Design, synthesis and discovery of picomolar selective α4β2 nicotinic acetylcholine receptor ligands

Article

pubs.acs.org/jmc

Design, Synthesis and Discovery of Picomolar Selective α4β2

Nicotinic Acetylcholine Receptor Ligands

Venkata M. Yenugonda,^†,∥Yingxian Xiao,^‡Edward D. Levin,^§Amir H. Rezvani,^§Thao Tran,^‡

Nour Al-Muhtasib,^‡Niaz Sahibzada,^‡Teresa Xie,^‡Corinne Wells,^§Susan Slade,^§Joshua E. Johnson,^§

Sivanesan Dakshanamurthy,^†,∥Hye-Sik Kong,^†,∥York Tomita,^∥Yong Liu,^†,∥Mikell Paige,^†,∥

Kenneth J. Kellar,^‡and Milton L. Brown*^,†,∥

^†Center for Drug Discovery, Georgetown University Medical Center, 3970 Reservoir Road NW, Research Building, EP-07,

Washington, D.C. 20057, United States

^‡Department of Pharmacology and Physiology, Georgetown University School of Medicine, Washington, D.C. 20057, United States

^§Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, North Carolina 27710, United States

^∥Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3970 Reservoir Road

NW, Research Building, Washington, D.C. 20057, United States

S

* Supporting Information

ABSTRACT: Developing novel and selective compounds that desensitize α4β2

nicotinic acetylcholine receptors (nAChRs) could provide new eﬀective

treatments for nicotine addiction, as well as other disorders. Here we report a

new class of nAChR ligands that display high selectivity and picomolar binding

aﬃnity for α4β2 nicotinic receptors. The novel compounds have K_ivalues in the

range of 0.031−0.26 nM and properties that should make them good candidates

as drugs acting in the CNS. The selected lead compound 1 (VMY-2-95) binds

with high aﬃnity and potently desensitizes α4β2 nAChRs. At a dose of 3 mg/kg,

compound 1 signiﬁcantly reduced rat nicotine self-administration. The overall

results support further characterizations of compound 1 and its analogues in

preclinical models of nicotine addiction and perhaps other disorders involving

nAChRs.

assumed to mediate the pleasurable and rewarding eﬀects of

most drugs of abuse, including nicotine.¹¹There is convincing

evidence from in vivo studies that mesolimbic α4β2* nAChRs

play a pivotal role in nicotine self-administration in mice.¹²⁻¹⁴

Furthermore, diﬀerential activation and desensitization of

nAChR subtypes on dopamine and GABA neurons and

possibly glutamate neurons results in stimulated dopamine

release in the nucleus accumbens, leading to positive reinforce-

ment of nicotine.¹⁵

The α4β2 subtype of nAChR serves as an important target

for treating nicotine addiction as well as anxiety, depression,

and cognitive disorders, and thus these receptors have been

targeted with a wide array of compounds to develop potential

therapies (Figure 1A). Many of the new compounds are

structurally related to the natural nAChR ligands, including

nicotine, epibatidine, and cytisine.

INTRODUCTION

■

Nicotinic acetylcholine receptors (nAChRs) are pentameric

ligand gated ion channels with signiﬁcant potential as molecular

targets for drugs designed to treat a variety of central nervous

system disorders.¹⁻⁴In vertebrates, 12 neuronal nAChR

subunits have been identiﬁed including nine alpha subunits

(α2−α10) and three beta subunits (β2−β4).⁵These subunits

may coassemble as either heteromeric and homomeric

pentameric receptors, forming a theoretically large array of

receptor subtypes.⁶⁻⁸The predominant nAChRs in the CNS

are the heteromeric α4β2* subtype, composed of α4 and β2

subunits (the * indicates that some of these nAChRs may

contain one or more other subunits) and the homomeric α7

subtype. Certain areas of brain also contain a high density of

α3β4* nAChRs, and this subtype appears to be the

predominant nAChR in several autonomic nervous system

ganglia.⁹

In 2006, varenicline (Figure 1A) emerged as the newest drug

approved by U.S. FDA as a therapeutic aid for smoking

cessation.¹⁶Clinical studies indicate that varenicline is the most

eﬀective drug currently available to help people stop

Nicotine interacts with α4β2, α4β2α6*, and α7 nAChRs in

the dopaminergic mesolimbic pathway, which connects the

ventral tegmental area of the midbrain and the limbic system

via the nucleus accumbens,¹⁰and these eﬀects of nicotine on

brain dopaminergic systems are important in reinforcing drug

self-administration. The mesolimbic dopamine system is

Received: June 6, 2013

Published: September 18, 2013

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Figure 1. (A) Selected examples of natural and synthetic nAChR ligands. (B) Design strategy for compound 1. (C) General structures of the present

series of compounds.

smoking,¹⁷⁻²⁰but its eﬀects may be temporary.^17,18Further-

more, varenicline shows notable adverse side eﬀects that limit

the use of this compound.²¹These side eﬀects of varenicline are

thought to be mediated predominantly through its action at

non-α4β2 receptors, most likely the α3β4* and α7 sub-

types.^22,23Pharmacological studies indicate that the interactions

of varenicline with α4β2 nAChRs play essential roles in the

clinical eﬀect of varenicline as a smoking cessation aid. In

addition, varenicline also interacts with nAChRs containing α6

and β2 subunits, which may also contribute to its eﬀect.²⁴

Xiao, et al. previously reported the synthesis and

pharmacological properties of sazetidine (Saz-A) (Figure 1B),

which potently and selectively desensitizes β2 containing

nicotinic receptors, especially α4β2 nAChRs, as measured by

whole cell patch clamp and ion eﬄux assays.^25,26In addition to

its ability to cause long lasting desensitization of α4β2

receptors, Saz-A also has agonist activity at the receptor

subtype. It is highly interesting that Saz-A showed full agonist

activity at the (α4)₂(β2)₃receptors but nearly no agonist

activity at the (α4)₃(β2)₂receptors.²⁷In the animal model, Saz-

A reduced nicotine self-administration,²⁸decreased alcohol

intake,²⁹and improved performance in tests of attention.³⁰Saz-

A was also found to produce behaviors consistent with potential

antidepressant and/or antianxiety eﬀects in rats and mice.³¹⁻³³

These promising in vivo results suggest that Saz-A is an

excellent starting compound for developing additional potent

and subtype-selective drugs that desensitize α4β2 nAChRs.

Recent in vivo studies showed a low concentration of Saz-A in

rat brain,^33,34suggesting that optimization of Saz-A phys-

icochemical properties might enhance the in vivo CNS eﬃcacy

of this group of compounds.

In this report, we optimized the Saz-A ligand and provide the

design, synthesis, and evaluation of a new class of picomolar

active α4β2 selective compounds. These compounds could lead

to more eﬀective treatments for nicotine addiction as well as

other conditions that involve nAChRs.

RESULTS

■

Design and Synthesis of the New Nicotinic Ligands.

Utilizing homologous series, we began our optimization of Saz-

A by replacing the alkyl hydroxyl with a benzene group

resulting in the design of compound 1 (Figure 1B). We

anticipated that compound 1 would have an enhanced CNS

proﬁle based on the physicochemical properties of PSA

(34.15), lipophilicity (log P = 2.36), and log BB (0.22).

From our modeling studies (Figure 2), we envisioned that the

benzene ring would provide more rigidity and favorable

interactions with the α4β2 nAChR subtype. Aromatic

optimization of the benzene ring of compound 1 provided

the compounds in series 1 (Figure 1C).

The compounds from route 1 were synthesized as shown in

Scheme 1. Compound 18 was formed from a reaction of the 5-

bromopyridin-3-ol 16 and the Boc-protected alcohol 17 under

Mitsunobu conditions. Aromatic coupling of ethynyltrimethyl-

silane 19 to 18 resulted in the formation of 20. Deprotection of

TMS group in compound 20 resulted in the (S)-3-(azetidin-2-

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Figure 2. Predicted structural models of α4β2 nAChR. (A) Atomic level interactions between compound 1 with α4β2 nAChR. Binding site residues

are labeled and shown in a stick model rendering. Molecular modeling overlay of compound 1 (colored green) with S-nicotine (B), varenecline (C),

and Saz-A (D) in the α4β2 nAChR binding site. Residues are labeled and shown in a stick model rendering.

ylmethoxy)-5-(phenylethynyl)pyridine 21. Substituted aryl

iodides were added to the intermediate 21 under Sonogashira

reaction conditions to generate compounds 22, 23, 24, and 25.

Subsequent deprotection of the Boc group yielded the ﬁnal

compounds (compound 1, 2 (VMY-2-101), 3 (VMY-2-105),

and 4 (VMY-2-109) in series 1.

With the success of the multistep route synthetic strategy, we

optimized the chemical synthesis with a more eﬃcient one-pot

Sonogashira reaction³⁵(Scheme 1, route 2) to generate the

compounds in route 2. Synthesis of compounds 12 (VMY-2-

161), 13 (VMY-2-177), and 14 (VMY-2-191) was accom-

plished by route 2 (Scheme 2A−C).

Compound 1 analogues were also designed and synthesized

to investigate the importance of the azetidine ring, N-

substitution, and stereochemistry on the binding aﬃnity of

ligands for nAChRs (Scheme 2). The N-methyl azetidine (15,

VMY-2-205, Scheme 2D) was prepared by reductive methyl-

ation of the secondary amine with formaldehyde. Analogue

compound 16 (VMY-2-203), a previously reported compound,

was prepared as a standard to investigate the importance of a

spacer group between the benzene and pyridine rings.³⁶

Compound 16 (Scheme 3) was synthesized by applying the

Suzuki reaction to the Mitsunobu product (S)-tert-butyl 2-((5-

bromopyridin-3-yloxy)methyl)azetidine-1-carboxylate (18) and

phenylboronic acid (41), followed by Boc deprotection.

Binding Aﬃnities for nAChR Subtypes. The binding

aﬃnities of all compounds synthesized for the receptor

subtypes were examined in binding competition studies against

[³H]epibatidine. K_ivalues of these compounds at six deﬁned

subtypes of rat nAChRs are provided in Tables 1 and 2.

Included in the tables are the K_ivalues in rat forebrain, which

represents a mixture of more than one native nAChR.

All compounds in series 1 exhibited high aﬃnity for the rat

α4β2 nAChR subtype with K_ivalues ranging from 0.031 nM

compound 9 (VMY-2-131) to 0.26 nM compound 6 (VMY-2-

117). These compounds also showed high aﬃnities for the two

other subtypes containing β2 subunits, α2β2 and α3β2

nAChRs. In contrast, the binding aﬃnities of these compounds

for nAChR subtypes containing β4 subunits are much lower

than those for their β2 containing counterparts. As shown in

Table 1, the selectivity of these compounds for α4β2 receptors

over α3β4 receptors (K_iratio) were very high, ranging from

5400 times compound 6 to 87000 times compound 7 (VMY-2-

123). All of these ratios are much greater than that of nicotine

or varenicline. Similar to the low aﬃnities for α3β4 receptors,

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a

Scheme 1. Synthesis of Compounds in Series 1

a

Reagents and conditions: (a) DEAD, PPh₃, THF, O °C, 48 h; (b) 2 mol % Pd(PPh₃)₂Cl₂, 4 mol % PPh₃, 2 mol % CUI, iPr₂NH, toluene, 80 °C, 18

h; (c) KOH, MeOH/H₂O (20:1), 25 °C, 3 h; (d) 2 mol % Pd(PPh₃)₂Cl₂, 4 mol % PPh₃, 2 mol % CUI, iPr₂NH, toluene, 25 °C, 18 h; (e) TFA,

CH₂Cl₂, 0 °C−rt 4−6 h then 2 M NaOH methanol in water (9:1), methanol, 18 h; (f) 4 mol% Pd(PPh₃)₂Cl₂, 8 mol % PPh₃, 8 mol% CUI, iPr₂NH,

toluene, 80 °C, 18 h; (g) KOH, MeOH/H₂O (4:1), 25 °C, 3 h; (h) substituted aryl iodides, 25 °C, 16 h or substituted aryl bromide, 80 °C, 16 h; (i)

TFA, CH₂Cl₂, 0 °C−rt , 4−6 h then 2M NaOH methanol in water (9:1), methanol, 18 h.

these compounds showed very low aﬃnities for α7 homomeric

receptors (Table 1).

2-139), also showed high aﬃnities and selectivity for α4β2

receptors. However, the installation of CF₃at position 3

decreased the binding aﬃnity for the α4β2 receptor, as shown

by the binding proﬁle of 6.

Interestingly, two compounds in series 2, compound 12

(azetidine replaced with cyclobutane) and compound 13 (ring

opened analogue of azetidine), showed very low binding

aﬃnities to nAChRs (Table 2), indicating that the azetidine

ring is important for the high aﬃnity and selectivity binding

Because compound 1 showed a promising binding proﬁle, we

synthesized several analogues to evaluate the eﬀects of

substitutions on the benzene ring. Substitutions at position 2,

3, or 4 generated compounds 2, 3, 4, 7, and 8 (VMY-2-127).

These singly substituted analogues have binding proﬁles similar

to that of compound 1 (Table 1). The dual substituted

analogues 5 (VMY-2-113), 9, 10 (VMY-2-135), and 11(VMY-

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a

Scheme 2. Synthesis of Compounds in Series 2

a

Reagents and conditions: (a) DEAD, PPh₃, THF, 0 °C, 48 h; (b) 4 mol % Pd(PPh₃)₂Cl₂, 8 mol % PPh₃, 8 mol % CUI, iPr₂NH, toluene, 80 °C, 18

h; (c) KOH, MeOH/H₂O (4:1), 25 °C, 3 h; (d) iodobenzene, 25 °C, 16 h; (e) TFA, CH₂Cl₂, 0 °C−rt 4−6 h then 2 M NaoH in methanol in water

(9:1), methanol, 18 h; (f) HCHO, NaCNBH₃, PH = 4−5 (CH₃COOH:CH₃COONa), ethanol.

a

Scheme 3. Synthesis of Compound 16

a

Reagents and conditions: (a) Pd(PPh₃)₄, 2 M Na₂CO₃, toluene:ethanol (3:1), 90 °C, 18 h; (b) TFA, CH₂Cl₂, 0 °C−rt, 6 h then 2 M NaOH

methanol in water (9:1), methanol, 18 h.

proﬁle. In addition, the (S)-N-methyl compound 15 binds

nearly 25 times weaker than the corresponding (S)-N−H

compound 1, suggesting that N−H provides a potentially

important H-bond interaction. For comparing the diﬀerence

between stereotypes, compound 14, which is the (R)-form of

compound 1, was synthesized. The binding proﬁle of 14 is

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Table 1. Comparison of Binding Aﬃnities of Series 1 Compounds for Rat nAChR Subtypes with Those of Saz-A, Varenicline,

a

and Nicotine

b

K_i(nM)

compd ID

α2β2

α2β4

α3β2

α3β4

α4β2

α4β4

α7

forebrain

α3β4/α4β2

1

0.11 (0.01)

0.045 (0.016)

0.081 (0.021)

0.029 (0.005)

0.063 (0.011)

0.28 (0.04)

58 (4)

37 (7)

47 (6)

31 (5)

23 (1)

54 (7)

46 (4)

150 (1)

66 (3)

38 (4)

53 (2)

210 (30)

110 (20)

71 (5)

0.53 (0.08)

0.41 (0.09)

0.61 (0.07)

0.19 (0.03)

0.52 (0.34)

1.4 (0.6)

640 (40)

650 (20)

0.049 (0.007)

0.083 (0.029)

0.072 (0.007)

0.032 (0.012)

0.050 (0.009)

0.26 (0.04)

11 (1)

7.2 (0.7)

8.7 (0.5)

5.3 (0.2)

4.7 (0.6)

11 (2)

580 (50)

2,000 (800)

1100 (500)

720 (180)

1,800 (700)

1,300 (500)

200 (80)

0.5 (0.06)

0.50 (0.05)

0.58 (0.10)

0.44 (0.05)

0.73 (0.05)

3.5 (0.3)

13000

7800

2

3

580 (50)

8100

4

520 (30)

16000

28000

5400

5

540 (60)

6

1,400 (100)

4,000 (2,900)

3,400 (400)

1,000 (30)

1,100 (60)

1,300 (40)

1,900 (300)

440 (60)

7

0.053 (0.004)

0.19 (0.05)

0.34 (0.14)

1.6 (0.1)

0.046 (0.011)

0.093 (0.037)

0.031 (0.003)

0.043 (0.003)

0.076 (0.017)

0.062 (0.006)

10 (2)

12 (3)

1.1 (0.4)

87000

37000

32000

26000

17000

31000

44

8

17 (8)

