SYNTHESIS, CHARACTERIZATION AND PHARMACOLOGICAL EVALUATION OF SOME NEW 6-(3,5-SUBSTITUTED-2-BROMO/HYDROXY PHENYL)-1,2,4-TRIAZINE DERIVATIVES AS ANTICONVULSANT AGENTS

SYNTHESIS, CHARACTERIZATION AND PHARMACOLOGICAL EVALUATION OF SOME NEW 6-(3,5-SUBSTITUTED-2-BROMO/HYDROXY PHENYL)-1,2,4-TRIAZINE DERIVATIVES AS ANTICONVULSANT AGENTS

Rajeev Kumar ¹, Anita Singh ^*2 and Ram Kumar Roy ³

Devsthali Vidyapeeth College of Pharmacy ¹, Lalpur Kichha road, Rudrapur (Udham Singh Nagar) -263148, Uttarakhand, India

Department of Pharmacy, Kumaun University ², Bhimtal, Nainital-263136, Uttarakhand, India

Spectrum Institute of Pharmaceutical Science and Research ³, Greater Noida, Uttar Pradesh, India

ABSTRACT: 6-(2-Amino-3,5-substituted phenyl)-1,2,4-triazines were prepared by refluxing semicarbazones or thiosemicarbazones in presence of sodium hydroxide solution. 6-(3,5-Substituted-2-bromophenyl)-1,2,4-triazine derivatives and 6-(3,5-substituted-2-hydroxyphenyl)-1,2,4-triazine derivatives were prepared from 6-(2-amino-3,5-substituted phenyl)-1,2,4-triazine derivatives by sodium nitrite, hydrobromic acid, copper (I) bromide and sodium nitrite, sulfuric acid, respectively. The structures of the synthesized compounds were confirmed on the basis of their elemental analysis and spectral data (FT-IR and H¹-NMR). Amongst synthesized compounds, some displayed significantly active profile against the electrically induced seizures at a dose of 30 mg/kg after 0.5 h. At the same dose level, one compound also showed activity after 4.0 h. Two compounds exhibited protection at dose level of 100 mg/kg after 0.5 h. At 4.0 h, two compounds persisted to reveal anti-MES protection at same dose, whereas two other compounds indicated to prevent seizure spread at a higher dose of 300 mg/kg after 4h. One compound displayed protection at 300 mg/kg after 0.5 h. The neurotoxicity screening data revealed that two compounds were exhibited neurotoxicity at a dose level of 300 mg/kg after 0.5 h. Screened compounds displayed 40.59, 56.88 and 66.61 % increase in immobility time. Some compounds showed excellent anticonvulsant activity with no neurotoxicity and little CNS depressant effect and does not violated Lipinski’s rule, making them potentially promising agents for treatment of epilepsy

Anticonvulsant activity,

CNS depressant, semicarbazone

and 1,2,4-Triazine

INTRODUCTION: Epilepsy is one of the most common serious CNS disorder and characterized by the recurrent, unprovoked seizures.^1-2 A global campaign against epilepsy conducted by World Health Organization showed that about 50 million people worldwide are suffering from this CNS disorder.

Every year about 2.4 million new cases are added to this figures.^3-4 The currently available antiepileptic drugs (AEDs) in clinical practice produce effective seizure control only in 70% of patients.⁵ Moreover, several new AEDs (such as levetiracetam, pregabaline, topiramate) have proven to be effective in reducing seizures but their therapeutic efficacy is limited due to undesirable side effects such as anorexia, ataxia, headache, hepatotoxicity, gastrointestinal disturbance, hirsutism, and nausea.^6-7 Lamotrigine (containing phenyl 1,2,4-triazine basic moiety) is a broad spectrum antiepileptic drug acts by prolonging inactivation of voltage-sensitive Na⁺ channels.⁸ Aryl semicarbazones (like 4-bromobenzaldhyde semicarbazones) and various aryloxy semicarbazones revealed activity comparable with or exceeding that of phenytoin in the maximal electroshock (MES) induced seizure test in mice.^9-10

Based on the above facts, new 6-(3,5-substituted-2-bromo/hydroxy phenyl)-1,2,4-triazine derivatives represented by the following structure, were synthesized with anticipation of improvement in the anticonvulsant activity with little toxicity (Fig.1).

