Insights into the structural determinants required for high-affinity binding of chiral cyclopropane-containing ligands to α4β2-nicotinic acetylcholine receptors: An integrated approach to behaviorally active nicotinic ligands

Article abstract of DOI:10.1021/jm3008739

Structure-based drug design can potentially accelerate the development of new therapeutics. In this study, a cocrystal structure of the acetylcholine binding protein (AChBP) from Capitella teleta (Ct) in complex with a cyclopropane-containing selective α4β2-nicotinic acetylcholine receptor (nAChR) partial agonist (compound 5) was acquired. The structural determinants required for ligand binding obtained from this AChBP X-ray structure were used to refine a previous model of the human α4β2- nAChR, thus possibly providing a better understanding of the structure of the human receptor. To validate the potential application of the structure of the Ct-AChBP in the engineering of new α4β2-nAChR ligands, homology modeling methods, combined with in silico ADME calculations, were used to design analogues of compound 5. The most promising compound, 12, exhibited an improved metabolic stability in comparison to the parent compound 5 while retaining favorable pharmacological parameters together with appropriate behavioral end points in the rodent studies.

Full text of DOI:10.1021/jm3008739

Subscriber access provided by FONDREN LIBRARY, RICE UNIVERSITY
Article
Insights into the Structural Determinants Required for High Affinity Binding
of Chiral Cyclopropane-Containing Ligands to #4#2-Nicotinic Acetylcholine
Receptors; An Integrated Approach to Behaviorally Active Nicotinic Ligands
Han-Kun Zhang, J. Brek Eaton, Li-Fang Yu, Mieke Nys, Angelica Mazzolari, René van
Elk, August B. Smit, Vadim Alexandrov, Taleen Hanania, Emily Sabath, Allison Fedolak,
Daniela Brunner, Ronald J. Lukas, Giulio Vistoli, Chris Ulens, and Alan P. Kozikowski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3008739 • Publication Date (Web): 28 Aug 2012
Downloaded from http://pubs.acs.org on September 7, 2012
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 25
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Insights into the Structural Determinants Required for High Affinity Binding of
Chiral Cyclopropane-Containing Ligands to α4β2-Nicotinic Acetylcholine
Receptors; An Integrated Approach to Behaviorally Active Nicotinic Ligands
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†
‡
†
§
┴
║
HanꢀKun Zhang, J. Brek Eaton, LiꢀFang Yu, Mieke Nys, Angelica Mazzolari, René van Elk,
║
ꢁ
ꢁ
ꢁ
ꢁ
August B. Smit, Vadim Alexandrov, Taleen Hanania, Emily Sabath, Allison Fedolak, Daniela
,
ꢁ,#
,‡
,┴
,§
,†
Brunner,* Ronald J. Lukas,* Giulio Vistoli,* Chris Ulens,* and Alan P. Kozikowski*
†
Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of
‡
Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, Division of Neurobiology, Barrow
§
Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013, Laboratory of Structural
Neurobiology, Dept. of Cellular and Molecular Medicine, Herestraat 49, PB 601, Bꢀ3000 Leuven,
┴
Belgium, Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università degli Studi di Milano,
║
Via Mangiagalli 25, Iꢀ20133, Milan, Italy, Department of Molecular & Cellular Neurobiology, Center
for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, The
ꢁ
Netherlands, PsychoGenics, Inc., 765 Old Saw Mill River Road, Tarrytown, New York 10591, and
#Department of Psychiatry, Columbia University, NYSPI, 1051 Riverside Drive, New York 10032.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 25
ABSTRACT: Structureꢀbased drug design can potentially accelerate the development of new
therapeutics. In this study, a coꢀcrystal structure of the acetylcholine binding protein (AChBP) from
Capitella teleta (Ct) in complex with a cyclopropaneꢀcontaining, selective α4β2ꢀnicotinic acetylcholine
receptor (nAChR) partial agonist (compound 5) was acquired. The structural determinants required for
ligand binding obtained from this AChBP Xꢀray structure were used to refine our previous model of the
human α4β2ꢀnAChR, thus possibly providing a better understanding of the structure of the human
receptor. In order to validate the potential application of the structure of the CtꢀAChBP in the
engineering of new α4β2ꢀnAChR ligands, homology modeling methods, combined with in silico
ADME calculations, were used to design analogs of compound 5. The most promising compound 12,
exhibited an improved metabolic stability in comparison to the parent compound 5 while retaining
favorable pharmacological parameters together with appropriate behavioral endpoints in the rodent
studies.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Introduction
Nicotinic acetylcholine receptors (nAChRs) belong to the Cysꢀloop superfamily of ligandꢀgated ion
1
channels (LGICs) that also include 5ꢀHT , GABA , and glycine receptors. They are widely distributed
3
A/C
in the central and peripheral nervous systems, where they broadly participate in regulating major
physiological functions and pathophysiological processes associated with learning and memory, mood,
2
, 3
reward, motor control, arousal, analgesia, and inflammation.
Mammalian neuronal nAChRs are
homoꢀ or heteromeric pentamers assembled from diverse combinations of subunits (α2–α10 and β2–
β4). The variety in subtype stoichiometries results in neuronal nAChRs that exhibit highly variable
properties in regard to ligand pharmacology, activation and desensitization kinetics, cation permeability,
and subcellular and regional distribution. Each nAChR subunit consists of a large aminoꢀterminal
extracellular domain (ECD), a transmembrane domain comprising four αꢀhelices (M1–M4), and a
variable cytoplasmic domain between M3 and M4. Acetylcholine (ACh) binding sites are thought to be
formed between the subunit interfaces of the ECD that involve soꢀcalled loops A–C on the principle face
ACS Paragon Plus Environment

