3D-QSAR and 3D-QSSR models of negative allosteric modulators facilitate the design of a novel selective antagonist of human α4β2 neuronal nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.bmcl.2011.11.051

Subtype selective molecules for α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4β2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3β4 and a7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4β2 nAChRs and human α3β4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4β2 nAChRs.

Full text of DOI:10.1016/j.bmcl.2011.11.051

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1797–1813
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3D-QSAR and 3D-QSSR models of negative allosteric modulators facilitate
the design of a novel selective antagonist of human
nicotinic acetylcholine receptors
a4b2 neuronal
Brandon J. Henderson a, , Crina M. Orac b, Iwona Maciagiewicz b, Stephen C. Bergmeier b,
⇑
Dennis B. McKay a
a Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH (BJH, DBM), USA
b Department of Chemistry and Biochemistry, Ohio University, Athens, OH (CMO, IM, SCB), USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Subtype selective molecules for
sought as novel therapeutics for nicotine cessation.
a
4b2 neuronal nicotinic acetylcholine receptors (nAChRs) have been
4b2 nAChRs have been shown to be involved in
mediating the addictive properties of nicotine while other subtypes (i.e., 3b4 and 7) are believed to
Received 3 October 2011
Revised 11 November 2011
Accepted 14 November 2011
Available online 20 November 2011
a
a
a
mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to
understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes.
Here we present novel QSAR/QSSR models for negative allosteric modulators of human
a4b2 nAChRs and
Keywords:
human 3b4 nAChRs. These models support previous homology model and site-directed mutagenesis
a
Structure–activity relationship (SAR)
Qualitative structure–activity relationship
(QSAR)
Qualitative structure selectivity relationship
(QSSR)
studies that suggest a novel mechanism of antagonism. Additionally, information from the models pre-
sented in this work was used to synthesize novel molecules; which subsequently led to the discovery
of a new selective antagonist of human a4b2 nAChRs.
Ó 2011 Elsevier Ltd. All rights reserved.
Nicotinic acetylcholine receptor (nAChR)
a
a
4b2
3b4
Negative allosteric modulator (NAM)
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand
gated ion channels and members of the cys-loop family of recep-
tors. nAChRs are found both in the peripheral and central nervous
systems and are implicated in many diseases and disorders such
as: Alzheimer’s disease, epilepsy, autism, Parkinson’s disease,
depression, anxiety, and nicotine addiction.1,2 Worldwide, nicotine
addiction is a significant problem. Smoking is the primary cause of
preventable death worldwide and roughly 90% of the people who
non-competitive antagonists that target nAChRs.10,11 Mecamyl-
amine, a non-selective non-competitive nAChR antagonist, was
shown to promote 40% abstinence at the 1 year mark when used
as an agonist–antagonist therapy in combination with the nicotine
patch.10 In addition, Yoshimura et al. (2007)11 discovered a novel
negative allosteric modulator (NAM) that was selective for neuro-
nal nAChRs as opposed to the muscle nAChR which significantly
blocked nicotine self-administration on fixed and progressive ratio
schedules in rats. These data support the use of non-competitive
antagonists and NAMs as nicotine cessation therapies; however,
to produce new therapeutic molecules it is believed that nAChR
subtype selectivity must be pursued.12
attempt to quit are unable to do so.3 It is now known that
a4b2
nAChRs are primarily responsible for the addiction to tobacco re-
lated products.4–6 Current FDA approved treatments for tobacco
addiction are nicotine replacement, bupropion (ZybanÒ), and
varenicline (ChantixÒ). Each of these therapies has a modest suc-
cess of 20–30% abstinence 1 year after quit date.7,8 However, drugs
such as varenicline have been associated with severe adverse car-
diovascular effects.9 This combined with the low success rates of
therapies warrant the need for novel small molecules that can be
used in nicotine cessation. In an attempt to discover better thera-
peutics for nicotine cessation, some laboratories have proposed
Our laboratory has previously published the synthesis and
pharmacology of a novel class of NAMs.13–19 We have previously
reported a novel NAM, KAB-18, which shows selectivity for human
a
4b2 (Ha4b2) nAChRs and through SAR have identified several
chemical features important for its selectivity.19 One problem with
the study of non-competitive and allosteric agents is the fact that
most of these agents lack information concerning the site of inter-
action on their target receptor. To address this, we have con-
structed a homology model for the extracellular domain of the
⇑
Corresponding author. Tel.: +1 614 330 1463.
E-mail address: mckay.2@osu.edu (B.J. Henderson).
Ha4b2 nAChR and have identified the site in which these NAMs
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.051

