Synthesis, structure-affinity relationships, and modeling of AMDA analogs at 5-HT2A and H1 receptors: Structural factors contributing to selectivity

Article abstract of DOI:10.1016/j.bmc.2009.08.016

Histamine H₁ and serotonin 5-HT_2A receptors present in the CNS have been implicated in various neuropsychiatric disorders. 9-Aminomethyl-9,10-dihydroanthracene (AMDA), a conformationally constrained diarylalkyl amine derivative, has affinity for both of these receptors. A structure-affinity relationship (SAFIR) study was carried out studying the effects of N-methylation, varying the linker chain length and constraint of the aromatic rings on the binding affinities of the compounds with the 5-HT_2A and H₁ receptors. Homology modeling of the 5-HT_2A and H₁ receptors suggests that AMDA and its analogs, the parent of which is a 5-HT_2A antagonist, can bind in a fashion analogous to that of classical H₁ antagonists whose ring systems are oriented toward the fifth and sixth transmembrane helices. The modeled orientation of the ligands are consistent with the reported site-directed mutagenesis data for 5-HT_2A and H₁ receptors and provide a potential explanation for the selectivity of ligands acting at both receptors.

Full text of DOI:10.1016/j.bmc.2009.08.016

Bioorganic & Medicinal Chemistry 17 (2009) 6496–6504
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structure–affinity relationships, and modeling of AMDA analogs
at 5-HT2A and H1 receptors: Structural factors contributing to selectivity
Jitesh R. Shah a, Philip D. Mosier a, Bryan L. Roth b, Glen E. Kellogg a, Richard B. Westkaemper a,
*
a Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, PO Box 980540, Richmond, VA 23298, USA
b Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Histamine H1 and serotonin 5-HT2A receptors present in the CNS have been implicated in various neuro-
psychiatric disorders. 9-Aminomethyl-9,10-dihydroanthracene (AMDA), a conformationally constrained
diarylalkyl amine derivative, has affinity for both of these receptors. A structure–affinity relationship
(SAFIR) study was carried out studying the effects of N-methylation, varying the linker chain length
and constraint of the aromatic rings on the binding affinities of the compounds with the 5-HT2A and
H1 receptors. Homology modeling of the 5-HT2A and H1 receptors suggests that AMDA and its analogs,
the parent of which is a 5-HT2A antagonist, can bind in a fashion analogous to that of classical H1 antag-
onists whose ring systems are oriented toward the fifth and sixth transmembrane helices. The modeled
orientation of the ligands are consistent with the reported site-directed mutagenesis data for 5-HT2A and
H1 receptors and provide a potential explanation for the selectivity of ligands acting at both receptors.
Ó 2009 Published by Elsevier Ltd.
Received 21 April 2009
Revised 6 August 2009
Accepted 9 August 2009
Available online 13 August 2009
Keywords:
5-HT2A receptor
H1 receptor
Phenylethylamines
9-Aminoalkyl-9,10-dihydroanthracene
(AMDA)
G Protein-coupled receptor (GPCR)
Structure–activity relationship (SAR)
Structure–affinity relationship (SAFIR)
1. Introduction
on antagonists that are used for their antiallergic effects in the
periphery. However, first-generation H1 receptor antagonists
Sleep disorders and circadian rhythm abnormalities are preva-
lent, with 70 million Americans reporting disturbed sleep each
year. Epidemiological studies indicate that between 30% and 48%
of the global population find difficulty in initiating and maintaining
sleep.1 Serotonin (5-hydroxytryptamine, 5-HT), a major neuro-
transmitter found in the CNS and periphery, has been reported to
be involved in the control of sleep and waking states.2 5-HT2 recep-
tors belong to the superfamily of G protein-coupled receptors
(GPCRs) and consist of three subtypes: 2A, 2B, and 2C. 5-HT2A
receptors are involved mainly in non-rapid eye movement sleep
regulation and respiratory control,3 and selective antagonism of
the 5-HT2A receptor has emerged as a promising new mechanism
for the treatment of sleep disorders.4
Histamine released by tissue mast cells, basophils, and hista-
minergic neurons is also known to impact the ability to fall asleep
and stay asleep.5 It has been suggested that ligands acting at H1
receptors in the CNS may be useful in the treatment of neuropsy-
chiatric and sleep disorders.6 Histamine exerts its effect by inter-
acting with four different receptors H1–H4, which are GPCRs.7,8
Early research and development of H1 ligands has focused largely
(diphenhydramine, mepyramine, chlorpheniramine) also exhibit
high H1 receptor occupancy in the CNS due in part to their lipophil-
icity, leading to sedative effects.9–11 This led to the development of
second-generation antagonists (acrivastine, loratadine, terfena-
dine, cetirizine, etc.) that exhibited high selectivity and less seda-
tive potential due to the decrease in their ability to penetrate the
blood brain barrier.12,13 Some of these antagonists (doxepin, ketot-
ifen, epinastine, olopatadine), which have a tri- or tetracyclic fused
ring system, also showed affinity at 5-HT2A, adrenaline
a1, dopa-
mine D2 and muscarinic M1 receptors, indicative of their low selec-
tivity for the H1 receptor.14 Among the various chemical classes of
H1 antagonists, ligands that are zwitterionic (olopatadine, acrivas-
tine, fexofenadine) showed the highest degree of selectivity for H1,
suggesting that the carboxylate group of the ligand may interact
with a residue (K191)15 in the H1 receptor that is not present in
the other GPCRs. Introduction of a carboxylate moiety thus pro-
vides one potential means of designing peripherally-acting H1-
selective antagonists.
In this study, we set out to determine if H1 versus 5-HT2A selec-
tivity could be achieved through the systematic modification of the
uncharacteristically nonpolar16 structure of 9-(aminomethyl)-
9,10-dihydroanthracene (AMDA, 1a). We have previously reported
AMDA as the parent member of a potentially new class of
* Corresponding author. Tel.: +1 804 828 6449; fax: +1 804 828 7625.
