Chemistry and pharmacological studies of 3-alkoxy-2,5-disubstituted- pyridinyl compounds as novel selective α4β2 nicotinic acetylcholine receptor ligands that reduce alcohol intake in rats

Article abstract of DOI:10.1021/jm4000374

Neuronal acetylcholine receptors mediate the addictive effects of nicotine and may also be involved in alcohol addiction. Varenicline, an approved smoking cessation medication, showed clear efficacy in reducing alcohol consumption in heavy-drinking smokers. More recently, sazetidine-A, which selectively desensitizes α4β2 nicotinic receptors, was shown to significantly reduce alcohol intake in a rat model. To develop novel therapeutics for treating alcohol use disorder, we designed and synthesized novel sazetidine-A analogues containing a methyl group at the 2-position of the pyridine ring. In vitro pharmacological studies revealed that some of the novel compounds showed overall pharmacological property profiles similar to that of sazetidine-A but exhibited reduced agonist activity across all nicotinic receptor subtypes tested. In rat studies, compound (S)-9 significantly reduced alcohol uptake. More importantly, preliminary results from studies in a ferret model indicate that these novel nAChR ligands have an improved adverse side-effect profile in comparison with that of varenicline.

Full text of DOI:10.1021/jm4000374

Article
pubs.acs.org/jmc
Chemistry and Pharmacological Studies of 3‑Alkoxy-2,5-
Disubstituted-Pyridinyl Compounds as Novel Selective α4β2
Nicotinic Acetylcholine Receptor Ligands That Reduce Alcohol Intake
in Rats
Yong Liu,† Janell Richardson,‡ Thao Tran,‡ Nour Al-Muhtasib,‡ Teresa Xie,‡
Venkata Mahidhar Yenugonda,† Hannah G. Sexton,§ Amir H. Rezvani,§ Edward D. Levin,§
Niaz Sahibzada,‡ Kenneth J. Kellar,‡ Milton L. Brown,† Yingxian Xiao,*,‡ and Mikell Paige*,†
†Center for Drug Discovery, Georgetown University Medical Center, Research Building EP07, 3970 Reservoir Road, NW,
Washington, D.C. 20057, United States
‡Department of Pharmacology and Physiology, Georgetown University School of Medicine, 3970 Reservoir Road, NW, Washington,
D.C. 20057, United States
§Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, North Carolina 27710, United States
ABSTRACT: Neuronal acetylcholine receptors mediate the
addictive effects of nicotine and may also be involved in
alcohol addiction. Varenicline, an approved smoking cessation
medication, showed clear efficacy in reducing alcohol
consumption in heavy-drinking smokers. More recently, sazetidine-A, which selectively desensitizes α4β2 nicotinic receptors,
was shown to significantly reduce alcohol intake in a rat model. To develop novel therapeutics for treating alcohol use disorder,
we designed and synthesized novel sazetidine-A analogues containing a methyl group at the 2-position of the pyridine ring. In
vitro pharmacological studies revealed that some of the novel compounds showed overall pharmacological property profiles
similar to that of sazetidine-A but exhibited reduced agonist activity across all nicotinic receptor subtypes tested. In rat studies,
compound (S)-9 significantly reduced alcohol uptake. More importantly, preliminary results from studies in a ferret model
indicate that these novel nAChR ligands have an improved adverse side-effect profile in comparison with that of varenicline.
predominant nAChR subtype in mammalian CNS, which
contains both α4 and β2 subunits, is essential for nicotine
addiction.11−18 Several lines of evidence show that nAChRs are
involved in mediating alcohol consumption as well. The
classical noncompetitive antagonist of nAChRs, mecamylamine,
significantly reduced alcohol consumption in rats and
mice.6,19−21 Several other nicotinic ligands were reported to
attenuate alcohol seeking and consumption in rats.22,23
Furthermore, genetic studies showed that nicotinic receptor
subunits are directly involved in alcohol consumption in mouse
models.24−26
Varenicline, an nAChR ligand developed by Pfizer, was
approved by the US Food and Drug Administration (FDA) in
2006 as a smoking cessation medication.27−30 Interestingly,
smokers treated with varenicline reported that the medication
also reduced their alcohol consumption.31 In a double-blind,
placebo-controlled clinical trial, varenicline significantly reduced
alcohol self-administration in heavy-drinking smokers.32 Fur-
thermore, several studies using animal models showed that
varenicline effectively reduced alcohol consumption in rats and
mice.24,25,31,33 Although varenicline appears to be safe for most
INTRODUCTION
■
Tobacco use, mainly via cigarette smoking, and alcohol
consumption are among the leading preventable risk factors
for premature mortality in the world. In 2000, tobacco use was
the number one risk factor for mortality in the United States,
which resulted in 435,000 premature deaths. In that same year,
alcohol consumption was the number three risk factor, which
was responsible for 85,000 premature deaths.1 In addition to
being an immense medical and public health problem, tobacco
use and alcohol consumption also impose a huge social and
economic burden to society. Given the grave consequences of
nicotine and alcohol addiction, there is obviously a great need
for significant improvement in existing therapies for treating
these disorders.
Although alcohol and nicotine addictions are usually
categorized as separate disorders, heavy alcohol consumption
and smoking occur very often in the same individual.2,3 It has
been well established that there are potent interactions between
alcohol and nicotinic systems and that alcohol potentiates
nicotine reward and self-administration.4−7
Neuronal nicotinic acetylcholine receptors (nAChRs) are
found throughout the central nervous system (CNS) and
peripheral nervous system (PNS). The receptors mediate the
pharmacological effects of nicotine and play a key role in
regulating nicotine self-administration behavior.8−10 The
Received: January 9, 2013
Published: April 1, 2013
© 2013 American Chemical Society
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Figure 1. Development of nicotinic desensitizers with reduced agonist activity.
people, significant side effects such as nausea have been
reported.28,29,34
group, or the large volume of the group, is responsible for the
significant alteration in the pharmacological properties for this
structural class. To test this possibility, we then synthesized
compound (S)-9 (Figure 1), which contains a 2-methyl
substituent on the pyridine moiety in place of the CF3 group
in compound (S)-22. The 2-methyl group of compound (S)-9
provides an electron-donating substituent at the 2-position in
contrast to the CF3 group of compound (S)-22, as well as a
smaller volume than that of the CF3 group. We were delighted
that in the binding assays compound (S)-9 showed a high
binding affinity (Ki = 12 nM) to α4β2 nAChRs and high
selectivity for the α4β2 nAChR subtype over two other major
subtypes, α3β4 and α7 (Table 1). Furthermore, in comparison
with varenicline and sazetidine-A, compound (S)-9 exhibited
significantly lower agonist activities at both the α4β2 and α3β4
nAChRs (Table 2). This discovery indicated a possibility for
developing a series novel nicotinic ligands that selectively bind
to α4β2 nAChRs with high affinities and potently desensitize
the receptor subtype with very low agonist activities across all
major nAChR subtypes. To further study this new class of
nAChR ligands, we explored the importance of the ring size
and the stereocenter of the cyclic amine. The effect of changing
the terminal group of the alkynyl appendage was also
investigated.
In 2006, we proposed a new approach in the design of novel
nicotinic therapeutics, which is based on the ability of nicotinic
ligands to desensitize nAChRs.35 The actions of nicotine and
other nicotinic agonists to activate and then desensitize
nAChRs have been known for more than 100 years36 and
conceptualized for more than 50 years.37 A fundamental
question about nicotinic cholinergic signaling in the CNS is
how each of these two opposite actions contributes to the
overall pharmacological effects of nicotine. It has been well-
known that nicotine, as well as all other known nicotinic
agonists, is more potent in desensitizing nAChRs than in
activating them.38−45
Sazetidine-A is a highly selective ligand for nAChRs that
contain the β2 subunit, especially the α4β2 nAChR subtype.35
Sazetidine-A was shown to potently and selectively desensitize
α4β2 nAChRs (see Table 2). In studies using animal behavioral
models, sazetidine-A reduced nicotine self-administration,46,47
improved performance in tests of attention,48,49 and showed
antidepressant and/or antianxiety effects.50−52 Interestingly,
sazetidine-A also significantly decreased alcohol intake in rats.47
Herein we report the design, synthesis, and biological
characterization of a new class of sazetidine-A analogues that
have a reduced agonist effect on several nAChR subtypes and a
reduced adverse side-effect profile in ferrets as compared with
varenicline. These novel compounds could lead to more
effective treatments for alcohol addiction.