480 (140)

250 (93)

1.7 (1.4)

9

0.053 (0.012)

0.064 (0.007)

0.091 (0.011)

0.087 (0.014)

12 (2)

0.46 (0.22)

0.24 (0.02)

0.59 (0.01)

0.38 (0.07)

47 (11)

12 (2)

0.70 (0.37)

1.4 (0.5)

10

11

Saz-A

8.1 (0.4)

10 (0.3)

52 (2)

590 (250)

1,500 (100)

670 (220)

520 (140)

62 (11)

1.7 (0.3)

0.17 (0.02)

12 (2)

c

nicotine

40 (6)

varenicline

0.22 (0.02)

1.9 (0.1)

460 (40)

0.13 (0.01)

15 (0.2)

0.71 (0.04)

3500

a

Competition binding assays were carried out in membrane homogenates of stably transfected cells or rat forebrain tissue as described

previously.⁷²⁻⁷⁴The rat nAChRs were labeled with [³H]epibatidine. The K_dvalues for [³H]epibatidine used for calculating K_ivalues were 0.02 for

α2β2, 0.08 for α2β4, 0.03 for α3β2, 0.3 for α3β4, 0.04 for α4β2, 0.09 for α4β4, 1.8 for α7, and 0.05 for rat forebrain. K_ivalues of the compounds

b

c

shown are the mean of 3−5 independent measurements with SEM values included in brackets. The K_ivalues of (−)-nicotine was published

previously.⁷²

a

Table 2. Binding Aﬃnities of Series 2 and Series 3 Compounds for Rat nAChR Subtypes

b

K_i(nM)

compd ID

α2β2

α2β4

α3β2

α3β4

α4β2

α4β4

α7

forebrain

α3β4/α4β2

c

12

>100000

>5000

>50000

>250000

46 (16)

>25000

>10000

>1000000

>500000

>100000

>1000

>500000

>50000

>250000

>25000

>100000

>50000

1.1 (0.2)

30 (2)

c

13

14

15

16

0.19 (0.05)

2.9 (0.5)

0.22 (0.02)

0.92 (0.26)

26 (2)

1700 (130)

40,000 (4000)

1300 (200)

0.11 (0.03)

1.2 (0.2)

0.14 (0.01)

8.9 (1.8)

1,100 (100)

6.2 (0.3)

1,600 (500)

7,700 (3000)

11,000 (3000)

15000

33000

9285

4,100 (400)

36 (3)

5.0 (0.4)

1.1 (0.2)

a

Competition binding assays were carried out in membrane homogenates of stably transfected cells or rat forebrain tissue as described

previously.⁷²⁻⁷⁴The rat nAChRs were labeled with [³H]epibatidine. The K_dvalues for [³H]epibatidine used for calculating K_ivalues were 0.02 for

α2β2, 0.08 for α2β4, 0.03 for α3β2, 0.3 for α3β4, 0.04 for α4β2, 0.09 for α4β4, 1.8 for α7, and 0.05 for forebrain. K_ivalues of the compounds shown

are the mean of 3−5 independent measurements with SEM values included in brackets. The compound showed very low binding aﬃnity at all

nAChR subtypes. In the range of the concentration tested, no accurate K_ivalues could be determined. The numbers shown are estimated range of K_i

b

c

values.

similar to that of compound 1, although compound 14 has a

slightly lower aﬃnity for α4β2 receptors than compound 1. The

binding aﬃnity and drug-like properties of compound 1

prompted us to choose this molecule as a staring compound

in the new series developed in this report and to advance this

analogue in further in vitro and in vivo studies.

Binding Aﬃnities for Targets Other than nAChRs. To

determine the aﬃnity of compound 1 to molecular targets

other than neuronal nAChRs, 41 binding assays were evaluated

including CNS receptors and transporters. As shown in the

Supporting Information Table S1, the preliminary binding

assays using a single concentration of compound 1 at 10 μM

generated 32 “miss” (less than 50% inhibition of binding by

speciﬁcally labeled ligands) and 9 “hit” (more than 50%

inhibition of binding). The K_ivalues of compound 1 at these

nine targets were determined by performing secondary binding

assays using a series of concentrations of compound 1. As

shown in the Supporting Information Table S2, the compound

has low binding aﬃnities at these targets; thus, the binding

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a

Table 3. Activation and Inhibition of nAChR Function by Compound 1, Saz-A, Varenicline, and Nicotine

a

α4β2 nAChRs

α3β4 nAChRs

E_max(%)

b

c

d

compd

EC₅₀(nM)

10

E_max(%)

IC_50(10′)(nM)

EC₅₀(nM)

IC_50(10′)(nM)

e

1

2

24

40

3

2

5

16

11

2

4

ND

>10000

b

sazetidine-A

varenicline

24 12

950 30

ND

45

94 18

21,000 1,000

24,000 1,000

82

3

(−)-nicotine

2400 300

100

370 110

100

2

a

Functional properties of each compounds were determined in using stable cell lines expressing human α4β2 or rat α3β4 nAChRs. EC₅₀values

show potencies of agonist activities. E_maxvalues show relative eﬃcacies of agonist activities, which were normalized to the E_maxvalue of nicotine.

IC_{50(10 min)}values show potencies of desensitizer activities, which were determined by preincubated cells with various concentrations of test

compounds for 10 min then exposed to 100 μM nicotine. ND indicates no stimulation was detected. All values shown are means SEM of 3−9

c

d

e

independent experiments.

aﬃnity of compound 1 for α4β2 nAChRs is at least 3000 times

higher than that for any of those nine targets.

6-(3-pyridyl) hex-5-yn-1-ol group of Saz-A (Figure 2A,B).

These additional interactions and favorable entropy penalty

may likely confer potency to compound 1. The overlay of

compound 1 with (S)-nicotine, varenicline, and Saz-A (Figure

2B−D) suggests that compound 1 forms more favorable

hydrophobic interactions with the α4β2 nAChR and occupies

diﬀerent receptor space that may be critical for selective and

high aﬃnity binding.

Molecular Modeling Interactions of Compound 1 and

Other Known Ligands with the Agonist Form of the X-

ray Crystal Structure of the AChBP. To better understand

the binding interactions between α4β2 nAChR and compound

1, molecular models of compound 1 with the α4β2 nAChR

were constructed (Figure 2A) and overlaid with known

nicotinic ligands (S)-nicotine, varenicline, and Saz-A (Figure

2B−D). The docked structural models of the α4β2 nAChR/

ligand complex reveal that the binding mode interactions are

slightly diﬀerent from one another as reported previously.³⁷⁻⁴³

To provide a consistent model with the nicotine-AChBP (PDB:

1UW6), the docked position of each compound was remodeled

using a step-by-step manual docking methodology with

restrained molecular dynamic (MD) simulations followed by

energy minimization. In the restrained MD simulations, the

optimum van der Waals and H-bond distance constraints was

set between the ligand and the α4β2 nAChR ligand binding

domain residues. The ﬁnal binding complex is depicted in

Figure 2A−D.

The compound 1, nicotine, varenicline, and Saz-A are

predicted to be buried near aromatic rich residues such as

W147α, W55β, Y91α, Y188α, Y195α, and F117β (Figure 2A−

D) and are expected to occupy a similar binding region.

However, MD simulations suggest a slightly diﬀerent

orientation of the compounds in the α4β2 binding site. This

may be due to conformational adjustments inside the binding

site. The azetidine group in compound 1 and Saz-A are shown

forming stacking interactions with the amino acid W147α

(Figure 2A,D). However, positioning of the pyridine group is

slightly perturbed to compensate for the conformational

entropy penalty due to isomeric constraints. The hydroxy

group of Saz-A may form a hydrogen bond with Y188α,

whereas this residue likely forms a hydrogen bond with the

pyrazine ring nitrogen of varenicline, and this hydrogen bond is

absent in (S)-nicotine. Compound 1 and Saz-A have hydro-

phobic groups extending from the pyridine ring, which may

form additional favorable hydrophobic interactions with K76β,

K77β, Y112β, V109β, F117β, and L119β. These interactions

can be compared with the interactions of varenicline involving

V109β, F117β, and L119β (Figure 2C). Although Saz-A and

compound 1 are shown to have a similar pattern of interaction,

the benzene ring of the ethynylbenzene of compound 1 can

form a stronger stacking and lipophilic interaction with K76β,

K77β, Y112β, and V109β than the corresponding hex-5-yn-1-ol

group of Saz-A. Moreover, the 3-(2-phenylethynyl) pyridine

group of compound 1 is more rigid and may require a smaller

penalty in conformational entropy as compared to the ﬂexible

Eﬀects of Compound 1 on Functions of nAChRs. The

functional eﬀect of the lead compound 1 was assessed by

measuring agonist-stimulated ⁸⁶Rb⁺eﬄux from stably trans-

fected cells expressing nAChRs, either human α4β2 subtype or

rat α3β4 subtype. Its ability to desensitize nAChRs was

determined by measuring nicotine-stimulated ⁸⁶Rb⁺eﬄux after

cells were preincubated with compound 1 for 10 min. For

comparison, we also examined three other nicotinic ligands in

the same manner, Saz-A, varenicline, and (−)-nicotine.

As shown in Table 3, compound 1 did not show any

detectable agonist activity at rat α3β4 nAChRs in ⁸⁶Rb⁺eﬄux

assays. At human α4β2 receptors, the compound showed

agonist activity with an EC₅₀value as 10 nM and a maximal

eﬃcacy (E_max) relative to the maximal stimulation by nicotine

of 24%. In parallel experiments, varenicline also showed partial

agonist activity at α4β2 receptors (45%) and, at much higher

concentrations, near-full agonist activity at α3β4 nAChRs

(85%). The full activation and desensitization curves of

compound 1 are shown in Supporting Information Figure S1.

It is important to note that all four compounds studied,

compound 1, Saz-A, varenicline, and nicotine inhibited nicotine

activation of α4β2 receptors after preincubation with the cells

for 10 min, which desensitizes the receptors. As shown in Table

3, after a 10 min preincubation, compound 1 potently

desensitized α4β2 receptor function with an IC₅₀value

(IC_50(10″)) of 16 nM, indicating a potency for desensitization

of these receptors similar to that of Saz-A, but 6- and 23-times

more potent than varenicline and nicotine, respectively. In

contrast to their high potency to desensitize α4β2 nAChRs, the

IC_50(10″)values of these four compounds to desensitize α3β4

nAChRs were greater than 10000 nM (Table 3). All of these

compounds, especially compound 1 and Saz-A, are much more

potent desensitizers of α4β2 than α3β4 nAChRs.

To conﬁrm that compound 1 is a low eﬃcacy agonist of

α4β2 nAChRs, we evaluated the compound for its agonist

activity at α4β2 nAChRs using whole cell patch clamp. In all

cells studied (n = 3), the bath application of compound 1 (200

nM) produced a mean inward whole-cell current that was 12.61

1.8% of the control whole-cell response to 32 μM ACh. This

electrophysiological study conﬁrms that compound 1 has very

low agonist eﬃcacy for α4β2 nAChRs.

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Figure 3. (A) Acute compound 1 eﬀects on nicotine self-administration (0.03 mg/kg/infusion). Compound 1 (3 mg/kg) resulted in a signiﬁcant (p

< 0.025) decrease in nicotine self-administration relative to nicotine self-administration after control saline injections (mean SEM; n = 15). (B)

Compound 1 eﬀects on nicotine self-administration during each 15 min time period within session. (C) Acute compound 1 eﬀects on locomotor

activity. Eﬀect of compound 1 on locomotor activity in the ﬁgure-eight maze during the 1 h session (mean + SEM; n = 12). (D) Compound 1 eﬀects

on locomotor activity during each 15 min time period within session.

Table 4. Calculated Ligand Eﬃciency and Physicochemical Properties of Compounds in Series 1

binding aﬃnity K_i(nM)

α4β2

ligand eﬃciency kcal/mol

predicted physicochemical properties

a

b

c

d

compd ID

LE

MW

CLogP

PSA

log BB

1

0.049

0.083

0.072

0.032

0.050

0.26

0.70

0.65

0.66

0.68

0.64

0.54

0.67

0.65

0.60

0.64

0.60

0.73

0.85

1.14

264.32

282.31

300.30

332.32

278.35

298.77

325.38

296.34

312.34

260.33

211.26

162.23

3.712

3.855

3.998

4.595

4.211

4.425

4.191

4.354

3.914

1.47

34.15

37.39

34.15

43.38

54.38

37.81

16.13

0.2

0.22

0.24

0.33

0.27

0.30

0.22

0.29

0.1

2

3

4

5

6

7

0.046

0.093

0.031

0.043

0.076

0.062

0.12

8

9

10

11

Saz-A

varenecline

nicotine

−0.44

−0.28

−0.035

0.899

0.883

10

a

b

Ligand binding eﬃciency was calculated according to the Hopkins equation: LE = 1.372 × (−log K_i(moles))/N. Molecular weight and cLogP

were calculated from ChemBioDraw Ultra 11.0. Polar surface area (PSA) was calculated from www.chemicalize.org. Log BB was calculated from

c

d

the following equation: Log BB = −0.0148PSA + 0.152CLogP + 0.139.

Eﬀects of Compound 1 on Nicotine Self-Adminis-

tration and Locomotor Activity in Rats. The lever press

data from the 10 training sessions clearly shows that the

but not the lower doses, caused a signiﬁcant (p < 0.025)

decrease in the number of nicotine infusions compared with

vehicle. The 15 min time periods within each session were

analyzed (Figure 3B). There was a signiﬁcant (F(2,28) = 19.85,

p < 0.0005) main eﬀect of time period reﬂecting the commonly

seen initial higher rate of nicotine self-administration during

each session. However, there was no signiﬁcant interaction of

compound 1 multiplied by time period within session. The

eﬀect of repeated dosing was also analyzed. The entire range of

compound 1 doses and the saline control were given twice.

nicotine lever is preferred (active lever 12.6

1.2 presses/

session, inactive lever 3.2 0.3 presses per session, (F(1,14) =

28.55, p < 0.0005) and that it is stable over this period. We then

studied acute eﬀects of compound 1 on nicotine self-

administration in rats. As shown in Figure 3A, compound 1

signiﬁcantly (F(3,42) = 3.36, p < 0.05) decreased intravenous

nicotine self-administration. The 3 mg/kg dose of compound 1,

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Figure 4. Unbound fraction of each compound in the rat brain tissue and plasma protein using an equilibrium dialysis method. (A) Representative

percentage of free fraction of rat plasma and brain tissue against each compound tested at 5 μM for 4 h. (B) Representative data summary of free

fractions of the brain tissue and plasma protein against each compound.

There was no signiﬁcant eﬀect of the repeated testing and no

signiﬁcant interaction compound 1 with repeated dosing. These

data demonstrate that, similar to Saz-A,²⁶compound 1 at 3 mg/

kg eﬀectively reduces nicotine self-administration in rats.

Interestingly, the locomotor activity of rats was signiﬁcantly

increased by compound 1 (F(3,33) = 13.58, p < 0.0005). All

three doses of compound 1 signiﬁcantly (p < 0.0005 for 0.3 and

1 mg/kg and p < 0.005 for 3 mg/kg) increased the mean

activity over the 1 h test session (Figure 3C). Therefore, the

eﬀect of compound 1 in decreasing nicotine self-administration

did not appear to result from any drug induced sedative eﬀect.

Furthermore, the dose−eﬀect function of compound 1 on

locomotor activity did not correspond with the eﬀect on

nicotine self-administration, as the peak of increased locomotor

activity was at 1 mg/kg, at which dose there was no signiﬁcant

reduction of nicotine self-administration. There was a

signiﬁcant main eﬀect of 15 min time blocks within the session

(F(3,33) = 79.68, p < 0.0005), reﬂecting the normal habituation

of locomotor exploration (Figure 3D), but the drug treatment

multiplied by the block interaction was not signiﬁcant.

In this study, we used Clark’s equation to predict the log BB

values for compounds in series 1.⁴⁹In general, a log BB value

greater than zero is a favorable factor for BBB penetration. The

log BB values of all compounds in series 1 were in the positive

range. Ligand eﬃciency (LE) is an important metric in drug

discovery and has been used to measure the relationship of a

ligand’s aﬃnity with molecular size. LE is the ratio of the free

energy of binding over the number of heavy atoms in a

molecule.⁵⁰LE is a useful optimization tool to evaluate a

ligand’s ability to eﬀectively bind to the targeted protein.

Considering the binding aﬃnity (K_i) of the compounds in

series 1, we calculated the LE using Hopkin’s equation.⁵¹An LE

≥ 0.3 is considered favorable and suggests that a compound is

optimized for receptor occupancy. All compounds in series 1

have an LE value in the range of 0.6 to 1 kcal/mol (Table 4).

