Chemistry of Heterocyclic Compounds

, Volume 53, Issue 3, pp 329–334 | Cite as

2-Arylazetidines as ligands for nicotinic acetylcholine receptors

  • Leonardo Degennaro
  • Marina Zenzola
  • Annunziatina Laurino
  • Maria Maddalena Cavalluzzi
  • Carlo Franchini
  • Solomon Habtemariam
  • Rosanna Matucci
  • Renzo Luisi
  • Giovanni Lentini
Article
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 sensation 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 nonselective agonists of nAChR and muscarinic ACh receptors.2a,9
Figure 1

Structures of known nicotinic nAChR ligands 14, and biologically relevant, structurally related compounds.

In the present study, we hypothesized that 2-arylazetidines 5 might satisfy the need for novel nAChR ligands by being safer than the known agonists 1 and 2. 2-Arylazetidines 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 transmission. Additionally, we decided to characterize 2-aryl-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 5ag 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, cycloaddition reactions, rearrangements of larger rings, reduction of azetidin-2-ones, and aza-Michael addition of diethyl N-arylphosphoramidates to chalcones.15 2-Arylazetidines 5 were obtained through two alternative routes A and B (Scheme 1) for the construction of the fourmembered ring. The procedure A started from styrenes, while the procedure B used suitable 1-aryl-3-chloro-1-propanols as starting materials.

Scheme 1

We followed both routes to obtain compounds 5af,h, as shown in Scheme 2.16 Both protocols were practical and gave the title compounds in moderate to good yields. Procedure A allowed the introduction of various N-substituents, while the procedure B was more appropriate when N-methylazetidines were the target compounds. Furthermore, 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 orthodirecting 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.

Scheme 2

When tested as nAChR ligands, all of the obtained compounds 5aj 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 orthosubstituted azetidines 5g,h may suggest that steric hindrance hampers ligand binding either directly or by constraining the orientation of the aromatic ring in an unfavorable 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-chlorosubstituted analog 5d were the most interesting compounds of the series (Table 1). As representatives of the series, analogs 5a,c,h were also evaluated as MAOIs and AChEIs showing no activity at the highest tested concentrations (1 mM).
Table 1

Binding affinity values of 2-arylazetidines 5ai and azetidin-2-one 5j for nAChRs according to [3H]cytisine specific displacement assay and respective ligand efficiency metrics

Compound

pIC50 ± SEM*

GE**

LLE***

5a

5.39 ± 0.16

3.9

5b

5.05 ± 0.14

–0.47

3.1

5c

5.08 ± 0.09

–0.42

3.5

5d

5.74 ± 0.06

0.48

3.6

5e

5.50 ± 0.13

0.08

2.9

5f

<4

< –0.95

<2.6

5g

4.40 ± 0.14

–1.36

2.4

5h

<4

< –0.48

<1.3

5i

<4

< –0.32

<1.4

5j

<4

< –1.90

<3.2

Nicotine (1)

7.99 ± 0.14

7.3

Carbachol (4)

6.32 ± 0.05

10.2

* 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 *** 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-chlorosubstituted 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 proposed synthetic procedures, a wide and diverse set of 2-arylazetidines may be designed and prepared to allow exploration of structure–activity relationships and to perform fine tuning of toxico-pharmacological profile.

Experimental

IR spectra were recorded with a Perkin Elmer 283 spectrometer either for neat compounds as thin film on NaCl plate or for KBr pellets. 1H and 13C NMR spectra were recorded on Bruker Avance II (600 and 125 MHz, respectively), Varian Inova (400 and 100 MHz, respectively) spectrometers, all chemical shift values are reported in ppm relative to TMS. High-resolution mass spectrometry 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, 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 9ah were obtained by adapting our previously reported continuous flow process.16c 1-Methyl-2-arylazetidines 5aj were prepared as shown in Scheme 2, following a previously reported procedure.16a–c

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.

3-Chloro-1-(p-tolyl)propan-1-ol (9b). Yield 66%, white solid, mp 42–44°C. IR spectrum (KBr), ν, cm–1: 3306, 2917, 1416, 1286, 1049, 817. 1H 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).

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.

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, 2904, 1612, 1516, 1288, 1253, 1179, 1032, 834, 652. 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, 3051, 2962, 1597, 1511, 1281, 1070, 801, 778. 1H 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 mixture 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% 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-methylbenzene 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.