1798
B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
interact allosterically through blind docking and molecular
dynamics (MD) simulations.19 In this study, three-dimensional
qualitative structure–activity relationship (3D-QSAR) studies and
three-dimensional qualitative structure-selectivity relationship
(3D-QSSR) studies were completed to study the relationship be-
tween functional activity (e.g., IC50 values) and selectivity of NAMs
with their 3D structures. This study reports the construction and
analysis of models that predict the detailed structural interactions
SYBYL’s CoMSIA program, linear regression analyses were per-
formed to correlate the experimentally derived pIC50 values with
the computationally derived pIC50 values (Fig. S1). Fifty-five mole-
cules were chosen for the H
molecules were chosen for the H
(Table 1). Correlation coefficients (r2) for the H
a
4b2 QSAR model training set and 42
4b2 QSAR and the QSSR models
4b2 QSAR, H 3b4
a
a
a
QSAR and QSSR models were 0.766, 0.761, and 0.871 respectively
for the training set (Table 3, Fig. S1). Ten molecules were chosen
for the test set for all molecules (Table 2). Correlation coefficients
of this novel class of NAMs18,19 with their binding site on H
nAChRs and H 3b4 nAChRs. In addition to this, we propose a mod-
el which distinguishes the physiochemical features that are impor-
tant for selectivity for H 4b2 nAChRs versus H 3b4 nAChRs that
a4b2
a
(r2) for the H
a4b2 QSAR, Ha3b4 QSAR and QSSR models were
0.620, 0.621, and 0.480 respectively for the test set (Table 3, Fig. S1).
CoMSIA descriptors for steric (green/yellow field maps), electro-
static (blue/red field maps), hydrophobic (purple/gray field maps),
hydrogen bond donor (cyan/magenta field maps), and hydrogen
bond acceptors (orange/red field maps) were generated for the
a
a
also agree with previously reported homology modeling, SAR,
and site-directed mutagenesis studies.19,23 Finally, these models
were used in the generation of novel Ha4b2 nAChR antagonists.
To facilitate the presentation of data, four regions for the NAM
scaffold have been defined (Fig. 1B). These four regions were defined
from a pharmacophore model that was generated previously by
using KAB-18 and KAB-18 like molecules.19 This pharmacophore
model featured four hydrophobic regions and one hydrogen bond
acceptor region. Region 1 was defined as the substitution on the
nitrogen moiety of the piperidine ring containing hydrophobic do-
main 1 (Fig. 1B). Region 2 was defined as the ester acyl substitution
containing the biphenyl (Fig. 1B). Region 3 was the piperidine ring
which has been defined in the pharmacophore as the fourth hydro-
phobic region (Fig. 1B). Region 4 was the linkage between Regions 2
and 3, containing an ester bond with a hydrogen bond accepting do-
main (Fig. 1B). All of the NAMs presented in this manuscript contain
one or more stereiogenic centers. In construction of the QSAR and
QSSR models the selected conformation of compounds used in the
alignment play a pivotal role in determining the position of the field
contribution maps and validation of the model. The conformation of
our NAMs was determined by selecting the stereoisomers that
match those found to be most stable in previously conducted dock-
Ha4b2 QSAR, Ha3b4 QSAR, and QSSR models (Figs. 2–4, respec-
tively). For detailed description of the model construction process,
refer to the Methods section (see Supplementary data). KAB-18
was used as the structural scaffold for all field contribution maps
(Figs. 2–4) due to its selectivity for H
For the H 4b2 nAChR QSAR model, steric contributions were
a
4b2 nAChRs.19
a
important surrounding the phenyl rings of Regions 1 and 2 as well
as the piperidine of Region 3 (Fig. 2A). Important electrostatic
interactions were predicted surrounding the anthranilic moiety
(positive) and the phenyl (negative) of Region 2 (Fig. 2B). Hydro-
phobic contributions were shown to be important around the phe-
nyl rings of Regions 1 and 2 as well as the piperidine of Region 3
(Fig. 2C) Hydrophobic contributions were shown to be disfavored
around the propyl chain of Region 1 (Fig. 2C). The protonated
hydrogen of the piperidine nitrogen of Region 3 is predicted to
act as a hydrogen bond donor (Fig. 2D) and the carbonyl of Region
4 is predicted to act as a hydrogen bond acceptor (Fig. 2E). These
predictions support the binding mode of KAB-18 that has been re-
cently presented by molecular dynamics (MD) simulation on the
ing and MD simulations on the H
a
4b2 nAChR homology model.19
Ha
4b2 nAChR homology model.19 This MD simulation predicted
Enantiomers of NAMs at position C3 (respective of the piperidine
that the protonated hydrogen of the piperidine nitrogen is involved
in a stabilizing hydrogen bond with the carbonyl group of Glu60 on
the b2 subunit.19 This agrees with the favored hydrogen bond do-
ring, Region 3, Fig. 1B) have shown no functional difference in either
Ha4b2 or Ha
3b4 nAChRs.19 Choosing this set of conformations for
NAMs in the QSAR and QSSR models allow the results to be com-
pared to homology model data in addition to previous SAR data.19
The alignment of NAMs (Fig. 1A) and the construction of QSAR and
nor predicted to occur in Region 3 by the Ha4b2 QSAR where the
cyan field surrounds the piperidine nitrogen (Fig. 2D). The carbonyl
of Region 4 was also shown to be involved in another important
hydrogen bond with Thr58 on the b2 subunit and this interaction
was validated through site-directed mutagenesis.19,23 This agrees
with the favored hydrogen bond acceptor predicted in Region 4
of the Ha4b2 QSAR where the orange field surrounds the carbonyl
of KAB-18 (Fig. 2E). Additionally, the model predicts favorable
interactions between negative electron densities of ligands in
QSSR (Ha4b2/Ha3b4) models using CoMSIA are described in detail
in the Methods section (refer to Supplementary data). For every
molecule used in the construction of the models, 3D structures were
prepared and are also described in the Methods section. All func-
tional data for training set molecules are detailed in Table 1 and
the functional data for test set molecules are found in Table 2. Using
Figure 1. Alignment of the negative allosteric modulators. (A) Alignment of molecules used in QSAR and QSSR models (Figs. 2–4). (B) Regions of the NAM scaffold displayed
with scaffold molecule, KAB-18.