Brain Tissue Distribution and Plasma Binding Studies

for Compound 1. To study in vitro unbound brain-to-plasma

concentration ratio of compound 1, we applied an equilibrium

dialysis technique as described in the Experimental Procedures.

As shown in Figure 4A,B, compound 1 (5 μM) has a

signiﬁcantly less percentage of unbound drug fraction in the rat

Physicochemical Properties of the New Ligands.

Several key physicochemical properties of compounds may

inﬂuence the blood−brain barrier (BBB) penetration of CNS

drugs,^44,45including molecular weight (MW), polar surface area

(PSA), and lipophilicity (clogP). These parameters were

calculated for all series 1 compounds and are presented in

Table 4. In general, CNS drugs have a MW ≤ 450 Da. All the

compounds in this report have molecular weights less than 450

Da. In addition, all these compounds have clogP values <5,

suggesting a reasonable probability of good oral absorption and

intestinal permeability.⁴⁶In general, a polar surface area (PSA)

less than 60 Å²is predictive of good penetration of the blood−

brain barrier. As shown in Table 4, all the compounds in series

1 have PSA values less than than 60 Å². We also calculated the

log BB, which is a parameter commonly used to express the

extent of a drug passing through the blood−brain barrier.⁴⁷

Several QSAR models were developed to calculate the log BB.⁴⁸

brain tissue (%f _UBrain= 1.21) as compared to Saz-A (%f_UBrain

=

59.8) and varenicline (%f_UBrain= 89.7). We also found that the

observed percentage of unbound drug fraction in plasma

protein for compound 1 (%f _Uplasma= 8.40) is higher than that

of brain tissue (Figure 4A,B). These results support that

compound 1 may have better brain tissue distribution as

compared to Saz-A.

DISCUSSION AND CONCLUSIONS

■

Tobacco use and nicotine addiction imposes a huge health and

economic burden. Cytisine, a plant alkaloid, has been used for

aiding smoking cession in Eastern and Central European

countries since 1964.⁵²To date, there are only three classes of

medications that have been approved by the U.S. Food and

Drug Administration for smoking cessation: nicotine replace-

ment therapy, bupropion, and varenicline. Among the three,

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varenicline, which is a rational designed compound related to

cytisine, is considered superior in terms of relative eﬃcacy over

a period of 3 months, at which time ∼44% of the subjects were

abstinent. However, the percentage of subjects who remained

smoke-free for 12 months following treatment with varenicline

fell to ∼22%.^17,18Moreover, although varenicline appears to be

safe for most people, exacerbation of schizophrenia and manic

episodes associated with treatment with varenicline has been

reported.^53,54In addition, based on a very recent report,⁵⁵the

FDA issued a “notiﬁcation” warning that varenicline may be

associated with increased adverse cardiovascular events,

including angina and heart attack. More commonly, nearly

30% of participants taking varenicline in clinical trials reported

nausea and 18% reported vomiting.^17,18

Although varenicline was developed as a partial agonist at

α4β2 nAChRs,⁵⁶it is also nearly a full agonist at α3β4 nAChRs,

which predominate in autonomic ganglia and brainstem

autonomic centers, as well as at α7 nAChRs, another important

subtype in brain.²³However, it is not known if varenicline

reaches concentrations necessary to act at these receptors.

More recently, varenicline was found to be a potent agonist of

the human 5-hydroxytryptamine₃receptors (5-HT₃).⁵⁷The

side eﬀects of varenicline are most likely mediated through its

actions at receptors other than α4β2* nAChRs, including

α3β4* and/or α7 nAChRS and/or 5-HT₃receptors.

Given the grave health and economic consequences of

smoking, there is an obvious great need for signiﬁcant

improvement in the existing smoking cessation therapies. On

the basis of a study of Saz-A, which desensitizes α4β2 nAChRs

potently and with high selectivity, we previously proposed a

strategy to develop novel nicotinic therapeutic drugs, including

smoking cessation drugs, based on their ability to selectively

desensitize α4β2 nAChRs.²⁵

The high potency of Saz-A to desensitize α4β2 nAChRs in

cells in vitro suggested that it would produce important eﬀects

similar to some of those produced by nicotine but with much

more receptor selectivity. It would therefore potentially be a

drug candidate to help people overcome addiction to nicotine,

as well as to treat other CNS disorders. Since 2007, Saz-A

studies in rodents have found that it reduces nicotine self-

administration^28,58and alcohol intake²⁹and produces anti-

nociceptive eﬀects^59,60and preclinical eﬀects consistent with

anxiolytic³²and possible antidepressant activity.^31,33,61Interest-

ingly, it also increased attention and reduced errors in rats even

after pretreatment with scopolamine and dizocilpine, drugs that

normally disrupt attention.³⁰

eﬃcacy of nicotine in activating human α4β2 nAChRs (Table

3). Importantly, the potency of compound 1 to desensitize

α4β2 nAChRs is similar to that of Saz-A and much more potent

than varenicline or nicotine (Table 3).

Consistent with an excellent pharmacological property

proﬁle, improved physicochemical properties and brain tissue

distribution (Figure 4), compound 1 signiﬁcantly reduced

nicotine self-administration in the rat model (Figure 3). This

suggests that compound 1 has the potential to be developed

into a smoking cessation drug.

There were diﬀerences between the eﬀects of compound 1

and Saz-A on locomotor activity. Compound 1 caused a modest

but signiﬁcant increase in locomotor exploration in the ﬁgure-

eight apparatus at all three doses tested (1−3 mg/kg). In

contrast, in the same test, Saz-A did not show any apparent

eﬀect on locomotor activity at 1 and 3 mg/kg (Saz-A produced

a slight decrease in activity at the beginning of the test session).

Compound 1 may have less risk of sedative side eﬀects than

Saz-A.

To elucidate SAR for compound 1, in addition to

compounds in series 1 (compounds 1−11), we also synthesized

four compounds in series 2, including 12, 13, 14, and 15

(Scheme 2A−D). The very low binding aﬃnities of compounds

12 and 13 may be an indication that the azetidine ring is

important for good binding property proﬁles. The binding

aﬃnity of (S)-N-methyl compound 15 is 25 times lower than

that of corresponding (S)-N−H compound 1. It is conceivable

that the N−H provides a potentially important H-bond

interaction. It is interesting that the two enantiomers,

compound 14 and compound 1, showed a similar binding

proﬁle although compound 14 has a slightly lower aﬃnity for

α4β2 receptors than compound 1. In series 3, we replaced the

alkyne group and made a direct link to pyridine as shown in

compound 16 (scheme 3). The binding data suggested that a

spacer is necessary to retain activity similar to that of

compound 1.

It is important to note that all binding data in this study are

from rat nAChRs, as well as functional data for α3β4 receptor

subtype. Rat nAChRs were instrumental in the discovery of

clinically used nicotinic drugs. In the consideration of possible

species diﬀerence, we are currently studying these novel

compounds using human nAChR subtypes. We will report

the comparative results once the study is completed.

In screening studies, compound 1 did not show high binding

aﬃnity for more than 40 other CNS receptors (Tables S1 and

S2, Supporting Information), including 5-HT₃receptors, which

could mediate some of varenicline’s adverse side eﬀects,

including nausea.⁵⁶

In summary, genetic and pharmacological studies suggest

that α4β2 nAChRs are crucial to nicotine self-administration in

rodents and likely very important in nicotine addiction.

Developing novel drug candidates that selectively target the

α4β2 nAChR subtype may provide an important therapeutic

advance for treating nicotinic addiction/dependence. Com-

pound 1 represents the lead of a class of novel compounds that

maintain the excellent pharmacological proﬁle of Saz-A but

with improved physicochemical properties.

Recent studies found that the Saz-A reached only low

concentrations in brain, even after chronic administration.^33,34

Compound 1 emerged from an eﬀort to improve brain

penetration of drugs that could selectively target α4β2 nAChRs.

Thus, while the synthesis and structure of compound 1 are

based on the Saz-A scaﬀold, its physiochemical properties,

especially its PSA and clogP, favor improved entry into the

brain. The binding model of α4β2 nAChR with compound 1

suggests similar occupancy of the binding pocket as that of

nicotine and varenicline. Moreover, the phenylethynyl group at

the C-5 position of the pyridine in compound 1 occupies a

potentially critical space in the pocket to form favorable

hydrophobic interactions with the receptor (Figure 2A).

In binding studies, compound 1 and analogues (Scheme 1)

are highly selective for α4β2 nAChRs over α3β4 and α7

subtypes (Table 1). In studies of receptor function as assessed

in ⁸⁶Rb⁺eﬄux assays, compound 1 has less than 30% of the

EXPERIMENTAL PROCEDURES

■

Chemistry. All reagents and solvents used in this study were

commercially available and used as obtained. Puriﬁcation of

compounds was performed by chromatography using a Biotage SP-1

system with silica gel cartridges. NMR spectra were recorded on a

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1

Varian 400 MR spectrometer at 400 MHz for H and 100 MHz for

until the pH 9−10. The reaction mixture stirred for 18 h, upon which

dichloromethane (10−20 mL) was added and rotary evaporated. The

resulting residue was puriﬁed by column chromatography.

¹³C. Chemical shifts (δ) are expressed in ppm downﬁeld from

tetramethylsilane, and coupling constants (J-values) are reported in

hertz (Hz). Molecular mass spectra measured using a Waters Q-TOF

premier mass spectrometer. The purity of ﬁnal compounds was

evaluated by C, H, N analysis (Atlantic microlabs) and HPLC

methods for compounds 9 and 15 (Figure S2, Supporting

Information). The purities of all the ﬁnal compounds were conﬁrmed

to be ≥95% by combustion and HPLC analytical methods. The HPLC

method of purity (9 and 15) was conﬁrmed using two diﬀerent mobile

phases as described in the Supporting Information (Figure S2).

Detailed synthetic and characterization data of intermediate

compound 3-ﬂuoro-5-iodo-N,N-dimethylaniline (VMY-2-119) was

presented in Supporting Information experimental procedures.

General Procedure for the Mitsunobu Reaction (18). To a

mixture of 5-bromo-3-pyridinol (1.2 equiv) and Ph₃P (1.6 equiv) in

anhydrous THF taken in a ﬂame-dried ﬂask under N₂, N-Boc

protected alcohol (1 equiv) was added and the mixture was cooled to

−10 °C. Diethyl azodicarboxylate (40% w/v) in toluene (1.6 equiv)

was added dropwise to the mixture and was warmed gradually to the

room temperature. After 48 h, the reaction mixture was quenched with

1 mL of water and the solvent was removed under reduced pressure.

The resulting yellow oil was puriﬁed by column chromatography on

silica gel to yield 55−60% as the white solid.

General Procedure for Sonogashira Coupling Reaction.

Sequential Desilylation and Sonogashira Coupling of the TMS

Protection (22−25). Route 1 in Scheme 1: An oven-dried and

nitrogen-ﬁlled round-bottom ﬂask was charged with (S)-tert-butyl-2-

((5-((trimethylsilyl)ethynyl)pyridin-3-yloxy)methyl)azetidine-1 car-

boxylate (20, 1 equiv), KOH (2 equiv) methanol in water (20:1).

The whole reaction mixture was stirred 25 °C for 3 h. It was then

added to a second ﬂask, which contained a performed mixture of

Pd(PPh₃)₂Cl₂(0.02 equiv, 2 mol %), CuI (0.02 equiv, 2 mol %), PPh₃

(0.04 equiv, 4 mol %), i-Pr₂NH (1 mL), toluene (3−5 mL), and

substituted ﬂuoro-iodobenzene that had been prestirred at 25 °C for

30 min. The complete mixture was then stirred at 25 °C for 16 h. The

reaction mixture was quenched with a saturated NH₄Cl solution and

extracted with CH₂Cl₂. The combined organic layers were washed

with 2N HCl, water, and saturated NaCl solution. The organic phase

was separated and dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure to give crude product. The crude

product was puriﬁed by column chromatography.

One-Pot Synthesis (22, 26−32). Route 2 in Scheme 1: The

Mitsunobu adduct (18, 1 equiv), Pd(PPh₃)₂Cl₂(0.04 equiv, 4 mol %),

CuI (0.08 equiv, 8 mol %), and PPh₃(0.08 equiv, 8 mol %) were

placed in a oven-dried round-bottom ﬂask with nitrogen. After

addition of i-Pr₂NH (1 mL) and toluene (3−5 mL), the mixture was

stirred at room temperature for 5 min and (trimethylsilyl) acetylene

(19, 2.7 equiv) was added and stirred at rt for 10 min. The whole

reaction mixture was stirred at 80 °C for 18 h, a solution of KOH in

methanol and water (4:1) was added in one portion, and the mixture

was stirred for additional 3 h at 25 °C. A second substituted aryl iodide

was added and stirred continually for 16 h at 25 °C (80 °C in case of

substituted aryl bromide). The reaction mixture was quenched with

saturated NH₄Cl solution and extracted with CH₂Cl₂. The combined

organic layers were washed with 2 N HCl, water, and saturated NaCl

solution. The organic phase was separated and dried over anhydrous

sodium sulfate, ﬁltered, and concentrated under reduced pressure to

give the crude product. The crude product was puriﬁed by column

chromatography.

General Procedure for the Deprotection of the N-Boc

Precursors. Route 1, method e, and route 2, method i: To a stirred

solution of N-Boc protected compound in dichloromethane was added

triﬂuoro acetic acid (5−10 equiv) dropwise at 0 °C under a nitrogen

atmosphere. The reaction mixture was stirred at room temperature for

4−6 h (TLC showed complete deprotection of Boc after 5 h). The

solvent and excess of TFA was removed under reduced pressure. The

resulting residue was further apply the nitrogen to remove the traces of

TFA and was taken in 2−3 mL methanol followed by dropwise

addition 2 M NaOH solution in methanol and water (9:1) at 0 °C

(S)-3-(Azetidin-2-ylmethoxy)-5-(phenylethynyl)pyridine (1). Route

2 (method i) in Scheme 1 was used and generated a yield of 70%

(liquid). ¹H NMR (400 MHz, CDCl₃): δ 8.37 (s, 1H), 8.31−8.25 (m,

1H), 7.60−7.49 (m, 2H), 7.41−7.29 (m, 4H), 4.33−4.20 (m, 1H),

4.10−3.96 (m, 2H), 3.69 (q, J = 7.9, 1H), 3.45 (td, J = 4.8, 8.1, 1H),

2.45−2.19 (m, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 154.4, 144.5,

137.7, 131.6, 128.7, 128.4, 122.9, 122.4, 120.4, 92.3, 85.8, 72.7, 56.9,

44.2, 23.9. HRMS (ESI): exact mass calcd for C₁₇H₁₆N₂O [M + H]⁺,

23.4

265.1341; found, 265.1339. [α]_D= −6.0 (c = 1.1, CHCl₃). Anal.

Calcd for C₁₇H₁₆N₂O: C,77.25; H, 6.10; N, 10.60. Found: C, 77.22; H,

6.13; N, 10.51.

(S)-3-(Azetidin-2-ylmethoxy)-5-((4-ﬂuorophenyl)ethynyl)pyridine

(2). Route 1 (method e) in Scheme 1 was used and generated a yield

of 77% (liquid). ¹H NMR (400 MHz, CDCl₃): δ 8.27 (s, 1H), 8.19 (d,

J = 2.8, 1H), 7.47−7.39 (m, 2H), 7.23 (dd, J = 1.5, 2.7, 1H),7.00−6.93

(m, 2H), 4.20 (s, 1H), 4.04−3.90 (m, 2H), 3.62 (d, J = 7.2, 1H), 3.37

(s, 1H), 2.37−2.14 (m, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 162.7

(d, J_F−C= 249 Hz, C), 154.4, 144.5, 137.8,133.6 (d, J_F−C= 8.5), 122.8,

120.3, 118.6 (d, J_F−C= 3.5), 115.7 (d, J_F−C= 23 Hz), 91.2, 85.6, 72.8,

57.0, 44.2, 23.9. HRMS (ESI): exact mass calcd for C₁₇H₁₅FN₂O [M +

24.4

H]⁺, 283.1247; found, 283.1241. [α]_D= −6.1 (c = 0.54, CHCl₃) .

Anal. Calcd for C₁₇H₁₅FN₂O·0.1H₂O: C, 71.63; H, 5.41; N, 9.82.