FIG.1: RATIONAL CONCEPT TO DESIGN NEW 6-(3,5-SUBSTITUTED-2-BROMO/HYDROXY PHENYL)-1,2,4-TRIAZINE DERIVATIVES (5a-j AND 6a-j).

MATERIAL AND METHODS:

Chemicals and solvents for synthesis were purchased by Merck (India), Spectrochem chemicals (India) and S.D. Fine Chemicals (India). Thin layer chromatography (TLC) was run throughout the reactions to optimize the reactions for purity and completion. Developing solvent for TLC was ethyl acetate and n-hexane (50 %). Iodine vapors or UV light was used as visualizing agents. Digital melting point apparatus was used for determine melting points of newly synthesized compounds, were uncorrected.

FT-IR (KBr) spectra of the synthesized compounds were recorded on a Nicolet 5PC FTIR spectrophotometer (λ-max in cm^-1) and ¹H NMR was observed on a Brucker Model-300 NMR Spectrometer in DMSO-d₆ using tetramethylsilane (TMS) as the internal reference (chemical shifts in δ ppm). The details of physical properties of the synthesized compounds were presented in Table 1.         

1. Synthesis of isatin semicarbazones or isatin thiosemicarbazones (3a-j): Semicarbazones and thiosemicarbazones (3a-j) were synthesized according to the procedure reported in literature.^11-12
2. Synthesis of 6-(2-amino-3,5-substituted phenyl)-1,2,4-triazines (4a-j): 6-(2-Amino-3,5-substituted phenyl)-1,2,4-triazines (4a-j) were synthesized according to the procedure reported in literature.^{11, 13}
3. General procedure for synthesis of 6-(3,5-substituted-2-hydroxy phenyl)-1,2,4-triazine derivatives (5a-j): 6-(3,5-Substituted-2-hydroxy phenyl)-1,2,4-triazine derivatives 5a-j were synthesized according to the procedure specified in literature with some modifications as shown in synthetic Scheme 1.¹¹ Appropriate triazines (4a-j, 0.6 mmol) were dissolved in sodium hydroxide solution (10%, 5 mL) with heating. After cooling sodium nitrite (0.8 mmol) was added, the mixture was cooled in an ice bath to 5 °C, it was added drop-wise to the cold solution of sulphuric acid (96%, 3.5 mL) and water (3.5 mL), and stirring in an ice bath was continued for 15 min. The mixture was filtered and boiled for 30 min. The precipitate was collected through filtration, washed with water and dried.

SCHEME I: FOR SYNTHESIS OF 6-(3,5-SUBSTITUTED-2-BROMO/HYDROXY PHENYL)-1,2,4-TRIAZINE DERIVATIVES (5a-j and 6a-j)

TABLE 1: PHYSICAL DATA OF COMPOUNDS 5a-j AND 6a-j

6-(2-hydroxyphenyl)-1,2,4-triazine-3,5(2H,4H)-dione 5a: IR (KBr): n_max = 3431 (OH_Phenolic), 3224 (NH_Triazine), 1736 (C=O, Amide), 1610 (C=N) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d =5.70 (br, s, 1H, OH), 6.68-7.24 (m, 4H, ArH), 12.13 (s, 1H, =NNH), 12.47 (s, 1H, NH) ppm. Mass (ES+) m/z= 332.6 (M⁺+1). Anal. calcd for C₉H₇N₃O₃  (205.0487): C 52.67, H 3.41, N 20.46; found  C 52.65, H 3.43, N 20.42.

3,4-dihydro-6-(2-hydroxyphenyl)-3-thioxo-1,2,4-triazin-5(2H)-one 5b: IR (KBr): n_max = 3386 (OH_Phenolic), 3176 (NH_Triazine), 1733 (C=O, Amide), 1616 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d =5.34 (br, s, 1H, OH), 6.92-7.50 (m, 4H, ArH), 12.77 (s, 1H, =NNH), 14.61 (s, 1H, NH) ppm. Mass (ES+) m/z= 222.3 (M⁺+1). Anal. calcd for C₉H₇N₃O₂S  (221.0259): C 48.84, H 3.21, N 18.97; found  C 48.82, H 3.23, N 18.95.

