Synthesis, antimicrobial and anticonvulsant screening of small library of tetrahydro-2H-thiopyran-4-yl based thiazoles and selenazoles

Abstract

Synthesis and investigation of antimicrobial activity of 22 novel thiazoles and selenazoles derived from dihydro-2H-thiopyran-4(3H)-one are presented. Additionally, anticonvulsant activity of six derivatives is examinated. Among the derivatives, compounds 4a–f, 4i, 4k, 4 l, 4n, 4o–s and 4v have very strong activity against Candida spp. with MIC = 1.95–15.62 μg/ml. In the case of compounds 4a–f, 4i, 4k, 4 l, 4n, 4o, 4r and 4s, the activity is very strong against some strains of Candida spp. isolated from clinical materials, with MIC = 0.98 to 15.62 μg/ml. Additionally, compounds 4n-v are found to be active against Gram-positive bacteria with MIC = 7.81–62.5 μg/ml. The results of anticonvulsant screening reveal that compounds 4a, 4b, 4m and 4n demonstrate a statistically significant anticonvulsant activity in the pentylenetetrazole model, whereas compounds 4a and 4n showed protection in 6-Hz psychomotor seizure model. Noteworthy, none of these compounds impaired animals’ motor skills in the rotarod test. We also performed quantum chemical calculation of interaction and binding energies in complex of 4a with cyclodextrin.

Keywords:

• Anticonvulsant activity
• Candida spp.
• selenazole
• tetrahydro-4H-thiopyran-4-one
• thiazole

Introduction

Treatment of infectious diseases caused by bacteria and fungi is an increasing problem being fought in many research laboratories over the world1. This is due to the increasing number of multi-drug resistant microorganisms caused by widespread and irresponsible use of broad-spectrum antibiotics, anticancer, immunosuppressive and anti-HIV drugs2. People with serious underlying diseases are very susceptible to the attack of Candida spp., naturally occurring in the human body, and other very dangerous pathogens, such as Aspergillus spp. and Cryptococcus spp3. A possible solution to the observed drug-resistance of microorganisms is responsible, prudent and not speculative use of already existing drugs, and the search for innovative drugs possessing a different mechanism of action.

In the recent years, a strong link between infections of the central nervous system and various neurological disorders has been shown4–6. It was demonstrated that bacterial (typical bacterial meningitis, tuberculosis), viral (HSV, HHV-6), fungal (candidiasis, coccidiodomycosis) or parasitic (cerebral toxoplasmosis, neurocysticercosis, malaria) infections are one of the major risk factors associated with the onset of epilepsy4,6 that might be regarded as an under-recognized long-term complication of infections within the central nervous system5,6.

In general, epileptic seizures are characterized by recurrent and unprovoked seizure episodes that are triggered by numerous infectious and non-infectious conditions5. So far, more than 20 antiepileptic drugs (AEDs) have been identified, however, ∼30% of epileptic patients are resistant to available treatment7. Furthermore, adverse effects of AEDs and their poor tolerability often limit antiepileptic therapy8–10. Hence, there is still a need for finding novel compounds with antiepileptic activity and exploring novel drug targets for their pharmacological action7. Moreover, based on available knowledge on the pathogenic factors underlying epilepsy, it is particularly important to find ‘hybrid’ drugs which not only fight against neuroinfections, but also possess anticonvulsant efficacy.

Thiazole is easily metabolized by known biochemical reactions, and is non-cancerogenic in nature11 which makes it a good scaffold for a wide variety of antibacterial12,13, antifungal14–17 and anticonvulsant18,19 drugs. Introduction of selenium into this class of compounds, leading to selenazoles, in most cases influences their chemical properties and in turn changes their biological activity20.

In the recent years, selenazoles have been reported to have very unique chemical and biological properties such as inactivation of free radicals21, antioxidant22–25, cancer cell proliferation25–27 and protein kinase activation28. Selenium atom is also included in very important components of the human body such as selenoproteins29, and glutathione peroxidase-a key enzyme in antioxidant defense30. One of the most well known anti-cancer drugs containing a selenium atom is selenazofurin (2-β-D-ribofuranosylselenazole-4-carboxamide), five to ten times more potent in in vitro and in vivo anti-tumor screenings than thiazofurin31.

In our earlier study it has been shown that compounds possessing selenazol-2-yl-hydrazine pharmacophore and cyclohexanone moiety demonstrated high antifungal activity32,33. Keeping in mind the above facts and continuing our previous investigation on the synthesis and molecular interaction of biologically active azoles34–37, we decided to prepare twelve novel 2,4-disubstituted 1,3-thiazoles and ten novel 2,4-disubstituted 1,3-selenazoles containing tetrahydro-2H-thiopyran-4-yl pharmacophore to improve their biological properties and increase the possibility of their future use.

Based on our previous experience, we also inserted fluorine-, chlorine-, bromine-, iodine-, methoxy-, methyl-, trifluoromethyl-, nitro- and cyano- groups in the para-position in the phenyl ring. We also examined several derivatives with methyl-, -COOEt and -CH₂COOEt groups attached directly to the thiazole ring. All of these substituents are useful to modulate electronic effects, and may influence the hydrophilic and hydrophobic properties of the molecules.

Next, the synthesized compounds were evaluated for their antimicrobial activity against a panel of reference strains of 30 microorganisms, including Gram-positive bacteria, Gram-negative bacteria and fungi belonging to yeasts.

The microorganisms came from American Type Culture Collection (ATCC), routinely used for the evaluation of antimicrobials, and from clinical materials. In the in vivo part of this research anticonvulsant properties of three selected thiazoles and three selenazoles were assessed in the mouse model of pentylenetetrazole (PTZ)-induced seizures. Four compounds active in this test were additionally tested in the 6-Hz psychomotor seizure model of partial epilepsy and their influence on animals’ motor coordination was assessed in the rotarod test. We have also performed Density Functional Theory (DFT) calculation of interaction energy and binding energy of complex of selected thiazole derivative with β-cyclodextrin. Such complexes could increase bioavailability of proposed drugs.

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. FAB(+)-MS was performed by the Laboratory for Analysis of Organic Compounds and Polymers of the Center for Molecular and Macromolecular Studies of the Polish Academy of Science in Łódź. MS spectra were recorded on a Finnigan MAT 95 spectrometer. Elemental analysis was performed on ELEMENTAR Vario MACRO CHN. 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. Dihydro-2H-thiopyran-4(3H)-one, ethyl 4-chloro-3-oxobutanoate, ethyl 2-chloro-3-oxobutanoate, 1-chloro-propan-2-one, potassium selenocyanate, hydrazine hydrate and appropriate bromoketones were commercial materials (Sigma Aldrich, Poznań, Poland).

2-(Tetrahydro-4H-thiopyran-4-ylidene)hydrazinecarbothioamide (2)

Thiosemicarbazide (1.57 g, 17.21 mmol) was added to a stirred solution of dihydro-2H-thiopyran-4(3H)-one (1) (2.0 g, 17.21 mmol) in absolute ethyl alcohol (20 ml) and then (0.9 ml) of acetic acid was added. The reaction mixture was stirred under reflux for 20 h, cooled to room temperature and separate precipitate was collected by filtration to yield 2.49 g (76%); m.p. 136–137 °C; eluent: dichloromethane/methanol (90:10), R_f = 0.64. ¹H NMR (400 MHz, DMSO-d₆):

δ (ppm) 2.53–2.57 (m, 2H, CH₂); 2.68–2.80 (m, 6H, 3CH₂); 7.61 (bs, 1H, NH₂); 8.04 (bs, 1H, NH₂); 10.29 (bs, 1H, NH).

¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 28.11; 29.53; 30.16; 37.05; 154.03; 179.34. Anal. Calcd. for C₆H₁₁N₃S₂:

C, 38.07; H, 5.86; N, 22.20. Found: C, 38.09; H, 5.83; N, 22.24.

2-(Tetrahydro-4H-thiopyran-4-ylidene)hydrazinecarboselenoamide (3)

Hydrazine hydrate, 80% (0.6 ml, 0.01 mmol), concentrated hydrochloric acid (0.86 ml, 0.01 mmol) and a solution of potassium selenocyanate (1.44 g, 0.01 mol) in water (5 ml) were added to a mixture of ethyl alcohol (40 ml) and water (5 ml). Subsequently, dihydro-2H-thiopyran-4(3H)-one (1) (1.35 g, 0.01 mol) was added and the mixture was heated to reflux for 2 h under nitrogen atmosphere. After this time, the solution was filtered while hot to remove the remaining selenium. The filtrate was concentrated under vacuum to 20 ml giving a pale yellow oil which solidified on cooling. The solid was next isolated by filtration, washed with water and left to dry under vacuum. The dry solid was then dissolved in chloroform (60 ml), filtered through celite and the filtrate was evaporated to dryness. The crude product was recrystallized two times from benzene to give a pure product: 0.94 g, (34%); m.p. 129–133 °C; eluent: dichloromethane/methanol (95:5), R_f=0.67. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.49–2.54 (m, 2H, CH₂); 2.69–2.74 (m, 4H, 2CH₂); 2.75–2.79 (m, 2H, CH₂); 8.09 (bs, 1H, NH₂); 8.47 (bs, 1H, NH₂); 10.59 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 28.17; 29.49; 30.52; 37.11; 156.25; 174.81. Anal. Calcd. for C₆H₁₁N₃SSe: C, 30.51; H, 4.69; N, 17.79. Found: C, 30.52; H, 4.71; N, 17.82.