Found: C, 71.21; H, 5.41; N, 9.76

(S)-3-(Azetidin-2-ylmethoxy)-5-((2-ﬂuorophenyl)ethynyl)pyridine

(3). Route 1 (method e) in Scheme 1 was used and generated a yield

of 81% (liquid). ¹H NMR (400 MHz, CDCl₃): δ 8.31 (d, J = 1.0, 1H),

8.21 (dd, J = 2.7, 0.9, 1H), 7.49−7.41 (m, 1H), 7.31−7.23 (m, 2H),

7.11−7.00 (m, 2H), 4.29−4.14 (m, 1H), 4.05−3.91 (m, 2H), 3.63 (q, J

= 7.9, 1H), 3.39 (td, J = 7.8, 5.0, 1H), 2.41−2.11 (m, 3H). ¹³C NMR

(100 MHz, CDCl₃): δ 162.5 (d, J_F−C= 251 Hz), 154.4, 144.5, 138.1,

133.3 (d, J_F−C= 1.0), 130.5 (d, J_F−C= 8.0), 124.0 (d, J_F−C= 3.7),

122.8, 120.0, 115.5 (d, J_F−C=21 Hz), 111.0(d, J_F−C=16 Hz), 90.7 (d,

J_F−C= 3.3), 85.6, 72.7, 56.9, 44.1, 23.8. HRMS (ESI): exact mass calcd

24.7

for C₁₇H₁₅FN₂O [M + H]⁺, 283.1247; found. 283.1243. [α]_D

=

−11.5 (c = 0.87, CHCl₃). Anal. Calcd for C₁₇H₁₅FN₂O: C, 72.32; H,

5.36; N, 9.92. Found: C, 71.82; H, 5.38; N, 9.77.

(S)-3-(Azetidin-2-ylmethoxy)-5-((3-ﬂuorophenyl)ethynyl)pyridine

(4). Route 1 (method e) in Scheme 1 was used and generated a yield

of 78% (liquid). ¹H NMR (399 MHz, CDCl₃): δ 8.30 (d, J = 1.6, 1H),

8.22 (d, J = 2.8, 1H), 7.30−7.23 (m, 3H), 7.20−7.13 (m, 1H), 7.07−

6.95 (m, 1H), 4.29−4.15 (m, 1H), 3.98 (qd, J = 9.5, 5.5, 2H), 3.65 (q,

J = 7.9, 1H), 3.46−3.35 (m, 1H), 2.42−2.13 (m, 3H). ¹³C NMR (100

MHz, CDCl₃): δ 162.3 (d, J_F−C= 246 Hz). 154.4, 144.6, 138.0, 130.0

(d, J_F−C=9 Hz), 127.5 (d, J_F−C= 3.1 Hz), 124.2 (d, J_F−C= 9 Hz),

123.0, 120.0, 118.4 (d, J_F−C=23 Hz), 116.1(d, J_F−C=21 Hz), 116.0,

90.9 (d, J_F−C= 3.4), 72.8, 57.0, 44.2, 23.9. HRMS (ESI): exact mass

calcd for C₁₇H₁₅FN₂O [M + H]⁺, 283.1247; found, 283.1236.

[α]_D^24.8= −8.4 (c = 0.8, CHCl₃). Anal. Calcd for C₁₇H₁₅FN₂O: C,

72.32; H, 5.36; N, 9.92. Found: C, 72.03; H, 5.46; N, 9.78

(S)-3-(Azetidin-2-ylmethoxy)-5-((3,5-diﬂuorophenyl)ethynyl)-

pyridine (5). Route 2 (method i) in Scheme 1 was used and generated

1

a yield of 79% (liquid). H NMR (400 MHz, CDCl₃): δ 8.29 (d, J =

1.6,1H), 8.24 (d, J = 2.8, 1H), 7.25 (dd, J = 1.7, 2.8, 1H), 7.03−6.94

(m, 2H), 6.77 (tt, J = 2.3, 8.9, 1H), 4.23 (s, 1H), 3.99 (qd, J = 5.5, 9.5,

2H), 3.66 (d, J = 7.6, 1H), 3.39 (s, 1H), 2.48−1.79 (m, 3H).¹³CNMR

(100 MHz, CDCl₃): δ 163.9 (d, J_F−C= 13 Hz), 161.4 (d, J_F−C=13

Hz), 154.4, 144.6, 138.4, 125.1(t), 123.0, 119.5, 114.7 (d, J_F−C= 8

Hz), 114.5 (d, J_F−C= 7.7), 104.9 (t), 89.8 (t, J_F−C= 3.9), 87.7, 72.9,

56.9, 44.2, 23.9. HRMS (ESI): exact mass calcd for C₁₇H₁₄F₂N₂O [M

+ H]⁺, 301.1152; found, 301.1155. [α]_D^24.5= −8.0 (c = 0.8, CHCl₃).

Anal. Calcd for C₁₇H₁₄F₂N₂O·0.06H₂O: C, 67.74; H, 4.72; N, 9.28.

Found: C, 67.35; H, 4.68; N, 9.10.

(S)-3-(Azetidin-2-ylmethoxy)-5-((3-(triﬂuoromethyl)phenyl)-

ethynyl)pyridine (6). Route 2 (method i) in Scheme 1 was used and

1

generated a yield of 78% (liquid). H NMR (400 MHz, CDCl₃): δ

8.31 (d, J = 1.6, 1H), 8.24 (d, J = 2.8, 1H), 7.74 (s, 1H), 7.64 (d, J =

7.7, 1H), 7.55 (d, J = 7.9, 1H), 7.43 (t, J = 7.8, 1H), 7.27 (dd, J = 1.7,

8414

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2.8, 1H), 4.23 (td, J = 7.5, 12.3, 1H), 3.99 (qd, J = 5.5, 9.5, 2H), 3.66

(dd, J = 8.1, 15.9, 1H), 3.40 (dt, J = 4.9, 7.6, 1H), 2.52−1.90 (m, 3H).

¹³C NMR (100 MHz, CDCl₃): δ 154.4, 144.6, 138.2, 134.6 (d, J =

1.1), 131.0 (q), 128.9, 128.4 (d, J = 3 Hz), 125.2 (d, J = 3.7), 123.4,

123.0, 119.8, 90.6, 87.3, 72.8, 57.0, 44.2, 23.9. HRMS (ESI): exact

mass calcd for C₁₈H₁₅F₃N₂O [M + H]⁺, 333.1215; found, 333.1216.

25.0), 91.1 (d, J_F−C= 4.2), 86.4, 72.8, 57.0, 55.6, 44.2, 23.9. HRMS

(ESI): exact mass calcd for C₁₈H₁₇FN₂O₂[M + H]⁺, 313.1352; found,

25.2

313.1358. [α]_D

= −8.3 (c = 0.4, CHCl₃). Anal. Calcd for

C₁₈H₁₇FN₂O₂·0.04H₂O: C, 68.89; H, 5.47; N, 8.92. Found: C,

67.51; H, 5.51; N, 8.60.

3-(Cyclobutylmethoxy)-5-(phenylethynyl)pyridine (12). Methods

of b, c, d in Scheme 2A was used and generated a yield of 65% (liquid).

¹H NMR (400 MHz, CDCl₃): δ 8.36 (d, J = 1.5, 1H), 8.26 (d, J = 2.8,

24.7

[α]_D= −7.8 (c = 4.2, CHCl₃). Anal. Calcd for C₁₈H₁₅F₃N₂O: C,

65.06; H, 4.55; N, 8.43. Found: C, 64.74; H, 4.45; N, 8.30.

S)-3-(Azetidin-2-ylmethoxy)-5-(m-tolylethynyl)pyridine (7). Route

2 (method i) in Scheme 1 was used and generated a yield of 68%

(liquid). ¹H NMR (400 MHz, CDCl₃): δ 8.26 (t, J = 2.9, 1H), 8.17 (d,

J = 2.8, 1H), 7.30−7.21 (m, 3H), 7.15 (t, J = 7.6, 1H), 7.10−7.05 (m,

1H), 4.24−4.12 (m, 1H), 3.94 (qd, J = 9.5, 5.5, 2H), 3.60 (q, J = 8.1,

1H), 3.36 (td, J = 8.2, 4.4, 1H), 2.44 (s, 1H), 2.35−2.09 (m, 5H). ¹³C

NMR (100 MHz, CDCl₃): δ 154.4, 144.5, 138.0, 137.6, 132.2, 129.6,

128.7, 128.29, 122.9, 122.2, 120.5, 92.6, 85.5, 72.7, 57.0, 44.2, 23.8,

21.2. HRMS (ESI): exact mass calcd for C₁₈H₁₈N₂O [M + H]⁺,

1H), 7.60−7.50 (m, 2H), 7.40−7.33 (m, 3H), 7.34−7.28 (m, 1H),

3.98 (d, J = 6.6, 2H), 2.88−2.73 (m, 1H), 2.23−2.10 (m, 2H), 2.07−

1.82 (m, 4H). ¹³C NMR (100 MHz, CDCl₃): δ 154.7, 144.3, 137.8,

131.6, 128.7, 128.4, 122.8, 122.5, 120.4, 92.2, 85.9, 72.4, 34.4, 24.7,

18.5. HRMS (ESI): exact mass calcd for C₁₈H₁₇NO [M + H]⁺,

264.1388, found 264.1396. Anal. Calcd for C₁₈H₁₇NO: C, 82.10; H,

6.51; N, 5.32. Found: C, 81.94; H, 6.44; N, 5.31.

N-(2-(5-(Phenylethynyl)pyridin-3-yloxy)ethyl)propan-1-amine

(13). Method e in Scheme 2B was used and generated a yield of 87%

(solid). ¹H NMR (400 MHz, CDCl₃): δ 8.29 (d, J = 1.5, 1H), 8.19 (d,

J = 2.8, 1H), 7.50−7.41 (m, 2H), 7.33−7.26 (m, 3H), 7.26−7.22 (m,

1H),4.05 (t, J = 5.2, 2H), 2.96 (t, J = 5.2, 2H), 2.58 (t, J = 7.2, 2H),

1.47 (td, J = 14.6, 7.3, 3H), 0.87 (t, J = 7.4, 3H).¹³C NMR (100 MHz,

CDCl₃): δ 154.3, 144.6, 137.7, 131.6, 128.7, 128.4, 122.8, 122.4, 120.5,

92.3, 85.8, 68.0, 51.7, 48.5, 23.1, 11.7. HRMS (ESI): exact mass calcd

for C₁₈H₂₀N₂O [M + H]⁺, 281.1654; found, 281.1645. Anal. Calcd for

C₁₈H₂₀N₂O: C, 77.11; H, 7.19; N, 9.99. Found: C, 76.81; H, 7.12; N,

9.84.

25.5

279.1497; found, 279.1512. [α]_D= −8.4 (c = 1.9, CHCl₃). Anal.

Calcd for C₁₈H₁₈N₂O·0.06CH₂Cl₂: C, 76.19; H, 6.60; N, 9.87. Found:

C, 75.84; H, 6.34, N, 9.75.

(S)-3-(Azetidin-2-ylmethoxy)-5-((3-chlorophenyl)ethynyl)pyridine

(8). Route 2 (method i) in Scheme 1 was used and generated a yield of

1

72% (liquid). H NMR (400 MHz, CDCl₃): δ 8.27 (d, J = 1.6, 1H),

8.20 (d, J = 2.8, 1H), 7.44 (t, J = 1.7, 1H), 7.33 (dt, J = 7.4, 1.4, 1H),

7.27−7.17 (m, 3H), 4.26−4.14 (m, 1H), 4.04−3.89 (m, 2H), 3.62 (q, J

= 8.1, 1H), 3.43−3.32 (m, 1H), 2.38−2.11 (m, 3H). ¹³C NMR (100

MHz, CDCl₃): δ 154.4, 144.5, 138.0, 134.2, 131.4, 129.7, 129.6, 128.9,

124.1, 122.9, 119.9, 90.7, 86.9, 72.8, 56.9, 44.2, 23.8. HRMS (ESI):

(R)-3-(Azetidin-2-ylmethoxy)-5-(phenylethynyl)pyridine (14).

Method e in Scheme 2C was used and generated a yield of 75%

(liquid). ¹H NMR (400 MHz, CDCl₃): δ 8.28 (s, 1H), 8.19 (d, J = 2.8,

1H), 7.51−7.42 (m, 2H), 7.27 (ddd, J = 10.0, 6.0, 0.8, 4H), 4.26−4.14

(m, 1H), 3.96 (qd, J = 9.5, 5.7, 2H), 3.62 (q, J = 8.0, 1H), 3.38 (td, J =

8.1, 4.4, 1H), 2.38−2.12 (m, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

154.4, 144.6, 137.7, 131.6, 128.7, 128.4, 122.9, 122.4, 120.5, 92.3, 85.8,

exact mass calcd for C₁₇H₁₅ClN₂O [M + H]⁺, 299.0951; found,

25.6

299.0965. [α]_D

= −8.1 (c = 1.6, CHCl₃). Anal. Calcd for

C₁₇H₁₅ClN₂O·0.06H₂O: C, 67.84; H, 5.04; N, 9.30. Found: C,

67.70, H, 5.03; N, 9.11.

(S)-3-((5-(Azetidin-2-ylmethoxy)pyridin-3-yl)ethynyl)-5-ﬂuoro-

N,N-dimethylaniline (9). Route 2 (method i) in Scheme 1 was used

and generated a yield of 72% (liquid). ¹H NMR (400 MHz, CDCl₃): δ

8.29 (d, J = 1.6, 1H), 8.21 (t, J = 4.5, 1H), 7.26 (dd, J = 2.8, 1.7, 1H),

6.58−6.46 (m, 2H), 6.32 (dt, J = 12.5, 2.3, 1H), 4.21 (d, J = 15.8, 1H),

3.99 (qd, J = 9.5, 5.5, 2H),3.65 (q, J = 7.9, 1H), 3.41 (dd, J = 12.0, 8.3,

1H), 2.93−2.85 (s, 6H), 2.40−2.13 (m, 2H), 2.00 (d, J = 19.3, 1H).

¹³C NMR (100 MHz, CDCl₃): δ 163.5 (d, J_F−C= 241 Hz), 154.4,

72.7, 57.0, 44.2, 23.9. HRMS (ESI): exact mass calcd for C₁₇H₁₆N₂O

24.1

[M + H]⁺, 265.1341; found, 265.1349. [α]_D

= +13.6 (c = 0.7,

CHCl₃). Anal. Calcd for C₁₇H₁₆N₂O·0.6H₂O: C, 74.21; H, 6.30; N,

10.18. Found: C, 74.50; H, 6.20; N, 10.07.

(S)-3-((1-Methylazetidin-2-yl)methoxy)-5-(phenylethynyl)pyridine

(15). Method f in Scheme 2D was used and generated a yield of 48%

(liquid). (S)-3-(Azetidin-2-ylmethoxy)-5-(phenylethynyl) pyridine

(compound 1, 0.12 mmol) was taken in a 2 mL of ethanol. Formalin

(37%, 0.3 mL) was added, and the acidity was adjusted to pH 5 with

the addition of acetic acid and sodium acetate. The reaction mixture

was stirred for 15 min. Sodium cyanoborohydride (0.38 mmol) was

added. The whole reaction mixture was allowed to stir for 18 h at

room temperature. The solvent was evaporated, and the crude product

151.6 (d, J_F−C= 11 Hz), 144.6, 137.8, 123.8 (d, J_F−C=12 Hz), 123.0,

120.3, 111.1 (d, J_F−C= 2.0, 1H), 105.9 (d, J_F−C=24 Hz), 100.1 (d, J_F−C

= 26 Hz), 92.3 (d, J_F−C= 4 Hz), 85.2, 72.7,57.0, 44.2, 40.3, 23.9.

HRMS (ESI): exact mass calcd for C₁₉H₂₀FN₃O [M + H]⁺, 326.1669;

25.5

found, 326.1668. [α]_D= −18.5 (c = 0.18, CHCl₃).