6-(5-fluoro-2- hydroxyphenyl) - 1, 2, 4 -triazine-3,5(2H,4H)-dione 5c: IR (KBr): n_max = 3453 (OH_Phenolic), 3226 (NH_Triazine), 1742 (C=O, Amide), 1605 (C=N) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d =5.70 (br, s, 1H OH), 6.68-7.24 (m, 3H, ArH), 12.10 (s, 1H, =NNH), 12.48 (s, 1H, NH) ppm. Mass (ES+) m/z= 223.1 (M⁺+1). Anal. calcd for C₉H₆FN₃O₃  (223.0393): C 48.46, H 2.69, N 18.80; found  C 48.49, H 2.67, N 18.78.

6-(5-fluoro-2-hydroxyphenyl)-3, 4 – dihydro - 3-thioxo-1,2,4-triazin-5(2H)-one 5d: IR (KBr): n_max = 3386 (OH_Phenolic), 3176 (NH_Triazine), 1730 (C=O, Amide), 1615 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d =5.34 (br, s, 1H, OH), 6.93-7.50 (m, 3H, ArH), 12.75 (s, 1H, =NNH), 14.65 (s, 1H, NH) ppm. Mass (ES+) m/z= 240.2 (M⁺+1). Anal. calcd for C₉H₆FN₃O₂S  (239.0165): C 45.21, H 2.55, N 17.59; found  C 45.23, H 2.52, N 17.61.

 6-(5-chloro-2-hydroxyphenyl)-1, 2, 4 - triazine-3,5(2H,4H)-dione 5e: IR (KBr): n_max = 3396 (OH_Phenolic), 3191 (NH_Triazine), 1741 (C=O, Amide), 1642 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d =5.79 (br, s, 1H, OH) 7.68-7.84 (m, 3H, ArH), 11.97 (s, 1H, NH), 12.79 (s, 1H, NH) ppm. Mass (ES+) m/z= 240.2 (M⁺+1). Anal. calcd for C₉H₆ClN₃O₃  (239.0098): C 45.14, H 2.54, N 17.52; found C 45.16, H 2.57, N 17.50.

6-(5-chloro-2-hydroxyphenyl)- 3, 4 - dihydro-3-thioxo-1,2,4-triazin-5(2H)-one 5f: IR (KBr): n_max = 3363 (OH_Phenolic), 3197 (NH_Triazine), 1733 (C=O, Amide), 1629 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d =5.54 (br, s, 1H, OH), 7.12-7.82 (m, 3H, ArH), 12.75 (s, 1H, =NNH), 14.11 (s, 1H, NH) ppm. Mass (ES+) m/z= 256.6 (M⁺+1). Anal. calcd for C₉H₆ClN₃O₂S (254.9869): C 42.26, H 2.41, N 16.41; found C 42.24, H 2.39, N 16.45.

6-(5-bromo-2-hydroxyphenyl)-1, 2, 4 - triazine -3,5(2H,4H)-dione 5g: IR (KBr): n_max = 3396 (OH_Phenolic), 3187 (NH_Triazine), 1741 (C=O, Amide), 1643 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d =5.21 (br, s, 1H, OH), 7.68-7.84 (m, 3H, ArH ), 11.97 (s, 1H, NH), 12.76 (s, 1H, NH) ppm. Mass (ES+) m/z= 285.6 (M⁺+1). Anal. calcd for C₉H₆BrN₃O₃ (282.9593): C 38.10, H 2.15, N 14.75; found C 38.12, H 2.13, N 14.77.

6-(5-bromo-2-hydroxyphenyl)-3, 4 - dihydro - 3-thioxo-1,2,4-triazin-5(2H)-one 5h: IR (KBr): n_max = 3362 (OH_Phenolic), 3193 (NH_Triazine), 1739 (C=O, Amide), 1625 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d =5.12 (br, s, 1H, OH), 7.13-7.82 (m, 3H, ArH), 12.76 (s, 1H, =NNH), 14.08 (s, 1H, NH) ppm. Mass (ES+) m/z= 300.6 (M⁺+1). Anal. calcd for C₉H₇ClN₄OS (298.9364): C 36.06, H 2.08, N 14.02; found C 36.08, H 2.12, N 13.98.