E-mail address: rbwestka@vcu.edu (R.B. Westkaemper).
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.08.016

J. R. Shah et al. / Bioorg. Med. Chem. 17 (2009) 6496–6504
6497
high-affinity 5-HT2A antagonists.17,18 This work builds on our ear-
lier studies19 by creating a ‘matrix’ of AMDA-like compounds in
which the aromatic ring system, degree of N-methylation and
length of the aliphatic linker are systematically varied. A series of
9-(aminoalkyl)-9,10-dihydroanthracenes (DHAs) and an analogous
series of diphenylalkylamines (DPAs) were synthesized and their
binding affinities at H1 and 5-HT2A determined. Our results show
that AMDA and related compounds exhibit varying degrees of
affinity for the H1 receptor. Homology models using the human
b2-adrenoceptor (b2-AR)20 as a template provide receptor-based
explanations for the observed structure–affinity relationships (SA-
FIR) among the AMDA analogs. To our knowledge this is also the
first report of a comparison of 5-HT2A and H1 homology models
generated from the b2-adrenergic structure.
nature of the two required aromatic groups, the terminal basic
amine, and the aliphatic linker between these two features.32,33
In this work, we examined similar structural variations using
AMDA as the parent structure. These compounds closely resemble
well-established H1 antagonists: replacement of the ether oxygen
of diphenhydramine by a methylene unit results in compound 6c
and replacement of the pyridin-2-yl group of the H1 antagonist
pheniramine with a phenyl group results in 5c.
Compound affinities for the 5-HT2A and H1 receptors are given
in Table 1 and displayed graphically in Figure 1. The most signifi-
cant differences in affinity were observed between the 9-(aminoal-
kyl)-9,10-dihydroanthracene (DHA) compounds (Fig. 1a) and their
corresponding diphenylalkylamine (DPA) congeners (Fig. 1b).
Ring-opening of the tricyclic system uniformly produced decreases
in affinity for both 5-HT2A and H1 receptors. No DPA compound
exhibited high affinity (Ki <100 nM) at 5-HT2A, and only three
showed high affinity for H1 receptors (5b, Ki = 64 nM; 5c,
Ki = 75 nM; 6c, Ki = 70 nM). In contrast, many of the DHA analogs
were found to have high affinity at both receptors, demonstrating
that dihydroanthracene is a privileged34 structure. For compounds
interacting with the H1 receptor, progressively increasing either
the number of methylene units in the linker or the number of N-
methyl groups consistently retained or enhanced affinity in both
the DHA and the DPA series. H1 receptor affinity was insensitive
to chain length for the unsubstituted amine analogs of AMDA
(1a, Ki = 197 nM; 2a, Ki = 137 nM; 3a, Ki = 175 nM). In contrast, H1
affinity increased with increasing chain length for both N-methyl-
ated (1b, Ki = 189 nM; 2b, Ki = 48 nM; 3b, Ki = 3 nM) and N,N-dime-
thylated analogs (1c, Ki = 25 nM; 2c, Ki = 6 nM; 3c, Ki = 0.5 nM). For
the DHA analogs, N,N-dimethylation and a three-methylene linker
was optimal for H1 affinity (3c, Ki = 0.5 nM). The trends in affinity
at 5-HT2A are less uniform. Like the H1 receptor, the affinity of
the DPA compounds for 5-HT2A generally increased with increasing
linker length or degree of N-methylation though, as noted above,
no DPA compound was found to have substantial (Ki <700 nM)
affinity for the 5-HT2A receptor. As observed previously for
DHA19 and compounds with a one-methylene linker, 5-HT2A affin-
ity decreased as N-methylation degree increased (1a, Ki = 20 nM;
1b, Ki = 52 nM; 1c, Ki = 540 nM). This trend was not observed for
the other linker lengths. Additionally, for DHA compounds with
no N-methylation, linker lengths of one (1a, Ki = 20 nM) and three
(3a, Ki = 32 nM) methylene units demonstrated significantly higher
affinity than the two-methylene linker (2a, Ki = 480 nM) com-
pound. Affinities were more uniform for compounds with one N-
methyl group (1b, Ki = 52 nM; 2b, Ki = 92 nM; 3b, K = 13 nM), and
for those with two N-methyl groups, affinity increased with linker
length by 25-fold (1c, Ki = 540 nM; 2c, Ki = 84 nM; 3c, Ki = 22 nM).
In general, 5-HT2A receptor affinity is less sensitive to N-methyla-
tion and chain length variation than H1 receptor affinity.
2. Results and discussion
2.1. Chemistry
9-(Aminomethyl)-9,10-dihydroanthracene (1a), 9-(2-amino-
ethyl)-9,10-dihydroanthracene (2a), and 9-(2-aminopropyl)-9,10-
dihydroanthracene (3a) were prepared as previously described.19,21
N-Alkylated analogs 1b, 1c, 3b, and 3c have been reported by us.19 N-
Methylated analogs of 2a were synthesized (Scheme 1) using 9-
hydroxymethylanthracene 7 as the starting material. Halogena-
tion22 followed by cyanation23 of the alcohol gave 2-(anthracen-9-
yl)acetonitrile 9 that was further hydrolyzed to the corresponding
acid 10. Anthracene ring reduction using sodium metal in n-penta-
nol gave 2-(9,10-dihydroanthracen-9-yl)acetic acid (11). Sequential
conversion of the acid 11 to its amide 12 via acid chloride, followed
by reduction, afforded 2-(9,10-dihydroanthracen-9-yl)-N-methyle-
thanamine (2b). 2-(9,10-Dihydroanthracen-9-yl)-N,N-dimethyle-
thanamine (2c) was obtained by reductive amination24 of 2-(9,10-
dihydroanthracen-9-yl)acetaldehyde (15), which was obtained by
the oxidation of 2-(9,10-dihydroanthracen-9-yl)ethanol (14)
(Scheme 2). N-Methyl-2,2-diphenylethanamine25 (4b) was ob-
tained by the N-methylation of the respective amine as reported.26
N,N-Dimethyl-3,3-diphenylpropan-1-amine27 (5c) was obtained
by the same method adopted for the synthesis of 2c from
commercially available 3,3-diphenylpropanal. 4,4-Diphenylbutan-
1-amine28 (6a) was obtained by the BH3ÁTHF reduction of commer-
cially available 4,4-diphenylbutanenitrile. Compounds 4a, 4c, 5a,
5b, 6b, and 6c were commercially obtained.