(S)-3-(Azetidin-2-ylmethoxy)-5-((3-ﬂuoro-5-methylphenyl)-

ethynyl)pyridine (10). Route 2 (method i) in Scheme 1 was used and

1

was puriﬁed by column chromatography to yield pure 15. H NMR

1

(400 MHz, CDCl₃): δ 8.30 (d, J = 1.4, 1H), 8.20 (d, J = 2.8, 1H),

7.52−7.43 (m, 2H), 7.32−7.28 (m, 3H), 7.25 (dd, J = 2.7, 1.7, 1H),

4.06−3.95 (m, 2H), 3.51−3.35 (m, 2H), 2.86 (dd, J = 15.9, 8.6, 1H),

2.37 (s, 3H), 2.05 (td, J = 8.7, 6.0, 2H). ¹³C NMR (100 MHz, CDCl₃):

δ 154.3, 144.6, 137.7, 131.6, 128.7, 128.4, 123.0, 122.4, 120.5, 92.3,

85.8, 71.5, 66.0, 53.4, 44.8, 20.3. HRMS (ESI): exact mass calcd for

generated a yield of 73% (liquid). H NMR (400 MHz, CDCl₃): δ

8.33 (d, J = 1.6, 1H), 8.29−8.24 (m, 1H), 7.29 (dd, J = 2.8, 1.7, 1H),

7.12 (dt, J = 2.1, 0.7, 1H), 7.05−6.98 (m, 1H), 6.87 (dddd, J = 9.6, 2.3,

1.4, 0.7, 1H), 4.28 (tt, J = 12.3, 6.2, 1H), 4.03 (qd, J = 9.5, 5.5, 2H),

3.70 (dd, J = 15.9, 8.2, 1H), 3.45 (ddd, J = 7.6, 7.1, 4.4, 1H), 2.43−2.34

(m, 2H), 2.33 (t, J = 1.5, 3H), 2.25 (ddd, J = 16.4, 11.1, 8.2, 1H). ¹³C

NMR (100 MHz, CDCl₃): δ 162.3 (d, J_F−C= 246.3), 154.4, 144.6,

24.5

C₁₈H₁₈N₂O [M + H]⁺, 279.149; found, 279.1513. [α]_D= −32.2 (c

= 0.3, CHCl₃).

140.5 (d, J_F−C= 8.6), 138.0, 128.2 (d, J_F−C= 2.7), 123.7 (d, J_F−C

=

(S)-3-(Azetidin-2-ylmethoxy)-5-phenylpyridine (16). Methods of a

and b in Scheme 3 was used and generated a yield of 71% (liquid). To

a solution of (S)-tert-butyl 2-((5-bromopyridin-3-yloxy)methyl)-

azetidine-1-carboxylate (18, 0.58 mmol) in 9 mL of toluene and 3

mL of ethanol was added phenyl boronic acid (41, 0.69 mmol)

followed by 2 mL of 2 M Na₂CO₃and tetrakis(triphenylphosphine)-

palladium (0) (0.03 mmol). The reaction was stirred for 12 h at 90 °C

under nitrogen. The reaction was cooled to room temperature, diluted

with water, and extracted three times with ethyl acetate. The combined

organic layers dried over anhydrous sodium sulfate, ﬁltered, and

concentrated in vacuo to aﬀord a crude product, which was

subsequently puriﬁed by column chromatography. The resulting

pure compound was subjected to Boc deprotection followed by

puriﬁcation of column chromatography to yield a ﬁnal pure compound

16. ¹H NMR (400 MHz, CDCl₃): δ 8.37 (d, J = 1.7, 1H), 8.22 (d, J =

10.3), 122.9, 120.1, 116.8 (d, J_F−C= 21.1), 115.4 (d, J_F−C= 23.1), 91.2

(d, J_F−C= 3.7), 86.2, 72.7, 57.0, 44.2, 23.8, 21.1. HRMS (ESI): exact

mass calcd for C₁₈H₁₇FN₂O [M + H]⁺, 297.1403; found, 297.1403.

25.0

[α]_D

= −7.2 (c = 1.8, CHCl₃). Anal. Calcd for C₁₈H₁₇FN₂O·

0.04H₂O: C, 72.59; H, 5.76; N, 9.40. Found: C, 70.86; H, 5.72; N,

9.01.

(S)-3-(Azetidin-2-ylmethoxy)-5-((3-ﬂuoro-5-methoxyphenyl)-

ethynyl)pyridine (11). Route 2 (method i) in Scheme 1 was used and

1

generated a yield of 66% (liquid). H NMR (400 MHz, CDCl₃): δ

8.29 (d, J = 1.6, 1H), 8.22 (d, J = 2.8, 1H), 7.25 (dd, J = 2.8, 1.7, 1H),

6.83−6.72 (m, 2H), 6.57 (dt, J = 10.6, 2.3, 1H), 4.22 (s, 1H), 4.06−

3.92 (m, 2H), 3.75 (s, 3H), 3.64 (s, 1H), 3.39 (s, 1H), 2.54−1.91 (m,

3H).¹³C NMR (100 MHz, CDCl₃): δ 163.1 (d, J_F−C= 245.6), 160.7

(d, J_F−C= 12.1), 154.4, 144.6, 138.1, 124.3 (d, J_F−C= 12.1), 123.0,

119.9, 112.8 (d, J_F−C= 2.9), 110.9 (d, J_F−C= 23.6), 103.2 (d, J_F−C

=

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2.7, 1H), 7.49 (dd, J = 5.1, 3.8, 2H), 7.38 (t, J = 7.6, 2H), 7.31 (ddd, J

= 7.7, 4.1, 0.5, 2H), 4.31−4.14 (m, 1H), 4.02 (qd, J = 9.5, 5.6, 2H),

3.63 (q, J = 8.0, 1H), 3.39 (td, J = 8.2, 4.4, 1H), 2.39−2.13 (m, 3H).

¹³C NMR (100 MHz, CDCl₃): δ 155.1, 140.7, 137.6, 137.2, 136.5,

(s, 1H), 4.08 (dd, J = 10.1, 2.8, 1H), 3.82 (t, J = 7.5, 2H), 2.41−2.12

(m, 2H), 1.37 (s, 9H). ¹³C NMR (100 MHz, CDCl₃): δ 162.3 (d, J_F−C

= 246 Hz), 156.1, 154.5, 144.7, 138.2, 130.0 (d, J_F−C= 8.6, 7H), 127.5

(d, J_F−C= 3.1), 124.3 (d, J_F−C= 9 Hz), 123.0, 120.1, 118.4 (d, J_F−C

=

23 Hz), 116.1 (d, J_F−C=21 Hz), 91.0 (d, J_F−C= 3.4), 86.6, 79.7, 68.8,

60.0, 47.1, 28.4, 19.0. HRMS (ESI): exact mass calcd for C₂₂H₂₃FN₂O₃

[M + H]⁺, 383.1771; found, 383.1778.

128.9, 128.1, 127.1, 119.7, 72.8, 57.1, 44.2, 24.0. HRMS (ESI): exact

mass calcd for C₁₅H₁₆N₂O [M + H]⁺, 241.1341; found, 241.1348.

[α]_D^25.5= −4.69(c = 1.4, CHCl₃). Anal. Calcd for C₁₅H₁₆N₂O·0.6H₂O:

C, 71.74; H, 6.90; N, 11.15. Found: C, 71.73; H, 6.76; N, 10.88.

(S)-tert-Butyl-2-((5-bromopyridin-3-yloxy)methyl)azetidine-1-car-

boxylate (18). Mitsunobu reaction conditions were applied and

generated a yield of 55% (white solid). ¹H NMR (400 MHz, CDCl₃):

δ 8.25−8.19 (m, 2H), 7.36 (s, 1H), 4.44 (d, J = 5.3, 1H), 4.32−4.20

(m, 1H), 4.06 (dd, J = 2.8, 10.1, 1H), 3.81 (t, J = 7.5, 2H), 2.36−2.14

(m, 2H), 1.36 (s, 9H). ¹³C NMR (100 MHz, CDCl₃): δ

156.1,155.4,143.1, 136.7, 124.0, 120.3, 79.8, 69.0, 59.9, 47.1, 28.3,

18.95. HRMS (ESI): exact mass calcd for C₁₄H₁₉BrN₂O₃[M + H]⁺,

343.0657; found, 343.0670.

(S)-tert-Butyl-2-((5-((3,5-diﬂuorophenyl)ethynyl)pyridin-3 yloxy)-

methyl)azetidine-1-carboxylate (26). Route 2 (methods of f, g, h)

in Scheme 1 was used and generated a yield of 60% (liquid). ¹H NMR

(400 MHz, CDCl₃): δ 8.30 (s, 1H), 8.26 (d, J = 2.8, 1H), 7.30 (s, 1H),

7.02−6.93 (m, 2H), 6.76 (tt, J = 8.9, 2.2, 1H), 4.54−4.40 (m, 1H),

4.29 (s, 1H), 4.15−4.03 (m, 1H), 3.82 (t, J = 7.6, 2H), 2.40−2.12 (m,

2H), 1.36 (s, 9H). ¹³C NMR (100 MHz, CDCl₃): δ 163.9 (d, J_F−C

=

13 Hz), 161.4 (d, J_F−C= 13 Hz), 156.1, 154.4, 144.7, 138.5, 125.1(t),

123.0, 119.5, 114.6 (d, J_F−C= 7.7), 114.4 (d, J_F−C= 7.6), 104.9 (t),

89.8, 87.6, 79.7, 68.8, 60.0, 47.1, 28.3, 18.9. HRMS (ESI): exact mass

calcd for C₂₂H₂₂F₂N₂O₃[M + H]⁺, 401.1677; found, 401.1692.

(S)-tert-Butyl-2-((5-((3-(triﬂuoromethyl)phenyl)ethynyl)pyridin-

3yloxy)methyl)azetidine-1-carboxylate (27). Route 2 (methods of f,

g, h) in Scheme 1 was used and generated a yield of 50% (liquid). ¹H

NMR (400 MHz, CDCl₃): δ 8.39 (d, J = 1.4, 1H), 8.33 (d, J = 2.8,

1H), 7.80 (s, 1H), 7.70 (d, J = 7.8, 1H), 7.61 (d, J = 7.9, 1H), 7.50 (t, J

= 7.8, 1H), 7.40 (d, J = 1.8, 1H), 4.53 (dd, J = 5.4, 8.1, 1H), 4.37 (s,

1H), 4.22−4.10 (m, 1H), 3.90 (t, J = 7.6, 2H), 2.49−2.17 (m, 2H),

1.55−1.32 (m, 9H). ¹³C NMR (100 MHz, CDCl₃): δ 156.0, 154.4,

144.6, 138.4, 134.6, 130.9, 128.9, 128.3, 125.1, 123.4, 122.9, 119.7,

90.6, 87.2, 79.6, 68.7, 60.0,47.1, 28.3, 18.9. HRMS (ESI): exact mass

calcd for C₂₃H₂₃F₃N₂O₃[M + H]⁺, 433.1739; found, 433.1749.

(S)-tert-Butyl-2-((5-(m-tolylethynyl)pyridin-3-yloxy)methyl)-

azetidine-1-carboxylate (28). Route 2 (methods of f, g, h) in Scheme

1 was used and generated a yield of 56% (liquid). ¹H NMR (400 MHz,

CDCl₃): δ 8.38 (d, J = 1.6, 1H), 8.30 (d, J = 2.8, 1H), 7.40−7.33 (m,

3H), 7.29−7.24 (m, 1H), 7.19 (d, J = 7.6, 1H), 4.59−4.48 (m, 1H),

4.36 (s, 1H), 4.22−4.12 (m, 1H), 3.90 (t, J = 7.6, 2H), 2.43−2.33 (s,

3H), 1.44 (s, 9H). ¹³C NMR (100 MHz, CDCl₃): δ 156.0, 154.4,

144.7, 138.0, 137.8, 132.2, 129.6, 128.7, 128.3, 122.9, 122.2, 120.6,

92.6, 85.4, 79.7, 68.7, 60.0, 47.0, 28.4, 21.1, 19.0. HRMS (ESI): exact

mass calcd for C₂₃H₂₆N₂O₃[M + H]⁺, 379.2022; found, 379.2031.

(S)-tert-Butyl-2-((5-((3-chlorophenyl)ethynyl)pyridin-3yloxy)-

methyl)azetidine-1-carboxylate(29). Route 2 (methods of f, g, h) in

(S)-tert-Butyl-2-((5-((trimethylsilyl)ethynyl)pyridin-3-yloxy)-

methyl)azetidine-1-carboxylate (20). Sonogashira coupling method

(route 1 (method b) in Scheme1) was applied and generated a yield of

1

62% (liquid). H NMR (400 MHz, CDCl₃): δ 8.04 (s, 2H), 7.08 (d,

1H), 4.26 (s, 1H), 4.09 (s, 1H), 3.89 (s,1H), 3.63 (s, 2H), 2.07 (d,

2H), 1.18 (s, 9H), 0.16 (s, 9H). ¹³C NMR (100 MHz, CDCl₃): δ

156.2, 154.5, 145.2, 138.4, 123.4, 120.5, 101.5, 98.1, 79.7, 68.9, 60.2,

47.3, 28.6, 19.2, 0.3.

(S)-tert-Butyl-2-((5-ethynylpyridin-3-yloxy)methyl)azetidine-1-

carboxylate (21). Route 1 (method c) in Scheme 1 was applied.

¹HNMR (400 MHz, CDCl₃): δ 8.36 (s, 2H), 7.35 (s, 1H), 4.65−4.46

(m, 1H), 4.35 (s, 1H), 4.16 (dd, J = 2.9, 10.1, 1H), 3.91 (t, J = 7.6,

2H), 3.22 (s, 1H), 2.50−2.19 (m, 2H), 1.45 (s, 9H). ¹³C NMR (100

MHz, CDCl₃): δ 156.1, 155.5, 145.2, 138.5, 123.7, 80.4, 80.1, 79.8,

68.8, 60.0, 47.1, 28.4, 19.0.

(S)-tert-Butyl-2-((5-(phenylethynyl)pyridin-3-yloxy)methyl)-

azetidine-1-carboxylate (22). Both routes 1 and 2 in Scheme 1 were

1

used and generated a yield of 54% (liquid). H NMR (400 MHz,

CDCl₃): δ 8.39 (s, 1H), 8.31 (s, 1H), 7.59−7.51 (m, 2H), 7.37 (ddd, J

= 1.5, 3.4, 5.9, 4H), 4.62−4.44 (m, 1H), 4.36 (s, 1H), 4.16 (dd, J = 2.9,

10.1, 1H), 3.90 (t, J = 7.6, 2H), 2.51−2.19 (m, 2H), 1.44 (s, 9H). ¹³C

NMR (100 MHz, CDCl₃): δ 156.1, 154.5, 144.8, 137.9, 131.6, 128.8,

128.4, 122.9, 122.4, 120.6, 92.4, 85.8, 79.7, 68.8, 60.0, 47.0, 28.4, 19.0.

HRMS (ESI): exact mass calcd for C₂₂H₂₄N₂O₃[M + H]⁺, 365.1870;

found, 365.1870.

1

Scheme1 was used and generated a yield of 65% (liquid). H NMR

(400 MHz, CDCl₃): δ 8.29 (d, J = 1.5, 1H), 8.24 (d, J = 2.8, 1H), 7.44

(dd, J = 2.5, 0.9, 1H), 7.33 (dt, J = 7.4, 1.5, 1H), 7.28 (ddd, J = 3.4, 2.4,

1.5, 1H), 7.25 (dd, J = 2.0, 1.4, 1H),7.24−7.18 (m, 1H), 4.56−4.38

(m, 1H), 4.28 (s, 1H), 4.08 (dd, J = 10.1, 2.9, 1H), 3.81 (t, J = 7.6,

2H), 2.37−2.12 (m, 2H), 1.35 (s, 9H). ¹³C NMR (100 MHz, CDCl₃):

δ 156.0, 154.4, 144.7, 138.2, 134.2, 131.4, 129.7, 129.6, 128.9,

124.1,122.9, 120.0, 90.8, 86.9, 79.7, 68.7, 60.0, 47.0, 28.3, 19.02.

HRMS (ESI): exact mass calcd for C₂₂H₂₃ClN₂O₃[M + H]⁺,

399.1475; found, 399.1456.

(S)-tert-Butyl-2-((5-((4-ﬂuorophenyl)ethynyl)pyridin-3-yloxy)-

methyl)azetidine-1-carboxylate (23). Route 1 (method d) in Scheme

1 was used and generated a yield of 63% (liquid). ¹H NMR (400 MHz,

CDCl₃): δ 8.37 (d, J = 1.0, 1H), 8.31 (d, J = 2.8, 1H), 7.57−7.50 (m,

2H), 7.40−7.35 (m, 1H),7.12−7.03 (m, 2H), 4.58−4.50 (m, 1H), 4.36

(s, 1H), 4.16 (dd, J = 2.9, 10.1, 1H), 3.90 (t, J = 7.6, 2H), 2.53−2.18

(m, 2H), 1.44 (s, 9H). ¹³C NMR (100 MHz, CDCl₃): δ 162.7(d, J_F−C

= 250 Hz), 156.0, 154.5, 144.6, 137.9, 133.6 (d, J_F−C= 8.4 Hz,), 122.9,

120.4, 118.5 (d, J_F−C= 3.6), 115.7(d, J_F−C= 22), 91.3, 85.5 (d, J_F−C

=

(S)-tert-Butyl-2-((5-((3-(dimethylamino)-5-ﬂuorophenyl)ethynyl)-

pyridin-3-yloxy)methyl)azetidine-1-carboxylate (30). Route 2

(methods of f, g, h) in Scheme 1 was used and generated a yield of

1.3) 79.7, 68.7, 60.0, 47.1, 28.3, 19.0. HRMS (ESI): exact mass calcd

for C₂₂H₂₃FN₂O3 [M + H]⁺, 383.1771; found, 383.1767.