4-(4-Fluorophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-thiazole (4a). Typical procedure

Carbothioamide 2 (0.189 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 (15 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=0.74) to afford the desired product: 0.30 g, 97%; m.p. 191–193 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 2.53–2.60 (m, 2H, CH₂); 2.70–2.82 (m, 6H, 3CH₂); 7.20–7.25 (m, 3H, 3CH); 7.80–7.90 (m, 2H, 2CH); 11.03 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.83; 29.39; 30.41; 36.78; 104.05; 115.97 (d, 2C, J_C–F=22.0 Hz); 128.20 (d, 2C; J_C–F=9.0 Hz); 128.48 (d, J_C–F=9.0 Hz); 147.66; 154.57; 161.00; 170.36. FAB(+)-MS (m/z, %): 308.1 [(M⁺+1), 100], 194.0 (28), 185.1 (24), 152.0 (12), 116.1 (12). Anal. Calcd. for C₁₄H₁₄FN₃S₂: C, 54.70; H, 4.59; N, 13.67. Found: C, 54.73; H, 4.58; N, 13.70.

4-(4-Chlorophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-1,3-thiazole (4b)

2-Bromo-1-(4-chlorophenyl) ethanone was reacted with 2. Yield: 0.31 g, 99%, (dichloromethane/methanol, 90:10, R_f=0.84); m.p. 175–176 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 2.56–2.60 (m, 2H, CH₂); 2.72–2.82 (m, 6H, 3CH₂); 7.32 (s, 1H, CH); 7.45 (d, 2H, 2CH, J = 9 Hz); 7.85 (d, 2H, 2CH, J = 9 Hz); 11.05 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.82; 29.39; 30.28; 36.79; 104.89; 127.76 (2C); 129.08 (2C); 132.49; 133.56; 148.56; 153.60; 170.46. FAB(+)-MS (m/z, %): 324.1 [(M⁺+1), 100], 210.0 (28), 209.0 (12), 116.0 (24). Anal. Calcd. for C₁₄H₁₄ClN₃S₂: C, 51.92; H, 4.36; N, 12.97. Found: C, 51.94; H, 4.39; N, 13.00.

4-(4-Iodophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-thiazole (4c)

2-Bromo-1-(4-iodophenyl)ethanone was reacted with 2. Yield: 0.41 g, 99%, (dichloromethane/methanol, 95:5, R_f=0.62); m.p. 186–189 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.55–2.58 (m, 2H, CH₂); 2.72–2.80 (m, 6H, 3CH₂); 7.32 (s, 1H, CH); 7.62 (d, 2H, 2CH, J = 9 Hz); 7.75 (d, 2H, 2CH, J = 9 Hz); 11.07 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27,82; 29,39; 30,28; 36,79; 104,89; 127,76 (2C); 129,08 (2C); 132,49; 133,56; 148,56; 153,60; 170,46. FAB(+)-MS (m/z, %): 416.1 [(M⁺+1), 100], 302.0 (12), 185.1 (20), 174.0 (12), 116.0 (16). Anal. Calcd. For C₁₄H₁₄IN₃S₂: C, 40.49; H, 3.40; N, 10.12. Found: C, 40.47; H, 3.39; N, 10.15.

4-(4-Methoxyphenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-thiazole (4d)

2-Bromo-1-(4-methoxyphenyl)ethanone was reacted with 2. Yield: 0.30 g, 94%, (dichloromethane/methanol, 90:10, R_f=0.82); m.p. 166–168 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 2.56–2.63 (m, 2H, CH₂); 2.73–2.83 (m, 6H, 3CH₂); 3.78 (s, 3H, CH₃); 6.97 (d, 2H, 2CH, J = 9 Hz); 7.10 (s, 1H, CH); 7.75 (d, 2H, 2CH, J = 9 Hz); 11.02 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.85; 29.38; 30.45; 36.78; 55.68; 102.21; 114.53 (2C); 127.58 (2C); 127.65; 147.75; 155.10; 159.61; 170.20. FAB(+)-MS (m/z, %): 320.1 [(M⁺+1), 100], 206.0 (16), 185.0 (12). Anal. Calcd. for C₁₅H₁₇N₃OS₂: C, 56.40; H, 5.36; N, 13.15. Found: C, 56.40; H, 5.33; N, 13.17.

4-(4-Methylphenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-1,3-thiazole (4e)

2-Bromo-1-(4-methylphenyl)ethanone was reacted with 2. Yield: 0.33 g, 99%, (dichloromethane/methanol, 90:10, R_f=0.84); mp 171–174 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 2.31 (s, 3H, CH₃); 2.56–2.61 (m, 2H, CH₂); 2.73–2.82 (m, 6H, 3CH₂); 7.17 (s, 1H, CH); 7.20 (d, 2H, 2CH, J = 8 Hz); 7.72 (d, 2H, 2CH, J = 8 Hz); 11.07 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 21.32; 27.85; 29.38; 30.48; 36.77; 103.43; 126.12 (2C); 129.71 (2C); 130.81; 137.91; 147.85; 155.24; 170.21. FAB(+)-MS (m/z, %): 304.1 [(M⁺+1), 100], 190.0 (12), 185.1 (16). Anal. Calcd. for C₁₅H₁₇N₃S₂: C, 59.37; H, 5.65; N, 13.85. Found: C, 59.34; H, 5.66; N, 13.88.

4-(4-Bromophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-1,3-thiazole (4f)

2-Bromo-1-(4-bromophenyl)ethanone was reacted with 2. Yield: 0.37 g, 99%, (dichloromethane/methanol, 90:10, R_f=0.77); m.p. 177–179 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 2.56–2.60 (m, 2H, CH₂); 2.72–2.82 (m, 6H, 3CH₂); 7.33 (s, 1H, CH); 7.59 (d, 2H, 2CH, J = 9 Hz); 7.79 (d, 2H, 2CH, J = 9 Hz); 11.08 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.81; 29.40; 30.22; 36.80; 104.90; 120.97; 128.03 (2C); 131.98 (2C); 134.17; 149.05; 153.16; 170.48. FAB(+)-MS (m/z, %): 368.1 [(M⁺+1), 92], 256.0 (16), 245.0 (12), 185.1 (44), 174.0 920), 149.0 (16), 116.0 (20). Anal. Calcd. for C₁₄H₁₄BrN₃S₂: C, 45.65; H, 3.83; N, 11.41. Found: C, 45.62; H, 3.80; N, 11.45.

2-(2-(Tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-4-(4-(trifluoromethyl)phenyl)-1,3-thiazole (4g)

2-Bromo-1-(4-trifluoromethylphenyl)ethanone was reacted with 2. Yield: 0.33 g, 92%, (dichloromethane/methanol, 90:10, R_f=0.80); m.p. 179–180 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 2.55–2.61 (m, 2H, CH₂); 2.73–2.83 (m, 6H, 3CH₂); 7.50 (s, 1H, CH); 7.75 (d, 2H, 2CH, J = 8 Hz); 8.05 (d, 2H, 2CH, J = 8 Hz); 11.04 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.82; 29.40; 30.26; 36.81; 106.84; 123.45; 126.04 (2C); 126.54 (2C); 128.88; 138.64; 148.67; 153.36; 170.62. FAB(+)-MS (m/z, %): 358.1 [(M⁺+1), 100], 244.0 (12), 185.1 (16). Anal. Calcd. for C₁₅H₁₄F₃N₃S₂: C, 50.41; H, 3.95; N, 11.76. Found: C, 50.42; H, 3.93; N, 11.78.

4-(4-Nitrophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-1,3-thiazole (4h)

2-Bromo-1-(4-nitrophenyl)ethanone was reacted with 2. Yield: 0.31 g, 94%, (dichloromethane/methanol, 95:5, R_f=0.80); m.p. 183–184 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 2.56–2.62 (m, 2H, CH₂); 2.73–2.84 (m, 6H, 3CH₂); 7.66 (s, 1H, CH); 8.10 (d, 2H, 2CH, J = 9 Hz); 8.27 (d, 2H, 2CH, J = 9 Hz); 11.03 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.81; 29.39; 30.22; 36.81; 108.89; 124.57 (2C); 126.76 (2C); 141.23; 146.60; 148.70; 153.10; 170.78. FAB(+)-MS (m/z, %): 335.1 [(M⁺+1), 28], 185.1 (100), 149.0 (28), 117.0 (12). Anal. Calcd. for C₁₄H₁₄N₄O₂S₂: C, 50.28; H, 4.22; N, 16.75. Found: C, 50.26; H, 4.20; N, 16.78.

4-(2-(2-(Tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-1,3-thiazol-4-yl)benzonitrile (4i)

4-(Bromoacetyl)benzonitrile was reacted with 2. Yield: 0.30 g, 99%, (dichloromethane/methanol, 95:5, R_f=0.74); m.p. 209–211 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 2.56–2.61 (m, 2H, CH₂); 2.73–2.83 (m, 6H, 3CH₂); 7.57 (s, 1H, CH); 7.86 (d, 2H, 2CH, J = 9 Hz); 8.02 (d, 2H, 2CH, J = 9 Hz); 11.08 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.82; 29.40; 30.24; 36.81; 107.88; 109.96; 119.45; 126.57 (2C); 133.14 (2C); 139.24; 148.86; 153.20; 170.68. FAB(+)-MS (m/z, %): 315.1 [(M⁺+1), 100], 255.2 (20), 219.2 (20), 201.0 (16), 185.1 (28), 173.1 (68), 147.0 (28), 136,0 (40), 131.0 (32), 107.0 (24). Anal. Calcd. for C₁₅H₁₄N₄S₂: C, 57.30; H, 4.49; N, 17.82. Found: C, 57.31; H, 4.49; N, 17.85.