B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
1799
Table 1
Observed and predicted IC50 values of NAMs used in QSAR and QSSR models (training set molecules)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
Pred. pIC50
QSSR (Ha4b2/Ha3b4)
a
a
Obs. pIC50
Obs. pIC50
Obs. [SI]a
Pred. [SI]
O
O
O
APB-1
4.99
5.00
4.96
4.86
N
CH3
O
O
O
O
O
O
APB-10
N
O
O
CH3
O
O
APB-4b
4.84
4.75
4.81
4.87
0.02
À0.08
O
N
Cl
O
O
O
APB-8
4.84
4.86
4.65
4.13
0.19
0.79
N
CH3
O
O
O
COB-1b
5.15
5.16
5.20
4.93
5.09
5.32
5.41
5.21
0.06
À0.19
À0.07
O
N
H
O
COB-2b
À0.16
O
N
CH3
O
COB-3b
5.62
5.04
5.67
5.03
5.19
5.18
4.82
5.07
0.43
0.73
O
N+
CH3
H3C
O
COB-8
À0.14
À0.05
N
H
O
O
DDR-13b
5.22
5.20
3.48
3.43
1.74
1.78
N
O
(continued on next page)

1800
B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
Table 1 (continued)
Structure
QSAR (H
a
4b2)
QSAR (H
a
3b4)
Pred. pIC50
QSSR (Ha4b2/Ha3b4)
a
a
Obs. pIC50
Pred. pIC50
Obs. pIC50
Obs. [SI]a
Pred. [SI]
O
DDR-15b
5.37
5.23
5.74
5.57
À0.38
À0.26
O
N
N3
O
O
DDR-18b
5.19
5.32
3.41
3.36
1.78
1.97
N
N
N
N
N
N
O
N
DDR-19b
5.55
5.59
5.37
5.34
0.19
0.34
O
N
O
N
O
DDR-20
4.94
4.89
4.86
3.64
0.08
1.07
O
O
H3C
CH3
CH3
O
O
O
N
O
DDR-21
4.96
4.99
4.51
4.65
0.45
0.19
N
O
O
H3C
CH3
O
N
O
DDR-25
5.02
5.00
CH3
O
O
H3C
CH3
H3C

B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
1801
Table 1 (continued)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
Pred. pIC50
QSSR (H
a
4b2/H
a3b4)
a
a
Obs. pIC50
Obs. pIC50
Obs. [SI]a
Pred. [SI]
CH3
O
O
O
N
O
DDR-26
4.92
5.06
4.11
4.66
0.81
0.15
N
CH3
O
O
H3C
CH3
CH3
O
O
O
N
O
DDR-27
5.12
5.05
N
CH3
O
O
H3C
CH3
H3C
O
NH
DDR-3b
DDR-4
DDR-5b
4.78
4.68
4.70
4.80
4.64
4.66
4.84
4.39
1.00
5.14
4.55
0.68
À0.06
À0.54
À0.05
4.00
N
Ph
0.29
N
Ph
N
O
Ph
O
NH
3.70
N
O
O
FFB-1
4.93
5.04
4.2
3.75
0.73
1.29
N
CH3
O
O
O
N
O
IB-2b
4.98
5.15
4.96
4.84
0.02
0.31
N
(continued on next page)

