Introduction

Epilepsy, an ubiquitous disease characterized by recurrent seizures that affects more than 60 million people worldwide and 2.5 million people in USA, according to epidemiological studies (Wasterlain et al., 1989; Loscher, 1998). Drugs presently available in the market for the treatment of epilepsy do not provide a complete cure and have an effect only in 60–70% of patients. Moreover, these drugs cause various side effects such as drowsiness, gastrointestinal disturbance, hepatotoxicity, and megaloblastic anemia (Perucca, 1996; Lin and Kadaba, 1997), including some life-threatening conditions (Al-Soud et al., 2003).

2-Azetidinones, commonly known as β-lactams, are heterocyclic compounds well known to organic and medicinal chemists. The activities of widely used antibiotics such as penicillins, cephalosporins, and carbapenems are attributed to the presence of 2-azetidinone ring (Singh, 2004). Literature survey reveals that beside their antibacterial activity (Patel and Mehta, 2006) 2-azetidinones also possess various biological activities that include anticonvulsant (El-Helby and Wahab, 2003), carbonic anhydrase inhibition (Russo et al., 1994), local anesthetic (Costakes and Tsatsa, 1978), hypoglycemic (Chernykh and Sidorenko, 1981), and anti-inflammatory (Sreenivasa et al., 2005). The β-lactams also serve as synthons for many biologically important classes of organic compounds (Deshmukh et al., 2004). Thus, various activities attributed to 2-azetidinone ring hold the potential in the development of novel antiepileptic agents (Singh, 2004). Therefore, the potential therapeutic importance of azetidinone rings prompted us to develop selective molecules in which a substituent could be arranged in a pharmacophoric pattern to display higher pharmacological activities.

Result and discussion

Synthesis

The most common method for the synthesis of 2-azetidinones is the Staudinger keteneimine cycloaddition, which involves the reaction of imines with acid chloride in the presence of a tertiary base (Staudinger, 1907). In the present work, we have synthesized N-[3-chloro-2-(substitutedphenyl)-4-oxo-azetidin-1-yl]isonicotinamide (2a–l) in a two-step procedure, as shown in Scheme 1. Various isonicotinic acids (substituted-benzylidene)-hydrazide (1a–l) were synthesized by treating isonicotinohydrazide with different substituted benzaldehydes in absolute ethanol. Compounds (1a–l) were refluxed with triethylamine and chloroacetaldehyde to form N-[3-chloro-2-(substitutedphenyl-4-oxo-azetidin-1-yl]isonicotinamides (2a–l) in moderate to good yields (60–80%). Thin layer chromatography (TLC) was used throughout to monitor and optimize the reactions for purity and completion. Synthesized compounds were characterized by IR, 1H NMR, mass spectroscopy, and elemental analysis.

Scheme 1
scheme 1

Protocol for synthesis of N-[3-chloro-2-(substitutedphenyl)-4-oxo-azetidin-1-yl]isonicotinamide derivatives. R: a = 4-OH, b = 2-OH, c = 2-OCH3, d = 4-N(CH3)2, e = 4-CH3, f = 3,4-di-OCH3, g = 3-OCH3-4OH, i = 2-NO2, j = 4-Cl, k = 2-Cl, l = H

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Pharmacology

All newly synthesized compounds were evaluated for their anticonvulsant activity. Neurotoxicity and antidepressant activity are shown in Tables 1 and 2. Most of the compounds showed significant anticonvulsant activity, except for ortho derivatives 2b, 2c, and 2k (no anticonvulsant activity at dosages up to 100 mg/kg, i.p.). One can suppose that ortho substituent may possibly block the azetidinone ring receptor interaction.