Ethyl 4-methyl-2-(2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-1,3-thiazole-5-carboxylate (4j)

Ethyl 2-chloro-3-oxobutanoate was reacted with 2. Yield: 0.26 g, 87%, (dichloromethane/methanol, 95:5, R_f=0.57); m.p. 203–205 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 1.24 (t, 3H, CH_3, J = 7 Hz); 2.44 (s, 3H, CH₃); 2.55–2.60 (m, 2H, CH₂); 2.72–2.83 (m, 6H, 3CH₂); 4.17 (q, 2H, CH_2, J = 7 Hz); 11.03 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 14.72; 16.70; 28.02; 29.49; 30.56; 36.92; 60.61; 108.09; 153.11; 157.41; 162.27; 169.68. FAB(+)-MS (m/z, %): 300.2 [(M⁺+1), 100], 114.0 (12). Anal. Calcd. for C₁₂H₁₇N₃O₂S₂: C, 48.14; H, 5.72; N, 14.03. Found: C, 48.15; H, 5.70; N, 14.04.

Ethyl (2-(2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-1,3-thiazol-4-yl)acetate (4k)

Ethyl 4-chloro-3-oxobutanoate was reacted with 2. Yield: 0.16 g, 53%, (dichloromethane/methanol, 95:5, R_f=0.54); m.p. 171–172 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 1.20 (t, 3H, CH_3, J = 7 Hz); 2.58–2.63 (m, 2H, CH₂); 2.74–2.84 (m, 6H, 3CH₂); 3.66 (s, 2H, CH₂); 4.09 (q, 2H, CH_2, J = 7 Hz); 6.71 (s, 1H, CH); 11.03 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 14.54; 28.01; 29.44; 31.24; 34.63; 36.81; 61.14; 107.34; 159.30; 169.49; 169.69. FAB(+)-MS (m/z, %): 300.2 [(M⁺+1), 100]. Anal. Calcd. for C₁₂H₁₇N₃O₂S₂: C, 48.14; H, 5.72; N, 14.03. Found: C, 48.12; H, 5.73; N, 14.03.

4-Methyl-2-(2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-1,3-thiazole (4l)

1-Chloropropan-2-one was reacted with 2. Yield: 0.14 g, 61%, (dichloromethane/methanol, 95:5, R_f=0.66); m.p. 148–151 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 2.12 (s, 3H, CH₃); 2.52–2.57 (m, 2H, CH₂); 2.68–2.80 (m, 6H, 3CH_2); 6.22 (s, 1H, CH), 10.70 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 17.17; 27.89; 29.46; 30.03; 36.81; 101.64; 146.11; 152.51; 169.78. EI(+)-MS (m/z, %): 228.0 [(M⁺), 100], 116.0 (20), 113.9 (40). Anal. Calcd. for C₉H₁₃N₃S₂: C, 47.55; H, 5.76; N, 18.48. Found: C, 47.52; H, 5.73; N, 18.51.

4-(4-Fluorophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-selenazole (4m)

2-Bromo-1-(4-fluorophenyl)ethanone was reacted under nitrogen atmosphere with 3. Yield: 0.24 g, 94%, (dichloromethane/methanol, 95:5, R_f=0.71); m.p. 199–200 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.57–2.60 (m, 2H, CH₂); 2.74–2.77 (m, 2H, CH₂); 2.78–2.82 (m, 4H, 2CH₂); 7.20–7.24 (m, 2H, 2CH); 7.55 (s, 1H, CH); 7.80–7.84 (m, 2H, 2CH); 11.04 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.92; 29.40; 30.95; 36.83; 107.16; 116.01 (d, 2C, J_C–F=22.0 Hz); 128.54 (d, 2C; J_C–F=8.0 Hz); 130.47; 157.09; 161.03; 163.47; 173.62. FAB(+)-MS (m/z, %): 356.0 [(M⁺+1), 100], 352.0 (20), 353.0 (24), 354.0 (50), 358.0 (20), 242.0 (10), 185.1 (16), 116.0 (12). Anal. Calcd. for C₁₄H₁₄FN₃SSe: C, 47.46; H, 3.98; N, 11.86. Found: C, 47.49; H, 4.00; N, 11.88.

4-(4-Chlorophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-selenazole (4n)

2-Bromo-1-(4-chlorophenyl)ethanone was reacted under nitrogen atmosphere with 3. Yield: 0.35 g, 95%, (dichloromethane/methanol, 95:5, R_f=0.72); m.p. 206–207 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.57–2.60 (m, 2H, CH₂); 2.74–2.77 (m, 2H, CH₂); 2.78–2.82 (m, 4H, 2CH₂); 7.45 (d, 2H, 2CH, J = 9 Hz); 7.65 (s, 1H, CH); 7.81 (d, 2H, 2CH, J = 9 Hz); 11.05 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.90; 29.42; 30.73; 36.87; 107.97; 128.03 (2C); 129.06 (2C); 132.45; 133.80; 147.60; 155.40; 173.45. FAB(+)-MS (m/z, %): 372.1 [(M⁺+1), 100], 367.9 (18), 369.1 (20), 370.0 (52), 374.0 (48), 185.0 (12), 116.0 (12). Anal. Calcd. for C₁₄H₁₄ClN₃SSe: C, 45.35; H, 3.81; N, 11.33. Found: C, 45.38; H, 3.78; N, 11.36.

4-(4-Iodophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-selenazole (4o)

2-Bromo-1-(4-iodophenyl)ethanone was reacted under nitrogen atmosphere with 3. Yield: 0.32 g, 96%, (dichloromethane/methanol, 95:5, R_f=0.81); m.p. 191–193 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.56–2.59 (m, 2H, CH₂); 2.73–2.76 (m, 2H, CH₂); 2.77–2.81 (m, 4H, 2CH₂); 7.60 (d, 2H, 2CH, J = 9 Hz); 7.66 (s, 1H, CH); 7.74 (d, 2H, 2CH, J = 9 Hz); 11.05 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.90; 29.42; 30.75; 36.86; 108.06; 128.40 (2C); 134.32; 137.83 (2C); 138.19; 147.66; 155.54; 173.46. FAB(+)-MS (m/z, %): 464.0 [(M⁺+1), 100], 459.9 (20), 461.0 (20), 462.0 (56), 466.0 (20), 349.9 (40), 374.9 (20), 222.0 (60), 116.0 (76), 114.0 (80). Anal. Calcd. for C₁₄H₁₄IN₃SSe: C, 36.38; H, 3.05; N, 9.09. Found: C, 36.36; H, 3.06; N, 9.10.

4-(4-Methoxyphenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-selenazole (4p)

2-Bromo-1-(4-methoxyphenyl)ethanone was reacted under nitrogen atmosphere with 3. Yield: 0.21 g, 80%, (dichloromethane/methanol, 95:5, R_f=0.67); m.p. 179–182 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.60–2.63 (m, 2H, CH₂); 2.75–2.79 (m, 2H, CH₂); 2.79–2.84 (m, 4H, 2CH₂); 3.78 (s, 3H, CH₃); 6.99 (d, 2H, 2CH, J = 9 Hz); 7.38 (s, 1H, CH); 7.68 (d, 2H, 2CH, J = 9 Hz); 11.02 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.92; 29.38; 31.08; 36.80; 55.74; 104.97; 114.62 (2C); 125.53; 127.85 (2C); 144.45; 158.45; 159.82; 173.76. FAB(+)-MS (m/z, %): 368.1 [(M⁺+1), 100], 364.0 (20), 365.0 (20), 366.0 (50), 370.1 (24), 254.0 (16), 114.0 (12). Anal. Calcd. for C₁₅H₁₇N₃OSSe: C, 49.18; H, 4.68; N, 11.47. Found: C, 49.19; H, 4.68; N, 11.50.

4-(4-Methylphenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-selenazole (4q)

2-Bromo-1-(4-methylphenyl)ethanone was reacted under nitrogen atmosphere with 3. Yield: 0.32 g, 91%, (dichloromethane/methanol, 95:5, R_f=0.79); m.p. 204–205 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.30 (s, 3H, CH₃); 2.56–2.59 (m, 2H, CH₂); 2.73–2.76 (m, 2H, CH₂); 2.78–2.80 (m, 4H, 2CH₂); 7.19 (d, 2H, 2CH, J = 8 Hz); 7.44 (s, 1H, CH); 7.67 (d, 2H, 2CH, J = 8 Hz); 11.06 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 21.27; 27.93; 29.40; 30.98; 36.83; 106.13; 126.34 (2C); 129.73 (2C); 129.97; 138.11; 145.58; 157.97; 173.56. FAB(+)-MS (m/z, %): 352.0 [(M⁺+1), 100], 348.0 (21), 349.0 (20), 350.0 (50), 354.1 (22). Anal. Calcd. for C₁₅H₁₇N₃SSe: C, 51.42; H, 4.89; N, 11.99. Found: C, 51.40; H, 4.90; N, 12.01.

4-(4-Bromophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-selenazole (4r)

2-Bromo-1-(4-bromophenyl)ethanone was reacted under nitrogen atmosphere with 3. Yield: 0.28 g, 93%, (dichloromethane/methanol, 95:5, R_f=0.86); m.p. 198–199 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.56–2.59 (m, 2H, CH₂); 2.73–2.76 (m, 2H, CH₂); 2.78–2.81 (m, 4H, 2CH₂); 7.57 (d, 2H, 2CH, J = 9 Hz); 7.67 (s, 1H, CH); 7.75 (d, 2H, 2CH, J = 9 Hz); 11.08 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.91; 29.16; 29.40; 30.91; 108.48; 121.41; 128.44 (2C); 132.03 (2C); 132.34; 146.12; 156.70; 173.64. FAB(+)-MS (m/z, %): 416.0 [(M⁺+1), 100], 411.9 (15), 412.9 (15), 413.9 (50), 418.0 (76), 303.9 (16), 301.9 (24), 221.9 (36), 185.1 912), 112.0 (40), 114.0 (44). Anal. Calcd. for C₁₄H₁₄BrN₃SSe: C, 40.50; H, 3.40; N, 10.12. Found: C, 40.51; H, 3.40; N, 10.13.