The new sazetidine-A analogues designed in our study that
contain a 2-methyl group on the pyridine moiety were
synthesized by an optimized three-step procedure as shown
in Scheme 1. Each enantiomer of Boc-protected 2-azetidinyl-
methanol or 2-pyrrolidinylmethanol was reacted with 5-bromo-
2-methylpyridin-3-ol via standard Mitsunobu coupling con-
ditions to afford bromides 3 and 4. The alkynyl appendage was
RESULTS
■
Chemistry. Previous studies have shown that O-substitution
at the 3-position of pyridine with a 2(S)-azetinylmethanol
substituent and the presence of an aliphatic chain at the 5-
position, such as that found in sazetidine-A, are key structural
components for the selective desensitization of α4β2
nAChRs.35 During our studies of sazetidine-A and related
analogues, it became clear that the in vivo stability of these
compounds should be an important consideration for clinical
development. Recently, the Baran and the MacMillan groups
independently reported that the innate susceptibility for
heteroaromatic hydroxylation could be investigated under
oxidative conditions in the presence of a trifluoromethylating
reagent.53,54 We subjected sazetidine-A (Figure 1) to the
conditions reported by Baran and co-workers and observed that
trifluoromethylation occurred at the 2-position of the pyridine
ring, albeit in 10% yield. This result suggested that the 2-
position of the pyridine moiety could be susceptible to P450-
mediated oxidation. We hypothesized that blocking the 2-
position of the pyridine ring of sazetidine-A with a CF3 group
could result in a compound with increased stability. Therefore,
a
Scheme 1. Synthesis of Analogues 9−12
we synthesized compound (S)-22, a sazetidine-A analogue that
incorporates a CF3 group at the 2-position of the pyridine
moiety (Figure 1). However, this analogue exhibited a
tremendous decrease in binding affinity to the target receptors
(Table 1). It is conceivable that the electronegativity of the CF3
a
Reagents and conditions: (a) DEAD, PPh3, THF, 0 °C-rt, 48 h, 68−
77%; (b) 1-hexyne, Pd(PPh3)2Cl2 (cat.), CuI (cat.), PPh3 (cat.), Et3N,
DMSO, sealed tube, 95 °C, 60 h, 89−98%; (c) method A, TFA,
CH2Cl2, 0 °C-rt, 3 h then 10% aqueous NaOH solution−methanol,
75−83%; method B, HCl in methanol, 0 °C-rt, 3 h, 87−92%.
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a
Scheme 2. Synthesis of Analogues 16−17
a
Reagents and conditions: (a) p-TsCl, DMAP, Et3N, DCM, 0 °C-rt, overnight, 80%; (b) Ph3P, CCl4, cat. NaHCO3, sealed tube, 80 °C, 48 h, 69%;
(c) Kryptofix 2.2.2, KF, THF, reflux, overnight, 85%; (d) HCl in methanol, 0 °C-rt, 3 h, 90%.
a
incorporated by Sonogashira homologation in the presence of
triphenylphosphine in triethylamine with DMSO as a
cosolvent. The N-Boc protecting group was then removed by
treatment with trifluoroacetic acid or hydrogen chloride to
afford the analogues 9 through 12 as the corresponding salt.
Analogues 16 and 17, which contain a terminal halogen
substituent, were synthesized as outlined in Scheme 2. The
hydroxide group of compound 12 was tosylated by treatment
with p-toluenesulfonyl chloride in methylene chloride at 0 °C.
The resulting product was then treated with potassium fluoride
in the presence of Kryptofix 2.2.2 in anhydrous THF to afford
fluoroalkane 15.55 Compound 16 was obtained as the
hydrochloride salt after deprotection of the N-Boc group by
treatment with hydrogen chloride in methanol. The chlor-
oalkane analogue 17 was originally afforded by a side reaction
during the tosylation step when compound 12 was treated with
p-toluenesulfonyl chloride in pyridine at room temperature. We
have optimized the synthesis of the chloroalkane analogue by
treating compound 12 with triphenylphosphine and carbon
tetrachloride in a sealed tube at 80 °C for 48 h.56 The
deprotection step of the chlorinated product 14 was
accomplished by treatment with hydrogen chloride in methanol
to afford the hydrochloride salt of compound 17.
The 2-trifluoromethylpyridinyl analogue 22 was synthesized
as shown in Scheme 3. The methyl aryl ether 18 was treated
with hydrogen bromide in acetic acid at elevated temperatures
to afford phenol 19.57 Mitsunobu homologation of phenol 19
with alcohol 1 then gave aryl bromide 20. Homologation of 1-
hexyne to compound 20 was accomplished following the
Sonogashiro protocol as outlined in Scheme 1. However, for
this substrate the reaction was accomplished at lower
temperature and shorter reaction time than for the 2-
methylpyridinyl substituted substrates, presumably because
the electron-withdrawing properties of the trifluoromethyl
substituent assists in the initial oxidative insertion of the
palladium between the carbon and bromine atoms. It is
Scheme 3. Synthesis of Analogue 22
a
Reagents and conditions: (a) HBr, AcOH, 110 °C, sealed tube,
overnight, 79%; (b) DEAD, PPh3, THF, 0 °C-rt, 24 h, 88%; (c) 1-
hexyne, Pd(PPh3)2Cl2 (cat.), CuI (cat.), PPh3 (cat.), Et3N, DMSO, 50
°C, overnight, 91%; (d) HCl in methanol, 0 °C-rt, 3 h, 73%.
important to note that higher temperatures and longer reaction
times resulted in diminished yields. Deprotection of the N-Boc
group was then effected by treatment with hydrogen chloride in
methanol to afford compound 22 as the hydrogen chloride salt.
In Vitro Binding Affinities for nAChR Subtypes. The
binding affinities of the novel 2,5-disubstituted sazetidine-A
analogues for the defined rat nAChR subtypes as well as for
native nAChRs of rat forebrain were examined in binding
competition studies against [3H]-epibatidine. For comparison,
binding affinities of (−)-nicotine, varenicline, and sazetidine-A
were obtained from parallel binding experiments. To determine
the selectivities of these compounds in binding assays among
the three predominant nAChR subtypes, α3β4, α4β2, and α7,
the ratios of the corresponding Ki values (α3β4/α4β2 and α7/
α4β2) were determined.
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Table 1. Comparison of the Binding Affinities of Nicotine, Varenicline, and Sazetidine-A for Rat nAChR Subtypes to Those of
the 3-Alkoxy-2,5-Substituted-Pyridyl Compounds
a
Ki values of the compounds shown are the mean of 3 to 5 independent measurements (for clarity, SEM values were omitted, which were within
20% of the mean in most cases).
As reported previously35 and shown in Table 1, sazetidine-A
bound to α4β2 nAChRs with a very high affinity (Ki = 0.062
nM), which was 31,000 and 11,000 times higher than its
affinities for the α3β4 and α7 subtypes, respectively. The data
suggest that sazetidine-A bound to each β2-containing nAChR
subtype with a much higher affinity than to the corresponding
β4-containing counterparts having the same α subunit.
As shown in Scheme 1, the incorporation of a methyl group
at the 2-position of the pyridine moiety in sazetidine-A afforded
analogue (S)-12. The Ki value of this compound for α4β2
nAChRs is 4.3 nM, representing a lower binding affinity than
that of sazetidine-A (Table 1). However, the binding affinity of
(S)-12 for α4β2 nAChRs is 2-fold higher than that of
(−)-nicotine. More importantly, analogue (S)-12 retained the
excellent selectivity pattern seen with sazetidine-A with a
slightly more favorable selectivity for the α4β2 nAChRs over
the α3β4 and α7 subtypes.
We then synthesized four analogues of compound (S)-12.
Replacing the hydroxyl group at the terminal end of the alkyl
chain with a fluorine atom (analogue (S)-16) or a chlorine
atom (analogue (S)-17) did not lead to significant changes in
the binding property profiles (Table 1). Similarly, removing the
hydroxyl group (analogue (S)-9) or replacing the end of the
side chain with a cyclopropyl ring (analogue (S)-11) resulted in
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little change in the binding profiles for this class of compounds
(Table 1).
compounds for 10 min. For comparison, nicotine, varenicline,
and sazetidine-A were included in these experiments.