2.2. Structure–affinity relationships
Several reviews describing structure–activity relationship stud-
ies of diphenhydramine-like antihistamines have been reported.29–31
The features that have been systematically varied include the
NC
Br
HOOC
HO
c
a
b
10
7
8
9
NH
O
HOOC
N
H
g
e, f
d
11
12
2b
Scheme 1. Reagents and conditions: (a) PBr3, toluene 0 °C, 1 h; (b) KCN, DMSO, 70 °C, 1 h; (c) KOH, ethylene glycol/H2O (1:1), reflux, 12 h; (d) Na, 1-pentanol, reflux, 30 min;
(e) SOCl2, dry benzene, reflux, 2 h; (f) methylamine–THF, 25 °C, 6 h; (g) BH3ÁTHF, THF reflux, 6 h.

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J. R. Shah et al. / Bioorg. Med. Chem. 17 (2009) 6496–6504
O
OH
N
OH
H
c
b
a
15
13
14
2c
Scheme 2. Reagents and conditions: (a) Na2K silica gel, THF, 1 h; (b) Dess–Martin reagent, CH2Cl2, 1 h; (c) Me2NHÁHCl, Ti(O-i-Pr)4, NEt3, abs EtOH, 25 °C, 9 h, NaBH4, 25 °C,
10 h.
Table 1
a
Observed binding affinities for 9-(aminoalkyl)-9,10-dihydroanthracene (DHA) and
diphenylalkylamine (DPA) analogs at 5-HT2A and H1 receptors
5-HT2A
H1
10.0
R1
N
9.0
8.0
7.0
R2
n
X
Compd
X
n
R1
R2
Ki (nM)
H1
4
5-HT2A
6.0
5.0
3
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–H, –H
–H, –H
–H, –H
–H, –H
–H, –H
–H, –H
–H, –H
–H, –H
–H, –H
1
1
1
2
2
2
3
3
3
1
1
1
2
2
2
3
3
3
–H
–H
–CH3
–H
–H
–CH3
–H
–H
–CH3
–H
–H
–CH3
–H
–H
–CH3
–H
–H
–H
20a
197
189
25
137
48
6
175
3
2
–CH3
–CH3
–H
–CH3
–CH3
–H
–CH3
–CH3
–H
–CH3
–CH3
–H
–CH3
–CH3
–H
–CH3
–CH3
52b
540b
480b
92
2
1
1
N
0
0
84
32b
13
22
0.5
4610c
>10,000
7356
>10,000
1498
1636
2589
754
>10,000
>10,000
5172
2758
64
b
5-HT2A
H1
75
10.0
1670
386
70
–CH3
1151
9.0
8.0
7.0
From Westkaemper, et al. 2001.21
From Runyon, et al. 2001.19
From Runyon, et al. 2002.17
a
b
c
4
2.3. Modeling receptor–ligand interactions
2.3.1. H1 and 5-HT2A receptor models
6.0
5.0
3
2
When considering the selectivity of a particular ligand for one
receptor versus another, it is useful to analyze the differences in
amino acids that comprise the binding sites of the two receptors.
The alignment of the human H1 and 5-HT2A receptor sequences
with that of human b2-AR is presented in Figure 2. The H1 receptor
is phylogenetically related to the 5-HT2 receptor subtypes, with
higher sequence homology found primarily in the transmem-
brane-spanning regions.35 Based on the primary sequences, bind-
ing site analysis of both 5-HT2A and H1 receptors indicates that
the major variations in amino acid constitution occur at positions
3.33, xl2.52, xl2.54, 5.39, 5.42, 6.55, and 7.35 (Fig. 2). Differences
in steric and electrostatic sidechain properties at these and other
positions are likely responsible for directing the ligand selectivity
of the two receptors. The H1 receptor amino acid residue
K1915.39, not commonly found at this position among aminergic
GPCRs, could be potentially exploited for the design of highly selec-
tive H1 ligands. The three-dimensional arrangement of these resi-
dues is shown in Figure 3. The residues that are conserved
2
1
1
N
0
0
Figure 1. 3D bar graph showing the affinities at 5-HT2A and H1 for the (a) 9-
(aminoalkyl)-9,10-dihydroanthracene (DHA) and (b) diphenylalkylamine (DPA)
analogs listed in Table 1.
between H1 and 5-HT2A are those that tend to be highly conserved
among all aminergic GPCRs. These residues, primarily located in
the inner region of the binding cavity, include W3.28, the amine
counter-ion D3.32, S3.36, and T3.37, and the ‘aromatic cluster’36–39
residues W6.48 and F6.52, and Y7.43 (an H-bonding partner for D3.32).
