2-Arylazetidines as ligands for nicotinic acetylcholine receptors

Article abstract of DOI:10.1007/s10593-017-2061-5

Alternative and complementary procedures were adopted for preparing 2-arylazetidine derivatives in moderate to good yields. Preliminary biological evaluation of 2-arylazetidines as ligands of nicotinic acetylcholine receptors allowed to identify chloro-substituted analogs as the most interesting congeners. The title compounds may be considered as suitable hit compounds for developing new nicotinic acetylcholine receptor ligands that may be safer than the currently available drugs targeting nicotinic acetylcholine receptors. Our described synthetic approaches enable facile access to a large number of diversely decorated azetidines for studying the structure-activity relationships and for refining the toxico-pharmacological profile of these agents.

Full text of DOI:10.1007/s10593-017-2061-5

DOI 10.1007/s10593-017-2061-5
Chemistry of Heterocyclic Compounds 2017, 53(3), 329–334
2-Arylazetidines as ligands for nicotinic acetylcholine receptors
Leonardo Degennaro1, Marina Zenzola1, Annunziatina Laurino2,
Maria Maddalena Cavalluzzi1, Carlo Franchini1, Solomon Habtemariam3,
Rosanna Matucci2, Renzo Luisi1*, Giovanni Lentini1*
1 Department of Pharmacy – Drug Sciences, University of Studies of Bari Aldo Moro,
via Orabona 4, Bari 70126, Italy; е-mail: giovanni.lentini@uniba.it; renzo.luisi@uniba.it
2 Department of Neuroscience, Area del Farmaco e Salute del Bambino (NEUROFARBA),
Viale Pieraccini, Firenze 650139, Italy; e-mail: rosanna.matucci@unifi.it
3 Pharmacognosy Research Laboratories, Medway School of Science, University of Greenwich,
Chatham-Maritime, Kent ME4 4TB, United Kingdom;
e-mail: s.habtemariam@herbalanalysis.co.uk
Submitted January 16, 2017
Accepted after revision March 10, 2017
Published in Khimiya Geterotsiklicheskikh Soedinenii,
2017, 53(3), 329–334
Alternative and complementary procedures were adopted for preparing 2-arylazetidine derivatives in moderate to good yields.
Preliminary biological evaluation of 2-arylazetidines as ligands of nicotinic acetylcholine receptors allowed to identify chloro-substituted
analogs as the most interesting congeners. The title compounds may be considered as suitable hit compounds for developing new
nicotinic acetylcholine receptor ligands that may be safer than the currently available drugs targeting nicotinic acetylcholine receptors.
Our described synthetic approaches enable facile access to a large number of diversely decorated azetidines for studying the structure–
activity relationships and for refining the toxico-pharmacological profile of these agents.
Keywords: azetidine, ligand efficiency metrics, lipophilicity, neurodegeneration, nicotinic acetylcholine receptors, pain, schizophrenia,
smoking cessation.
Nicotinic acetylcholine receptors (nAChRs) play an
important role in neurodegenerative diseases such as
Parkinson's disease1,2a and Alzheimer's disease,2 the sensa-
tion of pain,2,3 and CNS disorders including anxiety and
depression,2a,4 addiction,2a,5 neurodevelopmental disorders6
such as Tourette's syndrome,5,7 attention deficit and
hyperactivity disorder,2a autism,2b and schizophrenia.2,8 The
alteration of nAChR physiology in neuronal tissues is now
understood to be the pathological hallmark of these
diseases and provides a promising therapeutic target.2,9 As
a result, over the last decade there has been a growing
interest toward the development of full and partial nAChR
agonists. Despite extensive efforts, the currently available
drugs targeting nAChRs have limited efficacy and/or are
associated with severe side effects.5,10 Thus, only nicotine (1)
and varenicline (2) (Fig. 1) have entered the market as
systemic drugs. Generally, nAChR agonists are structurally
related, with an easily protonated secondary or tertiary
nitrogen atom corresponding to the quaternary ammonium
head of ACh (3) and carbachol (4) – classical full but nonselec-
tive agonists of nAChR and muscarinic ACh receptors.2a,9
In the present study, we hypothesized that 2-arylazeti-
dines 5 might satisfy the need for novel nAChR ligands by
being safer than the known agonists 1 and 2. 2-Aryl-
azetidines and the corresponding azetidinones are emerging
as highly appealing structural motifs in medicinal chemistry, as
evidenced by their occurrence in several drug molecules
(a few examples are given at the bottom of Fig. 1).11,12
Given the structural similarity of 2-arylazetidines 5 with
tranylcypromine (6), a well-known irreversible inhibitor of
monoamine oxidase (MAOI)13 and antidepressant, we also
employed an in vitro MAO inhibition assay to verify
possible (undesirable) effects on monoaminergic transmis-
sion. Additionally, we decided to characterize 2-aryl-
0009-3122/17/53(03)-0329©2017 Springer Science+Business Media New York
329

Chemistry of Heterocyclic Compounds 2017, 53(3), 329–334
N
cycloaddition reactions, rearrangements of larger rings,
Me
N+
Me
N
N
N
O
R
X–
reduction of azetidin-2-ones, and aza-Michael addition of
diethyl N-arylphosphoramidates to chalcones.15 2-Aryl-
azetidines 5 were obtained through two alternative routes A
and B (Scheme 1) for the construction of the four-
membered ring. The procedure A started from styrenes,
while the procedure B used suitable 1-aryl-3-chloro-
1-propanols as starting materials.