2-(2-(Tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-4-(4- (trifluoromethyl)phenyl)-1,3-selenazole (4s)

2-Bromo-1-(4-trifluoromethylphenyl)ethanone was reacted under nitrogen atmosphere with 3. Yield: 0.19 g, 67%, (dichloromethane/methanol, 95:5, R_f=0.82); m.p. 199–203 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.56–2.60 (m, 2H, CH₂); 2.73–2.77 (m, 2H, CH₂); 2.78–2.81 (m, 4H, 2CH₂); 7.74 (d, 2H, 2CH, J = 8 Hz); 7.86 (s, 1H, CH); 8.02 (d, 2H, 2CH, J = 8 Hz); 11.02 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 24.27 (2C); 35.79 (2C); 94.46; 123.37; 126.07; 126.40 (2C); 127.14 (2C); 128.73 (q, J_C–F=32.0 Hz); 138.69; 153.73; 182.31. FAB(+)-MS (m/z, %): 406.2 [(M⁺+1), 100], 402.2 (20), 403.2 (20), 404.2 (52), 408.2 (20), 292.0 (16), 112.0 (24), 114.0 (28). Anal. Calcd. for C₁₅H₁₄F₃N₃SSe: C, 44.56; H, 3.49; N, 10.39. Found: C, 44.58; H, 3.47; N, 10.40.

4-(4-Nitrophenyl)-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-selenazole (4t)

2-Bromo-1-(4-nitrophenyl)ethanone was reacted under nitrogen atmosphere with 3. Yield: 0.26 g, 95%, (dichloromethane/methanol, 95:5, R_f=0.69); m.p. 206–208 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.56–2.59 (m, 2H, CH₂); 2.73–2.76 (m, 2H, CH₂); 2.77–2.81 (m, 4H, 2CH₂); 8.05 (s, 1H, CH); 8.08 (d, 2H, 2CH, J = 9 Hz); 8.24 (d, 2H, 2CH, J = 9 Hz); 11.03 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.88; 29.41; 30.73; 36.87; 113.03; 124.51 (2C); 127.12 (2C); 140.87; 146.52; 147.21; 155.05; 173.80. FAB(+)-MS (m/z, %): 383.1 [(M⁺+1), 100], 379.1 (20), 380.1 (20), 381.1 (50), 385.2 (20), 338.4 (40), 219.2 (40), 185.1 (28), 173.1 (36), 147.0 (28), 136.0 (32), 114.0 (44), 112.0 (40). Anal. Calcd. for C₁₄H₁₄N₄O₂SSe: C, 44.10; H, 3.70; N, 14.69. Found: C, 44.11; H, 3.71; N, 14.72.

4-(2-(2-(Tetrahydro-4H-thiopyran-4-ylidene)hydrazinyl)-1,3-selenazol-4-yl)benzonitrile (4u)

4-(Bromoacetyl)benzonitrile was reacted under nitrogen atmosphere with 3. Yield: 0.25 g, 96%, (dichloromethane/methanol, 95:5, R_f=0.72); mp 211–212 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 2.55–2.58 (m, 2H, CH₂); 2.73–2.76 (m, 2H, CH₂); 2.77–2.81 (m, 4H, 2CH₂); 7.83 (d, 2H, 2CH, J = 8 Hz); 7.95 (s, 1H, CH); 8.00 (d, 2H, 2CH, J = 8 Hz); 11.01 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 27.89; 29.42; 30.72; 36.88; 109.94; 111.68; 119.43; 126.90 (2C); 133.12 (2C); 139.36; 148.19; 155.02; 173.67. FAB(+)-MS (m/z, %): 363.0 [(M⁺+1), 100], 359.0 (20), 361.0 (52), 365.0 (20), 219.2 (16), 185.1 (20), 136.0 (16), 114.0 (12), 112.0 (12). Anal. Calcd. for C₁₅H₁₄N₄SSe: C, 49.86; H, 3.91; N, 15.51. Found: C, 49.84; H, 3.93; N, 15.53.

Ethyl 4-methyl-2-(2-(tetrahydro-4H-thiopyran-4-ylidene) hydrazinyl)-1,3-selenazole-5-carboxylate (4v)

Ethyl 2-chloro-3-oxobutanoate was reacted under nitrogen atmosphere with 3. Yield: 0.16 g, 62%, (dichloromethane/methanol, 95:5, R_f=0.44); m.p. 199–202 °C. ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) 1.21 (t, 3H, CH₃, J = 7 Hz); 2.34 (s, 3H, CH₃) 2.55–2.59 (m, 2H, CH_2,); 2.69–2.73 (m, 2H, CH₂); 2.77–2.80 (m, 2H, CH₂); 2.85–2.88 (m, 2H, CH₂); 4.13 (q, 2H, CH_2, J = 7 Hz); 11.10 (bs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 14.70; 15.50; 27.44; 29.70; 31.25; 37.09; 60.70; 101.64; 149.95; 162.02; 163.39; 167.64. FAB(+)-MS (m/z, %): 348.1 [(M⁺+1), 100], 344.1 (20), 345.1 (20), 346.1 (56), 350.1 (20), 114.0 (18). Anal. Calcd. for C₁₂H₁₇N₃O₂SSe: C, 41.62; H, 4.95; N, 12.13. Found: C, 41.60; H, 4.95; N, 12.15.

Microbiology

The examined compounds 4a–v were screened in vitro for antibacterial and antifungal activities using the broth microdilution method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST)38 and Clinical and Laboratory Standards Institute guidelines39 against a panel of reference strains of microorganisms, including Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10876, Micrococcus luteus ATCC 10240), Gram-negative bacteria (Bordetella bronchiseptica ATCC 4617, Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella pneumoniae ATCC 13883, Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 9027) and fungi belonging to yeasts (Candida albicans ATCC 2091, Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019) and molds (Aspergillus niger ATCC 16404). These microorganisms came from American Type Culture Collection (ATCC), routinely used for the evaluation of antimicrobials. In the study of antifungal activity of the compounds 4a–v 13 clinical strains of yeasts from Candida species, that is C. albicans, C. dubliniensis, C. famata, C. inconspicua, C. krusei, C. lambica, C. lusitaniae, C. parapsilosis, C. pulcherrima, C. sake, C. kefyr, C. glabrata and C. tropicalis were also used. These fungi were extracted from different clinical materials isolated from upper respiratory tract of hospitalized patients including cancer persons (i.e. with non-small cell lung cancer or hematological malignancies). Some patients were after pre- or post-operative chemotherapy (treated with Vepesid or cis-platin given in doses according to the standard procedures), patients with chronic hepatitis C (undergoing peginterferon and ribavirin therapy or without antiviral therapy), patients with diabetes, elderly people, aged of 65 years old or older, staying in close population, such as a care center and people staying outside the home care. The Ethical Committee of the Medical University of Lublin approved the study protocol (No. KE-0254/75/2011). The isolates were identified by standard diagnostic methods – biochemical microtest, e.g. API 20 C AUX, ID 32 C, API Candida (bioMérieux, Warszawa, Poland) on the basis of assimilation of various substrates.

All the used microbial cultures were first subcultured on nutrient agar or Sabouraud agar at 35 °C for 18–24 h or 30 °C for 24–48 h for bacteria and fungi, respectively. The surface of Mueller-Hinton agar (for bacteria) and RPMI 1640 with MOPS (for fungi) were inoculated with the suspensions of bacterial or fungal species. Microbial suspensions were prepared in sterile saline (0.85% NaCl) with an optical density of McFarland standard scale 0.5 – ∼1.5 × 10⁸ CFU (Colony Forming Units)/ml for bacteria and 0.5 McFarland standard scale – ∼5 × 10⁵ CFU/ml) for fungi.

Samples containing examined compounds 4a–v were dissolved in 1 ml dimethyl sulphoxide (DMSO). Furthermore, bacterial and fungal suspensions were put onto Petri dishes with solid media containing 2 mg/ml of the tested compounds followed incubation at 37 °C for 24 h and 30 °C for 48 h for bacteria and fungi, respectively. The inhibition of microbial growth was judged by comparison with a control culture prepared without any tested sample. Ciprofloxacin, or fluconazole (Sigma Aldrich, Poznań, Poland) were used as reference antibacterial or antifungal compounds, respectively.

Subsequently MIC (Minimal Inhibitory Concentration) of the compounds was examined by the microdilution broth method, using their two-fold dilutions in Mueller-Hinton broth (for bacteria) and RPMI 1640 broth with MOPS (for fungi) prepared in 96-well polystyrene plates. Final concentrations of the compounds ranged from 1000 to 0.488 μg/ml. Microbial suspensions were prepared in sterile saline (0.85% NaCl) with an optical density of 0.5 McFarland standard. Next 2 μl of each bacterial or fungal suspension was added per each well containing 200 μl broth and various concentrations of the examined compounds. After incubation (37 °C, 24–48 h), the MIC was assessed spectrophotometrically as the lowest concentration of the samples showing complete bacterial or fungal growth inhibition. Appropriate DMSO, growth and sterile controls were carried out. The medium with no tested substances was used as control.

