Synthesis, Pharmacological Characterization, and Structure-Activity Relationships of Noncanonical Selective Agonists for α7 nAChRs

Article abstract of DOI:10.1021/acs.jmedchem.9b01467

A lack of selectivity of classical agonists for the nicotinic acetylcholine receptors (nAChR) has prompted us to identify and develop a distinct scaffold of α7 nAChR-selective ligands. Noncanonical 2,4,6-substituted pyrimidine analogues were framed around compound 40 for a structure-activity relationship study. The new lead compounds activate selectively the α7 nAChRs with EC₅₀'s between 30 and 140 nM in a PNU-120596-dependent, cell-based calcium influx assay. After characterizing the expanded lead landscape, we ranked the compounds for rapid activation using Xenopus oocytes expressing human α7 nAChR with a two-electrode voltage clamp. This approach enabled us to define the molecular determinants governing rapid activation, agonist potency, and desensitization of α7 nAChRs after exposure to pyrimidine analogues, thereby distinguishing this subclass of noncanonical agonists from previously defined types of agonists (agonists, partial agonists, silent agonists, and ago-PAMs). By NMR, we analyzed pK_a values for ionization of lead candidates, demonstrating distinctive modes of interaction for this landscape of ligands.

Full text of DOI:10.1021/acs.jmedchem.9b01467

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Article
Synthesis, pharmacological characterization, and structure-activity
relationships of non-canonical selective agonists for #7 nAChRs
Gisela Andrea Camacho-Hernandez, Clare Stokes, Brendan M. Duggan, Katarzyna
Kaczanowska, Stefania Brandao-Araiza, Lisa Doan, Roger L. Papke, and Palmer Taylor
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01467 • Publication Date (Web): 01 Nov 2019
Downloaded from pubs.acs.org on November 2, 2019
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Synthesis, pharmacological characterization, and structure-activity
relationships of non-canonical selective agonists for 7 nAChRs
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Gisela Andrea Camacho-Hernandez†, Clare Stokes‡, Brendan M. Duggan†, Katarzyna Kaczanowska†,
Stefania Brandao-Araiza†, Lisa Doan†, Roger L. Papke‡, and Palmer Taylor†*.
†Department of Pharmacology, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California-San Diego,
La Jolla, CA 92093-0751. ‡Department of Pharmacology & Therapeutics, University of Florida, PO Box 100267, Gaines
ville FL, 32610-0267.
KEYWORDS Selective agonists, substituted pyrimidines, nicotinic acetylcholine receptors, ligand-gated ion channels, NMR
pKa analysis
ABSTRACT: A lack of selectivity of classical agonists for the nicotinic acetylcholine receptors (nAChR) has prompted us to
identify and develop a distinct scaffold of α7 nAChR-selective ligands. Non-canonical 2,4,6-substituted pyrimidine
analogues were framed around compound 40 for a structure-activity relationship study. The new lead compounds activate
selectively the α7 nAChRs with EC50’s between 30-140 nM in a PNU-120596-dependent, cell-based calcium influx assay.
After characterizing the expanded lead landscape, we ranked the compounds for rapid activation using Xenopus oocytes
expressing human α7 nAChR with a two-electrode voltage clamp. This approach enabled us to define the molecular
determinants governing rapid activation, agonist potency, and desensitization of α7 nAChRs after exposure to pyrimidine
analogues, thereby distinguishing this subclass of non-canonical agonists from previously defined types of agonists (agonists,
partial agonists, silent agonists, and ago-PAMs). By NMR, we analyzed pKa values for ionization of lead candidates,
demonstrating distinctive modes of interaction for this landscape of ligands.
INTRODUCTION
attention.14-15 Activation of this subtype of nAChR can
enhance attention and cognition in animal models.10,15 More
recently the α7 nAChR has been explored as a target for
inflammation via metabotropic activation in immune cells
where it regulates the cholinergic anti-inflammatory pathway
(CAP).11,16-18
Nicotinic acetylcholine receptors (nAChRs) belong to a
pentameric ligand-gated ion channel superfamily and are
among the most well-studied allosteric transmembrane
signaling proteins.1-2 They are sub-classified as muscle and
neuronal receptors, and the latter are widely distributed in the
peripheral autonomic and the central nervous systems, as well
as in some non-neuronal cell types. nAChRs are
predominantly found in brain on presynaptic terminals
modulating the release of other neurotransmitters.3 The
orthosteric and allosteric sites and functions of nAChRs have
made them potential candidate targets for treating progressive
brain disorders of aging and development such as
Alzheimer’s,4-6 Parkinson’s,7-8 and schizophrenia,9-10 as well as
inflammation.11 One of the most abundant neuronal nAChRs is
the homopentameric α7 subtype. As such, it is comprised of
five identical α7 subunits with five potential orthosteric
binding sites, which are located at the respective subunit
interfaces of the extracellular domain. The α7 nAChR has
unique pharmacological recognition and electrophysiological
response properties when compared to other subtypes; for
example, it retains relatively low affinity for nicotine or
acetylcholine in the desensitized state, is activated by choline,
displays high permeability for calcium, and exhibits rapid
desensitization when exposed to high concentrations of
agonist.12-13 The α7 nAChR is known to be highly expressed in
the hippocampus and prefrontal cortex, where it modulates
cognition, sensory processing, working memory, and
Ligand binding to the orthosteric binding site of neuronal and
non-neuronal nAChRs requires a cationic center and usually a
hydrogen bond donor or acceptor.19 Horenstein et al. reported
three chemical motifs to achieve α7 nAChR selectivity: the
benzylidene motif, tropane motif, and choline motif.20
Incorporating a hydrophobic feature, such as a single methyl,
as with tropane, or a large aromatic group such as the
benzylidene in GTS-21,5,9 in addition to a cationic center can
convert a non-selective agonist into a more -selective drug.
Despite these considerations, more detailed clinical studies
have often resulted in leads lacking sufficient selectivity
among other nAChR subtypes and a 5-hydroxytryptamine (5-
HT3A) ion channel receptor. Accordingly, critical side effects
or limited efficacy at the doses used become manifest. GTS-
21,5,9 MEM3454 (RG3487),15,21 Encenicline (EVP-6124),22
and TC-561910,23 are examples of leads with these
characteristics that have been brought to Phase 1 and 2 clinical
trials (Figure 1).
In previous work,24 we identified a small sub-group of 4,6-
disubstituted-2-aminopyrimidines that interact with the
acetylcholine binding protein (AChBP)25-26 and selectively
activate 7 nAChRs in a cell-based calcium influx assay.27
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Moreover, these ligands are structurally distinct from classical was required for signal detection. An open question remains as
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nicotinic agonists, where the nitrogens residing on the
pyrimidine and pyridine rings all are weakly basic. We
hypothesize, based on the structures, when compared to
prototypical agonists, that these ligands show limited
protonation at physiological pH, thus minimizing classical
cation- interactions.28 Furthermore, a cell-based calcium
influx assay has a prolonged time-frame, so inclusion of a type
II positive allosteric modulator (PAM), such as PNU-120596
to whether these compounds activate the 7 nAChR in the
absence of PNU-120596 and, accordingly, differ from silent
agonists18,29 or ago-PAMs.30-31 Our current measurements in
the oocyte system not only indicate agonist activity without
the presence of PNU-120596, but also reveal that the kinetics
of activation, efficacy, and recovery from desensitization
differ within this pyrimidine sub-family from the classic 7
nAChR agonists described to date.
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Figure 1. Examples of nAChR ligands: a) (-)-Nicotine, b) ()-Epibatidine, c) Varenicline, d) Cytisine. Examples of nAChR ligands
with selective α7 nAChR agonist activation: e) GTS-21,5,9 f) MEM3454 (RG3487),15,21 g) Encenicline (EVP-6124),22 h) TC-
5619.10,23
Scheme 1. Synthesis of compounds 56-66 (for structures see Table 1).
Reagents and conditions: a) di-(2-picolyl) amine, DIPEA, DMF, 80 C; b) boronic acid, Pd(dppf)Cl2, K2CO3, DMA, 149 C; c)
boronic acid, Pd(PPh3)4, Na2CO3, THF, reflux; d) 1 eq. or 2 eq. of m-CPBA in DCM, 0 C; e) morpholine, DIPEA, 2-propanol,
microwave irradiation, 160 C.
RESULTS AND DISCUSSION
with varying electronic influences. Starting with symmetric
commercially available pyrimidine precursors, aromatic
nucleophilic substitution was performed with 4,6-dichloro-2-
(methylthio)pyrimidine, 4,6-dichloropyrimidine, or 4,6-
Synthetic strategies and distinguishing chemistry. Based
on our crystallographic studies with AChBP,24 in complex
with the di-(2-picolyl) amine ligand series, we identified key
ligand features for influencing AChBP affinity. Along with
di-(2-picolyl) amine system capturing carbonyl and indole
regions of Trp143 of LsAChBP, major contributions were
attributed to the pyrimidine ring nitrogen and the pyrimidine
position 2 NH2 substituent. With the understanding of
importance of the two latter positions, we modified the 2
position by introducing relatively small functional groups
dichloro-2-(trifluoromethyl)pyrimidine
to
obtain
intermediate B-1 (91% yield), B-2 (64% yield), or
intermediate B-3 (93% yield), respectively. Cross-coupling
Suzuki reactions utilizing a choice of boronic acid,
Pd(dppf)Cl2, and as a base K2CO3 in N,N-dimethylacetamide
(DMA) yielded compounds 56-59, 62-63 (Table 1). For
compounds 60-61 (Table 1) different coupling conditions
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were used, namely Pd(PPh3)4 as a catalyst, Na2CO3 as a base
in THF. A total of eight compounds were synthesized via
1
2
3
these two steps. Compounds 64-65 (Table 1) required an
additional oxidation step using m-CPBA (Scheme 1).
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Recognizing the importance of the di-(2-picolyl) amine
substituent, we confirmed the need for both pyridines that
maintain an axis of symmetry in this part of the structure.
Following this concept, we synthesized compound 75
(Scheme 2, Table 1). An additional step was added to
assemble N-benzyl-1-(pyridin-2-yl)methanamine by a direct
reductive amination that afforded B-4. Purification of this
amine presented a challenge, where the best option seemed
to be utilizing oxalic acid to generate the protonated
secondary amine. After isolation in 67% yield, the amine
was reacted with 2-amino-4,6-dichloropyrimidine to give
intermediate B-5 in 36% yield. Suzuki coupling was
performed utilizing Pd(dppf)Cl2 and K2CO3 in DMA to yield
83% of a white solid compound 75 (Scheme 2). Based on the
previous lead, compound 40, we continued to expand our
landscape with modifications in the position 6. Initially, we
sampled mostly the -ortho position of the aromatic
substituent in combination with different variants of the -
para substituents. Our intent was to increase hydrophobicity
in this part of the structure, as, according to the earlier
studies, this substitution emerged as an important feature to
achieve selectivity with respect to other nicotinic receptors.20
Synthesis of B-21 intermediate has been previously
described.24 Coupling condition (b) was utilized to obtain
compounds 67-69 (Table 1) and 71-72 (Table 1) in good to
excellent yields. In the case of compound 70, no traces of the
expected product were observed leading to reaction
conditions modification by applying Pd(PPh3)4 as a catalyst,
and Na2CO3 as a base in THF. Compound 70 (Table 1) was
successfully obtained albeit in low, 27% yield. Similar
coupling conditions were used to produce compounds 73-74
(Table 1)
Scheme 3. Modification at pyrimidine position 4.
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Reagents and conditions: a) benzylamine, NaBH4 EtOH, 0
C; b) 2-amino-4,6-dichloropyrimidine, DIPEA, DMF, 80
C; c) 2-methoxyphenyl boronic acid, Pd(dppf)Cl2, K2CO3,
DMA, 149 C.
Effects of divergent substitutions, using nAChR cell-based
neurotransmitter fluorescent engineered reporters (CNiFERs)
for structure-activity analysis.
As a screen of moderate throughput, and as a first analyzing
step, we utilized HEK stable cell lines expressing 7 and
42 nAChRs and 5-HT3A receptors with the TN-XXL cal-
cium reporter biosensor.27 The CNiFERs measure changes in
fluorescence by Ca2+ binding as a result of Ca2+ influx from
agonist activation. Due to the desensitizing profile of 7
nAChR, PNU-120596 was required in initial 7 nAChR
screens. Saturation of genetically encoded fluorescence
biosensor can occur, and this may lead to a misinterpretation
of efficacy and potency; therefore, we treated this evaluation
as a first step to detect well-suited candidates for
electrophysiologic analysis.
