Discovery of benzamide analogs as negative allosteric modulators of human neuronal nicotinic receptors: Pharmacophore modeling and structure-activity relationship studies

Article abstract of DOI:10.1016/j.bmc.2013.03.082

The present study describes our ongoing efforts toward the discovery of drugs that selectively target nAChR subtypes. We exploited knowledge on nAChR ligands and their binding site that were previously identified by our laboratory through virtual screenings and identified benzamide analogs as a novel chemical class of neuronal nicotinic receptor (nAChR) ligands. The lead molecule, compound 1 (4-(allyloxy)-N-(6-methylpyridin-2-yl)benzamide) inhibits nAChR activity with an IC₅₀ value of 6.0 (3.4-10.6) μM on human α4β2 nAChRs with a ～5-fold preference against human α3β4 nAChRs. Twenty-six analogs of compound 1 were also either synthesized or purchased for structure-activity relationship (SAR) studies and provided information relating the chemical/structural properties of the molecules to their ability to inhibit nAChR activity. The discovery of subtype-selective ligands of nAChRs described here should contribute significantly to our understanding of the involvement of specific nAChR subtypes in normal and pathophysiological states.

Full text of DOI:10.1016/j.bmc.2013.03.082

Bioorganic & Medicinal Chemistry 21 (2013) 4730–4743
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of benzamide analogs as negative allosteric modulators
of human neuronal nicotinic receptors: Pharmacophore modeling
and structure–activity relationship studies
Bitna Yi a, , Sihui Long b, , Tatiana F. González-Cestari a, Brandon J. Henderson a,à, Ryan E. Pavlovicz c,
Karl Werbovetz b, Chenglong Li b,c, Dennis B. McKay a,
⇑
a Division of Pharmacology, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA
b Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
c Biophysics Program, The Ohio State University, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study describes our ongoing efforts toward the discovery of drugs that selectively target
nAChR subtypes. We exploited knowledge on nAChR ligands and their binding site that were previously
identified by our laboratory through virtual screenings and identified benzamide analogs as a novel
chemical class of neuronal nicotinic receptor (nAChR) ligands. The lead molecule, compound 1 (4-(allyl-
oxy)-N-(6-methylpyridin-2-yl)benzamide) inhibits nAChR activity with an IC50 value of 6.0 (3.4–10.6)
Received 9 January 2013
Revised 22 March 2013
Accepted 29 March 2013
Available online 6 April 2013
l
M on human
a
4b2 nAChRs with a ꢀ5-fold preference against human
a3b4 nAChRs. Twenty-six analogs
Keywords:
of compound 1 were also either synthesized or purchased for structure–activity relationship (SAR) stud-
ies and provided information relating the chemical/structural properties of the molecules to their ability
to inhibit nAChR activity. The discovery of subtype-selective ligands of nAChRs described here should
contribute significantly to our understanding of the involvement of specific nAChR subtypes in normal
and pathophysiological states.
Neuronal nicotinic receptors
Negative allosteric modulators
Structure–activity relationship studies
Pharmacophore modeling
Ó 2013 Published by Elsevier Ltd.
1. Introduction
human genes that encode nAChR subunits (a2–a10 excluding a8
and b2–b4). These subunits assemble to form multiple subtypes
that are characterized by distinct biochemical properties (e.g., ion
selectivity, conductance, mean channel open time, permeability
to Ca2+, and desensitization rate).4–6 Importantly, each subunit is
expressed in unique spatial and developmental patterns, suggest-
ing the possibility that individual subtypes may play distinct roles
in certain physiological processes.6,7 Knowledge of these roles will
pave the way toward the identification of novel therapeutic targets
and allow the development of more targeted pharmacotherapy
with reduced side effects. Therefore, drugs that specifically target
nAChR subtypes can represent valuable research tools by providing
insight into the roles that different nAChR subtypes play in physi-
ological and pathological states.
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-
gated ion channels that mediate the physiological effects elicited
by the endogenous neurotransmitter, acetylcholine (ACh). nAChR
signaling influences and regulates a number of physiologically
important processes (e.g., cognition, arousal, anxiety, pain process-
ing, food intake, and reward).1,2 Furthermore, disruption or alter-
ation of nAChR activity has been implicated in many diseases
and disorders (Alzheimer’s disease, Parkinson’s disease, depres-
sion, schizophrenia, and addiction).3 Therefore, nAChRs hold con-
siderable promise as therapeutic targets.
A major challenge in the study of the nAChRs is the diversity
and complexity of these pentameric receptors. There are eleven
Orthosteric binding sites of nAChRs (sites of ACh binding) con-
tain a high level of sequence homology. At the interface between
a
Abbreviations: NAM, negative allosteric modulator; nAChRs, neuronal nicotinic
acetylcholine receptors; HBK, HEPES-buffered Krebs; LBVS, ligand-based virtual
screening; SBVS, structure-based virtual screening; SAR, structure–activity rela-
tionship; nh, Hill coefficient.
and b subunits, ACh interacts with the ‘aromatic nest’, which is a
group of five aromatic amino acid residues (i.e., Trp56, Trp61,
Trp87, Trp150, and Tyr199). (The numbers used in this manuscript
are based on the first transcribed protein). These residues are com-
pletely conserved in all nAChR subunits.18 In addition, amino acid
residues surrounding the orthosteric site (Cys 193, Cys 194, and
Tyr 94) are also completely conserved or highly conserved
(75–100% amino acid identity across nAChR subunits). We believe
⇑
Corresponding author. Address: Division of Pharmacology, The Ohio State
University, College of Pharmacy, 500 West 12th Avenue, Columbus, Ohio 43210,
USA. Tel.: +1 614 292 3771; fax: +1 614 292 9083.
E-mail address: Mckay.2@osu.edu (D.B. McKay).
These authors contributed equally to the work.
Present address: Division of Biology, California Institute of Technology, Pasadena,
à
CA 91125, USA.
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.03.082

B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
4731
that this extensive level of sequence homology makes it difficult to
discover and develop drugs that interact with the orthosteric bind-
ing site and pharmacologically discriminate one subtype from oth-
ers. Our laboratory is addressing the issue of subtype-selectivity by
targeting ‘non-orthosteric’ sites of nAChRs previously identified by
our laboratory (e.g., allosteric sites).16 Our hypotheses are that (1)
structural variations within allosteric binding sites can provide a
foundation to develop subtype-selective ligands of nAChRs and
(2) a multidisciplinary approach can direct the design and synthe-
sis of new molecules with optimized properties (i.e., selectivity and
potency). The present study is a proof-of-concept study to docu-
ment the utilization of allosteric binding sites to develop drugs
with specificity for nAChR subtypes using a combination of multi-
ple approaches. In particular, we focused on the subtype-selectiv-
(4-(allyloxy)-N-(6-methylpyridin-2-yl)benzamide) that acts as a
NAM of nAChRs. Furthermore, 26 analogs of compound 1 were
either synthesized or purchased to determine the structural fea-
tures that are relevant for potency and relative selectivity toward
nAChRs.
2. Chemistry
Target compounds 8–15 were obtained through reaction be-
tween commercially available 6-substituted-2-aminopyridines
6a–e with 4-propoxybenzoyl chloride (7a) or 4-allyloxybenzoyl
chloride (7b) as shown in Scheme 1. Benzoyl chlorides 7a,b were
readily prepared by heating 4-propoxybenzoic acid or 4-allyloxy-
benzoic acid to reflux in thionyl chloride.19 4-Allyloxybenzoic acid
(18) was made through the reaction between 4-hydroxybenzoic
acid (16) and allyl bromide. Since ester 17 was also formed as an
undesired side product of the reaction above, 17 was converted
to the desired acid 18 by treatment with NaOH in ethanol/water
(Scheme 2).
ity for human
nAChRs. As one of the main subtypes expressed in the central ner-
vous system (CNS), h 4b2 nAChRs have been implicated in various
brain diseases and disorders (e.g., nicotine addiction, anxiety, and
depression).8–12 On the other hand, h
3b4 nAChR is largely ex-
a4b2 (ha4b2) nAChRs over human a3b4 (ha3b4)
a
a
pressed in the peripheral autonomic ganglia where it modulates
Target compounds 19–30 were synthesized in high yield by
reacting a variety of aliphatic and aromatic amines with bromo-
pyridine 12 through Buchwald-Hartwig amination20 (Scheme 3).
In contrast, Buchwald–Hartwig reactions using chloropyridine 10
failed to give the desired products. Also, Buchwald–Hartwig cou-
pling using compound 9 as a precursor in an attempt to produce
compounds 34 and 35 was unsuccessful, likely due to the pres-
ence of the allyl group in 9. An alternative route was thus required
to synthesize target compounds 34 and 35. 2-Amino-6-bromopyr-
idine (6b) was first protected by acylation to provide 31, followed
by Buchwald-Hartwig amination with either propylamine or
butylamine to yield protected alkylaminopyridines 32 and 33,
respectively. The acetyl group was removed by treatment with so-
dium hydroxide, followed by acylation of the resulting primary
aromatic amines with 7b to afford target compounds 34 and 35
(Scheme 4).
the release of multiple neurotransmitters. Due to the roles of
ha3b4 nAChR in the autonomic ganglia, activity of nicotinic drugs
on this subtype has been postulated to mediate a wide range of
autonomic side effects, as exemplified by those reported from
usage of the classical autonomic blocker, mecamylamine (e.g., con-
stipation, urinary retention, dilation of the pupils, and postural
hypotension).1–3 Therefore, with interest in developing safer drugs
for CNS application, subtype-selectivity was pursued for the h
nAChRs over the h 3b4 nAChRs.
a4b2
a
In this study, we report the discovery of a novel chemical class
of nAChR antagonists that allosterically modulate receptor activity
using multiple approaches. As a rational drug design strategy, we
utilized knowledge gained from our previous studies13–18 by incor-
porating ligand-based modeling, structure-based modeling, phar-
macology, and medicinal chemistry. Previously, our laboratory
has identified several classes of novel negative allosteric modula-
tors (NAMs) of nAChRs.13–16 Effects of those NAMs were insur-
mountable with increasing concentrations of the orthosteric
agonist, epibatidine, suggesting their non-competitive mecha-
nisms of action. Importantly, some of these NAMs showed selectiv-
3. Results
3.1. Pharmacophore and ligand-based virtual screening
ity for ha4b2 nAChRs over ha3b4 nAChRs. Utilizing a combination
In order to identify novel chemical entities that exhibit subtype-
selectivity, information obtained from our previous studies was
utilized.13–18 Four NAMs (i.e., KAB-18, DDR-5, DDR-13, and DDR-
18) previously identified by our laboratory16 were selected based
of homology modeling, blind docking, and site-directed mutagene-
sis, an allosteric binding site where these NAMs bind was also
identified.16–18 This site is located approximately 10 Å from the
orthosteric site at the interface between
a and b subunits. Since
on their preference for ha4b2 nAChRs against ha3b4 nAChRs and
major amino acid residues comprising this site are located at the
b subunit, we named this site the ‘b subunit site’. The ‘b subunit
site’ is sequentially and structurally diverse among subtypes,
which provides an explanation for the relative subtype-selectivity
shown with some of the NAMs17: among residues positioned with-
in the ‘b subunit site’, Thr58, Ser133, Ser138, Ser142, Phe118, and
Ser137 showed reduced sequence conservation (less than 26% ami-
no acid identity across nAChR subunits). Other amino acids (Glu60
and Ser97) that contribute to interactions between ligands and
receptors occurring through the ‘b subunit site’ also share a limited
degree of sequence conservation among subunits (amino acid
identity of 63% and 39%, respectively).17 The diversity within the
‘b subunit site’ and the identification of subtype-selective NAMs
acting on this site provide support for our approach of targeting
allosteric binding sites to develop selective nAChR antagonists. In
this study, the physicochemical properties of NAMs that selectively
used here to generate an initial pharmacophore (Fig. 1). In parallel,
structure-based virtual screening (SBVS) using the allosteric bind-
ing sites for these four NAMs (i.e., b subunit site) was performed
and led to the identification of four novel scaffolds that inhibit
the activity of nAChRs with relative selectivity for ha
4b2 nAChRs.14
Among top hits from this SBVS, Hit 2 ((5-amino-N-(6-methylpyri-
din-2-yl)-2-(piperidin-1-yl)benzamide) shares structural similari-
ties with the four NAMs used to generate the initial
pharmacophore (Fig. 1). In addition, Hit 2 has lower molecular
weight by lacking the substitutions linked to the piperidine ring
and thereby possesses potential to exhibit improved bioavailability
(Fig. 1). As Hit 2 shows a preference for ha4b2 nAChRs, shares
structural similarity with the four NAMs used for initial pharmaco-
phore development, and possesses more desirable drug properties
with regard to in vivo bioavailability, the initial pharmacophore
was then refined here using Hit 2 from the SBVS. The refined phar-
macophore reported here features three hydrophobic regions
(HYD1, HYD2, and HYD3) and one hydrogen bond acceptor (HBA)
(Fig. 2). This refined pharmacophore model was utilized for li-
gand-based virtual screening (LBVS) as described below.
target ha4b2 nAChRs as well as the biochemical characteristics of
their binding sites (i.e., the ‘b subunit site’) mentioned above were
exploited. Utilization of ligand-based and structure-based
approaches using the previously obtained knowledge led to the
discovery of a lead compound of this study (i.e., Compound 1,