The MBC (Minimal Bactericidal Concentration) or MFC (Minimal Fungicidal Concentration) are defined as the lowest concentration of the compounds that is required to kill a particular bacterial or fungal species. MBC/MFC was determined by removing 20 μl of the culture using for MIC determinations from each well and spotting onto appropriate agar medium. The plates were incubated for 37 °C for 24 h and 30 °C for 48 h for bacteria and fungi, respectively. The lowest compounds concentrations with no visible growth observed was assessed as a bactericidal/fungicidal concentration. All the experiments were repeated three times and representative data are presented40.

In this study, no bioactivity was defined as a MIC > 1000 μg/ml, mild bioactivity as a MIC in the range 501–1000 μg/ml, moderate bioactivity with MIC from 126 to 500 μg/ml, good bioactivity as a MIC in the range 26–125 μg/ml, strong bioactivity with MIC between 10 and 25 μg/ml and very strong bioactivity as a MIC < 10 μg/ml41. The MBC/MIC or MFC/MIC ratios were calculated in order to determine bactericidal/fungicidal (MBC/MIC ≤ 4, MFC/MIC ≤ 4) or bacteriostatic/fungistatic (MBC/MIC > 4, MFC/MIC > 4) effect of the tested compounds.

In vivo pharmacology

Animals and housing conditions

In the in vivo tests adult male Albino Swiss (CD-1) mice weighing between 18 and 22 g were used. 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 were kept consistent throughout all the tests. For behavioral experiments the animals were selected randomly. Each group consisted of 6–9 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.

The 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

For in vivo assays the test compounds and the reference drugs (ethosuximide, ICN Polfa Rzeszow, Poland and levetiracetam, Sigma Aldrich, Poznań, Poland) were suspended in 1% Tween 80 (Polskie Odczynniki Chemiczne, Gliwice, Poland) and administered by the intraperitoneal route (ip) 60 min before the test. PTZ (Sigma Aldrich, Poznań, Poland) was prepared in 0.9% natrium chloride solution. Control mice received 1% Tween 80.

PTZ seizure test

The test was performed according to literature42 with some minor modification43. 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 comparison of latency time to first clonus and mortality rate in vehicle-treated and drug-treated groups were indicatives of the anticonvulsant activity of the test compounds.

6-Hz test

This test was performed according to literature44 with some modification45. It is an alternative electroshock paradigm that involves low-frequency (6 Hz), long-duration (3 s) electrical stimulation. Corneal stimulation (0.2 ms-duration monopolar rectangular pulses at 6 Hz for 3 s) was delivered by a constant-current device. During the stimulation mice were manually restrained and released into the observation cage immediately after current application. At the time of drug administration a drop of 0.5% tetracaine hydrochloride (Alcon) was applied into the eyes of all animals. Prior to the placement of corneal electrodes, a drop of 0.9% saline was applied on the eyes. In this model seizures manifest in ‘stunned’ posture associated with rearing, forelimb automatic movements and clonus, twitching of the vibrissae and Straub-tail. At the end of the seizure episode the animals resume their normal exploratory behavior. In this test the protection against the seizure episode is considered as the end point. The animals are considered to be protected if they resume their normal exploratory behavior within 10 s after electrical stimulation.

Rotarod test

The test was performed according to the method recently described46. The mice were trained daily for three consecutive days on the rotarod apparatus (Rotarod apparatus, May Commat RR0711, 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 last training trial. Briefly, 60 min before the rotarod test the mice were ip pretreated with the test 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 the in vivo tests was performed 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. In each case p < 0.05 was considered significant.

Computational details

Interaction within the complex of 4a with β-cyclodextrin was investigated. Due to its large size, the Density Functional Theory (DFT) approximation was chosen. In order to recognize minima on the interaction energy surface, four different orientations of 4a with respect to the cyclodextrin were examined. We started with four starting points (one per orientation) with the β-cyclodextrin geometrical parameters adopted from the work47, where β-cyclodextrin–resveratrol complex was investigated. Geometrical parameters of the complexes were optimized using the B3LYP functional and the 6–31G** basis set. Next, eight other starting points were examined within the same approximation (two per orientation), this time taking the starting geometrical parameters of the cyclodextrin from the present optimizations. Calculation of interaction and binding energies was carried out for the energetically most stable conformer recognized for each of the four orientations using the M06–2X functional and the 6–31G** and 6–311G** basis sets. The supermolecular approach was used, and the results were counterpoise-corrected to account for the basis set superposition error (BSSE). All calculations were carried out using the Gaussian 09 program (Gaussian 09, Wallingford, CT)48.

Results and discussion

Chemistry

Tetrahydro-4H-thiopyran-4-one (1) was used as starting key for the synthesis of 2,4-disubstituted thiazole and selenazole derivatives. Heating of compound 1 with thiosemicarbazide in absolute ethanol in the presence of catalytic amount of glacial acetic acid led to 2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazinecarbothioamide (2). However, 2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazinecarboselenoamide (3) was synthesized using potassium selenocyanate and hydrazine hydrate in the presence of hydrochloric acid in ethanol and under reflux. The products 2 and 3 were obtained with 76% and 34% yield, respectively. In the next step Hantzsch cyclization protocol is used. Synthesis of compounds 4a–v consists of the condensation of appropriate para-substituted bromoacetophenones, ethyl 4-chloroacetoacetate, ethyl 2-chloroacetoacetate, and chloroacetone with thiosemicarbazone (2), and seleno-semicarbazone (3) afforded the corresponding thiazoles 4a–4 l and selenazoles 4m–4v with good yield (53–99%) and with high chemical purity. The reaction pathway has been summarized in Scheme 1.

Scheme 1. Synthesis of the target compounds 4a–v.

All obtained products were purified on silica gel column chromatography, and fully characterized spectroscopically using ¹H and ¹³C NMR, FAB(+)-MS, and elemental analyzes. The mass spectra of all compounds are fully consistent with the assigned structures. In all cases [M⁺+1] peaks were observed. The typical abundances for selenium isotopes ⁷⁶Se (9.37%), ⁷⁷Se (7.63%), ⁷⁸Se (23.77%), ⁸⁰Se (49.61%) and ⁸²Se (8.73%) are present in the MS spectra49,50. The molecular ion, accompanied by the isotopic peaks confirms the molecular weight of the selenazole derivatives [i.e., in the MS spectra for compound 4m, all the isotopic peaks for selenium are present, 352 (⁷⁶Se), 353 (⁷⁷Se), 354 (⁷⁸Se), 356 (⁸⁰Se), 358 (⁸²Se)]. The ¹H NMR spectrum of 2 and 3 presents typical three proton signals of NH₂ and NH groups at 7.61, 8.04, 10.29 and 8.09, 8.47, 10.59 ppm, respectively. These three signals are due to the exchange of H between the terminal NH₂ group and sulphur atom. ¹H NMR spectra of azole derivatives showed singlet at

δ (6.22–8.05) due to thiazole-5H/selenazole-5H proton and singlet at δ (10.70–11.10) indicating the presence of hydrazine NH proton, which confirms the conversion of substrates into the expected products. The elemental analyzes 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.

Biological evaluation

Antifungal activity

According to the results presented in Table 1, on the basis of minimal inhibitory concentration values (MIC) obtained by the broth microdilution method, it was shown that compounds 4a–f, 4i, 4k, 4 l, 4n, 4o, 4p, 4r, 4s and 4v possess strong or very strong activity against Candida spp. with MIC = 1.95–15.62 μg/ml. The minimal fungicidal concentration values of these substances ranged from 3.91 μg/ml to 62.5 μg/ml. These compounds showed fungicidal effect (MFC/MIC = 1–4) against yeasts except 4v, which shows fungistatic activity towards C. albicans ATCC 2091 and C. parapsilosis ATCC 22019 (MFC/MIC = 16).

Table 1. The antifungal activity data expressed as MIC [μg/ml] and, in parentheses, as MFC [μg/ml] for compounds 4a–v.

