, C17H14O2 requires 250.0994), 249 (38%), 235 (52%),
133 (50%). Anal. (C17H14O2) C, H.
2-Acetyl-4-p r op ylp h en yl Ben soa te (Sch em e 1; I Lea d -
in g to 4). The compound was prepared according to the
procedure described for I leading to 3. 2-Acetyl-4-propylphenol
was used instead of 2-acetyl-4-ethylphenol. The crude product
was purified by chromatography (heptane:ethyl acetate 9:1)
and obtained as a yellow oil in 93% yield. 1H NMR (CDCl3):
0.98 (t, 3H, J ) 7.3), 1.69 (sext., 2H, J ) 7.3), 2.55 (s, 3H),
2.66 (t, 2H, J ) 7.3), 7.15 (d, 1H, J ) 8.2), 7.39 (dd, 1H, J )
8.2, 2.3), 7.52 (m, 3H), 7.81 (d, 1H, J ) 2.3), 8.23 (d, 2H, J )
8.1). 13C NMR (CDCl3): 14.2, 24.9, 30.3, 37.7, 124.0, 129.3,
126.3, 129.3, 130.5, 130.7, 130.7, 131.0, 134.0, 134.2, 131.2,
147.8, 165.7, 198.2. MS m/z (% rel int): 282 (M+, 8%), 105
(100%), 77 (34%).
1-(2-Hyd r oxy-5-p r op ylp h en yl)-3-p h en ylp r op a n e-1,3-d i-
on e (Sch em e 1; II Lea d in g to 4). The compound was
prepared according to the procedure described for II leading
to 3. The product was purified by chromatography (heptane:
ethyl acetate 15:1) to give yellow crystals in a yield of 58%;
mp 58-60 °C. Only the enol form was obtained. 1H NMR
(CDCl3): 0.97 (t, 3H, J ) 7.3), 1.65 (sext., 2H, J ) 7.3), 2.61
(t, 2H, J ) 7.3), 6.84 (s, 1H), 6.96 (d, 1H, J ) 8.3), 7.29 (dd,
1H, J ) 8.3, 2.2), 7.52 (m, 3H), 7.86 (m, 3H), 11.91 (s, 1H),
15.60 (s, 1H). 13C NMR (CDCl3): 13.7, 24.7, 37.2, 92.3, 118.5,
126.8, 126.8, 127.7, 128.7, 128.7, 128.8, 132.3, 133.1, 133.7,
136.3, 160.6, 177.4, 195.7. MS m/z (% rel int): 282 (M+, 49%),
105 (100%), 77 (38%).
interactions, S4 and S5. In terms of substituted flavones,
the lipophilic L2 pocket optimally accommodates sub-
stituents of the size of an ethyl group. 2′-Hydroxy sub-
stitution has been shown to give a significant increase
in affinity, which in terms of the pharmacophore model
straightforwardly can be interpreted in terms of hydro-
gen bond interaction with a previously proposed hydro-
gen bond-accepting site A2. The receptor region in the
vicinity of the 3′-position is most likely a partly lipophilic
region with possibilities for hydrogen bonding and
possibly close to a channel at the interface between an
R- and a γ-subunit in the GABAA receptor. On the basis
of the results of these studies and the refined pharma-
cophore model, we have designed and synthesized 5′-
bromo-2′-hydroxy-6-methylflavone, with a Ki value of 0.9
nM, the highest affinity flavone derivative reported. As
mentioned in the Introduction, different GABAA recep-
tor subtypes mediate different pharmacological actions.
It remains to be investigated if the compounds discussed
in the present work display subtype selectivity.
Exp er im en ta l Section
1
Ch em istr y. H NMR and 13C NMR were recorded at room
temperature with a Bruker ARX300, Bruker DRX400, or
Bruker ARX500 spectrometer. The spectra were recorded in
CDCl3, DMSO-d6, and CD3OD, and the solvent signals (7.27
and 77.23, 2.50 and 39.52, 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). EI mass spectra were recorded at 70
eV with a J EOL SX102 spectrometer. Concentrations were
made using rotary evaporation with bath temperatures at or
below 40 °C. Anhydrous MgSO4 was used as a drying agent
for the organic extracts in the work up procedures. Thin-layer
chromatography (TLC) was performed on silica gel 60 F254
plates (Merck). Column chromatography was performed on
SiO2 (Matrex LC-gel: 60A, 35-70 MY, Grace) using the flash
technique. Melting points (uncorrected) were determined with
a Reichert microscope. Compound 5 (isopropylflavone) was
purchased from Carbocore Inc.
6-P r op ylfla von e (4). The compound was prepared accord-
ing to the procedure described for 3. The crude product was
crystallized from acetone to give white needles in a yield of
48%; mp 97-99 °C. 1H NMR (CDCl3): 0.96 (t, 3H, J ) 7.3),
1.71 (sext., 2H J ) 7.3), 2.71 (t, 2H, J ) 7.3), 6.82 (s, 1H), 7.53
(m, 5H), 7.92 (d, 2H, J ) 8.1), 8.06 (d, 1H, J ) 2.3). 13C NMR
(CDCl3): 14.1, 24.8, 37.8, 107.9, 118.3, 124.1, 125.0, 126.7,
126.7, 129.5, 129.5, 131.9, 132.4, 134.9, 140.4, 155.2, 163.7,
179.1. MS m/z (% rel int): 264.1150 (M+, 55, C18H16O2 requires
264.1150), 236 (21%), 235 (100%), 133 (64%). Anal. (C18H16O2)
C, H.
2-Acetyl-6-m eth ylp h en yl Ben soa te (Sch em e 1; I Lea d -
in g to 6). The compound was prepared according to the
procedure described for I leading to 3. 2-Acetyl-6-methylphenol
was used instead of 2-acetyl-4-ethylphenol. The crude product
was purified by chromatography (heptane:ethyl acetate 5:1)
to give white crystals in 44% yield. Spectroscopic data were
identical with those previously reported.31
1-(2-H yd r oxy-3-m et h ylp h en yl)-3-p h en ylp r op a n e-1,3-
d ion e (Sch em e 1; II Lea d in g to 6). The compound was
prepared according to the procedure described for II leading
to 3. Purification was made by recrystallization from EtOH
to give yellow crystals in a yield of 35%. Spectroscopic data
were identical with those previously reported.32
8-Meth ylfla von e (6). The compound was prepared accord-
ing to the procedure described for 3. The crude product was
recrystallized from acetone to give beige crystals in a yield of
69%. Spectroscopic data were identical with those previously
reported.33 Anal. (C16H12O2) C, H.
2-Acetyl-4-eth ylp h en yl Ben soa te (Sch em e 1; I Lea d in g
to 3). To a stirred solution of 1-(5-ethyl-2-hydroxy-phenyl)-
ethanone (1.59 g, 9.68 mmol) in anhydrous pyridine (1.94 mL),
benzoyl chloride (1.63 g, 0.012 mol) was added dropwise over
a period of 10 min. The solution was stirred at 50 °C for 30
min and then poured into a mixture of ice (9.7 g) and 1 M
HCl(aq) (20 mL). The organic phase was extracted with diethyl
ether and washed with brine, dried, and evaporated. The
product was purified by chromatography (heptane:ethyl ac-
1
etate 9:1) and obtained as a transparent oil in 85% yield. H
NMR (CDCl3): 1.30 (t, 3H, J ) 7.6), 2.55 (s, 3H), 2.74 (q, 2H,
J ) 7.6) 7.16 (d, 1H, J ) 8.3), 7.42 (dd, 1H, J ) 8.3, 2.3), 7.55
(m, 3H), 7.69 (d, 1H, J ) 2.3), 8.23 (d, 2H, J ) 8.2). 13C NMR
(CDCl3): 15.9, 28.7, 30.4, 124.1, 129.1, 129.1, 129.3, 129.9,
130.7, 130.7, 131.0, 133.3, 134.2, 142.7, 147.8, 165.8, 198.3.
MS m/z (% rel int): 268 (M+, 10%), 105 (100%), 77 (43%).
1-(5-Eth yl-2-h yd r oxyp h en yl)-3-p h en ylp r op a n e-1,3-d i-
on e (Sch em e 1; II Lea d in g to 3). 2-Acetyl-1-benzoyloxy-4-
ethylbenzene (2.68 g, 9.97 mmol) was dissolved in 10 mL of
anhydrous pyridine and heated to 50 °C. Finely powdered
potassium hydroxide (0.78 g, 14.0 mmol) was added in small
portions over a period of 20 min, and stirring was continued
for an additional 1.5 h at 50 °C. After the mixture was cooled
to room temperature and 15 mL of 10% acetic acid(aq) was
added, the precipitate formed was collected by filtration and
recrystallized from EtOH to give slightly yellow-brown needles
(yield 46%); mp 62-65 °C. In CDCl3, the product is present
1
as the enol form. H NMR (CDCl3): 1.25 (t, 3H, J ) 7.3), 2.63
(q, 2H, J ) 7.3), 6.84 (s, 1H), 6.93 (d, 1H, J ) 8.5), 7.32 (dd,
1H, J ) 8.5, 2.3), 7.53 (m, 3H), 7.84 (d, 2H, J ) 8.1), 7.90 (d,
1H, J ) 2.3), 11.94 (s, 1H), 15.58 (s, 1H). 13C NMR (CDCl3):
2-Acetyl-4-m eth ylp h en yl 4-Meth ylben soa te (Sch em e 1;
I Lea d in g to 7). The compound was prepared according to

4194 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Kahnberg et al.
the procedure described for I leading to 3. 4-Methylbenzoyl
chloride was made from 4-methylbenzoic acid. The acid (2.20
g, 16.2 mmol) was dissolved in SOCl2 (1.77 mL, 24.3 mmol), a
drop of DMF was added, and the solution was refluxed for 2.5
h. The acid chloride was purified by distillation using high
vacuum. 2-Acetyl-4-methylphenol was used instead of 2-acetyl-
4-ethylphenol. The product was purified by recrystallization
from MeOH to give white crystals in a yield of 82%. Spectro-
scopic data were identical with those previously reported.34
1-(2-Hyd r oxy-5-m eth ylp h en yl)-3-(4-m eth ylp h en yl)p r o-
p a n e-1,3-d ion e (Sch em e 1; II Lea d in g to 7). The compound
was prepared according to the procedure described for II
leading to 3. Purification was made twice by recrystallization
from EtOH to give yellow crystals in a yield of 96%. Spectro-
scopic data were identical with those previously reported.34
4′,6-Dim eth ylfla von e (7). The compound was prepared
according to the procedure described for 3. The product was
recrystallized from acetone to give beige crystals in a yield of
91%. Spectroscopic data were identical with those previously
reported.34 Anal. (C17H14O2) C, H.
2-Acetyl-4-methylphenyl 3,4-Dimethylbensoate (Scheme
1; I Lea d in g to 8). The compound was prepared according to
the procedure described for I leading to 7. The crude product
was purified by recrystallization from MeOH to give white
crystals in a yield of 93%; mp 67-69 °C. 1H NMR (CDCl3):
2.35 (s, 3H), 2.36 (s, 3H), 2.41 (s, 3H), 2.52 (s, 3H), 7.10 (d,
1H, J ) 8.2), 7.29 (d, 1H, J ) 8.5), 7.38 (dd, 1H, J ) 8.2, 2.3),
7.65 (d, 1H, J ) 2.3), 7.95 (m, 2H). 13C NMR (CDCl3): 20.0,
20.4, 21.1, 30.2, 123.8, 126.9, 128.1, 130.2, 130.7, 131.2, 131.5,
134.2, 136.1, 137.4, 143.7, 147.6, 165.8, 198.2. MS m/z (% rel
int): 282 (M+, 3%), 150 (12%), 133 (100%), 105 (34%), 77 (16%).