Since basicity of the pyrimidine nitrogen in the series should
be affected by the adjacent substituent in position 6, we
introduced a non-aromatic moiety to increase the pKa in the
pyrimidine (calculations performed in MarvinSketch 17.27).
Consequently, in compound 66 (Table 1), we introduced a
morpholine as a substituent via an aromatic substitution
performed under microwave irradiation.32
Out of twenty-three present compounds, nineteen activated
the human 7 nAChRs; accordingly activation was
normalized to a 316 nM response of ()-Epibatidine (Figure
2). As a preliminary structure-activity relationship (SAR)
analysis and to explore how different substitutions in the 2-
position affect the response, we compared compound 4024
(EC50= 70 nM) with compounds 56 (EC50= 110 nM), 58
(EC50= 140 nM), and 62 (EC50= 530 nM). A decrement of 2-
to 8-fold in potency and a modest decrement in the efficacy
were observed (Table 1, Figure 2A). In our previous study,24
utilizing the structural surrogate Ls-AChBP in complex with
compound 40 (PDB ID 5j5h), we observed a 2.5-3.5Å
hydrogen bonding distance from the conserved Tyr 164 in
the 7 nAChR to the NH2 group. Although the same
interaction might not be present in compounds 56 and 58,
compounds 62, 64, and 65 should form hydrogen bonds,33-34
perhaps constrained by parameters of distance, geometry,
and molecular dimensions. Compounds 57 (EC50= 1.8 M),
59 (EC50= 2.1 M), and 63 (EC50= 2.5 M), when compared
to compound 4424 (EC50= 500 nM), present a ~4-fold
Scheme 2. Modifications at pyrimidine position 6.
Reagents and conditions: a) di-(2-picolyl amine), DIPEA,
DMF, 80 C; b) boronic acid, Pd(dppf)Cl2, K2CO3, DMA,
149 C; c) boronic acid, Pd(PPh3)4, Na2CO3, THF, reflux.
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decrease in potency, underlining sensitivity of this region of necessarily antagonism. When the 2-NH2 group was replaced
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the complex and appropriate heterocyclic aromatic group in
the position 6, and the stabilization by an additional
hydrogen bond in position 2 to increase potency. When the
aromatic group was replaced by the non-aromatic
heterocyclic moiety in 66, a diminished interaction was
detected in all the three different cell lines, despite increased
basicity of the pyrimidine ring nitrogen compared to analogs
58-61, (MarvinSketch pKa 6.75 for 66, 4.56 for 58, 4.71 for
59, 3.53 for 60 and 3.48 for 61). This suggested that basicity
of the pyrimidine ring nitrogen does not suffice for this
series to maintain affinity/potency to the studied receptors.
Rather, the ligand system requires additional hydrophobic
interactions offered by aromatic substituents in the position 6
of the pyrimidine ring. A similar loss of agonist activity was
found by symmetry disruption in the di-2-picolyl system. As
demonstrated for compound 75, activation of 7 nAChRs
was abolished up to concentrations as high as 13.3 M,
confirming the need for a bidentate system, such as the di-(2-
picolyl) group, to achieve activation. Interestingly,
compound 75 inhibited both 42 nAChR and 5-HT3A
receptors. The di-(2-picolyl) moiety appears as a key
pharmacophore feature for 7 nAChR activation but not
by hydrophobic moieties in 56 and 62, antagonism of 42
nAChR and 5-HT3A serotonin receptors increased.
Compounds 64 and 65 exhibited approximately 7-fold
decreases in potency and maintained the selectivity to some
degree, suggesting that large polar substituents at position 2
of the pyrimidine reduce potency (Table 1, Figure 2A).
An increase in hydrophobicity and atom size at the -ortho
position of the phenyl substituent in position 6 of the
pyrimidine ring led to an increment in antagonism of 42
nicotinic and 5-HT3A receptors, as well as a loss in potency
in 7 nAChRs (40 vs 71 and 72, Table 1, Figure 2B).
Substitution with fluorine in compound 74 was aimed at
maintaining hydrogen bond accepting properties at the -
ortho position while modulating lipophilicity of the ligand.
Ligand 74 gave increased potency compared to 41, with an
EC50 of 30 nM. The key replacement in compound 74 was a
swap of a methoxy group for a fluorine. Fluorine can act as a
bio-isostere of a methoxy group if the binding pocket can
accommodate the CH3 group.35 A fluorine substitution often
leads to improved permeability and metabolic stability, both
parameters are of great importance to drug development.35-36
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A) 1.2
B)
1.2
EPI
EPI
1.1
1.0
0.9
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0.5
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-0.1
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-0.1
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-10
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-6
-5
-4
-10
-9
-8
-7
-6
-5
-4
log[Agonist]
log[Agonist]
Figure 2. 7 nAChR concentration-response curves in the presence of PNU-120596 of a series of 2,4,6-trisubstituted pyrimidines
performed with HEK cells containing a calcium-sensitive fluorescence reporter.
TABLE 1. Functional parameters, 7 nAChR EC50, and antagonist dissociation constants, KA, for 42 nAChR and 5-HT3A receptors.
Agonist
EC50  SD
(M)
Antagonist
KA  SD
(M)
R1
R2
R3
Compound
40*
5-HT3A
7 nAChR
42 nAChR
NH2
>10
0.07  0.01
6.3  1.5
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1
2
NH2
41*
>10
0.07  0.01
2.5  0.8
3
4
5
6
NH2
44*
56
>10
>10
0.5  0.1
SCH3
0.11  0.02
1.1  0.2
2.8  0.6
7
8
9
SCH3
H
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58
1.8  0.5
3.4  1.5
7.9  0.9
2.2  0.8
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>10
0.14  0.02
H
H
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>10
>10
2.1  0.2
0.069  0.0003
3.0  1.2
5.4  1.1
H
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62
0.36  0.07
0.53  0.06
5.6  1.1
1.7  0.3
8.4  1.1
3.7  0.8
CF3
CF3
63
64
2.5  0.3
0.96  0.2
7.2  1.9
3.6  1.3
SO2CH3
>10
0.69  0.06
SOCH3
65
>10
>10
0.92  0.2
H
66
67
>13.3
>10
>10
>10
>10
NH2
0.23  0.05
NH2
NH2
68
69
>10
>10
5.4  1.0
7.1  0.9
4.8  0.8
0.12  0.04
NH2
NH2
70
71
>10
>10
0.24  0.03
0.89  0.07
4.3  1.5
1.5  0.4
NH2
NH2
NH2
72
73
74
1.6  0.5
0.16  0.03
0.030.009
1.3  0.4
3.4  0.9
3.2  1.3
5.5  0.5
>10
5.3  0.5
NH2
75
>13.3
1.3  0.6
4.1  0.6
1Dissociation constants are based on non-competitive antagonism. 42 nAChR analysis was made without distinguishing subunit
stoichiometry. Responses are measured from at least three independent experiments in HEK cells containing a fluorescence reporter
and plated as monolayers on 96-well plate. * Previously described compounds.24
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0.84  0.03 of the ACh maximum, followed by compound
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40 with an EC50 of 0.35  0.11M and Imax= 0.98  0.08.
Compound 60 showed a partial agonist profile with an EC50
of 0.60  0.15 M and Imax= 0.53  0.04.
6
7-nAChR
Electrophysiological analysis
5
Based on CNiFERs results, four compounds were selected
for fast activation analysis: compounds 58, 60, and 74, in
addition to the previously described24 compound 40.
Electrophysiological characterization was performed in
Xenopus oocytes expressing human neuronal 7 nAChR
with two-electrode voltage clamp. Initially, the four
compounds were tested at 30 M (Figure 3); the test
responses were normalized to the average of two 60 M
ACh pre-control responses for each oocyte. A single-
concentration analysis, when an appropriate concentration is
used, can provide information of potency and efficacy based
on the peak-current to net-charge ratio, and facilitates a
further concentration-response analysis.37 The four
compounds activated 7 nAChR at the standard single
concentration; peak current measurements were compared,
and the responses were significantly larger for all
compounds than for the 60 M ACh controls (Figure 3),
reflecting high potency of the four compounds.37 After a
washing period of 181 s with Ringer's solution, 60 M ACh
was applied, and minimal activation of the receptor was
detected for all compounds (Figure 3), suggesting a low
recovery of the receptor and potentially a stabilization of a
closed channel, desensitized state. The type II PAM PNU-
120596 affects the peak-current responses and the kinetics of
the agonist response by binding to the transmembrane
domain and destabilizing some of the desensitized states.38
Therefore, to demonstrate whether the pyrimidine analogs
induced stabilization of a PAM-sensitive closed, desensitized
state (Ds), 10 M PNU-120596 was then applied. As shown
in Figure 3, the receptors desensitized by the four
compounds responded to PNU-120596, with compounds 58,
60, and 74 showing marked increase over the 60 M ACh
pre-control responses. Despite the stabilization of a PAM-
sensitive Ds state by these compounds, thereby resembling
silent agonists, the pyrimidines differ by having significant
activation when PNU-120596 is absent.
6
7
8
9
5
40
58
4
60
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
74
3
2
1
0
h
U
M
C
µ
N
A
P
0
3
M
M
s
µ
µ
e
0
6
0
n
i
1
d
i
m
i
r
y
P
Figure 3. Peak current responses to 30 M of selected lead
compounds 40, 58, 60, and 74 followed by application of 60
M ACh and then 10 M PNU-120596 performed in
Xenopus oocytes expressing human 7 nAChRs. Compound
applications were 12 sec duration, followed by 211-second
washout periods. Experimental values are the mean
responses (± SEM) of six cells, each scaled to their
respective ACh pre-controls.
The least potent compound analyzed electrophysiologically
was compound 58, with an EC50 of 3.35  0.68 M showing
a full agonistic profile with an Imax = 0.89  0.07. All
compounds followed the same potency order in single-
concentration analysis and the calcium CNiFERs assay,
suggesting that the NH2 group in position 2 enhances
potency. Characterization of the inhibition was made by
adding a single concentration of ACh, 60 M, post-
application of compounds (Figure 4b). Inhibition followed
the potency order: compound 74 with an IC50= 0.47  0.20,
40 IC50=0.37  0.15, 60 IC50= 1.3  0.30 and, 58 IC50= 3.5 
1.0, confirming the binding of these compounds in the
orthosteric site.
An alternative allosteric binding site in the extracellular
vestibule of the  nAChR has been suggested to be
involved with PNU-dependent reactivation of desensitized
receptors39. This site was first identified as being one of two
binding sites recognized by the ago-PAM GAT107, the other
site being a site in the transmembrane domains that is shared
with other PAMs such as PNU-120596 or TQS.40 A further
detailed electrophysiological analysis, outside the scope of
the present study, indicates that the 2,4,6-substituted
pyrimidine analogues may also depend on this allosteric
activation site. Concentration-response curves of selected
compounds are shown in Figure 4a, based on net-charge,
rather than peak-current, analysis. Due to the rapid
desensitization of  receptors by high agonist
concentrations, peak currents often occur before completing
application of drug, so that increases in peak current do not
reflect increased receptor activation, but rather
synchronization of channel opening events.41 Net charge
analysis avoids this artifact. These results are consistent
with single-concentration analysis; compound 74 was the
To increase our selectivity profile and confirm previous
CNiFERs results with 42 nAChR, all compounds were
assayed at 30 M against four heteromeric receptors: the
stoichiometry differing between the human high sensitivity
(HS) and low sensitivity (LS) forms of 42,42 , and
the mouse muscle 11 nAChRs, all expressed in
Xenopus oocytes utilizing the two-electrode voltage clamp
system. The four compounds showed high selectivity for the
7 subtype with minimal to no activation on heteromeric
nAChRs (Figure 5), confirming our previous findings.
Compound 40 showed more inhibition of the four receptors
when co-applied with ACh, especially in mouse muscle
11 and human  nAChR (Figure 5). Structurally,
pyrimidine analogs 40 and 74, with NH2 in the position 2,
most potent with an EC50 of 0.11  0.03 M with and Imax
=
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Journal of Medicinal Chemistry
selected compounds 40, 58, 60, and 74, when tested by a
appeared to inhibit the 11 and  nAChRs more than
1
2
3
4
5
6
7
8
their two congeners, 58 and 60. There was no notable
preference for inhibition of one of the two isoforms of 42
n-AChR. When recovery of the receptor was assessed by
control application of ACh (30M for 11, 100 M for
, 100 M for LS 42, and 10 M for HS 42), 
and the LS and HS forms of 42 nAChRs appeared to
recover faster than the muscle receptor, which remained
inhibited after the wash period (figure 5).
post application of 60 M ACh (see Figure 3 for details).