NH
Me
Me
O
Acetylcholine 3
1
2
Nicotine
Varenicline
R = Me, X = counterion
Carbachol 4,
R = NH2, X = counterion
R2
N
NH2
We followed both routes to obtain compounds 5a–f,h, as
shown in Scheme 2.16 Both protocols were practical and
gave the title compounds in moderate to good yields. Pro-
cedure A allowed the introduction of various N-substi-
tuents, while the procedure B was more appropriate when
N-methylazetidines were the target compounds. Further-
more, route B may be used to obtain optically active
2-arylazetidines.16c Our recently developed regioselective
ortho-C–H functionalization procedure could be used as a
complementary strategy. In this latter case, the ortho-
directing ability of an already installed azetidine ring was
exploited.16,17 The o-tolyl-substituted analog 5g was obtained
using such approach. The structures of the obtained
compounds were confirmed by spectrometric analyses.
When tested as nAChR ligands, all of the obtained
compounds 5a–j were less potent than the reference
compounds 1 and 4 (Table 1). The N-Boc-azetidine 5i and
azetidin-2-one 5j showed no affinity at all (half-maximal
inhibitory concentration, IC50 >100 μM), thus confirming
that a nitrogen atom capable of being protonated may be
necessary for nAChR affinity. The different activity of the
p-methyl-substituted derivative 5b compared to ortho-
substituted azetidines 5g,h may suggest that steric
hindrance hampers ligand binding either directly or by con-
straining the orientation of the aromatic ring in an unfavor-
able position. The most active compound was the p-chloro
congener 5d followed by its 3,4-dichloro-substituted
analog 5e and 5a. However, when considering ligand
efficiency metrics18 such as group efficiency (GE = ΔΔG/
Δnumber of non-hydrogen atoms = Δ1.37pIC50/ΔHA)18a
and lipophilic ligand efficiency (LLE = pIC50 − cLog P),18
the unsubstituted 2-phenylazetidine 5a and its p-chloro-
substituted analog 5d were the most interesting compounds
of the series (Table 1). As representatives of the series, ana-
logs 5a,c,h were also evaluated as MAOIs and AChEIs show-
ing no activity at the highest tested concentrations (1 mM).
R1
6
5
( )-Tranylcypromine
OH
2-Arylazetidines
N
O
N
OH
F
NH
N
R2
R1
N
F
O
R1 = Me, NH2; R2 = F, Cl
CDK8 inhibitors
Ezetimibe,
hypocholesterolemic agent
anticancer agents
Figure 1. Structures of known nicotinic nAChR ligands 1–4,
and biologically relevant, structurally related compounds.
azetidines 5 as potential acetylcholinesterase inhibitors
(AChEIs), since nAChRs and AChE share some common
structural features.14 Even though inhibition of AChE is a
therapeutic approach for some of the above-mentioned
neurological disorders, primarily Alzheimer's disease, such
an effect would augment systemic cholinergic transmission
and hence complicate the toxicological profile of nAChR
agonists. In this paper, we describe the preparation of 2-aryl-
1-methylazetidines 5a–g and their analogs 5i,j and report
their preliminary biological evaluation as nAChR ligands,
MAOIs, and AChEIs.
2-Arylazetidines are generally obtained in cyclization
reactions initiated by nucleophilic substitution of amine
nucleophiles, cyclizations involving C–C bond formation,
Scheme 1
Scheme 2
330

Chemistry of Heterocyclic Compounds 2017, 53(3), 329–334
Table 1. Binding affinity values of 2-arylazetidines 5a–i
visualization under 254 nm UV light or by spraying with a
solution of 5% (w/v) ammonium molybdate and 0.2%
(w/v) cerium(III) sulfate in 100 ml of 17.6% (w/v) aqueous
sulfuric acid and heating to 200°C until the emergence
of blue spots. Merck silica gel 60 with 0.04–0.063 mm
particle size was used for preparative flash chromatography.