Page 3 of 25
Journal of Medicinal Chemistry
of an αꢀtype subunit and the adjacent, complementary face of the neighboring subunit involving loops
D–F. Known heteromeric pentamers have a minimum of two αꢀtype subunits whereas homopentamers
have five, resulting in two to five AChꢀbinding sites within the ECD, the occupancy of which by ACh or
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
other agonists leads to global allosteric transitions of the receptor protein that mediate channel opening.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The diversity of nAChR subtypes and their putative, distinctive roles in different functions provides an
opportunity, in principle, to produce subtypeꢀspecific ligands that may serve to treat a variety of
conditions. However, to date, the high sequence homology across individual subtypes provides a
substantial challenge for the development of subtypeꢀselective nicotinic drugs. Nicotinic ligands lacking
sufficient subtypeꢀselectivity may cause adverse side effects. For instance, nicotine (1) has wellꢀknown
and epibatidine (2) has potential therapeutic benefits for a number of nervous system disorders (Figure
1
). However, the therapeutic potential of these two natural products is compromised by their
unacceptable side effects arising from their affinity to almost all subtypes of nAChRs, albeit with
different potencies. Furthermore, although varenicline (3) has been launched and marketed as an α4β2ꢀ
nAChR partial agonist for the indication of tobacco addiction, peripheral and central side effects, such as
4
, 5
nausea, gastrointestinal symptoms, and changes in mood have been observed in some patients.
Compound 3 also functions as a full agonist at α7ꢀ and α3β4*ꢀnAChRs (the asterisk denotes the
6
possible integration of other subunits into the pentamer), and also as a 5ꢀHT agonist.
3
Thus, understanding the detailed atomic structure remains crucial to the design of novel nicotinic
agents with minimum side effects that selectively target designated receptor subtypes and thus the
related nervousꢀsystem pathologies to which they are linked. Highꢀresolution crystal structures of
integral membrane protein, mammalian nAChRs are not yet available. Nevertheless, the invertebrate
pentameric acetylcholine binding proteins (AChBPs), which are structural and functional surrogates for
the Nꢀterminal, extracellular ligandꢀbinding domain (LBD) of nAChRs, are more readily crystallized,
and they provide wellꢀstudied structural models that provide insights into ligand recognition sites and
7, 8
other elements of nAChR ECDs. To date, AChBPs from several different snail species have been
identified, and more than fifty different ligandꢀbound coꢀcrystal structures have been determined,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 25
facilitating both mechanistic studies and nicotinic drug development. The majority of these structures
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
have been obtained with AChBP from Aplysia californica (Ac) or Lymnaea stagnalis (Ls), which
exhibit substantial differences in affinities for nicotinic ligands and thereby mimic distinct subtypes of
9, 10
nAChR.
Recently, a novel AChBP has been identified from the marine annelid Capitella teleta (Ct),
which more closely mimics α4β2ꢀnAChRs and their high affinity binding of several ligands, including
compound 3, thus providing a potentially useful model to study structural determinants of ligand
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1, 12
recognition at this receptor subtype.
H
Cl
N
N
N
N
NH
N
N
(-)-nicotine, 1
epibatidine, 2
varenicline, 3
OH
O
OR
HN
O
HN
N
N
sazetidine-A, 4
5, R = -H
6, R = -CONHCH3
Figure 1. Selected examples of nAChR ligands.
Accumulating evidence suggests that drugs targeting α4β2*ꢀnAChRs, which are the predominant
1
3, 14
subtype of neuronal nAChRs, may prove useful in the management of major depression.
In pursuit
of antidepressants that exhibit fewer side effects and act pharmacologically in novel ways, we recently
reported the introduction of a chiral cyclopropane ring in place of the acetylene bond in sazetidineꢀA (4)
15, 16
that furnished a series of cyclopropaneꢀcontaining ligands selective for α4β2ꢀnAChRs.
Their
superior subtype selectivity makes these compounds stand out among all of the other α4β2ꢀnAChR
agonists reported to date, including the marketed drug 3. One of the most promising compounds, 5,
displays subꢀnanomolar binding affinity at α4β2ꢀnAChRs and excellent subtype selectivity over α3β4*ꢀ
and α7ꢀnAChRs, while acting as a potent α4β2ꢀnAChR partial agonist. Its favorable antidepressantꢀlike
effect was demonstrated in the mouse forced swim test, suggesting that compound 5 represents a
promising lead for the treatment of depression, and a good progenitor to other potential candidates to
advance through the drug discovery pipeline. To provide insight into the structural determinants of
compound 5 that confer its remarkable nAChR subtype selectivity, partial agonism, and antidepressantꢀ
ACS Paragon Plus Environment

Page 5 of 25
Journal of Medicinal Chemistry
like properties, both structural studies and the comprehensive pharmacological analysis of compound 5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
were carried out.
Results and Discussion
First, we determined binding affinities (K ) of selected cyclopropane ligands for Acꢀ and CtꢀAChBP
i
3
using [ H]epibatidine binding competition assays to evaluate the validity of using these proteins as
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
models for high affinity binding at α4β2ꢀnAChRs. These binding data (Table 1) reveal high affinities of
the two cyclopropane ligands for the CtꢀAChBP, which are approximately 80ꢀfold higher than those for
the AcꢀAChBP. Moreover, the K values of compound 5 are about 2ꢀfold lower than those of compound
i
16
6
for both Acꢀ and CtꢀAChBP, which is in agreement with the previously observed binding data for
these two compounds at heterologously expressed α4β2ꢀnAChRs or rat forebrain α4β2*ꢀnAChRs.
Taken together, these data demonstrate that compounds 5 and 6 bind with higher affinity to CtꢀAChBP
than to AcꢀAChBP, indicating that CtꢀAChBP is more suitable for structural studies of ligand
recognition using Xꢀray crystallography.
a
Table 1. Ligand dissociation constants (K , nM) for Acꢀ and CtꢀAChBP
i
Ligand
AcꢀAChBP
979±364
CtꢀAChBP
12.8±0.4
22±3
α4β2
0.1
α4β2*
0.5
5
6
1
1927±458
598±108
0.6
1.7
496±30
4.9
9.8
a
Experimental procedures see Supporting Information. The binding data for compounds 1, 5, and 6 at
α4β2ꢀ and α4β2*ꢀnAChRs were obtained from reference 16. α4β2*ꢀnAChRs were prepared from rat
forebrain.
The Xꢀray crystal structure of CtꢀAChBP in complex with compound 5 (Figure 2) was determined at a
resolution of 2.3 Å (Table S1). Figure 2a shows a cartoon representation of CtꢀAChBP along the fiveꢀ
fold symmetry axis with the Cꢀterminus of the protein pointing toward the viewer. Difference electron
density in all five ligand binding sites of the pentamer unambiguously revealed the occupancy of
compound 5 (shown in sphere representation in Figure 2a). Figure 2b shows a magnified stereo view of
compound 5 as well as two interacting water molecules (w1 and w2) built into 2F –F electron density
o
c
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 25
(
grey mesh, contoured at 1.2σ). Similar to other nicotinic ligands, compound 5 is bound at the interface
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
between two subunits where it forms interactions with amino acids of loops A, B, and C on the principal
subunit and loops D, E, and F on the complementary subunit (in Figure 2c principal and complementary
subunits are shown in white and blue, respectively). The azetidinyl moiety of the compound (shown in
yellow) protrudes deep inside the binding pocket, whereas the hydroxyethyl group attached to the
cyclopropane ring points outward. The nitrogen atom of the azetidinyl moiety forms hydrogen bonds
with the carbonyl oxygen of W153 (loop B) and an ordered water molecule (w2), which forms part of a
chain of water molecules (w2–w5) extending into the core of the βꢀsandwich. In addition, the azetidinyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nitrogen forms cationꢀπ interactions with W153 (loop B) and Y201 (loop C). Hydrophobic interactions
are formed with the side chains of V154 (loop B) and Y194, Y201 (loop C) and the vicinal disulfide
bond between C196 and C197 (loop C). The nitrogen atom of the pyridine ring is involved in a
hydrogen bond with a water molecule (w1), which forms a bridge to interact with the peptide backbone
atoms of Q116 and I128 (loop E) on the complementary face of the binding pocket. Hydrophobic
interactions are formed with the side chains of I118 and F128 (loop E). These interactions are
remarkably similar to those observed in the structure of CtꢀAChBP in complex with compound 3
(Figure 2d), which also binds with high affinity to CtꢀAChBP and acts as a partial agonist at the α4β2ꢀ
1
2
nAChRs. In the compound 3ꢀbound structure, the ligand interactions also include water molecules that
occupy nearly identical positions and bridge interactions within the complementary (w1) and principal
(w2–w5) binding sites (Figure 2d). In addition, compound 3 and compound 5 stabilize loop C in
similarly identical conformation, which is intermediate between the contracted and extended
1
7
conformation observed with most agonists and antagonists, respectively. A notable difference between
the two structures is the different side chain orientation of F102 (loop A), namely a gꢀ versus tꢀrotamer
in the compound 5ꢀ and compound 3ꢀbound structures, respectively. Such rotamer differences of the
loop A aromatic residue have also been observed in crystal structures of AcꢀAChBP (Y91) and Lsꢀ
18
AChBP (Y89). Remarkably, a superposition of the compound 3ꢀ and compound 5ꢀbound structures
(Figure 2e) demonstrates that both ligands adopt overlapping binding poses and that the two nitrogen
ACS Paragon Plus Environment