1.2
1.2
34-nAChR
11-nAChR
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
40
58
40
58
60
74
60
74
9
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40
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42
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A)
1.2
7-nAChR
h
n
40
58
60
74
1.1
M
h
n
M
o
C
A
µ
i
o
i
t
C
A
µ
t
0
a
c
0
a
3
d
3
d
c
i
e
l
i
s
e
l
i
l
s
i
l
p
e
p
e
p
p
p
a
n
p
p
p
a
-
n
i
i
-
d
a
-
d
i
a
-
o
c
i
o
c
r
1.0
o
m
o
m
i
i
C
r
r
C
r
e
y
e
t
f
y
t
f
P
P
A
A
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.9
(4)2(2)3-nAChR
(4)3(2)2-nAChR
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.8
ACh
40
58
40
58
60
74
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
60
74
h
C
n
o
i
h
C
n
M
M
o
µ
µ
0
i
t
t
A
A
0
a
a
c
i
3
d
e
c
3
s
d
e
i
l
l
s
i
i
l
l
p
p
e
e
n
p
p
p
p
a
-
o
n
i
i
d
p
a
p
a
-
d
a
i
i
m
-
-
o
o
c
o
c
r
m
i
i
r
C
r
C
r
e
e
t
y
y
P
t
f
f
P
A
A
-8
-7
-6
-5
-4
-3
Figure 5. Electrophysiological evaluation of compounds 40,
58, 60, and 74 in heteropentameric nAChRs performed in
Xenopus oocytes. Compound applications were 6 seconds
duration, followed by a 271-second washout period. The
control concentrations of ACh were 30 M for 11, 100
M for , 100 M for LS 42, and 10 M for HS
42. After experimental drug applications, follow-up
control applications of ACh were made to test prior
pyrimidine exposures.
log[Agonist]
B)
1.7
7-nAChR
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
40
58
60
74
pKa determinations and in silico phisico-chemical properties
pKa values were determined utilizing 1H-NMR by
measuring chemical shifts when neighboring nuclei
protonate43-44. This technique has been reported to be a
highly reliable alternative for pKa assessments when
compared
to
UV-spectroscopy,
potentiometric
measurements, or in silico estimates.45-46 Our samples were
prepared in phosphate buffer over pH ranges of 1.85-9.56 for
nicotine, 2.62-8.51 for compounds 40 and 74, and 2.65-7.53
for compound 60. No D2O to suppress the H2O signals was
employed to avoid changes in the chemical shifts.
Compound 40 was prepared at a final concentration of 1 mM
and compounds 60 and 74 at 0.25 mM. Nicotine was used as
a control; final concentration over the pH range 9.56-3.28
was 20 mM; 10 mM of nicotine was utilized from pH 3.61 to
1.85. Chemical shifts were plotted against pH forming a
sigmoidal curve (supplemental information). pKa’s were
obtained from the inflection points as previously described;44
pKa’s and standard deviations are reported as means when 2
or more vicinal hydrogens were plotted. We did not measure
values below pH 2.65 to avoid compound degradation. This
-8
-7
-6
-5
-4
log[Agonist]
Figure 4. a) Concentration-response curves of 7 nAChR
for selected compounds 40, 58, 60, and 74. The responses
are normalized to 60 M ACh; experimental values are the
averaged responses (±SEM) of six cells. b) Inhibition
concentration-response curves (iCRC) of 7 nAChR for
7
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Page 8 of 18
did not allow us to reach the plateau of the sigmoidal curve results with in silico analysis utilizing Marvin Sketch
software.47 In silico profiles suggested protonation occurring
on the endocyclic nitrogens of the pyrimidine system.
Previous studies by Schiebel et al.48 demonstrated
protonation by nuclear diffraction, most likely to occur in an
endocyclic nitrogen when exocyclic nitrogen is also
available. The pKa’s of ionizable moieties in the three
compounds exhibited marked differences when compared to
nicotine.
1
2
3
4
5
6
7
8
for compound 60 (supplemental information).
Compounds 40 and 74 containing the 2-amino moiety
showed a pKa between 6.85 and 6.68, respectively from the
pyrimidine system; our results argue that only a small
fraction of the two compounds will be protonated at
physiological pH. When fluorine (compound 74) was
introduced in the ortho-position, a decrease on the pKa of
the vicinal pyrimidine took place, as suggested in previous
reports.36 When the 2-NH2 entity was substituted by a
hydrogen in compound 60, the pKa of the pyrimidine
underwent a major drop of ~3.25, underlying the 2-NH2
entity as a contributor to increase basicity in the pyrimidine
system (Table 2).
Drug-likeliness of analogues 40, 60 and 74 were determined
in silico utilizing Marvin Sketch software (Table 2).47 Clog
P, hydrogen bond donors (HBD) and acceptors (HBA), and
molecular weight (MW) of analyzed analogues are in the
range of the “Lipinski’s rule of 5”.49 The polar surface area
(PSA) of 2-NH2 analogues 40 and 74 is enhance when
9
10
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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
Two hydrogens of the pyridines in compounds 40 and 74
were followed by NMR. pKa from pyridine system in the
three compounds varied from 4.1-4.3. We compared our
compared
to
compound
60.
TABLE 2. pKa values for nicotine and compounds 40, 60 and 74 acquired by 1H-NMR spectroscopy and physico-chemical properties.
COMPOUND
1H-NMR
pKa
pyrimidine
ring
1H-NMR
pKa
pyridine
ring
1H-NMR
pKa
pyrrolidine
ring
*pKa
pyrimidine
ring
*pKa
*pKa
*CLog *HBD *HBA
P
*MW
*PSA
pyridine pyrrolidine
ring
ring
meanSD
meanSD
meanSD
Nicotine
40
-
-
2.70
8.58
-
1.16
3.65
0
1
2
7
162.12 17.33
398.19 90.05
3.380.03 8.170.01
6.98
-
-
-
6.05 (-0.64)
4.39
(4.76)
4.310.02
60
74
~3.25
6.68
~4.14
3.53 (-1.62)
5.95 (-0.73)
4.04
(4.53)
-
-
3.95
3.80
0
1
6
7
401.17 60.03
416.18 90.05
4.39
(4.75)
4.260.03
*In silico values calculated utilizing Marvin Sketch software.47
CONCLUSIONS
Herein we present the synthesis and pharmacological
characterization of a non-canonical structural landscape of
ligands for activation of  nAChRs that contains a
pyrimidine center to which functionalization of positions 2,
4, 6 was made. Amino substitution in position 2 is important
for enhancing activity of the series (Table 1), yet removal or
substitution of the 2-amino motif does not eliminate sub-
micromolar agonist responses (Table 1). For 7 agonist
activity a required di(2-picolyl) amine substitution was made
at the 4-position of the pyrimidine ring. The symmetry of the
di-(2-picolyl) amine moiety is critical for activation of the
7 nAChR as seen in compound 75. Functionalization of
aromatic substitution in position 6 plays an important role in
modulating the activity and selectivity.
Electrophysiological measurements confirmed activation of
 nAChR without the presence of PNU-120596, differing
from the responses of silent agonists. Nevertheless,
evaluation revealed stabilization of a desensitized PNU-
sensitive closed state of  nAChR by the leads. These
results, as well as the fact that the structures differ from
classical agonists, opens the possibility of occupation of both
the orthosteric and potential allosteric binding sites.39
A
more detailed electrophysiologic analysis beyond the scope
of this article, directed to the chemical characterization of the
series, is necessary to address these alternatives.
Nevertheless, it is clear that this family of ligands differ from
8
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Journal of Medicinal Chemistry
other agonist classifications in terms of activation and using an Agilent 6230 time of flight mass spectrometer with
1
2
3
4
5
6
7
8
desensitization.
Jetstream electrospray ionization source.
Syntheses.
Multiple endocyclic (pyrimidine and pyridine) and exocyclic
nitrogens at the 2 and 4 positions are found in this landscape.
Our titrations of chemical shifts in 1H-NMR reveal that all of
the nitrogens exist predominantly as neutral, non-ionized
species at physiological pH values (Table 2). This contrasts
with the classical nicotinic agonists with a separated two ring
system, like nicotine, cytisine or epibatidine, one of which
contains a strong base and the other a hydrogen bond donor
or acceptor (Figure 1). It appears from the titration data that
the 2-NH2 group on the pyrimidine will contribute to
incremental basicity in the pyrimidine system (Table 2). In
silico analysis utilizing Marvin Sketch (Version 17.27.0)
suggests that protonation may occur at endocyclic nitrogens
(supporting information), as supported by previously
published studies,48-50 where calculation and nuclear
diffraction identified endocyclic nitrogens in 2-
aminopyridine and 2-aminopyrimidine as the more stable
tautomer. Also, the lack of a dominant cationic species with
a high pKa should facilitate disposition of these compounds
at tissue target sites and the potential crossing of the blood-
brain barrier to achieve CNS activities and perhaps possible
side effects, physico-chemical properties of lead compounds
are within threshold values for membrane permeation and
oral bioavailability.49-51
6-chloro-2-(methylthio)-N,N-bis(pyridin-
2ylmethyl)pyrimidin-4-amine (B-1). Di-(2-picolyl)amine
(1.12 g, 5.63 mmol) was added to a solution 4,6-dichloro-2-
(methylthio)pyrimidine (1.00 g, 5.01 mmol) and N,N-
diisopropylethylamine (0.73 mL, 5.63 mmol) in DMF (8.0
mL) and the solution was stirred at 80 ºC for 3.5 hours
(reaction monitored by TLC). The solvent was removed
under reduced pressure, brine was added, and the mixture
was extracted with ethyl acetate (3 x 40 mL). The organic
layers were washed with brine, dried over anhydrous sodium
sulfate and filtered. The filtrate was concentrated on a rotary
evaporator, and the product was purified by column
chromatography using silica gel (hexanes/EtOAc 3:2) or by
crystallization. After purification a white solid was obtained
9
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
1
in a 91% yield. H NMR (600 MHz, Chloroform-d)  2.37
(s, 3H), 4.74 (s, 2H), 5.14 (s, 2H), 6.22 (s, 1H), 7.26 – 7.12
(m, 4H), 7.64 (td, J = 7.7, 1.6 Hz, 2H), 8.54 (s, 2H). HR-
ESI-TOFMS: [C17H17ClN5S]+ 358.0885
6-chloro-N,N-bis(pyridin-2-ylmethyl)pyrimidin-4-amine
(B-2). Di-(2-picolyl)amine (1.45 mL, 8.0 mmol) was added
to a solution of 4,6-dichloropyrimidine (1.00 g, 6.70 mmol)
and N,N-diisopropylethylamine (1.40 mL, 8.00 mmol) in
DMF (9.0 mL) and the solution was stirred at 80 ºC for 1.5
hours (reaction monitored by TLC). The solvent was
removed under reduced pressure, brine was added and the
mixture was extracted with ethyl acetate (3 x 40 mL). The
organic layers were washed with brine, dried over anhydrous
sodium sulfate and filtered. The filtrate was concentrated on
a rotary evaporator, and the product was purified by column
chromatography using silica gel and hexanes-EtOAc (1:3).
After purification a white solid was obtained in a 64% yield.
1H NMR (600 MHz, Chloroform-d)  4.77 (s, 2H), 5.12 (s,
2H), 6.54 (s, 1H), 7.30 – 7.11 (m, 4H), 7.65 (td, J = 7.7, 1.7
Hz, 2H), 8.44 (s, 1H), 8.57 (s, 2H). 13C-APT NMR (151
MHz, Chloroform- d)  53.7 (CH2), 102.2 (CH), 102.2 (CH),
122.9 (CH), 137.2 (CH), 158.3 (CH), 160.0 (C), 160.3 (C),
163.4 (C). HR-ESI-TOFMS: [C16 H15 Cl N5]+ 312.1007
A combination of a screen of moderate throughput, followed
by detailed electrophysiologic measurements of currents, has
enabled us to identify a series of non-canonical selective
nAChR agonists. Certainly, this new library of compounds
affords some interesting new possibilities and refreshes the
concepts of ligand design for nAChRs.