Table 1 Anticonvulsant and neurotoxicity screening of N-substituted-3-chloro-2-azetidinones
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Table 2 CNS depressant study on selected active compounds by forced swim pool test method
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Analyzing the activities of all synthesized derivatives 2a–l, the following structure–activity relationship (SAR) was observed. It was found that the azetidinone derivatives having CH3 group showed maximum anticonvulsant activity in comparison to OH, OCH3, NO2, N(CH3)2, Cl, and H group/atom on the whole level. The position of the substituents on the phenyl ring greatly influenced the anticonvulsant activity, the activity order is p > p–m > o. The p-CH3 derivative 2e exhibited the highest at minimum dose 30 mg/kg in maximum electroshock seizure (MES) at both 0.5 and 4.0 h and 100 and 30 mg/kg, i.p. in subcutaneous pentylenetetrazole (scPTZ) at 0.5 and 4.0 h, respectively. The ortho and meta derivative 2f and 2g exhibited weaker activity than para substituted derivatives 2a, 2d, 2e, 2h, and 2j. In this series, most of the tested compounds showed no neurotoxicity except for compounds 2a and 2h, which showed neurotoxicity at the highest dose (300 mg/kg) after 4.0 h, possibly because the presence of highly polar group at para position.

Compound 2d, 2e, 2f, 2j, and 2l showed significant anticonvulsant activity and were also tested for their CNS depressant effect (Table 2). The tested compounds showed 49.8, 21.6, 33.8, 54.5, and 11.5% increase in immobility time with respect to controls whereas the standard drug carbamazepine showed 30.6% increase in the immobility time, indicating a lower CNS depressant effect for compounds 2e and 2l.

Conclusion

A series of N-[3-chloro-2-(substitutedphenyl)-4-oxo-azetidin-1-yl]isonicotinamide derivatives were synthesized. The anticonvulsant effect and the neurotoxicity of the compounds were calculated with MES test, Sc-PTZ, and rotarod tests with intraperitoneally injected mice. Among the synthesized it was found that N-[3-chloro-2-(4-methyl-phenyl)-4-oxoazetidin-1-yl]isonicotinamide (2e) possessed significant anticonvulsant activity in comparison to standard drug phenytoin and carbamazepine (showed activity at 30 mg/kg body weight without neurotoxicity). On the basis of these results it can be concluded that the anticonvulsant activity was affected by two factors. Firstly the introduction of electron withdrawing/releasing groups, where anticonvulsant activity decreases with the introduction of electron withdrawing groups like OH, NO2, Cl and significant increases of anticonvulsant activity were observed with the substitution of electron donating groups like CH3, OCH3, N(CH3)2, and H. Moreover, the molecular symmetry is the 2nd factor influencing the biological activity because unsubstituted and para-substituted compounds showed good activity as compared to ortho- and meta-substituted compounds. Compound 2e showed excellent anticonvulsant activity due to substitution of electron releasing group (p-CH3) which increases the lipophilicity and on the other hand, para substitution facilitates molecule receptor interactions.

Experimental

General chemistry

All chemicals used in the synthesis were supplied by E. Merck and S.D. Fine Chemicals. Melting points are determined by open capillary tube method and are uncorrected. Purity of the compound was checked on TLC using iodine vapors as visualizing agents. IR spectra were obtained on a Perkin-Elmer 1720 FT-IR spectrometer (KBr pellets). 1H NMR spectra were obtained on a Bruker DRX-300 (300 MHz FT NMR) spectrometer in CDCl3 using tetramethylsilane (TMS) as the internal reference (chemical shifts in δ ppm). Elemental analysis (C, H, and N) were undertaken with Perkin-Elmer model 240C analyzer. Mass spectra were recorded at Jeol SX-102 spectrometer.

General procedure for synthesis of isonicotinic acid (substituted-benzylidene)-hydrazide (1a–l)

To a solution of isoniazide (0.01 mol) in absolute ethanol (30 ml) few drops of glacial acetic acid and substituted benzaldehyde (0.01 mol) were added. The reaction mixture was refluxed for 3 h on water bath. The reaction mixture was poured onto crushed ice, a solid mass separated was filtered, washed with water and recrystallized from ethanol.

Isonicotinic acid (4-hydroxy-benzylidene)-hydrazide (1a)

Yield 67%; m.p.: 242–245°C; IR (KBr, cm−1): 3546 (OH), 3447 (NH), 2917 (NCH), 1671 (C=O); 1H NMR (CDCl3), δ (ppm): 11.9 (bs, 1H, CONH), 9.31 (s, 1H, OH), 8.52 (s, 1H, N–CH), 8.1–8.4 (m, 4H, pyridine), 7.1–7.69 (m, 4H, Ar–H); MS: m/z 241 (M+); Anal. Calcd for C13H11N3O2: C, 64.72; H, 4.60; N, 17.42; Found: C, 64.61; H, 4.43; N, 17.48.