4732
B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
O
X
N
X
O
a
Cl
+
R1
NH2
R1
N
-
N
H
OR2
6a e
-
7a b
,
8
15
OR2
a:
a:
8:
R1 = CH3, X = CH
R2 = (CH2)2CH3
R1 = CH3, R2 = (CH2)2CH3, X = CH
9: R1 = Br, R2 = CH2CH=CH2, X = CH
R1 = Cl, R2 = CH2CH=CH2, X = CH
b: R1 = Br, X = CH
c: R1 = Cl, X = CH
b: R2 = CH2CH=CH2
10:
d:
R1 = CH2CH3, X = CH
11: R1 = CH2CH3, R2 = CH2CH=CH2, X = CH
e: R1 = H, X = N
12:
R1 = Br, R2 = (CH2)2CH3, X = CH
13: R1 = Cl, R2 = (CH2)2CH3, X = CH
14:
R1 = H, R2 = (CH2)2CH3, X = N
15: R1 = CH2CH3, R2 = (CH2)2CH3, X = CH
Scheme 1. Reagents: (a) DCM, Et3N.
b
+
O
O
a
c
7b
HO
CO2H
O
O
CO2H
16
17
18
Scheme 2. Reagents: (a) NaOH, allyl bromide, H2O; (b) NaOH, EtOH/H2O; (c) thionyl chloride.
O
a
12
HN
R
N
N
H
O
19 30
-
19
23
27
: R = 4-Cl-Ph
: R = (CH2)2CH3
: R = Ph
20: R = (CH2)3CH3
21: R = (CH2)4CH3
24: R = 2-F-Ph
25: R = 3-F-Ph
28: R = 4-OCH3-Ph
29: R = CH2Ph
22:
26
30:
R = H = (CH2)5
R = (CH2)5CH3
: R = 4-F-Ph
Scheme 3. Reagents: (a) aliphatic or aromatic amine, Pd2(dba)3, (rac)-BINAP, NaO-
t-Bu, toluene.
O
a
b
Br
N
NH2
O
Br
N
N
H
6b
31
O
c, d
HN
R
N
N
H
HN
R
N
N
Figure 1. Structures of the compounds used for pharmacophore generation.
H
O
32, 33
34, 35
tsA201 cells stably expressing either ha4b2 nAChRs or ha3b4 nAC-
32, 34: R = (CH2)2CH3
33, 35:
hRs (data not shown). One of the top ranked hits from this LBVS
(4-(allyloxy)-N-(6-methylpyridin-2-yl)benzamide, 1) is the focus
of this study. As a lead molecule, compound 1 inhibited the
R1 = (CH2)3CH3
Scheme 4. Reagents: (a) acetyl chloride, DCM, Et3N; (b) Pd2(dba)3, (rac)-BINAP,
NaOH-t-Bu, toluene, propylamine or butylamine; (c) NaOH, H2O; (d) 7b, DCM,Et3N.
activity of h
a4b2 nAChRs with an IC50 value of 6.0 (3.4–10.6)
lM
with ꢀ5-fold preference against h
a
3b4 nAChRs (Fig. 3A and B;
Table 1). On the other hand, compound 8 (4-(6-methylpyridin-2-
yl)-4-propoxybenzamide), in which the propene moiety in the
alkoxy portion of compound 1 was replaced with the propane moi-
ety, showed no preference for either subtype; compound 8 pro-
3.2. Lead compounds
Chembridge’s CNS diversity set of small molecules (ꢀ10,000)
was virtually screened using the refined pharmacophore (Fig. 2)
with 220 hits being identified. The hits were ranked based on a
query fit (QFIT) score and pharmacological activity of the top 10
hits was evaluated as described in the Section 6 using HEK
duced antagonistic activity on both h
a
4b2 and h
a
3b4 nAChRs
with IC50 values of 9.5 (3.7–24.1) M and 11.1 (8.2–15.2)
l
l
M,
respectively (Fig. 3A and C; Table 1). Based on their potency and/
or selectivity, compounds 1 and 8 were selected as lead molecules

B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
4733
shown). With regard to their mechanisms of action, effects of com-
pounds 1 and 8 were not surmountable with increasing concentra-
tions of epibatidine but led to a decrease in the maximum effect of
epibatidine (Fig. 4). This suggested that compounds 1 and 8 are not
competing with epibatidine for the orthosteric binding site but in-
stead act as non-competitive antagonists of nAChRs.
3.3. Analyses of SAR studies on analogs of compound 1
In the first series of SAR studies, chemical modifications were
made primarily to the alkoxy portion of the lead molecule, com-
pound 1, to determine functional effects of those structural
changes. Concerning modifications in the alkoxy portion of com-
pound 1, replacement of the propene group with propane moiety
(compound 8) resulted in an increase in potency on h
with no change in potency on h 4b2 nAChRs; thus, this modifica-
tion led to loss of relative selectivity for h 4b2 nAChRs (Table 1).
a3b4 nAChRs
a
a
Introduction of a methyl moiety (compound 2) and replacement
of the propene with a propyne (compound 3) induced changes in
functional activity on nAChR in a subtype-specific manner. While
those changes led to decreases in potency on h
same changes resulted in a trend to increased potency on h
a
4b2 nAChRs, the
Figure 2. Proposed pharmacophore describing features of subtype selective NAMs
of nAChRs. The features of the pharmacophore model generated using GASP are
illustrated. Three hydrophobic (HYD1, HYD2, and HYD3) and a hydrogen bonding
acceptor (HBA1) feature of the pharmacophore are marked. This pharmacophore
was developed as described in the Section 6.
a3b4
nAChRs. However, the increases in potency were not statistically
significant (compound 1 vs 2, p = 0.261; compound 1 vs 3,
p = 0.143). Compound 4 had structural modifications on both the
pyridyl and alkoxy portions of compound 1; a methyl group was
introduced to the methylpyridine in the pyridyl portion and the
propene moiety in the alkoxy portion was replaced by an ethyl res-
idue. These modifications led to a decrease in potency on h
nAChRs, whereas they caused an increase in potency on h
nAChRs. Overall, these structural changes decreased the selectivity
ratio and compound 4 inhibited the activity of h 4b2 and h 3b4
nAChRs with comparable potency. The pyridyl portion of
and structural modifications were made at the pyridyl or alkoxy
portions of these compounds as outlined in the chemistry section
(Fig. 3A). Analyses of structure activity relationship (SAR) studies
with 25 analogs of these lead compounds were then performed
to determine how modifications are related to the compounds’
activity on nAChRs. None of the compounds reported here exhib-
a
a
4b2
3b4
a
a
ited agonist activity on either ha4b2 or ha3b4 nAChRs (Data not
Figure 3. Concentration–response effects of compounds 1 and 8 on h
primary substitutions for SAR studies. The concentration-response effects of compound 1 (B) and 8 (C) were investigated on h
described in the Section 6. Values represent means SEMs (n = 4–7). Data are reported as a percentage of peak fluorescence responses for 1
a
4b2 nAChRs and h
a
3b4 nAChRs. (A) Structure of lead compounds 1 and 8 with the location of the
4b2 nAChRs (j) and h 3b4 nAChRs (h) as
M epibatidine. IC50 values of
a
a
l
compounds 1 and 8 are reported in Table 1.