1-(2-Hyd r oxy-5-m eth ylp h en yl)-3-(3,4-d im eth ylp h en yl)-
p r op a n e-1,3-d ion e (Sch em e 1; II Lea d in g to 8). The
compound was prepared according to the procedure described
for II leading to 3. Purification of the crude product was made
twice by recrystallization from EtOH to give yellow needles
in a yield of 74%; mp 113-115 °C. The product was present
as the enol form in CDCl3. 1H NMR (CDCl3): 2.36 (s, 3H), 2.36
(s, 3H), 2.37 (s, 3H), 6.80 (s, 1H), 6.92 (d, 1H, J ) 8.4), 7.28
(m, 2H), 7.56 (d, 1H, J ) 2.2), 7.70 (dd, 1H, J ) 8.1, 2.2), 7.74
(d, 1H, J ) 2.1), 11.97 (s, 1H), 15.57 (s, 1H). 13C NMR
(CDCl3): 20.3, 20.6, 21.0, 92.1, 118.9, 119.1, 124.9, 128.3, 128.5,
128.6, 130.5, 131.6, 137.1, 137.6, 142.4, 160.7, 178.5, 195.7.
MS m/z (% rel int): 282 (M+, 40%), 135 (13%), 133 (100%),
105 (18%), 77 (13%).
6.81 (s, 1H), 6.92 (d, 1H, J ) 8.4), 7.21 (s, 1H), 7.29 (dd, 1H,
J ) 8.4, 2.2), 7.57 (m, 3H), 11.95 (s, 1H), 15.66 (s, 1H). 13C
NMR (CDCl3): 20.8, 21.5, 21.5, 92.5, 118.8, 124.9, 124.9, 128.3,
128.4, 134.0, 134.4, 134.4, 137.0, 138.7, 138.7, 160.6, 178.4,
195.7. MS m/z (% rel int): 282 (M+, 45%), 135 (15%), 134 (13%),
133 (100%), 105 (18%).
3′,5′,6-Tr im eth ylfla von e (9). The compound was prepared
according to the procedure described for 3. The crude product
was purified by chromatography (heptane:ethyl acetate 5:1)
to give white crystals in a yield of 75%; mp 143-145 °C. 1H
NMR (CDCl3): 2.42 (s, 6H), 2.48 (s, 3H), 6.79 (s, 1H), 7.18 (s,
1H), 7.53 (m, 4H), 8.03 (d, 1H, J ) 2.0). 13C NMR (CDCl3):
21.2, 21.6, 21.6, 107.6, 118.1, 123.9, 124.3, 124.3, 125.2, 132.1,
133.4, 135.1, 135.3, 138.9, 138.9, 154.8, 163.9, 178.8. MS m/z
(% rel int): 264.1151 (M+, 100, C18H16O2 requires 264.1151),
265 (19%), 236 (20%), 134 (52%). Anal. (C18H16O2) C, H.
2′-Am in o-6-m eth ylfla von e (10). Sn (89 mg, 0.749 mmol)
was added to a solution of 6-methyl-2′-nitroflavone14 (40 mg,
0.142 mmol) in EtOH (2 mL), and the solvent was heated to
reflux. A 12 M amount of HCl (0.64 mL, 7.68 mmol) was added
to the solution, and after 20 min, the mixture was cooled to
room temperature. The solution was made weakly basic with
2 M NaOH(aq), extracted with diethyl ether, and washed with
brine. The organic phase was dried and evaporated. The crude
product was purified by chromatography (toluene:acetone 5:1)
to give yellow-white crystals in 70% yield; mp 173-175 °C.
1H NMR (CDCl3): 2.49 (s, 3H), 4.46 (bs, 2H), 6.66 (s, 1H), 6.81
(d, 1H, J ) 8), 6.87 (t, 1H, J ) 8), 7.29 (t, 1H, J ) 8), 7.40 (d,
1H, J ) 8.4), 7.47 (d, 1H, J ) 8), 7.50 (dd, 1H, J ) 8.4, 2.2),
8.02 (d, 1H, J ) 2.2). 13C NMR (CDCl3): 20.9, 110.3, 117.1,
117.1, 117.6, 118.5, 123.5, 125.1, 129.6, 132.1, 134.9, 135.3,
145.2, 154.5, 164.7, 178.3. MS m/z (% rel int): 251.0948 (M+,
44, C16H13NO2 requires 251.0946), 117 (100%). Anal. (C16H13
NO2) C, H, N.
-
3′-Am in o-6-m eth ylfla von e (11). 6-Methyl-3′-nitroflavone14
was reacted according to the procedure described for 6-methyl-
2′-nitroflavone leading to 10. The compound was purified by
chromatography (toluene:acetone 5:1) to give yellow-white
crystals in 65% yield; mp 194-198 °C. 1H NMR (DMSO-d6):
2.42 (s, 3H), 5.41 (bs, 2H), 6.76 (s, 1H), 6.79 (ddd, 1H, J ) 7.2,
2.1, 2.1), 7.20 (m, 3H), 7.59 (d, 1H, J ) 8.6), 7.62 (dd, 1H, J )
8.6, 1.8), 7.82 (d, 1H, J ) 1.8). 13C NMR (DMSO-d6): 20.5,
106.4, 110.9, 113.8, 117.2, 118.1, 123.1, 124.1, 129.7, 131.8,
135.0, 135.3, 149.3, 153.9, 163.5, 177.0. MS m/z (% rel int):
251.0948 (M+, 100, C16H13NO2 requires 251.0946), 117 (36%).
Anal. (C16H13NO2) C, H, N.
3′,4′,6-Tr im eth ylfla von e (8). The compound was prepared
according to the procedure described for 3. The product was
recrystallized from acetone to give light yellow crystals in a
4′-Am in o-6-m eth ylfla von e (12). 6-Methyl-4′-nitroflavone14
was reacted according to the procedure described for 6-methyl-
2′-nitroflavone leading to 10. The crude product was purified
by chromatography (toluene:acetone 5:1) to give yellow-white
crystals in 76% yield; mp 194-197 °C. 1H NMR (CDCl3): 2.47
(s, 3H), 4.16 (bs, 2H), 6.70 (s, 1H) 6.77 (d, 2H, J ) 8.9), 7.44
(d, 1H, J ) 8.5), 7.49 (dd, 1H, J ) 8.5, 2.1), 7.76 (d, 2H, J )
8.9), 8.02 (bs, 1H). 13C NMR (CDCl3): 21.1, 105.0, 105.1, 114.9,
117.8, 121.3, 123.8, 125.1, 125.2, 128.2, 134.8, 135.0, 150.0,
154.6, 164.1, 178. MS m/z (% rel int): 251.0946 (M+, 100,
1
yield of 94%; mp 142-144 °C. H NMR (CDCl3): 2.33 (s, 3H),
2.35 (s, 3H), 2.46 (s, 3H), 6.77 (s, 1H), 7.24 (d, 1H, J ) 8.2),
7.47 (m, 2H), 7.67 (m, 2H), 8.00 (d, 1H, J ) 2.1). 13C NMR
(CDCl3): 19.9, 20.0, 20.9, 106.7, 117.8, 123.6, 123.8, 124.9,
127.2, 129.3, 130.2, 134.8, 135.0, 137.4, 140.9, 154.5, 163.6,
178.6. MS m/z (% rel int): 264.1148 (M+, 100, C18H16O2
requires 264.1150), 249 (28%), 236 (23%), 134 (42%), 115 (14%).
Anal. (C18H16O2) C, H.
C
16H13NO2 requires 251.0946), 117 (47%). Anal. (C16H13NO2)
C, H, N.
2-Acetyl-4-m eth ylp h en yl 2-Meth oxyben soa te (Sch em e
2-Acetyl-4-methylphenyl 3,5-Dimethylbensoate (Scheme
1; I Lea d in g to 9). The compound was prepared according to
the procedure described for I leading to 7. The crude product
was purified by chromatography (heptane:ethyl acetate 8:1)
1; I Lea d in g to 13 a n d 14). The compound was prepared
according to the procedure described for I leading to 7. The
acid chloride was commercially available. The product was
purified by chromatography (heptane:ethyl acetate 6:1) as
1
to give white crystals (yield 50%); mp 115-117 °C. H NMR
(CDCl3): 2.41 (s, 6H), 2.43 (s, 3H), 2.54 (s, 3H), 7.10 (d, 1H, J
) 8.2), 7.29 (s, 1H), 7.39 (dd, 1H, J ) 8.2, 2.1), 7.66 (d, 1H, J
) 2.1), 7.96 (m, 2H). 13C NMR (CDCl3): 21.0, 21.4, 21.4, 30.1,
123.8, 128.2, 128.2, 129.3, 130.7, 131.2, 134.2, 135.7, 136.1,
138.6, 138.6, 147.5, 165.9, 198.4. MS m/z (% rel int): 282 (M+,
7%), 134 (10%), 133 (100%), 105 (18%).
1
white crystals in 87% yield; mp 72-74 °C. H NMR (CDCl3):
2.42 (s, 3H), 2.56 (s, 3H), 3.94 (s, 3H), 7.05 (d, 1H, J ) 8), 7.08
(dt, 1H, J ) 1.5, 8), 7.13 (d, 1H, J ) 8.2), 7.37 (dd, 1H, J )
8.2, 2.3), 7.57 (ddd, 1H, J ) 8.4, 7.6, 1.9), 7.64 (d, 1H, J )
2.3), 8.10 (dd, 1H, J ) 7.7, 1.9). 13C NMR (CDCl3): 21.0, 30.1,
56.2, 112.4, 119.0, 120.6, 123.9, 130.6, 131.4, 132.8, 134.1,
134.7, 135.9, 147.5, 160.2, 164.5, 198.3. MS m/z (% rel int):
284 (M+, 2%), 136 (7%), 135 (100%), 92 (12%), 77 (18%).
1-(2-Hyd r oxy-5-m eth ylp h en yl)-3-(3,5-d im eth ylp h en yl)-
p r op a n e-1,3-d ion e (Sch em e 1; II Lea d in g to 9). The com-
pound was prepared according to the procedure described for
II leading to 3. The crude product was purified by chroma-
tography (heptane:ethyl acetate 12:1) to give yellow crystals
(yield 95%); mp 99-101 °C. Only the enol form was present
in CDCl3. 1H NMR (CDCl3): 2.37 (s, 3H), 2.42 (d, 6H, J ) 0.5),
1-(2-H yd r oxy-5-m et h ylp h en yl)-3-(2-m et h oxyp h en yl)-
p r op a n e-1,3-d ion e (Sch em e 1; II Lea d in g to 13 a n d 14).
The compound was prepared according to the procedure

Pharmacophore Model for Flavone Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4195
described for II leading to 3. The product was isolated in 76%
yield as yellow crystals, mp 102-104 °C, in CDCl3, as the enol
white crystals. The mother liquor was purified by chromatog-
raphy (heptane:ethyl acetate 6:1) to give the product in 85%
yield; mp 120-122 °C. 1H NMR (CDCl3): 2.46 (s, 3H), 3.89 (s,
3H), 6.79 (s, 1H), 7.06 (dd, 1H, J ) 8.3, 2.4), 7.48 (m, 5H),
8.01 (d, 1H, J ) 2.4). 13C NMR (CDCl3): 21.1, 55.6, 107.9,
111.9, 117.3, 118.0, 118.9, 123.8, 125.1, 130.3, 133.4, 135.2,
135.4, 154.7, 160.2, 163.2, 178.7. MS m/z (% rel int): 267 (18%),
266.0937 (M+, 100, C17H14O3 requires 266.0943), 265 (12%),
134 (30%). Anal. (C17H14O3) C, H.
1
isomer. H NMR (CDCl3): 2.34 (s, 3H), 4.00 (s, 3H), 6.92 (d,
1H, J ) 8.4), 7.04 (m, 2H), 7.23 (s, 1H), 7.28 (dd, 1H, J ) 8.4,
1.8), 7.51 (m, 2H), 7.80 (dd, 1H, J ) 7.8, 1.9), 11.98 (s, 1H),
15.61 (s, 1H). 13C NMR (CDCl3): 20.9, 56.1, 97.7, 112.0, 118.6,
119.1, 121.1, 122.9, 128.2, 128.6, 130.2, 133.3, 136.8, 158.7,
160.6, 175.4, 196.1. MS m/z (% rel int): 284 (M+, 21%), 235
(100%), 77 (18%).