Alcohols 9a–h were obtained by adapting our previously
reported continuous flow process.16c 1-Methyl-2-arylazeti-
dines 5a–j were prepared as shown in Scheme 2, following
a previously reported procedure.16a–c
and azetidin-2-one 5j for nAChRs according to [3H]cytisine
specific displacement assay and respective ligand efficiency
metrics
Compound
pIC50 SEM*
GE**
LLE***
5.39 0.16
5.05 0.14
5.08 0.09
5.74 0.06
5.50 0.13
< 4
–
3.9
3.1
5a
–0.47
–0.42
0.48
5b
3.5
5c
3.6
5d
3-Chloro-1-phenylpropan-1-ol (9a). Yield 94%,
colorless oil. IR spectrum (thin film), ν, cm–1: 3060, 2878,
1620, 1612, 1596, 1452, 1215, 1190, 1042, 1020, 988.
1H NMR spectrum (600 MHz, CDCl3), δ, ppm (J, Hz): 1.96
(1H, br. s, exchange with D2O); 2.07–2.13 (1H, m); 2.22–
2.28 (1H, m); 3.54–3.59 (1H, m); 3.72–3.77 (1H, m); 4.95
(1H, dd, J = 8.5, J = 4.6); 7.28–7.38 (5H, m). 13C NMR
spectrum (125 MHz, CDCl3), δ, ppm: 41.7; 41.9; 71.6;
126.0; 128.1; 128.9; 143.9.
0.08
2.9
5e
< –0.95
–1.36
< –0.48
< –0.32
< –1.90
–
< 2.6
2.4
5f
4.40 0.14
< 4
5g
< 1.3
< 1.4
< 3.2
7.3
5h
5i
< 4
< 4
5j
Nicotine (1)
Carbachol (4)
7.99 0.14
6.32 0.05
–
10.2
3-Chloro-1-(p-tolyl)propan-1-ol (9b). Yield 66%,
white solid, mp 42–44°C. IR spectrum (KBr), ν, cm–1:
* Negative decimal logarithm of half-maximal inhibitory concentration (M).
** Group efficiency: GE = ΔΔG/Δnumber of non-hydrogen atoms =
= Δ1.37pIC50/ΔHA, with respect to compound 5a.18a
1
3306, 2917, 1416, 1286, 1049, 817. H NMR spectrum
(600 MHz, CDCl3), δ, ppm (J, Hz): 1.86 (1H, br. s,
exchange with D2O); 2.05–2.12 (1H, m); 2.21–2.28 (1H,
m); 2.36 (3H, s); 3.54–3.59 (1H, m); 3.71–3.76 (1H, m);
4.92 (1H, dd, J = 8.3, J = 4.6); 7.19 (2H, d, J = 7.8); 7.27
(2H, d, J = 7.8). 13C NMR spectrum (125 MHz, CDCl3),
δ, ppm: 21.3; 41.5; 41.9; 71.3; 125.9; 129.5; 137.8; 140.9.
3-Chloro-1-(4-fluorophenyl)propan-1-ol (9c). Yield
75%, colorless oil. IR spectrum (thin film), ν, cm–1: 3381,
2032, 1891, 1715, 1605, 1511, 1224, 1054, 836, 661.
1H NMR spectrum (600 MHz, CDCl3), δ, ppm (J, Hz):1.82
(1H, br. s, exchange with D2O); 2.02–2.09 (1H, m); 2.18–
2.25 (1H, m); 3.52–3.57 (1H, m); 3.71–3.76 (1H, m); 4.94
(1H, dd, J = 8.6, J = 4.5); 7.05 (2H, t, J = 8.6); 7.34 (2H,
dd, J = 8.6, J = 5.3). 13C NMR spectrum (125 MHz,
CDCl3), δ, ppm (J, Hz): 29.7; 41.5; 41.6; 70.7; 115.5 (d,
J = 21.3,); 127.5; 139.5; 162.0 (d, J = 247.9).
*** Lipophilic ligand efficiency (LLE = pIC50 − cLog P).18
Early, it has been demonstrated that high potency is not
necessarily required for therapeutic cholinergic activity.19
Thus, the unsubstituted 2-phenylazetidine 5a and its p-chloro-
substituted analog 5d may be considered as good starting
points to developing nAChR ligands, which may be safer
than the currently available cholinergic agents.
In conclusion, two facile alternative methods were
developed to enable the preparation of a series of azetidine
congeners that are potential nAChR agonists. Considering
the wide commercial availability of suitable starting
materials and complementary versatility of the two pro-
posed synthetic procedures, a wide and diverse set of 2-aryl-
azetidines may be designed and prepared to allow explo-
ration of structure–activity relationships and to perform
fine tuning of toxico-pharmacological profile.