Isonicotinic acid (2-hydroxy-benzylidene)-hydrazide (1b)

Yield 73%; m.p.: 174–176°C; IR (KBr, cm−1): 3521 (OH), 3398 (NH), 2924 (NCH), 1676 (C=O); 1H NMR (CDCl3), δ (ppm): 10.03 (bs, 1H, CONH), 9.43 (s, 1H, OH), 8.34 (s, 1H, N–CH), 7.64–7.91 (m, 4H, pyridine), 7.1–7.59 (m, 4H, Ar–H); MS: m/z 241 (M+); Anal. Calcd for C13H11N3O2: C, 64.72; H, 4.60; N, 17.42; Found: C, 64.57; H, 4.42; N, 17.41.

Isonicotinic acid (2-methoxy-benzylidene)-hydrazide (1c)

Yield 70%; m.p.: 131–133°C; IR (KBr, cm−1): 3425 (NH), 3028 (NCH), 1668 (C=O); 1H NMR (CDCl3), δ (ppm): 12.07 (bs, 1H, CONH), 8.7 (s, 1H, N–CH), 7.8–8.4 (m, 4H, pyridine), 7.4–7.76 (m, 4H, Ar–H), 2.98 (s, 3H, OCH3); MS: m/z 255 (M+); Anal. Calcd for C14H13N3O2: C, 65.87; H, 5.13; N, 16.46; Found: C, 65.74; H, 5.02; N, 16.34.

Isonicotinic acid (4-dimethylamino-benzylidene)-hydrazide (1d)

Yield 75%; m.p.: 192–194°C; IR (KBr, cm−1): 3475 (NH), 3012 (NCH), 1659 (C=O); 1H NMR (CDCl3), δ (ppm): 12.18 (bs, 1H, CONH), 8.3 (s, 1H, N–CH), 8.2–8.5 (m, 4H, pyridine), 6.9–7.75 (m, 4H, Ar–H), 2.89 (s, 6H, 2CH3); MS: m/z 268 (M+); Anal. Calcd for C15H16N4O: C, 67.15; H, 6.01; N, 20.88; Found: C, 67.04; H, 6.11; N, 20.69.

Isonicotinic acid (4-methyl-benzylidene)-hydrazide (1e)

Yield 70%; m.p.: 212–214°C; IR (KBr, cm−1): 3425 (NH), 3028 (NCH), 1668 (C=O); 1H NMR (CDCl3), δ (ppm): 11.07 (bs, 1H, CONH), 8.6 (s, 1H, N–CH), 7.9–8.7 (m, 4H, pyridine), 6.9–7.76 (m, 4H, Ar–H), 2.50 (s, 3H, CH3); MS: m/z 239 (M+); Anal. Calcd for C14H13N3O: C, 70.28; H, 5.48; N, 17.56; Found: C, 70.18; H, 5.32; N, 17.35.

Isonicotinic acid (3,4-dimethoxy-benzylidene)-hydrazide (1f)

Yield 68%; m.p.: 167–169°C; IR (KBr, cm−1): 3425 (NH), 3028 (NCH), 1679 (C=O); 1H NMR (CDCl3), δ (ppm): 11.37 (bs, 1H, CONH), 8.51 (s, 1H, N–CH), 7.8–8.3 (m, 4H, pyridine), 6.8–7.2 (m, 3H, Ar–H), 3.87 (s, 6H, 2OCH3); MS: m/z 285 (M+); Anal. Calcd for C15H15N3O3: C, 63.15; H, 5.30; N, 14.73; Found: C, 62.98; H, 5.13; N, 14.58.

Isonicotinic acid (4-hydroxy-3-methoxy-benzylidene)-hydrazide (1g)

Yield 65%; m.p.: 187–189°C; IR (KBr, cm−1): 3387 (NH), 3087 (NCH), 1675 (C=O); 1H NMR (CDCl3), δ (ppm): 12.25 (bs, 1H, CONH), 9.6 (s, 1H, OH), 8.42 (s, 1H, N–CH), 7.6–8.2 (m, 4H, pyridine), 6.9–7.73 (m, 3H, Ar–H), 3.76 (s, 3H, OCH3); MS: m/z 271 (M+); Anal. Calcd for C14H13N3O3: C, 61.99; H, 4.83; N, 15.49; Found: C, 61.83; H, 4.71; N, 15.29.