1802
B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
Table 1 (continued)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
Pred. pIC50
QSSR (H
a
4b2/H
a3b4)
a
a
Obs. pIC50
Obs. pIC50
Obs. [SI]a
Pred. [SI]
CH3
O
O
O
N
O
IB-4b
5.05
5.15
5.02
4.84
0.02
0.31
N
H3C
O
O
O
N
O
JHB-7
5.25
5.36
4.13
4.13
1.12
1.18
N
N
H
O
O
JHB-9b
4.75
4.86
4.99
5.01
0.24
À0.35
N
CH3
O
O
O
N
JHB-11
5.15
5.24
N
O
CF3
H3C
O
O
O
N
O
JHB-12
5.27
4.99
N
H3C
O
O
O
N
O
JHB-13
4.40
4.53
4.07
4.09
0.33
0.24
N
CH3
O
O
O
N
O
KAB-9
4.18
4.39
4.92
4.60
À0.75
À0.32
N
O
CH3
O

B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
1803
Table 1 (continued)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
Pred. pIC50
QSSR (Ha4b2/Ha3b4)
a
a
Obs. pIC50
Obs. pIC50
Obs. [SI]a
Pred. [SI]
CH3
O
O
O
N
O
KAB-10
4.61
5.01
5.38
4.94
À0.77
À0.08
N
CH3
O
O
O
N
O
KAB-11
4.53
4.60
N
CH3
O
O
O
N
O
KAB-13
4.95
4.80
4.73
4.97
0.22
À0.04
N
O
CH3
CH3
O
O
O
N
O
KAB-15
4.66
4.70
4.94
4.90
0.14
0.11
N
O
Cl
CH3
O
O
O
N
O
KAB-16
5.41
5.26
4.80
4.73
0.61
0.55
N
O
CH3
O
O
O
N
O
KAB-17
5.13
5.05
5.17
5.18
À0.04
À0.06
N
O
O
O
KAB-18b
4.87
5.03
1.00
1.73
3.87
3.30
N
(continued on next page)

1804
B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
Table 1 (continued)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
Pred. pIC50
QSSR (H
a
4b2/H
a3b4)
a
a
Obs. pIC50
Obs. pIC50
Obs. [SI]a
Pred. [SI]
O
O
HN
CH3
O
KAB-19b
5.26
5.31
5.37
5.42
À0.11
0.04
N
O
O
O
N
KAB-20b
5.30
5.22
5.23
5.13
0.07
0.05
O
O
O
N
N
N
O
KAB-21b
5.20
5.03
5.14
5.11
4.73
4.64
4.79
4.74
0.47
0.39
0.43
0.34
O
Cl
KAB-22b
CH3
O
O
O
KAB-23b
5.10
5.10
5.26
5.14
4.92
5.26
4.93
4.71
0.19
0.34
0.37
N
O
CF3
O
KAB-24b
À0.16
N
CH3
O
O
O
N
O
KAB-28
5.14
5.24
4.57
4.78
0.57
0.44
N
O
Ph
CH3
CH3
O
O
O
N
KAB-30b
5.31
5.26
5.30
5.17
0.01
0.07
O
N

B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
1805
Table 1 (continued)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
Pred. pIC50
QSSR (Ha4b2/Ha3b4)
a
a
Obs. pIC50
Obs. pIC50
Obs. [SI]a
Pred. [SI]
CH3
O
O
O
N
O
KAB-32
5.77
4.77
4.84
5.24
4.75
4.84
N
O
CH3
O
O
O
N
O
KAB-33
N
O
CH3
O
O
O
N
O
KAB-35
N
O
CH3
O
CH3
O
O
O
N
O
KAB-37
4.91
5.09
4.13
4.22
0.78
0.56
N
O
Cl
Cl
CH3
O
O
O
N
O
KAB-38
4.76
4.63
5.04
4.76
À0.28
À0.03
N
Cl
Cl
O
NEB-2
5.38
5.29
O
N
H
Cl
O
O
O
N
O
PPB-1
4.97
4.96
4.84
4.81
0.14
0.23
N
(continued on next page)

1806
B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
Table 1 (continued)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
Pred. pIC50
QSSR (H
a
4b2/H
a3b4)
Obs. pIC50
Obs. pIC50
Obs. [SI]a
Pred. [SI]
a
a
O
O
O
N
PPB-3
5.29
5.22
4.48
4.71
0.81
0.58
O
N
O
O
O
N
PPB-6
5.05
5.11
O
N
H3C
CH3
CH3
O
O
O
N
O
SMB-1
5.08
4.92
2.70
3.10
2.38
1.89
N
O
CH3
CH3
O
O
CH3
O
O
O
N
O
SMB-2
5.06
4.93
N
CH3
O
O
O
CH3
a
ÀLog of geometric mean, n = 4–10.
b
Previously reported by Henderson et al.19
Table 2
Observed and predicted IC50 values of NAMs used in QSAR and QSSR models (test set molecules)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
QSSR (H
Obs. [SI]a
a
4b2/H
a3b4)
a
a
Obs. pIC50
Obs. pIC50
Pred. pIC50
Pred. [SI]
CH3
O
O
O
N
O
O
KAB-39
4.90
5.02
4.38
4.32
0.68
0.82
N
O
CH3
O
CH3

