Synthesis and anticonvulsant activities of novel 2-(cyclopentylmethylene)hydrazinyl-1,3-thiazoles in mouse models of seizures

Abstract

Synthesis, characterization and investigation of in vivo anticonvulsant activities of 13 novel cyclopentanecarbaldehyde-based 2,4-disubstituted 1,3-thiazoles are presented. Their structures were determined using ¹H and ¹³C NMR, FAB(+)-MS, HRMS and elemental analyses. The results of anticonvulsant screening reveal that seven intraperitoneally administered compounds: 3a, 3b, 3d, 3e, 3f, 3k and 3m containing F-, Cl-, Br-, CF₃-, CH₃- and adamantyl substituents demonstrated significant anticonvulsant activity in the pentylenetetrazole model with median effective doses (ED₅₀) ≤ 20 mg/kg, respectively, which was approximately seven-fold lower than that reported for the reference drug, ethosuximide. Noteworthy, none of these compounds impaired animals’ motor skills in the rotarod test.

• Anticonvulsant drugs
• epilepsy
• PTZ model
• rotarod test
• thiazoles

Introduction

Epilepsy is a chronic neurological disorder characterized by recurrent and unprovoked seizure episodes. Although numerous clinically effective antiepileptic drugs (AEDs) are available, it is estimated that ∼30% of epileptic patients are drug-resistant. Furthermore, adverse effects and poor tolerability of so-called first- and second-generation of AEDs necessitate the search for novel anticonvulsant active agents1–3.

Thiazole derivatives are among of the most intensively studied classes of aromatic five-membered heterocycles because of their diverse pharmacological activities. Examples of antifungal (ravuconazole)4, antineoplastic (tiazofurin)5, anti-HIV (ritonavir)6, antiparasitic (nitazoxanide)7, as well as anti-inflammatory drugs (fentiazac, meloxicam)8 and MAO inhibitors9,10 can be found in this group.

Finally, it has been demonstrated that 3-(4-(4-bromophenyl)-1,3-thiazol-2-ylamino)-4-(2-methylphenyl)-4,5-dihydro-1H-1,2,4-triazole-5-thione showed high anticonvulsant activity in the maximal electroshock seizure (MES) model and in pentylenetetrazole (PTZ) test with median effective doses (ED₅₀) of 13.4 mg/kg and 81.6 mg/kg, respectively11. Also imidazolyl derivative, 1-((2-(4-chlorophenyl)thiazol-4-yl)methyl)-1H-imidazole and triazolyl derivative, 1-((2-phenylthiazol-4-yl)methyl)-1H-1,2,4-triazole showed the highest anticonvulsant activity in both MES and PTZ models with protection range of 33–100%12. Several triazolyl-thiazole derivatives were obtained and tested as anticonvulsant agents in the PTZ model in mice. The results showed that these derivatives protect from seizures and reduce mortality rate in this model13.

Considering that the thiazole derivatives can exhibit various biological properties, and continuing our previous investigation14–18 we decided to incorporate cyclopentylmethylidene moiety into the thiazol-2-yl-hydrazine pharmacophore containing various electron-donating and electron-withdrawing substituents. These substituents are useful to modulate electronic effects, and may influence the hydrophilic and hydrophobic properties of the molecules.

Consequently, we prepared 13 novel thiazole derivatives and evaluated their in vivo anticonvulsant properties in mouse models of chemically and electrically induced seizures. Additionally, the effect of the test compounds on animals’ motor coordination was assessed using the rotarod test.

Experimental

Materials and methods

All experiments were carried out under air atmosphere unless stated otherwise. Reagents were generally the best quality commercial-grade products and were used without further purification. ¹H NMR (700 and 400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on a Bruker Avance III multinuclear instrument (Toruń, Poland). FAB(+)-MS and HRMS analyses were performed by the Laboratory for Analysis of Organic Compounds and Polymers of the Centre for Molecular and Macromolecular Studies of the Polish Academy of Sciences in Łódź, Poland. MS spectra were recorded on a Finnigan MAT 95 spectrometer. Elemental analysis was performed on ELEMENTAR Vario MACRO CHN (Toruń, Poland). Melting points were determined in open glass capillaries and are uncorrected. Analytical TLC was performed using Macherey-Nagel Polygram Sil G/UV₂₅₄ 0.2 mm plates (Sigma Aldrich, Poznań, Poland). Cyclopentanecarbaldehyde, ethyl 4-chloro-3-oxobutanoate, ethyl 2-chloro-3-oxobutanoate, 1-chloropropan-2-one and appropriate bromoketones were commercial materials (Sigma Aldrich, Poznań, Poland).