(S)-tert-Butyl-2-((5-((2-ﬂuorophenyl)ethynyl)pyridin-3-yloxy)-

methyl)azetidine-1-carboxylate (24). Route 1 (method d) in Scheme

1 was used and generated a yield of 77% (liquid). ¹H NMR (400 MHz,

CDCl₃): δ 8.32 (d, J = 4.1, 1H), 8.24 (dd, J = 2.8, 5.7, 1H), 7.45 (d, J =

5.4, 1H), 7.29 (d, J = 16.2, 2H), 7.06 (dd, J = 9.0, 10.1, 2H), 4.45 (s,

1H), 4.28 (s, 1H), 4.08 (dd, J = 3.4, 6.6, 1H), 3.88−3.72 (m, 2H), 2.25

(m, 2H), 1.35 (s, 9H). ¹³C NMR (100 MHz, CDCl₃): δ 162.61 (d,

J_F−C=251 Hz), 156.0, 154.4, 144.7, 138.2, 133.4 (d, J_F−C= 0.9), 130.5

(d, J_F−C= 8.0), 124.0 (d, J_F−C= 3.8), 122.9,120.1, 115.5 (d, J_F−C=21

Hz), 111.1(d, J_F−C=15 Hz), 90.7 (d, J_F−C= 3.2 Hz), 85.7, 79.7, 68.7,

60.0, 28.3, 47.1, 19.0. HRMS (ESI): exact mass calcd for C₂₂H₂₃FN₂O₃

[M + H]⁺, 383.1771; found. 383.1784.

(S)-tert-Butyl-2-((5-((3-ﬂuorophenyl)ethynyl)pyridin-3-yloxy)-

methyl)azetidine-1-carboxylate (25). Route 1 (method d) in Scheme

1 was used and generated a yield of 48% (liquid). ¹H NMR (400 MHz,

CDCl₃): δ 8.30 (s, 1H), 8.24 (d, J = 2.6, 1H), 7.26 (ddd, J = 12.6, 8.1,

1.1, 3H), 7.20−7.12 (m, 1H), 7.05−6.94 (m, 1H), 4.45 (s, 1H), 4.29

1

14% (liquid). H NMR (400 MHz, CDCl₃): δ 8.35 (s, 1H), 8.27 (s,

1H), 7.35 (s, 1H), 6.60 (s, 1H), 6.54 (d, J = 8.7, 1H), 6.37 (dt, J =

12.4, 2.2, 1H), 4.49 (s, 1H), 4.33 (s, 1H), 4.19−4.08 (m, 1H), 3.87 (t,

J = 7.6, 2H), 2.94 (d, J = 9.7, 6H), 2.43−2.20 (m, 2H), 1.40 (s, 9H).

¹³C NMR (100 MHz, CDCl₃): δ 163.5 (d, J_F−C= 241 Hz), 156.1,

154.4, 151.6 (d, J_F−C= 12 Hz), 144.8, 138.0, 123.8 (d, J_F−C= 12 Hz),

123.0, 111.13 (d, J_F−C= 2.0), 106.45 (d, J_F−C= 76 Hz), 100.1 (d, J_F−C

= 26 Hz), 92.42 (d, J_F−C= 4.2), 85.1, 79.7, 68.8 (d, J_F−C= 3 Hz), 60.0,

47.0, 40.2, 28.4, 19.0.

(S)-tert-Butyl-2-((5-((3-ﬂuoro-5-methylphenyl)ethynyl)pyridin-3-

yloxy)methyl)azetidine-1-carboxylate (31). Route 2 (methods of f, g,

1

h) in Scheme 1 was used and generated a yield of 65% (liquid). H

NMR (400 MHz, CDCl₃): δ 8.28 (d, J = 1.5, 1H), 8.22 (d, J = 2.8,

1H), 7.28 (dd, J = 2.6, 1.7, 1H), 7.06 (d, J = 0.5, 1H), 6.94 (dd, J = 9.1,

0.5, 1H), 6.80 (d, J = 9.5, 1H), 4.51−4.39 (m, 1H), 4.27 (s, 1H),

4.12−4.00 (m, 1H), 3.81 (t, J = 7.6, 2H), 2.35−2.09 (m, 5H), 1.35 (s,

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9H). ¹³C NMR (100 MHz, CDCl₃): δ 162.3 (d, J_F−C= 245 Hz), 156.1,

151.4, 144.7, 140.5 (d, J_F−C= 8 Hz), 138.1, 128.2 (d, J_F−C= 2.8, 3H),

123.7 (d, J_F−C= 11 Hz), 123.0, 120.1, 116.8 (d, J_F−C= Hz), 115.4 (d,

J_F−C= 23 Hz), 91.3 (d, J_F−C3.7), 86.1, 79.7, 68.8, 60.0, 47.2, 28.3, 21.1,

19.0. HRMS (ESI): exact mass calcd for C₂₃H₂₅FN₂O₃[M + H]⁺,

397.1927; found, 397.1929.

possible side chain atom contacts with the neighboring residues,

diﬀerent rotamer states of the residue were assigned, and then local

side chain atom dynamics followed by minimization were performed.

Minimization and molecular dynamics simulations were carried out

using the SANDER module of AMBER 10.0⁶⁵with default parameters.

The homology modeled structure was validated with PROCHECK⁶⁶

and WHATIF program.⁶⁷

(S)-tert-Butyl-2-((5-((3-ﬂuoro-5-methoxyphenyl)ethynyl)pyridin-

3-yloxy)methyl)azetidine-1-carboxylate (32). Route 2 (methods f, g,

Molecular docking was carried out using SurFlexDock Module of

Sybyl-X (Tripos Inc. St. Louis, USA). However, to be consistent with

the nicotine structural conformation (PDB: 1UW6), a manual

intervention followed by constrained molecular dynamics simulations

were carried out. This procedure was applied to compound 1,

varinicline, and sazetidine A. The α4β2 nAChR compound complexes

were reﬁned by molecular dynamics simulation using the Amber

10.0⁶⁵with the PARM98 force-ﬁeld parameter. The charge and force

ﬁeld parameters of the compounds were obtained using the most

recent Antechamber module in the Amber 10.0 (4), where compounds

were minimized at the MP2/6-31G* level using Gaussian 09.⁶⁸The

SHAKE algorithm⁶⁹was used to keep all bonds involving hydrogen

atoms rigid. Weak coupling temperature and pressure coupling

algorithms⁷⁰were used to maintain constant temperature and pressure,

respectively. Electrostatic interactions were calculated with the Ewald

particle mesh method⁷¹with a dielectric constant at 1R_ijand a

nonbonded cutoﬀ of 12 Å for the real part of electrostatic interactions

and for van der Waals interactions. The total charge of the system was

neutralized by addition of a chloride ion. The system was solvated in a

12 Å cubic box of water where the TIP3P model was used. 5000 steps

of minimization of the system were performed in which the α4β2

nAChR was constrained by a force constant of 75 kcal/mol/Å. After

minimization, a 20 ps simulation was used to gradually raise the

temperature of the system to 298 K while the complex was constrained

by a force constant of 20 kcal/mol/Å. Another 20 ps equilibration run

was used where only the backbone atoms of the complex were

constrained by a force constant of 5 kcal/mol/Å. Final production run

of 200 ps was performed with no constraints. When applying

constraints, the initial complex structure was used as a reference

structure. The PME method was used and the time step was 5 fs, and a

neighboring pairs list was updated every 25 steps.

General Procedure for in Vitro Studies. Cell Lines and Cell

Culture. The cell line expressing rat α3β4 nAChRs, KXα3β4R2, was

established previously by stably transfecting HEK 293 cells with

combinations of rat nAChR α3 and β4 subunit genes.^72,73The cell line

expressing human α4β2 nAChRs, YXα4β2H1, were established

recently (Tuan et al., 2012, manuscript in preparation). These cell

lines were maintained in minimum essential medium (MEM)

supplemented with 10% fetal bovine serum, 100 units/mL penicillin

G, 100 mg/mL streptomycin, and selective antibiotics at 37 °C with

5% CO₂in a humidiﬁed incubator. Tissue culture medium and

antibiotics were obtained from Invitrogen Corporation (Carlsbad,

CA), unless otherwise stated. Fetal bovine serum and horse serum

were provided by Gemini Bio-Products (Woodland, CA).

1

h) in Scheme 1 was used and generated a yield of 37% (liquid). H

NMR (400 MHz, CDCl₃): δ 8.27 (s, 1H), 8.21 (d, J = 2.7, 1H), 7.31−

7.24 (m, 1H), 6.74 (ddd, J = 1.2, 2.1, 9.9, 2H), 6.54 (dt, J = 2.3, 10.6,

1H), 4.47−4.38 (m, 1H), 4.25 (s, 1H), 4.09−4.02 (m, 1H), 3.79 (t, J =

7.6, 2H), 3.72 (s, 3H), 2.32−2.13 (m, 2H), 1.32 (s, 9H). ¹³C NMR

(100 MHz, CDCl₃): δ 163.1 (d, J_F−C= 245.8), 160.7 (d, J_F−C= 12.1),

156.1, 154.5, 144.8, 138.2, 124.34 (d, J_F−C= 12.1), 123.0, 120.0, 112.8

(d, J_F−C= 2.9), 110.9 (d, J_F−C= 23.5), 103.2 (d, J_F−C= 25.0), 91.2,

86.3, 79.7, 68.8, 60.0, 55.6, 47.3, 28.4, 19.05. HRMS (ESI): exact mass

calcd for C₂₃H₂₅FN₂O₄[M + H]⁺, 413.1877; found, 413.1887.

3-Bromo-5-(cyclobutylmethoxy)pyridine (34). Method a in

Scheme 2A was used and generated a yield of 69% (liquid). ¹H

NMR (400 MHz, CDCl₃): δ 8.18 (dd, J = 11.0, 2.1, 2H), 7.28 (dd, J =

2.5, 1.9, 1H), 3.88 (dd, J = 6.4, 3.4, 2H), 2.78−2.64 (m, 1H), 2.14−

2.02 (m, 2H), 1.99−1.74 (m, 4H). ¹³C NMR (100 MHz, CDCl₃): δ

155.4, 142.3, 136.3, 123.4, 120.1, 72.3, 34.1, 24.5, 18.40. HRMS (ESI):

exact mass calcd for C₁₀H₁₂BrNO [M + H]⁺, 242.0181; found,

242.0181.

tert-Butyl-2-(5-bromopyridin-3-yloxy)ethyl(propyl)carbamate

(36). Method a in Scheme 2B was used and generated a yield of 62%

(liquid). ¹H NMR 400 MHz, CDCl₃): δ 8.21 (s, 1H), 8.15 (d, J = 2.5,

1H), 7.29 (s, 1H), 4.06 (s, 2H), 3.51 (s, 2H), 3.16 (s, 2H), 1.56−1.43

(m, 2H), 1.39 (s, 9H), 0.82 (t, J = 7.4, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 155.0, 142.7, 136.3, 123.4, 120.1, 79.3, 67.0, 50.4, 46.6, 28.2,

21.8, 11.0. HRMS (ESI): exact mass calcd for C₁₅H₂₃BrN₂O₃[M +

H]⁺, 359.0970; found, 359.0978.

tert-Butyl-2-(5-(phenylethynyl)pyridin-3-yloxy)ethyl(propyl)-

carbamate (37). Methods of b, c, d in Scheme 2B was used and

generated a yield of 53% (solid). ¹H NMR (400 MHz, CDCl₃): δ 8.37

(s, 1H), 8.25 (d, J = 2.8, 1H), 7.59−7.49 (m, 2H), 7.37−7.28 (m, 4H),

4.14 (s, 2H), 3.59 (s, 2H), 3.25 (s, 2H), 1.64−1.51 (m, 2H), 1.47 (s,

9H), 0.89 (t, J = 7.4, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 155.6,

154.2, 144.5, 137.6, 131.6, 128.7, 128.3, 122.6, 122.4, 120.5, 92.4, 85.8,

79.5, 66.9, 50.5, 46.8, 28.3, 21.9, 11.1. HRMS (ESI): exact mass calcd

for C₂₃H₂₈N₂O₃[M + H]⁺, 381.2187; found, 381.2169

(R)-tert-Butyl-2-((5-bromopyridin-3-yloxy)methyl)azetidine-1-car-

boxylate (39). Method a in Scheme 2C was used and generated a yield

of 30% (solid). ¹H NMR (400 MHz, CDCl₃): δ 8.20 (d, J = 2.4, 2H),

7.35 (t, J = 1.9, 1H), 4.48−4.40 (m, 1H), 4.26 (s, 1H), 4.05 (dd, J =

10.1, 2.7, 1H), 3.86−3.76 (m, 2H), 2.70−2.01 (m, 2H), 1.35 (s, 9H).

¹³C NMR (100 MHz, CDCl₃): δ 156.0, 155.4, 143.1, 136.6, 124.0,

120.3, 79.7, 69.0, 59.9, 47.0, 28.3, 18.9. HRMS (ESI): exact mass calcd

for C₁₄H₁₉BrN₂O₃[M + H]⁺, 343.0657; found, 343.0680.

(R)-tert-Butyl-2-((5-(phenylethynyl)pyridin-3-yloxy)methyl)-

azetidine-1-carboxylate (40). Methods of b, c, d in Scheme 2C were

[³H]Epibatidine Radioligand Binding Assay. Stably transfected cell

lines, tissue culture conditions, membrane preparation procedures, and

binding assays were described previously.⁷²⁻⁷⁴Brieﬂy, cultured cells at

>80% conﬂuence were removed from their ﬂasks (80 cm²) with a

disposable cell scraper and placed in 10 mL of 50 mM Tris·HCl buﬀer

(pH 7.4, 4 °C). The cell suspension was centrifuged at 10000g for 5

min, and the pellet was collected. The cell pellet was then

homogenized in 10 mL of buﬀer with a polytron homogenizer and

centrifuged at 36000g for 10 min at 4 °C. The membrane pellet was

resuspended in fresh buﬀer, and aliquots of the membrane preparation

were used for binding assays. The concentration of [³H]epibatidine

used was ∼500 pM for competition binding assays. Nonspeciﬁc

binding was assessed in parallel incubations in the presence of 300 μM

nicotine. Bound and free ligands were separated by vacuum ﬁltration

through Whatman GF/C ﬁlters treated with 0.5% polyethylenimine.

The ﬁlter-retained radioactivity was measured by liquid scintillation

counting. Speciﬁc binding was deﬁned as the diﬀerence between total

binding and nonspeciﬁc binding. Data from competition binding

1

used and generated a yield of 74% (liquid). H NMR (400 MHz,

CDCl₃): δ 8.31 (s, 1H), 8.23 (s, 1H), 7.52−7.41 (m, 2H), 7.36−7.24

(m, 4H), 4.45 (d, J = 5.4, 1H), 4.28 (s, 1H), 4.08 (dd, J = 10.2, 2.6,

1H), 3.82 (t, J = 7.6, 2H), 2.28 (ddd, J = 25.0, 14.3, 7.1, 2H), 1.36 (d, J

= 0.5, 9H). ¹³C NMR (100 MHz, CDCl₃): δ 156.1, 154.5, 144.7,

137.8, 131.6, 128.7, 128.4, 123.0, 122.4, 120.6, 92.4, 85.7, 79.7, 68.8,

47.0, 60.0, 28.4, 19.0. HRMS (ESI): exact mass calcd for C₂₂H₂₄N₂O₃

[M + H]⁺, 365.1865; found, 343.1893.

Computational Studies. To study (S)-(−)-nicotine and (R)-

(−)-deschloroepibatidine binding with the α4β2 nAChR in atomic

detail, a structural model of the LBD of human α4β2 nAChR was built

by using the Homology Model software MODELLERX9.10.⁶²The

reference template is the X-ray crystal structure of the AChBP (PDB

entry of 1UW6) and the rat α4β2 X-ray structure (PDB: 1OLE).

Multiple sequence alignment was generated by Psi-BLAST⁶³and

ClusterW.⁶⁴Upon construction of the model, appropriate ionization

states were maintained, the side chains were relaxed to remove

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assays were analyzed using Prism 5 (GraphPad Software, San Diego,

CA).

test compound concentration in protein-free compartment, f _UBrain= 1/

D { (1/f _Umeans) − 1 + 1/D}.