All closely related S enantiomers, including (S)-9, (S)-11,
(S)-16, and (S)-17, showed binding property profiles similar to
that of (S)-12. In contrast, compound (R)-9 showed
significantly lower binding affinities and reduced nAChR
subtype selectivities than those of compound (S)-9 (Table
1). Consistent with this finding, all analogues containing an R
stereocenter in this study showed lower binding affinities and
less nAChR subtype selectivities than analogues containing the
S stereochemistry.
It is important to note that compound (S)-10, which has a
five-membered ring pyrrolidine group, showed much lower
affinities and nAChR subtype selectivites than those of
compound (S)-9, which has a four-member ring azetidinyl
group (Table 1). However, compound (S)-10 still had binding
affinities at α4β2 nAChRs in the nanomolar range and
maintained a greater than 1,000-fold selectivity for the α4β2
nAChR subtype over the α3β4 or α7 subtypes. In line with our
emerging SAR, the R enantiomer of compound 10 showed
reduced binding affinity and nAChR subtype selectivities in
comparison to the corresponding S enantiomer.
Compound 22, which contains a trifluoromethyl group at the
2-position of the pyridine ring, showed minimal binding affinity
to every nAChR subtype tested. This may indicate that
reducing the electron density in the pyridine ring and/or
increasing steric bulk at the 2-position of the pyridine moiety
can lead to lower binding affinities.
In Vitro Effects on nAChR Function. Six new compounds
showing high binding affinities for α4β2 nAChRs and high
selectivities for this subtype over α3β4 and α7 receptors in
binding assays were chosen for functional studies (Table 2).
The agonist activities for these analogues were assessed by
measuring stimulated 86Rb+ efflux from stably transfected cells,
either expressing human α4β2 nAChRs or rat α3β4 receptors.
The ability of the analogues to desensitize the two nAChR
subtypes were determined by measuring nicotine-stimulated
86Rb+ efflux after cells were preincubated with the test
As expected, nicotine showed full agonist activities at both
human α4β2 and rat α3β4 nAChRs (Table 2). Consistent with
previous reports,27,30,58 in comparison with (−)-nicotine our
studies show that varenicline had 45% of the efficacy in
stimulating efflux from cells expressing the α4β2 nAChRs and
90% of the efficacy from cells expressing the α3β4 nAChRs.
Sazetidine-A showed lower efficacy at the α4β2 nAChRs than
that of varenicline. In contrast, we found that sazetidine-A’s
agonist activity at the α3β4 nAChRs was lower than that of
(−)-nicotine or varenicline. Consistent with the purposes of
developing this line of novel ligands, all 6 novel compounds
tested showed much lower agonist activities than those of
sazetidine-A at both receptor subtypes. Compounds (S)-9, (S)-
11, and (S)-12 had less than 20% of the agonist efficacy at the
human α4β2 nAChRs in comparison to sazetidine-A. There-
fore, though these three new compounds showed partial
agonist activities at the α4β2 nAChR subtype, their efficacies of
activations were much lower than that of varenicline.
Compounds (S)-10, (S)-16, and (S)-17 showed no detectable
agonist activity at the human α4β2 receptor subtype.
Additionally, all 6 compounds showed no detectable agonist
activity at the rat α3β4 nAChRs.
Consistent with our previous report,35 (−)-nicotine,
varenicline, and sazetidine-A potently and selectively desensi-
tize α4β2 nAChRs with IC50(10′) values in the nanomolar range
(Table 2). To our delight, compounds (S)-9, (S)-11, (S)-12,
(S)-16, and (S)-17 selectively desensitized the α4β2 nAChRs.
The IC50(10′) values for these compounds to desensitize the
α4β2 nAChRs ranged from 51 nM for analogue (S)-12 to 260
nM for analogue (S)-17. In contrast, the IC50(10′) values for
these compounds in desensitizing α3β4 nAChRs were higher
than 10,000 nM.
Analogue (S)-10 has the lowest potency in desensitizing the
α4β2 nAChRs among all of the new compounds studied.
Furthermore, the potency of analogue (S)-10 to desensitize
α4β2 nAChRs is only slightly higher than that to desensitize
α3β4 nAChRs. This observation confirmed the importance of
the four-member ring azetidinyl group in sazetidine-A, (S)-9,
(S)-11, (S)-12, (S)-16, and (S)-17 for selective desensitization
of the α4β2 nAChRs.
Table 2. Comparison of Activation and Desensitization of
nAChR Function by Ligands
a
a
α4β2 nAChRs
α3β4 nAChRs
In Vivo Effects on Alcohol Intake in Rats. In our initial
behavioral studies in an animal model, effects of compound (S)-
9 on alcohol self-administration in selectively bred alcohol-
preferring rats (P rats) were assessed (Figure 2). Three doses of
compound (S)-9 were tested: 0.33, 1, and 3 mg/kg (s.c.).
Compared with the control vehicle, compound (S)-9
significantly reduced alcohol intake in a dose-dependent
manner. At 0.33 mg/kg, compound (S)-9 did not show a
significant effect at any time point. The medium dose of 1 mg/
kg reduced alcohol intake significantly (p < 0.05) over the 24-h
time point after administration. At 3 mg/kg, the compound had
significant effects on alcohol intake at both the 6-h (p < 0.05)
and 24-h time points (p < 0.0005) (Figure 2).
Emetic Effects of Compound (S)-9 in Ferrets. The
effects of compound (S)-9 on causing nausea and emesis were
assessed in a ferret model. Varenicline was used as a control.
Administration of compound (S)-9 (1 to 30 mg/kg; s.c.) did
not elicit any emetic episodes or stereotypical behaviors that are
indicative of nausea (n = 6). In general, all animals remained
alert and active with periods of inactivity that mirrored their
baseline behavior within the 1-h observation period.
b
c
d
EC50
Emax
IC50(10′)
(nM)
EC50
Emax
IC50(10′)
(nM)
compd
(nM)
(%)
(nM)
(%)
(−)-nicotine
varenicline
sazetidine-A
(S)-9
2,400
950
24
100
45
370
94
23,000
21,000
30,000
NA
100
82
>10,000
>10,000
>10,000
>10,000
6,400
40
11
12
310
15
260
1,600
110
51
NA
NA
NA
NA
NA
NA
e
(S)-10
NA
NA
17
NA
(S)-11
160
450
NA
NA
NA
>10,000
>10,000
>10,000
>10,000
(S)-12
20
NA
(S)-16
NA
NA
130
260
NA
(S)-17
NA
a
Functional properties of each compound were determined using
stable cell lines expressing human α4β2 or rat α3β4 nAChRs. EC50
values show potencies of agonist activities. Emax (%) values show
relative efficacies of agonist activities, which were normalized to the
Emax value of nicotine. IC50(10′) values show potencies of desensitizer
activities. NA indicates that no significant stimulated efflux was
detected. All values shown are the means of 3 to 9 independent
experiments (for clarity, SEM values were omitted, which were within
20% of the mean in most cases).
b
c
d
e
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to its ability to cause long lasting desensitization of α4β2
receptors, Zwart and co-workers reported that sazetidine-A also
elicited agonist activity at the α4β2 receptors.60 It is very
interesting that this agonist effect is specific to the
stoichiometry of the subunits. Of the two stoichiometric
forms of the α4β2 receptors, sazetidine-A showed full agonist
activity at the (α4)2(β2)3 receptors but nearly no agonist
activity at the (α4)3(β2)2 receptors.61 Thus, sazetidine-A, like
nicotine itself, has both agonist and desensitizer effects on β2-
containing nAChRs, but in contrast to nicotine, its desensitizing
effects appear to predominate and be long lasting.35,59,62 For
example, initial activation of the α4β2 nAChRs in vitro by
sazetidine-A is brief, lasting only seconds, whereas the receptor
desensitization that immediately follows activation lasts more
than an hour even with continual washing of the cells.