6-(3,5-dibromo-2-hydroxyphenyl)-1,2,4-triazine-3,5(2H,4H)-dione 5i: IR (KBr): n_max = 3367 (OH_Phenolic), 3167 (NH_Triazine), 1734 (C=O, Amide), 1638 (C=N) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d =5.20 (br, s, 1H, OH), 6.79-7.15 (m, 2H, ArH), 12.33 (s, 1H, =NNH), 12.68 (s, 1H, NH) ppm. Mass (ES+) m/z= 362.9 (M⁺+1). Anal. calcd for C₉H₆Br₂N₄O₂  (360.8698): C 29.82, H 1.41, N 13.24; found C 29.84, H 1.43, N 13.26.

 6-(3,5-dibromo-2-hydroxyphenyl)-3, 4 - dihydro-3-thioxo-1,2,4-triazin-5(2H)-one 5j: IR (KBr): n_max = 3357 (OH_Phenolic), 3123 (NH_Triazine), 1726 (C=O, Amide), 1608 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d  = 5.10 (br, s, H, OH), 6.80-7.31 (m, 2 H, ArH), 12.88 (s, 1H, =NNH), 13.90 (s, 1H, NH) ppm. Mass (ES+) m/z= 378.0 (M⁺+1). Anal. calcd for C₉H₆Br₂N₄OS  (376.8469): C 28.50, H 1.35, N 11.02; found C 28.48, H 1.37, N 11.05.

1. General procedure for synthesis of 6-(3, 5-substituted - 2 - bromophenyl) - 1, 2, 4 - triazine derivatives (6a-j):

6-(3,5-Substituted-2-bromophenyl)-1,2,4-triazine derivatives (6a-j) were synthesized according to the procedure specified in literature with some modifications as shown in synthetic Scheme 1.¹¹ Triazines (4a-j, 0.8 mmol) were dissolved in sodium hydroxide solution (10%, 5 mL) with heating. After cooling sodium nitrite (0.9 mmol) was added, the resultant mixture was cooled in an ice bath to 5 °C and added drop-wise to an ice cold mixture of hydrobromic acid (2.5 mL), water (2.5 mL) and toluene (2.5 mL). After stirring for 15 min, the resulting diazonium salt solution was carefully added to a solution of copper (I) bromide (0.5 mmol) in hydrobromic acid (48%, 5 mL) and water (5 mL). The mixture was stirred at room temperature overnight. The formed product was filtered, washed with water and dried.

6-(2-bromophenyl)-1,2,4-triazine-3, 5 (2H,4H) -dione 6a: IR (KBr): n_max = 3168 (NH_Triazine), 1735 (C=O, Amide), 1620 (C=N) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d = 6.78-7.14 (m, 4H, ArH), 12.32 (s, 1H, =NNH), 12.69 (s, 1H, NH) ppm. Mass (ES+) m/z= 266.9 (M⁺+1). Anal. calcd for C₉H₆BrN₃O₂  (366.9643): C 40.30, H 2.23, N 15.55; found C 40.28, H 2.21, N 15.57.

6-(2-bromophenyl)-3,4-dihydro-3-thioxo -1, 2, 4-triazin-5(2H)-one 6b: IR (KBr): n_max = 3129 (NH_Triazine), 1723 (C=O, Amide), 1631 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d  = 6.82-7.34 (m, 4H, ArH), 12.83 (s, 1H, =NNH), 13.89 (s, 1H, NH) ppm. Mass (ES+) m/z= 285.1 (M⁺+1). Anal. calcd for C₉H₆BrN₃OS  (282.9415): C 38.10, H 2.15, N 14.81; found C 38.12, H 2.16, N 14.83.

6-(2-bromo-5-fluorophenyl)-1, 2, 4- triazine -3, 5 (2H, 4H)-dione 6c: IR (KBr): n_max = 3226 (NH_Triazine), 1739 (C=O, Amide), 1660 (C=N) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d = 6.68-7.24 (m, 3H, ArH), 12.09 (s, 1H, =NNH), 12.43 (s, 1H, NH) ppm. Mass (ES+) m/z= 286.1 (M⁺+1). Anal. calcd for C₉H₅BrFN₃O₂   (284.9549): C 37.77, H 1.78, N 14.72; found  C 37.75, H 1.80, N 14.74.