Page 7 of 25
Journal of Medicinal Chemistry
atoms involved in water molecule interactions (w1 and w2, respectively) are in nearly identical
locations. This result indicates that compounds with distinct chemical structures can engage in similar
interactions, which critically rely on the presence of two cationic centers spaced at ~5.8Å from each
other (cationic centers are indicated with arrows in Figure 2e). Together, the results from our CtꢀAChBP
crystal structures reveal a common mode of interaction for compound 5 and compound 3, which both act
as partial agonists at α4β2ꢀnAChRs.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Xꢀray crystal structure of the CtꢀAChBP in complex with compound 5 or compound 3. (a)
Crystal structure of CtꢀAChBP in complex with compound 5 as observed along the fiveꢀfold symmetry
axis. (b) Stereographic view of compound 5 based on its 2F ꢀF electron density contour at a 1.2σ level.
o
c
Carbon = yellow, oxygen = red, nitrogen = blue, sulfur = green in all figures. (c) CtꢀAChBP amino acid
interactions with compound 5. (d) CtꢀAChBP amino acid interactions with compound 3. (e) A
superposition of bound compound 3 (orange) and compound 5 (yellow).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 25
With the resolved CtꢀAChBP structure in hand, and given the greater similarity between human
receptor and CtꢀAChBP binding data, a computational study was undertaken to generate a CtꢀAChBP
based homology model for the ligand binding domain of the human α4β2ꢀnAChR. The use of the Ctꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
AChBP structure as the template is also justified by its high homology with the human α4β2 receptor
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(as computed by ClustalX: α4/CtꢀAChBP = 64.6% and β2/CtꢀAChBP = 62.1%) as well as by the
acceptable conservation of the key residues involved in ligand recognition (Figure S1). Figure 3 reveals
a ribbon structure for the ECD of the modeled, human α4β2 dimer (Figure 3A), which superimposes
well on the structure of the CtꢀAChBP (Figure 3B). Each monomer shows the typical, tenꢀstranded βꢀ
sandwich capped by an Nꢀterminal αꢀhelix. The two modeled βꢀsandwich cores can be nicely
superimposed on that of CtꢀAChBP, whereas some divergence is observed in the superimposition of
their peripheral loops, likely due to differences between their sequences and lengths.
Figure 3. Homology model of the human α4β2ꢀnAChR extracellular domain (ECD) including the
ligand binding interface. (3A) Ribbon structure representation colored by subunit (yellow = α4, azure =
β2) for the human α4β2ꢀnAChR ECD. (3B) Superimposition of the modeled structure (in blue) with the
experimental template from the CtꢀAChBP (in red).
The α4β2ꢀnAChR ligand binding site is located at the dimer interface and is lined by residues from
loops A–C from the α4 subunit and loops D–E from the β2 subunit, including Y98 (loop A), W154
ACS Paragon Plus Environment

Page 9 of 25
Journal of Medicinal Chemistry
(
loop B), Y195 and Y202 (loop C) as well as W57 (loop D), V111 and L121 (loop E). As depicted in
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure S1, the key residues are well conserved in the CtꢀAChBP apart from W57, which is replaced by
I64 in the CtꢀAChBP, and this induces a deeper arrangement of the W153 side chain in the CtꢀAChBP
structure compared to that of the homologue W154 residue due to reduced steric hindrance of I64
compared to W57. As documented by Figure S2, the key residues involved in ligand recognition show
similar spatial arrangements between CtꢀAChBP and human α4β2ꢀnAChR, thus suggesting a generally
conserved architecture of the binding sites which reflects on superimposable poses of the docked and
resolved compound 5 (see below).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The reliability of the generated model and its interaction properties were then analyzed by docking a
set of reported compound 4 derivatives (as compiled in Table S2), including compound 5, which was
coꢀcrystallized with the CtꢀAChBP. The best complex obtained for compound 5 within the binding
cavity of the human α4β2 model is in encouraging agreement with the Xꢀray structure (Figure 4A and
Figure S2). Indeed, the azetidine ring protrudes deep inside the binding pocket, where it can stabilize
pivotal Hꢀbonds with the backbone carbonyl group of S153 and W154 (loop B). The main difference
between the computed complex and the resolved one concerns F102 of the CtꢀAChBP, which is
replaced by Y98 (loop A) in the α4β2ꢀnAChR model, whose hydroxyl group is involved in a clear Hꢀ
bond with the charged nitrogen atom and may replace the ordered water molecule (w2) as seen in the Ctꢀ
AChBP complex. Again, the charged ring stabilizes a set of cationꢀπ interactions (plus various
hydrophobic interactions) with the already mentioned Y98 and W153 as well as with W57 (loop D),
Y92, (loop A) Y195 and Y202 (loop C). The nitrogen atom of the pyridine ring is involved in an Hꢀbond
with the backbone atoms of L121 (loop E), and considering the distance between them (3.3 Å), it might
occur both directly and through a bridging water as seen in the CtꢀAChBP structure. Finally, the
hydroxyethyl group attached to the cyclopropane ring shows an arrangement remarkably similar to that
observed in the Xꢀray complex since it points outward without stabilizing significant polar interactions.
In detail, the cyclopropane ring elicits apolar contacts with V111, F119 (loop E) and A199 (loop C),
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 25
whereas the hydroxyl group approaches loop C but does not stabilize significant polar interactions apart
from a weak Hꢀbond with the backbone atoms of A199. Such a pose confirms that the region at the
interface between α4 and β2 subunits can accommodate quite large moieties and this is in agreement
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with the recent human α4β2 models proposed by Li and coꢀworkers who indeed postulated the existence
1
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of a negative allosteric site in this region.
A
B
Compound 5
Compound 12
Figure 4. Docking of compounds 5 and 12 at the ligand binding site of the modeled, human α4β2ꢀ
nAChR extracellular domain. The protein backbone is colored in green for the α4 and in azure for the
β2 subunit. Oxygen = red, nitrogen = blue, sulfur = yellow, fluorine = brown in all figures. (4A) Main
interactions stabilizing the computed complex between compound 5 and the α4β2ꢀnAChR model. (4B)
Main interactions stabilizing the computed complex between compound 12 and the α4β2ꢀnAChR
model.
When analyzing the best complexes as generated for the set of docked compound 4 congeners, one
may note that most compounds assume a binding mode superimposable to that of compound 5, since in
all monitored complexes, the moieties corresponding to the cyclopropane tail are unable to elicit
significant polar interactions with the β2 subunit and remain in the subpocket flanked by loop C. Even
the compounds bearing large moieties (as seen for some carbamate derivatives, such as compound 6)
show similar binding modes although these moieties cannot be completely harbored below loop C and
ACS Paragon Plus Environment