2′-Meth oxy-6-m eth ylfla von e (13). The compound was
prepared according to the procedure described for 3. The crude
product was recrystallized from acetone to give white crystals
3′-Hyd r oxy-6-m eth ylfla von e (16). The compound was
prepared according to the procedure described for 13 leading
to 14. BBr3, 1.1 equiv, was added. The reaction time was 18 h
before addition of MeOH. Under the work up procedure, the
organic phase was removed and the water phase was acidified
with 2 M HCl(aq). New diethyl ether was added, and the
organic phase was washed with water. The organic phase was
dried and evaporated. White crystals were obtained in a yield
of 92%; mp 196-198 °C. 1H NMR (DMSO-d6): 2.41 (s, 3H),
6.86 (s, 1H), 7.00 (dd, 1H, J ) 8.1, 2.4), 7.36 (t, 1H, J ) 8),
7.41 (t, 1H, J ) 2), 7.48 (d, 1H, J ) 7.8), 7.62 (m, 2H), 7.81 (d,
1H, J ) 2.4), 9.89 (s, 1H). 13C NMR (DMSO-d6): 20.5, 106.8,
112.8, 117.1, 118.2, 118.8, 123.0, 124.1, 130.2, 132.5, 135.0,
135.3, 153.9, 157.9, 162.5, 177.0. MS m/z (% rel int): 253 (18%),
252.0795 (M+, 100, C16H12O3 requires 352.0786), 251 (14%),
224 (18%), 135 (15%), 134 (37%). Anal. (C16H12O3) C, H.
3′-Bu toxy-6-m eth ylfla von e (17). K2CO3 (134 mg, 0.97
mmol) followed by 1-brombutane (1.0 mL, 8.33 mmol) was
added to a solution of 16 (175 mg, 0.69 mmol) in DMF (6 mL)
at room temperature. After 48 h, the mixture was extracted
with diethyl ether and washed with brine, dried, and evapo-
rated. The crude product was purified by chromatography
(heptane:ethyl acetate 6:1) to give a yield of 88%; mp 87-90
°C. 1H NMR (CDCl3): 1.00 (t, 3H, J ) 7.3), 1.53 (sext., 2H,
J ) 7), 1.82 (quint. 2H, J ) 7), 2.47 (s, 3H), 4.04 (t, 2H, J )
6.5), 6.79 (s, 1H), 7.05 (dd, 1H, J ) 8.1, 2.1), 7.47 (m, 5H),
8.01 (d, 1H, J ) 2.1). 13C NMR (CDCl3): 14.2, 19.6, 21.2, 31.6,
68.4, 108.0, 112.7, 118.0, 118.3, 118.9, 123.6, 125.4, 130.4,
133.6, 135.4, 135.6, 154.9, 160.0, 163.6, 179.0. MS m/z (% rel
int): 308.1412 (M+, 100, C20H20O3 requires 308.1412), 308
(60%), 252 (100%), 224 (15%), 134 (21%). Anal. (C20H20O3)
C, H.
3′-(2-Eth ylbu tyloxy)-6-m eth ylfla von e (18). A solution of
2-ethyl-1-butanol (15 mg, 0.149 mmol) and triphenylphosphine
(39 mg, 0.149 mmol) in dry THF (5 mL) was added dropwise
to a solution of diethyl azodicarboxylate (26 mg, 0.149 mmol)
and 16 (25 mg, 0.099 mmol) in dry THF (1 mL) at room
temperature under N2. The mixture was stirred for 16 h before
20 mL of 1 M NaOH(aq) was added, and the mixture was
extracted with diethyl ether. The organic phase was dried and
evaporated. The crude product was purified by chromatogra-
phy (heptane:ethyl acetate 6:1), and the product was obtained
as white crystals in a yield of 45%; mp 93-95 °C. 1H NMR
(CDCl3): 0.97 (t, 6H, J ) 7.4), 1.52 (m, 4H), 1.73 (m, 1H), 2.48
(s, 3H), 3.94 (d, 2H, J ) 5.7), 6.82 (s, 1H), 7.08 (dd, 1H, J )
8.1, 2.2), 7.40 (d, 1H, J ) 7.8), 7.49 (m, 4H), 8.03 (d, 1H, J )
2.2). 13C NMR (CDCl3): 11.4, 21.1, 23.6, 41.1, 70.6, 107.8,
112.5, 117.9, 118.1, 118.7, 123.8, 125.3, 130.2, 133.4, 135.2,
135.4, 154.8, 160.0, 163.4, 178.8. MS m/z (% rel int): 336.1726
(M+, 37, C22H24O3 requires 336.1725), 253 (42%), 252 (100%),
224 (15%). Anal. (C22H24O3) C, H.
2-Meth oxy-5-m eth ylben za ld eh yd e (Lea d in g to 19 a n d
20). K2CO3 (2.84 g, 20.6 mmol) and MeI (1.28 mL, 20.6 mmol)
were added to a solution of 2-hydroxy-5-methylbenzaldehyde
(2.0 g, 14.7 mmol) in DMF (100 mL). After it was stirred for
20 h at room temperature, 100 mL of 0.5 M NaOH(aq) was
added to the solution and the reaction mixture was extracted
with diethyl ether and washed with brine, dried, and evapo-
rated. The product was obtained as a yellowish oil in 98% yield.
Spectroscopic data were identical with those previously re-
ported.35
1
in 96% yield; mp 108-110 °C. H NMR (CDCl3): 2.47 (s, 3H),
3.94 (s, 3H), 7.05 (d, 1H, J ) 8.4), 7.11 (m, 2H), 7.47 (m, 3H),
7.90 (dd, 1H, J ) 7.8, 1.7), 8.03 (bs, 1H). 13C NMR (CDCl3):
21.1, 55.9, 112.0, 112.7, 112.7, 118.0, 120.9, 121.2, 123.7, 125.2,
129.5, 132.5, 134.0, 155.0, 158.2, 160.9, 179.2. MS m/z (% rel
int): 266.0956 (M+, 80, C17H14O3 requires 266.0943), 135
(100%), 134 (33%), 132 (19%), 131 (26%), 78 (17%). Anal.
(C17H14O3) C, H.
2′-Hyd r oxy-6-m eth ylfla von e (14). BBr3, 3.6 mL (1 M in
CH2Cl2), was added to 13 (0.20 g, 0.75 mmol) dissolved in
freshly distilled CH2Cl2 (30 mL) at -78 °C and under inert
conditions (N2). After the addition, the cooling bath was
removed and a beige precipitate was formed. The reaction
mixture was left at room temperature for 48 h whereafter
MeOH (4 mL) was added slowly. The reaction mixture was
poured into a separatory funnel with 40 mL of 1 M NaOH(aq)
and washed four times with CH2Cl2 to remove starting
material. The mixture was acidified with concentrated HCl,
extracted with CH2Cl2, and washed with brine. After the
organic phase was dried and concentrated, the residue was
purified by flash chromatography (heptane:ethyl acetate 2:1,
pure ethyl acetate) to give the product as white crystals in a
yield of 63%; mp 264-266 °C. 1H NMR (CD3OD): 2.46 (s, 3H),
6.95 (d, 1H, J ) 8.3), 6.98 (ddd, 1H, J ) 8, 7, 1.1), 7.32 (ddd,
1H, J ) 8.3, 7.4, 1.7), 7.36 (s, 1H), 7.50 (d, 1H, J ) 8.6), 7.55
(dd, 1H, J ) 8.6, 2.0), 7.89 (dd, 1H, J ) 7.9, 1.7), 7.92 (d, 1H,
J ) 2.0). 13C NMR (CD3OD): 21.2, 111.9, 117.5, 118.7, 118.9,
120.3, 123.6, 125.1, 129.4, 133.2, 135.9, 136.0, 155.6, 157.5,
163.1, 181.0. MS m/z (% rel int): 253 (36%), 252.0786 (M+,
100, C16H12O3 requires 352.0790), 135 (27%), 134 (54%). Anal.
(C16H12O3) C, H.
2-Acetyl-4-m eth ylp h en yl 3-Meth oxyben soa te (Sch em e
1; I Lea d in g to 15-18). The compound was prepared accord-
ing to the procedure described for I leading to 7. The product
was purified by recrystallization in 80% MeOH(aq) to give
white crystals. The mother liquor was purified by chromatog-
raphy (heptane:ethyl acetate 3:1). The product was isolated
as white needles in 91% yield; mp 72-75 °C. 1H NMR
(CDCl3): 2.42 (s, 3H), 2.52 (s, 3H), 3.88 (s, 3H), 7.11 (d, 1H,
J ) 8.2), 7.19 (dt, 1H, J ) 1.5, 8), 7.39 (dd, 1H, J ) 8.2, 2.3),
7.65 (d, 1H, J ) 0.9), 7.70 (d, 1H, J ) 2.3), 7.81 (d, 1H, J )
8.2). 13C NMR (CDCl3): 21.2, 30.3, 55.9, 114.9, 119.6, 120.8,
123.1, 124.0, 130.1, 131.0, 133.6, 134.4, 136.4, 147.6, 160.1,
165.6, 199.4. MS m/z (% rel int): 284 (M+, 11%), 135 (100%),
107 (21%), 92 (16%), 77 (20%).
1-(2-H yd r oxy-5-m et h ylp h en yl)-3-(3-m et h oxyp h en yl)-
p r op a n e-1,3-d ion e (Sch em e 1; II Lea d in g to 15-18). The
compound was prepared according to the procedure described
for II leading to 3. The product was purified by chromatogra-
phy (heptane:ethyl acetate 10:1) to give yellow crystals in 85%
yield; mp 89-91 °C. In CDCl3, the product is a 9:1 enol:keto
mixture. Data are given for the major enol form. 1H NMR
(CDCl3): 2.35 (s, 3H), 3.90 (s, 3H), 6.82 (s, 1H), 6.92 (d, 1H,
J ) 8.5), 7.11 (dd, 1H, J ) 8.2, 2.3), 7.29 (dd, 1H, J ) 8.5,
2.3), 7.41 (t, 1H, J ) 8), 7.48 (dd, 1H, J ) 2.3, 1.9), 7.53 (m,
2H), 11.90 (s, 1H), 15.63 (s, 1H). 13C NMR (CDCl3): 20.8, 55.7,
92.8, 112.3, 118.3, 118.3, 118.8, 119.4, 128.4, 130.0, 135.3,
137.2, 137.2, 160.1, 160.6, 177.4, 195.8. MS m/z (% rel int):
284 (M+, 40%), 135 (100%), 107 (18%), 77 (23%).
2-Meth oxy-5-m eth ylben zoic Acid (Lea d in g to 19 a n d
20). A solution of 2-methoxy-5-methylbenzaldehyde (2.19 g,
14.6 mmol) in MeOH (125 mL) was warmed to 60 °C where-
after 2 M KOH(aq) (30 mL) followed by 35% H2O2(aq) (40 mL)
3′-Meth oxy-6-m eth ylfla von e (15). The compound was
prepared according to the procedure described for 3. The crude
product was purified with recrystallization from acetone to give

4196 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Kahnberg et al.
was added. Gas evolution was observed. After 2 h of reflux,
the solution was cooled and the solvent was evaporated. The
residue was extracted with diethyl ether. The aqueous phase
was acidified with diluted HCl(aq) and extracted with dichlo-
romethane, which was dried and evaporated to give 2-methoxy-
5-methylbenzoic acid as white crystals in 88% yield. The
spectroscopic data were identical with those previously re-
ported.36
2-Acetyl-4-m eth ylp h en yl 2-Meth oxy-5-m eth ylben soa te
(Sch em e 1; I Lea d in g to 19 a n d 20). The compound was
prepared according to the procedure described for I leading to
7. The crude product was purified by chromatography (hep-
tane:ethyl acetate 5:1-2:1) to give white crystals in 66% yield;
mp 74-76 °C. 1H NMR (CDCl3): 2.37 (s, 3H), 2.42 (s, 3H),
2.56 (s, 3H), 3.91 (s, 3H), 6.95 (d, 1H, J ) 8.5), 7.12 (d, 1H,
J ) 8.2), 7.36 (m, 2H), 7.64 (d, 1H, J ) 1.9), 7.89 (d, 1H, J )
2.4). 13C NMR (CDCl3): 20.5, 21.0, 30.1, 56.3, 112.5, 118.6,
123.9, 129.9, 130.5, 131.5, 132.9, 134.1, 135.3, 135.9, 147.5,
158.2, 164.7, 198.4. MS m/z (% rel int): 298.1207 (M+, 65,
C18H18O4 requires 298.1205), 150 (100%), 149 (100%), 106
(72%), 91 (100%), 78 (59%).