Isonicotinic acid (4-nitro-benzylidene)-hydrazide (1h)

Yield 75%; m.p.: 155–157°C; IR (KBr, cm−1): 3467 (NH), 3014 (NCH), 1678 (C=O); 1H NMR (CDCl3), δ (ppm): 11.76 (bs, 1H, CONH), 8.32 (s, 1H, N–CH), 7.92–8.56 (m, 4H, CH-pyridine), 6.75–7.54 (m, 4H, Ar–H); MS: m/z 270 (M+); Anal. Calcd for C13H10N4O3: C, 57.78; H, 3.73; N, 20.73; Found: C, 57.65; H, 3.57; N, 20.58.

Isonicotinic acid (2-nitro-benzylidene)-hydrazide (1i)

Yield 76%; m.p.: 123–125°C; IR (KBr, cm−1): 3464 (NH), 3011 (NCH), 1673 (C=O); 1H NMR (CDCl3), δ (ppm): 10.76 (bs, 1H, CONH), 8.56 (s, 1H, N–CH), 8.21–8.49 (m, 4H, CH-pyridine), 6.76–7.54 (m, 4H, Ar–H); MS: m/z 270 (M+); Anal. Calcd for C13H10N4O3: C, 57.78; H, 3.73; N, 20.73; Found: C, 57.69; H, 3.61; N, 20.59.

Isonicotinic acid (4-chloro-benzylidene)-hydrazide (1j)

Yield 73%; m.p.: 225–227°C; IR (KBr, cm−1): 3399 (NH), 2969 (NCH), 1653 (C=O), 679 (C–Cl); 1H NMR (CDCl3), δ (ppm): 12.27 (bs, 1H, CONH), 8.52 (s, 1H, N–CH), 7.3–8.1 (m, 4H, pyridine), 6.91–7.73 (m, 4H, Ar–H); MS: m/z 259 (M+), 261 (M++2); Anal. Calcd for C13H10ClN3O: C, 60.12; H, 3.88; N, 16.18; Found: C, 60.01; H, 3.67; N, 16.04.

Isonicotinic acid (2-chloro-benzylidene)-hydrazide (1k)

Yield 67%; m.p.: 177–179°C; IR (KBr, cm−1): 3403 (NH), 2976 (NCH), 1643 (C=O), 674 (C–Cl); 1H NMR (CDCl3), δ (ppm): 12.07 (bs, 1H, CONH), 8.41 (s, 1H, N–CH), 7.3–8.0 (m, 4H, pyridine), 6.81–7.72 (m, 4H, Ar–H); MS: m/z 259 (M+), 261 (M+ + 2); Anal. Calcd for C13H10ClN3O: C, 60.12; H, 3.88; N, 16.18; Found: C, 59.97; H, 3.71; N, 16.21.

Isonicotinic acid benzylidene-hydrazide (1l)

Yield 72%; m.p.: 102–104°C; IR (KBr, cm−1): 3425 (NH), 3028 (NCH), 1668 (C=O); 1H NMR (CDCl3), δ (ppm): 12.07 (bs, 1H, CONH), 7.95 (s, 1H, N–CH), 7.63–7.89 (m, 4H, pyridine), 6.92–7.75 (m, 5H, Ar–H); MS: m/z 225 (M+); Anal. Calcd for C13H11N3O: C, 69.32; H, 4.92; N, 18.66; Found: C, 69.17; H, 4.71; N, 18.54.

General procedure for synthesis of N-[3-chloro-2-(substitutedphenyl)-4-oxo-azetidin-1-yl]isonicotinamide derivatives (2a–l)

A solution of coumpound 1a–l, triethylamine (0.02 mol) and chloroacetylchloride (0.02 mol) in DMF (40 ml) was refluxed for 3 h on water bath. The reaction mixture was poured onto crushed ice, a solid mass separated out which was filtered, washed with water and recrystallized from ethanol.