Page 11 of 25
Journal of Medicinal Chemistry
thus partially protrude toward the β2 subunit without stabilizing polar contacts. Indeed, the
cyclopropane tail remains in a region around loop C that, as evidenced by Figure S3 for the human
model, is surrounded by apolar residues only without contacting polar residues of both α4 and β2
subunits. This suggests that the hydroxyethyl group attached to the cyclopropane ring can be modified
by more hydrophobic moieties without producing steric hindrance.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Rational Design and Synthesis of Fluorine-Containing Cyclopropane nAChR Ligands
Based upon our analysis of the CtꢀAChBP–compound 5 coꢀcrystal structure together with the
molecular docking studies, it is apparent that no strong hydrogen bonding interactions are observed for
the hydroxyl group at the terminus of the rightꢀhand side chain, suggesting that this hydroxyl group does
not constitute an essential pharmacophoric element for receptor recognition, and may therefore be
further modified using other functional groups. Moreover, the free hydroxyl group in compound 5
represents a possible metabolic liability due to potential for oxidation, glucuronidation, sulfation, and
2
0
other bioconjugations. Consequently, small and hydrophobic functional groups might be able to
replace the polar and hydrophilic hydroxyl group thus yield new cyclopropane ligands with improved
drugꢀlike properties. In the field of medicinal chemistry, aside from the introduction or presence of
nitrogen atoms in the drug scaffolds, the incorporation of fluorine atoms likely constitutes the second
most favored heteroꢀatom that has been incorporated into biologically active molecules. The unique
nature of fluorine, including its small size, high electronegativity, and high energy of bond formation to
carbon, imparts a variety of advantages to fluorineꢀcontaining drugs. These advantages include
properties such as enhanced binding interactions, improved metabolic stability, lack of alkylating
activity (in contrast to chlorides or bromides), changes in physicochemical properties, and possibly
enhanced subtypeꢀselectivity. It is not surprising that about 20% of all drugs currently on the market
2
1, 22
contain at least one fluorine atom, with many more to come.
The length of the C–F bond (1.41 Å) is
similar to that of the C–O bond (1.43 Å), suggesting that these atoms are bioisosteres. In addition, it is
well accepted that the C–F unit may participate in electrostatic interactions responsible for strengthening
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 25
2
3
ligandꢀprotein binding interactions. With these considerations in mind, we designed analogs of
compound 5 in which the hydroxyl group is substituted with either hydrogen or fluorine atoms
(compounds 10, 12, and 13). As shown in Table S3, the bloodꢀbrain barrier (BBB) permeability of these
new analogs, as estimated by their calculated logBB values, increases from ꢀ0.5 to about ꢀ0.1, which is
similar to the logBB of compound 3. Drug permeability across the BBB is, of course, essential to
obtaining therapeutic drug levels in the brain, as this is where the targeted α4β2ꢀnAChRs are
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
expressed. These numbers suggest a beneficial effect of the replacement of the hydroxyl group with
either hydrogen or fluorine atoms on BBB penetration and subsequent bioavailability.
Docking simulations of the three proposed compounds confirm the reliability of such modifications
since all derivatives assume poses almost superimposable to that of compound 5 and stabilize reinforced
hydrophobic interactions with the region surrounding loop C. Thus, Figure 4B illustrates the best
complex for compound 12, showing that it stabilizes the same interaction pattern already observed for
compound 5 involving the azetidine and the pyridine rings. Notably, Figure 4B shows that the fluorine
atom is inserted in a markedly hydrophobic niche lined by V111, F119, and the characteristic disulphide
bridge of loop C (C197–C198) thus confirming the relevance of the apolar contacts elicited by this
moiety.
The synthesis of compounds 10, 12, and 13 is described in Scheme 1. For the terminal methyl
16
compound, the optically pure alcohol 7 was acylated with isobutyric anhydride and the benzyl group
was removed by hydrogenolysis, followed by installation of the azetidine moiety by a modified
Mitsunobu reaction to obtain the intermediate 8. After removal of the isobutyryl group, the alcohol
intermediate was subjected to standard Swern oxidation and Wittig reaction to furnish the olefin 9.
Successive hydrogenation of the terminal double bond and removal of the Boc group smoothly gave the
target compound 10 as its trifluoroacetate salt. For the fluorineꢀcontaining analogs, the starting alcohol
1
6
1
1 was tosylated followed by reaction with nꢀtetrabutylammonium fluoride. Boc deprotection of the
resulting fluoride yielded compound 12 as its trifluoroacetate salt. Similarly, the aldehyde prepared from
the same alcohol 11 by Swern oxidation was converted to gemꢀdifluoride using the stable and mild
ACS Paragon Plus Environment

Page 13 of 25
Journal of Medicinal Chemistry
reagent, (diethylamino)difluorosulfonium tetrafluoroborate (XtalFluorꢀE), in conjunction with
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
5
Et N•3HF as a promoter. After removal of the Boc group, the resulting gemꢀdifluoride 13 was
3
obtained as its trifluoroacetate salt.
a
Scheme 1. Synthesis of compounds 10, 12, and 13
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BnO
O
N
d, e
OH a, b, c
O
Boc
N
N
7
8
O
O
O
O
N
b, f
HN
Boc
Boc
N
N
10
9
OH
R1
N
g or h
f
HN
R2
N
N
12: R1 = F, R = H
2
11
3: R1 = F, R = F
2
1
a
Conditions: (a) isobutyric anhydride, cat. DMAP, Et N, CH Cl , 0 °C to rt; (b) 10% Pd/C, H ,
3
2
2
2
EtOAc/MeOH, rt; (c) 1ꢀ(tertꢀbutoxycarbonyl)ꢀ(2S)ꢀazetidinylmethanol, azodicarbonyldipiperidide
(ADDP), P(nꢀBu) , PhMe, 0 °C to rt; (d) NaOMe, MeOH, 40 °C; (e) i. (COCl) , DMSO, Et N, CH Cl ,
3
2
3
2
2
ꢀ
78 °C; ii. Ph P=CH , THF, 0 °C; (f) CF COOH, CH Cl , rt; (g) i. TsCl, Et N, CH Cl , 0 °C to rt; ii. nꢀ
3 2 3 2 2 3 2 2
Bu NF, THF, rt; (h) i. (COCl) , DMSO, Et N, CH Cl , ꢀ78 °C; ii. XtalFluorꢀE, Et N•3HF, CH Cl , rt.
4
2
3
2
2
3
2
2
In Vitro Radioligand Binding and Functional Studies.
The K values of all of the synthesized cyclopropane compounds, 10, 12, and 13 were evaluated by
i
3
[
H]epibatidine binding competition assays at seven heterologously expressed rat nAChR subtypes. As
shown in Table 2, compounds 10, 12, and 13 exhibited subnanomolar to low nanomolar binding
affinities for both the α4β2ꢀ and α4β2*ꢀnAChRs, which are comparable to those of compound 5. This
confirms that the interactions elicited by the cyclopropyl tail are restricted to apolar contacts only and
the hydroxyl group does not take part in the binding process. All three of these compounds demonstrated
good selectivity for nAChRs containing β2 subunits (α2β2ꢀ, α3β2ꢀ, α4β2ꢀ, and α4β2*ꢀnAChRs) over
β4 subunits (α3β4ꢀ, α2β4ꢀ, and α4β4ꢀnAChRs). As it is known that α3β4*ꢀnAChRs mediate
autonomic nicotinic signaling, interaction with these receptors is likely to contribute to adverse side
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 25
1
, 26
effects.
With the exception of compound 10, the selectivity for α4β2ꢀ over α3β4ꢀnAChRs of
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compounds 12 and 13 are greater than 16000ꢀfold, suggesting that few if any peripheral side effects
should be observed.
a
Table 2. Binding affinities of cyclopropane ligands and nicotine at seven nAChR subtypes
K (nM)
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compd.
α2β2
0.1
α2β4
249
α3β2
3.0
α3β4
6520
α4β2
0.1
α4β2*
0.5
α4β4
82.6
5
1
1
1
0
2
3
0.4
0.4±0.1
0.3±0.1 85.4±15.1
5.5 70
41.8±9
137.6
10.3±1.8 852.1 0.4±0.1 1.2±0.2 14.8±2.8
6.1±1.9
6.9±1
29
3224
1976
260
0.2
0.1
4.9
0.9±0.1 39.1±5.6
0.8±0.2
9.8
23±3.1
23
1
a
Experimental procedures see PDSP Assay Protocol Book (http://pdsp.med.unc.edu/). α4β2*ꢀnAChRs
were prepared from rat forebrain. The binding data for compounds 1 and 5 were obtained from reference
1
6. SEM values are not provided for K values > 100 nM.
i
Next, the functional activity of compounds 10, 12, and 13 at the human α4β2ꢀ, α3β4*ꢀ, and α1β1γδꢀ
8
6
+
nAChRs was determined using the Rb ion flux assays and SHꢀEP1, SHꢀSY5Y, and TE671/RD cells,
27ꢀ30
respectively (Table 3, Figure S4).
All three of these ligands were found to be highly potent, partial
agonists at the mixture of high sensitivity (HS) and low sensitivity (LS) α4β2ꢀnAChRs having weak if
any agonist activity at α3β4*ꢀ or α1β1γδꢀnAChRs (Figure S4). Their efficacies ranged from 68–76% of
the response to a maximallyꢀefficacious concentration of the full agonist, carbamylcholine, for actions at
HS α4β2ꢀnAChRs. At LS α4β2ꢀnAChRs, these ligands had no measurable efficacy as agonists. Their
inactivation of nAChR function (following 10 min preꢀexposure; blockade of carbamylcholine
stimulation) was most potent for α4β2ꢀnAChRs and weak or absent out to 3 ꢂM for actions at α3β4*ꢀ
or α1β1γδꢀnAChRs, respectively. Consistent with the binding data, the fluorineꢀcontaining compounds
1
2 and 13 were about 2ꢀfold more potent than compound 10, both in agonism and functional
inactivation at α4β2ꢀnAChRs, which is similar to that seen for parent compound 5. The absence or
weakness of agonist or antagonist activity at ganglionic α3β4*ꢀ or muscleꢀtype α1β1γδꢀnAChRs
indicates that the occurrence of peripheral side effects is unlikely. When taking all the present data into
ACS Paragon Plus Environment