B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
1807
Table 2 (continued)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
QSSR (H
Obs. [SI]a
a4b2/Ha3b4)
a
a
Obs. pIC50
Obs. pIC50
Pred. pIC50
Pred. [SI]
CH3
O
O
O
O
N
O
PPB-5
4.41
4.87
4.54
4.66
À0.13
À0.28
N
O
O
O
N
PPB-9
5.35
5.12
4.93
4.55
0.42
0.29
O
N
CH3
O
O
O
N
PPB-11
5.35
5.24
4.93
4.78
0.42
0.51
O
N
O
O
O
N
O
COB-5
5.29
5.35
5.43
4.99
À0.14
À0.44
N
O
APB-21
4.90
4.85
4.81
4.38
0.09
0.35
N
N
O
O
COB-4
5.09
5.54
5.00
5.17
4.98
5.14
5.05
5.10
0.11
0.39
0.65
0.13
O
N
N
DDR-17
N
N
CH3
O
O
O
N
O
KAB-8
4.84
5.03
4.97
4.75
À0.14
0.26
N
(continued on next page)

1808
B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
Table 2 (continued)
Structure
QSAR (H
a
4b2)
Pred. pIC50
QSAR (H
a
3b4)
QSSR (H
Obs. [SI]a
a4b2/Ha3b4)
a
a
Obs. pIC50
Obs. pIC50
Pred. pIC50
Pred. [SI]
CH3
O
O
O
N
O
KAB-14
4.82
4.95
4.76
4.81
0.06
À0.01
N
O
Cl
Cl
a
ÀLog of geometric mean, n = 4–10.
Region 1 (Fig. 2B). This has been observed previously with an ana-
log of KAB-18 that included a carbonyl in Region 1,19 which lead to
Table 3
QSAR and QSSR analysis results
a 3-fold increase in potency for H
a4b2 nAChRs.
Dependent variable
Data set
QSAR (H
The H 3b4 nAChR QSAR model predicted that steric features in
a
QSAR (H
a4b2)
a3b4)
QSSR
both Regions 1 and 2 had a positive effect on potency (Fig. 3A). Elec-
trostatic field maps predicted that negative electrostatics contribute
to potency in Region 1 and positive electrostatics contribute to po-
tency in Region 2 (Fig. 3B). Hydrophobic features were shown to
be disfavored in Regions 1 and 2 (Fig. 3C). Hydrogen bond donors
were shown to be favorable with the piperidine of Region 3 and
the propyl chain in Region 1 (Fig. 3D). Hydrogen bond acceptors in
r2 (training set)
standard error
F value
0.856
0.15
26.13
6
0.761
0.57
15.1
6
0.871
0.57
14.3
6
Components
r2 (test set)
0.620
0.621
0.480
Figure 2. CoMSIA models for Ha4b2 nAChRs. Steric (A), electrostatic (B), hydrophobic (C), hydrogen bond donor (D), and hydrogen bond acceptor (E) field contribution maps
are shown with KAB-18. Bottom right, color key to field contribution maps. Greater potency (lower IC50 values) is correlated with: less bulk near yellow/gray, more bulk near
green/purple, more hydrogen bond acceptors near blue/red, less hydrogen bond acceptors near red, More hydrogen bond donors near cyan and less hydrogen bond donors
near magenta.