EXPERIMENTAL SECTION
Materials. Epibatidine (Tocris, batch No. 14B/181649),
PNU-120596 (Tocris, batch No. 3A/210007 and
3A/210046), MLA (Tocris, batch No: 20A/164724), 5-HT
(Tocris, batch No: 2A/201353 and 2B/226517), Tropisetron
(Tocris, batch No. 2B/214070), DHE (Tocris, batch No.
11A/219484), FBS (Gibco, Lot: 834471), Glutamine (Gibco,
Lot: 1894162). Reagents and solvents used for chemical
synthesis and for buffer preparation were purchased from
commercial sources in the highest purity available and used
without further purification. All final compounds submitted
for screening had a purity of 95% or higher (HPLC-MS), and
chemical structures were confirmed by 1H and 13C NMR and
HRMS (Supporting Information). For NMR assessment a
600MHz NMR spectrometer and a Bruker TopSpin 2.1.6
software was used. An Agilent 1260 liquid chromatography
(LC) system coupled with a Thermo LCQdeca mass
spectrometer was employed for LC-MS analysis. The
electrospray ionization (ESI) source was operated under
positive ion mode. An Agilent Zorbax SB-C18 column (ID
4.6 mm × length 150 mm, particle size 3.5 µm) was utilized
for LC separation using water with 0.05 % TFA as mobile
phase A and acetonitrile with 0.05 % TFA as mobile phase
B. The LC flow rate was set at 1.0 mL/minute. The LC
gradient setting is as followed: 0 minute: 5% mobile phase
B, 12 minute: 95% mobile phase B, 14 minute: 95% mobile
phase B, 15 minute: 5% mobile phase B, 20 minute: 5%
mobile phase B. The total runtime was 20 minutes. High
resolution mass spectrometry (HR-MS) data was acquired by
6-chloro-N,N-bis(pyridin-2-ylmethyl)-2-
(trifluoromethyl)pyrimidin-4-amine
(B-3).
Di-(2-
picolyl)amine (0.22 mL, 1.20 mmol) was added to a solution
of 4,6-dichloro-2-(trifluoromethyl)pyrimidine (0.22 g, 1.00
mmol) and N,N-diisopropylethylamine (0.21 mL, 1.20
mmol) in DMF (1.5 mL) and the solution was stirred at 80
ºC for 3.5 hours (reaction monitored by TLC). The solvent
was removed under reduced pressure, brine was added and
the mixture was extracted with ethyl acetate (3 x 20 mL).
The organic layers were washed with brine, dried over
anhydrous sodium sulfate and filtered. The filtrate was
concentrated on a rotary evaporator, and the product was
purified by column chromatography using silica gel
(hexanes/EtOAc 7:3). After purification a white solid was
obtained in a 93% yield. 1H NMR (600 MHz, Chloroform-d)
 4.87 (s, 2H), 5.15 (s, 2H), 6.66 (s, 1H), 7.17 (d, J = 7.1 Hz,
1H), 7.26 – 7.19 (m, 2H), 7.42 (d, J = 7.1 Hz, 1H), 7.70 –
7.62 (m, 2H), 8.57 (d, J = 38.8 Hz, 2H). 13C-APT NMR (151
MHz, Chloroform-d)  55.2 (CH2), 104.0 (CH), 116.5-
1
121.95 (q, JC-F= 276.3 Hz, CF3), 121.3 (CH), 123.1 (CH),
123.2 (CH), 123.5 (CH), 149.6 (CH), 150.4 (CH), 155. 3 (C),
9
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Page 10 of 18
2
= 4.0 Hz, 2H). 13C-APT NMR (150 MHz, Chloroform-d) 
53.2 (CH2), 92.7 (CH), 122.4 (CH), 136.8 (CH), 149.6 (CH),
160.4 (C), 162.1 (C), 164.1 (C), 170.1 (C). HR-ESI-MS:
[C16H16N6Cl]+ 327.1124.
156.5-155.7 (q, JC-F = 36.96 Hz, C), 156.4 (C), 160.8 (C),
163.6 (C). HR-ESI-TOFMS: [C17H14ClF3N5]+ 380.0879.
HPLC: 99% pure.
1
2
3
N-benzyl-1-(pyridin-2-yl)methanamine (B-4). To
a
4
5
6
7
8
9
solution of benzylamine (0.54 g, 5.00 mmol) in 4 mL of
ethanol, pyridine-2-carboxaldehyde (0.54 g, 5.00 mmol) in
4.0 mL of ethanol was added. The solution was stirred
overnight and boiled gently for 30 min. NaBH4 (0.25 g, 6.50
mmol) was slowly added at 0 °C. After 2 hours, 7 mL of
H2O was slowly added following by addition of 20 mL of a
saturated solution of NH4Cl, bring to pH 7 the solution, the
mixture was extracted with ethyl acetate (3 x 30 mL). The
filtrate was concentrated under reduced pressure and the
crude oil was purified. Oxalic acid was added (0.64 g, 5.04
mmol) in 15 mL of ethanol. The salt was filtered and
dissolved in 20 mL of H2O following by addition of NaOH
(30%), extractions with EtOAc was made. The organic
solvent was removed and B-5 (oil) was isolated in a 67%
6-(2-methoxyphenyl)-2-(methylthio)-N,N-bis(pyridin-2-
ylmethyl)pyrimidin-4-amine(56).
2-Methoxyphenyl
boronic acid (0.59 g, 3.91 mmol) was added to a solution of
6-chloro-2-(methylthio)-N,N-bis(pyridin-2-
ylmethyl)pyrimidin-4-amine (B-1) (1.00 g, 2.80 mmol) in
DMA (22 mL), 2 M aqueous potassium carbonate (4.19 mL)
was added following the addition of Pd(dppf)Cl2 (0.20 g,
0.28 mmol). The resulting mixture was stirred in a capped
glass vial at 149 °C overnight. The solvent was removed
under reduced pressure, brine was added and the mixture
was extracted with ethyl acetate (3 x 50 mL). The organic
layers were dried over anhydrous magnesium sulfate, and
filtered. The filtrate was concentrated under reduced pressure
and the crude product was filtrate through silica gel and
recrystallized in ethyl acetate to give white solid in a 53%
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
1
yield. H NMR (600 MHz, Chloroform-d)  2.52 (s, 1H),
1
3.86 (s, 2H), 3.94 (s, 2H), 7.20 – 7.14 (m, 1H), 7.26 (t, J =
7.2 Hz, 1H), 7.34 (td, J = 8.4, 7.9, 2.0 Hz, 3H), 7.38 (d, J =
7.5 Hz, 2H), 7.65 (td, J = 7.6, 1.8 Hz, 1H), 8.57 (ddd, J =
4.8, 1.6, 0.8 Hz, 1H). HR- ESI-TOFMS: [C13H15N2]+
199.1223
yield. H NMR (600 MHz, Chloroform-d)  2.47 (s, 3H),
3.61 (s, 3H), 4.85 (s, 2H) , 5.22 (s, 2H), 6.85 (s, 1H), 6.88 (d,
J = 8.3 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 7.20 (dd, J = 7.1,
5.1 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.65 (t, J = 7.6 Hz,
2H), 7.93 (dd, J = 7.7, 1.7 Hz, 1H), 8.57 (s, 2H). 13C-APT
NMR (151 MHz, Chloroform-d)  14.2 (CH3), 53.6 (CH2),
55.3 (CH3), 99.5 (CH), 111.3 (CH), 114.2 (C), 120.0 (CH),
120.9 (CH), 122.3 (CH), 126.9 (C), 130.8 (CH), 130.9 (CH),
136.9 (CH), 157.6 (C), 161.5 (C), 162.0 (C), 170.63 (C).
HR-ESI-TOFMS: [C24H24N5OS]+ 430.1692. HPLC: 99%
pure.
N4-benzyl-6-chloro-N4-(pyridin-2-ylmethyl)pyrimidine-
2,4-diamine (B-5). N-benzyl-1-(pyridin-2-yl)methanamine
(0.50 g, 2.50 mmol) was added to a solution of 2-amino-4,6-
dichloropyrimidine (0.37 g, 2.20 mmol) and N,N-
diisopropylethylamine (0.44 mL, 2.50 mmol) in DMF (3.4
mL) and the solution was stirred at 80 ºC overnight. The
solvent was removed under reduced pressure, brine was
added and the mixture was extracted with ethyl acetate (3 x
40 mL). The organic layers were washed with brine, dried
over anhydrous magnesium sulfate, and filtered. The filtrate
was concentrated on the rotary evaporator, and the product
was purified by column chromatography using silica gel
(EtOAc/MeOH 9:1). After purification a white solid was
obtained in a 36% yield. 1H NMR (600 MHz, Chloroform-d)
 4.96 (s, 6H), 5.94 (s, 1H), 7.20 (dd, J = 13.8, 6.6 Hz, 3H),
7.28 (t, J = 7.2 Hz, 2H), 7.33 (t, J = 7.3 Hz, 2H), 7.64 (td, J =
7.7, 1.8 Hz, 1H), 8.57 (d, J = 4.3 Hz, 1H). 13C-APT NMR
(151 MHz, Chloroform-d)  52.7 (CH2), 92.8 (CH), 110.4
(CH), 120.2 (CH), 122.6 (CH), 127.7 (CH), 129.0 (CH),
137.1 (CH), 149.8 (CH), 160.6 (C), 162.3 (C), 164.4(C).
HR-ESI-TOFMS: [C17H17ClN5]+ 326.1165.
2-(methylthio)-N,N-bis(pyridin-2-ylmethyl)-6-(thiophen-
3-yl)pyrimidin-4-amine(57). 3-Thienylboronic acid (0.04 g,
0.30 mmol) was added to a solution of 6-chloro-2-
(methylthio)-N,N-bis(pyridin-2-ylmethyl)pyrimidin-4-amine
(B-1) (0.10 g, 0.3 mmol) in DMA (2.0 mL), 2 M aqueous
potassium carbonate (0.3 mL) was added following the
addition of Pd(dppf)Cl2 (22 mg, 0.030 mmol). The resulting
mixture was stirred in a capped glass vial at 149°C
overnight. The solvent was removed under reduced pressure,
brine was added and the mixture was extracted with ethyl
acetate (3 x 50 mL). The organic layers were dried over
anhydrous magnesium sulfate, and filtered. The filtrate was
concentrated under reduced pressure and the product was
isolated by column chromatography on silica gel using
1
Hex/EtOAc gradient (pale solid, 40% yield). H NMR (600
6-chloro-N4,N4-bis(pyridin-2-ylmethyl)pyrimidine-2,4-
diamine (B-21). B-21 was synthesized as previously
describe.23 Di-(2-picolyl)amine (0.44 mL, 2.20 mmol) was
added to a solution of 2-amino-4,6-dichloropyrimidine (0.33
g, 2.00 mmol) and N,N-diisopropylethylamine (0.38 mL,
2.20 mmol) in DMF (3.0 mL) and the solution was stirred at
80 ºC for 3.5 hours (reaction monitored by TLC). The
solvent was removed under reduced pressure, brine was
added and the mixture was extracted with ethyl acetate (3 x
20 mL). The organic layers were washed with brine, dried
over anhydrous sodium sulfate and filtered. The filtrate was
concentrated on a rotary evaporator, and the product was
purified by column chromatography using silica gel
(hexanes/EtOAc 7:3) or by crystallization (the compound
crystalized using ethyl acetate as solvent). After purification
MHz, Chloroform-d)  2.47 (s, 3H), 4.86 (s, 2H), 5.18 (s,
2H), 6.47 (s, 1H), 7.19 (dd, J = 7.0, 5.2 Hz, 3H), 7.36 – 7.26
(m, 2H), 7.49 – 7.44 (m, 1H), 7.63 (t, J = 7.2 Hz, 2H), 7.94
(d, J = 2.1 Hz, 1H), 8.57 (s, 2H). 13C-APT NMR (151 MHz,
Chloroform-d)  14.2 (CH3), 53.7 (CH2), 94.2 (CH), 122.5
(CH), 125.9 (CH), 126.0 (CH), 126.2 (CH), 127.3 (CH),
127.8 (CH), 136.9 (CH), 140.7 (C), 159.1 (C), 162.4 (C),
171.3 (C). HR-ESI-TOFMS: [C21H20N5S2]+ 406.1152 HPLC:
95% pure.