Page 15 of 25
Journal of Medicinal Chemistry
account, the presence of a fluorine at the terminus of the side chain appendage leads to a compound with
an improved pharmacological profile compared to a hydrogen atom, as not only is high affinity for
α4β2ꢀnAChRs maintained, but so is the excellent selectivity over α3β4*ꢀnAChRs.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Table 3. Potencies and efficacies of ligand agonism and inactivation at α4β2ꢀnAChRs
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Agonism
Efficacy at
HS (%)
92±6
Inactivation
Efficacy
(%)
Compd.
EC50
nM)
Hill
IC50
Hill
(
slope
(nM)
Slope
5
10.2×/÷1.7
19.1×/÷1.2
11.3×/÷1.4
8.8×/÷1.2
290×/÷1.1
1.0±0.4
1.1±0.2
1.3±0.5
1.4±0.4
1.0±0.1
9.4×/÷1.1
22.2×/÷1.4
10.2×/÷1.3
8.6×/÷1.3
430×/÷1.1
62±1
ꢀ1.4±0.2
ꢀ1.1±0.3
ꢀ1.2±0.4
ꢀ1.1±0.3
ꢀ0.8±0.09
1
0
68±4
87±4
12
76±6
81±4
1
3
75±7
86±4
1
88±2*
92±2
a
Experimental procedures see reference16. Potencies (EC or IC values) and inactivation efficacies
5
0
50
were measured for actions at a mixture of highꢀsensitivity (HS) and lowꢀsensitivity (LS) α4β2ꢀnAChRs.
Due to the semiꢀlogarithmic nonlinear regression analysis, the SEM for EC50 or IC50 values is not
symmetric in linear space. EC or IC values are therefore reported with a multiplying/dividing factor.
5
0
50
Agonism efficacy values were extrapolated using sazetidineꢀA defined as a full agonist at the HS α4β2ꢀ
nAChR (see Supporting Information for details). Results for compounds 1 and 5 were obtained from
reference 16. *The efficacy of agonism for compound 1 was available only for a mixed population of
HS and LS α4β2ꢀnAChRs. The efficacy of agonism for all other compounds at LS α4β2ꢀnAChRs was
approximately zero.
Behavioral Pharmacology and Metabolic Stability Test
To acquire preliminary in vivo evaluation of our nicotinic ligands for behavioral effects relevant to
®
31
psychiatric disease, we used SmartCube , an automated system in which behaviors of compoundꢀ
treated mice are captured by digital video and analyzed with computer algorithms. We compared the
behavioral signature of a test compound to a database of behavioral signatures obtained using a large set
of diverse reference compounds. In this way, the neuropharmacological effects of a test compound can
be predicted by similarity to major classes of compounds, such as antipsychotics, anxiolytics, and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 25
antidepressants. As shown in Figure 5, all of the selected nicotinic ligands produced, in most cases,
doseꢀdependent behavioral signatures that are quite different from that of the vehicle, and contained an
antidepressantꢀanxiolytic component and other therapeutic signatures. All of the compounds showed no
or a minor profile of side effects.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
00%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Vehicle
Unknown
8
0%
0%
0%
0%
Undesirable/Side Effect
Other Therapeutic
Antidepressant/Anxiolytic
6
4
2
0
%
5
10
5
10
5
10
5
10
5
10 Dose (mg/kg)
5
6
10
12
13
Compound#
Figure 5. All of the cyclopropaneꢀcontaining α4β2ꢀnAChRs partial agonists 5, 6, 10, 12, and 13
produced a signature of activity suggesting potential antidepressantꢀlike effect. The drug was injected
i.p., 15 min before testing.
Next, the antidepressantꢀlike properties of compounds 10, 12, and 13 were further demonstrated in the
3
2
classical mouse forced swim test, an assay in which mice are placed into a beaker of water and the
time the mouse spends passively floating in the water (immobility) is recorded. Most traditional
antidepressants decrease the amount of time the mouse spends immobile. Mice were administered
compound, 10, 12 or 13, or the selective serotonin reuptake inhibitor, sertraline, as a positive control (20
mg/kg). Compounds 10 and 13, the least potent and the most potent ligands in vitro, had no significant
effect in the forced swim test when administered intraperitoneally at doses of 3 or 10 mg/kg. In contrast,
compound 12 exhibited an antidepressantꢀlike effect when administered intraperitoneally or orally, with
a significant reduction in immobility at the minimal dose of 3 mg/kg (Figure 6).
1
6
Additionally, we note that the carbamate derivative 6 reported in our previous publication was
metabolically more stable than the hydroxyl compound 5 in the microsomal stability assays (98% vs
8
0% of the tested compound remained unchanged after 1 h of incubation with a starting concentration of
1
ꢃM). In this context, the fluorinated compound 12 is likely to possess enhanced metabolic stability
ACS Paragon Plus Environment