B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
1809
Region 4 were shown to be unfavorable around the carbonyl of Re-
gion 4 and favorable with the phenyl of Region 2 (Fig. 3E). One fea-
ture that supports findings from previous SAR data,19 concerns
aromatic-containing features in Regions 1 and 2 (e.g., phenylpropyl
substitution in Region 1) that contribute to a decrease of potency on
specifically at the phenyl ring positions, contribute to selectivity
for H 4b2 nAChRs while hydrophobics in Region 4 decrease selec-
tivity (Fig. 4C). Donor contribution maps predicted that Hydrogen
bond donors in Region 4 contribute to selectivity on H 4b2 nAChRs
(Fig. 4D). Acceptor maps predicted that hydrogen bond acceptors
are favored in Region surrounding the ester carbonyl of
(Fig. 4E). Many of these findings predicted by the QSSR model sup-
port findings of our previous SAR data.19 As mentioned earlier, the
presence of aromatic containing features in both Regions 1 and 2
a
a
Ha
3b4 nAChRs. This agrees with the unfavored hydrophobics that
are predicted by the H 3b4 QSAR where the gray fields surround
the phenylpropyl of KAB-18 ( Fig. 3C). One finding that differs
significantly between the H 4b2 and H 3b4 QSAR is in the lack
of a favored hydrogen bond acceptor in Region 3 of the H 3b4 QSAR
(Fig. 3D). Also, the hydrophobics and hydrogen bond acceptor field
maps are opposing when compared between the H 4b2 and
3b4 QSAR models (Figs. 2C, E and 3C, E). Altogether, this suggests
that this class of NAMs may have significantly different binding
interactions between H 4b2 and H 3b4 nAChRs.
The QSSR model predicted that steric interactions are important
for selectivity on H 4b2 nAChRs surrounding the phenyl rings of
4
a
a
a
a
were shown to be important for the selectivity on Ha4b2 nAChRs.
The predictions of the steric and hydrophobic QSSR field contribu-
tion maps, which show both green and purple fields surrounding
KAB-18’s phenylpropyl (Region 1) and phenyl group (Region 2),
support this (Fig. 4A and C). As mentioned earlier, SAR data sup-
ported the importance of the specific orientation of the carbonyl
in Region 4. The acceptor field contribution maps support this find-
ing as well as the favored HBA field (orange field) surrounds the
carbonyl group of KAB-18. This suggests that the atom acceptor’s
placement (flanking the biphenyl of Region 2) is important for
selectivity (Fig. 4E). Many of the features highlighted in this QSSR
model correlate blind docking and MD simulation studies with
a
Ha
a
a
a
Regions 1, 2, and 3 (Fig. 4). The field contribution maps also
showed that sterics surrounding the propyl chain of Region 1 de-
crease selectivity (Fig. 4A). Thus, the net effect of removing steric
contributions in Region 1 may lead to an increase in selectivity.
Electrostatic field maps predicted that positive electrostatics in Re-
gions 1 (around the phenylpropyl of KAB-18) and 4 (ester Region of
an Ha
4b2 nAChR homology model.19 The regions shown to be
important in the QSSR model overlap with amino acid residues
that have been proposed to interact with KAB-18 (i.e., Phe118 in
Region 1 and Thr58 in Region 4 [both on b2 subunit]). These resi-
KAB-18) contribute to selectivity for Ha4b2 nAChRs while negative
electrostatics near Region 2 (biphenyl of KAB-18) decrease selec-
tivity ( Fig. 4B). Hydrophobic maps predicted that hydrophobic
features in Regions 1, 2, and 3 (piperidine region of KAB-18),
dues are not conserved in the H
a3b4 nAChR and may be important
for mediating selectivity for H
a
4b2 nAChRs. Previous SAR studies19
Figure 3. CoMSIA models for Ha3b4 nAChRs. Steric (A), electrostatic (B), hydrophobic (C), hydrogen bond donor (D), and hydrogen bond acceptor (E) field contribution maps
are shown with KAB-18. Bottom right, color key to field contribution maps. Greater potency (lower IC50 values) is correlated with: less bulk near yellow/gray, more bulk near
green/purple, more hydrogen bond acceptors near blue/red, less hydrogen bond acceptors near red, More hydrogen bond donors near cyan and less hydrogen bond donors
near magenta.

1810
B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
Figure 4. 3D-QSSR CoMSIA models of NAMs. Steric (A), electrostatic (B), hydrophobic (C), hydrogen bond donor (D), and hydrogen bond acceptor (E) field contribution maps
are shown with KAB-18. Bottom right, color key to field contribution maps. Greater selectivity (more potent on H 4b2 vs H 3b4) is correlated with: less bulk near yellow/
a
a
gray, more bulk near green/purple, more hydrogen bond acceptors near blue/red, less hydrogen bond acceptors near red, More hydrogen bond donors near cyan and less
hydrogen bond donors near magenta.
R-NH2 =
molecules that have improved potency on H
preserve selectivity for H 4b2 nAChRs. Novel molecules were syn-
thesized (Scheme 1) and tested for inhibitory activity on both
4b2 and H 3b4 nAChRs (Table 4).
a4b2 nAChRs; but still
a
NH2
NH2
Ha
a
NH2
NH2
Ph
O
Ph
N
R
CO2H
EDCI, HOBT
CH2Cl2
MW (140 ºC, 10 min)
+
R NH2
N
H
NH2
N
N
NH2
Scheme 1. Synthesis of novel biphenyl amides.
Two molecules (COB-170 and COB-171) were found to be 2-
fold more potent than lead molecule, KAB-18. The phenylmethyl
analog (COB-170) had an IC50 of 7.5 and 8.5 M on H 4b2 and
3b4 nAChRs, respectively (Table 4). The phenyl analog (COB-
171) had an IC50 of 6.9 and 10.7 M on H 4b2 and H 3b4 nAC-
hRs, respectively (Table 4). The naphthylmethyl (COB-172) pro-
duced no change in potency on H 4b2 nAChRs (IC50 value,
13.9 M, Table 4); but maintained selectivity for H 4b2 nAChRs.
The pyridinyl analog (COB-173) had an IC50 value of 13.6 and
20.0 M on H 4b2 and H 3b4 nAChRs, respectively (Table 4).
The pyrimidyl analog (COB-174) had an IC50 value of 25.1 and
39.6 M on H 4b2 and H 3b4 nAChRs, respectively (Table 4). Fi-
nally the naphthyl analog (COB-175) had an IC50 value of 10.7 and
18.2 M on H 4b2 and H 3b4 nAChRs, respectively (Table 4).
Concerning selectivity for H 4b2 nAChRs over H 3b4 nAChRs,
COB-170, COB-171, COB-173, and COB-174 are all nonselective
have identified three regions of importance for the selectivity of
NAMs on H 4b2 nAChRs: (1) The phenyl in Region 2; (2) the place-
ment of the carbonyl group in Region 4; and (3) the presence of an
aromatic feature in Region 1. When the aromatic feature is
removed from Region 1, a loss of selectivity is observed; however,
these molecules show an increase in potency up to 5-fold.19 This is
l
a
a
Ha
l
a
a
a
l
a
supported by the H
model also predicted that hydrophobic features were important
for H 4b2 selectivity in Regions 1 and 3 ( Fig. 4C) while steric
features in Region 1 may decrease selectivity for H 4b2 nAChRs
(Fig. 4A). Therefore, increasing the hydrophobics in Region 3 may
improve selectivity for H 4b2 nAChRs. We hypothesized that by
a4b2 nAChR QSAR model (Fig. 2C). The QSSR
l
a
a
a
a
l
a
a
a
l
a
a
combining these two findings (incorporating reduced sterics in Re-
gion 1 and increased hydrophobics in Region 3) we would obtain
a
a