6-(2-bromo – 5 - fluorophenyl)-3, 4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one 6d: IR (KBr): n_max = 3171 (NH_Triazine), 1722 (C=O, Amide), 1610 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d = 6.96-7.57 (m, 3H, ArH), 12.71 (s, 1H, =NNH), 14.54 (s, 1H, NH) ppm. Mass (ES+) m/z= 303.1 (M⁺+1). Anal. calcd for C₉H₅BrFN₃OS (300.9321): C 35.76, H 1.65, N 13.93; found  C 35.74, H 1.63, N 13.95.

6-(2-bromo-5-chlorophenyl)-1, 2, 4 - triazine- 3, 5(2H,4H)-dione 6e: IR (KBr): n_max = 3185 (NH_Triazine), 1726 (C=O, Amide), 1640 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d = 7.68-7.86 (m, 3H, ArH), 11.96 (s, 1H, NH), 12.74 (s, 1H, NH) ppm. Mass (ES+) m/z= 303.5 (M⁺+1). Anal. calcd for C₉H₅BrClN₃O₂  (238.6305): C 35.71, H 1.69, N 13.87; found C 35.70, H 1.71, N 13.86.

6-(2-bromo-5-chlorophenyl) - 3, 4- dihydro - 3-thioxo-1,2,4-triazin-5(2H)-one 6f: IR (KBr): n_max = 3193 (NH_Triazine), 1735 (C=O, Amide), 1619 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d = 7.12-7.83 (m, 3H, ArH), 12.74 (s, 1H, =NNH), 14.10 (s, 1H, NH), ppm. Mass (ES+) m/z= 319.5 (M⁺+1). Anal. calcd for C₉H₅BrClN₃OS (316.9025): C 33.90, H 1.60, N 13.21; found C 33.91, H 1.62, N 13.23.

6-(2,5-dibromophenyl)-1,2,4-triazine-3,5(2H,4H) - dione 6g: IR (KBr): n_max = 3116 (NH_Triazine), 1726 (C=O, Amide), 1642 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d = 6.56 -7.27 (m, 3H, ArH), 12.18 (s, 1H, =NNH), 12.93 (s, 1H, NH) ppm. Mass (ES+) m/z= 347.8 (M⁺+1). Anal. calcd for C₉H₅Br₂N₃O₂  (344.9629): C 31.20, H 1.42, N 12.13; found C 31.22, H 1.40, N 12.12.

6-(2,5-dibromophenyl)-3,4-dihydro – 3 - thioxo-1,2,4-triazin-5(2H)-one 6h: IR (KBr): n_max = 3184 (NH_Triazine), 1736 (C=O, Amide), 1651 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d = 6.72-7.33 (m, 3H, ArH), 12.37 (s, 1H, =NNH), 14.10 (s, 1H, NH) ppm. Mass (ES+) m/z= 364.2 (M⁺+1). Anal. calcd for C₉H₅Br₂N₃OS  (360.8520): C 29.80, H 1.41, N 11.55; found C 29.82, H 1.43, N 11.53.

6-(2,3,5-tribromophenyl)-1,2,4 – triazine - 3, 5 (2H,4H)-dione 6i: IR (KBr): n_max = 3167 (NH_Triazine), 1730 (C=O, Amide), 1630 (C=N) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) 6.75-7.15 (m, 2H, ArH), 12.38 (s, 1H, =NNH), 12.65 (s, 1H, NH) ppm. Mass (ES+) m/z= 426.4 (M⁺+1). Anal. calcd for C₉H₄Br₃N₃O₂  (422.7854): C 25.40, H 0.98, N 9.85; found C 25.42, H 0.97, N 9.83.

6-(2,3,5-tribromophenyl)-3, 4 -dihydro-3-thioxo-1,2,4-triazin-5(2H)-one 6j: IR (KBr): n_max = 3125 (NH_Triazine), 1724 (C=O, Amide), 1631 (C=N str) cm^-1. ¹H NMR (300 MH_Z, DMSO-d₆) d  = 6.81-7.31 (m, 2H, ArH), 12.87 (s, 1H, =NNH), 13.97 (s, 1H, NH) ppm. Mass (ES+) m/z= 442.6 (M⁺+1). Anal. calcd for C₉H₄Br₃N₃OS  (438.7625): C 24.44, H 0.94, N 9.53; found C 24.42, H 0.95, N 9.55.