3-Chloro-1-(4-chlorophenyl)propan-1-ol (9d). Yield
80%, colorless oil. IR spectrum (thin film), ν, cm–1: 3380,
2963, 1904, 1709, 1597, 1491, 1287, 1198, 1063, 927, 827,
735. 1H NMR spectrum (600 MHz, CDCl3), δ, ppm (J, Hz):
1.87 (1H, br. s, exchange with D2O); 2.03–2.09 (1H, m);
2.17–2.24 (1H, m); 3.53–3.58 (1H, m); 3.72–3.77 (1H, m);
4.95 (1H, dd, J = 8.6, J = 4.6); 7.32 (2H, d, J = 8.6); 7.34
(2H, d, J = 8.6). 13C NMR spectrum (125 MHz, CDCl3),
δ, ppm: 41.6; 41.7; 70.8; 127.3; 128.9; 133.7; 142.3.
Experimental
IR spectra were recorded with a Perkin Elmer 283
spectrometer either for neat compounds as thin film on
1
NaCl plate or for KBr pellets. H and 13C NMR spectra
were recorded on Bruker Avance II (600 and 125 MHz,
respectively), Varian Inova (400 and 100 MHz, respecti-
vely) spectrometers, all chemical shift values are reported
in ppm relative to TMS. High-resolution mass spectro-
metry analyses were performed using a Bruker microTOF
QII mass spectrometer equipped with an electrospray ion
source (ESI-TOF) operated in positive ion mode. Mass
spectra (ESI) were recorded on a LC/MSD trap system VL.
GC-MS spectrometry analyses were carried out on a gas
chromatograph (dimethylsilicon capillary column, 30 m,
0.25 mm i.d.) equipped with a mass selective detector
operating at 70 eV (EI). Melting points were uncorrected.
Analytical thin-layer chromatography (TLC) was carried out
on precoated 0.25 mm thick plates of Kieselgel 60 F254,
3-Chloro-1-(3,4-dichlorophenyl)propan-1-ol
(9e).
Yield 66%, white solid, mp 44–47°C. IR spectrum (KBr),
ν, cm–1: 3391, 2917, 1564, 1468, 1396, 1285, 1201, 1131,
1030, 885, 824, 666. 1H NMR spectrum (600 MHz,
CDCl3), δ, ppm (J, Hz): 1.59 (1H, br. s, exchange with
D2O); 2.02–2.09 (1H, m); 2.14–2.21 (1H, m); 3.54–3.59
(1H, m); 3.73–3.78 (1H, m); 4.96 (1H, dd, J = 8.6, J = 4.2);
7.21 (1H, dd, J = 8.2, J = 2.0); 7.44 (1H, d, J = 8.2); 7.49
(1H, d, J = 2.0). 13C NMR spectrum (125 MHz, CDCl3),
δ, ppm: 41.3; 70.1; 125.1; 127.8; 130.6; 131.7; 132.8; 144.0.
3-Chloro-1-(4-methoxyphenyl)propan-1-ol (9f). Yield
75%, colorless oil. IR spectrum (KBr), ν, cm–1: 3293, 2960,
331

Chemistry of Heterocyclic Compounds 2017, 53(3), 329–334
2904, 1612, 1516, 1288, 1253, 1179, 1032, 834, 652.
aqueous NaOH solution (10 ml) was added carefully. The
aqueous phase was extracted with Et2O (3×10 ml), dried over
anhydrous Na2SO4, and evaporated under reduced pressure.
Preparation of 2-aryl-N-methylazetidines 5a–f,h
(Method B). The synthesis of 1-methyl-2-(p-tolyl)azetidine
(5b) is described as an example. A solution of SOCl2 (3.54 g,
30 mmol, 3 equiv) in CH2Cl2 (3 ml) was added dropwise to
a solution of 3-chloro-1-(p-tolyl)propan-1-ol (9b) (1.84 g,
10 mmol, 1 equiv) in CH2Cl2 (10 ml) at 25°C. After stirring
for 2 h at 25°C, the reaction mixture was poured into water
(5 ml) and 15% aqueous NaOH solution (10 ml) was added
slowly. The aqueous phase was extracted with CH2Cl2
(3×15 ml), and the combined organic layers were dried
over anhydrous Na2SO4, filtered, and evaporated under
reduced pressure to give 1-(1,3-dichloropropyl)-4-methyl-
benzene that was employed without further purification.