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B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
Table 1
SAR studies on the alkoxy portion of Compound 1
Compound
h
a
4b2 nAChRs
h
a
3b4 nAChRs
M)
Selectivityc
b
b
IC50 valuea
(
l
M)
nh
IC50 valuea
(
l
nh
O
N
N
H
6.0 (3.4–10.6)
9.5 (3.7–24.1)
19.6 (16.1–23.9)
ꢁ0.7
ꢁ1.6
ꢁ1.0
31.7 (22.8–44.0)
11.1 (8.2–15.2)
24.7 (17.8–33.9)
ꢁ1.1
ꢁ1.5
ꢁ1.5
5.3
1.2
O
1
O
N
N
N
H
O
8
O
N
H
1.3
1.1
O
2
O
N
N
N
H
19.4 (15.7–24.0)
17.1 (11.2–26.0)
ꢁ1.1
ꢁ0.9
21.7 (15.3–31.0)
17.4 (14.9–20.4)
ꢁ1.6
ꢁ0.8
O
3
O
1.0
N
H
O
4
a
b
c
Values represent geometric means (confidence limits), n = 5–10.
nh, Hill coefficient.
Selectivity; fold ratio of IC50-h3a4b nAChRs/IC50-ha4b2 nAChRs.
Figure 4. Concentration-response effects of epibatidine in the absence and presence of compounds 1 and 8. The concentration–response effects of epibatidine were
investigated in the absence (h) and the presence (j) of compounds 1 (A) and 8 (B) on human
Values represent means SEM (n = 4).
a4b2 nAChRs, using calcium accumulation assays described in the Section 6.
compound 1 was also modified using several different substitu-
tions and functional effects of those changes were investigated (Ta-
ble 2). Replacement of 2-methylpyridine with pyridine (compound
(compound 11) led to a decrease in potency on h
a4b2 nAChRs.
However, potency on h 3b4 nAChRs was not affected by this sub-
a
stitution, leading to a decrease in the selectivity ratio. On the other
hand, replacement of 2-methylpyridine with N-propylpyridin-2-
amine (compound 34) or N-butylpyridin-2-amine (compound 35)
5) caused decreases in potency for both h
a
4b2 and h
a
3b4 nAChRs.
4b2,
Compound still maintained relative selectivity for
5
ha
although the selectivity ratio decreased to ꢀ3-fold. The introduc-
resulted in decreases in potency on h
creases in potency on h 3b4 nAChRs.
a4b2 nAChRs and slight in-
tion of halogen atoms (e.g., chlorine and bromine) led to decreases
a
in potency on both h
(compound 9) resulted in ꢀ11-fold and ꢀ2-fold decreases in po-
tency on h 4b2 and h 3b4 nAChRs, respectively, while the chlori-
nated analog 10 showed no activity up to 100 M on both
subtypes. Replacement of 2-methylpyridine with 2-ethylpyridine
a4b2 and ha3b4 nAChRs. Bromine addition
3.4. Analyses of SAR studies on analogs of compound 8
a
a
l
In the second series of SAR studies, compound 8 was used as the
basis of comparison and functional effects of different substitutions

B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
4735
Table 2
SAR studies on the pyridyl portion of Compound 1
Compound
h
a4b2 nAChRs
h
a3b4 nAChRs
Selectivityc
b
b
IC50 valuea
(l
M)
nh
IC50 valuea
(l
M)
nh
O
N
N
H
6.0 (3.4–10.6)
ꢁ0.7
31.7 (22.8–44.0)
ꢁ1.1
5.3
3.2
O
1
O
N
N
H
19.3 (14.5–25.7)
70.1 (53.9–91.2)
>100d
ꢁ1.0
ꢁ1.1
61.4 (40.6–92.8)
65.5 (54.4–78.9)
>100d
ꢁ1.3
ꢁ0.9
O
5
O
Br
N
N
H
0.9
O
9
O
e
e
e
Cl
N
N
H
ꢀ
ꢀ
ꢀ
O
10
O
N
N
H
20.5 (15.5–27.3)
ꢁ1.3
ꢁ2
27.4 (14.6–51.2)
ꢁ0.9
ꢁ2.1
1.3
0.8
O
11
O
HN
N
N
H
14.8 (10.6–20.8)
22.4 (12.4–40.4)
11.7 (8.2–16.8)
19.2 (13.7–26.9)
O
34
O
HN
N
N
H
ꢁ1.6
ꢁ1.7
0.9
O
35
a
b
c
Values represent geometric means (confidence limits), n = 5–10.
nh, Hill coefficient.
Selectivity; fold ratio of IC50-h3a4b nAChRs/IC50-ha4b2 nAChRs.
d
e
No activity up to 100
lM.
Could not be determined.
on the pyridyl portion of compound 8 were explored (Table 3). Sim-
ilar to the SAR studies on compound 1, introduction of halogen
atoms to the pyridyl portion of compound 8 produced significant
decreases in potency for both subtypes. Analogs with bromine or
chlorine substitutions (compounds 12 and 13) either showed weak
4. Discussion
Computer aided drug discovery (CADD) has become an increas-
ingly useful tool to facilitate many steps of the drug discovery pro-
cess such as identifying hits, enabling de novo design of ligands,
and modeling ADMET (absorption, distribution, metabolism, excre-
tion, and toxicity) properties.21–23 In particular, virtual screening
(VS) has been widely used to guide lead identification by employ-
ing various statistical analyses and algorithms designed to search
large chemical libraries in silico.24 Ligand-based virtual screening
(LBVS) takes advantage of available information for known ligands
such as structures, shape of individual fragments, and electrostatic
properties; whereas SBVS exploits the knowledge of the structure
of the target protein.25,26 In this study, a novel scaffold (compound
1) was identified through a virtual screening, using ligand-based
and structure-based approaches. Previously, our laboratory identi-
fied NAMs (i.e., KAB-18, DDR-5, DDR-13, and DDR-18) that showed
inhibitory activity (IC50 >50
subtypes. Replacement of 2-methylpyridine with pyrazine (com-
pound 14) resulted in a ꢀ3-fold decrease in potency on h 4b2 nAC-
hRs and 3b4 nAChRs.
lM) or no effects up to 100 lM on both
a
a
ꢀ2-fold decrease in potency on
ha
Substitution of 2-ethylpyridine at the pyridyl portion of compound
8 (compound 15) led to ꢀ5-fold and ꢀ2-fold decreases in potency
on ha4b2 and ha3b4 nAChRs, respectively. Four analogs that have
different alkylamine substitutions at the pyridine ring (compounds
19–22) provided insight into the functional effects of chain length
on the pyridyl portion of compound 8. Analogs incorporating longer
alkylamine side chains showed lower potency on both h
3b4 nAChRs, suggesting inverse correlation between the chain
length and potency. Introduction of structurally bulky groups to
a4b2 and
ha
relative selectivity for ha4b2 nAChRs against ha3b4 nAChRs
(Fig. 1).16 Furthermore, a combination of homology modeling, blind
the pyridine ring led to loss of activity on both ha4b2 and ha3b4
nAChRs (compounds 23–30).
docking, and site-directed mutagenesis identified the ‘b subunit

4736
B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
Table 3
SAR studies on the pyridyl portion of Compound 8
Compound
h
a
4b2 nAChRs
M)
h
a
3b4 nAChRs
Selectivityc
b
b
IC50 valuea
(
l
nh
IC50 valuea
(
l
M)
nh
O
N
N
H
7.5 (3.6–15.5)
ꢁ1.3
ꢁ1.0
11.5 (8.0–15.2)
ꢁ0.8
1.5
O
8
O
e
e
>100d
>100d
ꢀ
ꢀ
Br
Cl
N
N
N
H
80.7 (49.3–132.0)
O
12
O
>100d
ꢀ
ꢀ
ꢀ
e
e
e
N
H
O
13
N
O
N
N
23.2 (16.4–32.6)
ꢁ1.2
22.5 (12.0–42.0)
ꢁ0.9
1.0
H
O
14
O
N
N
H
37.6 (22.6–62.7)
15.4 (10.5–22.6)
ꢁ1.7
ꢁ1.4
29.2 (27.1–31.5)
12.6 (6.4–24.6)
ꢁ1.6
ꢁ1.1
0.8
0.8
O
15
O
HN
N
N
H
O
19
O
HN
HN
N
N
H
54.1 (24.7–118.0)
ꢁ0.8
77.8 (42.2–143.0)
ꢁ0.7
1.4
O
20
O
N
N
H
>100d
ꢀ
>100d
ꢀ
ꢀ
e
e
e
O
21
O
HN
N
N
H
e
e
e
>100d
ꢀ
>100d
ꢀ
ꢀ
O
22
O
HN
N
N
H
e
e
e
>100d
ꢀ
>100d
>100d
ꢀ
ꢀ
O
O
23
O
HN
N
F
N
H
e
e
e
>100d
ꢀ
ꢀ
ꢀ
24