6-(3,4-dimethoxyphenyl)-N4,N4-bis(pyridin-2ylmethyl)
pyrimidine-2,4-diamine (58). 2-Methoxyphenylboronic
acid (0.05 g, 0.30 mmol) was added to a solution of 6-
chloro-N,N-bis(pyridin-2-ylmethyl)pyrimidin-4-amine (B-2)
(0.075 g, 0.24 mmol) in DMA (2.0 mL), 2 M aqueous
potassium carbonate (0.25 mL) was added following the
addition of Pd(dppf)Cl2 (17 mg, 0.024 mmol). The resulting
mixture was stirred in a capped glass vial at 149 °C
overnight. DMA was removed under reduced pressure, brine
1
a white solid was obtained in a 63% yield. H NMR (600
MHz, Chloroform-d)  4.74 (bs, 2H), 4.99 (s, 4H), 5.94 (s,
1H), 7.12-7.23 (m, 4H), 7.55–7.69 (m, 2H), 8.52–8.57 (d, J
10
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Page 11 of 18
Journal of Medicinal Chemistry
was added and the mixture was extracted with ethyl acetate
3.08 Hz, C), 160.3 (C), 161.1 – 162.8 (d, 1J = 251.79 Hz, C),
3
1
2
3
4
5
6
7
8
(3 x 30 mL). The organic layers were dried over anhydrous
magnesium sulfate, and filtered. The filtrate was
concentrated under reduced pressure and the crude product
was purified by column chromatography on silica gel with
CH3Cl/MeOH/NH4OH 100/1.2/0.2 to give the product as a
white solid in a 49% yield. 1H NMR (600 MHz, Chloroform-
d)  3.62 (s, 3H), 4.95 (s, 3H), 6.92 – 6.87 (m, 1H), 7.10 (d,
J = 1.1 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H), 7.20 (dd, J = 7.0,
5.3 Hz, 2H), 7.40 – 7.28 (m, 2H), 7.65 (td, J = 7.7, 1.7 Hz,
2H), 7.86 (dd, J = 7.7, 1.8 Hz, 1H), 8.58 (d, J = 4.4 Hz, 2H),
8.78 (s, 1H). 13C NMR (151 MHz, Chloroform-d)  53.5
(CH2), 55.4 (CH3), 103.8 (CH), 111.4 (CH), 121.0 (CH),
122.5 (CH), 127.1 (C), 129.1 (CH), 130.8 (CH), 130.9 (CH),
137.0 (CH), 149.7 (CH), 156.4 (C), 157.4 (C), 158.2 (CH),
161.6 (C), 162.4 (C). HR-ESI-TOFMS: [C23H22N5O]+
384.1820 HPLC: 99% pure.
162.2 – 162.3 (d, J = 11.53Hz, C), 162.63 (C). HR-ESI-
TOFMS: [C23 H21FN5O]+ 402.1720 HPLC: 95% pure.
6-(2-fluorophenyl)-N,N-bis(pyridin-2-
ylmethyl)pyrimidin-4-amine (61). 2-fluorophenyl boronic
acid (0.13 g, 0.90 mmol) was added to a solution of 6-
chloro-N,N-bis(pyridin-2-ylmethyl)pyrimidin-4-amine (B-2)
(0.2 g, 0.64 mmol) in THF (4.6 mL), 2 M aqueous sodium
carbonate (0.96 mL) was added following the addition of
Pd(PPh3)4 (0.07 g, 0.06 mmol). The resulting mixture was
vigorously stirred in reflux overnight. THF was removed
under reduced pressure, brine was added and the mixture
was extracted with ethyl acetate (3 x 20 mL). The organic
layers were dried over anhydrous magnesium sulfate, and
filtered. The crude was purified using a biotage ZIP 10g
Si cartridge and Hex/EtOAc gradients. White solid in a 81%
9
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
1
yield. H NMR (600 MHz, Chloroform-d)  8.78 (s, 1H),
N,N-bis(pyridin-2-ylmethyl)-6-(thiophen-3-yl)pyrimidin-
4-amine (59). 3-Thienylboronic acid (0.08 g, 0.62 mmol)
was added to a solution of 6-chloro-N,N-bis(pyridin-2-
ylmethyl)pyrimidin-4-amine (B-2) (0.15 g, 0.62 mmol) in
DMA (3.0 mL), 2 M aqueous potassium carbonate (0.72 mL)
was added following the addition of Pd(dppf)Cl2 (35 mg,
0.05 mmol). The resulting mixture was stirred in a capped
glass vial at 149 °C overnight. DMA was removed under
reduced pressure, brine was added and the mixture was
extracted with ethyl acetate (3 x 30 mL). The organic layers
were dried over anhydrous magnesium sulfate, and filtered.
The compound was purified using silica gel columns
8.59 (d, J = 4.5 Hz, 2H), 7.99 (td, J = 7.8, 1.5 Hz, 1H), 7.65
(t, J = 7.6 Hz, 2H), 7.37 (q, J = 7.5, 7.0 Hz, 1H), 7.30 – 7.17
(m, 5H), 7.09 (dd, J = 11.4, 8.3 Hz, 1H), 7.01 (s, 1H), 5.05
(s, 4H). 13C-DEPTQ NMR (151 MHz, Chloroform-d)  53.6
4
(CH2), 103.3 – 103.4 (d, J = 10.17 Hz, CH), 116.3 – 116.6
2
4
(d, J = 22.88 Hz, CH), 122.6 (CH), 124.6 (d, J = 3.66Hz,
2
CH), 126.1 – 126.2 (d, J = 10.68 Hz, C), 130.8 – 130.9 (d,
3J= 2.6 Hz, CH), 131.4 –131.5 (d, J = 8.6 Hz, CH), 137.0
3
3
(CH), 149.8 (CH), 158.5 (CH), 159.1 – 159.2 (d, J = 2.44
Hz, C), 160.1 – 161.7 (d, 1J = 251.56 Hz, C), 162.6 (C). HR-
ESI-TOFMS: [C22H19FN5]+ 372.1619 HPLC: 99% pure.
6-(2-methoxyphenyl)-N,N-bis(pyridin2-ylmethyl)-
1
Hex/EtAcO 1:4 to give a white solid in a 74% yield. H
2(trifluoromethyl)
pyrimidin-4-amine(62).
2-
NMR (600 MHz, Chloroform-d)  8.70 – 8.67 (m, 1H), 8.59
(d, J = 4.4 Hz, 2H), 7.96 – 7.93 (m, 1H), 7.64 (td, J = 7.7,
1.7 Hz, 2H), 7.48 (dd, J = 5.1, 1.2 Hz, 1H), 7.34 (dd, J = 5.1,
3.0 Hz, 1H), 7.25 (s, 1H), 7.20 (dd, J = 7.1, 5.2 Hz, 3H),
6.78 (s, 1H), 5.08 (d, J = 57.2 Hz, 4H). 13C-APT NMR (151
MHz, Chloroform-d)  53.8 (CH2), 95.8(CH), 122.7 (CH),
125.9 (CH), 126.1(CH), 126.7(CH), 137.2 (CH), 141.0 (C),
149.9 (C), 158.7 (CH), 159.1 (C), 163.0 (C). HR-ESI-
TOFMS: [C20H18N5S]+ 360.1279 HPLC: 98% pure.
Methoxyphenylboronic acid (0.05 g, 0.34 mmol) was added
to a solution of 6-chloro-N,N-bis(pyridin-2-ylmethyl)-2-
(trifluoromethyl)pyrimidin-4-amine (B-3) (0.10 g, 0.34
mmol) in DMA (2.0 mL), 2 M aqueous potassium carbonate
(0.20 mL) was added following the addition of Pd(dppf)Cl2
(19 mg, 0.030 mmol). The resulting mixture was stirred in a
capped glass vial at 149 °C overnight. DMA was removed
under reduced pressure, brine was added and the mixture
was extracted with ethyl acetate (3 x 30 mL). The organic
layers were dried over anhydrous magnesium sulfate, and
filtered. The filtrate was concentrated under reduced pressure
and the crude product was purified by column
chromatography on silica gel with Hex/EtOAc 1:4 to give
6-(2-fluoro-4-methoxyphenyl)-N,N-bis(pyridin-2-
ylmethyl)pyrimidin-4-amine
methoxybenzene boronic acid (0.12 g, 0.67 mmol) was
added to solution of 6-chloro-N,N-bis(pyridin-2-
(60).
2-fluoro-4-
a
1
the product as a white solid in a 38% yield. H NMR (600
ylmethyl)pyrimidin-4-amine (B-2) (0.15 g, 0.48 mmol) in
THF (3.5 mL), 2 M aqueous sodium carbonate (0.72 mL)
was added following the addition of Pd(PPh3)4 (0.06 g, 0.048
mmol). The resulting mixture was vigorously stirred in
reflux for 6 hours. THF was removed under reduced
pressure, brine was added and the mixture was extracted
with ethyl acetate (3 x 20 mL). The organic layers were dried
over anhydrous magnesium sulfate, and filtered. The crude
was purified using a biotage ZIP 10 g Si cartridge
MHz, Chloroform-d)  3.61 (s, 3H), 4.92 (s, 2H), 5.23 (s,
2H), 6.90 (d, J = 8.2 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 7.21
(s, 3H), 7.25 (s, 1H), 7.40 – 7.33 (m, 1H), 7.52 (s, 1H), 7.67
(s, 2H), 8.02 – 7.98 (m, 1H), 8.58 (d, J = 25.7 Hz, 2H). 13C-
APT NMR (151 MHz, Chloroform-d)  54.26 (CH2), 55.6
(CH3), 105.3 (CH), 105.5 (CH), 111.6 (CH), 118.4 –121.8
1
(q, JC-F = 276.3 Hz, CF3), 121.3 (CH), 122.8 (CH), 123.0
(CH), 126.1 (C), 131.3 (CH), 131.6 (C), 137.1(CH), 155.6 –
1
2
Hex/EtOAc gradients. White solid in a 68% yield. H NMR
156.3 (q, JC-F = 36.2 Hz, C) 156.2 (CH), 157.8 (C), 161.9
(C), 162.9 (C). HR-ESI-TOFMS: [C24H21F3N5O]+ 452.1689
HPLC: 95% pure.
(600 MHz, Chloroform-d)  8.74 (s, 1H), 8.59 (d, J = 4.3 Hz,
2H), 8.00 (t, J = 8.9 Hz, 1H), 7.65 (td, J = 7.7, 1.5 Hz, 2H),
7.26 (s, 2H), 7.20 (dd, J = 7.0, 5.3 Hz, 2H), 6.97 (s, 1H),
6.79 (dd, J = 8.8, 2.5 Hz, 1H), 6.65 – 6.59 (m, 1H), 5.14 (s,
4H), 3.83 (s, 3H).13C-DEPTQ NMR (151 MHz, Chloroform-
N,N-bis(pyridin-2-ylmethyl)-6-(thiophen-3-yl)-2-
(trifluoromethyl)pyrimidin-4-amine
Thienylboronic acid (0.07 g, 0.51 mmol) was added to a
solution of 6-chloro-N,N-bis(pyridin-2-ylmethyl)-2-
(63).