N-[3-Chloro-2-(4-hydroxy-phenyl)-4-oxoazetidin-1yl]isonicotinamide (2a)

Yield 70%; m.p.: 292–294°C; IR (KBr, cm−1): 3425 (NH), 3019 (CH), 1671 (C=O), 657 (C–Cl); 1H NMR (CDCl3), δ (ppm): 11.24 (bs, 1H, CONH), 9.32 (s, 1H, OH), 8.41–8.79 (m, 4H, pyridine), 7.67–7.98 (m, 4H, Ar–H), 7.13 (d, 1H, N–CH), 5.49 (d, 1H, CH–Cl); MS: m/z 317 (M+), 319 (M+ + 2); Anal. Calcd for C15H12ClN3O3: C, 56.70; H, 3.81; N, 11.16; Found: C, 56.58; H, 3.69; N, 10.98.

N-[3-Chloro-2-(2-hydroxy-phenyl)-4-oxoazetidin-1yl]isonicotinamide (2b)

Yield 73%; m.p.: 174–176°C; IR (KBr, cm−1): 3412 (NH), 3029 (CH), 1656 (C=O), 677 (C–Cl); 1H NMR (CDCl3), δ (ppm): 11.04 (bs, 1H, CONH), 9.21 (s, 1H, OH), 8.38–8.79 (m, 4H, pyridine), 7.77–7.93 (m, 4H, Ar–H), 7.08 (d, 1H, N–CH), 5.48 (d, 1H, CH–Cl); MS: m/z 317 (M+), 319 (M+ + 2); Anal. Calcd for C15H12ClN3O3: C, 56.70; H, 3.81; N, 11.16; Found: C, 56.59; H, 3.67; N, 11.21.

N-[3-Chloro-2-(2-methoxy-phenyl)-4-oxoazetidin-1yl]isonicotinamide (2c)

Yield 70%; m.p.: 160–162°C; IR (KBr, cm−1): 3425 (NH), 3023 (CH), 1670 (C=O), 687 (C–Cl); 1H NMR (CDCl3), δ (ppm): 11.91 (bs, 1H, CONH), 7.75–8.79 (m, 4H, pyridine), 7.3 (d, 1H, N–CH), 6.54–6.95 (m,4H, Ar–H), 5.47 (d, 1H, CH–Cl), 3.67 (s, 3H, OCH3); MS: m/z 331 (M+) 333 (M+ + 2); Anal. Calcd for C16H14ClN3O3: C, 57.93; H, 4.25; N, 12.67; Found: C, 57.76; H, 4.13; N, 12.81.

N-[3-Chloro-2-(4-dimethylamino-phenyl)-4-oxoazetidin-1-yl]isonicotinamide (2d)

Yield: 70%; m.p.: 230–232°C; IR (KBr, cm−1): 3460 (NH), 3052 (CH), 1655 (C=O), 671 (C–Cl); 1H NMR (CDCl3), δ (ppm): 11.63 (bs, 1H, CONH), 8.31 (d, 1H, N–CH), 7.77–8.29 (m, 4H, pyridine), 6.54–6.95 (m, 4H, Ar–H), 5.43 (d, 1H, CH–Cl), 2.74 (s, 6H, 2CH3); MS: m/z 344 (M+), 346 (M+ + 2); Anal. Calcd for C17H17ClN4O2: C, 59.22; H, 4.97; N, 16.25; Found: C, 59.03; H, 4.76; N, 16.11.

N-[3-Chloro-2-(4-methyl-phenyl)-4-oxoazetidin-1-yl]isonicotinamide (2e)

Yield: 60%; m.p.: 140–142°C; IR (KBr, cm−1): 3387 (NH), 3012 (CH), 1684 (C=O), 676 (C–Cl); 1H NMR (CDCl3), δ (ppm): 11.86 (bs, 1H, CONH), 8.42 (d, 1H, N–CH), 7.89–8.39 (m, 4H, pyridine), 7.54–7.95 (m, 4H, Ar–H), 5.49 (d, 1H, CH–Cl), 2.54 (s, 3H, CH3); MS: m/z 315 (M+), 317 (M+ + 2); Anal. Calcd for C16H14ClN3O2: C, 60.86; H, 4.47; N, 13.31; Found: C, 60.67; H, 4.29; N, 13.17.