(E)-2-(Cyclopentylmethylidene)hydrazinecarbothioamide (2)

Thiosemicarbazide (1.86 g, 20.38 mmol) was added to a stirred solution of cyclopentanecarbaldehyde1 (2.00 g, 20.38 mmol) in absolute ethyl alcohol (20 ml) and then acetic acid (1 ml) was added. The reaction mixture was stirred under reflux for 20 h. After that, the reaction mixture was poured on the crushed ice and kept aside. The product was filtered off and subsequently washed with water to yield 2.72 g, 78%; mp 76–78 °C; eluent: dichloromethane/methanol (90:10), R_f=0.73. ¹H NMR (DMSO-d₆); δ (ppm); 1.45–1.68 (m, 6H, 3CH₂); 1.69–1.82 (m, 2H, CH₂); 2.57–2.68 (m, 1H, CH); 7.36 (d, 1H, CH, J = 6.0 Hz); 7.41 (s, 1H, NH); 7.94 (s, 1H, NH); 10.99 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.39 (2C); 30.39 (2C); 42.28; 151.01; 178.06. Anal. Calcd. for C₇H₁₂N₂S: C, 49.09; H, 7.65; N, 24.54. Found: C, 49.11; H, 7.67; N, 24.56.

(E)-2-(2-(Cyclopentylmethylene)hydrazinyl)-4-(4-fluorophenyl)thiazole (3a). Typical procedure

Carbothioamide 2 (0.171 g, 1.0 mmol) was added to a stirred solution of 2-bromo-1-(4-fluorophenyl)ethanone (0.217 g, 1.0 mmol) in absolute ethyl alcohol (10 ml). The reaction mixture was stirred at room temperature for 20 h. The separated precipitate was purified on silica gel column chromatography (230–400 mesh) using (dichloromethane/methanol, 90:10, R_f = 0.77) to afford the desired product: 0.18 g, 62%; mp 145–146 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.45–1.70 (m, 6H, 3CH₂); 1.73–1.88 (m, 2H, CH₂); 2.60–2.73 (m, 1H, CH); 7.16–7.26 (m, 3H, 3CH); 7.31 (d, 1H, CH, J = 6.0 Hz); 7.81–7.89 (m, 2H, 2CH); 11.54 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.32 (2C); 30.62 (2C); 42.44; 103.08; 115.82 (d, 2C, J = 21.0 Hz); 127.89 (d, 2C, J = 8.0 Hz); 131.92; 149.61; 160.78; 163.21; 169.11. FAB(+)-MS (m/z, %): 290.2 [(M⁺+1), 100], 185.1 (20). HRMS (m/z) calculated for C₁₅H₁₆FN₃S: 289.1049. Found: 289.1052. Anal. Calcd. for C₁₅H₁₆FN₃S: C, 62.26; H, 5.57; N, 14.52. Found: C, 62.24; H, 5.56; N, 14.55.

(E)-4-(4-Chlorophenyl)-2-(2-(cyclopentylmethylene)hydrazinyl)thiazole (3b)

2-Bromo-1-(4-chlorophenyl)ethanone was reacted with 2. Yield: 0.12 g, 38%, (dichloromethane/methanol, 90:10, R_f=0.86); mp 155–156 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.47–1.70 (m, 6H, 3CH₂); 1.74–1.86 (m, 2H, CH₂); 2.61–2.73 (m, 1H, CH); 7.29 (s, 1H, CH); 7.34 (d, 1H, CH, J= 6.0 Hz); 7.45 (d, 2H, 2CH, J = 9.0 Hz); 7.83 (d, 2H, 2CH, J = 9.0 Hz); 11.60 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.33 (2C); 30.55 (2C); 42.44; 104.45; 127.81 (2C); 129.07 (2C); 132.61; 133.19; 148.12; 151.16; 169.21. FAB(+)-MS (m/z, %): 306.2 [(M⁺+1), 100], 212.0 (15), 210.1 (40). HRMS (m/z) calculated for C₁₅H₁₆ClN₃S: 305.0753. Found: 305.0760. Anal. Calcd. for C₁₅H₁₆ClN₃S: C, 58.91; H, 5.27; N, 13.74. Found: C, 58.90; H, 5.28; N, 13.76.