Page 17 of 25
Journal of Medicinal Chemistry
compared to the hydroxyl compound 5, as the hydroxyl group of the latter compound is likely to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
undergo oxidation and/or bioconjugation reactions. Incubation with mouse, rat, dog, and human liver
3
4
microsomes demonstrated that no less than 98% of compound 12 remained unchanged after 1 h of
incubation at a starting concentration of 2 ꢃM, thereby supporting the greater metabolic stability of the
fluorinated compound 12 relative to its parent compound 5.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
00
300
Water (i.p.)
A
Water (p.o.)
Sertraline (20 mg/kg,i.p.)
Compound 12 (3 mg/kg, i.p.)
Compound 12 (10 mg/kg,i.p.)
B
Sertraline (20 mg/kg, i.p.)
Compound 12 (3 mg/kg, p.o.)
Compound 12 (10 mg/kg, p.o.)
Compound 12 (30 mg/kg, p.o.)
250
2
50
200
200
150
1
50
*
*
*
100
*
100
*
*
50
0
50
*
0
Figure 6. Mouse forced swim data for compound 12. The selective serotonin reuptake inhibitor,
sertraline, produced the expected decrease in immobility. (ANOVAs: F (3,36) = 12.38, p < 0.001 (left);
F (4,44) = 38.97, p < 0.001 (right). *Fisher’s PLSD postꢀhoc test: ps < 0.05 vs vehicle). n = 9—
1
0/group.
Conclusions
Herein we have used the Xꢀray structure of the α4β2ꢀnAChR partial agonist 5 in complex with Ctꢀ
AChBP to refine our homology model of the human α4β2ꢀnAChR. By combining homology modeling
methods with in silico ADME calculations, we in turn designed and synthesized several new analogs of
compound 5. The in vitro binding and functional data as well as the in vivo behavioral efficacy obtained
for the fluorinated compound 12 were found to be comparable to the same parameters obtained for
parent hydroxyl containing compound 5. Additionally, as expected based upon the timeꢀtested principles
of medicinal chemistry, the metabolic stability of analog 12 is better than that of 5. The more limited
metabolic stability of 5, which is subject to both oxidation and bioconjugation reactions, conspires to
make this compound a suboptimal drugꢀlike molecule. Of greatest significance is the structural
information gleaned from the coꢀcrystal structure of compound 5 in complex with CtꢀAChBP. This
particular structure has enabled us to refine our homology model of the human α4β2ꢀnAChR, and as
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 25
such we anticipate that we will be able to design other chemical scaffolds with which to interrogate
these important brain receptors, thus providing the opportunity to create improved therapeutics for a host
of pathophysiological mechanisms.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Experimental Section
®
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedures for SmartCube . Drugs were injected 15 min before the test, during which
multiple challenges were presented over the course of the test session. At least 12 mice were used in
each treatment group. Digital videos of the subjects were processed with computer vision algorithms to
extract over 2000 dependent measures including frequency and duration of behavioral states such as
grooming, rearing, etc., and many other features obtained during the test session. These data were
compared against a standard database of therapeutic class signatures obtained with the same
experimental protocol. The database comprises 14 classes of drugs with some of the major classes, such
as the antidepressant class, comprising several subclasses with representatives of most of the drugs in
the market. Using machine learning techniques, the reference database was combed to find reference
drug class signatures. The best performing techniques were chosen from our evaluation tests and used to
build classifiers that capture the therapeutic class signatures. The behavioral signatures of the test
compound were then assessed quantitatively with these classifiers to predict total and specific potential
31, 35
therapeutic utility.
We present the major signatures in four general groups: antidepressants and
anxiolytics, other therapeutics effects, side effects and signatures not recognized by the classifiers, and
similarity to vehicle (inactivity).
General Chemistry. All chemicals were purchased from SigmaꢀAldrich or ChemꢀImpex, and solvents
were used as obtained from Fisher Scientific or SigmaꢀAldrich without further purification. Anhydrous
THF and CH Cl were obtained by distillation over sodium wire or CaH , respectively. All nonꢀaqueous
2
2
2
reactions were run under an argon atmosphere with exclusion of moisture from reagents, and all reaction
vessels were ovenꢀdried. The progress of reactions was monitored by TLC on SiO . Spots were
2
visualized by their quenching of the fluorescence of an indicator admixed to the SiO layer, or by
2
dipping into I /SiO mixture. Products were purified by column chromatography on 230−400 mesh SiO .
2
2
2
ACS Paragon Plus Environment

Page 19 of 25
Journal of Medicinal Chemistry
Proton and carbon NMR spectra were recorded at spectrometer frequencies of 400 MHz and 100 MHz,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
respectively. NMR chemical shifts were reported in δ (ppm) using the δ 7.26 signal of CHCl ( H
3
1
13
NMR), the δ 4.80 signal of HDO ( H NMR), and the δ 77.23 signal of CDCl ( C NMR) as internal
3
1
3
standards. C NMR spectra in D O were not adjusted. Optical rotation was detected on an Autopol IV
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
automatic polarimeter. Mass spectra were measured in the ESI mode at an ionization potential of 70 eV
with an LCꢀMS MSD (Hewlett Packard). The final compounds were purified by preparative HPLC,
which was carried out on an ACE 5 AQ column (150 × 20 mm), with detection at 254 and 280 nm on a
Shimadzu SPDꢀ10A VP detector; flow rate = 17.0 mL/min; gradient of 0 to 50% methanol in water
(
both containing 0.05 vol% of CF COOH) in 30 min. Purities of final compounds (> 98%) were
3
established by both elemental analysis and by analytical HPLC, which was carried out on an Agilent
100 HPLC system with a Synergi 4 ꢃm HydroꢀRP 80A column, with detection at 254 or 280 nm on a
1
variable wavelength detector G1314A; flow rate = 1.4 mL/min; gradient of 0 to 100% methanol in water
(both containing 0.05 vol% of CF COOH) in 18 min.
3
3
-[(2(S)-Azetidinyl)methoxy]-5-((1S,2S)-2-ethylcyclopropyl)pyridine Trifluoroacetate (10).
1
H NMR (D O): δ 8.29 (s, 1H), 8.19 (s, 1H), 7.81 (s, 1H), 4.95 (m, 1H), 4.50 (d, J = 4.0 Hz, 2H), 4.17ꢀ
2
4
.02 (m, 2H), 2.68 (q, J = 8.4 Hz, 2H), 1.88 (m, 1H), 1.48ꢀ1.35 (m, 2H), 1.25 (m, 1H), 1.08 (m, 2H),
1
3
0
.95 (t, J = 7.2 Hz, 3H); C NMR (D O): δ 162.6 (TFA), 156.2, 147.3, 132.2, 128.2, 125.4, 116.2
2
2
0
(
TFA), 67.5, 58.6, 43.6, 27.6, 26.3, 20.2, 19.9, 17.1, 12.4; [α]D = +32.7 (c 0.22, MeOH); HPLC purity
9
9.2%, t = 11.9 min; Anal. Calcd for C H N O•2.25CF COOH•0.4H O: C, 44.79; H, 4.68; F, 25.85;
R
14 20
2
3
2
N, 5.65. Found: C, 44.67; H, 4.53; F, 25.79; N, 5.62.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-(2-fluoroethyl)cyclopropyl]pyridine
12).
Trifluoroacetate
(
1
H NMR (D O): δ 8.32 (s, 1H), 8.22 (s, 1H), 7.85 (s, 1H), 4.95 (m, 1H), 4.66 (t, J = 6.0 Hz, 1H), 4.55ꢀ
2
4
2
.50 (m, 3H), 4.10 (m, 2H), 2.68 (q, J = 8.4 Hz, 2H), 1.99 (m, 1H), 1.82 (m, 2H), 1.37 (m, 1H), 1.17 (m,
13
H); C NMR (D O): δ 162.1 (TFA), 155.8, 146.0, 132.0, 128.1, 125.4, 115.8 (TFA), 84.3 (d, J
=
2
CꢀF
1
9
157.7 Hz), 67.1, 58.2, 43.2, 33.2 (d, JCꢀF = 18.9 Hz), 21.1, 19.8, 19.2, 15.8; F NMR (D O): δ ꢀ75.7, ꢀ
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 25
2
0
2
17.8. [α]D = +33.8 (c 0.26, MeOH); HPLC purity 99.4%, t = 6.4 min; Anal. Calcd for
R
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C H FN O•2.0CF COOH•0.8H O: C, 43.87; H, 4.62; F, 26.99; N, 5.68. Found: C, 43.51; H, 4.23; F,
1
4
19
2
3
2
2
6.80; N, 5.58.
-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-(2,2-difluoroethyl)cyclopropyl]pyridine
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Trifluoroacetate (13).
1
H NMR (D O): δ 8.34 (s, 1H), 8.25 (s, 1H), 7.87 (s, 1H), 6.04 (tt, JHꢀF = 56.4 Hz, JHꢀH = 4.0 Hz, 1H),
2
4
.97 (m, 1H), 4.51 (d, J = 4.0 Hz, 2H), 4.10 (m, 2H), 2.68 (t, JHꢀF = 8.4 Hz, 2H), 2.09ꢀ1.92 (m, 3H), 1.38
1
3
(
m, 1H), 1.22 (m, 2H); C NMR (D O): δ 162.2 (TFA), 155.9, 145.3, 132.2, 128.3, 125.7, 117.1 (t, J
2
CꢀF
=
235.9 Hz), 115.8 (TFA), 67.1, 58.2, 43.2, 36.5 (t, JCꢀF = 20.9 Hz), 19.8, 19.0, 17.2 (t, JCꢀF = 7.4 Hz),
1
9
20
D
1
5.0; F NMR (D O): δ ꢀ75.8, ꢀ116.5. [α] = +25.0 (c 0.2, MeOH); HPLC purity 99.3%, t = 6.3 min;
2
R
Anal. Calcd for C H F N O•2.7CF COOH•1.2H O: C, 38.98; H, 3.89; F, 32.10; N, 4.69. Found: C,
1
4
18
2
2
3
2
38.91; H, 3.58; F, 31.81; N, 4.65.
ASSOCIATED CONTENT
Supporting Information
Synthetic procedures and compound characterizations, crystallization and data collection, computational
methods, and behavioral studies. This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
PDB code: 4B5D for the Xꢀray crystal structure of CtꢀAChBP in complex with compound 5.
AUTHOR INFORMATION
Corresponding Author
*
To whom correspondence should be addressed. Eꢀmail: Ronald.Lukas@DignityHealth.org
[
pharmacology], dani.brunner@psychogenics.com [rodent behavior], giulio.vistoli@unimi.it
[modeling], chris.ulens@med.kuleuven.be [Xꢀray], and kozikowa@uic.edu [medicinal chemistry].
ACS Paragon Plus Environment