A solution of MeNH2 (33% solution in EtOH, 0.312 g,
12.5 ml) was added to a solution of 1-(1,3-dichloropropyl)-
4-methylbenzene (2.0 g, 10 mmol, 1 equiv) in EtOH
(12.5 ml) and Et3N (2.02 g, 20 mmol, 2 equiv) at 25°C. The
reaction mixture was heated for 24 h at 70°C and then
allowed to cool to room temperature. The solvent was
removed in vacuo and aqueous 15% solution of NaOH
(20 ml) was added. The aqueous phase was extracted with
CH2Cl2 (3×20 ml), dried over anhydrous Na2SO4, filtered,
and evaporated under reduced pressure.
1-Methyl-2-phenylazetidine (5a). Yield 0.362 g (88%,
method A), 0.808 g (55%, method B), colorless oil.
IR spectrum (thin film), ν, cm–1: 2959, 2934, 2826, 1452,
1190, 964, 745, 699. 1H NMR spectrum (400 MHz,
CDCl3), δ, ppm (J, Hz): 2.12 (1H, quint, J = 9.2); 2.26 (1H,
dtd, J = 9.4, J = 7.3, J = 1.7); 2.32 (3H, s); 2.84 (1H, dt,
J = 9.7, J = 7.0); 3.41–3.46 (1H, m); 3.85 (1H, t, J = 8.2);
7.20–7.26 (1H, m); 7.32 (2H, t, J = 7.6); 7.36 (2H, d, J = 7.1).
13C NMR spectrum (150 MHz, toluene-d8), δ, ppm: 28.0;
44.2; 53.0; 71.4; 126.9; 127.3; 128.5; 144.0. Mass
spectrum, m/z (Irel,%): 147 [M]+ (12), 146 [M–H]+ (37),
119 (22), 118 (100), 104 (46). Found, m/z: 170.0938
[M+Na]+. C10H13NNa. Calculated, m/z: 170.0940.
1H NMR spectrum (600 MHz, CDCl3), δ, ppm (J, Hz): 1.90
(1H, br. s, exchange with D2O); 2.04–2.10 (1H, m); 2.22–
2.27 (1H, m); 3.52–3.56 (1H, m); 3.69–3.75 (1H, m); 3.81
(3H, s); 4.89 (1H, dd, J = 8.2, J = 4.9); 6.90 (2H, d, J = 8.5);
7.29 (2H, d, J = 8.5). 13C NMR spectrum (125 MHz,
CDCl3), δ, ppm: 41.3; 41.7; 55.3; 70.9; 114.1; 127.0;
135.7; 159.3.
3-Chloro-1-(naphtalen-1-yl)propan-1-ol (9h).21 Yield
39%, colorless oil. IR spectrum (thin film), ν, cm–1: 3382,
1
3051, 2962, 1597, 1511, 1281, 1070, 801, 778. H NMR
spectrum (600 MHz, CDCl3), δ, ppm (J, Hz): 2.06 (1H, br.
s, exchange with D2O); 2.18–2.37 (2H, m); 3.62–3.70 (1H,
m); 3.85–3.93 (1H, m); 5.85 (1H, dd, J = 9.0, J = 3.8);
7.42–7.54 (3H, m); 7.65 (1H, d, J = 7.1); 7.77 (1H, d,
J = 7.7); 7.87 (1H, d, J = 7.4); 8.10 (1H, d, J = 7.1).
13C NMR spectrum (125 MHz, CDCl3), δ, ppm: 40.9; 42.5;
68.1; 122.9; 123.1; 125.7; 125.9; 126.5; 128.5; 129.2;
130.2; 134.0; 139.7.
Preparation of 1-methyl-2-phenylazetidine (5a)
(Method A). N-Chlorosulfonyl isocyanate (0.846 g, 6 mmol,
1.2 equiv) was added dropwise over 10 min to a solution of
styrene (7) (0.520 g, 5 mmol, 1.0 equiv) in anhydrous
diethyl ether (5 ml) at room temperature under an inert
atmosphere. The mixture was stirred at room temperature
for 2 h, and the solvent was removed under reduced
pressure. The residue was taken up in diethyl ether (10 ml)
and added dropwise over 10 min to a vigorously stirred
solution of sodium carbonate (1.70 g, 16 mmol, 3.3 equiv)
and sodium sulfite (0.882 g, 7 mmol, 1.4 equiv) in water
(10 ml) containing ice (10 g). The solution was stirred for 1 h
and filtered. The organic layer was separated, while the
aqueous layer was extracted with diethyl ether (3×10 ml).