Many studies to further understand the in vitro pharmaco-
logical properties of sazetidine-A and its behavioral effects in
animal models have been reported.47−50,52,63−69 The results
from these studies are highly supportive of our proposal to
develop novel nicotinic therapeutics based on their ability to
selectively desensitize α4β2 nAChRs. In the present study, we
investigated the agonist and desensitization properties of these
new analogues at human α4β2 nAChRs and rat α3β4 nAChRs
(Table 2). For the efficacies of agonist activities at α4β2
nAChRs in comparison to nicotine, compounds (S)-9, (S)-11,
and (S)-12 showed very low relative Emax values at the α4β2
nAChRs as follows: 15%, 17%, and 20%, respectively.
Compounds (S)-10, (S)-16, and (S)-17 did not show any
measurable agonist activities at this subtype, and none of the
compounds tested exhibited agonist activities at rat α3β4
nAChRs. It is important to note that all of the new analogues
tested showed significantly lower agonist activities at both α4β2
and α3β4 receptor subtypes than those of varenicline or
sazetidine-A. With regards to desensitization activities, com-
pounds (S)-9, (S)-11, (S)-12, (S)-16, and (S)-17 potently and
selectively desensitized the α4β2 nAChRs after a 10-min
incubation period (Table 2). These in vitro pharmacological
functional properties indicate the possibility for these
compounds to desensitize α4β2 nAChRs in vivo.
As shown in Figure 2, compound (S)-9 significantly reduced
alcohol intake in a dose-dependent manner in the animal
efficacy model using selectively bred alcohol-preferring rats. On
the basis of the in vitro pharmacological property profile of
compound (S)-9, we speculate that the ability of compound
(S)-9 in reducing alcohol intake resulted mainly from its α4β2
nAChR desensitization properties. In our previous postdepri-
vation studies,47 sazetidine-A at 3 mg/kg significantly reduced
alcohol intake at the 2-, 4-, and 6-h time points but not at the
24-h time point. Interestingly, (S)-9 showed a different pattern
of the inhibition, significantly reducing the alcohol intake at the
6- and 24-h time points but not at the 2- and 4-h points. It is
important to note that though the binding affinity of (S)-9 to
α4β2 nAChRs is lower than that of sazetidine-A (Table 1),
both compounds reduced alcohol intake at 3 mg/kg. It is
conceivable that (S)-9 might have reached a higher brain
concentration than sazetidine-A at the same dose given
systematically.
Figure 2. Effect of acute administration of compound (S)-9 on alcohol
intake in P-rats. Alcohol intakes (g/kg) were measured at 2, 4, 6, and
24 h after an injection of (S)-9. All values are expressed as the mean
SEM (n = 18).
Varenicline (0.5 mg/kg; s.c.; n = 5) was administered to
ferrets to assess its effects on emesis or stereotypical behaviors
associated with nausea. Whereas no emetic episodes were
observed, drug-induced nausea as evidenced by gagging
(retching; mean # of episodes 2.80 + 1.63) were evident
following varenicline administration in the majority of the
animals (n = 4/5) tested. In one animal, this behavior was
accompanied by oral stereotypy (licking), whereas in two
animals it was interspersed by episodes of backward walking.
On the average, these behaviors manifested around ∼2.20 min.
In general, the locomotor behavior of these animals was
noticeably reduced at ∼1.05 min postinjection and remained so
for the duration of the observation period (∼1 h). Occasionally,
when animals tried to ambulate they would immediately cease
their movement after each attempt. This would be followed by
a momentary increase in breathing giving the impression that
the animal was in distress.
DISCUSSION AND CONCLUSIONS
■
We have developed a series of sazetidine-A analogues that
contain a methyl group at the 2-position of the pyridine ring.
Similar to sazetidine-A, compounds (S)-9, (S)-10, (S)-11, (S)-
12, (S)-16, and (S)-17 are highly selective to the α4β2 nAChR
subtype over the α3β4 and α7 subtypes in binding assays. In
vitro pharmacological studies to assess their functional effects at
nicotinic receptors showed that these compounds selectively
desensitize α4β2 nAChRs. Particularly noteworthy, we found
that these compounds exhibit much lower agonist activities at
all nAChR subtypes tested in comparison with that of
sazetidine-A. In rat modeling, compound (S)-9 significantly
reduced alcohol self-administration. It is important to note that
preliminary studies in a ferret model show that compound (S)-
9 has a better adverse effects profile than that of varenicline.
In our previous studies, sazetidine-A exhibited much higher
binding affinity for α4β2 nAChRs than for α3β4 or α7
receptors.35,59 After cells expressing α4β2 nAChRs were
exposed to sazetidine-A for 10 min, sazetidine-A potently
inhibited nicotine-stimulated function mediated by the α4β2
nAChRs. Consistent with its high selectivity for the α4β2
receptor, sazetidine-A at concentrations up to 10 μM had no
effect on the function of α3β4 or α7 nAChRs. These
observations are consistent with our hypothesis that
sazetidine-A selectively desensitizes α4β2 receptors. In addition
Varenicline is marketed in the U.S. and many other countries
as a smoking cessation aid. Clinical trials before and after its
approval as well as postmarketing reports demonstrated its
superior efficacy compared with placebo or other marketed
smoking cessation therapeutics.28,29,70−72 In addition to its use
as a smoking cessation drug, the significant efficacy of
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was removed under reduced pressure. The residue was purified by
column chromatography on silica gel using a gradient of hexane−ethyl
acetate (10:1 to 5:1) as the eluent to give the product in 68−90%
yield.
varenicline in reducing alcohol consumption in heavy-drinking
smokers indicated the possibility for it to be used to treat
alcohol use disorders.32 However, notable adverse events
related to varenicline have been reported,28,29,34 which resulted
in the recent ruling by the FDA that varenicline carry a black
box label for suicidal ideation and suicidal behavior (FDA Drug
Safety Newsletter 2009). The most common adverse events
reported for varenicline are nausea and vomiting. For example,
30−40% of people taking varenicline reported nausea in
randomized clinical trials, which is greater than 3 times that
reported for the placebo group.73 The adverse side effects of
varenicline are thought to be mediated through its strong
agonist activities at the α3β4 and α7 nAChR subtypes.58 As
shown in Table 2, compound (S)-9 did not show any agonist
activity at the rat α3β4 nAChRs. In addition, preliminary
studies using whole cell current measurements showed that
compound (S)-9 did not elicit any detectable current in cells
expressing rat α7 nAChRs (Sahibzada et al., data not shown).
On the basis of a comparison of the in vitro pharmacological
property profile of compound (S)-9 with that of varenicline, it
is conceivable that compound (S)-9 might be much less likely
to induce nausea and vomiting than varenicline. The animal
studies in a ferret nausea and emetic model showed a clear
difference between varenicline and compound (S)-9 in their
potential to induce nausea and vomiting. Varenicline at 0.5 mg/
kg (s.c., n = 5) caused evident signs related to nausea and
vomiting in 4 ferrets. In contrast, compound (S)-9 at doses up
to 30 mg/kg (s.c.) did not elicit any emetic episodes or
stereotypical behaviors that are indicative of nausea (n = 6).
In summary, a series of novel sazetidine-A analogues were
designed and synthesized. The in vitro pharmacological
evaluations revealed that these compounds were selective
α4β2 nAChR desensitizers. Analogue (S)-9, our lead
compound, showed significant efficacy in reducing alcohol
intake in a rat model. More importantly, analogue (S)-9 did not
induce nausea and vomiting up to 30 mg/kg in ferrets,
exhibiting a potentially better adverse side effect profile than
that of varenicline. Therefore, we concluded that this series of
analogues should be further studied in the development of new
treatments for alcohol use disorders.
General Procedure for the Sonogashira Coupling Reaction. A
mixture of the Mitsunobu adduct (1.0 equiv), alkyne (4.0 equiv),
Pd(PPh3)2Cl2 (5 mol %), CuI (10 mol %), and PPh3 (10 mol %) in
Et3N/DMSO (10:1, 0.12 M) was heated to 95 °C under a nitrogen
atmosphere in a sealed tube for 60 h. The cooled reaction mixture was
taken up in ethyl acetate, and the organic phase was washed with
water, brine, and then dried over Na2SO4. The extract was
concentrated under reduced pressure, and the residue was purified
by column chromatography on silica gel using a gradient of CH2Cl2−
ethyl acetate (16:1 to 10:1) as the eluent to give the product in 88−
98% yield.