	MIC (MFC) [μg/ml] of the compounds
Species	4a	4b	4c	4d	4e	4f	4g	4h	4i	4k	4l	FLU
A. niger ATCC 16404	62.5 (250)	62.5 (62.5)	250 (>1000)	125 (1000)	62.5 (250)	62.5 (250)	31.25 (62.5)	31.25 (250)	15.62 (31.25)	125 (125)	62.5 (250)	–
C. albicans ATCC 2091	7.81 (15.62)	7.81 (15.62)	7.81 (15.62)	7.81 (15.62)	7.81 (31.25)	7.81 (15.62)	125 (250)	1000 (>1000)	15.62 (31.25)	3.91 (15.62)	7.81 (15.62)	0.24
C. albicans ATCC 10231	7.81 (15.62)	1.95 (3.91)	7.81 (31.25)	3.91 (15.62)	3.91 (7.81)	15.62 (62.5)	15.62 (62.5)	250 (>1000)	7.81 (15.62)	1.95 (3.91)	3.91 (7.81)	0.98
C. parapsilosis ATCC 22019	7.81 (15.62)	3.91 (7.81)	15.62 (15.62)	15.62 (15.62)	7.81 (15.62)	7.81 (15.62)	125 (500)	500 (>1000)	15.62 (15.62)	3.91 (7.81)	3.91 (7.81)	1.95
C. albicans	3.91 (7.81)	0.98 (15.62)	1.95 (31.25)	3.91 (31.25)	1.95 (3.91)	0.98 (1.95)	15.62 (62.5)	15.62 (500)	3.91 (3.91)	1.95 (3.91)	3.91 (7.81)	0.03
C. parapsilosis	7.81 (7.81)	3.91 (7.81)	3.91 (7.81)	7.81 (31.25)	3.91 (7.81)	1.95 (3.91)	15.62 (250)	31.25 (250)	7.81 (15.62)	1.95 (3.91)	3.91 (7.81)	0.24
C. sake	3.91 (3.91)	3.91 (15.62)	15.62 (500)	15.62 (250)	3.91 (15.62)	15.62 (250)	62.5 (1000)	15.62 (>1000)	3.91 (250)	3.91 (3.91)	3.91 (7.81)	0.24
C. dubliniensis	3.91 (3.91)	1.95 (3.91)	1.95 (15.62)	3.91 (3.91)	0.98 (1.95)	0.98 (1.95)	15.62 (15.62)	15.62 (15.62)	1.95 (3.91)	0.98 (0.98)	3.91 (7.81)	0.03
C. famata	1.95 (3.91)	1.95 (3.91)	1.95 (15.62)	1.95 (15.62)	0.98 (3.91)	0.98 (1.95)	7.81 (15.62)	15.62 (62.5)	0.98 (15.62)	1.95 (3.91)	3.91 (7.81)	0.98
C. lusitaniae	1.95 (3.91)	1.95 (3.91)	3.91 (31.25)	3.91 (15.62)	3.91 (7.81)	1.95 (3.91)	15.62 (15.62)	500 (>1000)	3.91 (15.62)	7.81 (7.81)	7.81 (7.81)	0.004
C. krusei	3.91 (7.81)	1.95 (62.5)	7.81 (62.5)	3.91 (31.25)	1.95 (7.81)	1.95 (62.5)	15.62 (62.5)	15.62 (>1000)	3.91 (15.62)	0.98 (3.91)	0.98 (3.91)	0.122
C. inconspicua	0.98 (7.81)	0.98 (7.81)	3.91 (62.5)	3.91 (31.25)	0.98 (3.91)	0.98 (7.81)	7.81 (62.5)	7.81 (1000)	0.98 (15.62)	0.98 (3.91)	0.49 (1.95)	0.49
C. kefyr	125 (500)	250 (500)	1000 (1000)	62.5 (500)	125 (250)	500 (1000)	500 (1000)	500 (>1000)	125 (500)	7.81 (15.62)	62.5 (125)	0.49
C. pulcherrima	500 (>1000)	1000 (>1000)	1000 (1000)	1000 (>1000)	1000 (>1000)	1000 (1000)	1000 (>1000)	1000 (>1000)	500 (1000)	125 (250)	250 (250)	0.12
C. lambica	1000 (>1000)	500 (1000)	1000 (>1000)	1000 (>1000)	500 (1000)	1000 (>1000)	1000 (1000)	1000 (>1000)	1000 (1000)	125 (250)	250 (500)	0.98
C. glabrata	250 (1000)	1000 (1000)	1000 (>1000)	1000 (1000)	500 (>1000)	1000 (>1000)	1000 (>1000)	1000 (1000)	1000 (>1000)	62.5 (125)	250 (>250)	0.06
C. tropicalis	250 (1000)	500 (1000)	1000 (1000)	1000 (>1000)	500 (>1000)	1000 (1000)	1000 (1000)	1000 (>1000)	500 (1000)	125 (250)	250 (500)	0.06
	4m	4n	4o	4p	4q	4r	4s	4t	4u	4v	FLU
A. niger ATCC 16404	–	125 (250)	125 (125)	125 (1000)	125 (1000)	62.5 (500)	125 (500)	62.5 (250)	–	250 (>1000)	–
C. albicans ATCC 2091	1000 (>1000)	15.62 (31.25)	7.81 (15.62)	15.62 (62.5)	31.25 (31.25)	15.62 (31.25)	15.62 (31.25)	125 (250)	500 (500)	15.62 (250)	0.24
C. albicans ATCC 10231	1000 (>1000)	7.81 (15.62)	7.81 (15.62)	15.62 (15.62)	31.25 (31.25)	7.81 (15.62)	15.62 (15.62)	62.5 (125)	500 (500)	15.62 (62.5)	0.98
C. parapsilosis ATCC 22019	500 (>1000)	15.62 (15.62)	15.62 (15.62)	15.62 (31.25)	15.62 (31.25)	15.62 (15.62)	15.62 (15.62)	31.25 (125)	125 (250)	15.62 (250)	1.95
C. albicans	–	3.91 (7.81)	1.95 (7.81)	7.81 (250)	31.25 (62.5)	7.81 (31.25)	1.95 (31.25)	125 (125)	500 (1000)	15.62 (250)	0.03
C. parapsilosis	–	7.81 (15.62)	3.91 (7.81)	7.81 (62.5)	31.25 (31.25)	7.81 (31.25)	3.91 (7.81)	31.25 (125)	500 (1000)	62.5 (125)	0.24
C. sake	62.5 (>1000)	7.81 (7.81)	3.91 (31.25)	7.81 (62.5)	15.62 (15.62)	7.81 (15.62)	3.91 (15.62)	31.25 (62.5)	125 (500)	31.25 (125)	0.24
C. dubliniensis	31.25 (>1000)	3.91 (7.81)	0.98 (7.81)	3.91 (15.62)	15.62 (31.25)	3.91 (7.81)	0.98 (0.98)	125 (125)	500 (500)	15.62 (31.25)	0.03
C. famata	–	3.91 (7.81)	1.95 (7.81)	3.91 (15.62)	31.25 (62.5)	7.81 (31.25)	1.95 (15.62)	125 (125)	500 (1000)	15.62 (500)	0.98
C. lusitaniae	15.62 (>1000)	3.91 (3.91)	3.91 (7.81)	31.25 (250)	15.62 (31.25)	7.81 (7.81)	1.95 (15.62)	125 (250)	500 (1000)	7.81 (7.81)	0.004
C. krusei	–	3.91 (15.62)	3.91 (31.25)	31.25 (125)	15.62 (31.25)	15.62 (31.25)	3.91 (31.25)	62.5 (62.5)	250 (500)	31.25 (125)	0.122
C. inconspicua	–	3.91 (7.81)	3.91 (15.62)	62.5 (62.5)	15.62 (15.62)	7.81 (15.62)	3.91 (31.25)	31.25 (31.25)	250 (500)	31.25 (125)	0.49
C. kefyr	15.62 (>1000)	31.25 (31.25)	62.5 (62.5)	125 (125)	31.25 (31.25)	31.25 (31.25)	62.5 (125)	62.5 (125)	500 (>1000)	125 (250)	0.49
C. pulcherrima	–	62.5 (62.5)	125 (125)	250 (250)	62.5 (62.5)	62.5 (62.5)	125 (250)	250 (250)	1000 (>1000)	500 (>1000)	0.12
C. lambica	–	62.5 (62.5)	125 (125)	250 (500)	62.5 (62.5)	62.5 (62.5)	250 (250)	62.5 (125)	–	500 (>1000)	0.98
C. glabrata	–	62.5 (62.5)	62.5 (125)	250 (>250)	62.5 (62.5)	62.5 (62.5)	250 (250)	125 (125)	1000 (>1000)	125 (500)	0.06
C. tropicalis	–	62.5 (62.5)	125 (125)	250 (500)	62.5 (62.5)	62.5 (62.5)	125 (125)	250 (250)	1000 (1000)	500 (>1000)	0.06

The other compounds: 4 g, 4 h, 4m, 4q, 4t and 4u showed different antifungal activity against yeasts ranging from strong to mild effect with MIC = 15.62–1000 μg/ml and MFC = 31.25 – ≥ 1000 μg/ml.

Moreover, the growth of reference strains of molds, i.e. A. niger ATCC 16404 was inhibited by some of the compounds, too. The highest fungicidal activity with strong effect against this microorganism indicated 4i (MIC = 15.62 μg/ml, MFC = 31.25 μg/ml, MFC/MIC = 2). Compounds: 4a, 4b, 4d–h, 4k, 4 l, 4n–t showed good activity with fungicidal or fungistatic effect, MIC = 31.25–125 μg/ml and MFC = 62.5–1000 μg/ml (MFC/MIC = 1–8). In turn, the minimal inhibitory concentration of compounds 4c and 4v which inhibited growth of this fungus was 250 μg/ml and MFC > 1000 μg/ml. Our results indicated also that the substance 4j had no influence on the growth of all reference strains of fungi, whereas compounds 4m and 4u were not active against A. niger ATCC 16404 (Table 2).

Table 2. The antibacterial activity data expressed as MIC [μg/ml] and, in parentheses, as MBC [μg/ml] for compounds 4a–v.