B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
4737
Table 3 (continued)
Compound
h
a
4b2 nAChRs
M)
h
a
3b4 nAChRs
Selectivityc
b
b
IC50 valuea
(
l
nh
IC50 valuea
(
l
M)
nh
O
HN
HN
N
N
H
e
e
e
>100d
ꢀ
ꢀ
>100d
ꢀ
ꢀ
ꢀ
ꢀ
O
O
25
F
O
N
N
H
e
e
e
>100d
>100d
26
O
F
HN
N
N
N
H
e
e
e
>100d
ꢀ
ꢀ
>100d
ꢀ
ꢀ
ꢀ
ꢀ
O
O
27
O
Cl
HN
N
H
e
e
e
>100d
>100d
28
O
O
HN
N
N
e
e
e
>100d
ꢀ
ꢀ
>100d
ꢀ
ꢀ
ꢀ
ꢀ
H
O
29
O
N
N
N
e
e
e
>100d
>100d
H
O
30
a
b
c
Values represent geometric means (confidence limits), n = 5–10.
nh, Hill coefficient.
Selectivity; fold ratio of IC50-h3a4b nAChRs/IC50-ha4b2 nAChRs.
d
e
No activity up to 100
lM.
Could not be determined.
site’, a novel allosteric binding site on the receptor where these
NAMs interact.16–18 In contrast to the orthosteric binding site, the
‘b subunit site’ showed reduced sequence conservation. The ‘b sub-
unit site’ was then used for SBVS with a hypothesis that its sequen-
tial and structural diversity could provide molecular foundations to
develop selective nAChR drugs. Supporting this hypothesis, hits
using the structure of Hit 2, one of the hits from SBVS14, as it (1)
shows relative selectivity for h 4b2 nAChRs over h 3b4 nAChRs,
a
a
(2) displays structural similarity to the four NAMs used to develop
the initial pharmacophore, and (3) has physicochemical properties
associated with more desirable ADMET properties. The refined
pharmacophore was subsequently applied to the LBVS and led to
the identification of compound 1, a lead molecule of this study.
In particular, with an aim of developing CNS applicable drugs, we
performed the LBVS using the Chembridge’s CNS diversity set. Mol-
ecules contained in this library generally possess favorable physi-
cochemical properties with regard to CNS bioavailability due to
multiple strict sets of property filters. However, the strict criteria
for drug-like properties result in the small-sized library with de-
creased molecular diversity. This might have limited the potential
of identifying more potent and/or selective drugs, as well as chem-
ical diversity of hit molecules.
from this SBVS showed preference for ha
4b2 nAChRs.14 Interest-
ingly, one of the hits from the SBVS, (5-amino-N-(6-methylpyri-
din-2-yl)-2-(piperidin-1-yl)benzamide; Hit 2) shares the
following structural features with the previously identified four
subtype-selective NAMs (Fig. 1): (1) The carbonyl group is attached
to the aryl ring and (2) the aryl ring has a six-membered ring at the
ortho position. In order to identify a novel chemical scaffold with
relative selectivity for ha4b2 nAChRs, the above mentioned infor-
mation obtained from the previous studies was utilized. Initially,
chemical and structural features of four subtype-selective NAMs
(i.e., KAB-18, DDR-5, DDR-13, and DDR-18) were used to generate
a pharmacophore. This initial pharmacophore was then refined
In the present study, we used HEK tsA201 cells stably express-
ing ha4b2 or ha3b4 nAChRs for pharmacological evaluations of the

4738
B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
analogs. As reported in our previous paper, pharmacological prop-
erties of classical agonists (epibatidine and nicotine) and antago-
nists (mecamylamine and d-tubocurarine) determined using
these cells lines are in good agreement with previously reported
values.16 In particular, the non-competitive nAChR antagonist mec-
the chemical/structural properties of the molecules pertaining to
antagonistic activity on nAChRs, we have obtained 27 benzamide
analogs and performed SAR studies on these analogs. It is notable
that among the 27 analogs, two analogs (i.e., compounds 1 and
5) exhibit preference for ha4b2 nAChRs, although the selectivity
amylamine showed inhibitory activity with IC50 values of 0.6
and 0.7 M on h 4b2 nAChRs and h 3b4 nAChRs, respectively.
The competitive antagonist d-tubocurarine displayed antagonist
activity with IC50 values of 9.2 and 6.8 M on h 4b2 nAChRs and
3b4 nAChRs, respectively. Unlike these non-selective nAChR
antagonsits, the lead molecule (compound 1) exhibited h 4b2
nAChR-selective antagonism. Compound 1 showed antagonistic
activity with an IC50 value of 6.0 (3.4–10.6) M on h 4b2 nAChRs
with ꢀ5-fold preference against h 3b4 nAChRs (Table 1). Its non-
l
M
ratios remain modest.
l
a
a
The study described here documents the successful utilization
of a multifaceted approach for rational drug discovery employing
computational modeling, pharmacology, and medicinal chemistry,
which has led to the identification of a novel chemical class of
nAChR NAMs including molecules that show relative selectivity
l
a
ha
a
for ha4b2 nAChRs. The discovery of subtype-selective NAMs of
l
a
nAChRs described here should contribute significantly to our
understanding of the involvement of specific nAChR subtypes in
normal and pathophysiological states and may hold clinical prom-
ise for diseases linked to nAChRs.
a
competitive mechanism of action was described by a decrease in
maximum effects of the orthosteric ligand epibatidine (Fig. 4A).
In order to determine effects of structural modifications to the
pyridyl or alkoxy portions of the molecules with regard to their
functional activities, analyses of the SAR studies on analogs of com-
pounds 1 and 8 were performed. The most significant finding from
the SAR studies of analogs of compound 1 is that modification of
propene to propane (compound 1 vs compound 8) led to loss of
6. Experimental
6.1. Materials
The calcium sensitive fluorescent probe, Calcium 5 NW dye, was
obtained from Molecular Devices (Sunyvale, CA). Dulbecco’s Mod-
ified Eagle Medium (DMEM), penicillin, streptomycin and L-gluta-
preference for ha4b2, suggesting the importance of a double bond
in the alkoxy portion of the molecules with regard to subtype-
mine were purchased from Invitrogen Corporation (Grand Island,
NY). The nAChR agonist, epibatidine was obtained from Sigma–Al-
drich (St. Louis, MO). All other reagents were obtained from Fisher
Scientific (Pittsburg, PA).
selectivity. Another analog containing the propene moiety (com-
pound 5) also exhibited ꢀ3-fold preference for h
a
4b2 nAChRs
against h 3b4 nAChRs. However, relative selectivity for h
a
a
4b2
nAChRs was not observed for other analogs that also have the pro-
pene moiety (compounds 9–11, 34, and 35). Introduction of halo-
gen atoms, an ethyl moiety, and alkyl amines (compounds 9–11,
34, and 35) to the pyridyl portion of the molecules abolished the
subtype-selectivity. Comparison with their counterparts contain-
ing the propane moiety (compounds 12, 13, and 15) suggested that
functional activities were mainly determined by the pyridyl por-
tion of the molecules for this series of compounds and modification
of propene to propane did not affect either potency or subtype-
selectivity. Structural modifications to both the pyridyl and alkoxy
portions of the molecules led to an analog (compound 4) that has
6.2. Pharmacophore generation
A pharmacophore model was generated using a procedure pre-
viously reported by our laboratory.16,30 The four NAMs (i.e. KAB-18,
DDR-5, DDR-13, and DDR-18) that selectively target ha4b2 nAChRs
were aligned using GASP (Genetic Algorithm Similarity Program,
SYBYL 7.1) with default settings (population size, 125; the allele
mutate weight, 96; the fitness increment, 0.02). Alignments for
each set of molecules were repeated for 10 runs, followed by the
optimal model selection through visual inspection. This initial
pharmacophore model was then refined through observations of
a novel chemical structure identified by SBVS.14 One of the hits
from this SBVS contained three of the four hydrophobic pharmaco-
phore elements. Therefore, a hydrophobic domain of the initial
pharmacophore was eliminated using the ‘edit pharmacophore’
option in SYBYL 7.1.
comparable potency on h
these data suggest that both portions of the molecules contribute
to the selectivity for h 4b2 nAChRs.
a4b2 and ha3b4 nAChRs. Collectively,
a
The SAR studies on analogs of compound 8 also provide insight
into the functional effects of chemical modifications on the pyridyl
portion of the molecules. With the aim of exploring the appropriate
chain length linked to the pyridine ring, a series of compounds
(compounds 8, and 19–22) were synthesized. SAR studies on these
compounds suggested that steric hindrance exists at the portion of
the receptor-binding pocket where the pyridine moiety interacts.
Increasing the chain length resulted in decreases in potency for
both subtypes. Similarly, substitutions with structurally bulky
moieties to the pyridine ring (compounds 23–30) led to loss of
activity on both subtypes.
6.3. Ligand-based virtual screening
Ligand-based virtual screening (LBVS) was performed with the
above-mentioned pharmacophore. Chembridge’s CNS diversity
set of small molecules (ꢀ10,000) was virtually explored with an
aim of identifying molecules that contain chemical and geometri-
cal descriptors defined by the pharmacophore model. The LBVS
was performed with UNITY (SYBYL 7.1), using the default settings
of FlexX (the 3D dynamic setting) search options. In order to max-
imize the diversity in molecular scaffolds, the LBVS was performed
with Lipinski filter turned ‘off’. The hits were then scored in UNITY
based on their ability to fit the spatial and chemical features de-
scribed by the pharmacophore.
5. Conclusion
For the past several years, our laboratory has focused on discov-
ering small molecules that modulate nAChR activity with several
chemical classes of ligands being reported.13–18,27–29 Here, we de-
scribed the identification of a novel chemical class of NAMs of nAC-
hRs through the application of multiple approaches. Iterative
cycles of virtual screening using ligand-based and structure-based
approaches led to the identification of the lead molecule, com-
6.4. Calcium accumulation assay
For pharmacological evaluation of the synthesized analogs, cal-
cium accumulation assay was performed with HEK tsA201 cells
pound
the lead, compound 1 inhibits the activity of h
ꢀ5-fold preference against h 3b4 nAChRs. To gain insight into
1
(4-(allyloxy)-N-(6-methylpyridin-2-yl)benzamide). As
stably expressing either h
a4b2 nAChRs or ha3b4 nAChRs (obtained
a4b2 nAChRs with
from Professor Jon Lindstrom, University of Pennsylvania,
a