Models of human H1 and 5-HT2A receptors were generated with
40
the MODELLER program using a high-resolution crystal structure of

J. R. Shah et al. / Bioorg. Med. Chem. 17 (2009) 6496–6504
6499
Figure 2. Alignment of the human b2-adrenergic, H1 and 5-HT2A receptor sequences. Sequence positions highlighted in pink indicate highly conserved amino acids among the
Class A GPCR family that serve as reference positions in the Ballesteros–Weinstein52 numbering system, as well as the cysteine residues of the disulfide bridge tethering EL2
to TM3. The traditional numbering shown corresponds to the hH1 (top) and h5-HT2A (bottom) sequences. b2-Adrenergic residues highlighted in purple indicate positions in
the third intracellular loop (IL3) that were mutated to glycine in the hH1 and h5-HT2A sequences; these were retained in subsequent hH1 and h5-HT2A models. Other residues
highlighted in the hH1 and h5-HT2A sequences represent the residues of the binding site and correspond to those positions that are within 5.0 Å of the carazolol ligand bound
in the b2AR homolog. The color indicates the degree of similarity between the hH1 and h5-HT2A residue at a particular position as defined by the Gonnet PAM250 similarity
matrix (green = identical; yellow = strong or weak conservation; red = no conservation). The figure was created using ALSCRIPT.53
the human b2-AR20 as the template (see Section 4). Inspection of
putative receptor binding sites and ligand binding modes in our
homology models (Figs. 4 and 5) indicate two common interactions
that occur for each of the compounds listed in Table 1 at both 5-
HT2A and H1 receptors: 1) the highly conserved aspartic acid resi-
due at position41 3.32 (H1, D1073.32; 5-HT2A, D1553.32) is able to
interact with the protonated amine of the docked ligand; and 2)
hydrophobic residues present in transmembrane helix 6 (TM6)
comprising the aromatic cluster42 are able to interact with the aro-
matic rings of the ligands. In other binding site locations variability
of amino acid residues at equivalent positions in the hH1 and h5-
HT2A receptors influence the way the aromatic rings are oriented
in the receptor, which in turn provides an explanation for the ob-
served differences in affinity for AMDA and its analogs with vary-
ing chain lengths. The presence of hydrophobic residues
surrounding the conserved D3.32 in H1 and in 5-HT2A, together with
the aromatic cluster region in H1 and in 5-HT2A, provide sites of
favorable interaction with the ligands for each receptor.
2.3.2. Binding mode analysis
Figure 3. Illustration of the differences in binding site residue composition of the
hH1 and h5-HT2A receptors within the context of the b2AR-T4L crystal structure.
Compounds listed in Table 1 were docked into the receptor
binding sites of the H1 and 5-HT2A receptor models using the
Residues are truncated at the Cb carbon atom (ball-and-stick representation) and
GOLD automated docking routine.43 The GOLD scoring function (ste-
are colored based on residue similarity as described in Figure 2. For those positions
ric and electrostatic interactions) was used to select the favored
whose residue identity differs, the H1 residue is listed first, followed by the 5-HT2A
residue.
ligand conformation. In addition, we carried out HINT44 (Hydro-

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J. R. Shah et al. / Bioorg. Med. Chem. 17 (2009) 6496–6504
Figure 5. Docked h5-HT2A–ligand complexes. (a) 3c. (b) 6c. (c) 1a. Residues within
5 Å of the bound ligand are displayed.
pathic INTeraction) analysis to characterize the nature of the
binding site interactions. HINT is a free-energy-based method
that considers atom–atom interactions in a bimolecular complex
using a parameter set derived from octanol/water partition coef-
ficients.45 Modeling observations indicate that the compounds
prefer to be oriented within the binding pockets of the two recep-
tor models in similar, but distinct binding modes (see Figs. 4 and
5). The well-known aspartate D3.32 residue was found to interact
Figure 4. Docked hH1–ligand complexes. (a) 3c. (b) 6c. (c) 1a. Residues within 5 Å
of the bound ligand are displayed.

J. R. Shah et al. / Bioorg. Med. Chem. 17 (2009) 6496–6504
6501
with the basic amine in both receptors for each docked ligand,
and the ligand aromatic rings were consistently oriented in the
binding site surrounded by TM4, TM5, and TM6. In general, for
the short-chain linker ligands there is a relatively small amount
of ligand surface area that may interact with the surrounding
hydrophobic environment in the binding pocket, accounting for
the observed decreased affinity of these compounds at both 5-
HT2A and H1 (Figs. 4c and 5c). However, for AMDA (1a), the ob-
served high affinity could be due to the presence of an alternate
binding mode.46
The tricyclic DHA ring system in 3c showed strong hydropho-
bic interactions (Y1083.33
, , , , and
L1634.61 F168xl2.38 F1905.38
F4356.55), with the phenyl rings oriented toward TM5 and the
aliphatic linker located deep in the H1 binding pocket. In con-
trast, in the 5-HT2A model, 3c is oriented such that the phenyl
rings are facing TM6 (F3396.51, F3406.52, and N3436.55) and the
aliphatic linker is positioned closer to extracellular loop
2
(EL2). For both H1 and 5-HT2A, the compound with the longest
linker and highest degree of methylation (3c) was found to have
the highest (H1) or one of the highest (5-HT2A) affinities among
the compounds tested. The methylene linkers in the H1 receptor
are able to interact with residues L1043.29 and Y4316.51, which
explains the comparatively higher affinity of 3c for H1 than for
5-HT2A. Further, the observed differences in ligand orientation
within the receptor binding site can account for the preference
for N-methylation at H1, with the methyl groups more closely
surrounded by hydrophobic residues as compared to 5-HT2A
(Figs. 4a and 5a).
For the unbridged DPA analogs, the increased conformational
flexibility (as compared to DHA) and intramolecular steric repul-
sion produce a twisted ring orientation. This results in more unfa-
vorable receptor–ligand interactions (steric clashes and polar–
nonpolar interactions) involving the ring system and the surround-
ing residues (Figs. 4b and 5b).