N-[3-Chloro-2-(3,4-dimethoxyl-phenyl)-4-oxoazetidin-1-yl]isonicotinamide (2f)

Yield: 80%; m.p.: 180–182°C; IR (KBr, cm−1): 3416 (NH), 3021 (CH), 1674 (C=O), 665 (C–Cl); 1H NMR (CDCl3), δ (ppm): 12.07 (bs, 1H, CONH), 8.35 (d, 1H, N–CH), 7.79–8.11 (m, 4H, pyridine), 7.14–7.55 (m, 3H, Ar–H), 5.23 (d, 1H, CH–Cl), 3.79 (s, 6H, 2OCH3); MS: m/z 361 (M+), 363 (M+ + 2); Anal. Calcd for C17H16ClN3O4: C, 56.44; H, 4.46; N, 11.61; Found: C, 56.23; H, 4.28; N, 11.47.

N-[3-Chloro-2-(4-hydroxy-3-methoxy-phenyl)-4-oxoazetidin-1-yl]isonicotinamide (2g)

Yield: 70%; m.p.: 211–213°C; IR (KBr, cm−1): 3405 (NH), 3029 (CH), 1651 (C=O), 685 (C–Cl); 1H NMR (CDCl3), δ (ppm): 12.13 (bs, 1H, CONH), 10.23(s, 1H, OH), 8.05 (d, 1H, N–CH), 7.76–8.18 (m, 4H, pyridine), 7.24–7.57 (m, 3H, Ar–H), 5.73 (d, 1H, CH–Cl), 3.73 (s, 3H, OCH3); MS: m/z 347 (M+), 349 (M+ + 2); Anal. Calcd for C16H14ClN3O4: C, 55.26; H, 4.06; N, 12.08; Found: C, 55.03; H, 3.89; N, 12.18.

N-[3-Chloro-2-(4-nitrophenyl)-4-oxoazetidin-1-yl]isonicotinamide (2h)

Yield: 80%; m.p.: 192–194°C; IR (KBr, cm−1): 3415 (NH), 3011 (CH), 1661 (C=O), 672 (C–Cl); 1H NMR (CDCl3), δ (ppm): 11.97 (bs, 1H, CONH), 8.11 (d, 1H, N–CH), 7.76–7.98 (m, 4H, pyridine), 7.14–7.58 (m, 4H, Ar–H), 5.62 (d, 1H, CH–Cl); MS: m/z 346 (M+) 348 (M+ + 2); Anal. Calcd for C15H11ClN4O4: C, 51.96; H, 3.20; N, 16.16; Found: C, 51.75; H, 3.04; N, 16.01.

N-[3-Chloro-2-(2-nitrophenyl)-4-oxoazetidin-1-yl]isonicotinamide (2i)

Yield: 74%; m.p.: 170–172°C; IR (KBr, cm−1): 3434 (NH), 2978 (CH), 1660 (C=O), 668 (C–Cl); 1H NMR (CDCl3), δ (ppm): 10.95 (bs, 1H, CONH), 8.12 (d, 1H, N–CH), 7.75–7.87 (m, 4H, pyridine), 7.17–7.53 (m, 4H, Ar–H), 5.49 (d, 1H, CH–Cl); MS: m/z 346 (M+) 348 (M+ + 2); Anal. Calcd for C15H11ClN4O4: C, 51.96; H, 3.20; N, 16.16; Found: C, 51.79; H, 3.14; N, 16.23.

N-[3-Chloro-2-(4-chlorophenyl)-4-oxoazetidin-1-yl]isonicotinamide (2j)

Yield: 72%; m.p.: 270–272°C; IR (KBr, cm−1): 3398 (NH), 3017 (CH), 1679 (C=O), 681 (C–Cl); 1H NMR (CDCl3), δ (ppm): 11.67 (bs, 1H, CONH), 8.17 (d, 1H, N–CH), 7.78–8.01 (m, 4H, pyridine), 7.34–7.79 (m, 4H, Ar–H), 5.47 (d, 1H, CH–Cl); MS: m/z 335 (M+) 337 (M+ + 2); Anal. Calcd for C15H11Cl2N3O2: C, 53.59; H, 3.30; N, 12.50; Found: C, 53.47; H, 3.19; N, 12.39.