3-
2
d)  53.6 (CH2), 55.8 (CH3), 102.1 –102.3 (d, J=26.84 Hz,
4
CH), 102.2 (CH), 102.4 – 102.5 (d, J = 11.54 Hz, CH),
(trifluoromethyl)pyrimidin-4-amine (B-3) (0.15 g, 0.40
mmol) in DMA (2.6 mL), 2 M aqueous potassium carbonate
(0.59 mL) was added following the addition of Pd(dppf)Cl2
(29 mg, 0.040 mmol). The resulting mixture was stirred in a
4
2
110.5 – 110.6 (d, J = 2.86 Hz, CH), 118.4 – 118.5 (d, J =
3
10.74 Hz, C), 122.6 (CH), 131.5 – 131.6 (d, J = 4.58 Hz,
CH), 137.0 (CH), 149.8 (CH), 158.4 (CH), 159.0 (d, J =
3
11
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Journal of Medicinal Chemistry
Page 12 of 18
capped glass vial at 149 °C for 3.5 hours. DMA was 6-morpholino-N,N-bis(pyridin-2-ylmethyl)pyrimidin-4-
1
2
3
4
5
6
7
8
removed under reduced pressure, brine was added and the
mixture was extracted with ethyl acetate (3 x 20 mL). The
organic layers were dried over anhydrous magnesium
sulfate, and filtered. Purification, using column
chromatography on silica gel (hexanes/EtOAc 1:4), afforded
the product (pale solid) in a 95% yield. 1H NMR (600 MHz,
Chloroform-d)  8.64 – 8.50 (m, 2H), 8.03 (dd, J = 3.0, 1.1
Hz, 1H), 7.65 (s, 2H), 7.48 (dd, J = 5.1, 1.1 Hz, 2H), 7.34
(dd, J = 5.1, 3.1 Hz, 1H), 7.21 (dd, J = 7.1, 5.2 Hz, 3H), 6.84
(s, 1H), 5.22 (s, 2H), 4.94 (s, 2H). 13C-DEPTQ NMR (151
MHz, Chloroform-d)  54.0 (CH2), 99.6 (CH), 116.9 – 122.4
(C, q, 1JC-F = 276.1 Hz, CF3), 122.7 (CH), 125.7 (CH), 126.5
(CH), 126.8 (CH), 128.5 (CH), 136.9 (CH), 139.7 (C),
145.37 (CH), 155.8 – 156.5 (C, q 2JC-F = 35.5 Hz, CF3)159.5
(C), 163.1 (C). HR-ESI-TOFMS [C21H17F3N5S]+ 428.1152
HPLC: 99% pure.
amine (66). To a solution of 6-chloro-N,N-bis(pyridin-2-
ylmethyl)pyrimidin-4-amine (B-2) (0.15 g, 0.48 mmol) in 2-
propanol (0.96 mL) morpholine (0.05 g, 0.57 mmol) and
N,N-diisopropylethylamine (0.12 g, 0.95 mmol) was added.
The resulting mixture was stirred in microwave sealed vial
for 20 min at 160 °C. Solvent was removed at reduced
pressure, brine was added and the mixture was extracted
with ethyl acetate. The organic layers were dried over
anhydrous magnesium sulfate, and filtered. The filtrate was
concentrated under reduced pressure. The crude was purified
using a biotage ZIP 10 g Si cartridge and Hex/EtOAc
gradients. Compound was isolated with EtOAc 100%. Pale
powder in a 58% yield. 1H NMR (600 MHz, Chloroform-d) 
8.56 (d, J = 4.7 Hz, 2H), 8.29 (s, 1H), 7.64 (td, J = 7.7, 1.6
Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 7.19 (dd, J = 7.2, 5.1 Hz,
2H), 5.57 (s, 1H), 4.96 (s, 4H), 3.74–3.69 (m, 4H), 3.46–3.40
(m, 4H). 13C-DEPTQ NMR (151 MHz, Chloroform-d) 
44.6 (CH2), 53.9 (CH2), 66.7 (CH2), 81.9 (CH), 121.6 (CH),
122.5 (CH), 137.1 (CH), 149.6 (CH), 157.7 (CH), 158.0(C),
163.0 (C), 163.5 (C). HR-ESI-TOFMS [C20H23N6O]+
363.1930 HPLC: 99% pure.
9
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
6-(2-methoxyphenyl)-2-(methylsulfonyl)-N,N-bis(pyridin-
2 ylmethyl)pyrimidin-4-amine(64). 56 (0.30 g, 0.70 mmol)
was dissolved in (1.5 mL) DCM, following by slow addition
of m-CPBA (0.25 g, 1.50 mmol) at 0 °C. After 3 hours,
saturated solution of Na2S2O3 was added and the mixture
was extracted with ethyl acetate (2 x 40 mL). The organic
layers were washed with saturated solution of NaHCO3 and
the mixture was extracted with ethyl acetate (3 x 40 mL).
The organic layers were dried over anhydrous magnesium
sulfate, and filtered. The filtrate was concentrated under
reduced pressure and the crude product was purified by
column chromatography on silica gel with Hex/EtOAc 1:4 to
give white solid in a 31% yield. 1H NMR (600 MHz,
Chloroform-d)  8.59 (d, J = 46.9 Hz, 2H), 8.07 (dd, J = 7.8,
1.9 Hz, 1H), 7.68 (s, 2H), 7.56 (s, 1H), 7.42 – 7.38 (m, 1H),
7.37 (s, 1H), 7.26 – 7.16 (m, 3H), 7.06 (t, J = 7.6 Hz, 1H),
6.92 (d, J = 8.3 Hz, 1H), 5.24 (s, 2H), 4.96 (s, 2H), 3.64 (s,
3H), 3.29 (s, 3H). 13C-APT NMR (151 MHz, Chloroform-d)
 38.9 (CH3), 54.5 (CH2), 55.5 (CH3), 105.9 (CH), 111.5
(CH), 120.7 (CH), 121.2 (CH), 123.3 (CH), 125.2 (C), 131.3
(CH), 131.9 (CH), 137.2 (CH), 149.9 (CH), 156.3 (C), 158.0
(C), 161.7(C), 163.1 (C), 165.0 (C). HR-ESI-TOFMS:
[C24H24N5O3S]+ 462.1592 HPLC: 95% pure.
6-(2-methoxypyridin-3-yl)-N4,N4-bis(pyridin-2-
ylmethyl)pyrimidine-2,4-diamine
pyridinephenyl boronic acid (0.11 g, 0.73 mmol) was added
to solution of
6-chloro-N4,N4-bis(pyridin-2-
(67).
2-methoxy-3-
a
ylmethyl)pyrimidine-2,4-diamine (B-21) (0.20 g, 0.60
mmol) in DMA (5.0 mL), 2 M aqueous potassium carbonate
(0.92 mL) was added following the addition of Pd(dppf)Cl2
(45 mg, 0.06 mmol). The resulting mixture was stirred in a
capped glass vial at 149 °C for 18 hours. DMA was removed
under reduced pressure, brine was added and the mixture
was extracted with ethyl acetate (3 x 20 mL). The organic
layers were dried over anhydrous magnesium sulfate, and
filtered. The filtrate was concentrated under reduced pressure
and the crude product was isolated by silica gel column, the
compound came out with EtOAc/MeOH 9:1, pale powder,
1
73% yield. H NMR (600 MHz, Chloroform-d)  8.6 (d, J =
4.4 Hz, 2H), 8.2 (d, J = 9.4 Hz, 1H), 8.2 (d, J = 6.9 Hz, 1H),
7.7 (t, J = 8.5 Hz, 2H), 7.3 (s, 2H), 7.2 (dd, J = 7.1, 5.1 Hz,
2H), 7.0 (dd, J = 7.4, 4.9 Hz, 1H), 6.7 (s, 1H), 5.1 (s, 3H),
4.8 (s, 2H), 3.8 (s, 3H).13C-DEPTQ NMR (151 MHz,
Chloroform-d)  53.5 (CH3), 95.4 (CH), 117.2 (CH), 122.1
(C), 122.3 (CH), 136.9 (CH), 139.1 (CH), 147.4 (CH), 161.1
(C), 161.4 (C), 162.3 (C), 162.8 (C), 163.9 (C).HR-ESI-
TOFMS: [C22H22N7O]+ 400.1883 HPLC: 98% pure.
6-(2-methoxyphenyl)-2(methylsulfinyl)-N,N-bis(pyridin-
2-ylmethyl)pyrimidin-4-amine(65). 56 (0.10 g, 0.23 mmol)
was dissolved in (1.0 mL) DCM, followed by slow addition
of m-CPBA (0.04g, 0.23 mmol) at 0 °C. After 3 hours, a
saturated solution of Na2S2O3 was added and the mixture
was extracted with ethyl acetate (2 x 20 mL). The organic
layers were washed with saturated solution of NaHCO3 and
the mixture was extracted with ethyl acetate (3 x 20 mL).
The organic layers were dried over anhydrous magnesium
sulfate, and filtered. The filtrate was concentrated under
reduced pressure and the crude product was purified by
column chromatography on silica gel with EtOAc/MeOH 9:1
6-(2,6-dimethoxyphenyl)-N4,N4-bis(pyridin-2-
ylmethyl)pyrimidine-2,4-diamine
dimethoxyphenylboronic acid (0.17 g, 0.92 mmol) was
added to
solution of 6-chloro-N4,N4-bis(pyridin-2-
(68).
2,6-
a
ylmethyl)pyrimidine-2,4-diamine (B-21) (0.20 g, 0.61
mmol) in DMA (3.8 mL), 2 M aqueous potassium carbonate
(0.92 mL) was added following the addition of Pd(dppf)Cl2
(45 mg, 0.06 mmol). The resulting mixture was stirred in a
capped glass vial at 149 °C for 24 hours. DMA was removed
under reduced pressure, brine was added and the mixture
was extracted with ethyl acetate (3 x 30 mL). The organic
layers were dried over anhydrous magnesium sulfate, and
filtered. The crude was purified using a biotage ZIP 10 g
Si cartridge and Hex/EtOAc gradients. Pale powder, 42%
1
to give white solid in a 79% yield. H NMR (600 MHz,
Chloroform-d)  2.88 (s, 3H) 3.64 (s, 3H), 4.92 (s, 2H), 5.27
(d, J = 27.6 Hz, 2H), 6.90 (d, J = 8.2 Hz, 1H), 7.04 (t, J = 8.0
Hz, 1H), 7.21 (s, 2H), 7.23 (s, 2H), 7.39 – 7.34 (m, 1H), 7.53
(s, 1H), 7.67 (t, J = 7.1 Hz, 2H), 8.06 (dd, J = 7.8, 1.8 Hz,
1H), 8.61 (s, 2H).13C-APT NMR (151 MHz, Chloroform-d)
 39.9(CH3), 54.0 (CH2), 55.5 (CH3), 104.2 (CH), 111.5
(CH), 121.2 (CH), 122.7 (CH), 125.8 (C), 131.4 (CH), 131.7
(CH), 137.1 (CH), 157.9 (C), 162.0 (C), 163.2 (C), 172.0
(C). HR-ESI-TOFMS [C24H24N5O2S]+ 446.1645 HPLC: 99%
pure.
1
yield. H NMR (600 MHz, Chloroform-d)  8.5 (d, J = 4.5
Hz, 2H), 7.7 (t, J = 7.2 Hz, 2H), 7.3 (s, 2H), 7.2 (t, J = 8.4
Hz, 1H), 7.19 – 7.15 (m, 2H), 6.6 (d, J = 8.4 Hz, 2H), 5.9 (s,
12
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Journal of Medicinal Chemistry
1H), 4.9 (s, 4H), 4.8 (s, 2H), 3.7 (s, 6H).13C- DEPTQ NMR
under reduced pressure, brine was added, and the mixture
1
2
3
4
5
6
7
8
was extracted with ethyl acetate (3 x 20 mL). The organic
layers were dried over anhydrous magnesium sulfate, and
filtered. The filtrate was concentrated under reduced pressure
and the crude product was isolated by silica gel column,
ethyl acetate was use to isolate as a pale powder in a 57% of
(151 MHz, Chloroform-d)  56.1 (CH3), 83.9 (C), 96.9 (CH),
104.4 (CH), 107.0 (CH), 122.2 (CH), 129.8 (CH), 132.9
(CH), 136.8 (CH), 157.9 (C), 163.5 (C). HR-ESI-TOFMS:
[C24H25N6O2]+ 429.2029 HPLC: 99% pure.
6-(4-fluoro-2-methoxyphenyl)-N4,N4-bis(pyridin-2-
1
yield. H NMR (600 MHz, Chloroform-d)  8.55 (d, J = 4.6
ylmethyl)pyrimidine-2,4-diamine
methoxyphenyl boronic acid (0.104 g, 0.6 mmol) was added
to solution of
6-chloro-N4,N4-bis(pyridin-2-
(69).
4-fluoro-2-
Hz, 2H), 7.86 (dd, J = 7.7, 1.7 Hz, 1H), 7.64 (t, J = 7.5 Hz,
2H), 7.28 (dd, J = 15.5, 1.7 Hz, 3H), 7.17 (dd, J = 7.1, 5.2
Hz, 2H), 6.99 (t, J = 7.5 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H),
6.70 (s, 1H), 5.03 (s, 3H), 4.86 (s, 2H), 4.47 (hept, J = 6.0
Hz, 1H), 1.09 (d, J = 6.1 Hz, 6H).13C- DEPTQ NMR (151
MHz, Chloroform-d)  22.0 (CH3), 70.7 (CH), 95.5 (CH),
114.5 (CH), 120.9 (CH), 122.3 (CH), 128.9 (C), 130.3 (CH),
130.9 (CH), 136.9 (CH), 145.1 (CH), 155.9 (C), 162.8 (C),
a
ylmethyl)pyrimidine-2,4-diamine (B-21) (0.15 g, 0.45
mmol) in DMA (3.0 mL), 2 M aqueous potassium carbonate
(0.35 mL) was added following the addition of Pd(dppf)Cl2
(22 mg, 0.036 mmol). The resulting mixture was stirred in a
capped glass vial at 149 °C overnight. DMA was removed
under reduced pressure, brine was added and the mixture
was extracted with ethyl acetate (3 x 30 mL). The organic
layers were dried over anhydrous magnesium sulfate, and
filtered. The filtrate was concentrated under reduced pressure
and the crude product was isolated by silica gel
chromatography with Hex/EtOAc gradients to afford a white
9
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
163.2
(C),
163.7
(C).