Page 21 of 25
Journal of Medicinal Chemistry
ACKNOWLEDGMENTS. This research was supported by award No. U19MH085193 from the
National Institute of Mental Health. We thank the PDSP program for assistance through providing the
binding affinity data. CU, ABS, and RvE have received funding from the European Union Seventh
Framework Programme under grant agreement n° HEALTHꢀF2ꢀ2007ꢀ202088 ("NeuroCypres" project).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ABBREVIATIONS USED
nAChR(s), nicotinic acetylcholine receptor(s); LGICs, ligandꢀgated ion channels; ECD, extracellular
domain; ACh, acetylcholine; AChBP(s), acetylcholine binding protein(s), LBD, ligandꢀbinding domain;
Ac, Aplysia californica; Ls, Lymnaea stagnalis; Ct, Capitella teleta; BBB, bloodꢀbrain barrier;
XtalFluorꢀE, (diethylamino)difluorosulfonium tetrafluoroborate; HS, high sensitivity; LS, low
sensitivity; TFA, trifluoroacetic acid.
REFERENCES
1
. Gotti, C.; Clementi, F.; Fornari, A.; Gaimarri, A.; Guiducci, S.; Manfredi, I.; Moretti, M.; Pedrazzi,
P.; Pucci, L.; Zoli, M. Structural and functional diversity of native brain neuronal nicotinic receptors.
Biochem. Pharmacol. 2009, 78, 703ꢀ711.
2
. Jensen, A. A.; Frolund, B.; Liljefors, T.; KrogsgaardꢀLarsen, P. Neuronal nicotinic acetylcholine
receptors: structural revelations, target identifications, and therapeutic inspirations. J. Med. Chem. 2005,
4
8, 4705ꢀ4745.
3
. D'Hoedt, D.; Bertrand, D. Nicotinic acetylcholine receptors: an overview on drug discovery. Expert
Opin. Ther. Targets 2009, 13, 395ꢀ411.
. Taly, A.; Corringer, P. J.; Guedin, D.; Lestage, P.; Changeux, J. P. Nicotinic receptors: allosteric
4
transitions and therapeutic targets in the nervous system. Nat. Rev. Drug Discovery 2009, 8, 733ꢀ750.
5. Leung, L. K.; Patafio, F. M.; Rosser, W. W. Gastrointestinal adverse effects of varenicline at
maintenance dose: a metaꢀanalysis. BMC Clin. Pharmacol. 2011, 11, 15ꢀ22.
6
. Lummis, S. C.; Thompson, A. J.; Bencherif, M.; Lester, H. A. Varenicline is a potent agonist of the
human 5ꢀhydroxytryptamine3 receptor. J. Pharmacol. Exp. Ther. 2011, 339, 125ꢀ131.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 25
7
. Brejc, K.; van Dijk, W. J.; Klaassen, R. V.; Schuurmans, M.; van Der Oost, J.; Smit, A. B.; Sixma, T.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
K. Crystal structure of an AChꢀbinding protein reveals the ligandꢀbinding domain of nicotinic receptors.
Nature 2001, 411, 269ꢀ276.
8
. Rucktooa, P.; Smit, A. B.; Sixma, T. K. Insight in nAChR subtype selectivity from AChBP crystal
structures. Biochem. Pharmacol. 2009, 78, 777ꢀ787.
. Celie, P. H.; van RossumꢀFikkert, S. E.; van Dijk, W. J.; Brejc, K.; Smit, A. B.; Sixma, T. K.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal
structures. Neuron 2004, 41, 907ꢀ914.
1
0. Hansen, S. B.; Talley, T. T.; Radic, Z.; Taylor, P. Structural and ligand recognition characteristics of
an acetylcholineꢀbinding protein from Aplysia californica. J. Biol. Chem. 2004, 279, 24197ꢀ24202.
11. McCormack, T.; Petrovich, R. M.; Mercier, K. A.; DeRose, E. F.; Cuneo, M. J.; Williams, J.;
Johnson, K. L.; Lamb, P. W.; London, R. E.; Yakel, J. L. Identification and functional characterization
of a novel acetylcholineꢀbinding protein from the marine annelid Capitella teleta. Biochemistry 2010,
49, 2279ꢀ2287.
1
2. Billen, B.; Spurny, R.; Brams, M.; van Elk, R.; ValeraꢀKummer, S.; Yakel, J. L.; Voets, T.;
Bertrand, D.; Smit, A. B.; Ulens, C. Molecular actions of smoking cessation drugs at alpha4beta2
nicotinic receptors defined in crystal structures of a homologous binding protein. Proc. Natl. Acad. Sci.
U.S.A. 2012, 109, 9173ꢀ9178.
1
3. Philip, N. S.; Carpenter, L. L.; Tyrka, A. R.; Price, L. H. Nicotinic acetylcholine receptors and
depression: a review of the preclinical and clinical literature. Psychopharmacology (Berl) 2010, 212, 1ꢀ
1
1
2.
4. Mineur, Y. S.; Picciotto, M. R. Nicotine receptors and depression: revisiting and revising the
cholinergic hypothesis. Trends Pharmacol. Sci. 2010, 31, 580ꢀ586.
5. Kozikowski, A. P.; Eaton, J. B.; Bajjuri, K. M.; Chellappan, S. K.; Chen, Y.; Karadi, S.; He, R.;
1
Caldarone, B.; Manzano, M.; Yuen, P. W.; Lukas, R. J. Chemistry and pharmacology of nicotinic
ACS Paragon Plus Environment