(E)-2-(2-(Cyclopentylmethylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole (3c)

2-Bromo-1-(4-methoxyphenyl)ethanone was reacted with 2. Yield: 0.25 g, 82%, (dichloromethane/methanol, 95:5, R_f=0.83); mp 109–112 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.45–1.70 (m, 6H, 3CH₂); 1.75–1.85 (m, 2H, CH₂); 2.60–2.73 (m, 1H, CH); 3.78 (s, 3H, CH₃); 6.94 (d, 2H, 2CH, J= 9.0 Hz); 7.03 (s, 1H, CH); 7.30 (d, 1H, CH, J = 6.0 Hz); 7.75 (d, 2H, 2CH, J = 9.0 Hz); 11.50 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.33 (2C); 30.63 (2C); 42.43; 55.58; 101.19; 114.38 (2C); 127.27 (2C); 128.13; 149.47; 150.53; 159.19; 168.90. FAB(+)-MS (m/z, %): 302.3 [(M⁺+1), 100], 232.1 (20), 206.1 (30). HRMS (m/z) calculated for C₁₆H₁₉N₃OS: 301.1249. Found: 301.1239. Anal. Calcd. for C₁₆H₁₉N₃OS: C, 63.76; H, 6.35; N, 13.94. Found: C, 63.79; H, 6.33; N, 13.95.

(E)-4-(4-Bromophenyl)-2-(2-(cyclopentylmethylene)hydrazinyl)thiazole (3d)

2-Bromo-1-(4-bromophenyl)ethanone was reacted with 2. Yield: 0.23 g, 66%, (dichloromethane/methanol, 90:10, R_f=0.84); mp 155–156 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.45–1.71 (m, 6H, 3CH₂); 1.74–1.87 (m, 2H, CH₂); 2.61–2.73 (m, 1H, CH); 7.30 (s, 1H, CH); 7.33 (d, 1H, CH, J = 6.0 Hz); 7.58 (d, 2H, 2CH, J = 9.0 Hz); 7.77 (d, 2H, 2CH, J = 9.0 Hz); 11.60 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.60 (2C); 30.69 (2C); 42.44; 104.51; 121.16; 128.10 (2C); 131.98 (2C); 133.67; 148.34; 151.04; 169.20. FAB(+)-MS (m/z, %): 350.2 [(M⁺+1), 100], 256.1 (15), 174.1 (30). HRMS (m/z) calculated for C₁₅H₁₆BrN₃S: 349.0248. Found: 349.0238. Anal. Calcd. for C₁₅H₁₆BrN₃S: C, 51.43; H, 4.60; N, 12.00. Found: C, 51.42; H, 4.60; N, 12.03.

(E)-2-(2-(Cyclopentylmethylene)hydrazinyl)-4-(4-(trifluoromethyl)phenyl)thiazole (3e)

2-Bromo-1-(4-trifluoromethylphenyl)ethanone was reacted with 2. Yield: 0.16 g, 49%, (dichloromethane/methanol, 95:5, R_f=0.74); mp 146–147 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.46–1.70 (m, 6H, 3CH₂); 1.75–1.86 (m, 2H, CH₂); 2.61–2.73 (m, 1H, CH); 7.34 (d, 1H, CH, J = 6.0 Hz); 7.47 (s, 1H, CH); 7.74 (d, 2H, 2CH, J = 8.0 Hz); 8.03 (d, 2H, 2CH, J = 8.0 Hz); 11.60 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.32 (2C); 30.56 (2C); 42.43; 106.29; 125.99 (m, 2C); 126.54 (2C); 128.03 (q, J = 32.0 Hz); 128.87; 138.56; 148.65; 150.51; 169.31. FAB(+)-MS (m/z, %): 340.3 [(M⁺+1), 100], 244.2 (15). HRMS (m/z) calculated for C₁₆H₁₆F₃N₃S: 339.1017. Found: 339.1016. Anal. Calcd. for C₁₆H₁₆F₃N₃S: C, 56.62; H, 4.75; N, 12.38. Found: C, 56.59; H, 4.77; N, 12.40.

(E)-2-(2-(Cyclopentylmethylene)hydrazinyl)-4-p-tolylthiazole (3f)

2-Bromo-1-(4-methylphenyl)ethanone was reacted with 2. Yield: 0.24 g, 85%, (dichloromethane/methanol, 95:5, R_f=0.74); mp 156–157 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.48–1.70 (m, 6H, 3CH₂); 1.75–1.86 (m, 2H, CH₂); 2.32 (s, 3H, CH₃); 2.62–2.74 (m, 1H, CH); 7.16 (s, 1H, CH); 7.21 (d, 2H, 2CH, J = 8.0 Hz); 7.39 (d, 1H, CH, J = 6.0 Hz); 7.69 (d, 2H, 2CH, J = 8.0 Hz); 11.50 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 21.29; 25.36 (2C); 30.49 (2C); 42.46; 103.11; 126.21 (2C); 129.70 (2C); 138.07; 147.37; 150.88; 152.86; 169.16. FAB(+)-MS (m/z, %): 286.3 [(M⁺+1), 100], 190.1 (15). HRMS (m/z) calculated for C₁₆H₁₉N₃S: 285.1300. Found: 285.1303. Anal. Calcd. for C₁₆H₁₉N₃S: C, 67.33; H, 6.71; N, 14.72. Found: C, 67.31; H, 6.70; N, 14.75.