General Procedure (Method A) for Deprotection of the N-Boc
Group. To a stirred solution of the Sonogashira adduct (1.0 equiv) in
dichloromethane (0.10 M) was added trifluoroacetic acid (32 equiv)
dropwise at 0 °C under a nitrogen atmosphere. The reaction mixture
was stirred for 3 h at room temperature. The solvent and excess TFA
was then removed under reduced pressure. To the residue was added
2−3 mL of methanol followed by dropwise addition of 10% aqueous
NaOH solution at 0 °C until the pH value of the mixture was 9−10.
After the mixture was stirred at room temperature for 30 min, the
solution was taken up in dichloromethane, and the organic phase was
washed with brine and dried over Na2SO4. The extract was
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel using a gradient of CH2Cl2−
methanol (20:1 to 10:1) as the eluent to give the product in 75−83%
yield.
General Procedure (Method B) for Deprotection of the N-Boc
Group. To the Boc-protected compound (1.0 mmol), a solution of 2
M HCl in methanol (10 mL) was added at 0 °C under nitrogen
atmosphere. The reaction mixture was allowed to warm to room
temperature and stirred for 3 h. The reaction mixture was
concentrated under reduced pressure, and the residue was purified
by column chromatography on silica gel using a gradient of CH2Cl2−
methanol (10:1 to 5:1) as the eluent to give the product as a white
solid in 73−92% yield.
(S)-tert-Butyl 2-((5-bromo-2-methylpyridin-3-yloxy)methyl)-
azetidine-1-carboxylate ((S)-3). Yield: 69% (light red solid). 1H
NMR (CDCl3, 400 MHz): δ 8.15 (s, 1H), 7.26 (s, 1H), 4.52 (m, 1H),
4.33 (m, 1H), 4.05 (m, 1H), 3.90 (m, 2H), 2.45 (s, 3H), 2.34 (m, 2H),
1.41 (s, 9H).
(R)-tert-Butyl 2-((5-bromo-2-methylpyridin-3-yloxy)methyl)-
azetidine-1-carboxylate ((R)-3). Yield: 77% (light red solid). 1H
NMR (CDCl3, 400 MHz): δ 8.16 (s, 1H), 7.26 (s, 1H), 4.53 (m, 1H),
4.34 (m, 1H), 4.06 (m, 1H), 3.91 (m, 2H), 2.45 (s, 3H), 2.35 (m, 2H),
1.42 (s, 9H).
EXPERIMENTAL SECTION
■
General Chemistry Methods. All solvents and reagents were
used as obtained from commercial sources unless otherwise indicated.
All reactions were performed under nitrogen atmosphere unless
(S)-tert-Butyl 2-((5-bromo-2-methylpyridin-3-yloxy)methyl) pyr-
1
1
otherwise noted. The H and 13C NMR spectra were recorded on a
rolidine-1-carboxylate ((S)-4). Yield: 68% (light red solid). H NMR
1
Varian 400MR spectrometer operating at 400 MHz for H and 100
(CDCl3, 400 MHz): δ 8.13 (s, 1H), 7.26 (br s, 1H), 4.15 (m, 2H),
4.00 (m, 0.5H), 3.85 (m, 0.5H), 3.41 (m, 2H), 2.41 (s, 3H), 1.96 (m,
4H), 1.48 (s, 9H).
MHz for 13C. Deuterated chloroform or deuterated methanol was used
as the solvent for NMR experiments. 1H Chemical shifts values (δ) are
referenced to the residual nondeuterated components of the NMR
solvents (δ = 7.26 ppm for CHCl3 and δ = 3.31 ppm for CHD2OD).
The 13C chemical shifts (δ) are referenced to CDCl3 (central peak, δ =
77.0 ppm) or CD3OD (δ = 49.0 ppm) as the internal standard. Optical
rotation was detected on an ADP220 Automatic polarimeter from
Bellingham & Stanley Limited. Mass spectra were measured in positive
mode electrospray ionization (ESI). The HRMS data were obtained
on a Waters Q-TOF Premier mass spectrometer. TLC was performed
on silica gel 60 F254 plastic sheets. Column chromatography was
performed using silica gel (35−75 mesh). Combustion analyses were
performed at Atlantic Microlabs, Inc. Norcross, GA.
(R)-tert-Butyl 2-((5-bromo-2-methylpyridin-3-yloxy)methyl) pyr-
1
rolidine-1-carboxylate ((R)-4). Yield: 70% (light red solid). H NMR
(CDCl3, 400 MHz): δ 8.12 (s, 1H), 7.26 (br s, 1H), 4.14 (m, 2H),
3.99 (m, 0.5H), 3.84 (m, 0.5H), 3.41 (m, 2H), 2.40 (s, 3H), 1.95 (m,
4H), 1.47 (s, 9H).
(S)-tert-Butyl 2-((5-(hex-1-ynyl)-2-methylpyridin-3-yloxy)methyl)
azetidine-1-carboxylate ((S)-5). Yield: 95% (light yellow oil). 1H
NMR (CDCl3, 400 MHz): δ 8.12 (d, 1H, J = 1.2 Hz), 7.10 (d, 1H, J =
1.2 Hz), 4.52 (m, 1H), 4.32 (m, 1H), 4.05 (dd, 1H, J = 10, 2.8 Hz),
3.90 (m, 2H), 2.48 (s, 3H), 2.41 (t, 2H, J = 7.2 Hz), 2.33 (m, 2H),
1.59 (m, 2H), 1.48 (m, 2H), 1.41 (s, 9H), 0.95 (t, 3H, J = 7.2 Hz).
(R)-tert-Butyl 2-((5-(hex-1-ynyl)-2-methylpyridin-3-yloxy)methyl)
azetidine-1-carboxylate ((R)-5). Method B was used. Yield: 93%
(light yellow oil). 1H NMR (CDCl3, 400 MHz): δ 8.10 (d, 1H, J = 1.2
Hz), 7.09 (d, 1H, J = 1.2 Hz), 4.50 (m, 1H), 4.30 (m, 1H), 4.03 (dd,
1H, J = 10, 2.8 Hz), 3.89 (m, 2H), 2.46 (s, 3H), 2.39 (t, 2H, J = 7.2
General Procedure for the Mitsunobu Reaction. To a mixture of
the N-Boc protected alcohol (1.0 equiv), the 5-halogen-3-pyridinol
(1.0 equiv), and Ph3P (1.3 equiv) in anhydrous THF (0.10 M) was
added DEAD (1.3 equiv) dropwise at 0 °C under nitrogen
atmosphere. After stirring for 2 days at room temperature, the solvent
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Hz), 2.31 (m, 2H), 1.57 (m, 2H), 1.46 (m, 2H), 1.39 (s, 9H), 0.94 (t,
3H, J = 7.2 Hz).
(S)-3-(Azetidin-2-ylmethoxy)-5-(cyclopropylethynyl)-2-methylpyr-
idine Dihydrochloride ((S)-11). Method B was used. Yield: 91% (off-
1
white solid). H NMR (CD3OD, 400 MHz): δ 8.18 (s, 1H), 7.72 (s,
(S)-tert-Butyl 2-((5-(hex-1-ynyl)-2-methylpyridin-3-yloxy)methyl)
1
pyrrolidine-1-carboxylate ((S)-6). Yield: 98% (light yellow oil). H
1H), 4.93 (m, 1H), 4.47 (m, 2H), 4.11 (m, 2H), 2.71 (m, 2H), 2.60 (s,
3H), 1.54 (m, 1H), 0.95 (m, 2H), 0.81 (m, 2H). 13C NMR (CD3OD,
100 MHz): δ 154.4, 147.3, 140.2, 125.7, 122.8, 100.2, 71.5, 68.9, 60.3,
45.0, 21.9, 17.5, 9.3 (2C), 0.7. HRMS (ESI) m/z calcd for C15H18N2O
(M + H)+ 243.1497; found, 243.1509. [α]D26 = −8.1 (c 0.82, MeOH).
Anal. Calcd for C15H18N2O·2HCl·2.5H2O: C, 50.01; H, 6.99; N, 7.78.
Found: C, 49.64; H, 6.82; N, 8.12.
NMR (CDCl3, 400 MHz): δ 8.10 (s, 1H), 7.11 (br s, 1H), 4.14 (m,
2H), 4.00 (m, 0.5H), 3.83 (m, 0.5H), 3.42 (m, 2H), 2.44 (s, 3H), 2.41
(t, 2H, J = 7.2 Hz), 1.96 (m, 4H), 1.60 (m, 2H), 1.48 (m, 11H), 0.95
(t, 3H, J = 7.2 Hz).