HINT hydropathic interaction analysis provided additional sup-
port for the proposed orientation of the ligands in the binding
pocket. Figure 6 shows the HINT maps generated for compound
3c (which has the highest affinity for both receptors) in the binding
sites of the H1 and 5-HT2A receptors. In each case the tricyclic ring
system fits into the hydrophobic ‘aromatic cluster’ while the amine
group faces D1073.32 and is stabilized by both ionic and hydrogen
bonding interactions. However, both the aliphatic linker region
and the N-methyl groups engage in more extensive hydrophobic
Figure 6. HINT interaction maps for compound 3c in (a) H1 and (b) 5-HT2A binding
sites. Regions of favorable hydrophobic (green) and polar (magenta) intermolecular
interactions are shown as contours.
ity for the H1 receptor (44-fold). In addition, removing the con-
formational restriction of the dihydroanthracene tricyclic system
by ring-opening to provide a diphenyl system is detrimental to
the ligand affinity for both 5-HT2A and H1 receptors. Structure–
affinity relationships among these compounds show that N-
alkylation either decreases or has little effect on 5-HT2A affinity,
while the propylene linker is the optimum chain length between
the tricyclic system and amine for receptor affinity. Modeling
studies suggest that diaryl alkylamine analogs exhibit a common
binding mode within the 5-HT2A and H1 receptors. Hydropathic
analysis of the modeled complexes supports the proposed role
of the TM6 aromatic cluster in directing binding. The proposed
interactions in H1 than in 5-HT2A
.
In addition, the receptor–ligand complexes described here are
in general agreement with other studies that have implicated res-
idues that contribute to an antagonist binding site in H1 and 5-
HT2A. Besides the several key residues that have been reported
by site-directed mutagenesis studies in H1 receptor models15,47
(W1584.56, Y2005.48, F4246.44, W4286.48, F4326.52, and F4356.55
)
and 5-HT2A receptor models48 (F2435.47
, , ,
W3366.48 F3396.51
F3406.52), we found that Y1083.33 and I4547.39 were oriented to
favorably interact with ligands in the binding pocket of the H1
receptor, and the cognate residues V1563.33 and V3667.39 for the
5-HT2A receptor. The differences in the stereoelectronic character
of these residues likely contribute to the differences in the way
the ligand binds to these receptors, and consequently the observed
differences in binding affinity.
differences in the binding pocket of H1 (Y1083.33 and I4547.39
)
and 5-HT2A (V1563.33 and V3667.39) may determine the way li-
gands bind, which in turn may determine the selectivity of the
ligands for each of the two receptors. These preliminary model-
ing results provide a qualitative understanding of how AMDA
analogs might interact with the 5-HT2A and H1 receptors. This
study also provides potential insight into the mechanisms by
which differences in the structures of the receptors and ligands
determines receptor selectivity. We continue to synthesize and
test compounds and refine our models toward the development
3. Conclusions
Within the matrix of compounds synthesized and tested, the
N,N-dimethylated chain-lengthened propylene analog of AMDA
shows the highest affinity at both 5-HT2A and H1 receptors
(3c: 5-HT2A, Ki = 22 nM; H1, Ki = 0.5 nM) and the highest selectiv-
of
a more quantitative and predictive structure-based QSAR
model.

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J. R. Shah et al. / Bioorg. Med. Chem. 17 (2009) 6496–6504
mixture was concentrated under vacuum and the oily solution ob-
4. Experimental
4.1. Chemistry
tained was triturated with chloroform, dried (MgSO4), and concen-
trated to yield pure product 4 (0.76 g, 84%). 1H NMR (300 MHz,
CDCl3): d 2.8 (d, J = 7.8 Hz, 2H, CH2), 4.4 (t, J = 7.8 Hz, 1H, CH), 3.9
(d, J = 18.1 Hz, 2H, Ar-CH2-Ar), 4.1 (d, J = 18.1 Hz, 2H, Ar-CH2-Ar),
7.1–7.3 (m, 8H, Ar-H). 13C NMR (75 MHz, CDCl3): d 40.1, 41.2,
45.1, 126.8, 128.7, 138.4, 139.6, 177.4.
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra
were recorded using a Varian Gemini 300 spectrometer in CDCl3
using tetramethylsilane as an internal standard unless otherwise
specified. Melting points were determined using an OptiMelt melt-
ing point apparatus and are uncorrected. Elemental analyses were
performed by Atlantic Microlab, Inc., and determined values are
within 0.4% of theory. All reactions were maintained under a nitro-
gen atmosphere. Anhydrous solvents were purchased and stored
under nitrogen over molecular sieves. Medium-pressure column
chromatography was carried out using silica gel 60 Å, 0.040–
0.063 mm, (200–400 mesh), Sorbent Technologies.
4.1.5. 2-(9,10-Dihydroanthracen-9-yl)-N-methylacetamide (12)
Thionyl chloride (1.73 g, 14.6 mmol) was added under N2 to a
stirred solution of compound 11 (0.7 g, 2.9 mmol) in anhydrous
benzene (5 mL). The solution was heated at reflux (2 h), allowed
to cool and the excess benzene and thionyl chloride were removed
under reduced pressure to provide an oil. The oil obtained was dis-
solved in anhydrous THF (10 mL) and cooled in an ice bath (0 °C). A
methylamine/THF (2 M, 5.8 mmol) solution was added dropwise
into the stirred solution, and the mixture was stirred at rt (2 h).
The solvent was removed under reduced pressure to give a white
solid. Water (20 mL) was added, and the suspension was extracted
with EtOAc (3 Â 25 mL). The combined extracts were washed with
water, brine, and dried (MgSO4). Removal of solvent under reduced
pressure gave the crude amide as a viscous oil. The resulting amide
was purified using medium pressure chromatography (CH2Cl2/ace-
tone, 9:1), yield (75–80%). 1H NMR (300 MHz, CDCl3): d 2.45 (s, 3H,
CH3), d 2.67 (d, J = 7.8 Hz, 2H, CH2), 3.93 (d, J = 18.3 Hz, 2H, Ar-CH2-
Ar), 4.05 (d, J = 18.3 Hz, 2H, Ar-CH2-Ar), 4.56 (t, J = 7.5 Hz, 1H, CH),
7.16–7.37 (m, 8H, Ar-H). 13C NMR (75 MHz, CDCl3): d 34.79, 41.0,
41.6, 42.60 126.11, 127.47, 135.91, 138.24, 176.58.