N-[3-Chloro-2-(2-chlorophenyl)-4-oxoazetidin-1-yl]isonicotinamide (2k)

Yield: 85%; m.p.: 205–207°C; IR (KBr, cm−1): 3356 (NH), 3027 (CH), 1656 (C=O), 676 (C–Cl); 1H NMR (CDCl3), δ (ppm): 10.54 (bs, 1H, CONH), 8.03 (d, 1H, N–CH), 7.67–7.98 (m, 4H, pyridine), 7.32–7.76 (m, 4H, Ar–H), 5.43 (d, 1H, CH–Cl); MS: m/z 335 (M+) 337 (M+ + 2); Anal. Calcd for C15H11Cl2N3O2: C, 53.59; H, 3.30; N, 12.50; Found: C, 53.48; H, 3.14; N, 12.36.

N-(3-Chloro-2-oxo-4-phenyl-azetidin-1-yl)-isonicotinamide (2l)

Yield: 70%; m.p.: 131–133°C; IR (KBr, cm−1): 3402 (NH), 3023 (CH), 1686 (C=O), 661 (C–Cl); 1H NMR (CDCl3), δ (ppm): 12.08 (bs, 1H, CONH), 8.21 (d, 1H, N–CH), 7.68–8.11 (m, 4H, pyridine), 7.44–7.98 (m, 5H, Ar–H), 5.74 (d, 1H, CH–Cl); MS: m/z 301 (M+) 303 (M+ + 2); Anal. Calcd for C15H12ClN3O2: C, 59.71; H, 4.01; N, 13.93; Found: C, 59.63; H, 4.14; N, 13.49.

Pharmacology

All the compounds were screened for anticonvulsant activity adopting the anticonvulsant screening program (ASP) including maximal electroshock test (MES), the subcutaneous pentylene tetrazole (scPTZ), and evaluation of neurotoxicity (TOX) (Krall et al., 1978; Porter et al., 1984; Kupferberg, 1989). The mice weighing 18–25.5 g of either sex were used, and the number of animals used was six in each group. PEG-400 was used for dissolving the test compounds in MES, ScPTZ, and Minimal Motor Impairment Test. The compounds were administered intraperitoneally (0.1 ml/10 g) to mice, at doses of 30, 100, and 300 mg/kg body weight.

Maximal electroshock seizer

In the MES test, the mice were subjected to 50 mA of 60 Hz alternating current from a convulsiometer for 0.2 s through a pair of electrodes attached to each ear. The duration of the tonic hind limb extensor phase, and the number of animals protected from convulsions were noted. Phenytoin was used as standard drug.

Subcutaneous pentylene tetrazole

Pentylene tetrazole was used as convulsant, phenytoin and carbamazepine are used as standard drugs. Convulsion was induced 1 h after administration of the standard drug or the test compounds by i.p. injection of PTZ (30, 100, and 300 mg/kg) dissolved in saline to a volume of 0.1 ml/10 g body weight. The time needed for the development of unequivocal sustained clonic seizer activity involving the limbs (isolated myoclonic jerks or other preconvulsive chewing behavior were not counted) was carefully noted. Animals which did not meet this criteria were considered protected.

Minimal motor impairment (neurotoxicity)

The undesirable effects (toxicity) were studied by monitored the animals for overt sign of impaired neurological or muscular function. In mice, rotorod procedure was used, which rotate at a speed of 6 rpm. The dose at which animals fell off this rotating rod three times during a 1 min period was considered.

CNS depression study

The forced swim method (Porsolt’s swim pool test) is followed to study CNS depression (Porsolt et al., 1978). Mice are placed in a chamber (diameter 45 cm, height: 20 cm) containing water up to a height of 15 cm at 25 ± 2°C. Two swim sessions are conducted, an initial 15 min pre-test, followed by a 5 min test session 24 h later. The animals are administered an i.p. injection (30 mg/kg) of the test compounds 30 min before the test session. The period of immobility (passive floating without struggling, making only those movements which are necessary to keep its head above the surface of water) during the 5 min test period are measured.