(R)-tert-Butyl 2-((5-(hex-1-ynyl)-2-methylpyridin-3-yloxy)methyl)
pyrrolidine-1-carboxylate ((R)-6). Method B was used. Yield: 92%
1
(light yellow oil). H NMR (CDCl3, 400 MHz): δ 8.09 (s, 1H), 7.10
(S)-6-(5-(Azetidin-2-ylmethoxy)-6-methylpyridin-3-yl)hex-5-yn-1-
ol Dihydrochloride ((S)-12). Method B was used. Yield: 87% (white
(br s, 1H), 4.13 (m, 2H), 3.99 (m, 0.5H), 3.82 (m, 0.5H), 3.41 (m,
2H), 2.43 (s, 3H), 2.40 (t, 2H, J = 7.2 Hz), 1.95 (m, 4H), 1.59 (m,
2H), 1.48 (m, 11H), 0.95 (t, 3H, J = 7.2 Hz).
1
solid). H NMR (CD3OD, 400 MHz): δ 8.37 (s, 1H), 8.12 (s, 1H),
4.97 (m, 1H), 4.59 (m, 2H), 4.13 (m, 2H), 3.61 (t, 2H, J = 5.2 Hz),
2.71 (m, 5H), 2.56 (m, 2H), 1.71 (m, 4H). 13C NMR (CD3OD, 100
MHz): δ 155.6, 146.2, 136.2, 129.4, 124.4, 99.3, 75.5, 69.6, 62.3, 60.0,
44.9, 32.7, 25.8, 21.8, 19.9, 15.7. HRMS (ESI) m/z calcd for
(S)-tert-Butyl 2-((5-(cyclopropylethynyl)-2-methylpyridin-3-
yloxy)methyl)azetidine-1-carboxylate ((S)-7). Yield: 97% (light
1
yellow oil). H NMR (CDCl3, 400 MHz): δ 8.09 (d, 1H, J = 1.2
26
C16H22N2O2 (M + H)+ 275.1760; found, 275.1761. [α]D = −7.1 (c
Hz), 7.08 (d, 1H, J = 1.2 Hz), 4.51 (m, 1H), 4.29 (m, 1H), 4.03 (dd,
1H, J = 10, 2.8 Hz), 3.89 (m, 2H), 2.46 (s, 3H), 2.32 (m, 2H), 1.42
(m, 1H), 1.40 (s, 9H), 0.87 (m, 2H), 0.80 (m, 2H).
0.94, MeOH). Anal. Calcd for C16H22N2O2·2HCl·H2O: C, 52.61; H,
7.17; N, 7.67. Found: C, 52.54; H, 6.92; N, 7.92.
(S)-tert-Butyl 2-((5-(6-hydroxyhex-1-ynyl)-2-methylpyridin-3-
(S)-tert-Butyl 2-(2-methyl-5-(6-(tosyloxy) hex-1-ynyl) pyridine-3-
yloxy) methyl)azetidine-1-carboxylate ((S)-13). To a stirred solution
of 8 (374 mg, 1 mmol) in dry dichloromethane (25 mL) was added
DMAP (12 mg, 0.1 mmol), Et3N (0.34 mL, 2.4 mmol), and tosyl
chloride (228 mg, 1.2 mmol) at 0 °C under a nitrogen atmosphere.
The reaction mixture was stirred overnight at room temperature. The
mixture was taken up in dichloromethane, and the organic phase was
washed with saturated aqueous NaHCO3 solution, brine, and then
dried over Na2SO4. The extract was concentrated under reduced
pressure, and then the residue was purified by column chromatography
on silica gel using a gradient of hexane−ethyl acetate (6:1 to 3:1) as
the eluent to give the product (13) 422 mg as light yellow oil, Yield:
80%. 1HNMR (CDCl3, 400 MHz): δ 8.05 (s, 1H), 7.75 (d, 2H, J = 8.4
Hz), 7.30 (d, 2H, J = 8.0 Hz), 7.07 (s, 1H), 4.49 (m, 1H), 4.29 (m,
1H), 4.06 (t, 2H, J = 6.4 Hz), 4.01 (dd, 1H, J = 10.4, 2.4 Hz), 3.87 (m,
2H), 2.44 (s, 3H), 2.40 (s, 3H), 2.36 (t, 2H, J = 6.8 Hz), 2.28 (m, 2H),
1.80 (m, 2H), 1.61 (m, 2H), 1.37 (s, 9H).
yloxy) methyl) azetidine-1-carboxylate ((S)-8). Yield: 89% (yellow
1
oil). H NMR (CDCl3, 400 MHz): δ 8.04 (br s, 1H), 7.06 (s, 1H),
4.46 (m, 1H), 4.25 (m, 1H), 3.98 (dd, 1H, J = 10, 2.0 Hz), 3.84 (m,
2H), 3.62 (t, 2H, J = 6.0 Hz), 2.91 (br s, 1H), 2.41 (s, 3H), 2.39 (t,
2H, J = 6.0 Hz), 2.26 (m, 2H), 1.65 (m, 4H), 1.34 (s, 9H).
(S)-3-(Azetidin-2-ylmethoxy)-5-(hex-1-ynyl)-2-methylpyridine
((S)-9). Method A was used. Yield: 83% (light red oil). 1H NMR
(CDCl3, 400 MHz): δ 8.08 (d, 1H, J = 1.6 Hz), 7.04 (d, 1H, J = 1.6
Hz), 4.25 (m, 1H), 3.96 (m, 2H), 3.67 (m, 1H), 3.47 (m, 1H), 2.42 (s,
3H), 2.38 (m, 4H), 2.24 (m, 1H), 1.57 (m, 2H), 1.46 (m, 2H), 0.92 (t,
3H, J = 7.2 Hz). 13C NMR (CDCl3, 100 MHz): δ 152.3, 148.0, 143.1,
119.7, 118.8, 92.7, 77.4, 72.4, 57.1, 44.3, 30.6, 23.9, 22.0, 19.2, 19.1,
13.5. HRMS (ESI) m/z calcd for C16H22N2O (M + H)+ 259.1810;
24
found, 259.1813; [α]D = −4.3 (c 0.77, CHCl3). Anal. Calcd for
C16H22N2O·0.625H2O: C, 71.28; H, 8.69; N, 10.39; O, 9.64. Found:
C, 71.25; H, 8.46; N, 10.13; O, 9.89.
(R)-3-(Azetidin-2-ylmethoxy)-5-(hex-1-ynyl)-2-methylpyridine
((R)-9). Method A was used. Yield: 75% (light red oil). 1H NMR
(CDCl3, 400 MHz): δ 8.07 (d, 1H, J = 1.6 Hz), 7.04 (d, 1H, J = 1.6
Hz), 4.24 (m, 1H), 3.94 (m, 2H), 3.66 (m, 1H), 3.45 (m, 1H), 2.41 (s,
3H), 2.36 (m, 3H), 2.24 (m, 2H), 1.56 (m, 2H), 1.45 (m, 2H), 0.92 (t,
3H, J = 7.2 Hz). 13C NMR (CDCl3, 100 MHz): δ 152.3, 148.0, 143.1,
119.7, 118.8, 92.7, 77.4, 72.5, 57.1, 44.3, 30.6, 24.0, 22.0, 19.2, 19.0,
13.5. HRMS (ESI) m/z calcd for C16H22N2O (M + H)+ 259.1810;
(S)-tert-Butyl 2-((5-(6-chlorohex-1-ynyl)-2-methylpyridin-3-
yloxy)methyl)azetidine-1-carboxylate ((S)-14). A mixture of the
compound 8 (374 mg, 1 mmol), PPh3 (288 mg, 1.1 mmol), and
NaHCO3 (20 mg, 0.24 mmol) in dry CCl4 (15 mL) was heated to 80
°C under a nitrogen atmosphere in a sealed tube for 48 h. The cooled
reaction mixture was concentrated under reduced pressure, and the
residue was purified by column chromatography on silica gel using a
gradient of hexane−ethyl acetate (6:1 to 3:1) as the eluent to give the
product 14 (271 mg) as a light yellow oil, Yield: 69%. 1H NMR
(CDCl3, 400 MHz): δ 8.09 (d, 1H, J = 1.2 Hz), 7.08 (d, 1H, J = 1.2
Hz), 4.49 (m, 1H), 4.30 (m, 1H), 4.02 (dd, 1H, J = 10, 2.8 Hz), 3.88
(m, 2H), 3.57 (t, 2H, J = 6.4 Hz), 2.45 (s, 3H), 2.44 (t, 2H, J = 6.8
Hz), 2.31 (m, 2H), 1.92 (m, 2H), 1.74 (m, 2H), 1.38 (s, 9H).