(E)-4-(2-(2-(Cyclopentylmethylene)hydrazinyl)thiazol-4-yl)benzonitrile (3g)

4-(Bromoacetyl)benzonitrile was reacted with 2. Yield: 0.26 g, 87%, (dichloromethane/methanol, 95:5, R_f=0.76); mp 151–153 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.46–1.71 (m, 6H, 3CH₂); 1.74–1.86 (m, 2H, CH₂); 2.61–2.73 (m, 1H, CH); 7.34 (d, 1H, CH, J = 6.0 Hz); 7.54 (s, 1H, CH); 7.85 (d, 2H, 2CH, J = 9.0 Hz); 8.00 (d, 2H, 2CH, J = 8.0 Hz); 11.60 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.32 (2C); 30.57 (2C); 42.44; 107.33; 109.99; 119.44; 126.58 (2C);132.81; 133.10 (2C); 139.13; 150.39; 169.32. FAB(+)-MS (m/z, %): 297.2 [(M⁺+1), 100], 201.1 (15). HRMS (m/z) calculated for C₁₆H₁₆N₄S: 296.1096. Found 296.1096. Anal. Calcd. for C₁₆H₁₆N₄S: C, 64.84; H, 5.44; N, 18.90. Found: C, 64.81; H, 5.46; N, 18.91.

(E)-2-(2-(Cyclopentylmethylene)hydrazinyl)-4-(4-nitrophenyl)thiazole (3h)

2-Bromo-1-(4-nitrophenyl)ethanone was reacted with 2. Yield: 0.24 g, 75%, (dichloromethane/methanol, 95:5, R_f=0.54); mp 140–144 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.46–1.71 (m, 6H, 3CH₂); 1.74–1.87 (m, 2H, CH₂); 2.60–2.74 (m, 1H, CH); 7.34 (d, 1H, CH, J = 6.0 Hz); 7.62 (s, 1H, CH); 8.08 (d, 2H, 2CH, J = 9.0 Hz); 8.26 (d, 2H, 2CH, J = 9.0 Hz); 11.70 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.33 (2C); 30.58 (2C); 42.44; 108.33; 124.53 (2C); 126.76 (2C); 141.22; 146.62; 148.75; 150.23; 169.41. FAB(+)-MS (m/z, %): 317.3 [(M⁺+1), 100], 301.3 (30), 147.1 (30). HRMS (m/z) calculated for C₁₅H₁₆N₄O₂S: 316.0994. Found: 316.0986. Anal. Calcd. for C₁₅H₁₆N₄O₂S: C, 56.94; H, 5.10; N, 17.71. Found: C, 56.92; H, 5.09; N, 17.72.

(E)-2-(2-(Cyclopentylmethylene)hydrazinyl)-4-(3,4-dichlorophenyl)thiazole (3i)

2-Bromo-1-(3′4′-dichlorophenyl)ethanone was reacted with 2. Yield: 0.22 g, 65%, (dichloromethane/methanol, 95:5, R_f=0.81); mp 149–150 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.45–1.70 (m, 6H, 3CH₂); 1.73–1.86 (m, 2H, CH₂); 2.60–2.73 (m, 1H, CH); 7.32 (d, 1H, CH, J= 6.0 Hz); 7.44 (s, 1H, CH); 7.65 (d, 1H, CH, J = 8.0 Hz); 7.80 (dd, 1H, CH, J₁=2.0 Hz; J₂= 8.0); 8.04 (d, 1H, CH, J= 2.0 Hz); 11.50 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.32 (2C); 30.58 (2C); 40.43; 105.67; 126.01; 127.60; 130.06; 131.28; 131.84; 135.67; 147.99; 150.19; 169.24. FAB(+)-MS (m/z, %): 240.2 [(M⁺+1), 100]. HRMS (m/z) calculated for C₁₅H₁₅Cl₂N₃S: 339.0364. Found: 339.0352. Anal. Calcd. for C₁₅H₁₅Cl₂N₃S: C, 52.95; H, 4.44; N, 12.35. Found: C, 52.92; H, 4.41; N, 12.35.

(E)-Ethyl 2-(2-(2-(cyclopentylmethylene)hydrazinyl)thiazol-4-yl)acetate (3j)

Ethyl 4-chloro-3-oxobutanoate was reacted with 2. Yield: 0.14 g, 39%, (dichloromethane/methanol, 95:5, R_f=0.50); mp 109–110 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.18 (t, 3H, CH₃, J = 7.0 Hz); 1.43–1.68 (m, 6H, 3CH₂); 1.73–1.83 (m, 2H, CH₂); 2.60–2.71 (m, 1H, CH); 3.53 (s, 2H, CH₂); 4.07 (q, 2H, CH₂, J = 7.0 Hz); 6.53 (s, 1H, CH); 7.24 (d, 1H, CH, J = 6.0 Hz); 11.35 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 14.56; 25.30 (2C); 30.62 (2C); 37.54; 42.38; 60.64; 105.15; 145.30; 149.30; 168.85; 170.50. FAB(+)-MS (m/z, %): 282.3 [(M⁺+1), 100]. HRMS (m/z) calculated for C₁₃H₁₉N₃O₂S: 281.1198. Found: 295.1191. Anal. Calcd. for C₁₃H₁₉N₃O₂S: C, 55.49; H, 6.81; N, 14.93. Found: C, 55.52; H, 6.79; N, 14.95.