Binding Assays for Targets Other than nAChRS. All binding assays

for targets other than nAChRs were performed by the National

Institute of Mental Health’s Psychoactive Drug Screening Program

(PDSP) supported by NIMH grant HHSN-271-2008-00025-C (PI:

Bryan Roth). For experimental details (K_ideterminations, receptor

binding proﬁles, functional data, MDR1 data, etc., as appropriate),

refer to the PDSP Web site http://pdsp.med.unc.edu/.

General Procedures for Animal Behavioral Studies. Separate

sets of young adult female Sprague−Dawley rats were used for the

nicotine self-administration study (N = 15) and for the locomotor

activity study (N = 12). The studies were conducted in accordance

with the regulations outlined by the Duke University Animal Care and

Use Committee. The rats housed in approved standard laboratory

conditions in a Duke University vivarium facility near the testing room

to minimize stress induced by transporting the rats. The rats were kept

on a 12:12 reverse day/night cycle so that they were in their active

phase during behavioral testing. The rats in the drug iv self-

administration studies were singly housed to prevent them from

damaging each other’s catheters. The rats in the locomotor activity

studies were housed in groups of 2−3. All rats were allowed access to

water at all times; the rats in the nicotine-self-administration study

were fed daily approximately 30 min after completing the sessions

while those in the locomotor activity study had continuous access to

food.

Compound 1 Administration. Compound 1 was injected sc 10 min

before testing in a volume of 1 mL/kg of saline. The doses (0, 0.3, 1,

and 3 mg/kg) we given in a counterbalanced order with at least two

days between successive injections. For the nicotine self-administration

study, the compound 1 the acute dose−eﬀect study was tested twice

while for the locomotor activity study the dose−eﬀect function was

tested once.

Nicotine Self-Administration. Before beginning nicotine self-

administration, the rats were trained for three sessions on lever

pressing for food reinforcement. Then they were ﬁtted with iv

catheters, and they received nicotine infusions (0.03 mg/kg/infusion)

on an FR1 schedule for 10 sessions. The rats were trained to self-

administer nicotine (0.03 mg/kg/infusion, IV) via operant lever

response (FR1) with a visual secondary reinforcer. Two levers were

available to be pressed, and only one caused the delivery of nicotine on

an FR1 schedule. Pressing the lever on the active side resulted in the

activation of the feedback tone for 0.5 s and the immediate delivery of

one 50 μL infusion of nicotine in less than 1 s. Each infusion was

immediately followed by a 1 min period in which the cue lights went

out, the house light came on and responses were recorded but not

reinforced.²⁸

Locomotor Activity. Another set of rats (N = 12) was tested for

acute compound 1 eﬀects on locomotor activity in a ﬁgure-eight maze

over the course of a 1 h session. The mazes had continuous enclosed

alleys 10 cm × 10 cm in the shape of a ﬁgure eight.⁷⁶The dimensions

of the apparatus were 70 cm long and 42 cm wide, with a 21 cm × 16

cm central arena, a 20 cm high ceiling, and two blind alleys extending

20 cm from either side. Eight infrared photobeams, which crossed the

alleys, indexed locomotor activity. One photobeam was located on

each of the two blind alleys, and three were located on each of two

loops of the ﬁgure eight. Numbers of photobeam breaks were recorded

for 5 min blocks over the 1 h session. The repeated measures were

compound 1 dose and the repeated administration of each dose.

Signiﬁcant interactions were followed up by tests of the simple main

eﬀects. Alpha of p < 0.05 (two-tailed) was used as the threshold for

signiﬁcance.

⁸⁶Rb⁺Eﬄux Assay. Functional properties of compounds at nAChRs

expressed in the transfected cells were measured using ⁸⁶Rb⁺eﬄux

assays as described previously.^23,71In brief, cells were plated into 24-

well plates coated with poly-^D-lysine. The plated cells were grown at

37 °C for 18 to 24 h to reach 85−95% conﬂuence. The cells were then

incubated in growth medium (0.5 mL/well) containing ⁸⁶Rb⁺(2 μCi/

mL) for 4 h at 37 °C. The loading mixture was then aspirated, and the

cells were washed four times with 1 mL of buﬀer (15 mM HEPES, 140

mM NaCl, 2 mM KCl, 1 mM MgSO₄, 1.8 mM CaCl₂, 11 mM glucose,

pH 7.4). Then 1 mL of buﬀer with or without compounds to be tested

was added to each well. After incubation for 2 min, the assay buﬀer was

collected for measurements of ⁸⁶Rb⁺released from the cells. Cells were

then lysed by adding 1 mL of 100 mM NaOH to each well, and the

lysate was collected for determination of the amount of ⁸⁶Rb⁺that was

in the cells at the end of the eﬄux assay. Radioactivity of assay samples

and lysates was measured by liquid scintillation counting. Total loading

(cpm) was calculated as the sum of the assay sample and the lysate of

each well. The amount of ⁸⁶Rb⁺eﬄux was expressed as a percentage of

⁸⁶Rb⁺loaded. Stimulated ⁸⁶Rb⁺eﬄux was deﬁned as the diﬀerence

between eﬄux in the presence and absence of nicotine. For obtaining

EC₅₀and E_maxvalues, stimulation curves were constructed in which

eight diﬀerent concentrations of a ligand were included in the assay.

For obtaining an IC_50(10′)value, inhibition curves were constructed in

which eight diﬀerent concentrations of a compound were applied to

cells for 10 min before 100 μM nicotine was applied to measure

stimulated eﬄux. EC₅₀, E_max, and IC_50(10′)values were determined by

nonlinear least-squares regression analyses (GraphPad, San Diego,

CA).

Electrophysiological Studies. Whole-cell voltage clamp (holding

potential −70 mV) recordings were made from YXα4β2H1 cells. For

increasing functionality of cells, cells were treated with 1 mM

carbachol for 48 h before studies. The recordings were made with

patch electrodes (5−6 MΩ) containing a solution composed of (in

mM; pH 7.2): K gluconate (145), EGTA (5), MgCl₂(2.5), HEPES

(10), ATP·Na (5), and GTP·Na (0.2). Cells were continuously

perfused with recording solution having the following composition

(mM): NaCl (130), KCl (5), CaCl₂(2), MgCl₂(2), glucose (10), and

HEPES (10), pH 7.4, at a temperature of 24 °C. They were visually

identiﬁed by infrared-diﬀerential interference contrast (IR-DIC) optics

via a CCD camera (Dage S-75). A 60× water immersion objective

(Nikon) was used for identifying and approaching cells. Only one cell

per coverslip was recorded.

The patch pipet was coupled to an ampliﬁer (Axopatch 700B; Axon

Instruments Inc.) and its signal ﬁltered (5kHz), digitized (Digidata

1440A; Axon Instruments Inc.), and stored on a PC computer running

the pClamp 10 software (Axon Instruments Inc.) for later analysis.

Series resistance was typically <10 MΩ and was continuously

monitored with a 10 mV pulse.

Acetylcholine (32 μM) was delivered by rapid focal application (500

ms; pressure ∼15 psi), whereas compound 1 (200nM) was applied via

bath application. The whole-cell current response to compound 1 was

normalized in relation to two stable prior applications of the 32 μM

ACh-induced responses (interstimulus interval 5 min).

Brain Tissue and Plasma Protein Binding Studies. Brieﬂy, each

test compound at a concentration of 5 μM is incubated in plasma or

homogenized brain tissue in a RED equilibrium dialysis device and

dialyzed against PBS using the published procedure.⁷⁵After 4 h, each

side is analyzed for test agent by LC/MS/MS. From this, the free drug

in brain, free in plasma, and brain/plasma partitioning is calculated

using this equation f _Umeans= 1 − {(PC − PF)/PC} PC = test

compound concentration in protein-containing compartment, PF =

Physicochemical Properties and Ligand Eﬃciency. Molecular

properties of compounds in series 1 were calculated according the

available software tools. Ligand binding eﬃciency was calculated

according to the Hopkins equation: LE = 1.372 × (−log K_i(moles))/

N. Molecular weight and cLogP were calculated from Chembiodraw

Ultra 11.0. Polar surface area (PSA) was calculated from www.

chemicalize.org. Log BB was calculated from the following equation:

Log BB = −0.0148PSA + 0.152CLogP + 0.139.

ASSOCIATED CONTENT

■

S

* Supporting Information

Details of broad screening data, functional assay, and

experimental details of intermediate compound synthesis.

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containing the beta2 subunit are involved in the reinforcing properties

of nicotine. Nature 1998, 391, 173−177.

This material is available free of charge via the Internet at

http://pubs.acs.org.

(13) Marubio, L. M.; Gardier, A. M.; Durier, S.; David, D.; Klink, R.;

Arroyo-Jimenez, M. M.; McIntosh, J. M.; Rossi, F.; Champtiaux, N.;

Zoli, M.; Changeux, J. P. Effects of nicotine in the dopaminergic

system of mice lacking the alpha4 subunit of neuronal nicotinic

acetylcholine receptors. Eur. J. Neurosci. 2003, 17, 1329−1337.

(14) Maskos, U.; Molles, B. E.; Pons, S.; Besson, M.; Guiard, B. P.;

Guilloux, J. P.; Evrard, A.; Cazala, P.; Cormier, A.; Mameli-Engvall, M.;

Dufour, N.; Cloez-Tayarani, I.; Bemelmans, A. P.; Mallet, J.; Gardier,

A. M.; David, V.; Faure, P.; Granon, S.; Changeux, J. P. Nicotine

reinforcement and cognition restored by targeted expression of

nicotinic receptors. Nature 2005, 436, 103−107.

AUTHOR INFORMATION

Corresponding Author

*Phone: +1-202-687-8603. Fax: +1-202-687-7659. Email:

mb544@georgetown.edu.

■

Notes

The authors declare the following competing ﬁnancial

interest(s): A patent application has been ﬁled by Georgetown

University to claim commercial rights of inventions in this

work. The following authors were listed on the patent

application as inventors: V.M.Y., Y.X., E.D.L., A.H.R., M.P.,

K.J.K., and M.L.B.

(15) Dani, J. A.; Heinemann, S. Molecular and cellular aspects of

nicotine abuse. Neuron 1996, 16, 905−908.

(16) Rollema, H.; Coe, J. W.; Chambers, L. K.; Hurst, R. S.; Stahl, S.

M.; Williams, K. E. Rationale, pharmacology and clinical efficacy of

partial agonists of alpha4beta2 nACh receptors for smoking cessation.

Trends Pharmacol. Sci. 2007, 28, 316−325.

(17) Gonzales, D.; Rennard, S. I.; Nides, M.; Oncken, C.; Azoulay, S.;

Billing, C. B.; Watsky, E. J.; Gong, J.; Williams, K. E.; Reeves, K. R.

Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial

agonist, vs sustained-release bupropion and placebo for smoking

cessation: a randomized controlled trial. JAMA, J. Am. Med. Assoc.

2006, 296, 47−55.

(18) Jorenby, D. E.; Hays, J. T.; Rigotti, N. A.; Azoulay, S.; Watsky, E.

J.; Williams, K. E.; Billing, C. B.; Gong, J.; Reeves, K. R. Efficacy of

varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial

agonist, vs placebo or sustained-release bupropion for smoking

cessation: a randomized controlled trial. JAMA, J. Am. Med. Assoc.

2006, 296, 56−63.

(19) Tonstad, S.; Tonnesen, P.; Hajek, P.; Williams, K. E.; Billing, C.

B.; Reeves, K. R. Effect of maintenance therapy with varenicline on

smoking cessation: a randomized controlled trial. JAMA, J. Am. Med.

Assoc. 2006, 296, 64−71.

(20) Tsai, S. T.; Cho, H. J.; Cheng, H. S.; Kim, C. H.; Hsueh, K. C.;

Billing, C. B., Jr.; Williams, K. E. A randomized, placebo-controlled

trial of varenicline, a selective alpha4beta2 nicotinic acetylcholine

receptor partial agonist, as a new therapy for smoking cessation in

Asian smokers. Clin. Ther. 2007, 29, 1027−1039.

(21) Moore, T. J.; Furberg, C. D.; Glenmullen, J.; Maltsberger, J. T.;

Singh, S. Suicidal behavior and depression in smoking cessation

treatments. PLoS One 2011, 6, e27016.

(22) Rollema, H.; Chambers, L. K.; Coe, J. W.; Glowa, J.; Hurst, R.

S.; Lebel, L. A.; Lu, Y.; Mansbach, R. S.; Mather, R. J.; Rovetti, C. C.;

Sands, S. B.; Schaeffer, E.; Schulz, D. W.; Tingley, F. D., III; Williams,

K. E. Pharmacological profile of the alpha4beta2 nicotinic acetylcho-

line receptor partial agonist varenicline, an effective smoking cessation

aid. Neuropharmacology. 2007, 52, 985−994.

(23) Mihalak, K. B.; Carroll, F. I.; Luetje, C. W. Varenicline is a

partial agonist at alpha4beta2 and a full agonist at alpha7 neuronal

nicotinic receptors. Mol. Pharmacol. 2006, 70, 801−805.

(24) Bordia, T.; Hrachova, M.; Chin, M.; McIntosh, J. M.; Quik, M.

Varenicline is a potent partial agonist at alpha6beta2* nicotinic

acetylcholine receptors in rat and monkey striatum. J. Pharmacol. Exp.

Ther. 2012, 342, 327−334.

(25) Xiao, Y.; Fan, H.; Musachio, J. L.; Wei, Z. L.; Chellappan, S. K.;

Kozikowski, A. P.; Kellar, K. J. Sazetidine-A, a novel ligand that

desensitizes alpha4beta2 nicotinic acetylcholine receptors without

activating them. Mol. Pharmacol. 2006, 70, 1454−1460.

(26) Xiao, Y.; Yasuda, R. P.; Sahibzada, N.; Wolfe, B. B.; Horton, L.;

Paige, M.; Brown, M. L.; Kellar, K. J. Sazetidine-A selectively induces a

long lasting desensitization of α4β2 nicotinic acetylcholine receptors.

In (), 2010 Neuroscience Meeting, Society for Neuroscience, San

Diego, 2010, 38.10.

(27) Carbone, A. L.; Moroni, M.; Groot-Kormelink, P. J.; Bermudez,

I. Pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)

(3)(beta2)(2) nicotinic acetylcholine receptors: subunit arrangement

ACKNOWLEDGMENTS

■

This work was supported by the National Institutes of Health

grant DA02799 and by the Georgetown University Center for

Drug Discovery. We thank the National Institute of Mental

Health Psychoactive Drug Screening Program for performing

binding assays at targets other than nAChRs.

ABBREVIATIONS USED

■

nAChR(s), nicotinic acetylcholine receptor(s); CNS, central

nervous system; 5-HT, serotonin; GABA, γ-aminobutyric acid;

AChBP, acetylcholine binding protein; ADME, absorption,

metabolism, distribution, metabolism; SAR, structure−activity

relationship

REFERENCES

■

(1) Gotti, C.; Riganti, L.; Vailati, S.; Clementi, F. Brain neuronal

nicotinic receptors as new targets for drug discovery. Curr. Pharm. Des.

2006, 12, 407−428.

(2) Romanelli, M. N.; Gratteri, P.; Guandalini, L.; Martini, E.;

Bonaccini, C.; Gualtieri, F. Central nicotinic receptors: structure,

function, ligands, and therapeutic potential. ChemMedChem. 2007, 2,

746−767.

(3) Bunnelle, W. H.; Dart, M. J.; Schrimpf, M. R. Design of ligands

for the nicotinic acetylcholine receptors: the quest for selectivity. Curr.

Top. Med. Chem. 2004, 4, 299−334.

(4) Arneric, S. P.; Holladay, M.; Williams, M. Neuronal nicotinic

receptors: a perspective on two decades of drug discovery research.

Biochem. Pharmacol. 2007, 74, 1092−1101.

(5) Lindstrom, J.; Anand, R.; Peng, X.; Gerzanich, V.; Wang, F.; Li, Y.

Neuronal nicotinic receptor subtypes. Ann. N. Y. Acad. Sci. 1995, 757,

100−116.

(6) Boyd, R. T. The molecular biology of neuronal nicotinic

acetylcholine receptors. Crit. Rev. Toxicol. 1997, 27, 299−318.

(7) Gotti, C.; Clementi, F.; Fornari, A.; Gaimarri, A.; Guiducci, S.;

Manfredi, I.; Moretti, M.; Pedrazzi, P.; Pucci, L.; Zoli, M. Structural

and functional diversity of native brain neuronal nicotinic receptors.

Biochem. Pharmacol. 2009, 78, 703−711.