Page 23 of 25
Journal of Medicinal Chemistry
ligands based on 6ꢀ[5ꢀ(azetidinꢀ2ꢀylmethoxy)pyridinꢀ3ꢀyl]hexꢀ5ꢀynꢀ1ꢀol (AMOPꢀHꢀOH) for possible
use in depression. ChemMedChem 2009, 4, 1279ꢀ1291.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
16. Zhang, H.; Tückmantel, W.; Eaton, J. B.; Yuen, P. W.; Yu, L. F.; Bajjuri, K. M.; Fedolak, A.; Wang,
D.; Ghavami, A.; Caldarone, B.; Paterson, N. E.; Lowe, D. A.; Brunner, D.; Lukas, R. J.; Kozikowski,
A. P. Chemistry and behavioral studies identify chiral cyclopropanes as selective alpha4beta2ꢀnicotinic
acetylcholine receptor partial agonists exhibiting an antidepressant profile. J. Med. Chem. 2012, 55, 717ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
1
24.
7. Brams, M.; Pandya, A.; Kuzmin, D.; van Elk, R.; Krijnen, L.; Yakel, J. L.; Tsetlin, V.; Smit, A. B.;
Ulens, C. A structural and mutagenic blueprint for molecular recognition of strychnine and dꢀ
tubocurarine by different cysꢀloop receptors. PLoS. Biol. 2011, 9, e1001034.
18. Edink, E.; Rucktooa, P.; Retra, K.; Akdemir, A.; Nahar, T.; Zuiderveld, O.; van Elk, R.; Janssen, E.;
van Nierop, P.; van MuijlwijkꢀKoezen, J.; Smit, A. B.; Sixma, T. K.; Leurs, R.; de Esch, I. J. Fragment
growing induces conformational changes in acetylcholineꢀbinding protein: a structural and
thermodynamic analysis. J. Am. Chem. Soc. 2011, 133, 5363ꢀ5371.
1
9. Henderson, B. J.; Pavlovicz, R. E.; Allen, J. D.; GonzalezꢀCestari, T. F.; Orac, C. M.; Bonnell, A.
B.; Zhu, M. X.; Boyd, R. T.; Li, C.; Bergmeier, S. C.; McKay, D. B. Negative allosteric modulators that
target human alpha4beta2 neuronal nicotinic receptors. J. Pharmacol. Exp. Ther. 2010, 334, 761ꢀ774.
2
0. Shu, Y. Z.; Johnson, B. M.; Yang, T. J. Role of biotransformation studies in minimizing
metabolismꢀrelated liabilities in drug discovery. AAPS J. 2008, 10, 178ꢀ192.
21. O'Hagan, D. Understanding organofluorine chemistry. An introduction to the CꢀF bond. Chem. Soc.
Rev. 2008, 37, 308ꢀ319.
2
2. Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Fluorine in medicinal chemistry. Chem. Soc.
Rev. 2008, 37, 320ꢀ330.
2
3. Muller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: looking beyond intuition. Science
007, 317, 1881ꢀ1886.
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 25
2
4. Alavijeh, M. S.; Chishty, M.; Qaiser, M. Z.; Palmer, A. M. Drug metabolism and pharmacokinetics,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the bloodꢀbrain barrier, and central nervous system drug discovery. NeuroRx 2005, 2, 554ꢀ571.
25. L'Heureux, A.; Beaulieu, F.; Bennett, C.; Bill, D. R.; Clayton, S.; Laflamme, F.; Mirmehrabi, M.;
Tadayon, S.; Tovell, D.; Couturier, M. Aminodifluorosulfinium salts: selective fluorination reagents
with enhanced thermal stability and ease of handling. J. Org. Chem. 2010, 75, 3401ꢀ3411.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6. Sullivan, J. P.; Bannon, A. W. Epibatidine: Pharmacological Properties of a Novel Nicotinic
Acetylcholine Receptor Agonist and Analgesic Agent. CNS Drug Reviews 1996, 2, 21ꢀ39.
7. Lukas, R. J.; Norman, S. A.; Lucero, L. Characterization of Nicotinic Acetylcholine Receptors
Expressed by Cells of the SHꢀSY5Y Human Neuroblastoma Clonal Line. Mol. Cell Neurosci. 1993, 4,
ꢀ12.
2
1
28. Lukas, R. J. Cell lines as models for studies of nicotinic acetylcholine receptors. In Neuronal
Nicotinic Receptors: Pharmacology and Therapeutic Opportunities; WileyꢀLiss, Inc, New York: 1999;
pp 81ꢀ97.
29. Lukas, R. J.; Fryer, J. D.; Eaton, J. B.; Gentry, C. L. Some methods for studies of nicotinic
acetylcholine receptor pharmacology. In Nicotinic Receptors and the Nervous System; CRC Press, Boca
Raton: 2002; pp 3ꢀ27.
30. Eaton, J. B.; Peng, J. H.; Schroeder, K. M.; George, A. A.; Fryer, J. D.; Krishnan, C.; Buhlman, L.;
Kuo, Y. P.; Steinlein, O.; Lukas, R. J. Characterization of human alpha4beta2ꢀnicotinic acetylcholine
receptors stably and heterologously expressed in native nicotinic receptorꢀnull SHꢀEP1 human epithelial
cells. Mol. Pharmacol. 2003, 64, 1283ꢀ1294.
3
1. Roberds, S. L.; Filippov, I.; Alexandrov, V.; Hanania, T.; Brunner, D. Rapid, computer visionꢀ
enabled murine screening system identifies neuropharmacological potential of two new mechanisms.
Front. Neurosci. 2011, 5, 103.
3
2. Porsolt, R. D.; Bertin, A.; Jalfre, M. Behavioral despair in mice: a primary screening test for
antidepressants. Arch. Int. Pharmacodyn. Ther. 1977, 229, 327ꢀ336.
ACS Paragon Plus Environment

Page 25 of 25
Journal of Medicinal Chemistry
3
3. Walsh, J. S.; Miwa, G. T. Bioactivation of drugs: risk and drug design. Annu. Rev. Pharmacol.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Toxicol. 2011, 51, 145ꢀ167.
34. The metabolic stability studies were carried out by Cerep, Inc.
3
5. Houghten, R. A.; Pinilla, C.; Giulianotti, M. A.; Appel, J. R.; Dooley, C. T.; Nefzi, A.; Ostresh, J.
M.; Yu, Y.; Maggiora, G. M.; MedinaꢀFranco, J. L.; Brunner, D.; Schneider, J. Strategies for the use of
mixtureꢀbased synthetic combinatorial libraries: scaffold ranking, direct testing in vivo, and enhanced
deconvolution by computational methods. J. Comb. Chem. 2008, 10, 3ꢀ19.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TOC Graphic:
Ct-AChBP + compound 5
cationic centers
ACS Paragon Plus Environment