(E)-Ethyl 2-(2-(cyclopentylmethylene)hydrazinyl)-4-methylthiazole-5-carboxylate (3k)

Ethyl 2-chloro-3-oxobutanoate was reacted with 2. Yield: 0.18 g, 68%, (dichloromethane/methanol, 95:5, R_f=0.59); mp 143–147 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.25 (t, 3H, CH₃, J = 7.0 Hz); 1.47–1.68 (m, 6H, 3CH₂); 1.74–1.85 (m, 2H, CH₂); 2.44 (s, 3H, CH₃); 2.61–2.74 (m, 1H, CH); 4.18 (q, 2H, CH₂, J = 7.0 Hz); 7.38 (d, 1H, CH, J = 6.0 Hz); 11.93 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 14.76; 17.71; 25.32 (2C); 30.41 (2C); 42.41; 60.42; 112.29; 152.60; 162.43; 169.89. FAB(+)-MS (m/z, %): 282.3 [(M⁺+1), 100]. HRMS (m/z) calculated for C₁₃H₁₉N₃O₂S: 281.1198. Found: 281.1193. Anal. Calcd. for C₁₃H₁₉N₃O₂S: C, 55.49; H, 6.81; N, 14.93. Found: C, 55.47; H, 6.80; N, 14.95.

(E)-2-(2-(Cyclopentylmethylene)hydrazinyl)-4-methylthiazole (3l)

1-Chloropropan-2-one was reacted with 2. Yield: 0.15 g, 72%, (dichloromethane/methanol, 95:5, R_f=0.55); mp 70–75 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.44–1.67 (m, 6H, 3CH₂); 1.73–1.84 (m, 2H, CH₂); 2.12 (s, 3H, CH₃); 2.57–2.69 (m, 1H, CH); 6.27 (s, 1H, CH); 7.25 (d, 1H, CH, J = 6.0 Hz); 11.17 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 17.65; 25.31 (2C); 30.65 (2C); 42.41; 102.15; 148.00; 149.20; 168.63. FAB(+)-MS (m/z, %): 210.2 [(M⁺+1), 100]. HRMS (m/z) calculated for C₁₀H₁₅N₃S: 209.0987. Found: 209.0990. Anal. Calcd. for C₁₀H₁₅N₃S: C, 57.38; H, 7.22; N, 20.08. Found: C, 57.35; H, 7.20; N, 20.10.

(E)-2-(2-(Cyclopentylmethylene)hydrazinyl)-4-(adamant-1-yl)thiazole (3m)

1-Adamantyl bromomethyl ketone was reacted with 2. Yield: 0.24 g, 74%, (dichloromethane/methanol, 95:5, R_f=0.44); mp 153–154 °C. ¹H NMR (DMSO-d₆), δ (ppm): 1.48–1.68 (m, 11H, 6CH₂); 1.68–1.76 (m, 2H, CH₂); 1.76–1.90 (m, 7H, 4CH₂); 1.99–2.05 (m, 3H, 3CH); 2.66–2.78 (m, 1H, CH); 6.52–6.55 (m, 1H, CH); 7.63–7.70 (m, 1H, CH); 12.05 (bs, 1H, NH). ¹³C NMR (DMSO-d₆, 100 MHz), δ (ppm): 25.38 (2C); 28.09 (3C); 30.41 (2C); 35.82 (3C); 36.39 (3C); 40.80; 42.43; 101.32; 154.46; 156.05; 169.28. FAB(+)-MS (m/z, %): 230.3 [(M⁺+1), 100]. HRMS (m/z) calculated for C₁₉H₂₇N₃S: 329.1926. Found: 329.1935. Anal. Calcd. for C₁₉H₂₇N₃S: C, 69.26; H, 8.26; N, 12.75. Found: C, 69.30; H, 8.29; N, 12.78.