Pharmacology: Male albino mice (CF-1 strain, 18-25 g) were used in groups as experimental animals. The synthesized compounds and standard drug were suspended in polyethylene glycol, administered intraperitoneally. All experimental protocols were carried out with permission from the Institutional Animal Ethics Committee (838/ac/04/CPCSEA). Animals were obtained from the Central Animal House Facility, Dr. K. N. Modi Institute of Pharmaceutical Education and Research, Modinagar, Ghaziabad, Uttar Pradesh, India.

1. Anticonvulsant screening: Synthesized compounds (5a-j and 6a-j) were evaluated for their anticonvulsant activity by using maximal electroshock seizure (MES) model.^14-15 In MES screening, an electrical stimulus of 0.2 s in duration (50 mA at 60 Hz of alternating current) in mice was delivered by corneal electrodes. In anticonvulsant screening, each compound was administered intraperitoneal at three dose levels 30, 100, 300 mg/kg. In this test, anticonvulsant activity of the synthesized compounds was performed after 0.5 and 4h intervals after test sample administration. Protection against the spread of MES-induced seizures displayed by the abolition of the hind limb and tonic maximal extension component of the seizure.

3. Neurotoxicity screening: Neurological toxicity induced by a compound was detected in mice by the rotarod test.¹⁶ The mice were trained to stay on rotating rotarod (3.2 cm rod diameter, 6 rpm speed). The motor impairment effect was observed by the inability of the animal to maintain equilibrium for a given time (1 min) in each of the trials.

1. CNS depression study: The CNS depression effect of some anticonvulsant active compounds is observed by screening the immobility time of the mice during swimming phase following the Porsolt’s swim pool test.¹⁷ Mice were placed in chamber (height 20 cm, diameter 45 cm) containing water up to a height of 15 cm at 25±2° C. Two swim sessions were performed, an initial 15 min pre-test, followed by a 5 min test session 24 h later. Then test compounds were administered through an intraperitoneal route (100 mg kg^-1) in the trained animals, before 30 min the test session. The duration of passive floating without struggling was recorded during the 5 min test period as immobility time.

Computational study: (Calculation of physicochemical parameters): A computational study for prediction of ADME properties of titled compounds (5a-j and 6a-j) was performed. The degree of absorption is expressed by the percentage of absorption. Percentage absorption (% ABS) was calculated by using: % ABS = 109-(0.345 × TPSA).¹⁸  Violations of Lipinski’s rule of five,¹⁹ milog P, number of rotatable bonds, polar surface area (TPSA),²⁰ molecular volume, number of hydrogen donor and acceptor atoms were computed by MolSoft software.²¹ The drug-score, drug-likeness and theoretical toxicity risks were computed by Osiris Property Explorer.²²

RESULT AND DISCUSSION:

1. Designing of the novel anticonvulsant compounds: Substitution on the aryl ring by halogens has been found to increase potency in the MES screen.²³ According to Unverferth et al. study²⁴ essential pharmacophoric elements that are necessary for good anticonvulsant activity through blocking of voltage-gated sodium channels are (i) aryl ring center or the lipophilic group (A), (ii) an electron donor atom (D), (iii) a hydrogen bond acceptor (HA) and (iv) a hydrogen bond donor (HD) unit (Fig.2). ^25-26

FIG. 2: STRUCTURES OF PROPOSED GENERAL PHARMACOPHORE MODEL OF THE DESIGNED COMPOUNDS AND REPORTED MARKETED DRUGS

1. Anticonvulsant and CNS depressant activities: The MES model for generalized tonic-clonic seizure evaluation keyed out some clinical candidates that forbade seizure spread (Table 2). Amongst the plethora of the synthesized compounds, two compounds 5c and 6i displayed significantly active profile against the electrically induced seizures at a dose of 30 mg/kg after 0.5 h. At the same dose level, compound 6i also showed activity after 4.0 h. Compounds 5j and 6h exhibited protection at the dose level of 100 mg/kg after 0.5 h. At 4.0 h, compounds 5e and 6g persisted to reveal anti-MES protection at the same dose, whereas, compounds 5b and 6b indicated to prevent the seizure spread at a higher dose of 300 mg/kg after 4h. Compound 6c displayed protection at 300 mg/kg after 0.5 h. The remaining compounds were found to be inactive at the maximum administered dose of 300 mg/kg at both times (0.5 h and 4.0 h).