1-(2-H yd r oxy-5-m et h ylp h en yl)-3-(2-m et h oxy-5-m et h -
ylp h en yl)p r op a n e-1,3-d ion e (Sch em e 1; II Lea d in g to 19
a n d 20). The compound was prepared according to the
procedure described for II leading to 3. The crude product was
recrystallized from acetone to give yellow crystals. The yield
was 88%; mp 91-94 °C. In CDCl3, the product is a 2.5:1 enol:
keto mixture. Data are given for the major enol form. 1H NMR
(CDCl3): 2.33 (s, 3H), 2.37 (s, 3H), 3.97 (s, 3H), 6.91 (d, 1H,
J ) 8.4), 6.93 (d, 1H, J ) 8.4), 7.22 (s, 1H), 7.29 (m, 2H), 7.50
(brs, 1H), 7.76 (brs, 1H), 11.98 (s, 1H), 15.64 (s, 1H). 13C NMR
(CDCl3): 20.4, 20.6, 56.0, 97.5, 111.9, 118.4, 118.9, 122.3, 127.9,
128.4, 129.9, 130.2, 133.6, 136.5, 156.6, 160.3, 175.4, 195.8.
MS m/z (% rel int): 298.1208 (M+, 37, C18H18O4 requires
298.1205), 267 (10%), 150 (10%), 149 (100%), 135 (8%).
product was purified by flash chromatography (heptane:ethyl
acetate 5:1) to give white crystals in a yield of 79%; mp 104-
106 °C. 1H NMR (CDCl3): 2.42 (s, 3H), 2.54 (s, 3H), 3.95 (s,
3H), 7.12 (d, 1H, J ) 8.2), 7.40 (dd, 1H, J ) 8.2, 2.0), 7.62 (t,
1H, J ) 8), 7.68 (d, 1H, J ) 2.0), 8.31 (d, 1H, J ) 7.9), 8.39 (d,
1H, J ) 7.8), 8.85 (t, 1H, J ) 2). 13C NMR (CDCl3): 20.9, 29.5,
52.4, 123.6, 128.9, 129.9, 130.5, 130.8, 130.8, 131.9, 134.1,
134.6, 136.2, 146.9, 158.7, 164.6, 166.1, 197.4. MS m/z (% rel
int): 312 (M+, 9%), 163 (100%), 135 (16%).
Meth yl 3-[3-(2-Hyd r oxy-5-m eth ylp h en yl)-3-oxop r op a n -
oyl]ben zoa te (Sch em e 1; II Lea d in g to 21 a n d 22). The
compound was prepared according to the procedure described
for II leading to 3. The crude product was purified by
chromatography (heptane:ethyl acetate 3:1) to give yellow
crystals in a yield of 91%; mp 121-123 °C. Only the enol form
1
was present in CDCl3. H NMR (CDCl3): 2.37 (s, 3H), 3.99 (s,
3H), 6.88 (s, 1H), 6.93 (d, 1H, J ) 8.5), 7.31 (dd, 1H, J ) 8.5,
2.1), 7.58 (d, 1H, J ) 1.9), 7.59 (t, 1H, J ) 8), 8.16 (d, 1H, J )
7.9), 8.21 (d, 1H, J ) 7.8), 8.59 (t, 1H, J ) 2), 11.84 (s, 1H),
15.59 (s, 1H). 13C NMR (CDCl3): 21.0, 52.9, 93.1, 118.9, 119.0,
128.3, 128.7, 128.7, 129.5, 131.3, 131.5, 133.4, 134.6, 137.7,
160.9, 166.8, 176.3, 196.4. MS m/z (% rel int): 312 (M+, 43%),
163 (100%), 135 (48%), 134 (15%), 77 (20%).
Meth yl 3′-(6-Meth ylfla von e)ca r boxyla te (21). The com-
pound was prepared according to the procedure described for
3. The product was recrystallized from acetone to give white
1
crystals in a yield of 96%; mp 192-194 °C. H NMR (CDCl3):
2.48 (s, 3H), 3.99 (s, 3H), 6.87 (s, 1H), 7.52 (m, 2H), 7.61 (t,
1H, J ) 8), 8.02 (d, 1H, J ) 2.3), 8.10 (d, 1H, J ) 7.8), 8.20 (d,
1H, J ) 7.9), 8.60 (t, 1H, J ) 2). 13C NMR (CDCl3): 21.4, 52.9,
108.4, 118.4, 124.0, 125.5, 127.8, 129.7, 130.7, 131.6, 132.7,
132.8, 135.6, 135.9, 154.9, 162.5, 166.6, 178.8. MS m/z (% rel
int): 295 (19%), 294.0894 (M+, 100%, C18H14O4 requires
294.0892), 266 (29%), 263 (18%), 134 (43%), 106 (16%). Anal.
(C18H14O4) C, H.
2′-Meth oxy-5′,6-d im eth ylfla von e (19). The compound
was prepared according to the procedure described for 3. The
crude product was purified by chromatography (heptane:ethyl
acetate 3:1) and isolated in 98% yield as yellow crystals; mp
101-103 °C. 1H NMR (CDCl3): 2.38 (s, 3H), 2.46 (s, 3H), 3.90
(s, 3H), 6.93 (d, 1H, J ) 8.5), 7.13 (s, 1H), 7.27 (dd, 1H, J )
8.4, 2.3), 7.44 (d, 1H, J ) 8.4), 7.49 (dd, 1H, J ) 8.4, 2.1), 7.69
(d, 1H, J ) 2.1), 8.02 (d, 1H, J ) 2.3). 13C NMR (CDCl3): 20.7,
21.1, 55.9, 112.0, 112.6, 118.0, 120.7, 123.6, 125.1, 129.7, 130.2,
133.0, 134.9, 135.0, 155.0, 156.2, 161.1, 179.2. MS m/z (% rel
int): 281 (20%), 280.1093 (M+, 100, C18H16O3 requires 280.1099),
146 (34%), 145 (22%), 135 (100%). Anal. (C18H16O3) C, H.
3′-(6-Meth ylfla von e)ca r boxylic Acid (22). LiOH (30.7
mg, 1.28 mmol) was added to a solution of 21 (179 mg, 0.609
mmol) in a mixture of THF:H2O (1:1) (10 mL), and the reaction
was stirred at room temperature for 16 h. The solution was
acidified with 2 M HCl(aq), extracted with diethyl ether, and
washed with brine. The organic phase was dried and evapo-
rated. The crude product was purified by chromatography
(ethyl acetate) and isolated as white crystals in 67% yield; mp
249-252 °C. 1H NMR (CD3OD): 2.35 (s, 3H), 6.77 (s, 1H), 7.41
(d, 1H, J ) 8.5), 7.45 (dd, 1H, J ) 8.5, 2.0), 7.50 (t, 1H, J ) 8),
7.84 (d, 1H, J ) 2.0), 8.01 (dd, 1H, J ) 7.9, 1.9), 8.09 (dd, 1H,
J ) 7.8, 1.7), 8.50 (t, 1H, J ) 2). 13C NMR (CDCl3): 20.5, 107.1,
117.8, 122.9, 124.5, 124.5, 127.4, 129.0, 130.1, 131.7, 132.6,
135.4, 135.5, 154.4, 162.8, 167.5, 179.1. MS m/z (% rel int):
280.0734 (M+, 100%, C17H12O4 requires 280.0736), 252 (20%),
134 (41%). Anal. (C17H12O4) C, H.
3-(Isop r op oxyca r bon yl)ben zoic Acid (Lea d in g to 23).
The compound was prepared according to the procedure
described for 3-(methyl carboxy)benzoic acid from isophthalic
acid. i-Propanol was used instead of MeOH. After 44 h, the
mixture was washed with diethyl ether to remove the diester.
The water phase was acidified with 2 M HCl(aq) and extracted
with diethyl ether. The organic phase was washed with brine,
dried, and evaporated. The crude product was purified from
the diester and the starting material by chromatography
(toluene:acetone 7:3) as white crystals in a yield of 47%; mp
131-134 °C. 1H NMR (CDCl3): 2.82 (d, 6H, J ) 6.3), 5.31
(sept., 1H, J ) 6.3), 7.59 (t, 1H, J ) 8), 8.31 (dd, 2H, J ) 7.8,
1.7), 8.39 (dd, 2H, J ) 7.9, 1.9), 8.77 (t, 1H, J ) 2). 13C NMR
(CDCl3): 22.1, 69.3, 128.9, 129.8, 131.5, 131.8, 134.4, 134.9,
165.4, 171.6. MS m/z (% rel int): 208 (M+, 4%), 167 (40%),
149 (100%), 121 (17%), 65 (23%), 43 (16%).
2′-Hyd r oxy-5′,6-d im eth ylfla von e (20). The compound
was prepared according to the procedure described for 15
leading to 16. BBr3, 3 equiv, was added. The reaction time
was 20 h before addition of MeOH. The crude product was
purified by recrystallization from acetone to give white crystals
1
in a yield of 61%; mp 270-272 °C. H NMR (DMSO-d6): 2.29
(s, 3H), 2.43 (s, 3H), 6.96 (d, 1H, J ) 8.2), 7.11 (s, 1H), 7.20
(dd, 1H, J ) 8.2, 1.9), 7.63 (dd, 1H, J ) 8.5, 1.8), 7.67 (d, 1H,
J ) 8.5), 7.71 (d, 1H, J ) 1.9), 7.83 (d, 1H, J ) 1.8), 10.49 (bs,
1H). 13C NMR (DMSO-d6): 20.1, 20.5, 110.9, 117.0, 117.4,
118.3, 122.9, 124.0, 128.1, 128.4, 133.2, 134.8, 135.1, 154.2,
154.4, 160.7, 177.2. MS m/z (% rel int): 267 (22%), 266.0945
(M+, 100, C17H14O3 requires 266.0943), 135 (28%), 134 (21%),
132 (12%). Anal. (C17H14O3) C, H.
3-(Meth oxyca r bon yl)ben zoic Acid (Lea d in g to 21 a n d
22). Concentrated H2SO4 (0.5 mL) was added to a solution of
isophthalic acid (2.00 g, 12.0 mmol) in MeOH (15 mL) and THF
(45 mL). The mixture was heated to 65 °C. The reaction was
followed on TLC (CH2Cl2:MeOH:acetic acid 1:1:0.01) giving a
mixture of mono- and dimethylated acids. The solvents were
evaporated after 1.5 h, and the crude product was purified by
chromatography (pentane:diethyl ether 3:1 f 2:3) to give the
product as white crystals in 42% yield. Spectroscopic data were
identical with those previously reported.37
2-Acet yl-4-m et h ylp h en yl Isop r op yl Isop h t h a la t e
(Sch em e 1; I Lea d in g to 23). The compound was prepared
according to the procedure described for I leading to 7. The
crude product was purified by chromatography (heptane:ethyl
1
2-Acetyl-4-m eth ylph en yl Meth yl Isoph th alate (Sch em e
1; I Lea d in g to 21 a n d 22). The compound was prepared
according to the procedure described for I leading to 7. The
acetate 6:1) as a colorless oil in 60% yield. H NMR (CDCl3):
1.42 (d, 6H, J ) 6.3), 2.46 (s, 3H), 2.56 (s, 3H), 5.32 (sept., 1H,
J ) 6.3), 7.15 (d, 1H, J ) 8.2), 7.42 (dd, 1H, J ) 8.2, 1.9), 7.63

Pharmacophore Model for Flavone Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4197
(t, 1H, J ) 8), 7.69 (d, 1H, J ) 1.9), 8.34 (d, 1H, J ) 7.8), 8.40
(d, 1H, J ) 7.8), 8.86 (t, 1H, J ) 2). 13C NMR (CDCl3): 21.1,
22.1, 29.8, 69.2, 123.8, 128.4, 129.0, 129.2, 130.0, 130.8, 131.5,
131.9, 134.4, 134.8, 136.4, 147.2, 164.9, 165.3, 197.8. MS m/z
(% rel int): 340 (M+, 8%), 281 (15%), 191 (100%), 149 (92%),
121 (22%), 104 (25%), 76 (28%), 43 (15%).