4.1.1. 9-Bromomethylanthracene (8)22
Phosphorus tribromide (0.8 ml, 8.4 mmol) was added to a sus-
pension of 9-hydroxymethylanthracene 7 (1.5 g, 7.2 mmol) in tol-
uene (40 ml) at 0 °C via syringe. The mixture was stirred at 0 °C
for 1 h and then warmed to rt, during which the reaction became
homogeneous. Saturated Na2CO3 solution (15 mL) was added
slowly and the reaction mixture was stirred until it cooled to rt.
The phases were separated, and the organic phase was washed
with H2O (10 mL), brine (10 mL), and dried over MgSO4. The yellow
filtrate was concentrated to minimum volume, and then stored at
0 °C for crystallization. The yellow needle-like solid was collected
and dried in vacuum (1.24 g). The mother liquid was concentrated
and purified using medium pressure column chromatography
(0.6 g). The two parts were combined to give the product 8 (total
1.84 g, 94%) as yellow solid. Mp 143–146 °C. 1H NMR (300 MHz,
CDCl3): d 5.3 (s, 2H, CH2–Br), 7.2–8.3 (m, 9H, Ar-H). 13C NMR
(75 MHz, CDCl3): d 32.20, 125.50, 125.70, 125.80, 127.70, 128.10,
129.40, 131.60.
4.1.6. 2-(9,10-Dihydroanthracen-9-yl)-N-methylethanamine
(2b)
A borane–THF complex (1.0 M in THF, 0.537 g, 6.25 mmol) was
added in a dropwise manner to a stirred solution of 12 (0.35 g,
1.25 mmol) in anhydrous THF (5 mL) under N2 at 0 °C. The mixture
was slowly warmed to rt and heated at reflux (6 h). The reaction
mixture was allowed to cool to rt, and HCl (6.0 M, 3 mL) was added
with caution. The mixture was heated at reflux (1 h) and allowed to
cool to rt, and the solvent was removed under reduced pressure.
Water was added and the residue was extracted with ether
(25 mL). The aqueous portion was made basic with 10% NaOH
and extracted with CH2Cl2 (3 Â 25 mL). The organic layer was
washed with water and brine, dried (MgSO4), and concentrated un-
der reduced pressure to give 2b, which was then purified by med-
ium pressure column chromatography. CH2Cl2/MeOH (9:1) yield
(80–85%). Mp 202–205 °C (oxalate). 1H NMR (300 MHz, CDCl3): d
1.81 (q, J = 7.5 Hz, 2H, CH2), 2.38 (s, 3H, CH3), 2.56 (t, J = 7.5 Hz,
2H, CH2–NH), 3.89 (d, J = 18.3 Hz, 1H, Ar-CH2-Ar), 4.09 (d,
J = 18.3 Hz, 1H, Ar-CH2-Ar), 4.03 (t, J = 7.2 Hz, 1H, Ar-CH-Ar), 7.1–
7.3 (m, 8H, Ar-H). 13C NMR (75 MHz, CDCl3): d 35.30, 36.72,
37.40, 45.41, 50.20, 126.41, 128.10, 136.40, 140.61. Anal.
(C17H19NÁC2H2O4Á0.25H2O): C, H, N.
4.1.2. 2-(Anthracen-9-yl)acetonitrile (9)
A solution of 9-bromomethylanthracene 8 (1.5 g, 5.53 mmol) in
DMSO (15 mL) was added over 10 min to a stirred suspension of
KCN (0.54 g, 8.29 mmol) in DMSO (30 mL) at 70 °C under N2. The
mixture was stirred for an additional 40 min, cooled to rt, and di-
luted with H2O. The aqueous layer was saturated with NaCl and
then extracted with ether (3 Â 25 mL). The combined extracts were
washed with H2O, dried (MgSO4), filtered, and concentrated to
yield solid product 9 (0.96 g, 80%). Mp 154–156 °C. 1H NMR
(300 MHz, CDCl3): d 4.6 (s, 2H, CH2–CN), 7.2–8.5 (m, 9H, Ar-H).
13C NMR (75 MHz, CDCl3):
126.10, 128.20, 130.10, 131.60.
d 20.50, 125.20, 125.31, 124.60,
4.1.3. 2-(Anthracen-9-yl)acetic acid (10)
KOH (0.97 g, 17.48 mmol) in 10 ml of H2O was added to a sus-
pension of 2-(anthracen-9-yl)acetonitrile 9 (0.95 g, 4.37 mmol) in
ethylene glycol (50 ml). The mixture was heated at reflux for
24 h until homogenous. The hot solution was filtered, and the fil-
trate was acidified with dilute HCl to obtain the precipitated prod-
uct 10 (1.0 g, 100%). Mp 228–230 °C. 1H NMR (300 MHz, CDCl3): d
4.2 (s, 2H, CH2), 7.5–8.5 (m, 9H, Ar-H). 13C NMR (75 MHz, CDCl3): d
34.20, 124.60, 125.30, 125.60, 126.10, 128.20, 130.50, 131.90,
173.40.