The neurotoxicity screening data revealed that two compounds 5a and 6f exhibited neurotoxicity at a dose level of 300 mg/kg after 0.5 h. Compounds 5f and 6d displayed neurotoxicity at a maximum dose level of 300 mg/kg after 4.0 h. whereas rest the compounds were without any neurotoxicity.

Significantly anticonvulsant active compounds (5c, 6i and 6j) were further evaluated for their CNS depressant activity. With respect to control, screened compounds (5c, 6i and 6j) displayed 40.59, 56.88 and 66.61 % increase in immobility time, where as standard drug Carbamazepine revealed 58.63 % increase in the immobility time (Table 3). Putative binding site theory used for postulating the interaction of anticonvulsant compounds at a specific binding site, the molecule observed to interact with the protein receptor as shown in Fig. 3. ¹

74. Prediction of ADME properties: Computation study of the all compounds showed that none of the titled compounds violated lipinski’s rules and making them potentially promising agents for anticonvulsant therapy. All compounds exhibited a great % ABS ranging from 74.89 to 87.76% (Table 4). Positive drug likeness (1.06 to 5.22) and drug score (0.32 to 0.94) values of compounds were found to be similar or even better than some of the drugs currently used on the market [drug likeness (-0.88 to 4.20) and drug score (0.19 to 0.51) values] (Table 5). Theoretical toxicity risks data displayed that compounds (5b, 5d, 5f, 5h, 5j, 6b, 6d, 6f, 6h and 6j) showed only mutagenic and reproductive effective toxicities.

TABLE 2: ANTICONVULSANT AND MOTOR IMPAIRMENT SCREENING OF SYNTHESIZED COMPOUNDS (5a-j and 6a-j) USING MAXIMAL ELECTROSHOCK SEIZURE AND ROTAROD MODELS.

^aDoses of 30, 100, and 300 mg/kg were administered to albino mice through intraperitoneal route. The figures in the table indicate the minimum dose whereby bioactivity was demonstrated in half or more of mice. The dash (-) indicates an absence of activity at maximum dose administered (300 mg/kg). ^bData of Phenytoin and Carbamazepine (reference drugs) were obtained from the references.^{1, 27}

TABLE 3: DATA OF CNS DEPRESSANT ACTIVITY OF THE SELECTED COMPOUNDS (5c, 6i and 6j) PERFORMED IN MICE USING FORCED SWIM TEST.

The compounds were tested at a dose of 100 mg/kg. Each value represents the mean ± SEM of six mice. The CNS depressant effect was compared with respect to standard drug. *p < 0.0001. Data was analyzed by unpaired student’s t test.

TABLE 4: PHARMACOKINETIC PARAMETERS IMPORTANT FOR GOOD ORAL BIOAVAILABILITY OF COMPOUNDS 5a-j and 6a-j.

% ABS, percentage of absorption; TPSA, topological polar surface area; n-ROTB, number of rotatable bonds; MW, molecular weight; MV, molecular volume; n-OHNH, number of hydrogen bond donors; n-ON, number of hydrogen bond acceptors; miLogP, logarithm of compound partition coefficient between n-octanol and water.

TABLE 5:  DRUGLIKENESS, DRUG-SCORE AND IN SILICO TOXICITY RISKS OF TITLED COMPOUNDS 5a-j and 6a-j.

CONCLUSION: A computational study was also performed for the prediction of pharmacokinetic properties. Among the all screened compounds, compounds (5c), (6i) and (6j) revealed excellent protection against the electrically induced seizures at a dose of 30 mg/kg without neurotoxicity in rotarod test. Furthermore, all compounds also did not show Lipinski’s violation, making them potentially promising agent for epilepsy treatment.

ACKNOWLEDGEMENTS: Authors are grateful to the Director, Dr. K. N. Modi Institute of Pharmaceutical Education and Research, Modinagar, Ghaziabad, Uttar Pradesh, for providing laboratory facilities. The authors would like to express their gratitude to IIT, Delhi and CDRI, Lucknow for providing the spectral and elemental data.

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    How to cite this article:

    Kumar R, Singh A and Roy RK: Synthesis, Characterization and Pharmacological Evaluation of Some New 6-(3,5-Substituted-2-Bromo/Hydroxy Phenyl)-1,2,4-Triazine Derivatives as Anticonvulsant Agents. Int J Pharm Sci Res 2015; 6(8): 3490-00.doi: 10.13040/IJPSR.0975-8232.6(8).3490-00.

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