B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
4739
Philadelphia, PA), using a procedure previously reported by our
6.6.3. Preparation and characterization of compounds
6.6.3.1. N-(6-Methylpyridin-2-yl)-4-propoxybenzamide (8).
laboratory.13 Briefly, cells were plated 24 h prior to experiments
in clear pre-coated 96-well culture plates. Plates were incubated
at 37 °C in 5% CO2 in supplemented Dulbecco’s modified Eagle’s
medium and cells were allowed to form a ꢀ100% confluent mono-
layer (typically 24 h after plating). On the day of the experiment,
A
mixture of 2-amino-6-methylpyridine (6a) (0.61 g, 5.6 mmol) and trieth-
ylamine (4 mL) in dichloromethane (DCM) (10 mL) was added dropwise
to freshly made 4-propoxybenzoyl chloride (7a) (1.19 g, 6.0 mmol) in
DCM (5 mL) and the reaction mixture was heated to reflux overnight.
After cooling, the mixture was washed with water, brine and dried with
anhydrous MgSO4. The solvent was evaporated under reduced pressure,
then the crude product was further purified on a silica gel column using
hexanes/ethyl acetate (3:1) to yield the pure product (1.2 g, 79%) as a col-
orless solid, mp 86–89 °C. 1HNMR(CDCl3,300 MHz)d8.59 (s, 1H), 8.19 (d,
J = 8.1 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.64 (dd, J = 7.8 Hz, 1H), 6.96 (d,
J = 8.4 Hz, 2H), 6.91 (d, J = 7.5 Hz, 1H), 3.99 (t, J = 6.6 Hz, 2H), 2.46 (s,
3H), 1.85 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d
165.2, 162.4, 156.8, 151.1, 138.7, 129.1, 126.3, 119.2, 114.4, 110.9, 69.7,
24.0, 22.5, 10.5; ESI-MS m/z 271.11 (M+H, 100); Anal. Calcd for
cells were washed (100 lL) with HEPES-buffered Krebs (HBK) solu-
tion and incubated for 1 hour at room temperature with 50% Cal-
cium 5 NW dye (Molecular Devices). The plates were then placed
into a fluid handling integrated fluorescence plate reader (FlexSta-
tion II, Molecular Devices, Sunnyvale, CA) and treatment solutions
were added to the cells. Changes in intracellular calcium levels
were then simultaneously measured at excitation of 485 nm and
emission of 525 nm from the bottom of the plate every 1.5 s. For
each drug, six concentrations (0.1–100
full concentration–response curves and results are reported as the
concentration of drugs that reduced the effect of the 1 M epibati-
lM) were used to generate
l
C16H18N2O2 (270.14): C, 71.09; H, 6.71; N, 10.36. Found: C, 70.71; H,
dine control by 50% (IC50 values). All compounds were initially dis-
6.78; N, 10.06.
solved with 100% DMSO (0.01 M stocks) and further dilutions were
made in HEPES-buffered Krebs (HBK) solution (6100
l
M). The
6.6.3.2. 4-(Allyloxy)-N-(6-bromopyridin-2-yl)benzamide (9).
The
DMSO concentration at this compound concentration was less than
1% and had no effects on basal or agonist-induced increases in fluo-
rescence intensity.16 Due to solubility problems, compound con-
reaction was similar to the one above employing 2-amino-6-bromopyri-
dine (6b) (233 mg, 1.35 mmol) and 4-allyloxybenzoyl chloride (7b)
(265 mg, 1.35 mmol) to yield 9 (367 mg, 82%). Purification was carried
out on a silica gel column using hexanes/ethyl acetate (4:1) to provide
the pure compound as a colorless solid, mp 102–105 °C. 1H NMR (CDCl3,
300 MHz) d 8.52 (s, 1H), 8.35 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H),
7.60 (dd, J = 8.1 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.00 (d, J = 9.0 Hz, 2H),
6.07 (m, 1H), 5.45 (d, J = 17.4 Hz, 1H), 5.34 (d, J = 10.5 Hz, 1H), 4.62 (d,
J = 5.1 Hz, 2H); 13C NMR (CDCl3, 75 MHz) d 165.0, 162.1, 151.8, 140.7,
139.3, 132.4, 129.2, 125.9, 123.4, 118.3, 114.8, 112.4, 69.0; ESI-MS m/z
333.02 (M+H, 68).
centrations greater than 100
concentration–response studies.
lM
were not used in our
6.5. Calculations
Quantification of functional response was performed by deter-
mining the ability of compounds to inhibit the fluorescence signal
increases in response to epibatidine (1 lM). Curve fitting was per-
formed by Prism software (GraphPad, San Diego, CA) using the
equation for a single-site sigmoidal dose–response curve with a
variable slope. Functional data were calculated from the number
of observations (n) performed in triplicate. IC50 values are ex-
pressed as geometric means (95% confidence limits) of each exper-
iment. The Hill coefficients (nh) represented in the table were
calculated by taking arithmetic mean of each Hill coefficient from
individual experiments.
6.6.3.3.
(10).
4-(Allyloxy)-N-(6-chloropyridin-2-yl)benzamide
The reaction was similar to the one above employing 2-
amino-6-chloropyridine (6c) (60 mg, 0.47 mmol) and 4-ally-
loxybenzoyl chloride (7b) (67 mg, 0.34 mmol) to yield 10 (85 mg,
87%). Purification was carried out on a silica gel column using hex-
anes/ethyl acetate (3:1) to provide the pure compound as a colorless
solid, mp 98–100 °C. 1H NMR (CDCl3, 300 MHz) d 8.48 (s, 1H), 8.33
(d, J = 7.8 Hz, 1H), 7.89 (d, J = 9.0 Hz, 2H), 7.73 (dd, J = 7.8 Hz, 1H),
7.11 (d, J = 7.2 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H), 6.07 (m, 1H), 5.46 (d,
J = 17.4 Hz, 1H), 5.35 (d, J = 10.5 Hz, 1H), 4.64 (d, J = 5.1 Hz, 2H); 13C
NMR (CDCl3, 75 MHz) d 165.0, 162.1, 151.6, 149.0, 141.0, 132.4,
129.2, 125.9, 119.6, 118.3, 114.9, 112.1, 69.0; ESI-MS m/z 311.05
(M+Na, 100).
6.6. General procedure for the preparation of compounds
6.6.1. General experimental
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were re-
corded on a Bruker 300 UltraShield spectrometer. Electrospray ion-
ization mass spectrometry (ESI-MS) was performed by The Ohio
State University Mass Spectrometry and Proteomics Facility. Melt-
ing points were measured on a Fisher Scientific melting point
apparatus and were not corrected. Gravity and flash column chro-
matography were performed using type 60A silica gel (60–
230 mesh) from Fisher Scientific. Solid compounds were further
purified by recrystallization. Elemental analyses, performed by
Atlantic Microlabs, Norcross, GA, were carried out for all com-
6.6.3.4. 4-(Allyloxy)-N-(6-ethylpyridin-2-yl)benzamide (11).
The
reaction was similar to the one above employing 2-amino-6-ethylpyri-
dine (6d) (70 mg, 0.57 mmol) and 4-allyloxybenzoyl chloride (7b)
(112 mg, 0.57 mmol) to yield 11 (143 mg, 89%). Purification was carried
out on a silica gel column using hexanes/ethyl acetate (3:1) to provide the
pure compound as a colorless solid, mp 42–45 °C. 1H NMR (CDCl3,
300 MHz) d 8.61 (s, 1H), 8.19 (d, J = 8.1 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H),
7.67 (dd, J = 7.8 Hz, 1H), 6.99 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 7.5 Hz, 1H),
6.07 (m, 1H), 5.44 (d, J = 17.4 Hz, 1H), 5.33 (d, J = 10.5 Hz, 1H), 4.61 (d,
J = 5.4 Hz, 2H), 2.73 (q, J = 7.5 Hz, 2H), 1.29 (t, J = 7.5 Hz, 3H); 13C NMR
(CDCl3, 75 MHz) d 165.1, 162.1, 161.7, 151.0, 138.8, 132.6, 129.2, 126.7,
118.2, 117.9, 114.7, 111.2, 68.9, 31.0, 13.8; ESI-MS m/z 283.11 (M+H,
100); Anal. Calcd for C17H18N2O2 (282.14): C, 72.32; H, 6.43; N, 9.92.
Found: C, 72.36; H, 6.47; N, 9.65.
pounds prepared in our lab displaying an IC50 value of <50
well as for other selected target compounds.
lM as
6.6.2. Materials
All chemicals and solvents were purchased from Aldrich Chem-
ical Co., TCI, Alfa Aesar, Fisher Scientific, or Matrix Scientific and
were used without further purification. Compounds 1–5 were pur-
chased from Chembridge (San Diego, CA). These compounds were
obtained at P90% purity as determined by 1H NMR analyses as sta-
ted by the supplier. Compounds prepared in our lab were charac-
terized as free bases unless otherwise stated, but all were tested
as hydrochloric acid salts in the bioassay to aid in solubilizing
the compounds in the biological assay buffer.
6.6.3.5. N-(6-Bromopyridin-2-yl)-4-propoxybenzamide (12).
The
reaction was similar to the one above employing 2-amino-6-bromopyri-
dine (6b) (1.25 g, 7.2 mmol) and 4-propoxybenzoyl chloride (7a) (1.42 g,
7.2 mmol) to yield 12 (1.96 g, 81%). Purification was carried out on a silica
gel column using hexanes/ethyl acetate (3:1) to provide the pure