(S)-tert-Butyl 2-((5-(6-fluorohex-1-ynyl)-2-methylpyridin-3-yloxy)-
methyl)azetidine-1-carboxylate ((S)-15). A mixture of 13 (67 mg,
0.13 mmol), Kryptofix 2.2.2 (62 mg, 0.16 mmol), and KF (10 mg, 0.17
mmol) in dry THF (3 mL) was heated to reflux overnight under a
nitrogen atmosphere. The cooled reaction mixture was concentrated
under reduced pressure. The residue was purified by column
chromatography on silica gel using a gradient of hexane-ethyl acetate
(4:1) as the eluent to give the product 40 mg as light yellow oil, Yield:
85%. 1H NMR (CDCl3, 400 MHz): δ 8.11 (s, 1H), 7.10 (s, 1H), 4.56
(t, 1H, J = 6.0 Hz), 4.51 (m, 1H), 4.44 (t, 1H, J = 6.0 Hz), 4.31 (m,
1H), 4.05 (dd, 1H, J = 10, 2.8 Hz), 3.90 (m, 2H), 2.48 (m, 5H), 2.33
(m, 2H), 1.90 (m, 1H), 1.83 (m, 1H), 1.74 (m, 2H), 1.41 (s, 9H).
(S)-3-(Azetidin-2-ylmethoxy)-5-(6-fluorohex-1-ynyl)-2-methylpyr-
idine dihydrochloride ((S)-16). Method B was used. Yield: 92% (white
25
found, 259.1816; [α]D = +10.8 (c 0.62, CHCl3). Anal. Calcd for
C16H22N2O·0.375H2O: C, 72.49; H, 8.65; N, 10.57. Found: C, 72.72;
H, 8.67; N, 10.43.
(S)-5-(Hex-1-ynyl)-2-methyl-3-(pyrrolidin-2-ylmethoxy)pyridine
1
((S)-10). Method A was used. Yield: 80% (light red oil). H NMR
(CDCl3, 400 MHz): δ 8.07 (d, 1H, J = 1.6 Hz), 7.02 (d, 1H, J = 1.6
Hz), 3.84 (m, 2H), 3.52 (m, 1H), 2.98 (m, 2H), 2.42 (s, 3H), 2.38 (t,
2H, J = 7.2 Hz), 2.11 (br s, 1H), 1.93 (m, 1H), 1.78 (m, 2H), 1.56 (m,
3H), 1.45 (m, 2H), 0.92 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3, 100
MHz): δ 152.3, 147.8, 143.0, 119.6, 118.8, 92.6, 77.5, 71.6, 57.0, 46.7,
30.6, 28.0, 25.4, 22.0, 19.3, 19.0, 13.5. HRMS (ESI) m/z calcd for
25
C17H24N2O (M + H)+ 273.1967; found, 273.1964; [α]D = +6.4 (c
1.05, CHCl3). Anal. Calcd for C17H24N2O•0.375H2O: C, 73.15; H,
8.94; N, 10.04. Found: C, 73.29; H, 8.83; N, 9.99.
(R)-5-(Hex-1-ynyl)-2-methyl-3-(pyrrolidin-2-ylmethoxy) Pyridine
1
((R)-10). Method A was used. Yield: 83% (light red oil). H NMR
(CDCl3, 400 MHz): δ 8.06 (d, 1H, J = 1.6 Hz), 7.02 (d, 1H, J = 1.6
Hz), 3.84 (m, 2H), 3.52 (m, 1H), 2.97 (m, 2H), 2.66 (br s, 1H), 2.40
(s, 3H), 2.37 (t, 2H, J = 7.2 Hz), 1.93 (m, 1H), 1.78 (m, 2H), 1.55 (m,
3H), 1.44 (m, 2H), 0.91 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3, 100
MHz): δ 152.2, 147.8, 143.0, 119.6, 118.8, 92.6, 77.4, 71.4, 57.0, 46.6,
30.6, 27.9, 25.3, 21.9, 19.2, 19.0, 13.5. HRMS (ESI) m/z calcd for
1
solid). H NMR (CD3OD, 400 MHz): δ 8.09 (s, 1H), 7.49 (s, 1H),
4.91 (m, 1H), 4.54 (t, 1H, J = 6.0 Hz), 4.42 (t, 1H, J = 6.0 Hz), 4.41
(m, 2H), 4.11 (m, 2H), 2.69 (m, 2H), 2.53 (m, 5H), 1.89 (m, 1H),
1.82 (m, 1H), 1.73 (m, 2H). 13C NMR (CD3OD, 100 MHz): δ 153.6,
148.6, 143.1, 123.0, 121.5, 94.6, 84.5 (d, J = 163.1 Hz), 77.9, 68.4, 60.5,
45.0, 30.8 (d, J = 20.3 Hz), 25.6 (d, J = 4.9 Hz), 22.0, 19.8, 18.7. 19F
24
C17H24N2O (M + H)+ 273.1967; found, 273.1960; [α]D = −4.4 (c
0.85, CHCl3). Anal. Calcd for C17H24N2O·0.75H2O: C, 71.42; H, 8.99;
N, 9.80. Found: C, 71.58; H, 8.80; N, 9.51.
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NMR (CD3OD, 376 MHz): −220.6. HRMS (ESI) m/z calcd for
antibiotics at 37 °C with 5% CO2 in a humidified incubator. Fetal
bovine serum was provided by Gemini Bio-Products (Woodland, CA).
Tissue culture medium and antibiotics were obtained from Invitrogen
Corporation (Carlsbad, CA), unless otherwise stated.
25
C16H21FN2O (M + H)+ 277.1716; found, 277.1714. [α]D = −7.2 (c
1.16, MeOH). Anal. Calcd for C16H21FN2O·2HCl·2.5H2O: C, 48.74;
H, 7.16; N, 7.10. Found: C, 48.69; H, 6.84; N, 7.06.
[3H]-Epibatidine Radioligand Binding Assay. Membrane
preparation procedures and binding assays were described previ-
ously.74,75 Briefly, 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 10,000g 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 36,000g 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 concen-
tration 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 assays were analyzed using Prism 5 (GraphPad
Software, San Diego, CA). The Kd values for the [3H]-epibatidine used
for calculating Ki values of nAChR subtypes were 0.02 nM for α2β2,
0.08 nM for α2β4, 0.03 nM for α3β2, 0.3 nM for α3β4, 0.04 nM for
α4β2, 0.09 nM for α4β4, 1.8 nM for α7, and 0.05 for rat forebrain.
86Rb+ Efflux Assay. Functional properties of compounds at
nAChRs expressed in the transfected cells were measured using 86Rb+
efflux assays as described previously.35,75 In brief, cells expressing
human α4β2 or rat α3β4 nAChRs 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, and 11 mM glucose,
pH 7.4). One milliliter of buffer with or without compounds to be
tested was then 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 the 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 EC50 and Emax values, stimulation
curves were constructed in which 8 different concentrations of a ligand
were included in the assay. For obtaining an IC50(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, Emax, and IC50(10′)
values were determined by nonlinear least-squares regression analyses
(GraphPad, San Diego, CA).
(S)-3-(Azetidin-2-ylmethoxy)-5-(6-chlorohex-1-ynyl)-2-methylpyr-
idine Dihydrochloride ((S)-17). Method B was used. Yield: 90%
(white solid). 1H NMR (CD3OD, 400 MHz): δ 8.39 (s, 1H), 8.17 (s,
1H), 4.98 (m, 1H), 4.62 (m, 2H), 4.13 (m, 2H), 3.64 (t, 2H, J = 6.4
Hz), 2.73 (m, 5H), 2.58 (t, 2H, J = 6.8 Hz), 1.96 (m, 2H), 1.80 (m,
2H). 13C NMR (CD3OD, 100 MHz): δ 154.3, 144.8, 134.4, 128.4,
122.9, 97.5, 74.1, 68.2, 58.6, 43.8, 43.4, 31.4, 25.1, 20.3, 18.0, 14.1.