B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
1811
Table 4
Inhibition of new antagonists on Ha4b2 and Ha3b4 nAChRs
O
H
a4b2 nAChRs
H
a3b4 nAChRs
R
N
H
b
b
c
R
IC50 valuea
(lM)
nH
IC50 valuea
(lM)
nH
Fm
COB-170
7.5 (6.1–9.2)
6.9 (5.8–8.2)
À1.1
À1.3
7.6 (6.4–9.0)
À1.8
1.0
1.6
COB-171
COB-172
COB-173
10.7 (8.4–13.6)
>100d
À2.2
À0.4
13.9 (6.1–31.6)
À0.7
>10
13.6 (12.0–15.5)
25.1 (19.9–31.7)
À1.0
À1.5
17.5 (10.7–28.4)
39.6 (36.1–43.6)
À0.9
À0.9
1.3
1.6
N
N
COB-174
N
COB-175
10.7 (8.3–13.8)
À0.8
18.2 (9.9–33.5)
À1.2
1.8
a
Values represent geometric means (confidence limits), n = 3–7.
nH, Hill coefficient.
b
c
Fold difference in potency H
a3b4/H
a4b2.
d
No activity up to concentrations of 100
lM.
(Table 4). COB-175 showed a 2-fold preference for H
(Table 4). The methylnaphthyl analog (COB-172) is as potent and
a
4b2 nAChRs
The new amide-containing molecules (IMB-132, IMB-133,
IMB-134, IMB-135) were all more potent that their original
selective as lead molecule, KAB-18.
scaffolds (DDR-14, KAB-18, KAB-24, COB-4) on H
(Table 5). IMB-132 resulted in a 1.5-fold increase in potency on
4b2 nAChRs (IC50 value, 2.7 M, Table 5). IMB-134 resulted
in a 2.1-fold increase in potency on H 4b2 nAChRs (IC50 value,
3.9 M, Table 5). IMB-135 resulted in a 1.4 increase in potency
on H 4b2 nAChRs (IC50 value, 5.9 M, Table 5). Finally, IMB-133
resulted in a 2.4-fold increase in potency on H 4b2 nAChRs
(IC50 value, 5.7 M, Table 5). IMB-132 showed a 2-fold decrease
in potency on H 3b4 nAChRs compared to its scaffold molecule,
DDR-14 (IC50 value 6.3
a4b2 nAChRs
The Ha4b2 nAChR QSAR model shows that negative electron-
ics contribute to potency near the piperidine moiety of Region 3
contribute to potency (Fig. 2B). This is also supported by previous
data that shows the addition of an amide group leads to an in-
crease in potency.19 To determine the importance of this finding,
several new molecules (IMB-132, IMB-133, IMB-134, IMB-135)
containing amide groups in Region 1 were made from scaffolds
that have been previously reported19 (KAB-18, DDR-14, KAB-24,
COB-4) (Scheme 2).
Ha
l
a
l
a
l
a
l
a
l
M, Table 5). IMB-133, IMB-134, and
O
O
O
CF3
O
O
O
O
H
N
H
N
H
N
H
N
O
KAB-18
KAB-24
DDR-14
COB-4
O
O
O
CF3
O
O
O
O
O
N
N
N
N
O
O
O
O
IMB-134
IMB-133
IMB-132
IMB-135
Scheme 2. Novel compounds containing amide functionality in Region 1 and previously reported scaffolds. Synthetic details have been previously described.13,16,20–22