In vivo pharmacology

Animals and housing conditions

Adult male Albino Swiss (CD-1) mice weighing between 18 and 22 g were used for in vivo tests. The animals were housed in groups of 10 mice per cage at room temperature of 22 ± 2 °C, under light/dark (12:12) cycle. The animals had free access to food and tap water before the experiments. The ambient temperature of the experimental room and humidity was kept consistent throughout all the tests. For behavioral experiments, the animals were selected randomly. Each group consisted of 7–10 animals/dose, and each mouse was used only once. The experiments were performed between 8 a.m. and 2 p.m. Immediately after the in vivo assay the animals were euthanized by cervical dislocation. All procedures for animal maintenance and treatment were approved by the Local Ethics Committee of the Jagiellonian University in Krakow (ZI/862/2013).

Chemicals used in pharmacological tests

PTZ, pilocarpine hydrochloride and scopolamine butylbromide were provided by Sigma Aldrich (Poznań, Poland). For in vivo tests they were prepared in 0.9% saline (Polfa Kutno, Poland). Ethosuximide (ETX, ICN Polfa Rzeszow, Poland) dissolved in distilled water was used as a reference drug in the PTZ model. PTZ and pilocarpine were administered 60 min after the test compound or the vehicle. The test compounds were prepared in 1% Tween 80 (Polskie Odczynniki Chemiczne, Gliwice, Poland). Control mice received 1% Tween 80.

In behavioral assays to establish ED₅₀ value, a dose of 30 mg/kg was chosen as a starting dose for each compound.

The dose of 300 mg/kg was the maximal dose that was tested in vivo.

PTZ seizure test

The test was performed according to Łuszczki et al.19 with some minor modification20. Clonic convulsions were induced by the subcutaneous (sc) administration of PTZ at a dose of 100 mg/kg. After PTZ injection, the mice were placed separately into transparent Plexiglas cages (30 × 20 × 15) and were observed during the next 30 min for the occurrence of clonic seizures. Clonic seizures were defined as clonus of the whole body lasting more than 3 s, with an accompanying loss of righting reflex. The latency to first clonus, the number of seizure episodes and mortality rate were noted and compared with vehicle-treated and drug-treated groups.

Maximal electroshock seizure test

MES test was performed as previously described20. Briefly, vehicle-treated mice and drug-treated mice received a stimulus of 25 mA delivered by an electroshock generator (Hugo Sachs rodent shocker, March-Hugstetten, Germany) to induce maximal seizures (tonic extension) of hind limbs. Electroconvulsions were produced with the use of auricular electrodes and the stimulus duration was 0.2 s. The endpoint for this procedure was the tonic extension of the hind limbs.

Pilocarpine-induced seizures

The mice were intraperitoneally pretreated with the investigated compound or vehicle and 60 min later they received pilocarpine (400 mg/kg, ip). To avoid peripheral toxicity and diarrhea, masticatory and stereotyped movements, animals treated with pilocarpine also received scopolamine butylbromide (1 mg/kg, ip) which was injected 45 min before pilocarpine. After the administration of the convulsant, the mice were observed during 60 min for behavioral changes. Latency time to the onset of status epilepticus and latency time to death were noted in each experimental group21.

Rotarod test

In this test, only the highest, previously tested in the PTZ model, dose of each compound was assessed for its potential ability to impair motor coordination. The test was performed according to the method recently described22. Briefly the mice were trained daily for three days on the rotarod apparatus (Rotarod apparatus, May Commat RR0711, Ankara, Turkey; rod diameter: 2 cm) rotating at a constant speed of 18 rotations per minute (rpm). During each training session, the animals were placed on a rotating rod for 3 min with an unlimited number of trials. The proper experimentation was conducted 24 h after the final training trial. In order to establish the impact of the test compounds on animals’ motor coordination, 60 min before the rotarod test the mice were ip pretreated with each compound and then they were tested on the rotarod apparatus revolving at 6, 18 and 24 rpm. Motor impairments, defined as the inability to remain on the rotating rod for 1 min, were measured at each speed and the mean time spent on the rod was counted in each experimental group.

Data analysis

Data analysis of the results obtained in behavioral tests was provided by GraphPad Prism Software (ver. 5.0, La Jolla, CA). The results were statistically evaluated using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc comparison. p < 0.05 was considered significant.

To establish ED₅₀ value for each compound the log-probit method was applied23. In PTZ test, ED₅₀ was defined as the dose of the investigated compound that prolonged the latency to first clonus by 50%.

Results and discussion

Chemistry

Synthesis of desired thiazole compounds consists of two steps. In the first step, cyclopentanecarbaldehyde thiosemicarbazone 2 was synthesized through reaction of cyclopentanecarbaldehyde1 with thiosemicarbazide in absolute ethanol in the presence of catalytic amount of glacial acetic acid and under reflux with 78% yield. In the next step, Hantzsch cyclization reaction of thiosemicarbazone 2 with appropriate para-substituted bromoacetophenones, ethyl 4-chloroacetoacetate, ethyl 2-chloroacetoacetate, chloroacetone and 1-adamantyl bromomethyl ketone afforded the corresponding thiazoles 3a–3m with good yield (38–87%) and with high chemical purity. The reaction pathway has been summarized in Scheme 1.