2-Acetyl-4-m eth ylp h en yl Meth yl 5-Meth oxyisop h th a -
la te (Sch em e 1; I Lea d in g to 25). The compound was
prepared according to the procedure described for I leading to
7. The compound was purified by chromatography (heptane:
ethyl acetate 4:1) to give white crystals (yield 73%); mp 89-
91 °C. 1H NMR (CDCl3): 2.44 (s, 3H), 2.54 (s, 3H), 3.93 (s,
3H), 3.96 (s, 3H), 7.13 (d, 1H, J ) 8.2), 7.41 (dd, 1H, J ) 8.2,
2.2), 7.67 (d, 1H, J ) 2.2), 7.84 (d, 1H, J ) 2.7, 1.4), 7.90 (dd,
1H, J ) 2.7, 1.5), 8.46 (dd, 1H, J ) 1.5, 1.4). 13C NMR
(CDCl3): 21.1, 29.8, 52.7, 56.1, 120.0, 120.3, 123.7, 123.8, 130.8,
131.0, 131.3, 132.3, 134.3, 136.4, 147.2, 160.1, 164.7, 166.2,
197.7. MS m/z (% rel int): 342 (M+, 10%), 194 (11%), 193
(100%), 165 (13%), 150 (10%).
Meth yl 3-[3-(2-Hyd r oxy-5-m eth ylp h en yl)-3-oxop r op a n -
oyl]-5-m et h oxyb en zoa t e (Sch em e 1; II Lea d in g t o 25).
The compound was prepared according to the procedure
described for II leading to 3. The crude product was purified
by chromatography (heptane:ethyl acetate 6:1 f 4:1) to give
yellow crystals in a yield of 67%; mp 145-147 °C. Only the
enol form was present in CDCl3. 1H NMR (CDCl3): 2.37 (s,
3H), 3.94 (s, 3H), 3.99 (s, 3H), 6.87 (s, 1H), 6.93 (d, 1H, J )
8.5), 7.31 (dd, 1H, J ) 8.5, 2.1), 7.59 (d, 1H, J ) 2.1), 7.69 (dd,
1H, J ) 2.6, 1.6), 7.74 (dd, 1H, J ) 2.6, 1.4), 8.18 (dd, 1H, J )
1.6, 1.4), 11.81 (s, 1H), 15.62 (s, 1H). 13C NMR (CDCl3): 20.8,
52.8, 56.1, 93.1, 117.4, 118.0, 118.7, 118.8, 120.4, 128.5, 128.6,
132.2, 135.7, 137.5, 160.2, 160.7, 166.5, 176.0, 196.1. MS m/z
(% rel int): 342 (M+, 42%), 193 (100%), 135 (26%).
Isop r op yl 3-[3-(2-Hyd r oxy-5-m eth ylp h en yl)-3-oxop r o-
p a n oyl]ben zoa te (Sch em e 1; II Lea d in g to 23). The
compound was prepared according to the procedure described
for II leading to 3. Purification was made by chromatography
(heptane:ethyl acetate 6:1) to give red crystals in a yield of
84%; mp 88-90 °C. In CDCl3, the product is a 9:1 enol:keto
mixture. Data are given for the major enol form. 1H NMR
(CDCl3): 1.43 (d, 6H, J ) 6.3), 2.36 (s, 3H), 5.32 (sept., 1H,
J ) 6.3), 6.88 (s, 1H), 6.92 (d, 1H, J ) 8.4), 7.30 (d, 1H, J )
8.4), 7.56 (m, 2H), 8.14 (d, 1H, J ) 7.2), 8.21 (d, 1H, J ) 7.3),
8.59 (s, 1H), 11.80 (s, 1H), 15.62 (s, 1H). 13C NMR (CDCl3):
20.8, 22.1, 22.1, 69.3, 92.8, 118.7, 118.8, 127.9, 128.0, 128.5,
129.1, 131.0, 131.8, 133.0, 134.3, 137.4, 160.7, 165.6, 176.3,
196.1. MS m/z (% rel int): 340 (M+, 60%), 298 (17%), 281 (32%),
191 (48%), 149 (100%), 135 (52%), 134 (18%).
Isop r op yl 3′-(6-Meth ylfla von e)ca r boxyla te (23). The
compound was prepared according to the procedure described
for 3. The crude product was purified by chromatography
(heptane:ethyl acetate 6:1) to give white crystals in a yield of
81%; mp 147-149 °C. 1H NMR (CDCl3): 1.42 (d, 6H, J ) 6.3),
2.48 (s, 3H), 5.32 (sept. 1H, J ) 6.3), 6.88 (s, 1H), 7.50 (d, 1H,
J ) 8.3), 7.54 (dd, 1H, J ) 8.3, 1.9), 7.61 (t, 1H, J ) 8), 8.03
(d, 1H, J ) 1.9), 8.10 (dd, 1H, J ) 7.9, 1.9), 8.20 (dd, 1H, J )
7.8, 1.8), 8.58 (t, 1H, J ) 2). 13C NMR (CDCl3): 21.2, 22.1,
22.1, 69.3, 108.1, 108.2, 118.1, 123.8, 125.3, 127.5, 129.3, 130.3,
132.1, 132.4, 135.4, 135.6, 154.7, 162.4, 165.4, 178.7. MS m/z
(% rel int): m/z 323 (22%), 322.1206 (M+, 100, C20H18O4
requires 322.1205), 280 (33%), 263 (47%), 236 (18%), 134 (23%).
Anal. (C20H18O4) C, H.
Meth yl 3′-(5′-Meth oxy-6-m eth ylflavon e)car boxylate (25).
The compound was prepared according to the procedure
described for 3. The crude product was purified by chroma-
tography (heptane:ethyl acetate 1:1 f 1:3) to give white
1
crystals in a yield of 93%; mp 193-195 °C. H NMR (CDCl3):
2.49 (s, 3H), 3.95 (s, 3H), 3.99 (s, 3H), 6.86 (s, 1H), 7.50 (d,
1H, J ) 8.5), 7.52 (dd, 1H, J ) 8.5, 2.0), 7.60 (dd, 1H, J ) 2.3,
1.7), 7.70 (dd, 1H, J ) 2.5, 1.3), 8.01 (d, 1H, J ) 2.0), 8.17 (dd,
1H, J ) 1.7, 1.3). 13C NMR (CDCl3): 21.2, 52.8, 56.0, 108.4,
117.0, 117.1, 118.2, 120.0, 123.9, 125.3, 132.6, 133.8, 135.4,
135.7, 154.7, 160.3, 162.2, 166.3, 178.6. MS m/z (% rel int):
324.1004 (M+, 100, C19H16O5 requires 324.0998), 325 (21%),
293 (18%), 134 (27%). Anal. (C19H16O5) C, H.
2-Eth ylbu tyl 3′-(6-Meth ylfla von e)ca r boxyla te (24). The
compound was prepared according to the procedure described
for 18, from 22. The crude product was purified twice by
chromatography (heptane:ethyl acetate 4:1) to give white
crystals in a yield of 58%; mp 82-84 °C. 1H NMR (CDCl3):
0.99 (t, 6H, J ) 7.4), 1.50 (m, 4H), 1.73 (m, 1H), 2.49 (s, 3H),
4.33 (d, 2H, J ) 5.8) 6.87 (s, 1H), 7.50 (d, 1H, J ) 8.5), 7.55
(dd, 1H, J ) 8.5, 2.1), 7.63 (t, 1H, J ) 8), 8.04 (d, 1H, J ) 2.1),
8.11 (dd, 1H, J ) 7.9, 1.9), 8.21 (dd, 1H, J ) 7.8, 1.8), 8.61 (t,
1H, J ) 2). 13C NMR (CDCl3): 11.4, 11.4, 21.2, 23.8, 23.8, 40.7,
67.8, 108.1, 118.1, 123.8, 125.3, 127.6, 129.4, 130.4, 131.8,
132.5, 132.6, 135.4, 135.7, 154.7, 162.4, 166.1, 178. MS m/z
(% rel int): 364.1682 (M+, 71, C23H24O4 requires 364.1675),
281 (76%), 280 (100%), 263 (75%). Anal. (C23H24O4) C, H.
Meth yl 3′-(5′-Hydr oxy-6-m eth ylflavon e)car boxylate (26).
The compound was prepared according to the procedure
described for 14. BBr3, 2 equiv, was added and after 2 h, an
additional 2 equiv of BBr3 were added. After 6 more hours,
the reaction was quenched with 15 mL of water. The compound
was purified by chromatography (toluene:acetone 9:1) to
give white crystals (yield 52%); mp 255-257 °C. 1H NMR
(CDCl3 + 10%CD3OD): 2.39 (s, 3H), 3.87 (s, 3H), 6.74 (s, 1H)
7.47 (m, 3H), 7.54 (s, 1H), 7.88 (s, 1H), 8.02 (s, 1H). 13C NMR
(CDCl3 + 10%CD3OD): 20.9, 52.5, 107.3, 117.5, 118.1, 118.6,
119.7, 123.3, 124.8, 132.2, 133.1, 135.7, 135.8, 154.7, 158.0,
163.1, 166.7, 179.5. MS m/z (% rel int): 310.0843 (M+, 100,
Dim eth yl 5-Meth oxyisoph th alate (Leadin g to 25). 5-Hy-
droxy-isophthalic acid (2 g, 10.98 mmol), methyl iodide (7.79
g, 54.9 mmol), and potassium carbonate (6.07 g, 43.9 mmol)
were dissolved in 100 mL of N,N-dimethylformamide. The
mixture was heated to 40 °C, and after 7 h, more methyl iodide
(4.68 g, 32.9 mmol) was added. After it was stirred for 14 h,
200 mL of water was added and the mixture was extracted
three times with 100 mL of diethyl ether. The organic phase
was washed with 50 mL of 0.1 M NaOH(aq) followed by 50
mL of water, dried, and evaporated to give white crystals (yield
92%). Spectroscopic data were identical with those previously
reported.38
C
18H14O5 requires 310.0841), 282 (21%), 279 (19%), 134 (38%).
Anal. (C18H14O5) C, H.
3′-(5′-Meth oxy-6-m eth ylfla von e)ca r boxylic Acid (27).
The compound was prepared from 25 according to the proce-
dure described for 22. The crude product was purified by
chromatography (ethyl acetate with 1% acetic acid) to give
white-yellow crystals (yield 46%); mp 250-252 °C. 1H NMR
(CD3OD): 2.46 (s, 3H), 3.91 (s, 3H), 6.87 (s, 1H), 7.61 (m, 3H),
7.72 (s, 1H), 7.89 (s, 1H), 8.19 (s, 1H). 13C NMR (CD3OD): 21.1,
56.3, 108.1, 115.7, 119.0, 119.5, 121.0, 124.4, 125.6, 125.7,
133.8, 137.1, 137.3, 156.2, 161.7, 165.1, 165.4, 180.7. MS m/z
(% rel int): 310.0840 (M+, 100, C18H14O5 requires 310.0841),
282 (28%), 134 (51%), 106 (22%), 105 (20%), 78 (22%). Anal.
(C18H14O5) C, H.
3′-(5′-Hyd r oxy-6-m eth ylfla von e)ca r boxylic Acid (28).