1812
B. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1797–1813
Table 5
two of the six showed a higher preference for H
3b4 nAChRs (Table 4). Two molecules (COB-170 and COB-171)
were found to be 2-fold more potent than lead molecule, KAB-18.
The naphthylmethyl (COB-172) maintained potency on H 4b2
nAChRs and also maintained selectivity for H 4b2 nAChRs (Ta-
ble 4). This can be considered a significant improvement, as COB-
172’s scaffold is more ‘drug like’ when compared to KAB-18
(Table S1); but is still selective for Ha4b2 nAChRs. The novel syn-
thesis of the IMB compounds also shows strong evidence that
inclusion of amide groups may improve potency to a small degree;
but does so consistently. Altogether, this work presents novel QSAR
and QSSR models for nAChRs. These models present important
chemical features for a novel class of NAMS that promote both
a4b2 nAChRs over
Inhibition of new antagonists on Ha4b2 and Ha3b4 nAChRs
Ha
H
a
4b2 nAChRs
H
a
3b4 nAChRs
a
b
b
c
IC50 value (
l
M)a
nH
IC50 value (
l
M)a
nH
Fm
a
IMB-132
DDR-14d
IMB-133
KAB-18d
IMB-134
KAB-24d
IMB-135
COB-4d
4.3 (2.2–8.5)
6.5 (4.1–10.4)
5.6 (3.6–9.0)
13.5 (9.7–18.5)
3.9 (3.3–4.8)
8.0 (4.2–15.3)
5.9 (5.2–6.7)
8.1 (2.1–30.7)
À1.6
À1.6
À0.9
À1.4
À1.5
À0.8
À1.2
À0.8
6.0 (4.3–8.3)
2.7 (0.4–17.9)
>100e
À1.6
À1.4
ꢀ
1.5
2.4
2.1
1.4
>100e
ꢀ
6.3 (5.4–7.3)
5.5 (1.7–17.4)
9.4 (7.4–11.9)
10.5 (7.6–14.4)
À2.1
À0.8
À1.5
À1.0
a
b
c
Values represent geometric means (confidence limits), n = 3–5.
nH, Hill coefficient.
selectivity and potency on H
have aided in the design and synthesis of potent, novel antagonists
of H 4b2 and H 3b4 nAChRs as well as the discovery of a new,
a4b2 nAChRs. Finally, these models
Fold difference in potency carbonyl/non-carbonyl.
d
e
Previously reported data.19
No activity up to concentrations of 100 lM.
a
a
drug-like, selective antagonist of Ha4b2 nAChRs (COB-172).
IMB-135 showed no change in potency on
compared to its scaffolds (KAB-18, KAB-24, COB-4, respectively).
Ha3b4 nAChRs
Acknowledgments
The QSAR and QSSR models presented here describe the physio-
All stably-transfected human cell lines were kindly provided by
Dr. Jon M. Lindstrom, Department of Neuroscience School of Med-
icine, University of Pennsylvania, Philadelphia, PA. This work was
supported by the National Institutes of Health National Institute
on Drug Abuse [Grant DA029433]. Financial support for B.J.H. is
from the National Institutes of Health National Institute on Drug
Abuse Diversity Supplement. We also want to thank Phil Cruz of
Tripos for his help in providing technical support on SYBYL 7.1 fea-
tures and applications.
chemical interactions that are important for potency on H
a
4b2 and
4b2 nAChRs versus
3b4 nAChRs. The fact that these models agree with previously
H
a
3b4 nAChRs as well as selectivity for Ha
Ha
reported modeling and functional data support their strength and
validity. With the models presented here combined with the infor-
mation gathered from previous SAR, homology modeling, and site
directed mutagenesis, there are many physiochemical features
identified in this scaffold that mediate Ha4b2 nAChR selectivity.
These include steric/hydrophobic features in both Regions 1 and
2 as well as a hydrogen bond acceptor in Region 4. Previous SAR
has highlighted the importance of aromatic rings in Regions 1
and 2; but these models suggest that aromatics may not be neces-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.051.
sary to preserve potency or selectivity on Ha4b2 nAChRs. If true,
this allows for additional flexibility in the design of novel scaffolds.
However, this will need to be confirmed by designing and synthe-
sizing novel molecules containing distinct hydrophobic and steric
features as opposed to aromatic features to determine how this
will affect potency and selectivity. Previous SAR also points to
the importance of the carbonyl group in Region 4. The QSAR and
QSSR models suggest the importance of this position lies in the role
as a hydrogen bond acceptor. This finding correlates with the dock-
ing studies that show a stable hydrogen bond between Thr58 of the
b2 subunit and the carbonyl group in KAB-18’s Region 4.19,23 There
may be potential at this position for increasing selectivity by plac-
ing a feature that will enable stronger hydrogen bonding with
Thr58 (b2 subunit). Most importantly, this finding suggests that
maintaining an acceptor atom at this position is significant for both
potency and selectivity and should be remain in the design of fu-
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a
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