HRMS (ESI) m/z calcd for C16H21ClN2O (M + H)+ 293.1421; found,
25
293.1430. [α]D = −4.6 (c 0.72, MeOH). Anal. Calcd. for
C16H21ClN2O•2HCl•0.75H2O: C, 50.67; H, 6.51; N, 7.39. Found:
C, 50.31; H, 6.12; N, 7.59.
5-Bromo-2-(trifluoromethyl)pyridin-3-ol (19). A solution of 5-
bromo-3-methoxy-2-(trifluoromethyl)pyridine (18) (0.87 g, 3.4
mmol) in a mixture of acetic acid (6 mL) and 33% HBr in acetic
acid (12 mL) was stirred at 110 °C in a sealed tube overnight. Then,
potassium sodium tartrate tetrahydrate (7.8 g) was added slowly at 0
°C. The mixture was taken up in ethyl acetate after stirring for 10 min,
and the organic phase was washed with brine and dried over sodium
sulfate. The extract was concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel using a
gradient of hexane−ethyl acetate (1:2) as the eluent to give 0.65 g of
the product (19) as a gray solid with a yield of 79%. 1H NMR
(CD3OD, 400 MHz): δ 8.18 (s, 1H), 7.58 (s, 1H). 13C NMR
(CD3OD, 100 MHz): δ 154.6, 141.4, 134.7 (q, J = 34 Hz), 128.8,
125.2, 123.3 (q, J = 272 Hz). 19F NMR (CDCl3, 376 MHz): −68.0.
HRMS (ESI) m/z calcd for C6H3BrF3NO (M + H)+ 241.9428; found,
241.9434.
(S)-tert-Butyl 2-((5-bromo-2-(trifluoromethyl)pyridin-3-yloxy)-
methyl)azetidine-1-carboxylate ((S)-20). The general procedure for
1
the Mitsunobu Reaction was used. Yield: 88% (brown oil). H NMR
(CDCl3, 400 MHz): δ 8.31 (s, 1H), 7.62 (s, 1H), 4.55 (m, 2H), 4.11
(m, 1H), 3.88 (m, 2H), 2.36 (m, 2H), 1.41 (s, 9H).
(S)-tert-Butyl 2-((5-(hex-1-ynyl)-2-(trifluoromethyl)pyridin-3-
yloxy)methyl)azetidine-1-carboxylate ((S)-21). A mixture of com-
pound (20) (290 mg, 0.7 mmol), 1-hexyne (0.3 mL, 2.8 mmol),
Pd(PPh3)2Cl2 (25 mg, 0.036 mmol), CuI (13.5 mg, 0.071 mmol), and
PPh3 (18.6 mg, 0.071 mmol) in Et3N/DMSO (5 mL/0.5 mL) was
heated to 50 °C under a nitrogen atmosphere overnight. The cooled
reaction mixture was taken up in ethyl acetate, and the organic phase
was washed with water, brine, and then dried over Na2SO4. The extract
was concentrated under reduced pressure, and the residue was purified
by column chromatography on silica gel using a gradient of CH2Cl2−
ethyl acetate (50:1 to 20:1) as the eluent to give the product (265 mg)
1
as a yellow oil. Yield: 91%. H NMR (CDCl3, 400 MHz): δ 8.22 (s,
1H), 7.40 (s, 1H), 4.52 (m, 2H), 4.10 (m, 1H), 3.89 (m, 2H), 2.44 (t,
2H, J = 7.2 Hz), 2.36 (m, 2H), 1.59 (m, 2H), 1.48 (m, 2H), 1.41 (s,
9H), 0.96 (t, 3H, J = 7.2 Hz).
(S)-3-(Azetidin-2-ylmethoxy)-5-(hex-1-ynyl)-2-(trifluoromethyl)
Pyridine Hydrochloride ((S)-22). Method B was used. Yield: 73%
(white solid). 1H NMR (CD3OD, 400 MHz): δ 8.25 (s, 1H), 7.74 (s,
1H), 4.81 (m, 1H), 4.46 (m, 2H), 4.02 (m, 2H), 2.72 (m, 1H), 2.60
(m, 1H), 2.51 (t, 2H, J = 7.2 Hz), 1.63 (m, 2H), 1.52 (m, 2H), 0.98 (t,
3H, J = 7.2 Hz). 13C NMR (CD3OD, 100 MHz): δ 153.8, 144.3, 135.5
(q, J = 34 Hz), 127.4, 125.2, 123.1 (q, J = 272 Hz), 98.3, 77.1, 69.9,
59.7, 44.7, 31.6, 23.0, 22.1, 19.8, 13.9. 19F NMR (CD3OD, 376 MHz):
−67.3. HRMS (ESI) m/z calcd for C16H19F3N2O (M + H)+ 313.1528;
General Procedures for Effect on Alcohol Intake in Rats.
Adult male rats were obtained from a colony of selectively bred
alcohol-preferring rats (P rats) maintained at Indiana University
School of Medicine. Rats were housed in cages that were fitted with
two 100 mL Richter tubes for the recording of water and alcohol
intakes. Animals were kept under a constant room temperature of 22
1 °C and a 12:12 light−dark cycle (7:00 a.m.−7:00 p.m. dark).
Animals were fed 5001 Rodent Chow (Lab Diet, Brentwood, MO,
USA). All procedures were approved by the IACUC at Duke
University Medical Center.
25
found, 313.1525. [α]D = −3.9 (c 0.86, MeOH). Anal. Calcd for
C16H19F3N2O·HCl: C, 55.10; H, 5.78; N, 8.03. Found: C, 55.49; H,
5.65; N, 7.87.
Cell Lines and Cell Culture. The cell lines expressing defined rat
nAChR subtypes were established previously by stably transfecting
HEK 293 cells with rat nAChR subunit genes.74,75 The cell line
expressing human α4β2 nAChRs, YXα4β2H1, was established
recently. 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
After a week of handling and habituation, rats were given free access
to water in a graduated Richter tube for 1 day. Next, they were given
free access only to a solution of 10% (v/v) alcohol for 3 consecutive
days. Thereafter, rats were given free access to water and a solution of
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10% alcohol throughout the study. Water and alcohol intake were
indexed by graduated Richter drinking tubes.
varenicline though Global Compound Transfer Research, Pfizer
(Groton, CT). We thank Dr. Lawrence Lumeng of the
University of Indiana for providing P Rats (NIH- R24
AA015512). This research was supported by NIH grants
U19DA027990, R21DA032489, and R03DA025947.
An acute study was conducted to determine a dose−response for
compound (S)-9. After the establishment of a stable baseline for
alcohol and water intake, rats were injected subcutaneously with 0.33,
1, and 3 mg/kg of compound (S)-9 or the same volume of the vehicle
(1 mg/kg). Alcohol and water intakes were measured at, 2, 4, 6, and 24
h after drug administration. The preference for alcohol solution,
[(alcohol volume)/(alcohol + water volume) × 100], was calculated
for the same time points as alcohol and water intake.47 All animals (n =
18) received all treatments following a crossover design with random
assignment. The interval between injections was at least 3 days.
The solution of 10% (v/v) ethyl alcohol was prepared twice weekly
from a solution of 200% ethanol mixed with tap water. Solutions of
compound (S)-9 was prepared weekly in 100 mM HCl solution and
isotonic saline and was injected subcutaneously in a volume of 1 mL/
kg.
ABBREVIATIONS USED
nAChR, nicotinic acetylcholine receptors; CNS, central nervous
system; PNS, peripheral nervous system
■
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AUTHOR INFORMATION
Corresponding Authors
*(M.P.) Phone: +1-202-687-5341. E-mail: map65@
georgetown.edu.
■
*(Y.X.) Phone: +1-202-687-5390. E-mail: yxiao02@
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The authors declare the following competing financial
interest(s): Y.L., K.J.K., A.H.R., E.D.L., M.L.B., Y.X., and M.P.
are co-inventors on the novel compounds reported in this
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ACKNOWLEDGMENTS
■
We thank the Center for Drug Discovery, Georgetown
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partially supported by NIH/NCI grant P30-CA051008.
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