4740
B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
compound as a colorless solid, mp 128–132 °C. 1HNMR(CDCl3, 300 MHz)
d 8.52 (s, 1H), 8.36 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.61 (dd,
J = 8.1 Hz, 1H), 7.25 (d, J = 7.5 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 4.00 (t,
J = 6.6 Hz, 2H), 1.86 (m, 2H), 1.07 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3,
75 MHz) d 165.1, 162.7, 151.8, 140.7, 139.2, 129.2, 125.5, 123.4, 114.6,
112.4, 69.8, 22.5, 10.5; ESI-MS m/z357.02 (M+Na, 100), 335.04 (M+H, 39).
(m, 2H), 5.40 (d, J = 17.1 Hz, 2H), 5.28 (m, 2H), 4.80 (d, J = 5.7 Hz,
2H), 4.53 (d, J = 4.8 Hz, 2H). Compound 18: 1H NMR (CDCl3,
300 MHz) d 8.08 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.08
(m, 1H), 5.45 (d, J = 17.1 Hz, 1H), 5.35 (d, J = 10.5 Hz, 1H), 4.64 (d,
J = 5.1 Hz, 2H).
6.6.3.10. 4-Propoxy-N-(6-(propylamino)pyridin-2-yl)benzamide
6.6.3.6. N-(6-Chloropyridin-2-yl)-4-propoxybenzamide (13).
The
(19).
(50 mg, 0.85 mmol), Pd2(dba)3 (42 mg, 0.045 mmol), 2,20-
bis(diphenylphosphino)-1,10-binaphthyl
(rac-BINAP, 62 mg,
Compound 12 (212 mg, 0.63 mmol), propylamine
reaction was similar to the one above employing 2-amino-6-chloropyri-
dine (6c) (1.6 g, 12.5 mmol) and 4-propoxybenzoyl chloride (7a) (3.3 g,
16.7 mmol) to yield 13 (3.05 g, 84%). Purification was carried out on a sil-
ica gel column using hexanes/ethyl acetate (3:1) to provide the pure com-
pound as colorless solid, mp 123–125 °C. 1HNMR(CDCl3,300 MHz)d8.48
(s, 1H), 8.33 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 9.0 Hz, 2H), 7.72 (dd, J = 8.1 Hz,
1H), 7.10 (d, J = 7.8 Hz, 1H), 6.99 (d, J = 8.7 Hz, 2H), 4.01 (t, J = 6.6 Hz, 2H),
1.86 (m, 2H), 1.08 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d 165.1,
162.7, 151.6, 148.9, 141.0, 129.2, 125.5, 119.6, 114.6, 112.1, 69.8, 22.5,
10.5; ESI-MS m/z 313.06 (M+Na, 100).
0.10 mmol), NaO-t-Bu (320 mg, 3.29 mmol), and toluene (6 mL)
were added to an oven-dried reaction vessel that was purged with
argon for approximately 5 min. The reaction mixture was then
heated to 70 °C under argon until compound 12 was consumed
as determined by TLC. The reaction mixture was then allowed to
cool to room temperature and ethyl acetate (10 mL) was added.
The solid was removed by filtration and the filtrate was concen-
trated under reduced pressure to give the crude product. Purifica-
tion was carried out on a silica gel column using hexanes/ethyl
acetate (3:1) to provide the pure compound (152 mg, 77%) as an
off-white oil. 1H NMR (CDCl3, 300 MHz) d 8.36 (s, 1H), 7.85 (d,
J = 8.7 Hz, 2H), 7.62 (d, J = 7.8 Hz, 1H), 7.46 (dd, J = 7.8 Hz, 1H),
6.93 (d, J = 8.7 Hz, 2H), 6.16 (d, J = 8.1 Hz, 1H), 4.49 (t, J = 4.8 Hz,
1H), 3.96 (t, J = 6.6 Hz, 2H), 3.18 (q, J = 6.3 Hz, 2H), 1.83 (m, 2H),
1.60 (m, 2H), 1.05 (t, J = 7.2 Hz, 3H), 0.97 (t, J = 7.2 Hz, 3H); 13C
NMR (CDCl3, 75 MHz) d 165.0, 162.2, 157.8, 150.2, 139.7, 129.0,
126.6, 114.4, 102.2, 102.1, 69.7, 44.0, 22.8, 22.5, 11.6, 10.5; ESI-
6.6.3.7. 4-Propoxy-N-(pyrazin-2-yl)benzamide (14).
The
reaction was similar to the one above employing 2-aminopyrazine
(6e) (278 mg, 2.93 mmol) and 4-propoxybenzoyl chloride (7a)
(582 mg, 2.93 mmol) to yield 14 (127 mg, 17%). Purification was
carried out on a silica gel column using hexanes/ethyl acetate
(3:1) to provide the pure compound as a colorless solid, mp 137–
140 °C. 1H NMR (CDCl3, 300 MHz) d 9.73 (s, 1H), 8.57 (br, 1H),
8.38 (d, J = 2.4 Hz, 1H), 8.27 (br, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.01
(d, J = 8.8 Hz, 2H), 4.02 (t, J = 6.6 Hz, 2H), 1.87 (m, 2H), 1.08 (t,
J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d 164.9, 162.8, 148.5,
142.0, 140.1, 137.2, 129.4, 125.1, 114.7, 69.8, 22.5, 10.5; ESI-MS
m/z 258.10 (M + H, 100); Anal. Calcd for C14H15N3O2 (257.12): C,
65.35; H, 5.88; N, 16.33. Found: C, 65.01; H, 5.90; N, 15.95.
MS m/z 314.14 (M
+ H, 100); Anal. Calcd for C18H23N3O2
(313.18): C, 68.98; H, 7.40; N, 13.41. Found: C, 69.28; H, 7.79; N,
13.02.
6.6.3.11. 4-Propoxy-N-(6-(butylamino)pyridin-2-yl)benzamide
(20).
The reaction was similar to the one used to prepare
6.6.3.8. N-(6-Ethylpyridin-2-yl)-4-propoxybenzamide (15).
The
compound 19 with compound 12 (204 mg, 0.61 mmol), butyl-
reaction was similar to the one above employing 2-amino-6-ethylpyri-
dine (6d) (350 mg, 2.87 mmol) and 4-propoxybenzoyl chloride (7a)
(483 mg, 2.43 mmol) to yield 15 (428 mg, 62%). Purification was carried
out on a silica gel column using hexanes/ethyl acetate (3:1) to provide
the pure compound as colorless solid, mp 59–62 °C. 1H NMR (CDCl3,
400 MHz) d 8.56 (s, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 8.8 Hz, 2H),
7.66 (dd, J = 8.0 Hz, 2H), 6.96 (d, J = 8.8 Hz, 1H), 6.93 (d, J = 7.5 Hz, 1H),
3.98 (t, J = 6.5 Hz, 2H), 2.72 (q, J = 7.6 Hz, 2H), 1.85 (m, 2H), 1.28 (t,
J = 7.6 Hz, 3H), 1.06 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3, 100 MHz) d
165.6, 162.7, 162.5, 151.5, 139.1, 129.5, 126.7, 118.2, 114.8, 111.5,
70.1, 31.4, 23.1, 14.2, 10.9; ESI-MS m/z 285.13 (M+H, 100); Anal. Calcd
for C17H20N2O2 (284.12): C, 71.81; H, 7.09; N, 9.85. Found: C, 71.62; H,
7.40; N, 9.92.
amine (63 mg, 0.86 mmol), Pd2(dba)3 (47 mg, 0.068 mmol), 2,20-
bis(diphenylphosphino)-1,10-binaphthyl
(rac-BINAP,
68 mg,
0.11 mmol), NaO-t-Bu (321 mg, 3.29 mmol), and toluene (6 mL)
to yield 20 (155 mg, 78%). Purification was carried out on a silica
gel column using hexanes/ethyl acetate (3:1) to provide the pure
compound as an off-white oil. 1H NMR (CDCl3, 300 MHz) d 8.19
(s, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.1 Hz, 1H), 7.50 (dd,
J = 8.1 Hz, 1H), 6.97 (d, J = 8.7 Hz, 2H), 6.17 (d, J = 8.1 Hz, 1H),
4.37 (br, 1H), 3.99 (t, J = 6.3 Hz, 2H), 3.25 (q, J = 6.3 Hz, 2H), 1.85
(m, 2H), 1.61 (m, 2H), 1.44 (m, 2H), 1.07 (t, J = 7.5 Hz, 3H), 0.98
(t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d 165.0, 162.2, 157.8,
150.2, 139.8, 129.0, 126.6, 114.4, 102.1, 102.0, 69.7, 41.9, 31.7,
22.5, 20.2, 13.9, 10.5; ESI-MS m/z 328.16 (M+H, 100); Anal. Calcd
for C19H25N3O2 (327.19): C, 69.70; H, 7.70; N, 12.83. Found: C,
70.32; H, 7.72; N, 12.56.
6.6.3.9. Ally 4-(allyloxy)benzoate (17) and 4-(Allyloxy)benzoic
acid (18).
Allyl bromide (2.5 g, 20.7 mmol) was added to 4-
hydroxybenzoic acid (16) (700 mg, 5.07 mmol) and NaOH
(452 mg, 11.3 mmol) in 20 mL 1:1 H2O/ethanol (v/v). The solution
was heated to reflux overnight, and then solvent was removed un-
der reduced pressure to yield the crude products. Purification was
carried out on a silica gel column using hexanes/ethyl acetate (3:1)
to provide the pure compounds 17 (570 mg) and 18 (305 mg) as
colorless solids. Compound 17 (834 mg, 3.82 mmol) and NaOH
(153 mg, 3.83 mmol) were dissolved in 10 mL 1:1 H2O/ethanol
(v/v) and the solution was heated to reflux overnight. The solvent
was removed under reduced pressure and the solid was dissolved
in H2O and acidified. The aqueous solution was extracted with
ethyl acetate (3 ꢂ 30 mL), and then the organic layer was com-
bined and dried with anhydrous Na2SO4. Compound 18 was ob-
tained and further purified by recrystallization in 1:1 hexanes/
ethyl acetate (650 mg, 95%). Compound17: 1H NMR (CDCl3,
300 MHz) d 8.02 (d, J = 8.1 Hz, 2H), 6.91 (d, J = 8.1 Hz, 2H), 6.01
6.6.3.12. N-(6-(Pentylamino)pyridin-2-yl)-4-propoxybenzamide
(21).
The reaction was similar to the one used to prepare
compound 19 with compound 12 (203 mg, 0.61 mmol), amylamine
(63 mg, 0.72 mmol), Pd2(dba)3 (46 mg, 0.049 mmol), 2,20-
bis(diphenylphosphino)-1,10-binaphthyl
(rac-BINAP,
65 mg,
0.10 mmol), NaO-t-Bu (325 mg, 3.3 mmol), and toluene (5 mL) to
yield 21 (145 mg, 70%). Purification was carried out on a silica
gel column using hexanes/ethyl acetate (3:1) to provide the pure
compound as an off-white oil. 1H NMR (CDCl3, 300 MHz) d 8.22
(s, 1H), 7.87 (d, J = 8.1 Hz, 2H), 7.62 (d, J = 7.8 Hz, 1H), 7.50 (dd,
J = 8.1 Hz, 1H), 6.96 (d, J = 8.1 Hz, 2H), 6.17 (d, J = 8.1 Hz, 1H),
4.39 (br, 1H), 3.99 (t, J = 6.3 Hz, 2H), 3.24 (m, 2H), 1.85 (m, 2H),
1.62 (m, 2H), 1.38 (m, 4H), 1.07 (t, J = 7.2 Hz, 3H), 0.93 (m, 3H);
13C NMR (CDCl3, 75 MHz) d 165.0, 162.2, 157.8, 150.2, 139.8,
129.0, 126.6, 114.4, 102.1, 102.0, 69.7, 42.2, 29.3, 29.2, 22.5, 14.0,
10.5; ESI-MS m/z 342.16 (M + H, 100).