	MIC (MBC) [μg/ml] of the compounds
Species	4a	4d	4e	4g	4j	4k	4l	4m	4n	CIP
S. aureus ATCC 6538	500 (>1000)	–	–	–	250 (>1000)	500 (>1000)	250 (500)	–	31.25 (62.5)	0.24
S. aureus ATCC 25923	125 (500)	–	–	–	500 (>1000)	500 (>1000)	125 (250)	–	15.62 (62.5)	0.24
S. aureus ATCC 43300	250 (1000)	–	–	–	250 (>1000)	250 (250)	(62.5 (62.5)	–	15.62 (15.62)	0.49
S. epidermidis ATCC 12228	125 (500)	–	–	–	125 (1000)	62.5 (62.5)	62.5 (62.5)	–	7.81 (15.62)	0.12
M. luteus ATCC 10240	250 (500)	–	250 (>1000)	125 (125)	125 (250)	125 (250)	125 (125)	250 (1000)	31.25 (15.62)	0.98
B. subtilis ATCC 6633	500 (1000)	–	–	500 (>1000)	500 (>1000)	1000 (>1000)	250 (500)	–	125 (250)	0.03
B. cereus ATCC 10876	1000 (>1000)	–	–	500 (>1000)	500 (>1000)	500 (>1000)	250 (500)	–	125 (500)	0.06
B. bronchiseptica ATCC 4617	1000 (1000)	500 (1000)	125 (250)	–	–	31.25 (125)	15.62 (31.25)	–	250 (1000)	0.98
E. coli ATCC 25922	–	–	–	–	–	–	1000 (>1000)	–	–	0.004
P. mirabilis ATCC 12453	–	–	–	–	–	1000 (>1000)	1000 (>1000)	–	–	0.03
K. pneumoniae ATCC 13883	–	–	–	–	–	1000 (>1000)	500 (>1000)	–	250 (>1000)	0.12
S. typhimurium ATCC 14028	–	–	–	–	–	–	1000 (>1000)	–	–	0.06
P. aeruginosa ATCC 9027	–	–	–	–	–	–	1000 (>1000)	–	–	0.49
	4o	4p	4q	4r	4s	4t	4u	4v	CIP
S. aureus ATCC 6538	31.25 (62.5)	31.25 (125)	31.25 (62.5)	31.25 (125)	62.5 (125)	62.5 (125)	125 (250)	125 (500)	0.24
S. aureus ATCC 25923	15.62 (62.5)	31.25 (62.5)	15.62 (62.5)	31.25 (31.25)	31.25 (62.5)	31.25 (62.5)	62.5 (62.5)	125 (500)	0.24
S. aureus ATCC 43300	15.62 (15.62)	15.62 (31.25)	7.81 (15.62)	7.81 (15.62)	15.62 (31.25)	31.25 (62.5)	31.25 (62.5)	62.5 (125)	0.49
S. epidermidis ATCC 12228	7.81 (31.25)	15.62 (15.62)	15.62 (15.62)	15.62 (15.62)	7.81 (15.62)	15.62 (31.25)	7.81 (31.25)	15.62 (31.25)	0.12
M. luteus ATCC 10240	7.81 (31.25)	7.81 (15.62)	7.81 (15.62)	7.81 (15.62)	7.81 (31.25	7.81 (15.62)	31.25 (125)	62.5 (250)	0.98
B. subtilis ATCC 6633	125 (250)	125 (500)	62.5 (250)	62.5 (250)	62.5 (500)	125 (1000)	250 (1000)	1000 (>1000)	0.03
B. cereus ATCC 10876	250 (500)	500 (1000)	125 (250)	250 (1000)	250 (1000)	250 (500)	500 (>1000)	1000 (>1000)	0.06
B. bronchiseptica ATCC 4617	–	–	–	–	–	1000 (>1000)	–	–	0.98
E. coli ATCC 25922	–	–	–	–	–	–	–	–	0.004
P. mirabilis ATCC 12453	–	–	–	–	–	–	–	–	0.03
K. pneumoniae ATCC 13883	250 (>1000)	–	250 (500)	–	–	1000 (>1000)	–	–	0.12
S. typhimurium ATCC 14028	–	–	–	–	–	–	–	–	0.06
P. aeruginosa ATCC 9027	–	–	–	–	–	–	–	–	0.49

The tested compounds showed also strong and very strong fungicidal or fungistatic activity against some strains of Candida spp. isolated from clinical materials. The isolates belonging to C. albicans and non-albicans Candida spp. strains, i.e. C. parapsilosis, C. sake, C. dubliniensis, C. famata, C. lusitaniae, C. krusei or C. inconspicua were especially sensitive to substances 4a–f, 4i, 4k, 4 l, 4n, 4o, 4r and 4s. Minimum concentrations of compounds, which inhibited the growth of these yeasts ranged from 0.98 to 15.62 μg/ml and MFC = 0.98–500 μg/ml (MFC/MIC = 1–32). The activity of compounds 4 g, 4 h, 4m, 4p, 4q, 4t–v toward these strains was also high (MIC = 3.91–500 μg/ml and MFC = 7.81 – ≥1000 μg/ml). The remaining substances showed a lower activity (4m) or had no influence on the growth of these fungi (4j).

Moreover, the tested compounds showed much lower activity against remaining isolates of non-albicans Candida spp., i.e. C. tropicalis, C. glabrata, C. kefyr, C. lambica and C. pulcherrima with MIC ranged from 7.81 to >1000 μg/ml and MFC = 15.62 – ≥ 1000 μg/ml (Table 2).

Antibacterial activity

According to the data presented in Table 2, the newly synthesized compounds showed potential antibacterial activity mainly towards reference Gram-positive bacteria. Among them, compounds 4n–v exhibited especially high activity against strains of Staphylococcus spp. and Micrococcus spp. with good, strong or very strong effect. The opportunistic bacteria, such as S. epidermidis ATCC 12228 and M. luteus ATCC 10240 were particularly sensitive to these substances with MIC = 7.81–62.5 μg/ml and MBC = 15.62–250 μg/ml. The minimum concentrations of compounds 4n–v, which inhibited the growth of pathogenic staphylococci S. aureus ATCC were 7.81–125 μg/ml (MBC = 15.62–500 μg/ml). These compounds showed bactericidal effect against staphylococci and micrococci (MBC/MIC = 1–4). The activity towards reference strains of Bacillus spp. was lower with good, moderate or mild effect (MIC = 62.5–1000 μg/ml and MBC = 250 – ≥ 1000 μg/ml).

It was shown that remaining compounds 4a, 4j, 4k and 4 l exhibited smaller bioactivity against the same Gram-positive bacteria with MIC = 62.5–250 μg/ml and MBC = 62.5 – ≥ 1000 μg/ml. The bacteria S. epidermidis ATCC 12228 and M. luteus ATCC 10240 were also most susceptible to these substances. In turn, the compounds 4e, 4 g and 4m indicated only minor activity or no activity towards tested Gram-positive bacteria.

Only compounds: 4a, 4d, 4e, 4k, 4 l, 4n, 4o, 4q and 4t had some activity against Gram-negative bacteria. Among them, compound 4 l had the widest spectrum of activity against these microorganisms with strong bactericidal effect towards B. bronchiseptica ATCC 4617. The remaining substances 4b, 4c, 4f, 4 h and 4i had no inhibitory effect on the growth of all reference strains of bacteria.

To conclude, the antifungal activity of thiazole compounds (4a–f, 4i) containing -F, -Cl, -Br, -I, -OCH₃, -CH₃ and -CN substituents at 4th position in the phenyl ring showed the highest activity against reference strains. It is interesting that selenazole derivatives (4m, 4q, 4u) containing -F, -CH₃ and -CN substituents showed a lower activity. Thiazoles (4k, 4 l) with -CH₂COOEt and -CH₃ groups attached directly at 4th to the thiazole ring showed very strong activity. However, replacing the hydrogen atom in compound (4 l) at 5th position in the thiazole ring with -COOEt substituent results in total loss of activity (compound 4j). The replacement of the sulfur atom in compound (4j) with a selenium atom lead to substantial increase of activity (compound 4v). As can be easily seen, selenazole derivatives (4m–v) exhibit much lower activity against tested fungi than thiazole derivatives, but they have a good antibacterial activity missing in the case of thiazole derivatives.

In vivo pharmacology

In PTZ-induced seizures test 6 compounds (4a, 4b, 4e, 4m, 4n and 4q) at doses 30 mg/kg and 100 mg/kg were tested for their ability to prolong the latency to the onset of the first clonic seizure episode (Figure 1). At the dose 30 mg/kg only 4m delayed the onset of seizures in a statistically significant manner (p < 0.05). Compounds 4a, 4b and 4n were effective at 100 mg/kg (p < 0.01 for 4a and p < 0.001 for 4b and 4n), while 4e and 4q showed no protection. In this assay, ethosuximide (100 mg/kg, ip) was used as a reference drug. Compared to vehicle-treated mice, it prolonged the latency to the onset of seizures by 120% (significant at p < 0.01).

Figure 1. Influence of the test compounds 4a, 4b, 4e, 4m, 4n and 4q on latency to the first seizure episode in PTZ-induced seizures. Statistical analysis: one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc comparison. 4a: F[2,24] = 5.488, p < 0.05; 4b: F[2,23] = 18.22, p < 0.0001; 4e: F[2,24] = 1.965, p > 0.05; 4m: F[2,24] = 4.510, p < 0.05; 4n: F[2,23] = 9.384, p < 0.01; 4q: F[2,22] = 2.746, p > 0.05. Significance vs. vehicle-treated group: * p < 0.05, ** p < 0.01, *** p < 0.001.

In PTZ test, mortality rate was affected by the test compounds to a various degree. In vehicle-treated mice, the mean mortality rate was 50%. At the dose of 30 mg/kg 4m and 4n reduced mortality rate by 39% as compared to control.

At the dose of 100 mg/kg only 4e reduced mortality rate by 28% (vs. control). Ethosuximide (100 mg/kg) protected all animals from death.

In 6-Hz test, four compounds (4a, 4b, 4m and 4n) were tested at doses which were effective in the PTZ test: three of them (4a, 4b, and 4n) were tested at 100 mg/kg, while 4m was tested at 30 mg/kg. The anticonvulsant activity of the test compounds in the 6-Hz assay is shown in Table 3. Compounds 4a and 4n protected 50% of animals from electrically-induced psychomotor seizures. Compounds 4b and 4m were less active as they protected 13% and 25% of mice, respectively, from seizures. In this test, levetiracetam (10 mg/kg, ip) was used as a reference drug. Its anticonvulsant activity was tested at the previously established time of peak drug effect, i.e. 60 min after injection44. As shown in Table 3, this dose protected 50% of animals from seizures.