Scheme 1. Synthesis of the target compounds 3a–3m.

All obtained thiazoles were purified on silica gel column chromatography, and in all cases we obtained only pure, most stable (E)-isomers24,25. All compounds were fully characterized spectroscopically using ¹H and ¹³C NMR, FAB(+)-MS, HRMS and elemental analyses. The mass spectra of all compounds are fully consistent with assigned structures. The ¹H NMR spectrum of 2 presents typical three proton signals of NH₂ and NH groups at 7.41, 7.94 and 10.99 ppm, respectively. These three signals are due to the exchange of H between the terminal NH₂ group and sulfur atom.

¹H NMR spectra of azole derivatives showed singlet at δ (6.27–7.62) due to thiazole-5H and singlet at δ (11.17–12.05) indicating the presence of hydrazide NH proton, which confirms the conversion of substrates into the expected products. The proton of CH=N group occurs in the spectra around 7.24–7.39 ppm. The elemental analyses values are in good agreement with the calculated values confirming the formation of analytically pure products.

All reactions were repeated at least two times and are fully reproducible.

In vivo pharmacology

PTZ-induced seizures

In this screening test, 13 compounds (3a–3m) were tested for their ability to prolong latency to the onset of the first clonic seizure episode and to reduce the number of seizure episodes. For all of the compounds, except for 3c, 3i and 3j, median effective doses (ED₅₀ values) were established (Table 1). Noteworthy, ED₅₀ values obtained for the anticonvulsant active compounds were significantly lower than that reported for ETX (147.8 mg/kg)26.

Table 1. Anticonvulsant potencies of the test compounds 3a–3m and the reference drug (ETX) in the PTZ model expressed as ED₅₀.

Compound	ED50 (mg/kg)
3a	9.2 (7.5–11.3)
3b	18.8 (17.5–20.2)
3c	NE
3d	20.4 (18.4–22.5)
3e	12.0 (9.5–15.2)
3f	14.1 (11.8–16.8)
3g	65.3 (42.9–99.2)
3h	52.8 (41.7–66.9)
3i	NE
3j	NE
3k	12.5 (7.8–19.9)
3l	27.8 (20.3–37.9)
3m	17.6 (15.2–20.3)
ETX*	147.8 (130.7–167.3)

Values in parentheses are 95% confidence intervals determined by probit analysis.

NE indicates not established within the tested dose range (up to 300 mg/kg, ip).

* Data from literature26.

The test compounds also influenced the number of seizure episodes induced by PTZ administration. As shown in Figure 1, compounds 3a (30 mg/kg), 3b (25, 30 mg/kg), 3d (20 mg/kg), 3f (30 mg/kg), 3g (300 mg/kg) and 3h (100 mg/kg) decreased the mean number of seizure episodes in a statistically significant manner (p < 0.05).

Figure 1. Influence of the test compounds 3a–3g (a) and 3h–3m (b) on the number of seizure episodes in PTZ-induced seizure test. Statistical analysis: ANOVA, followed by Dunnett’s post hoc comparison. Significance versus vehicle-treated group: *p < 0.05.

In this assay, ETX (100 mg/kg, ip) reduced the mean number of seizure episodes by 64% (significant versus vehicle-treated mice at p < 0.05).

In the PTZ model, the mortality rate was affected by the test compounds to various degrees. In vehicle-treated mice, the mean mortality rate was 75%. ETX (100 mg/kg), 3a, 3d, 3e, 3j (30 mg/kg), 3b (20, 25 and 30 mg/kg), 3f (15 and 30 mg/kg), 3g (300 mg/kg), and 3h (30, 100 and 300 mg/kg) protected all animals from death. The influence of the test compounds on animals’ mortality is shown in Figure 2.

Figure 2. Influence of compounds 3a–3 g (a) and 3 h–3m (b) on mortality rate in the PTZ model. Data are shown as % of animals that died in each experimental group.

MES test

None of the investigated compounds within the tested dose range showed anticonvulsant activity in this model. Tonic hind limb extension was noted in experimental animals, which indicates that the investigated compounds 3a–3m do not protect animals from electrically induced seizures.

Pilocarpine-induced seizures

For extended studies on the anticonvulsant activity in the mouse model of status epilepticus the compound 3a at the dose of 30 mg/kg was chosen. Previously, in the PTZ model this dose not only significantly delayed the seizure onset, but it also decreased the number of seizure episodes and protected all experimental animals from death. In pilocarpine-induced seizures, the compound 3a was not effective. Compared with vehicle-treated mice it did not prolong the latency to status epilepticus (288 s ± 15) for the control group and 292 s ± 14 for 3a-treated mice). Latency to death was not affected either (369 s ± 34 and 368 s ± 11, respectively).