4.1.7. 2-(9,10-Dihydroanthracen-9-yl)ethanol (14)
Na2K silica gel (2 g) was added to a well-stirred solution of 2-
(anthracen-9-yl)ethanol 13 (0.75 g, 4.4 mmol) in anhydrous THF
and stirred continuously under nitrogen. The reaction mixture
was refluxed for 15 min then allowed to cool to rt and quenched
with H2O (50 mL). The solid precipitate was filtered and washed
with EtOAc (5 Â 25 mL). The filtrate was then collected followed
by extraction. The EtOAc portion was dried (MgSO4) and concen-
trated under reduced pressure to provide viscous yellow oil. The
resulting yellow oil was purified using medium pressure column
chromatography (CH2Cl2/MeOH, 9:1) to provide 14 (0.65 g, 86%)
as a yellow oil. 1H NMR (300 MHz, CDCl3): d 1.86 (m, 2H, CH2), d
3.61 (t, 2H, CH2-OH), 3.90 (d, J = 18.3 Hz, 2H, Ar-CH2-Ar), 3.95 (d,
J = 18.3 Hz, 1H, Ar-CH2-Ar), 4.0 (d, J = 18.3 Hz, 1H, Ar-CH2-Ar),
4.1.4. 2-(9,10-Dihydroanthracen-9-yl)acetic acid (11)
Sodium metal (10 equiv) was added slowly to a refluxing solu-
tion of 2-(anthracen-9-yl)acetic acid 10 (0.9 g, 3.8 mmol) in 1-
pentanol (20 ml). The reaction mixture was stirred for 10 min until
all of the sodium dissolved, then was cooled and H2O (10 mL) was
added. The solution was made acidic with 5% HCl. The reaction

J. R. Shah et al. / Bioorg. Med. Chem. 17 (2009) 6496–6504
6503
4.02 (t, J = 7.5 Hz, 1H, CH), 7.19–7.47 (m, 8H, Ar-H). 13C NMR
(75 MHz, CDCl3): d 31.29, 31.39, 52.1, 62.30 126.11, 128.47,
137.91, 138.24.
Ar-H). 13C NMR (75 MHz, CDCl3): d 32.30, 38.40, 42.30, 52.20,
126.80, 128.70, 129.40, 143.10. Anal. (C16H19NÁ0.5HCl): C, H, N.
4.2. Molecular modeling
4.1.8. 2-(9,10-Dihydroanthracen-9-yl)acetaldehyde (15)
A solution of (9,10-dihydroanthracene-9-yl)ethanol 14 (0.60 g,
1.5 mmol) dissolved in anhydrous CH2Cl2 (20 mL) was added to a
stirred mixture of Dess–Martin oxidant (0.954 g, 2.25 mmol) in
CH2Cl2. The reaction mixture was stirred for 1 h, then diluted with
ether (75 mL) and poured into 1.3 M NaOH (75 mL). The ether layer
was separated and extracted with 1.3 M NaOH (3 Â 15 mL) and
was washed with H2O (2 Â 20 mL), brine (20 mL), dried (MgSO4)
and concentrated under reduced pressure to yield an oil. The
resulting oil was purified using medium pressure column chroma-
tography (petroleum ether/EtOAc 9:1) to provide 15 (0.445 g, 75%)
as an oil. 1H NMR (300 MHz, CDCl3): d 2.69 (d, 2H, CH2–CHO), 3.87
(d, J = 18.3 Hz, 1H, Ar-CH2-Ar), 4.06 (d, J = 18.3 Hz, 1H, Ar-CH2-Ar),
4.55 (t, J = 6.9 Hz, 1H, CH), 7.19–7.34 (m, 8H, Ar-H) 9.71 (s, 1H,
CHO), 13C NMR (75 MHz, CDCl3): d 34.85, 40.77, 50.0, 126.19,
127.54, 137.21, 138.50.
Molecular modeling investigations were conducted using the
SYBYL 7.1 molecular modeling package (Tripos LP, St. Louis, MO)
on MIPS R14K- and R16K-based IRIX 6.5 Silicon Graphics Fuel
and Tezro workstations. Molecular mechanics-based energy mini-
mizations were performed using the Tripos Force Field with
Gasteiger–Hückel charges, a distance-dependent dielectric con-
stant (e = 4) and a non-bonded interaction cutoff of 8 Å and were
terminated at an energy gradient of 0.05 kcal/(mol Å). The hH1
(P35367) and h5-HT2A (P28233) receptor sequences were retrieved
from the ExPASy Proteomics Server (http://www.expasy.org/) and
aligned with a profile of several related class A GPCRs (human,
dopamine D3 (P35462), muscarinic cholinergic M1 (P11229), vaso-
pressin V1a (P37288), adrenergic b2 (P07550), d-opioid (P41143), 5-
HT2A (P28223), dopamine D2 (P14416), bovine rhodopsin (P02699)
using the CLUSTALX program.49 Within CLUSTALX, the slow-accurate
alignment algorithm was used, the BLOSUM matrix series was em-
ployed and the gap opening penalty was increased from 10.0 to
15.0 to help maintain the continuity of the transmembrane helical
segments. The alignment was carried out in two separate steps as
reported by Bissantz, et al.50 Manual adjustment of the CLUSTALX
alignment was required to properly align the disulfide-forming
cysteine residues in the EL2 loop. The result was an unambiguous
alignment in the transmembrane (TM) helical regions of both the
hH1 and h5-HT2A sequences with that of the b2-adrenoceptor. This
alignment, along with a file containing the atomic coordinates of
the adrenergic b2 receptor (PDB ID = 2RH1), was used as input to
the MODELLER40 software package to generate a population of 100 dif-
ferent hH1 or h5-HT2A homology models. Each of these receptors
was subsequently energy-minimized.
4.1.9. 2-(9,10-Dihydroanthracen-9-yl)-N,N-dimethylethan-
amine oxalate (2c)
2-(9,10-Dihydroanthracene-9-yl)acetaldehyde 15 (0.4 g, 1.79
mmol), dimethylamine hydrochloride (0.292 g, 3.59 mmol) and
titanium isopropoxide (1.02 g, 3.59 mmol) were added to a solu-
tion of triethylamine (0.363 g, 3.59 mmol) in absolute ethanol.