The combined organic extracts were dried over anhydrous
Na2SO4, filtered, and the solvent was evaporated under
reduced pressure to yield 4-phenylazetidin-2-one (8) (0.662 g,
4.5 mmol) in a very good yield (90%). The obtained
product was dissolved in anhydrous THF (20 ml), then
tetrabutylammonium bromide (0.128 g, 0.4 mmol, 0.1 equiv),
KOH (0.246 g, 4.4 mmol, 1.0 equiv), and MeI (0.852 g,
6.0 mmol, 1.4 equiv) were added and the solution was
stirred for 8 h at 25°C. The reaction mixture was filtered,
the filtrate was poured into water and extracted with Et2O.
The combined organic layers were dried over anhydrous
Na2SO4 and evaporated under reduced pressure to yield
0.507 g (70%) of 1-methyl-4-phenylazetidin-2-one (5j) as
white solid. 1H NMR spectrum (400 MHz, CDCl3), δ, ppm
(J, Hz): 2.73 (3H, t, J = 0.9); 2.79 (1H, ddd, J = 14.0, J = 2.3,
J = 0.9); 3.35 (1H, ddd, J = 14.0, J = 5.1, J = 0.9); 4.46
(1H, dd, J = 5.1, J = 2.3); 7.25–7.41 (5H, m). Intermediate
5j was employed without further purification in the
reduction reaction. LiAlH4 (0.319 g, 8.4 mmol, 3 equiv) was
carefully added to a solution of AlCl3 (1.11 g, 8.4 mmol,
3 equiv) in dry diethyl ether (15 ml) at 0°C. The reaction mix-
ture was stirred for 10 min at 0°C, then refluxed for 30 min.
1-Methyl-4-phenylazetidin-2-one (5j) (0.451 g, 2.8 mmol,
1 equiv) in dry diethyl ether (10 ml) was added slowly and,
after the addition was complete, reflux was maintained for
4 h. The reaction was cooled to room temperature and 5%
1-Methyl-2-(p-tolyl)azetidine (5b). Yield 1.29 g (80%,
method B), colorless oil, Rf 0.6 (Et2O). IR spectrum (KBr),
ν, cm–1: 2957, 2930, 2824, 2796, 1487, 1476, 1450, 1289,
1192, 968, 776, 699. 1H NMR spectrum (400 MHz,
CDCl3), δ, ppm (J, Hz): 2.04–2.16 (1H, m); 2.17–2.26 (1H,
m); 2.29 (3H, s); 2.31 (3H, s); 2.80 (1H, dt, J = 9.7,
J = 7.1); 3.38–3.46 (1H, m); 3.79 (1H, d, J = 8.2); 7.12
(2H, d, J = 7.8); 7.25 (2H, d, J = 7.8). 13C NMR spectrum
(150 MHz, CDCl3), δ, ppm: 21.1; 27.1; 44.4; 52.9; 71.1;
126.6; 129.0; 136.8; 140.0. Found, m/z: 184.1095 [M+Na]+.
C11H15NNa. Calculated, m/z: 184.1097.
2-(4-Fluorophenyl)-1-methylazetidine (5c). Yield
0.990 g (60%, method B), colorless oil, Rf 0.7 (Et2O).
IR spectrum (thin film), ν, cm–1: 2988, 2960, 2933, 2827,
1
2772, 1509, 1225, 1191, 837. H NMR spectrum (600 MHz,
CDCl3), δ, ppm (J, Hz): 2.08 (1H, quint, J = 8.9); 2.23–
2.27 (1H, m); 2.30 (3H, s); 2.84 (1H, dt, J = 9.6, J = 6.9);
3.43 (1H, t, J = 7.0); 3.82 (1H, t, J = 8.2); 7.00 (2H, t, J = 8.7);
7.34 (2H, dd, J = 8.3, J = 5.6). 13C NMR spectrum (150 MHz,
CDCl3), δ, ppm (J, Hz): 27.3; 44.4; 52.9; 70.6; 115.2 (d,
332

Chemistry of Heterocyclic Compounds 2017, 53(3), 329–334
J
CF = 21.6); 128.3 (d, JCF = 7.8); 138.9 (d, JCF = 2.4); 162.2
column chromatography on silica gel (CH2Cl2–MeOH,
90:10) gave yellow oil. Yield 0.040 g (50%). IR spectrum
(thin film), ν, cm–1: 2958, 2930, 2827, 2771, 1458, 1446,
(d, JCF = 245.0). Found, m/z: 188.0848 [M+Na]+.
C10H12FNNa. Calculated, m/z: 188.0846.
1
2-(4-Chlorophenyl)-1-methylazetidine (5d). Yield
1.36 g (75%, method B), yellow oil, Rf 0.65 (Et2O).