Rotarod test

All compounds at the highest dose that was previously tested in the PTZ model were additionally assessed for their influence on animals’ motor coordination in the rotarod test. The results obtained in this test demonstrated that none of the investigated compounds induced motor deficits in experimental animals (Figure 3).

Figure 3. Influence of the investigated compounds 3a–3m on motor coordination of experimental animals measured in the rotarod test revolving at 6 rpm (a), 18 rpm (b) and 24 rpm (c). Results are shown as the mean time spent on the rotating rod (±SEM). Statistical analysis: ANOVA, followed by Dunnett’s post hoc test: 6 rpm – F[13,52] = 0.6279, p > 0.05; 18 rpm – F[13,52] = 0.6168, p > 0.05; 24 rpm – F[13,52] = 0.6874, p > 0.05.

The search for new AEDs with better pharmacological efficacy and tolerability is still an important goal in the anticonvulsant drug discovery. This process strongly relies on the implementation of preclinical animal models before the anticonvulsant active agents are tested in clinical trials in humans2.

In the present research, three distinct mouse models of seizures were implemented in the in vivo tests. Two of them, i.e. MES test and PTZ test are regarded as simple seizure models that allow to test large numbers of compounds for their potential anticonvulsant activity in relatively short time2. In view of this, these easy-to-perform assays were used for the anticonvulsant screening of compounds 3a–3m to predict their potential clinical activity.

Noteworthy, these screening assays are not only used to identify novel AEDs, but they also enable to predict the efficacy of AEDs against different types of seizures in humans2. The PTZ model of clonic seizures generally refers to nonconvulsive (absence or myoclonic) seizures in humans, whereas MES test is predictive for generalized tonic–clonic seizures2. The efficacy of the test compounds in the PTZ model and the lack of anticonvulsant protection demonstrated in MES test suggest that these compounds might be potentially effective in absence seizures in humans but not in grand mal epilepsy.

The observed distinct efficacy of the compounds tested in PTZ test and MES test might also suggest a potential mechanism of their action. Drugs acting by blockade of voltage-gated sodium and to a lesser degree calcium channels (with the exception of ETX) are effective in MES test, and they show no anticonvulsant protection in the PTZ model, whereas numerous GABAergic AEDs and the T-type calcium channel blocker ETX are effective in the PTZ model. Mixed AEDs are active in both tests which is thought to be due to their multi-target profile of the anticonvulsant activity2,3. Bearing that in mind, it can be concluded that the influence of the test compounds on ion channels as a mechanism underlying their anticonvulsant activity is rather implausible. It should be however noted that the elucidation of the precise mechanism of action of compounds 3a–3m requires further extended studies. In view of the chemical similarity of these compounds to well-known MAO inhibitors9,10, this potential mechanism of the observed in vivo activity should be thoroughly considered.

Similarly to the PTZ model, pilocarpine-induced seizure test is also an animal model using a chemoconvulsant agent to induce seizures. In contrast to PTZ, pilocarpine evokes spontaneous recurrent seizures that mimic status epilepticus in humans and is regarded as a very useful tool to study drug-resistant epilepsy1. In this model, the compound 3a was not effective.

Motor coordination deficits are one of the most frequent adverse effects of AEDs and this effect might be a serious limitation of antiepileptic pharmacotherapy. We demonstrated that the test compounds at a dose range which effectively reduces seizure episodes do not cause motor deficits in experimental animals.

Conclusion

In summary, we have developed an efficient and economic method for the synthesis of 2-(cyclopentylmethylene)-hydrazinyl-1,3-thiazoles. In vivo pharmacological studies of the synthesized compounds have shown that the type of substituent in the para-position of benzene ring is very important for their anticonvulsant activity.

The results of the anticonvulsant screening reveal that seven compounds: 3a, 3b, 3d, 3e, 3f, 3k and 3m containing F-, Cl-, Br-, CF₃-, CH₃- and adamantyl substituents demonstrated the most significant anticonvulsant activity in the PTZ model, respectively. No effect was observed in electrically induced seizures, and no protection was shown in pilocarpine-induced seizures, either. Noteworthy, none of these compounds impaired animals’ motor skills in the rotarod test. Since the compounds 3a and 3b in a statistically significant manner delayed the onset of clonic seizures and reduced the number of seizure episodes in the PTZ test, they can be regarded as interesting novel lead structures in the search for new AEDs.

Declaration of interest

This study was supported by the Nicolaus Copernicus University (project no. 786/2014) and by the grant from the National Science Centre DEC-2012/05/B/NZ7/02705. The authors confirm that this article content has no conflicts of interest.