B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
4741
6.6.3.13. N-(6-(Hexylamino)pyridin-2-yl)-4-propoxybenzamide
(22). The reaction was similar to the one used to prepare
6.6.3.17.
oxybenzamide (26).
N-(6-((4-Fluorophenyl)amino)pyridin-2-yl)-4-prop-
The reaction was similar to the one used
compound 19 with compound 12 (204 mg, 0.61 mmol), hexyl-
to prepare 19 with compound 12 (114 mg, 0.34 mmol), 4-fluoroan-
amine (77 mg, 0.76 mmol), Pd2(dba)3 (43 mg, 0.045 mmol), 2,20-
iline (60 mg, 0.54 mol), Pd2(dba)3 (24 mg, 0.026 mmol), 2,20-
bis(diphenylphosphino)-1,10-binaphthyl
(rac-BINAP,
61 mg,
bis(diphenylphosphino)-1,10-binaphthyl
(rac-BINAP,
34 mg,
0.10 mmol), NaO-t-Bu (325 mg, 3.3 mmol), and toluene (5 mL) to
yield 22 (138 mg, 64%). Purification was carried out on a silica
gel column using hexanes/ethyl acetate (3:1) to provide the pure
compound as an off-white oil. 1H NMR (CDCl3, 300 MHz) d 8.26
(s, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.62 (d, J = 7.8 Hz, 1H), 7.49 (dd,
J = 8.1 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.16 (d, J = 8.1 Hz, 1H),
4.41 (br, 1H), 3.98 (t, J = 6.3 Hz, 2H), 3.22 (m, 2H), 1.85 (m, 2H),
1.59 (m, 2H), 1.35 (m, 6H), 1.06 (t, J = 7.2 Hz, 3H), 0.91 (t,
J = 6.9 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d 165.0, 162.2, 157.8,
150.2, 139.8, 129.0, 126.6, 114.4, 102.1, 102.0, 69.7, 42.2, 31.6,
29.6, 26.7, 22.6, 22.5, 14.0, 10.5; ESI-MS m/z 356.18 (M+H, 100).
0.055 mmol), NaO-t-Bu (186 mg, 1.91 mmol), and toluene (5 mL)
to yield 26 (86 mg, 69%). Purification was carried out on a silica
gel column using hexanes/ethyl acetate (3:1) to provide the pure
compound as a colorless solid, mp 135–138 °C. 1H NMR (CDCl3,
300 MHz) d 8.21 (s, 1H), 7.82 (d, 2H), 7.78 (d, 1H), 7.55 (m, 1H),
7.28(m, 2H), 7.05 (m, 4H), 6.52 (d, 1H), 6.25 (s, 1H), 4.02 (m, 2H),
1.86 (m, 2H), 1.11 (m, 3H); 13C NMR (CDCl3, 75 MHz) d 165.1,
162.4, 154.9, 150.2, 140.2, 129.1, 126.3, 123.0, 122.9, 116.1,
115.8, 114.5, 104.5, 103.9, 69.8, 22.5, 10.5; ESI-MS m/z 366.07
(M+H, 100).
6.6.3.18.
oxybenzamide (27).
N-(6-((4-Chlorophenyl)amino)pyridin-2-yl)-4-prop-
The reaction was similar to the one used
6.6.3.14. N-(6-(Phenylamino)pyridin-2-yl)-4-propoxybenzamide
to prepare 19 with compound 12 (115 mg, 0.34 mmol), 4-chloro-
(23).
The reaction was similar to the one used to prepare com-
aniline (61 mg, 0.48 mol), Pd2(dba)3 (25 mg, 0.027 mmol), 2,20-
pound 19 with compound 12 (112 mg, 0.33 mmol), aniline (100 mg,
1.08 mmol), Pd2(dba)3 (26 mg, 0.028 mmol), 2,20-bis(diphenylphos-
phino)-1,10-binaphthyl (rac-BINAP, 34 mg, 0.055 mmol), NaO-t-Bu
(192 mg, 2.0 mmol), and toluene (6 mL) to yield 23 (115 mg, 99%).
Purification was carried out on a silica gel column using hexanes/
ethyl acetate (3:1) to provide the pure compound as a colorless solid,
mp112–115 °C. 1H NMR (CDCl3, 300 MHz) d 8.49(s, 1H), 7.83 (m, 3H),
7.55 (dd, J = 8.1 Hz, 1H), 7.32 (m, 4H), 7.05 (m, 1H), 6.93 (d, J = 8.7 Hz,
2H), 6.76 (s, 1H), 6.63 (d, J = 8.1 Hz, 1H), 3.97 (t, J = 6.6 Hz, 2H), 1.85
(m, 2H), 1.07 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d 165.3,
162.3, 154.7, 150.4, 140.3, 140.1, 129.2, 129.1, 126.4, 122.8, 120.4,
114.4, 104.7, 104.3, 69.7, 22.5, 10.5; ESI-MS m/z 348.13 (M+H, 100).
bis(diphenylphosphino)-1,10-binaphthyl
(rac-BINAP,
35 mg,
0.056 mmol), NaO-t-Bu (187 mg, 1.92 mmol), and toluene (5 mL)
to yield 27 (76 mg, 58%). Purification was carried out on a silica
gel column using hexanes/ethyl acetate (3:1) to provide the pure
compound as a colorless solid, mp 143–146 °C. 1H NMR (CDCl3,
300 MHz) d 8.30 (s, 1H), 7.84 (m, 3H), 7.58 (dd, J = 7.8 Hz, 1H),
7.29 (m, 4H), 6.97 (d, J = 9.0 Hz, 2H), 6.55 (d, J = 8.1 Hz, 1H), 6.50
(s, 1H), 4.00 (t, J = 6.6 Hz, 2H), 1.86 (m, 2H), 1.08 (t, J = 7.5 Hz,
3H); 13C NMR (CDCl3, 75 MHz) d 165.1, 162.4, 154.1, 150.3,
140.2, 138.9, 129.2, 129.1, 127.5, 126.3, 121.3, 114.5, 105.0,
104.6, 69.8, 22.5, 10.5; ESI-MS m/z 382.09 (M + H, 100); Anal. Calcd
for C21H20ClN3O2 (381.86): C, 66.05; H, 5.28; N, 11.00. Found: C,
66.09; H, 5.24; N, 11.07.
6.6.3.15. N-(6-((2-Fluorophenyl)amino)pyridin-2-yl)-4-propoxyb-
6.6.3.19. N-(6-((4-Methoxyphenyl)amino)pyridin-2-yl)-4-prop-
enzamide (24).
The reaction was similar to the one used to
oxybenzamide (28).
The reaction was similar to the one used
prepare compound 19 with compound 12 (120 mg, 0.36 mmol),
2-fluoroaniline (80 mg, 0.72 mmol), Pd2(dba)3 (25 mg, 0.027 mmol),
2,20-bis(diphenylphosphino)-1,10-binaphthyl (rac-BINAP, 37 mg,
0.06 mmol), NaO-t-Bu (190 mg, 1.95 mmol), and toluene (6 mL) to
yield 24 (97 mg, 74%). Purification was carried out on a silica gel col-
umn using hexanes/ethyl acetate (3:1) to provide the pure compound
as a colorless solid, mp 87–90 °C. 1H NMR (CDCl3, 300 MHz) d 8.29 (s,
1H), 7.98 (dd, J = 8.1 Hz, 1H), 7.88 (dd, J = 8.1 Hz, 3H), 7.61 (dd,
J = 8.1 Hz, 1H), 7.14 (m, 2H), 7.01 (m, 3H), 6.60 (d, 1H), 6.50 (s, 1H),
4.01 (t, J = 6.3 Hz, 2H), 1.85 (m, 2H), 1.08 (t, J = 7.5 Hz, 3H); 13C NMR
(CDCl3, 75 MHz) d 165.1, 162.4, 153.8, 150.2, 140.1, 129.1, 126.4,
124.3, 124.2, 122.5, 122.4, 120.9, 115.5, 115.2, 114.5, 105.2, 69.8,
22.5, 10.5; ESI-MS m/z 366.12 (M+H, 100).
to prepare 19 with compound 12 (118 mg, 0.35 mmol), 4-
methoxyaniline (72 mg, 0.59 mol), Pd2(dba)3 (27 mg, 0.029 mmol),
2,20-bis(diphenylphosphino)-1,10-binaphthyl (rac-BINAP, 35 mg,
0.056 mmol), NaO-t-Bu (180 mg, 1.85 mmol), and toluene (6 mL)
to yield 28 (107 mg, 80%). Purification was carried out on a silica
gel column using hexanes/ethyl acetate (3:1) to provide the pure
compound as a colorless solid, mp 103–105 °C. 1H NMR (CDCl3,
300 MHz)
d 8.26 (s, 1H), 7.87 (d, J = 9.0 Hz, 2H), 7.75 (d,
J = 7.8 Hz, 1H), 7.53 (dd, J = 8.1 Hz, 1H), 7.25 (d, J = 8.7 Hz, 2H),
6.98 (d, J = 8.7 Hz,2H), 6.92 (d, J = 9.0 Hz, 2H), 6.44 (d, J = 8.4 Hz,
1H), 6.26 (s, 1H), 4.01 (t, J = 6.6 Hz, 2H), 3.84 (s, 3H), 1.85 (m,
2H), 1.08 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d 165.1,
162.3, 156.3, 155.9, 150.3, 140.1, 133.0, 129.1, 126.4, 124.1,
114.6, 114.4, 103.9, 103.2, 69.7, 55.5, 22.5, 10.5; ESI-MS m/z
378.15 (M+H, 81), 400.13 (M+Na, 100).
6.6.3.16. N-(6-((3-Fluorophenyl)amino)pyridin-2-yl)-4-propoxyb-
enzamide (25).
The reaction was similar to the one used to
prepare compound 19 with compound 12 (150 mg, 0.45 mmol),
3-fluoroaniline (90 mg, 0.81 mmol), Pd2(dba)3 (30 mg, 0.032 mmol),
2,20-bis(diphenylphosphino)-1,10-binaphthyl (rac-BINAP, 39 mg,
0.063 mmol), NaO-t-Bu (190 mg, 1.95 mmol), and toluene (5 mL) to
yield 25 (147 mg, 90%). Purification was carried out on a silica gel col-
umn using hexanes/ethyl acetate (3:1) to provide the pure compound
as a colorless solid, mp 108–110 °C. 1HNMR(CDCl3, 300 MHz)d8.24 (s,
1H), 7.88 (m, 3H), 7.62 (dd, J = 8.1 Hz, 1H), 7.28 (m, 2H), 7.02 (m, 3H),
6.74 (m, 1H), 6.62 (d, J = 8.1 Hz, 1H), 6.44 (br, 1H), 4.02 (t, J = 6.6 Hz,
2H), 1.87 (m, 2H), 1.08 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d
165.1, 162.4, 161.8, 153.7, 150.3, 142.0, 140.2, 130.3, 129.1, 126.3,
114.8, 114.5, 109.1, 106.6, 106.3, 105.3, 69.8, 22.5, 10.5; ESI-MS m/z
366.09 (M + H, 100); Anal. Calcd for C21H20FN3O2 (365.40): C, 69.03;
H, 5.52; N, 11.50. Found: C, 68.92; H, 5.48; N, 11.51.
6.6.3.20. N-(6-(Benzylamino)pyridin-2-yl)-4-propoxybenzamide
(29).
The reaction was similar to the one used to prepare 19
with compound 12 (113 mg, 0.34 mmol), benzylamine (70 mg,
0.65 mol), Pd2(dba)3 (24 mg, 0.026 mmol), 2,20-bis(diphenylphos-
phino)-1,10-binaphthyl (rac-BINAP, 38 mg, 0.06 mmol), NaO-t-Bu
(190 mg, 1.95 mmol), and toluene (5 mL) to yield 29 (83 mg,
68%). Purification was carried out on a silica gel column using hex-
anes/ethyl acetate (3:1) to provide the pure compound as a color-
less solid, mp 87–89 °C. 1H NMR (CDCl3, 300 MHz) d 8.33 (s, 1H),
7.86 (d, J = 8.7 Hz, 2H), 7.68 (d, J = 7.8 Hz, 1H), 7.48 (dd, J = 8.1 Hz,
1H), 7.35 (m, 4H), 7.30 (m, 1H), 6.95 (d, J = 9.0 Hz, 2H), 6.19 (d,
J = 8.1 Hz, 1H), 4.87 (br, 1H), 4.50 (d, J = 6.0 Hz, 2H), 3.98 (t,
J = 6.6 Hz, 2H), 1.85 (m, 2H), 1.08 (t, J = 7.5 Hz, 3H); 13C NMR
(CDCl3, 75 MHz) d 165.0, 162.2, 157.5, 150.2, 139.8, 139.3, 129.0,