IR spectrum (thin film), ν, cm–1: 2937, 2829, 1490, 1445,
1351, 1192, 967, 748. H NMR spectrum (400 MHz,
CDCl3), δ, ppm (J, Hz): 1.94 (1H, quint, J = 9.2); 2.21 (3H,
s); 2.32–2.43 (4H, m); 2.89–2.95 (1H, m); 3.32–3.56 (1H,
m); 4.06 (1H, t, J = 7.6); 7.08–7.15 (2H, m); 7.23 (1H, t,
J = 7.8); 7.59 (1H, d, J = 7.6). 13C NMR spectrum (100 MHz,
CDCl3), δ, ppm: 18.8; 26.7; 44.8; 53.2; 68.5; 125.4; 126.2;
126.6; 129.9; 135.4; 141.4. Mass spectrum, m/z (Irel, %):
161 [M]+, 132 (51), 118 (100), 117 (80), 115 (22), 91 (20),
44 (36). Found, m/z: 184.1087 [M+Na]+. C11H15NNa.
Calculated, m/z: 184.1097.
1
1190, 1087, 1014, 966, 832, 797, 774. H NMR spectrum
(600 MHz, CDCl3), δ, ppm (J, Hz): 2.07 (1H, quint, J = 8.8);
2.24–2.28 (1H, m); 2.31 (3H, s); 2.85 (1H, dt, J = 9.7, J = 6.7);
3.43 (1H, t, J = 7.3); 3.83 (1H, t, J = 8.2); 7.28–7.32 (4H,
m). 13C NMR spectrum (150 MHz, CDCl3), δ, ppm: 27.2;
44.5; 52.9; 70.5; 128.0; 128.6; 132.9; 141.7. Found, m/z:
182.0728 [M+H]+. C10H13ClN. Calculated, m/z: 182.0731.
2-(3,4-Dichlorophenyl)-1-methylazetidine (5e). Yield
1.87 g (87%, method B), colorless oil, Rf 0.7 (Et2O).
IR spectrum (thin film), ν, cm–1: 2937, 2829, 1491, 1444,
1190, 1087, 1014, 966, 832. 1H NMR spectrum (600 MHz,
CDCl3), δ, ppm (J, Hz): 1.95–2.10 (1H, m); 2.28–2.30 (1H,
m); 2.31 (3H, s); 2.85–2.90 (1H, m); 3.43 (1H, t, J = 7.2);
3.83 (1H, t, J = 8.1); 7.20 (1H, dd, J = 8.2, J = 2.0); 7.38
(1H, d, J = 8.2); 7.50 (1H, d, J = 2.0). 13C NMR spectrum
(150 MHz, CDCl3), δ, ppm: 27.1; 44.3; 52.7; 69.7; 125.8;
128.4; 128.6; 129.9; 130.2; 143.6. Found, m/z: 216.0338
[M+H]+. C10H12Cl2N. Calculated, m/z: 216.0341.
Preparation of N-(tert-butoxycarbonyl)-2-phenyl-
azetidine (5i). Lithium aluminum hydride (0.259 g,
6.8 mmol) was added portionwise to 4-phenyl-2-azeti-
dinone (8) (0.50 g, 3.4 mmol) in anhydrous Et2O (5 ml)
under nitrogen atmosphere at 0°C. After stirring at room
temperature for 20 min the mixture was refluxed for 4 h.
The reaction mixture was then cooled to room temperature,
20% aqueous sodium hydroxide (10 ml) was added and the
mixture was filtered. The filtrate was extracted with
dichloromethane (3×10 ml) and the combined organic
layers were dried over anhydrous Na2SO4. After filtration
and evaporation of solvent, the intermediate product was
used in the next step without further purification. Di-tert-
butyl dicarbonate (0.763 g, 3.5 mmol) was added to a
mixture of the intermediate product (0.452 g, 3.4 mmol)
and Et3N (1.03 g, 1.34 ml, 10.2 mmol) in CH2Cl2 (40 ml)
and the mixture was stirred for 16 h at room temperature.
Then water was added and, after extraction with Et2O,
drying over anhydrous Na2SO4, evaporation of the solvent,
and purification by silica gel column chromatography
(hexane–EtOAc, 9:1) N-(tert-butoxycarbonyl)-2-phenyl-
azetidine (5i) was isolated. Yield 657 mg (83%), colorless
oil. IR spectrum (thin film), ν, cm–1: 2974, 1701, 1389,
2-(4-Methoxyphenyl)-1-methylazetidine (5f). Yield
0.974 g (55%, method B), yellow oil. IR spectrum (thin
film), ν, cm–1: 2956, 2830, 1611, 1512, 1247, 1171, 1037,
833. 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz):
1.97–2.06 (1H, m); 2.09–2.16 (1H, m); 2.20 (3H, s); 2.71
(1H, td, J = 6.8, J = 9.6); 3.30–3.34 (1H, m); 3.64–3.71
(1H, m); 3.69 (3H, s); 6.75–6.79 (2H, m); 7.19–7.23 (2H, m).