HR-ESI-TOFMS:
[C25H27N6O]+427.2244 HPLC: 99% pure.
6-(2-isobutoxyphenyl)-N4,N4-bis(pyridin-2-
ylmethyl)pyrimidine-2,4-diamine (72). 2-isobutoxyphenyl
boronic acid (0.27 g, 1.37 mmol) was added to a solution of
6-chloro-N4,N4-bis(pyridin-2-ylmethyl)pyrimidine-2,4-
diamine (B-21) (0.3 g, 0.9 mmol) in DMA (7.5 mL), 2 M
aqueous potassium carbonate (1.365 mL) was added
following the addition of Pd(dppf)Cl2 (67 mg, 0.09 mmol).
The resulting mixture was stirred in a capped glass vial at
149 °C for 3 hours. DMA was removed under reduced
pressure, brine was added and the mixture was extracted
with ethyl acetate (3 x 20 mL). The organic layers were dried
over anhydrous magnesium sulfate, and filtered. The filtrate
was concentrated under reduced pressure and the crude
product was isolated with a silica gel column. The compound
came out as a white powder using pure ethyl acetate in a
41% yield. 1H NMR (600 MHz, Chloroform-d)  8.55 (d, J =
4.7 Hz, 2H), 7.78 (dd, J = 7.7, 1.6 Hz, 1H), 7.63 (t, J = 7.7
Hz, 2H), 7.36 – 7.22 (m, 3H), 7.19 – 7.14 (m, 2H), 7.00 (t, J
= 7.5 Hz, 1H), 6.91 – 6.86 (m, 1H), 6.64 (s, 1H), 4.96 (s,
3H), 4.81 (s, 2H), 3.68 – 3.64 (m, 2H), 1.80 (hept, J = 6.6
Hz, 1H). 13C-DEPTQ NMR (151 MHz, Chloroform-d) 
19.5 (CH3), 28.3 (CH), 52.9 (CH2), 75.1 (CH2), 95.2 (CH),
112.6 (CH), 120.7 (CH), 122.2 (CH), 128.4 (C), 130.3 (CH),
130.7 (CH), 136.9 (CH), 149.7 (CH), 157.1 (C), 162.8 (C),
163.6 (C), 163.7 (C).HR-ESI-TOFMS: [C26H29N6O]+
441.2396 HPLC: 99% pure.
1
solid in a 76% yield. H NMR (600 MHz, Chloroform-d) 
3.6 (s, 3H), 4.8 (s, 6H), 6.4 (s, 1H), 6.6 (dd, J = 11.0, 2.4 Hz,
1H), 6.7 (td, J = 8.3, 2.4 Hz, 1H), 7.2 (dd, J = 7.4, 5.7 Hz,
2H), 7.3 (d, J = 30.5 Hz, 2H), 7.6 (td, J = 7.7, 1.7 Hz, 2H),
7.7 (dd, J = 8.6, 7.1 Hz, 1H), 8.6 (d, J = 4.4 Hz, 2H).13C-APT
NMR (151 MHz, Chloroform-d)  55.8 (CH2), 55.8 (CH3),
95.5 (CH), 95.5 (CH), 99.7 (CH), 107.4 (CH), 107.6 (CH),
122.4 (CH), 124.2 (C), 131.9 (CH), 132.0 (CH), 137.0 (CH),
149.7 (CH), 158.8 (C), 158.8 (C), 162.4 (C), 162.9 (C),
163.4 (C), 163.
7
(C), 165.07 (C). HR-ESI-MS:
[C23H22N6OF]+ 417.1832 HPLC: 100% pure.
6-(2-(methylthio)phenyl)-N4,N4-bis(pyridin-2-
ylmethyl)pyrimidine-2,4-diamine(70).
2-(methylthio)
benzene boronic acid (0.062 g, 0.369 mmol) was added to a
solution
of
6-chloro-N4,N4-bis(pyridin-2-
ylmethyl)pyrimidine-2,4-diamine (B-21) (0.1 g, 0.30 mmol)
in THF (2.6 mL), 2 M aqueous sodium carbonate (0.46 mL)
was added following the addition of Pd(PPh3)4 (0.04 g, 0.03
mmol). The resulting mixture was vigorously stirred in
reflux overnight. THF was removed under reduced pressure,
brine was added, and the mixture was extracted with ethyl
acetate (3 x 20 mL). The organic layers were dried over
anhydrous magnesium sulfate, and filtered. The filtrate was
concentrated on a rotary evaporator. The crude was purified
in a silica gel column, using gradients of Hex/EtOAc. Pale
6-(2-chlorophenyl)-N4,N4-bis(pyridin-2-
ylmethyl)pyrimidine-2,4-diamine (73). 2-chlorophenil
boronic acid (0.20 g, 1.28 mmol) was added to a solution of
6-chloro-N4,N4-bis(pyridin-2-ylmethyl)pyrimidine-2,4-
diamine (B-21) (0.30 g, 0.91 mmol) in THF (7.5 mL), 2 M
aqueous sodium carbonate (1.37 mL) was added following
the addition of Pd(PPh3)4 (0.11 g, 0.09 mmol). The resulting
mixture was vigorously stirred in reflux overnight. THF was
removed under reduced pressure, brine was added, and the
mixture was extracted with ethyl acetate (3 x 30 mL). The
organic layers were dried over anhydrous magnesium
sulfate, and filtered. The filtrate was concentrated on a rotary
evaporator. The crude was purified in a silica gel column,
1
powder in a 27% yield. H NMR (600 MHz, Chloroform-d)
 8.5 (s, 2H), 7.6 (t, J = 7.5 Hz, 2H), 7.3 (dd, J = 12.0, 7.6
Hz, 4H), 7.2 (d, J = 7.9 Hz, 1H), 7.2 (dt, J = 15.0, 6.5 Hz,
3H), 6.2 (s, 1H), 5.0 (d, J = 104.2 Hz, 3H), 4.8 (s, 3H), 2.3
(s, 3H).13C- DEPTQ NMR (151 MHz, Chloroform-d)  16.5
(CH3), 94.8 (CH), 122.4 (CH), 124.8 (CH), 125.8 (CH),
129.3 (CH), 129.4 (CH), 136.9 (CH), 137.5 (C), 139.0 (C),
162.7 (C), 163.7 (C), 166.0 (C).HR-ESI-TOFMS:
[C23H23N6S]+ 415.1701 HPLC: 96% pure.
6-(2-isopropoxyphenyl)-N4,N4-bis(pyridin-2-
ylmethyl)pyrimidine-2,4-diamine
isopropoxyphenyl boronic acid (0.15 g, 0.85 mmol) was
added to
solution of 6-chloro-N4,N4-bis(pyridin-2-
(71).
2-
1
using gradients of Hex/EtOAc. Pale powder, 29% yield. H
NMR (600 MHz, Chloroform-d)  8.55 (d, J = 4.8 Hz, 2H),
7.64 (t, J = 6.9 Hz, 2H), 7.47 (dd, J = 6.9, 2.4 Hz, 1H), 7.37
– 7.34 (m, 1H), 7.30 – 7.24 (m, 4H), 7.19 – 7.15 (m, 2H),
6.17 (s, 1H), 4.85 (s, 6H).13C-DEPTQ NMR (151 MHz,
Chloroform-d)  95.7 (CH), 122.4 (CH), 127.0 (CH), 128.7
(CH), 129.9 (CH), 130.2 (CH), 130.8 (CH), 132.1 (C), 137.0
a
ylmethyl)pyrimidine-2,4-diamine (B-21) (0.20 g, 0.60
mmol) in DMA (5.0 mL), 2 M aqueous potassium carbonate
(0.91 mL) was added following the addition of Pd(dppf)Cl2
(45 mg, 0.06 mmol). The resulting mixture was stirred in a
capped glass vial at 149 °C for 18 hours. DMA was removed
13
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Journal of Medicinal Chemistry
Page 14 of 18
(CH), 138.6 (C), 149.7 (CH), 162.8 (C), 163.4 (C), 164.4
100 μL of cell suspension per well into black, transparent
(C). HR-ESI-TOFMS: [C22H20ClN6]+ 403.1429 HPLC 95%
pure.
flat-bottom, TC-treated 96-well plates (Thermo, Waltham,
MA; E&K, Greiner Campbell, CA). On the next day, media
was replaced with 100 μL of artificial cerebral spinal fluid
(aCSF, 121 mM NaCl, 5 mM KCl, 26 mM NaHCO3, 1.2
mM NaH2PO4·H2O, 10 mM glucose, 2.4 mM CaCl2, 1.3 mM
MgSO4, 5 mM HEPES, pH 7.4) buffer for cells expressing
42 and 5-HT3A receptors. For all assays performed on α7
nAChR CNiFERs, to aCSF buffer PNU-120596 was added
at a 10 μM final concentration. Plates were incubated with
buffer for 30 min at 37 °C and 6% CO2. Quick screen: The
tested compounds were prepared in aCSF for all receptors
except α7 nAChR, where 10 μM PNU-120596, a positive
allosteric modulator (PAM), was included. The prepared
compounds were added in a separate 96-well polypropylene
plate (Costar, Corning, NY). Experiments were conducted at
37 °C using 436 nm excitation wavelength. Emitted light
was collected at 485 and 528 nm. Basal fluorescence was
recorded for 30 s, followed by addition of 50 μL of ligand
(first addition). Measurements were made at 3.84 s intervals
for 2 min to measure the agonist responses, then followed by
application of the control agonist, which was 100 nM (±)-
epibatidine (Tocris Bioscience, Bristol, U.K.) for α7 and
α4β2 nAChRs, and 3 μM of 5-hydroxytryptamine (5-HT)
(Tocris Bioscience, Bristol, U.K.) for the 5HT3A receptor to
evaluate the antagonist responses of the test compounds.
Agonist and antagonist properties were screened at the final
concentration of 13.3 and 10 μM, respectively. Compounds
whose fraction of the maximal response (Δ/Δmax) was higher
than 0.20 were further evaluated to determine their EC50,
whereas compounds that inhibit Δ/Δmax more than 0.50 were
further characterized to determine the type of antagonism
and calculate the antagonist dissociation constant (KA).
1
2
3
4
5
6
7
8
6-(2-fluoro-4-methoxyphenyl)-N4,N4-bis(pyridin-2-
ylmethyl)pyrimidine-2,4-diamine
methoxybenzene boronic acid (0.22 g, 1.28 mmol) was
added to
solution of 6-chloro-N4,N4-bis(pyridin-2-
(74).
2-fluoro-4-
a
ylmethyl)pyrimidine-2,4-diamine (B-21) (0.3 g, 0.91 mmol)
in THF (7.5 mL), 2 M aqueous sodium carbonate (1.36 mL)
was added following the addition of Pd(PPh3)4 (0.106 g, 0.09
mmol). The resulting mixture was vigorously stirred in
reflux for 8 hours. THF was removed under reduced
pressure, brine was added and the mixture was extracted
with ethyl acetate (3 x 30 mL). The organic layers were dried
over anhydrous magnesium sulfate, and filtered. The crude
was purified in a silica gel column, compound came out
Hex/EtAcO 1:4. White solid, 74% yield. 1H NMR (600
MHz, Chloroform-d)  8.55 (d, J = 4.3 Hz, 2H), 7.86 (t, J =
8.9 Hz, 1H), 7.63 (t, J = 7.5 Hz, 2H), 7.17 (d, J = 12.1 Hz,
4H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 6.58 (dd, J = 13.1, 2.4
Hz, 1H), 6.39 (s, 1H), 4.82 (s, 6H), 3.80 (s, 3H).13C-DEPTQ
NMR (151 MHz, Chloroform-d)  55.8 (CH3), 60.6 (CH2),
9
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
4
2
94.3 – 94.4 (d, J = 10.56 Hz, CH), 101.9 – 102.01 (d, J =
26.85 Hz, CH), 110.3 – 110.4 (d, 4J = 2.89 Hz, CH), 119.1 –
119.2 (d, J = 11.24 Hz, C), 122.4 (CH), 131.3 (d, J = 4.68
Hz, CH), 136.9 (CH), 149.7 (CH), 160.4 (d, J = 2.96 Hz,
C), 160.9 – 162.5 (d, J = 251.41 Hz C), 161.8 (d, J =
11.36 Hz, C) 162.9 (C), 163.9 (C). HR-ESI-TOFMS:
[C23H22FN6O]+ 417.1829 HPLC: 98% pure.