The compound was prepared from 26 according to the proce-
dure described for 22. The crude product was purified by flash
chromatography (heptane:ethyl acetate 1:1, with 1% acetic
acid) to give pure product (yield 94%); mp 320-322 °C. 1H
NMR (DMSO-d6): 2.44 (s, 3H), 6.92 (s, 1H), 7.55 (dd, 1H, J )
2.1, 1.6), 7.66 (m, 2H), 7.71 (d, 1H, J ) 8.5), 7.85 (s, 1H), 8.0
(t, 1H, J ) 2). 13C NMR (DMSO-d6): 20.4, 107.3, 117.2, 117.7,
3-Meth oxy-5-(m eth oxyca r bon yl)ben zoic Acid (Lea d -
in g to 25). The compound was prepared according to the
procedure described for 22. The reaction was stirred at room
temperature for 2.5 h until TLC showed that diacid was
formed. The product was purified by chromatography (heptane:
ethyl acetate 4:1 with 1% acetic acid) and obtained as an oil
(yield 33%, 62% starting material was recovered). 1H NMR
(CDCl3 + 5%CD3OD): 3.86 (s, 3H), 3.91 (s, 3H), 7.72 (dd, 1H,
J ) 2.7, 1.5), 7.74 (dd, 1H, J ) 2.7, 1.5), 8.27 (dd, 1H, J ) 1.5,
1.5). 13C NMR (CDCl3 + 5%CD3OD): 52.5, 55.9, 119.5, 119.7,
123.5, 123.5, 131.8, 159.8, 166.6, 167.9. MS m/z (% rel int):
194 (M+, 5%), 149 (100%), 191 (33%).

4198 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Kahnberg et al.
118.3, 118.9, 123.0, 124.1, 132.9, 133.0, 135.2, 135.4, 153.9,
158.1, 161.7, 166.6, 177.0. MS m/z (% rel int): 296.0685 (M+,
100, C17H12O5 requires 296.0685), 297 (20%), 268 (22%), 134
(38%). Anal. (C17H12O5) C, H.
108.4, 118.2, 123.5, 125.1, 131.3, 131.3, 131.8, 133.0, 133.1,
135.7, 135.7, 136.0, 154.7, 161.5, 165.6, 165.6, 178.8. MS m/z
(% rel int): 352.0946 (M+, 100, C20H16O6 requires 352.0947),
324 (18%) 321 (23%), 134 (35%). Anal. (C20H16O6) C, H.
5′-Hydr oxym eth yl-3′-m eth oxy-6-m eth ylfla von e (29). Li-
AlH4 (9.83 mg, 0.26 mmol) was suspended in 5 mL of
anhydrous THF and cooled to 0 °C. 5′-Methoxy-6-methyl-3′-
methylcarboxyflavone (84 mg, 0.26 mmol) dissolved in 15 mL
of anhydrous THF was added dropwise to the suspension, and
the mixture was allowed to reach room temperature. After 4
and 15 h, more LiAlH4 (9.8 mg, 0.26 mmol and 20 mg, 0.53
mmol) was added. After the last addition of LiAlH4, the
solution was stirred for 4 h, and then, 10 mL of 0.1 M NaOH-
(aq) was added and the mixture was extracted four times with
30 mL of diethyl ether. The organic phase was washed with
20 mL of 0.1 M hydrochloric acid(aq). The compound was
purified by chromatography (heptane:ethyl acetate 1:1) to give
white-yellow crystals (yield 27%); mp 170-172 °C. 1H NMR
(CDCl3 + 10%CD3OD): 2.43 (s, 3H), 2.89 (bs, 1H), 3.84 (s, 3H),
4.67 (s, 2H), 6.74 (s, 1H), 7.04 (dd, 1H, J ) 2.4, 1.6), 7.28 (d,
1H, J ) 2.5), 7.43 (d, 1H, J ) 8.6), 7.46 (dd, 1H, J ) 1.6, 1.4),
7.49 (dd, 1H, J ) 8.6, 2.5), 7.92 (dd, 1H, J ) 2.4, 1.4). 13C NMR
(CDCl3 + 10%CD3OD): 21.0, 55.6, 64.3, 107.4, 111.0, 115.5,
117.2, 118.0, 123.5, 125.0, 133.1, 135.4, 135.6, 144.2, 154.7,
160.3, 163.7, 179.3. MS m/z (% rel int): 296.1051 (M+, 100,
3′,5′-(6-Meth ylfla von e)d ica r boxylic Acid (31). The com-
pound was prepared from 30 according to the procedure
described for 22. The product was purified by chromatography
(THF:CH2Cl2 2:3, with 1% acetic acid) to give white crystals
(yield 80%); mp 332-334 °C. 1H NMR (DMSO-d6): 2.45 (s, 3H),
3.33 (bs, 2H), 7.11 (s, 1H), 7.68 (dd, 1H, J ) 8.6, 1.9), 7.78 (d,
1H, J ) 8.6), 7.87 (d, 1H, J ) 1.9), 8.62 (t, 1H, J ) 1.5), 8.72
(d, 2H, J ) 1.5). 13C NMR (DMSO-d6): 20.9, 108.6, 118.9,
123.5, 124.6, 124.6, 130.9, 131.1, 131.1, 132.8, 133.0, 135.8,
136.0, 154.5, 161.3, 166.4, 166.4, 177.4. MS m/z (% rel int):
324.0632 (M+, 100, C18H12O6 requires 324.0634), 325 (21%),
296 (31%), 134 (59%). Anal. (C18H12O6) C, H.
Meth yl 3′-(5′-Hyd r oxym eth yl-6-m eth ylfla von e)ca r box-
yla te (32) a n d 3′,5′-Bis(h yd r oxym eth yl)-6-m eth ylfla von e
(33). The compounds were prepared from 30 according to the
procedure described for 29. The compounds were separated
and purified by chromatography (heptane:ethyl acetate 1:9)
to give light yellow crystals yield (22% monool and 19% diol).
1
32: mp 202-204 °C. H NMR (CDCl3 + 5%CD3OD): 2.42 (s,
3H), 3.44 (bs, 1H), 3.92 (s, 3H), 4.73 (s, 2H), 6.83 (s, 1H), 7.51
(m, 2H), 7.92 (d, 1H, J ) 1.9), 8.10 (m, 2H), 8.44 (t, 1H, J )
1.7). 13C NMR (CDCl3 + 10%CD3OD): 20.9, 52.5, 63.5, 107.7,
118.1, 123.4, 125.0, 126.3, 128.8, 130.7, 131.3, 132.4, 135.7,
135.8, 143.4, 154.7, 162.8, 166.6, 179.2. MS m/z (% rel int):
324.1000 (M+, 100, C19H16O5 requires 324.0998), 325 (21%),
293 (18%), 135 (16%), 134 (49%). Anal. (C19H16O5) C, H. 33:
C
18H16O4 requires 296.1049), 297 (20%), 135 (16%), 134 (30%).
Anal. (C18H16O4) C, H.
Tr im eth yl 1,3,5-Ben zen etr ica r boxyla te (Lea d in g to
30). The compound was prepared from 1,3,5-tribenzoic acid
according to the procedure described for 5-methoxy-1,3-dimeth-
ylbenzdioat. The product was isolated as white crystals (yield
84%). Spectroscopic data were identical with those previously
reported.39
3,5-Bis(m eth oxyca r bon yl)ben zoic Acid (Lea d in g to
30). The compound was prepared according to the procedure
described for 22. The reaction was stirred at room temperature
for 2.5 h until TLC indicated that the triacid had been formed.
The product was purified by chromatography (heptane:ethyl
acetate, 2:1, with 1% acetic acid) to give white crystals (yield
49% + 46% starting material). Spectroscopic data were identi-
cal with those previously reported.40
1
mp 203-205 °C. H NMR (CDCl3 + 5%CD3OD): 2.41 (s, 3H),
3.50 (bs, 2H), 4.66 (s, 4H), 6.76 (s, 1H), 7.47 (m, 3H), 7.77 (bs,
2H), 7.91 (m, 1H). 13C NMR (CDCl3 + 10%CD3OD): 20.9, 64.1,
64.1, 107.1, 118.0, 123.4, 123.7, 123.7, 124.9, 128.6, 131.9,
135.5, 135.6 142.8, 142.8, 154.8, 164.1, 179.5. MS m/z (% rel
int): 296.1041 (M+, 100, C18H16O4 requires 296.1049), 294
(27%), 135 (41%), 134 (62%). Anal. (C18H16O4) C, H.
3-(Meth oxyca r bon yl)-5-n itr oben zoic Acid (Lea d in g to
34 a n d 35). The compound was prepared according to the
procedure described for isophthalic acid leading to 21. The
product was purified by chromatography (pentane:diethyl
ether 3:1 f 2:3) to give the product in 29% yield; mp 167-
1-(2-Acet yl-4-m et h ylp h en yl) 3,5-Dim et h yl Ben zen e-
1,3,5-tr ica r boxyla te (Sch em e 1; I Lea d in g to 30). The
compound was prepared according to the procedure described
for I leading to 7. The crude product was purified by chroma-
tography (heptane:ethyl acetate 3:1) to give white crystals
1
169 °C. H NMR (CD3OD): 4.00 (s, 3H), 8.76 (s, 1H), 8.91 (s,
1H), 8.92 (s, 1H). 13C NMR (CD3OD): 52.5, 127.6, 128.0, 132.7,
133.7, 135.5, 148.9, 164.7, 165.4. MS m/z (% rel int): 225 (M+,
21%), 195 (34%), 194 (100%), 148 (31%), 120 (24%), 75 (31%),
74 (21%).
1
(yield 58%); mp 130-132 °C. H NMR (CDCl3): 2.45 (s, 3H),
2.54 (s, 3H), 4.00 (s, 6H), 7.13 (d, 1H, J ) 8.2), 7.41 (dd, 1H,
J ) 8.2, 1.8,), 7.68 (d, 1H, J ) 1.8), 8.94 (t, 1H, J ) 1.6), 9.02
(d, 2H, J ) 1.6). 13C NMR (CDCl3): 21.1, 29.5, 52.9, 52.9, 123.7,
130.4, 130.9, 131.2, 131.2, 131.7, 134.4, 135.4, 135.4, 135.4,
136.6, 147.0, 164.1, 165.5, 165.5, 197.6. MS m/z (% rel int):
370 (M+, 10%), 339 (7%), 222 (12%), 221 (100%), 193 (9%).
2-Acetyl-4-m eth ylp h en yl Meth yl 5-Nitr oisop h th a la te
(Sch em e 1; I Lea d in g to 34 a n d 35). The compound was
prepared according to the procedure described for I leading to
7. The product was purified by flash chromatography (heptane:
ethyl acetate 5:1) to give white crystals in a yield of 59%; mp
112-114 °C. 1H NMR (CDCl3): 2.44 (s, 3H), 2.53 (s, 3H), 4.01
(s, 3H), 7.13 (d, 1H, J ) 8.2), 7.42 (dd, 1H, J ) 8.2, 2.2), 7.70
(d, 1H, J ) 2.2), 9.07 (dd, 1H, J ) 2.2, 1.6), 9.11 (dd, 1H, J )
1.6, 1.6), 9.16 (dd, 1H, J ) 2.2, 1.6). 13C NMR (CDCl3): 21.0,
29.0, 53.2, 123.7, 128.7, 128.9, 129.5, 131.5, 132.2, 132.8, 134.6,
136.5, 136.8, 146.6, 148.6, 162.9, 164.2, 197.4. MS m/z (% rel
int): 357 (M+, 20%), 208 (100%), 162 (22%), 134 (21%), 75
(15%).
Dim eth yl 5-[3-(2-Hyd r oxy-5-m eth ylp h en yl)-3-oxop r o-
p a n oyl]isop h th a la te (Sch em e 1; II Lea d in g to 30). The
compound was prepared according to the procedure described
for II leading to 3. The crude product was purified by
chromatography (heptane:ethyl acetate 4:1) to give yellow
crystals (yield 59%); mp 182-184 °C. Only the enol form was
present in CDCl3. 1H NMR (CDCl3): 2.38 (s, 3H), 4.02 (s, 6H),
6.90 (s, 1H), 6.92 (d, 1H, J ) 8.5), 7.31 (dd, 1H, J ) 8.5, 2.1),
7.59 (d, 1H, J ) 2.1), 8.75 (d, 2H, J ) 1.5), 8.82 (t, 1H, J )
1.5), 11.76 (s, 1H), 15.60 (s, 1H). 13C NMR (CDCl3): 20.7, 52.9,
52.9, 93.4, 118.6, 118.9, 128.6, 128.7, 131.7, 131.9, 131.9, 133.8,
135.0, 135.0, 137.7, 160.8, 165.8, 165.8, 174.7, 196.4. MS m/z
(% rel int): 370 (M+, 46%), 221 (100%), 193 (15%), 135 (27%),
134 (22%).