4742
B. Yi et al. / Bioorg. Med. Chem. 21 (2013) 4730–4743
128.7, 127.4, 127.3, 126.5, 114.4, 102.7, 102.6, 69.7, 46.1, 22.5,
10.5; ESI-MS m/z 362.13 (M+H, 100).
7.77 (d, J = 8.1 Hz, 1H), 7.68 (dd, J = 8.4 Hz, 1H), 7.22 (br, 1H),
6.99 (d, J = 8.7 Hz, 2H), 6.54 (d, J = 8.4 Hz, 1H), 6.04 (m, 1H), 5.43
(d, 1H), 5.22 (d, J = 9.6 Hz, 1H), 4.59 (d, J = 5.4 Hz, 2H), 3.36 (m,
2H), 1.75 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3,
300 MHz) d 166.0, 162.7, 151.3, 145.1, 132.4, 130.6, 124.2, 118.3,
114.8, 101.0, 68.9, 44.6, 22.0, 11.3; MS m/z 312.11 (M+H, 100);
Anal. Calcd for C18H22ClN3O2 (347.56): C, 62.15; H, 6.37; N,
12.08. Found: C, 61.89; H, 6.34; N, 11.87.
6.6.3.21.
N-(6-(Piperidin-1-yl)pyridin-2-yl)-4-propoxybenza-
mide (30).
The reaction was similar to the one used to pre-
pare 19 with compound 12 (161 mg, 0.48 mmol), piperidine
(75 mg, 0.88 mol), Pd2(dba)3 (24 mg, 0.026 mmol), 2,20-bis(diphen-
ylphosphino)-1,10-binaphthyl (rac-BINAP, 42 mg, 0.07 mmol),
NaO-t-Bu (196 mg, 2.01 mmol), and toluene (5 mL) to yield 30
(106 mg, 65%). Purification was carried out on a silica gel column
using hexanes/ethyl acetate (3:1) to provide the pure compound
as a colorless solid, mp 89–92 °C. 1H NMR (CDCl3, 300 MHz) d
8.19 (s, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 7.5 Hz, 1H), 7.53
(dd, J = 7.8 Hz, 1H), 6.98 (d, J = 8.4 Hz, 2H), 6.42 (d, J = 8.1 Hz, 1H),
4.00 (t, J = 6.6 Hz, 2H), 3.52 (m, 4H), 1.86 (m, 2H), 1.66 (m, 6H),
1.08 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d 165.0, 162.1,
158.4, 150.0, 139.7, 129.0, 126.7, 114.4, 102.7, 101.9, 69.7, 46.2,
25.5, 24.7, 22.5, 10.5; ESI-MS m/z 340.10 (M+H, 100); Anal. Calcd
for C20H25N3O2 (339.43): C, 70.77; H, 7.42; N, 12.38. Found: C,
70.75; H, 7.37; N, 12.14.
6.6.3.26. 4-(Allyloxy)-N-(6-(butylamino)pyridin-2-yl)benzamide
(35).
Compound 33 (208 mg, 1.0 mmol) and NaOH (180 mg,
4.5 mmol) were heated to reflux in 10 mL 1:1 H2O/ethanol (v/v)
by the procedure used to hydrolyze ester 17 to yield pure N2-butyl-
pyridine-2,6-diamine. N2-butylpyridine-2,6-diamine (166 mg,
1 mmol) and 4-allyloxybenzoyl chloride (7b, 197.4 mg, 1 mmol)
were coupled using the procedure employed to prepare 8 to yield
35 (258 mg, 79%). Purification was carried out on a silica gel col-
umn using hexanes/ethyl acetate (3:1) to afford the pure com-
pound as an off-white oil. It was further converted into its
hydrochloric acid salt, mp 160–164 °C. 1H NMR (CDCl3, 300 MHz)
d 15.36 (s, 1H), 11.16 (s, 1H), 8.23 (d, J = 8.7 Hz, 2H), 7.84 (d,
J = 8.1 Hz, 1H), 7.74 (dd, J = 8.4 Hz, 1H), 7.01 (d, J = 8.7 Hz, 2H),
6.66 (br, 1H), 6.46 (d, J = 8.4 Hz, 1H), 6.06 (m, 1H), 5.44 (d,
J = 17.4 Hz, 1H), 5.33 (d, J = 10.5 Hz, 1H), 4.61 (d, J = 5.1 Hz, 2H),
3.39 (m, 2H), 1.71 (m, 2H), 1.47 (m, 2H), 0.98 (t, J = 7.5 Hz, 3H);
13C NMR (CDCl3, 75 MHz) d 165.6, 162.7, 151.3, 145.4, 145.2,
132.4, 130.5, 126.7, 124.2, 118.3, 114.9, 101.2, 68.9, 42.8, 30.7,
20.0, 13.9; ESI-MS m/z 326.11 (M+H, 100); Anal. Calcd for
6.6.3.22. N-(6-bromopyridin-2-yl)acetamide (31).
6-bromopyridine (6b) (730 mg, 4.22 mmol) and triethylamine
(588 L, 4.22 mmol) in 5 mL DCM was added to acetyl chloride
2-Amino-
l
(2 mL, 28 mmol) in DCM (5 mL). The solution was stirred over-
night, then was washed with saturated NaHCO3, H2O, brine and
dried with anhydrous Na2SO4. The crude product was obtained
by removing the solvent under reduced pressure and further puri-
fied by recrystallization in ethyl acetate (860 mg, 95%). 1H NMR
(CDCl3, 300 MHz) d 8.17 (d, J = 8.1 Hz, 1H), 7.98 (br, 1H), 7.57
(dd, J = 7.8 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 2.22 (s, 3H).
C19H24ClN3O2 (361.16): C, 63.06; H, 6.68; N, 11.61. Found: C,
63.11; H, 6.53; N, 11.45.
Author contributions
6.6.3.23. N-(6-(propylamino)pyridin-2-yl)acetamide (32).
The
Experimental design: Bitna Yi, Tatiana González-Cestari, Brandon
J. Henderson, Ryan E. Pavlovicz, Karl Werbovetz, Chenglong Li.
Pharmacological assays: Bitna Yi, Tatiana González-Cestari, Bran-
don J. Henderson.
Data analyses and interpretation: Bitna Yi, Tatiana González-Ces-
tari, Sihui Long, Karl Werbovetz, Dennis McKay.
Pharmacophore design: Brandon Henderson.
Ligand-based virtual screening: Brandon Henderson
Synthetic chemistry: Sihui Long
reaction was similar to the one used to prepare 19 with compound 31
(455 mg, 2.12 mmol), propylamine (144 mg, 2.44 mmol), Pd2(dba)3
(129 mg, 0.14 mmol), 2,20-bis(diphenylphosphino)-1,10-binaphthyl (rac-
BINAP, 195 mg, 0.31 mmol), NaO-t-Bu (1.07 g, 10.5 mmol), and toluene
(5 mL) to yield 32 (215 mg, 53%). Purification was carried out on a silica
gel column using hexanes/ethyl acetate (3:1) to provide the pure com-
pound as a colorless solid. 1H NMR (CDCl3, 300 MHz) d 8.23 (s, 1H), 7.43
(m, 2H), 6.13 (m, 1H), 4.48 (bs, 1H), 3.17 (m, 2H), 2.10 (s, 3H), 1.59 (m,
2H), 0.96 (t, J = 7.2 Hz, 3H).
Manuscript writing: Bitna Yi, Sihui Long, Karl Werbovetz, Dennis
McKay.
6.6.3.24. N-(6-(butylamino)pyridin-2-yl)acetamide (33).
The
reaction was similar to the one used to prepare 19 with compound
31 (400 mg, 1.86 mmol), butylamine (148 mg, 2.03 mmol), Pd2(dba)3
(127 mg, 0.14 mmol), 2,20-bis(diphenylphosphino)-1,10-binaphthyl
(rac-BINAP, 179 mg, 0.28 mmol), NaO-t-Bu (1.0 g, 10.5 mmol), and tol-
uene (5 mL) to yield 33 (207 mg, 54%). Purification was carried out on a
silica gel column using hexanes/ethyl acetate (3:1) to provide the pure
compound as a colorless solid. 1H NMR (CDCl3, 300 MHz) d 8.05 (s, 1H),
7.43 (m, 2H), 6.13 (m, 1H), 4.41 (bs, 1H), 3.21 (m, 2H), 2.12 (s, 3H), 1.59
(m, 2H), 1.39 (m, 2H), 0.94 (t, J = 7.5 Hz, 3H).
Acknowledgements
This work was supported by the National Institutes of Health
National Institute on Drug Abuse (Grant DA029433). All stably
transfected human cell lines were kindly provided by Dr. Jon M.
Lindstrom, Department of Neuroscience School of Medicine, Uni-
versity of Pennsylvania, Philadelphia, PA.
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