The reaction mixture was stirred at rt for 12 h. NaBH3 (0.1 g,
2.68 mmol) was then added and the mixture was stirred for 12 h.
The reaction was quenched by pouring the mixture into aqueous
ammonia (30 mL, 2 N). The resulting precipitate was filtered and
washed with CH2Cl2 (5 Â 20 mL). The filtrate was collected and ex-
tracted with CH2Cl2. The CH2Cl2 portion was dried (MgSO4) and
concentrated under reduced pressure to provide viscous yellow
oil. The resulting yellow oil was purified using medium pressure
column chromatography (CH2Cl2/MeOH, 9:1) to provide 2c
(0.248 g, 55%). The yellow oil was dissolved in anhydrous acetone
(10 mL) and oxalic acid (0.043 g, 0.47 mmol) was added until no
further precipitate formed. The oxalate salt was recrystallized from
methanol/ether to provide 2c oxalate as pale yellow crystals. Yield
(55%). Mp 183–185 °C (oxalate). 1H NMR (300 MHz, CDCl3): d 1.83
(q, J = 7.5 Hz, 2H, CH2), 2.26 (s, 6H, (CH3)2), 2.28–2.30 (m, 2H, CH2–
N), 3.94 (d, J = 18.6 Hz, 1H, Ar-CH2-Ar), 4.14 (d, J = 18.6 Hz, 1H, Ar-
CH2-Ar), 4.1 (t, J = 7.2 Hz, 1H, CH), 7.1–7.3 (m, 8H, Ar-H). 13C NMR
(75 MHz, CDCl3): d 32.30, 40.41, 45.32, 52.20, 56.21, 126.80,
128.71, 138.42, 140.10. Anal. (C18H21NÁC2H2O4Á0.5H2O): C, H, N.
43
The automated docking program GOLD version 3.01 (Cam-
bridge Crystallographic Data Centre, Cambridge, UK) was then used
to dock the classical antihistaminic diphenhydramine and the
high-affinity ligands 3c and AMDA (1a) from the synthesized dihy-
droanthracene matrix into each of the 100 receptor models using
the ChemScore fitness function. Based on the fitness function val-
ues, steric and electronic interactions of the docked poses and re-
ported site-directed mutagenesis data, one receptor model was
selected to represent the ligand binding site of the hH1 and h5-
HT2A receptors. These models were subsequently analyzed using
PROCHECK51 and the ProTable facility within SYBYL to assess the
geometric integrity of various structural elements (bond lengths,
torsion angles, etc.) of each receptor model. After checks for stereo-
chemical integrity, the receptor models were used for the docking
of all the target compounds. Ligand molecules were created within
SYBYL and energy-minimized using the same parameters as were
used for the receptor models. Basic amines were protonated to
form ammonium ions. GOLD was used to dock each resulting ligand
structure (using the parameter set defined by the ‘standard default
settings’ option) into the final receptor model. Each receptor–li-
gand complex was then energy-minimized with its best-ranked
docking pose.
The HINT scoring function was utilized (version 3.12) to explore
and visualize hydropathic interactions by analyzing the ligand–
receptor complexes generated by the automated docking program
GOLD. The interaction scores were calculated for the highest-ranked
ligand conformation. The receptors (5-HT2A and H1) and ligands
were partitioned as distinct molecules. The ‘all hydrogen atoms’
option was employed in the H-bonding model, and hydrogen
atoms at unsaturated positions and alpha to heteroatoms were
considered potential H-bond donors. The inferred solvent model,
which considers the partition of each residue based on its hydro-
4.1.10. N-Methyl-2,2-diphenylethanamine (4b)
Yield (50%). Mp 149–151 °C (oxalate) 1H NMR (300 MHz,
CDCl3): d 2.47 (s, 3H, CH3), 3.02 (d, J = 8.1 Hz, 2H, CH2), 4.3 (t,
J = 6 Hz, 1H, Ar-CH-Ar), 7.12–7.26 (m, 10H, Ar-H). 13C NMR
(75 MHz, CDCl3): d 37.20, 44.80, 60.20, 126.60, 128.50, 129.60,
143.0. Anal. (C15H17NÁC2H2O4): C, H, N.
4.1.11. N,N-Dimethyl-3,3-diphenylpropan-1-amine (5c)
Yield (20%). Mp 150–153 °C (oxalate) 1H NMR (300 MHz,
CDCl3): d 2.52–2.56 (m, 2H, CH2), 2.85 (s, 6H, (CH3)2), 3.0–3.02
(m, 2H, CH2–N), 4.2 (t, J = 7.4 Hz, 1H, Ar-CH-Ar), 7.2–7.4 (m, 10H,
Ar-H). 13C NMR (75 MHz, CDCl3): d 32.20, 45.80, 50.60, 58.33,
126.30, 128.20, 129.10, 143.15. Anal. (C17H21NÁC2H2O4Á0.5H2O): C,
H, N.
4.1.12. 4,4-Diphenylbutan-1-amine (6a)
Yield (60%). Mp 198–200 °C (HCl) 1H NMR (300 MHz, CDCl3): d
1.42–1.48 (m, 2H, CH2), 2.03–2.1 (m, 2H, CH2), 2.73 (t, J = 6.2 Hz
2H, CH2–NH2), 4.1 (t, J = 7.2 Hz, 1H, Ar-CH-Ar), 7.1–7.3 (m, 10H,

6504
J. R. Shah et al. / Bioorg. Med. Chem. 17 (2009) 6496–6504
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This work was supported by United States Public Health Service
Grant R01-MH57969 (RBW), R01-GM71894 (GEK), NIMH Psycho-
active Drug Screening Program (BLR) U19MH82441 (BLR) and
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