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, 2772, 1509, 1225, 1191, 837. 1H 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, JCF = 21.6); 128.3 (d, JCF = 7.8); 138.9 (d, JCF = 2.4); 162.2 (d, JCF = 245.0). Found, m/z: 188.0848 [M+Na]+. C10H12FNNa. Calculated, m/z: 188.0846.

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, 1190, 1087, 1014, 966, 832, 797, 774. 1H 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.

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, 1141, 978, 955, 801, 776. 1H NMR spectrum (400 MHz, 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 ofN-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 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, 1351, 1192, 967, 748. 1H 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.

Preparation ofN-(tert-butoxycarbonyl)-2-phenylazetidine (5i). Lithium aluminum hydride (0.259 g, 6.8 mmol) was added portionwise to 4-phenyl-2-azetidinone (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-tertbutyl 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-phenylazetidine (5i) was isolated. Yield 657 mg (83%), colorless oil. IR spectrum (thin film), ν, cm–1: 2974, 1701, 1389, 1364, 1132, 698. 1H NMR spectrum (600 MHz, CDCl3), 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.

Evaluation of the binding affinity to nicotinic acetylcholine receptors (nAChRs). Cytisine binding assays were performed as previously reported.20 Briefly, a 0.75 μM [3H]cytisine solution was incubated with membrane homogenates 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.

Acetylcholinesterase (AChE) inhibition assay. The AChE inhibition potential was assayed following a previously reported colorimetric method.22 Briefly, a mixture (200 ml) was prepared on a microtiter plate containing 25 ml of 0.26 U/ml AChE, 25 ml of test drugs in various concentrations, 25 ml of 15 mM ACh iodide, and 125 μl of 3 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) solution. The plates were incubated for 10 min at 37°C and absorbance at 405 nm was immediately measured using a Multiskan Plate Reader (Thermo Labsystems, UK).

Monoamine oxidase (MAO) inhibition assay. The AChE inhibition potential was assayed following a previously reported colorimetric method.23 Briefly, rat kidneys were homogenized (1:10) in ice-cold phosphate buffer (0.1 M, pH 7.4) containing sucrose (0.25 M). The homogenate was centrifuged (1000 g × 10 min at 4°C) in order to remove cell debris. The supernatant (sup-1) was used for enzyme determinations. Basal MAO activity was assayed radiochemically as previously described, using [14C]serotonin creatinine sulfate (1 μCi/ml, 100 μM; Amersham Biosciences, UK) or [14C]benzylamine (1 μCi/ml, 100 μM; Amersham Biosciences, UK) for measuring the activity of MAO-A and B, respectively. Test compounds (100 nM–1 mM) were preincubated for 30 min at 37°C in 100 μl of pH 7.4 phosphate buffer containing 40 μg of sup-1 proteins. The labeled substrates were then added and left in contact with enzyme preparations for 30 min at 37°C. The reactions were stopped by the addition of 3 M HCl (20 μl). The aldehyde produced by enzymatic reaction was extracted in ethyl acetate (300 μl). The organic phase was separated by brief spinning (1000 g × 5 min) and a 150-μl portion was tested for radioactivity in β-counter. MAO activity was then referred to as the radioactivity recovered in the organic phase corrected for the percentage of nonmetabolized substrate extracted in the organic phase. The results are expressed as nmol/mg of proteins/30 min. The extraction of labeled substrates in the organic phase was evaluated in reaction mixtures treated with 3 M HCl (20 μl) before the addition of substrates.

Supplementary information file containing 1H and 13C NMR, and HRMS spectra data is available at http://link.springer.com/journal/10593.

Notes

We thank the University of Bari for support. We are grateful to Dr. Laura Carroccia for precious contribution to the work.

Supplementary material

10593_2017_2061_MOESM1_ESM.pdf (1.4 mb)
ESM 1(PDF 1445 kb)

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Leonardo Degennaro
    • 1
  • Marina Zenzola
    • 1
  • Annunziatina Laurino
    • 2
  • Maria Maddalena Cavalluzzi
    • 1
  • Carlo Franchini
    • 1
  • Solomon Habtemariam
    • 3
  • Rosanna Matucci
    • 2
  • Renzo Luisi
    • 1
  • Giovanni Lentini
    • 1
  1. 1.Department of Pharmacy – Drug SciencesUniversity of Studies of Bari Aldo MoroBariItaly
  2. 2.Department of NeuroscienceArea del Farmaco e Salute del Bambino (NEUROFARBA)FirenzeItaly
  3. 3.Pharmacognosy Research Laboratories, Medway School of ScienceUniversity of GreenwichKentUnited Kingdom

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