Dim eth yl 3′,5′-(6-Meth ylflavon e)dicar boxylate (30). The
compound was prepared according to the procedure described
for 3. The crude product was recrystallized from acetone to
give white crystals (yield 85%); mp 204-206 °C. 1H NMR
(CDCl3 + 10%CD3OD): 2.50 (s, 3H), 4.03 (s, 6H), 6.95 (s, 1H),
7.57 (m, 2H), 8.04 (bs, 1H), 8.78 (d, 2H, J ) 1.5), 8.83 (t, 1H,
J ) 1.5). 13C NMR (CDCl3 + 10%CD3OD): 21.0, 52.9, 52.9,
Meth yl 3-[3-(2-Hyd r oxy-5-m eth ylp h en yl)-3-oxop r op a n -
oyl]-5-n itr oben zoa te (Sch em e 1; II Lea d in g to 34 a n d 35).
The compound was prepared according to the procedure
described for II leading to 3 and purified by recrystallization
from EtOH two times to give yellow needles in a yield of 64%;
mp 173-175 °C. In DMSO, the product is a 9:1 enol:keto
mixture. Data are given for the major enol form. 1H NMR
(DMSO-d6): 2.32 (s, 3H), 3.96 (3H), 7.08 (d, 1H, J ) 8.3), 7.46
(dd, 1H, J ) 8.3, 2.1), 7.61 (s, 1H), 8.04 (d, 1H, J ) 2.1), 8.62
(brs, 1H), 8.67 (bs, 1H), 8.71 (bs, 1H), 11.97 (s, 1H), 15.61 (s,
1H). 13C NMR (DMSO-d6): 20.0, 53.0, 101.1, 118.4, 120.3,
123.9, 124.9, 125.4, 130.9, 131.4, 132.2, 137.0, 145.6, 148.0,
148.0, 155.7, 164.4, 190.4. MS m/z (% rel int): 358 (17%), 357

Pharmacophore Model for Flavone Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4199
(M+, 84%), 340 (23%), 208 (84%), 177 (18%), 162 (20%), 135
(72%), 134 (100%), 106 (16%), 77 (19%).
J ) 271), 123.8 (quart, J ) 4), 128.5, 128.6, 128.8 (quart, J )
4), 129.6, 130.1, 131.6 (quart, J ) 33), 134.9, 137.6, 160.8,
175.3, 196.3. MS m/z (% rel int): 322 (M+, 72%), 173 (100%),
145 (30%), 135 (30%), 134 (35%).
Meth yl 3′-(6-Meth yl-5′-n itr ofla von e)ca r boxyla te (34).
The compound was prepared according to the procedure
described for 3. The product was recrystallized from acetone
to give white crystals in a yield of 95%; mp 210-212 °C. 1H
NMR (CD3OD): 2.28 (s, 3H), 3.85 (s, 3H), 6.79 (s, 1H), 7.40
(d, 1H, J ) 8.6), 7.42 (dd, 1H, J ) 8.6, 2.0), 7.75 (d, 1H, J )
2.0), 8.68 (t, 1H, J ) 2), 8.75 (t, 1H, J ) 2), 8.77 (t, 1H, J ) 2).
13C NMR (CD3OD): 20.5, 52.9, 108.5, 118.0, 123.0, 124.6,
124.7, 126.4, 132.2, 132.8, 134.0, 136.0, 136.2, 148.8, 154.4,
159.9, 164.2, 178.7. MS m/z (% rel int): 340 (22%), 339.0749
(M+, 100, C18H13N1O6 requires 339.0743), 311 (29%), 134 (45%),
106 (25%), 105 (16%), 78 (22%). Anal. (C18H13NO6) C, H, N.
3′-(6-Meth yl-5′-n itr ofla von e)ca r boxylic Acid (35). The
compound was prepared according to the procedure described
for 22. The crystals that precipitated by acidification were
filtered off, washed with water, and dried. The product was
obtained as white crystals in a yield of 94%; mp 310-313 °C.
1H NMR (DMSO-d6): 2.44 (s, 3H), 7.24 (s, 1H), 7.65 (dd, 1H,
J ) 8.5, 2.0), 7.75 (d, 1H, J ) 8.5), 7.83 (brs, 1H), 8.73 (brs,
1H), 8.82 (brs, 1H), 8.96 (brs, 1H). 13C NMR (DMSO-d6): 20.5,
108.9, 118.6, 123.1, 124.1, 124.7, 126.0, 132.3, 133.7, 134.0,
135.6, 135.8, 148.7, 154.0, 159.4, 165.0, 177.1. MS m/z (% rel
int): 325.0594 (M+, 100, C17H11NO6 requires 325.0586), 297
(15%), 134 (30%). Anal. (C17H11NO6) C, H, N.
Meth yl 3′-(5′-Am in o-6-m eth ylfla von e)ca r boxyla te (36)
a n d Eth yl 3′-(5′-Am in o-6-m eth ylfla von e)ca r boxyla te (37).
The compounds were prepared according to the procedure
described for 10. The compounds were separated and purified
by chromatography on silica gel, with petroleum ether:ethyl
acetate (1:1) and with toluene:acetone (2:1) to give both the
methyl ester 36 (33%) and the ethyl ester 37 (23%). Compound
36 was obtained as an oil. 1H NMR (CDCl3): 2.48 (s, 3H), 3.96
(s, 3H), 4.01 (bs, 2H), 6.81 (s, 1H), 7.38 (dd, 1H, J ) 1.8, 1.8),
7.50 (m, 2H), 7.53 (dd, 1H, J ) 8.6, 2.0), 7.97 (dd, 1H, J ) 1.8,
1.5), 8.02 (brs, 1H). 13C NMR (CDCl3): 21.2, 52.6, 108.1, 116.3,
117.8, 118.1, 118.7, 123.9, 125.3, 132.3, 133.5, 135.3, 135.6,
147.4, 154.7, 162.8, 166.7, 178.7. MS (70 eV): m/z 310 (21%),
309.0995 (M+, 100, C18H15NO4 requires 309.1001), 278 (12%),
175 (13%). Anal. (C18H15NO4) C, H, N. Compound 37 was
obtained as an oil. 1H NMR (CDCl3): 1.42 (t, 3H, J ) 7.0),
2.48 (s, 3H), 4.03 (bs, 2H), 4.42 (q, 2H, J ) 7.0), 6.80 (s, 1H),
7.36 (dd, 1H, J ) 1.8, 1.8), 7.49 (m, 2H), 7.52 (dd, 1H, J ) 8.6,
2.0), 7.97 (dd, 1H, J ) 1.8, 1.5), 8.03 (brs, 1H). 13C NMR
(CDCl3): 14.6, 21.2, 61.6, 108.1, 116.2, 117.7, 118.1, 118.7,
123.9, 125.3, 132.7, 133.5, 135.3, 135.6, 147.3, 154.7, 162.8,
166.2, 178.8. MS (70 eV): m/z 324 (22%), 323.1145 (M+, 100,
3′-Tr iflu or om eth yl-6-m eth ylfla von e (38). The compound
was prepared according to the procedure described for 3. The
compound was purified by flash chromatography (heptane:
ethyl acetate 8:1) to give light-yellow crystals (yield 87%); mp
147-149 °C. 1H NMR (CDCl3): 2.51 (s, 3H), 6.85 (s, 1H), 7.53
(m, 2H), 7.67 (dd, 1H, J ) 7.9, 7.8), 7.80 (d, 1H, J ) 7.8), 8.02
(brs, 1H), 8.09 (d, 1H, J ) 7.9), 8.19 (bs, 1H). 13C NMR
(CDCl3): 21.2, 108.5, 118.1, 123.3 (quart J ) 4), 123.9, 123.9
(quart J ) 271), 125.4, 128.2 (quart J ) 4), 129.6, 129.9, 132.0
(quart J ) 33), 133.2, 135.5, 135.8, 154.7, 161.7, 178.5. MS
m/z (% rel int): 304.0711(M+, 100, C17F3H11O2 requires
304.0711), 305 (18%), 276 (29%), 134 (52%). Anal. (C17F3H11O2)
C, H.
2-Acetyl-4-m eth ylp h en yl 3-Br om oben soa te (Sch em e 1;
I Lea d in g to 39). The compound was prepared according to
the procedure described for I leading to 3. The acid chloride
was (purchased from Aldrich) commercially available. The
product was purified by chromatography (heptane:ethyl ac-
etate 6:1) to give white crystals (yield 89%); mp 71-73 °C.1H
NMR (CDCl3): 2.44 (s, 3H), 2.54 (s, 3H), 7.11 (d, 1H, J ) 8.2),
7.41 (m, 2H), 7.67 (d, 1H, J ) 2.2), 7.78 (ddd, 1H, J ) 8.0, 2.0,
1.0), 8.15 (ddd, 1H, J ) 7.8, 1.7, 1.0), 8.35 (dd, 1H, J ) 2.0,
1.7). 13C NMR (CDCl3): 21.1, 29.7, 122.9, 123.7, 129.0, 130.4,
130.7, 131.0, 131.6, 133.4, 134.3, 136.4, 136.8, 147.1, 164.3,
197.7. MS m/z (% rel int): 332 (M+, 20%), 334 (20%), 185 (96%),
183 (100%), 157 (21%), 155 (21%).
1-(2-Hyd r oxy-5-m eth ylp h en yl)-3-(3-br om op h en yl)p r o-
p a n e-1,3-d ion e (Sch em e 1; II Lea d in g to 39). The com-
pound was prepared according to the procedure described for
II leading to 3. The crude product was purified by chroma-
tography (heptane:ethyl acetate 12:1) to give yellow crystals
(yield 69%); mp 122-124 °C. The product was present as the
enol form in CDCl3. 1H NMR (CDCl3): 2.37 (s, 3H), 6.80 (s,
1H), 6.93 (d, 1H, J ) 8.5), 7.32 (dd, 1H, J ) 8.5, 2.2), 7.39 (t,
1H, J ) 8), 7.56 (d, 1H, J ) 2.2), 7.69 (ddd, 1H, J ) 8.0, 2.0,
1.1), 7.89 (ddd, 1H, J ) 7.9, 1.7, 1.1), 8.09 (dd, 1H, J ) 2.0,
1.7), 11.83 (s, 1H), 15.53 (s, 1H). 13C NMR (CDCl3): 20.8, 93.0,
118.7, 118.9, 123.2, 125.6, 125.7, 128.5, 129.9, 130.5, 135.3,
136.0, 137.5, 160.7, 175.5, 196.1. MS m/z (% rel int): 332 (M+,
66%), 334 (66%), 185 (98%), 183 (100%), 135 (51%), 134 (38%).
3′-Br om o-6-m eth ylfla von e (39). The compound was pre-
pared according to the procedure described for 3. The crude
product was purified by chromatography (heptane:ethyl ac-
etate 5:1) to give white crystals (yield 75%). Spectroscopic data
were identical with those previously reported.25 Anal. (C16H11
BrO2) C, H.
-
C
19H17NO4 requires 323.1158), 309 (26%), 278 (19%), 251
(24%), 250 (17%). Anal. (C19H17NO4) C, H, N.