(8) Kellar, K. J.; Xiao, Y. Neuronal nicotinic receptors: one hundred

years of progress. In Handbook of Contemporary Neuropharmacology;

John Wiley & Sons, Inc: Hoboken, NJ, 2007; pp 107−145.

(9) Mao, D.; Yasuda, R. P.; Fan, H.; Wolfe, B. B.; Kellar, K. J.

Heterogeneity of nicotinic cholinergic receptors in rat superior cervical

and nodose Ganglia. Mol. Pharmacol. 2006, 70, 1693−1699.

(10) Pidoplichko, V. I.; DeBiasi, M.; Williams, J. T.; Dani, J. A.

Nicotine activates and desensitizes midbrain dopamine neurons.

Nature 1997, 390, 401−404.

(11) Nestler, E. J. Is there a common molecular pathway for

addiction? Nature Neurosci. 2005, 8, 1445−1449.

(12) Picciotto, M. R.; Zoli, M.; Rimondini, R.; Lena, C.; Marubio, L.

M.; Pich, E. M.; Fuxe, K.; Changeux, J. P. Acetylcholine receptors

8419

dx.doi.org/10.1021/jm4008455 | J. Med. Chem. 2013, 56, 8404−8421

Journal of Medicinal Chemistry

Article

determines functional expression. Br. J. Pharmacol. 2009, 156, 970−

981.

binding protein reveals the ligand-binding domain of nicotinic

receptors. Nature 2001, 411, 269−276.

(43) Celie, P. H.; Rossum-Fikkert, S. E.; van Dijk, W. J.; Brejc, K.;

Smit, A. B.; Sixma, T. K. Nicotine and carbamylcholine binding to

nicotinic acetylcholine receptors as studied in AChBP crystal

structures. Neuron 2004, 41, 907−914.

(44) Chico, L. K.; Van Eldik, L. J.; Watterson, D. M. Targeting

protein kinases in central nervous system disorders. Nature Rev. Drug

Discovery 2009, 8, 892−909.

(45) Pajouhesh, H.; Lenz, G. R. Medicinal chemical properties of

successful central nervous system drugs. NeuroRx 2005, 2, 541−553.

(46) Reichel, A. Addressing central nervous system (CNS)

penetration in drug discovery: basics and implications of the evolving

new concept. Chem. Biodiversity 2009, 6, 2030−2049.

(47) Ghose, A. K.; Herbertz, T.; Hudkins, R. L.; Dorsey, B. D.;

Mallamo, J. P. Knowledge-Based, Central Nervous System (CNS)

Lead Selection and Lead Optimization for CNS Drug Discovery. ACS

Chem. Neurosci. 2012, 3, 50−68.

(48) Goodwin, J. T.; Clark, D. E. In silico predictions of blood-brain

barrier penetration: considerations to “keep in mind”. J. Pharmacol.

Exp. Ther. 2005, 315, 477−483.

(49) Clark, D. E. Rapid calculation of polar molecular surface area

and its application to the prediction of transport phenomena. 2.

Prediction of blood−brain barrier penetration. J. Pharm. Sci. 1999, 88,

815−821.

(50) Abad-Zapatero, C.; Perisic, O.; Wass, J.; Bento, A. P.;

Overington, J.; Al-Lazikani, B.; Johnson, M. E. Ligand efficiency

indices for an effective mapping of chemico-biological space: the

concept of an atlas-like representation. Drug Discovery Today 2010, 15,

804−811.

(51) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a

useful metric for lead selection. Drug Discovery Today 2004, 9, 430−

431.

(52) Hajek, P.; McRobbie, H.; Myers, K. Eﬃcacy of cytisine in

helping smokers quit: systematic review and meta-analysis. Thorax.

2013, DOI: 10.1136/thoraxjnl-2012-203035.

(53) Freedman, R. Exacerbation of schizophrenia by varenicline. Am.

J. Psychiatry 2007, 164, 1269.

(54) Kohen, I.; Kremen, N. Varenicline-induced manic episode in a

patient with bipolar disorder. Am. J. Psychiatry 2007, 164, 1269−1270.

(55) Singh, S.; Loke, Y. K.; Spangler, J. G.; Furberg, C. D. Risk of

serious adverse cardiovascular events associated with varenicline: a

systematic review and meta-analysis. Can. Med. Assoc. J. 2011, 183,

1359−1366.

(56) Coe, J. W.; Brooks, P. R.; Vetelino, M. G.; Wirtz, M. C.; Arnold,

E. P.; Huang, J.; Sands, S. B.; Davis, T. I.; Lebel, L. A.; Fox, C. B.;

Shrikhande, A.; Heym, J. H.; Schaeffer, E.; Rollema, H.; Lu, Y.;

Mansbach, R. S.; Chambers, L. K.; Rovetti, C. C.; Schulz, D. W.;

Tingley, F. D., III; O’Neill, B. T. Varenicline: an alpha4beta2 nicotinic

receptor partial agonist for smoking cessation. J. Med. Chem. 2005, 48,

3474−3477.

(57) Lummis, S. C.; Thompson, A. J.; Bencherif, M.; Lester, H. A.

Varenicline is a potent agonist of the human 5-hydroxytryptamine3

receptor. J. Pharmacol. Exp. Ther. 2011, 339, 125−131.

(58) Johnson, J. E.; Slade, S.; Wells, C.; Petro, A.; Sexton, H.;

Rezvani, A. H.; Brown, M. L.; Paige, M. A.; McDowell, B. E.; Xiao, Y.;

Kellar, K. J.; Levin, E. D. Assessing the effects of chronic sazetidine-A

delivery on nicotine self-administration in both male and female rats.

Psychopharmacology (Berlin, Ger.) 2012, 222, 269−276.

(59) Cucchiaro, G.; Xiao, Y.; Gonzalez-Sulser, A.; Kellar, K. J.

Analgesic effects of sazetidine-A, a new nicotinic cholinergic drug.

Anesthesiology 2008, 109, 512−519.

(28) Levin, E. D.; Rezvani, A. H.; Xiao, Y.; Slade, S.; Cauley, M.;

Wells, C.; Hampton, D.; Petro, A.; Rose, J. E.; Brown, M. L.; Paige, M.

A.; McDowell, B. E.; Kellar, K. J. Sazetidine-A, a selective alpha4beta2

nicotinic receptor desensitizing agent and partial agonist, reduces

nicotine self-administration in rats. J. Pharmacol. Exp. Ther. 2010, 332,

933−939.

(29) Rezvani, A. H.; Slade, S.; Wells, C.; Petro, A.; Lumeng, L.; Li, T.

K.; Xiao, Y.; Brown, M. L.; Paige, M. A.; McDowell, B. E.; Rose, J. E.;

Kellar, K. J.; Levin, E. D. Effects of sazetidine-A, a selective

alpha4beta2 nicotinic acetylcholine receptor desensitizing agent on

alcohol and nicotine self-administration in selectively bred alcohol-

preferring (P) rats. Psychopharmacology (Berlin, Ger.) 2010, 211, 161−

174.

(30) Rezvani, A. H.; Cauley, M.; Sexton, H.; Xiao, Y.; Brown, M. L.;

Paige, M. A.; McDowell, B. E.; Kellar, K. J.; Levin, E. D. Sazetidine-A, a

selective alpha4beta2 nicotinic acetylcholine receptor ligand: effects on

dizocilpine and scopolamine-induced attentional impairments in

female Sprague-Dawley rats. Psychopharmacology (Berlin, Ger.) 2011,

215, 621−630.

(31) Kozikowski, A. P.; Eaton, J. B.; Bajjuri, K. M.; Chellappan, S. K.;

Chen, Y.; Karadi, S.; He, R.; Caldarone, B.; Manzano, M.; Yuen, P. W.;

Lukas, R. J. Chemistry and pharmacology of nicotinic ligands based on

6-[5-(azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-1-ol (AMOP-H-

OH) for possible use in depression. ChemMedChem 2009, 4, 1279−

1291.

(32) Turner, J. R.; Castellano, L. M.; Blendy, J. A. Nicotinic partial

agonists varenicline and sazetidine-A have differential effects on

affective behavior. J. Pharmacol. Exp. Ther. 2010, 334, 665−672.

(33) Caldarone, B. J.; Wang, D.; Paterson, N. E.; Manzano, M.;

Fedolak, A.; Cavino, K.; Kwan, M.; Hanania, T.; Chellappan, S. K.;

Kozikowski, A. P.; Olivier, B.; Picciotto, M. R.; Ghavami, A.

Dissociation between duration of action in the forced swim test in

mice and nicotinic acetylcholine receptor occupancy with sazetidine,

varenicline, and 5-I-A85380. Psychopharmacology (Berlin, Ger.) 2011,

217, 199−210.

(34) Hussmann, G. P.; Kellar, K. J. A new radioligand binding assay

to measure the concentration of drugs in rodent brain ex vivo. J.

Pharmacol. Exp. Ther. 2012, 343, 434−440.

(35) Severin, R.; Reimer, J.; Doye, S. One-pot procedure for the

synthesis of unsymmetrical diarylalkynes. J. Org. Chem. 2010, 75,

3518−3521.

(36) Holladay, M. W.; Bai, H.; Li, Y.; Lin, N. H.; Daanen, J. F.;

Ryther, K. B.; Wasicak, J. T.; Kincaid, J. F.; He, Y.; Hettinger, A. M.;

Huang, P.; Anderson, D. J.; Bannon, A. W.; Buckley, M. J.; Campbell,

J. E.; Donnelly-Roberts, D. L.; Gunther, K. L.; Kim, D. J.; Kuntzweiler,

T. A.; Sullivan, J. P.; Decker, M. W.; Arneric, S. P. Structure−activity

studies related to ABT-594, a potent nonopioid analgesic agent: effect

of pyridine and azetidine ring substitutions on nicotinic acetylcholine

receptor binding affinity and analgesic activity in mice. Bioorg. Med.

Chem. Lett. 1998, 8, 2797−2802.

(37) Huang, X.; Zheng, F.; Crooks, P. A.; Dwoskin, L. P.; Zhan, C. G.

Modeling multiple species of nicotine and deschloroepibatidine

interacting with alpha4beta2 nicotinic acetylcholine receptor: from

microscopic binding to phenomenological binding affinity. J. Am.

Chem. Soc. 2005, 127, 14401−14414.

(38) Karlin, A. Emerging structure of the nicotinic acetylcholine

receptors. Nature Rev. Neurosci. 2002, 3, 102−114.

(39) Karlin, A. A touching picture of nicotinic binding. Neuron 2004,

41, 841−842.

(40) Lester, H. A.; Dibas, M. I.; Dahan, D. S.; Leite, J. F.; Dougherty,

D. A. Cys-loop receptors: new twists and turns. Trends Neurosci. 2004,

27, 329−336.

(41) Unwin, N. Refined structure of the nicotinic acetylcholine

receptor at 4A resolution. J. Mol. Biol. 2005, 346, 967−989.

(42) Brejc, K.; van Dijk, W. J.; Klaassen, R. V.; Schuurmans, M.; van

Der, O. J.; Smit, A. B.; Sixma, T. K. Crystal structure of an ACh-

(60) AlSharari, S. D.; Carroll, F. I.; McIntosh, J. M.; Damaj, M. I. The

antinociceptive effects of nicotinic partial agonists varenicline and

sazetidine-A in murine acute and tonic pain models. J. Pharmacol. Exp.

Ther. 2012, 342, 742−749.

(61) Liu, J.; Eaton, J. B.; Caldarone, B.; Lukas, R. J.; Kozikowski, A. P.

Chemistry and pharmacological characterization of novel nitrogen

analogues of AMOP-H-OH (sazetidine-A, 6-[5-(azetidin-2-

8420

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Journal of Medicinal Chemistry

Article

ylmethoxy)pyridin-3-yl]hex-5-yn-1-ol) as alpha4beta2-nicotinic acetyl-

choline receptor-selective partial agonists. J. Med. Chem. 2010, 53,

6973−6985.

(62) Fiser, A.; Do, R. K.; Sali, A. Modeling of loops in protein

structures. Protein Sci. 2000, 9, 1753−1773.

(63) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J.

Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403−410.

(64) Chenna, R.; Sugawara, H.; Koike, T.; Lopez, R.; Gibson, T. J.;

Higgins, D. G.; Thompson, J. D. Multiple sequence alignment with the

Clustal series of programs. Nucleic Acids Res. 2003, 31, 3497−3500.

(65) Case, D. A.; Darden, T. A.; Cheatham, T. E.; Simmerling, C. L.;

Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Pearlman, D. A.; Crowley,

M.; Walker, R. C.; Zhang, W.; Wang, B.; Hayik, S.; Roitberg, A.;

Seabra, G.; Wong, K. F.; Paesani, F.; Wu, X.; Brozell, S.; Tsui, V.;

Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V.; Cui, G.;

Beroza, P.; Mathews, D. H.; Schafmeister, C.; Ross, W. S.; Kollman, P.

A. AMBER 9; University of California: San Francisco, 2006.

(66) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J.

M. PROCHECK: a program to check the stereochemical quality of

protein structure. J. Appl. Crystallogr. 1993, 26, 283−291.

(67) Vriend, G. WHAT IF: a molecular modeling and drug design

program. J. Mol. Graphics 1990, 8, 52−56.

(68) Frisch, M. J.; Trucks, G. W.; Cheeseman, J. R.; Scalmani, G.;

Caricato, M.; Hratchian, H. P.; Li, X.; Barone, V.; Bloino, J.; Zheng, G.;

Vreven, T.; Montgomery, Jr. J. A.; Petersson, G. A.; Scuseria, G. E.;

Schlegel, H. B.; Nakatsuji, H.; zmaylov, A. F.; Martin, R. L.;

Sonnenberg, J. L.; Peralta, J. E.; Heyd, J. J.; Brothers, E.; Ogliaro, F.;

Bearpark, M.; Robb, M. A.; Mennucci, B.; Kudin, K. N.; Staroverov, V.

N.; Kobayashi, R.; Normand, J.; Rendell, A.; Gomperts, R.;

Zakrzewski, V. G.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;

Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao O.; Nakai, H.

Gaussian 09; Gaussian, Inc.: Wallingford, CT, 2009.

(69) Hanson, R. N.; Lee, C. Y.; Friel, C. J.; Dilis, R.; Hughes, A.;

DeSombre, E. R. Synthesis and evaluation of 17alpha-20E-21-(4-

substituted phenyl)-19-norpregna-1,3,5(10),20-tetraene-3,17beta-diols

as probes for the estrogen receptor alpha hormone binding domain. J.

Med. Chem. 2003, 46, 2865−2876.

(70) Berendsen, H. J.C.; Postma, J. P. M.; vanGunsteren, W. F.;

DiNola, A.; Haak, J. R. Molecular dynamics with coupling to an

external bath. J. Chem. Phys. 1984, 81, 3684.

(71) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: An N·

log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993,

98, 10089.

(72) Xiao, Y.; Kellar, K. J. The comparative pharmacology and up-

regulation of rat neuronal nicotinic receptor subtype binding sites

stably expressed in transfected mammalian cells. J. Pharmacol. Exp.

Ther. 2004, 310, 98−107.

(73) Xiao, Y.; Meyer, E. L.; Thompson, J. M.; Surin, A.; Wroblewski,

J.; Kellar, K. J. Rat alpha3/beta4 subtype of neuronal nicotinic

acetylcholine receptor stably expressed in a transfected cell line:

pharmacology of ligand binding and function. Mol. Pharmacol. 1998,

54, 322−333.

(74) Xiao, Y.; Abdrakhmanova, G. R.; Baydyuk, M.; Hernandez, S.;

Kellar, K. J. Rat neuronal nicotinic acetylcholine receptors containing

alpha7 subunit: pharmacological properties of ligand binding and

function. Acta Pharmacol. Sin. 2009, 30, 842−850.

(75) Maurer, T. S.; Debartolo, D. B.; Tess, D. A.; Scott, D. O.

Relationship between exposure and nonspecific binding of thirty-three

central nervous system drugs in mice. Drug Metab. Dispos. 2005, 33,

175−181.

(76) Crofton, K. M.; Howard, J. L.; Moser, V. C.; Gill, M. W.; Reiter,

L. W.; Tilson, H. A.; MacPhail, R. C. Interlaboratory comparison of

motor activity experiments: implications for neurotoxicological

assessments. Neurotoxicol. Teratol. 1991, 13, 599−609.

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dx.doi.org/10.1021/jm4008455 | J. Med. Chem. 2013, 56, 8404−8421

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Design, synthesis and discovery of picomolar selective α4β2 nicotinic acetylcholine receptor ligands

DOI: 10.1021/jm4008455

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Article abstract of DOI:10.1021/jm4008455

Full text of DOI:10.1021/jm4008455

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