Table 3. Anticonvulsant activity of the test compounds in 6-Hz test.

Compound	Dose [mg/kg]	Result
Vehicle	–	0/8
4a	100.0	4/8
4b	100.0	1/8
4m	30.0	2/8
4n	100.0	4/8
Levetiracetam	10.0	4/8

The mice were tested 60 min after compound injection.

The results are presented as the number of animals protected/animals used in the test.

Four compounds (4a, 4b, 4m and 4n) with anticonvulsant activities in PTZ and 6-Hz models were additionally tested using the rotarod test to assess their impact on animals’ motor coordination at anticonvulsant active doses. None of them impaired motor skills of experimental animals as all animals were able to perform the test at each speed used.

Currently available AEDs were discovered as a result of a comprehensive screening process in which rodent models of seizures were used44. Despite a successful development of numerous novel AEDs in the last years, there is evidence that the efficacy and tolerability of pharmacological treatment of epilepsy has not fully improved7,9^,10,51. Hence, the continuous search for new AEDs with better pharmacological efficacy and reduced toxicity is still an important goal in the anticonvulsant drug discovery. Importantly, this process strongly relies on the implementation of preclinical animal models before anticonvulsant active agents are assessed in clinical trials9,51. Our earlier studies have shown that compounds containing -F, -Cl and -CH₃ substituents demonstrated significant anticonvulsant activity in pentylenetetrazole model with median effective doses (ED₅₀) ≤ 20 mg/kg36. Therefore, in our current research we decided to choose three thiazole and three selenazole derivatives containing -F, -Cl and -CH₃ substituents. In the present research two distinct mouse models of seizures were utilized in the in vivo tests. Noteworthy, these screening assays are not only used to identify novel AEDs through testing large numbers of compounds in relatively short time, but they also allow to predict the efficacy of AEDs against different types of seizures in humans9. The PTZ model of clonic seizures generally refers to nonconvulsive (absence or myoclonic) seizures in humans51, whereas the 6-Hz test is predictive for psychomotor partial seizures9,44 or therapy-resistant limbic seizures44. The efficacy of the test compounds 4a, 4b, 4m and 4n in PTZ model suggests that these compounds might be potentially effective in absence seizures in humans, whereas the effectiveness of 4a and 4n in the 6-Hz test might suggest their potential application in psychomotor seizures, as well.

The observed efficacy of the compounds tested in PTZ test and 6-Hz test might also suggest a potential mechanism of their action. Numerous GABAergic AEDs and the T-type calcium channel blocker ethosuximide are effective in PTZ model51, whereas drugs acting by blockade of voltage-gated sodium and to a lesser degree calcium channels (with the exception of ethosuximide) are not effective in PTZ model9,10. 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 should rather be excluded.

The 6-Hz psychomotor seizure model resembling psychomotor seizures occurring in human limbic epilepsy has been also regarded as a potential screen for AEDs to avoid that effective drugs such as levetiracetam (not active in PTZ or maximal electroshock seizure test even at 500 mg/kg) are falsely considered inactive9,10. The results of the present research have confirmed that compounds 4a and 4n at the dose of 100 mg/kg show anticonvulsant activity similar to that of levetiracetam at the dose 10-fold lower. 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 doses which effectively reduce seizure episodes do not cause motor deficits in experimental animals.

Among the derivatives, four compounds: 4a, 4b, 4m and 4n containing -F and -Cl substituents demonstrated a statistically significant anticonvulsant activity in the PTZ model. In addition, 4a and 4n protected 50% of experimental animals from limbic seizures in the 6-Hz test. Noteworthy, none of these compounds impaired animals’ motor skills in the rotarod test. Since these compounds delayed the onset of clonic seizures and showed protective activity in the 6-Hz test, they can be regarded as interesting novel lead structures in the search for new AEDs.

Calculation

One of the most important problems in development of new antimicrobial and anticonvulsant agents is their poor solubility in water. Currently, complexes with cyclodextrins are used in pharmaceutical industry to increase the solubility and bioavailability of drugs52. Cyclodextrins are cyclic oligosaccharides with a hydrophilic outer surface and a lipophilic central cavity. Due to the large number of hydrogen donors and acceptors, cyclodextrins are able to form inclusion complexes in aqueous solutions with drug molecule placed inside the central cavity. Theoretical studies of interaction of cyclodextrins with drugs are an important aspect in the drug design and development53. Therefore, we decided to perform Density Functional Theory (DFT) calculation of interaction energy and binding energy of complex of selected thiazole derivative with β-cyclodextrin.

Within the present work the interaction and binding energies of the complex of 4a with β-cyclodextrin are investigated. The large size of the studied system forces the use of DFT approximation and smaller among the available basis sets. To ensure possibly accurate description of the interaction, double- and triple-zeta quality basis sets are chosen, and polarization functions are used on all atoms. As the first step, optimization of geometrical parameters is carried out using the B3LYP functional and the 6–31G** basis set. Four starting structures are chosen for that purpose, corresponding to different orientations of 4a with respect to β-cyclodextrin (see Figure 2), with geometrical parameters of the latter adopted from the work of Troche-Pesqueira et al.47. Next, eight more starting points, corresponding to the same four orientations, with slightly different relative positions of the two molecules forming the complex, are examined within the same approximation. The starting geometrical parameters of β-cyclodextrin in these eight structures are taken from the complexes optimized within the present work.

Figure 2. The four investigated orientations of 4a with respect to the β-cyclodextrin.

In the further investigation, only the most stable recognized conformer per each of the four orientations is employed. The optimized structures of these four complexes are presented in Figure 3, and their relative energies, calculated with respect to the lowest-energy conformer, are presented in Table 4.

Table 4. The B3LYP/6–31G** relative energies, and the M06–2X/6–31G** and M06–2X/6–311G** interaction (ΔE) and binding (E_bind) energies of 4a–β-cyclodextrin complex.

Basis set	4a-C1	4a-C2	4a-C3	4a-C4
	Relative energy [kcal/mol]
6-31G**	0.00	2.82	7.42	4.87
	ΔE [kcal/mol]
6-31G**	−25.52	−16.87	−23.04	−21.32
6-311G**	−26.90	−18.13	−24.87	−23.31
	Ebind [kcal/mol]
6-31G**	−16.91	−11.02	−9.09	−14.72
6-311G**	−18.83	−12.40	−12.14	−17.57

Next, the interaction ΔE(AB) and binding E_bind (AB) energies are calculated using the supermolecular approach54: with E_G^B(S) being the energy of system S evaluated at the optimized geometry G using basis set B. Symbol AB denotes the complex, and A and B its two subunits. The M06–2X functional is chosen based on our earlier work55 where hydrogen bonded complexes were investigated using a number of different functionals and the M06–2X was shown to be the optimal choice. Due to the large computing cost of the present investigation, the relatively small 6–31G** and 6–311G** basis sets are used.

Figure 3. Optimized structures of 4a-β-cyclodextrin complex obtained within the DFT/B3LYP/6–31G** approximation.

Results are presented in Table 4. The 4a–C1 orientation is the one with the lowest energy, and the relative energies are calculated with respect to it. Interaction energies of the investigated complex are larger in absolute value than the corresponding binding energies, which is caused by deformation of monomer geometries during the formation of the complex from the isolated molecules. The interaction and binding energies for 4a–C1 are the largest among the four investigated orientations in the absolute value (−26.9 and −18.8 kcal/mol, respectively). The orientation in which fluorine atom is pointing towards the wide rim and thiazole ring towards the narrow rim thus seems much more probable than the other three examined here. The 4a–C2 with analogous relative orientation of the two subunits, but with the fluorobenzene ring outside the dextrin, is the second lowest-energy structure. Its interaction and binding energies are ∼9 and 7 kcal/mol lower in absolute value, respectively, compared to the 4a–C1. The presence of 4a–C3 and 4a–C4 in the mixture of different conformers evaluated based on the relative energy using Boltzmann statistics is negligibly small. While the interaction energy in 4a–C3, having the thiazole moiety pointing towards the narrow rim, is close to that in 4a–C1, its binding energy (−12.1 kcal/mol) is much closer to that of 4a–C2 (−12.4 kcal/mol). The difference of about 13 kcal/mol between the interaction and binding energies observed in the case of 4a–C3 is the largest among the investigated orientations, showing that 4a–C3 undergoes the most significant changes in geometries of the monomers during formation of the complex. Analogous difference for the lowest-energy 4a–C1 is in the order of 8 kcal/mol. Formation of stable complexes of the investigated novel compounds with cyclodextrins could increase drugs bioavailability, as suggested by other authors47.

Conclusion

In summary, we have developed an efficient and economic method for the synthesis of small library of tetrahydro-2H-thiopyran-4-yl based thiazoles and selenazoles. Our results indicate that newly synthesized compounds showed high antifungal and antibacterial activity. Also some compounds demonstrated a statistically significant anticonvulsant activity in the PTZ model and showed protective activity in the 6-Hz test. For this reason they can be considered as interesting new leading structures in the search of new antimicrobial and antiepileptic drugs. Considering the complex of 4a with β-cyclodextrin, the 4a–C1 orientation is the one with the lowest energy among the four investigated orientations, and simultaneously its interaction and binding energies (−26.9 and −18.8 kcal/mol, respectively) are the largest in the absolute value. Calculations showed that the obtained thiazoles could form stable complexes with cyclodextrin, which may contribute to increase drugs bioavailability. This will be probably the subject of our future research.

Declaration of interest

The authors confirm that this article content has no conflicts of interest.

Acknowledgements

This study was supported by the Nicolaus Copernicus University (project No. 786/2014).