13C NMR spectrum (100 MHz, CDCl3), δ, ppm: 27.2; 44.4;
52.9; 55.4; 71.0; 113.8; 128.0; 135.2; 159.1. Found, m/z:
178.1224 [M+H]+. C11H16NO. Calculated, m/z: 178.1226.
1-Methyl-2-(naphthalen-1-yl)azetidine (5h). Yield
0.142 g (72%, method B), yellow oil. IR spectrum (thin
film), ν, cm–1: 2930, 2827, 2770, 1509, 1443, 1328, 1190,
1
1364, 1132, 698. H NMR spectrum (600 MHz, CDCl3),
1
1141, 978, 955, 801, 776. H NMR spectrum (400 MHz,
δ, ppm (J, Hz): 1.32 (9H, br. s); 2.11–2.16 (1H, m); 2.59–
2.65 (1H, m); 3.99 (2H, t, J = 7.6); 5.18 (1H, t, J = 7.0);
7.24–7.35 (5H, m). 13C NMR spectrum (150 MHz, CD3OD,
mixture of rotamers), δ, ppm: 26.3; 26.7; 28.3; 28.5; 47.2;
48.4; 64.9; 66.0; 80.8; 81.0; 126.7; 127.2; 128.5; 128.6;
129.5; 129.6; 143.7; 143.8; 158.1; 158.2. Found, m/z: 256.1318
[M+H]+. C14H19NNaO2. Calculated, m/z: 256.1308.
CDCl3), δ, ppm (J, Hz): 2.09 (1H, quint, J = 9.3); 2.45 (3H,
s); 2.51–2.55 (1H, m); 2.59 (H, dt, J = 7.9, J = 5.9); 3.05
(1H, dt, J = 9.5, J = 7.0); 4.58 (1H, t, J = 8.1); 7.43–7.50
(3H, m); 7.72 (1H, d, J = 8.2); 7.75 (1H, dd, J = 7.1, J = 1.1);
7.82–7.88 (2H, m). 13C NMR spectrum (100 MHz, CDCl3),
δ, ppm: 28.0; 45.0; 53.5; 68.4; 122.6; 123.1; 125.5; 125.8;
126.0; 127.0; 128.9; 130.4; 133.8; 139.2. Found, m/z:
220.1097 [M+Na]+. C14H15NNa. Calculated, m/z: 220.1097.
Preparation of N-methyl-2-(o-tolyl)azetidine (5g).
A solution of hexyllithium (2.3 M in hexane, 0.60 g,
0.65 mmol, 1.3 equiv) was added dropwise to a stirred solution
of azetidine 5a (0.074 g, 0.5 mmol, 1 equiv) in Et2O (4 ml)
at 25°C and stirring was continued for 16 h. Then MeI
(0.092 g, 0.65 mmol, 50 μl, 1.3 equiv) was added to the
resulting deep orange solution. After 1 h, the reaction
mixture was poured into saturated aqueous NH4Cl solution
(10 ml) and extracted with Et2O (3×10 ml). The combined
organic layers were dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. The crude mixture was purified
by chromatography on silica gel (CH2Cl2–MeOH, 95:5) to
give 1-methyl-2-(o-tolyl)azetidine (5g) (0.072 g, 90%). Further
Evaluation of the binding affinity to nicotinic acetyl-
choline receptors (nAChRs). Cytisine binding assays were
performed as previously reported.20 Briefly, a 0.75 μM
[3H]cytisine solution was incubated with membrane homo-
genates from mouse brains (100–200 μg of protein) for
75 min at 4°C; 10 µM nicotine bitartrate was used to define
nonspecific binding. For equilibrium competition binding
assays, the concentration of unlabeled compounds varied from
0.1 to 1 mM. Bound and free fractions were separated by
rapid vacuum filtration through presoaked GF/B filters and
the radioactivity was determined by a liquid scintillation
counter. The IC50 values were determined by nonlinear
regression using a single sigmoid function in GraphPad
Prism 4.0 (San Diego, CA). The data are expressed as
mean values SEM of three independent experiments.
333

Chemistry of Heterocyclic Compounds 2017, 53(3), 329–334
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Supplementary information file containing 1H and
13C NMR, and HRMS spectra data is available at http://
link.springer.com/journal/10593.
We thank the University of Bari for support. We are
grateful to Dr. Laura Carroccia for precious contribution
to the work.
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