2-Acetyl-4-m eth ylp h en yl 3-(Tr iflu or om eth yl)ben soa te
(Sch em e 1; I Lea d in g to 38). The compound was prepared
according to the procedure described for I leading to 3. The
acid chloride was commercially available. The crude product
was purified by chromatography (heptane:ethyl acetate 5:1)
to give white crystals (yield 94%); mp 71-73 °C.1H NMR
(CDCl3): 2.45 (s, 3H), 2.55 (s, 3H), 7.13 (d, 1H, J ) 8.2), 7.42
(dd, 1H, J ) 8.2, 2.2), 7.68 (m, 2H), 7.91 (d, 1H, J ) 7.9), 8.41
(d, 1H, J ) 7.9), 8.48 (brs, 1H). 13C NMR (CDCl3): 21.1, 29.5,
123.8, 123.8 (quart, J ) 271), 127.4 (quart, J ) 4), 129.6, 130.3
(quart, J ) 4), 130.7, 131.1, 131.6 (quart, J ) 33), 133.7, 134.4,
134.4, 136.5, 147.1, 164.4, 197.6. MS m/z (% rel int): 322 (M+,
22%), 173 (100%), 145 (30%), 135 (9%).
2-Acet yl-4-m et h ylp h en yl (5-Br om o-2-m et h oxy)b en -
soa te (Sch em e 1; I Lea d in g to 40 a n d 41). The compound
was prepared according to the procedure described for I leading
to 7. The crystals were filtered off, rinsed with water and dried,
and obtained as white crystals in 88% yield; mp 110-112 °C.
1H NMR (CDCl3): 2.42 (s, 3H), 2.55 (s, 3H), 3.92 (s, 3H), 6.94
(d, 1H, J ) 8.9), 7.11 (d, 1H, J ) 8.2), 7.38 (dd, 1H, J ) 8.2,
2.2), 7.65 (m, 2H), 8.21 (d, 1H, J ) 2.2). 13C NMR (CDCl3):
21.1, 29.8, 56.6, 112.6, 114.3, 120.8, 123.9, 130.8, 131.0, 134.2,
135.1, 136.2, 137.2, 147.1, 159.2, 163.3, 198.0. MS m/z (% rel
int): 364 (5%), 362.0154 (M+, 5, C17H15Br1O4 requires 362.0154),
216 (7%), 215 (98%), 214 (7%), 213 (100%), 172 (5%), 170 (5%).
1-(2-Hyd r oxy-5-m eth ylp h en yl)-3-(5-br om o-2-m eth oxy-
p h en yl)p r op a n e-1,3-d ion e (Sch em e 1; II Lea d in g to 40
a n d 41). The compound was prepared according to the
procedure described for II leading to 3. The product was
isolated in 84% yield as yellow crystals; mp 124-126 °C. In
CDCl3, the product is a 3:1 enol:keto mixture. Data are given
1-(2-H yd r oxy-5-m et h ylp h en yl)-3-(3-t r iflu or om et h yl-
p h en yl)p r op a n e-1,3-d ion e (Sch em e 1; II Lea d in g to 38).
The compound was prepared according to the procedure
described for II leading to 3. The crude product was purified
by chromatography (heptane:ethyl acetate 10:1) to give yellow
crystals in a yield of 67%; mp 87-89 °C. Only the enol form
1
for the major enol form. H NMR (CDCl3): 2.35 (s, 3H), 4.00
1
was present in CDCl3. H NMR (CDCl3): 2.37 (s, 3H), 6.86 (s,
(s, 3H), 6.93 (m, 2H), 7.20 (s, 1H), 7.30 (dd, 1H, J ) 8.3, 2.1),
7.49 (d, 1H, J ) 1.6), 7.57 (dd, 1H, J ) 8.9, 2.6), 8.08 (d, 1H,
J ) 2.1), 11.90 (s, 1H), 15.49 (s, 1H). 13C NMR (CDCl3): 20.9,
56.5, 98.1, 113.5, 113.7, 118.6, 124.6, 128.3, 128.6, 130.0, 132.7,
135.6, 137.1, 157.7, 160.7, 173.4, 196.4. MS m/z (% rel int):
1H), 6.94 (d, 1H, J ) 8.4), 7.33 (dd, 1H, J ) 8.4, 1.8), 7.57 (d,
1H, J ) 1.8), 7.65 (dd, 1H, J ) 7.9, 7.8), 7.82 (d, 1H, J ) 7.8),
8.14 (d, 1H, J ) 7.9), 8.20 (brs, 1H), 11.79 (s, 1H), 15.58 (s,
1H). 13C NMR (CDCl3): 20.7, 93.1, 118.6, 118.9, 124.0 (quart

4200 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Kahnberg et al.
364 (60%), 362.0165 (M+, 59, C17H15BrO4 requires 362.0154),
333 (17%), 215 (100%), 213 (100%), 150 (48%), 135 (100%).
centrifuged for 10 min at 27 000g. The final pellet was
resuspended in 30 mL of buffer, and the preparation was
frozen and stored at -20 °C.
5′-Br om o-2′-m eth oxy-6-m eth ylfla von e (40). The com-
pound was prepared according to the procedure described for
3. The product was isolated in 91% yield as white crystals;
3H-Ro 15-1788 Bin d in g Assa y. The membrane preparation
was thawed and centrifuged at 0-4 °C for 10 min at 27 000g.
The pellet was washed twice with 20 mL of Tris-citrate (50
mM, pH 7.1) using an Ultra-Turrax homogenizer and centri-
fuged for 10 min at 27 000g. The final pellet was resuspended
in Tris-citrate (50 mM, pH 7.1, 500 mL buffer per gram of
original tissue), and then used for binding assays. Aliquots of
0.5 mL of membrane suspension were added to 25 µL of test
solution and 25 µL of 3H-Ro 15-1788 (0.7-1.5 nM, final
concentration in assay), mixed, and incubated for 40 min at
an ice-bath (0-4 °C). Nonspecific binding was determined
using Clonazepam (1 µM, final concentration) added to sepa-
rate samples. Following incubation, the samples were added
to 5 mL of ice-cold buffer and poured direcly onto Whatman
GF/C glass fiber filters under suction and immediately washed
with 5 mL of ice-cold buffer. The amount of radioactivity on
the filters was determined by conventional liquid scintillation
counting. Specific binding was total binding minus nonspecific
binding.
1
mp 151-155 °C. H NMR (CDCl3): 2.47 (s, 3H), 3.93 (s, 3H),
6.93 (d, 1H, J ) 9.0), 7.13 (s, 1H), 7.51 (m, 3H), 8.01 (m, 2H).
13C NMR (CDCl3): 21.1, 56.2, 113.1, 113.3, 113.8, 118.0, 122.8,
123.6, 125.2, 131.9, 134.9, 135.2, 135.3, 154.9, 157.2, 159.1,
179.0. MS m/z (% rel int): 346 (72%), 344.0048 (M+, 74, C17H13
-
79BrO3 requires 344.0048), 267 (25%), 163 (27%), 162 (35%),
135 (100%), 134 (33%), 113 (26%). Anal. (C17H13BrO3) C, H.
5′-Br om o-2′-h yd r oxy-6-m eth ylfla von e (41). The com-
pound was prepared according to the procedure described for
14. Only 2 equiv of BBr3 were added. The reaction time was 4
h before addition of MeOH. A beige solid, insoluble in CH2Cl2
and brine, was filtered off and recrystallized from acetone to
give white crystals in 72% yield; mp 285-287 °C. 1H NMR
(DMSO-d6): 2.43 (s, 3H), 7.02 (d, 1H, J ) 8.6), 7.12 (s, 1H),
7.55 (dd, 1H, J ) 8.6, 2.1), 7.63 (d, 1H, J ) 8.5), 7.70 (d, 1H,
J ) 8.5), 7.81 (brs, 1H), 8.02 (d, 1H, J ) 2.1). 13C NMR (DMSO-
d6): 30.5, 120.6, 121.4, 128.4, 129.3, 129.8, 132.8, 133.9, 140.5,
144.9, 144.9, 145.3, 164.1, 165.9, 169.0, 187.2. MS m/z (% rel
int): 333 (18%), 332 (98%), 331 (25%), 329.9888 (M+, 100,
Ca lcu la tion s. The IC50 values were calculated by (applied
test sustance concentration, µM) × 1/(C°/Cx - 1), where C° is
specific binding in control assay and Cx is the specific binding
in the test assay. Ki values were calculated from IC50 values
using the binding affinity constant, Kd ) 1.6 nM, for 3H-Ro
15-1788 binding.
Com p u ta tion a l Meth od s. Conformational analysis of the
flavones was performed by using the molecular mechanics
program MM3(92) developed by Allinger and co-workers.41,42
C
16H1179BrO3 requires 329.9892), 135 (28%), 134 (55%). Anal.
(C16H11BrO3) C, H.
2-Acetyl-4-m eth ylph en yl Nicotin ate (Sch em e 1; I Lead-
in g to 42). The compound was prepared according to the
procedure described for I leading to 3. Nicotinoyl chloride‚HCl
was purchased from Aldrich. The product was purified by
chromatography (heptane:ethyl acetate 5:1) to give white
crystals in a yield of 79%; mp 85-87 °C. 1H NMR (CDCl3):
2.45 (s, 3H), 2.55 (s, 3H), 7.13 (d, 1H, J ) 8.2), 7.41 (dd, 1H,
J ) 8.2, 2.2), 7.48 (dd, 1H, J ) 8.0, 4.9), 7.68 (d, 1H, J ) 2.2),
8.47 (ddd, 1H, J ) 8.0, 2.2, 1.9), 8.87 (dd, 1H, J ) 4.9, 1.9),
9.40 (d, 1H, J ) 2.2). 13C NMR (CDCl3): 21.1, 29.6, 123.8,
125.8, 130.5, 131.2, 134.4, 136.6, 137.8, 138.0, 146.9, 151.7,
154.2, 164.4, 197.6. MS m/z (% rel int): 255 (M+, 18%), 106
(100%), 78 (47%), 51 (16%).
Ack n ow led gm en t. This work was supported finan-
cially by the Swedish Research Board for Natural
Sciences and the NeuroScience PharmaBiotec Research
Center, Denmark.
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1-(2-Hyd r oxy-5-m eth ylp h en yl)]-3-p yr id in -3-ylp r op a n e-
1,3-d ion e (Sch em e 1; II Lea d in g to 42). The compound was
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1
are given for the major enol form. H NMR (CDCl3): 2.35 (s,
3H), 6.88 (s, 1H), 6.93 (d, 1H, J ) 8.5), 7.31 (dd, 1H, J ) 8.5,
1.9), 7.45 (dd, 1H, J ) 7.9, 4.9), 7.54 (bs, 1H), 8.22 (d, 1H, J )
7.9), 8.77 (dd, 1H, J ) 4.9, 1.8), 9.16 (d, 1H, J ) 1.8), 11.81 (s,
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118.7, 123.6, 128.3, 129.6, 134.1, 137.4, 148.0, 152.7, 160.5,
174.4, 196.0. MS m/z (% rel int): 255 (M+, 100%), 238 (14%),
135 (45%), 134 (32%), 107 (25%), 106 (100%), 79 (35%), 78
(55%), 77 (32%), 51 (20%).
3′-Aza -6-m eth ylfla von e (42). The compound was prepared
according to the procedure described for 3. The crude product
was recrystallized from acetone to give white crystals in a yield
1
of 97%; mp 156-159 °C. H NMR (CDCl3): 2.41 (s, 3H), 6.76
(s, 1H), 7.42 (m, 2H), 7.47 (dd, 1H, J ) 8.6, 2.2), 7.94 (d, 1H,
J ) 2.2), 8.13 (ddd, 1H, J ) 8.1, 1.9, 1.5), 8.71 (dd, 1H, J )
4.8, 1.5), 9.11 (d, 1H, J ) 1.9). 13C NMR (CDCl3): 21.0, 108.3,
117.9, 123.6, 123.7, 125.1, 128.0, 133.6, 135.4, 135.6, 147.6,
152.1, 154.5, 160.9, 178.1. MS m/z (% rel int): 237.0800 (M+,
100, C15H11NO2 requires 237.0790), 209 (13%), 134 (38%), 106
(13%). Anal. (C15H11NO2) C, H, N.
Tissu e P r ep a r a tion . Preparations were performed at 0-4
°C unless otherwise indicated. Cerebral cortex from male
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15 min, and the pellet was washed three times with buffer
(centrifuged at 27 000g for 10 min). The washed pellet was
homogenized in 20 mL of buffer and incubated on a water bath
(37 °C) for 30 min to remove endogenous GABA and then
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