2
3
3
1
3
N4-benzyl-6-(2-methoxyphenyl)-N4-(pyridin-2-
ylmethyl)pyrimidine-2,4-diamine
Methoxyphenylboronic acid (0.06 g, 0.37 mmol) was added
to
solution of N4-benzyl-6-chloro-N4-(pyridin-2-
(75).
2-
a
Agonist assays: Responses were measured in triplicate wells
with the FlexStation III (SoftMax Pro 5.2, Molecular
Devices) and run at 37 °C by monitoring TN-XXL FRET
ratios, emissions of citrine cp174 (527 nm) to eCFP (485
nm), over 120s with agonist injection at 30s. A sigmoidal
concentration−response (variable slope) regression of the
mean peak FRET ratios was fit to generate
concentration−response curves and obtain EC50 values
(GraphPad Prism 7 for Mac OS X); the plots were
normalized to a 316 μM response in α7 nAChR.
ylmethyl)pyrimidine-2,4-diamine (B-5) (0.10 g, 0.30 mmol)
in DMA (2.0 mL), 2 M aqueous potassium carbonate (0.45
mL) was added following the addition of Pd(dppf)Cl2 (22
mg, 0.030 mmol). The resulting mixture was stirred in a
capped glass vial at 149 °C for 3.5 hours. DMA was
removed under reduced pressure, brine was added and the
mixture was extracted with ethyl acetate (3 x 30 mL). The
organic layers were dried over anhydrous magnesium
sulfate, and filtered. The filtrate was concentrated under
reduced pressure and the crude product was filtered through
silica gel using ethyl acetate. After purification by column
chromatography on silica gel was performed utilizing ethyl
Antagonist assays: Tested compounds were prepared in
three different concentrations in aCSF, the 96-well plate was
divided in four sections (triplicates of each concentration
plus the agonist control). The media was replaced by aCSF
buffer (first 3 rows) at the 3 concentrations and was
incubated for 30 min at 37 °C and 6% CO2. Agonist was
subsequently added and responses were measured in
triplicate wells with the FlexStation III (SoftMax Pro 5.2,
Molecular Devices) and run at 37 °C by monitoring TN-
XXL FRET ratios, emissions of citrine cp174 (527 nm) to
eCFP (485 nm), over 120 s with agonist injection at 30s. A
sigmoidal concentration−response (variable slope) regression
of the mean peak FRET ratios was fit to generate
concentration−response curves of control agonist,
epibatidine for α7 and α4β2 nAChRs, and 5-HT for 5-
1
acetate to give a white solid in a 83% yield. H NMR (600
MHz, DMSO-d6)  8.53 (d, J = 4.3 Hz, 1H), 7.77 – 7.73 (m,
1H), 7.71 – 7.68 (m, 1H), 7.33 (q, J = 11.5, 9.7 Hz, 5H), 7.27
(dd, J = 10.4, 5.8 Hz, 3H), 7.02 – 6.93 (m, 2H), 6.39 (s, 1H),
6.08 (s, 2H), 5.02 (s, 3H), 3.56 (s, 3H). 13C-DEPTQ NMR
(151 MHz, DMSO-d6)  55.3 (CH2), 55.6 (CH3), 95.74 (CH),
111.6 (CH), 120.2 (CH), 120.2 (CH), 121.0 (CH), 121.5
(CH), 122.0 (CH), 122.3 (CH), 127.4 (CH), 128.4 (C), 128.8
(CH), 130.4 (CH), 130.6 (CH), 137.0 (CH), 145.0 (C), 149.7
(CH), 157.4 (C), 162.9 (C), 163.2 (C), 163.7 (C). HR-ESI-
TOFMS: [C24H24N5O]+ 398.1977 HPLC: 99% pure.
Cell-based functional assays. Generation of stable cell lines
previously described27 to express high levels of the specified
receptor and containing the CNiFER were cultured in 10 cm
plates with DMEM (Mediatech, Manassas, VA)
supplemented with 10% FBS (Gibco) and 1% Glutamine
(Gibco), and incubated at 37 °C with 6% CO2. Cells were
selected at 60% confluency and plated the day before using
HT3A.The
KA
for
competitive
antagonists
and
noncompetitive antagonists were calculated using the next
equations.52
KA = [A]/[DR − 1] (1)
KA = [A]/[(Δmax/Δ) − 1] (2)
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Journal of Medicinal Chemistry
where [A] is the concentration of compound, DR (dose ratio) comparisons of complex responses. Averages of the
1
2
3
4
5
6
7
8
is the EC50 ratio of tested compound over the control
compound, which is (±)-epibatidine for α7 and α4β2 nAChR,
and 5HT for 5HT3A, and Δ/Δmax is the fraction of the
maximal response. Mean values and standard deviations
were calculated from at least three independent experiments.
normalized data were calculated for each of the 10,322
points in each of the 206.44-second traces (acquired at 50
Hz), as well as the S.E.M. for those averages.
pKa sample preparation and 1H NMR parameters.
We utilized a modified version of a previously reported
method by Bezeçon et al.44 Samples were analyzed in 0.1 M
phosphate buffer (PB) at various pH between 4.0 and 8.5.
Compounds were pre-dissolved in DMSO to a 20 mM
concentration. Compound 40 was diluted in PB to a 1 mM
final concentration and compounds 60 and 74 to a 0.25 mM
final concentration in all pH samples. DMSO concentration
was 5% or less in every sample. 500 L of sample was
transferred to a 5 mm NMR tube (NORELL) for recording
NMR spectra. To avoid having to correct the pH for the
presence of deuterium, no deuterated solvent was added to
the compound solutions. To provide a lock signal for the
NMR spectrometer a sealed 1.7 mm capillary tube filled with
DMSO-d6 was placed in the solution inside the 5 mm NMR
tube. The DMSO in the capillary also acted as a chemical
Heterologous Expression of nAChRs in Xenopus
Oocytes. The cDNA clones of human nAChR and human
resistance-to-cholinesterase 3 (RIC-3) were provided by Dr.
Jon Lindstrom (University of Pennsylvania, Philadelphia
PA) and Dr. Millet Treinin (Hebrew University, Jerusalem,
Israel), respectively. Mouse muscle   and  cDNA
clones were provided by Dr. Jim Boulter (Salk Institute, La
Jolla CA) and the  by Dr. Paul Gardner (University of
Massachusetts Medical School, Worcester MA). After
linearization and purification of the plasmid cDNAs, RNAs
were prepared using the mMessage mMachine in vitro RNA
synthesis kit (Ambion, Austin, TX). Oocytes were surgically
removed from mature female Xenopus laevis frogs (Nasco,
Ft. Atkinson, WI) and injected with RNAs of nAChR and
RIC-3 as described previously.53 The RIC-3 chaperone
protein can improve and accelerate  expression with no
effects on the pharmacologic properties of the receptors.52
Frogs were maintained in the Animal Care Service facility of
the University of Florida, and all procedures were approved
by the University of Florida Institutional Animal Care and
Use Committee. All studies were carried out in accordance
with the Guide for the Care and Use of Laboratory Animals
as adopted and promulgated by the U.S. National Institutes
of Health.
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shift reference. The water peak in the H NMR spectra was
suppressed via pre-saturation or by using a jump-return pulse
sequence.55 Each spectrum was analyzed using Bruker
TopSpin 2.1.6. software. The chemical shifts were plotted
against pH using GraphPad Prism 7 and the curve was fitted
utilizing a sigmoidal concentration−response (variable slope)
equation. The inflection point of the sigmoidal curve gave
the pKa of the analyzed ionizable moiety.
ASSOCIATED CONTENT
Supporting Information
Two-Electrode Voltage-Clamp Electrophysiology of
Oocytes.
Experiments were conducted using OpusXpress 6000A
(Molecular Devices, Union City, CA).54 Both the voltage and
current electrodes were filled with 3 M KCl. Oocytes were
voltage-clamped at -60 mV. The oocytes were bath-perfused
with Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM
CaCl2, 10 mM HEPES, and 1 µM atropine, pH 7.2) with a
flow rate of 2 ml/min for  and 4 ml/min for heteromeric
nAChR. To evaluate the effects of experimental compounds
on nAChRs expressed in oocytes, two initial control
responses to applications of ACh were recorded before test
applications of experimental drugs alone or co-applied with
the ACh, and test responses were normalized to the average
of the two initial control responses for each oocyte. Drug
solutions were applied from a 96-well plate via disposable
tips. Drug applications were 12 seconds long, followed by a
211-second washout period, for  and 6 seconds long,
followed by a 271-second washout period, for heteromeric n-
AChR. The control concentrations of ACh were 60 µM for
wild-type , 30M for 11, 100M for , 100M
for LS 42, and 10M for HS 42. After experimental
drug applications, follow-up control applications of ACh
were made to determine primed potentiation, desensitization,
or rundown of the receptors. Data were collected at 50 Hz,
filtered at 20 Hz ( or 5 Hz (heteromeric nAChR), and
analyzed by Clampfit 9.2 or 10.0 (Molecular Devices) and
Excel (Microsoft, Redmond, WA). Data were expressed as
means ± S.E.M. from at least five oocytes for each
experiment and plotted with Kaleidagraph 4.5.2 (Abelbeck
Software, Reading, PA). Concentration-response data were
fit to the Hill equation using the Levenberg-Marquardt
algorithm. Multicell averages were calculated for
The Supporting Information is available free of charge on the
ACS Publications website.
Molecular formula strings (.csv)
pKa analysis and chemical characterization (PDF)
AUTHOR INFORMATION
Corresponding Author
* Palmer Taylor, pwtaylor@ucsd.edu
Author Contributions
The manuscript was written through input and contributions of
all authors. / All authors have given approval to the final version
of the manuscript.
Funding Sources
NIH GM18360, NIH R01 GM57481
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Franziska Schnarkowski, Von V Phan, Quynh My
Nguyen, Nila Rahmatyan and Joannie Ho, for insightful
discussions and contributions. The UCSD Chemistry and
Biochemistry Molecular MS facility. Bobby Lucero and Carlo
Ballatore for the use of the microwave for synthetic reactions.
ABBREVIATIONS
nAChR, nicotinic acetylcholine receptor; 5-HT, 5-
hydroxytryptamine; ACh, acetylcholine; AChBP, acetylcholine
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Page 16 of 18
(13) Bertrand, D.; Lee, C. H.; Flood, D.; Marger, F.; Donnelly-
Roberts, D. Therapeutic potential of α7 nicotinic acetylcholine
receptors. Pharmacol Rev. 2015, 67, 1025-1073.
binding protein; aCSF, artificial cerebrospinal fluid; KA,
antagonist dissociation constant; DMEM, Dulbecco’s modified
Eagles media; EtAcO, ethylacetate; DCM, dichloromethane;
DMF, dimethylformamide; DMA, dimethylacetamide; DMSO,
dimethylsulfoxide; DIPEA, N,N-diisopropylethylamine; m-
CPBA, meta-chloroperbenzoic acid; THF, tetrahydrofuran; LS,
low sensitivity form, HS, high sensitivity form; Pd(dppf)Cl2,
[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II);
Pd(PPh3)4, Tetrakis(triphenylphosphine)palladium(0); PAM,
positive allosteric modulator; Ago-PAM, agonist-positive
allosteric modulator; NMR, nuclear magnetic resonance; mm,
millimeter; M, molar; M, micromolar; nM, nanomolar; PB,
phosphate buffer; HEK, human embryonic kidney; CNiFERs,
cell-based neurotransmitter fluorescent engineered reporters;
13C-DEPTQ, 13C-distortionless enhancement by polarization
transfer quaternary carbons; 13C-APT, 13C-attached proton
test; HR-ESI-TOFMS, High resolution Electrospray ionization
time-of-flight mass spectrometry.
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