SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF SUBSTITUTED 1,3,4- THIADIAZOLE DERIVATIVES AS ANTICONVULSANT AGENTS

Author : Krishan Kumar, Kuntal Manna@, Hardik Bhatt*

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convulsions (P<0.01) and increased onset time for clonic convulsion (P<0.05) which was statistically significant in comparison to control. Study could further be investigated to design & identify lead compound. Keyword: Anticonvulsant agents, Carbamazepine, Convulsion, Epilepsy, Thiadiazole.    

Introduction

Epileptic seizures are responsible to cause brief injury of consciousness which leaves any individual at risk of injuring his/her own body and often interfering with day to day works, like family life, education, employment, etc. Available therapy for epileptic seizures and its consequences is symptomatic only as available drugs only inhibit seizures, but not effective as prophylaxis and not able to cure it completely. Major problem with epilepsy is compliance with medication and the reason behind this is need for long-term therapy without side effect [1]. One of the most common neurological disorder found in humans is epilepsy which affects around 2% of the global population. As per World Health Organization (WHO) data, 50 per 100 000 of the general population i.e. around 50 million people worldwide suffer from epileptic disorder. In United States of America, around 0.18 million new cases of epilepsy  are  observed  every year. Around 2.5 million patients experienced active epileptic episodes in recent 5 years. Overall, around 1% of child populations and around 0.5% of adults have an experience of recurrent seizures. Approximately 3% of the population experienced at least one seizure in lifetime. Lifelong medication is must for most patients. Apart from this, vagal nerve stimulation and surgery at defined respectable seizure foci are alternate options for treatment of epilepsy. Due to numerous economic and social issues with individuals having epileptic episode, patient gave little attention to disease as well as to treatment and makes quality of life poor. If these epileptic episodes become uncontrolled, then it results in substantial injury, mortality and economic burden to the patient. Healthcare industries costs at approximately $12.5 billion annually in USA [2]. Major causes of epilepsy includes; a brain injury (e.g. accident) infection or illness which affected development of brain of fetus during pregnancy; lack of oxygen during childbirth; encephalitis, meningitis or other infections affecting brain; brain tumours and lead or alcohol poisoning, etc. [3] A seizure is considered as a paroxysmal event happened to human due to abnormal, hyper or excessive synchronous discharge from central nervous system (CNS) neurons. Based upon distribution of discharges, this abnormal CNS activity can lead from dramatic convulsive activity to empirical phenomena not readily noticeable by spectator. Occasionally fit is considered as epileptic seizure and is   16                                                      SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF SUBSTITUTED…   defined as “a transient symptom of abnormal excessive or synchronous neuronal activity in the brain”. The superficial effect can be a rough movement (tonic-clonic seizure) or a brief loss of awareness. It marked the alteration of mental state, tonic or clonic actions, seizures, and various other cognitive signs. Sometimes, it is not accompanied by convulsions, but the person simply lost control of his/her body and collapse to ground. Medically, a condition of recurring, unprovoked seizures is termed as epilepsy, but seizures can arise in people who do not have epilepsy. Epileptic seizures are classified as, (i) partial seizures; (ii) primarily generalized seizures and (iii) unclassified seizures. [4-9] Contemporary treatment of seizures was started in 1850 with the introduction of bromides. In 1910, phenobarbital was found to have anti-seizure activity and became the drug of choice. In 1940, phenytoin (PHT) showed effect in epilepsy and since then, it was being used as major first-line antiepileptic drug (AED) in the treatment of partial and secondarily generalized seizures. Carbamazepine (CBZ) was initially approved for treatment of trigeminal neuralgia in1968 and then in 1974, it was approved for partial seizures. Ethosuximide was used as drug of choice in treatment of absence seizures (without generalized tonic-clonic seizures) since 1958. Valproate was used as important drug which was licensed in Europe in 1960 and then in USA in 1978. Now, it is widely used throughout the world. It became the drug of choice in primary generalized epilepsies and in the mid-1990s was approved for treatment of partial seizures. Even though, availability of many drugs for epilepsy and for seizures, mortality rates remains high and thus there is need to develop novel compounds which are useful, not only in reducing seizures, but also reduce mortality rate. Thus, efforts were given to synthesize and evaluate novel thiadiazole derivatives as anticonvulsant agents.

Results and Discussion Chemistry

Anticonvulsant agents, currently in use for treatment of epilepsy, have notable adverse effects and are inefficient in some types of seizures. Thus, there is clear need for safer and more effective antiepileptic drugs. Therefore, the development of new antiepileptic drug is major challenge for scientists working in the field of drug discovery. Acetazolamide          and methazolamide are the examples of 2,5- disubstituted-1,3,4- thiadiazole analogs. Various structures were studied for anticonvulsant activity and it was decided to synthesize novel substituted 1,3,4- thiadiazole derivatives and proved them as effective with anticonvulsant activity. In this study, sixteen new 1,3,4-thiadiazole derivatives was synthesized and evaluated for anticonvulsant activity against MES test. This title compounds were prepared from hydrazine hydrate and phenyl isothiocyanate or furan-2-carbonyl   NUJPS – 2019 | Vol. 6 | Issue 1                                                                                                                               17   isothiocyanate in presence of different thiourea and different aldehydes. In first step, isothiocyanate and hydrazine hydrate were reacted in presence of acetonitrile as a solvent to synthesize carbothiamide. In the second step, carbothiamide reacted with various substituted benzaldehyde and urea/thiourea to synthesize final derivatives, substituted 1,3,4-thiadiazole derivatives. Title compounds showed N–H and C-H stretching bands in the region of 3300- 3350 and 3100-3150 cm–1, respectively, which indicated presence of secondary amine. However IR spectra do not give complete idea about ring formation. In both series, title compound showed other bands like C-S stretching in the region of 691 cm–1, C=N stretching of thiadiazole in the 1500-1600 cm–1 region, C-N stretching at 1350-1300 cm–1 and aromatic C=C stretching band near 1600 cm–1 and 1450 cm–1 with overtone in the region of 1700- 2000 cm–1. Due to fermi resonance, overtone of N-H bending and C-N stretching, weak band was observed in the region of 3100 cm-1. Aliphatic C=C band observed only in case of second series, in

Anticonvulsant Activity

The anticonvulsant activity of the title compounds was evaluated by using Pentylenetetrazole (PTZ) induced convulsions model (60mg/kg) in mice and carbamazepine was used as a reference standard (100 mg/kg). From the first series of compounds, 6b, 6f, 6g, 6k and 6m showed 100% protection against 1hr and 24 hours mortality, which were statistically significant (P<0.05) in comparison to control. It also decreased number of clonic convulsions (P<0.05) and increased onset time for clonic convulsions (P<0.01), which was statistically significant in comparison to control. Similarly, in second series of compounds, 10a showed 66.66% protection against 1 hour mortality and 50% protection against 24 hours mortality as well as decreased number of clonic convulsions and increased onset for clonic convulsion which, however was not- significant in comparison to control. Data are shown in Table 1.   the region of 1600-1500 cm–1 which indicates the presence of styryl moiety. The structures of final synthesized derivatives were verified by mass spectral analysis, where molecular ion peaks (m/z value) were in complete arrangement with the calculated molecular weight for all compounds. The compounds with presence of halogen showed prominent peaks. 18                                                      SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF SUBSTITUTED…  

Table 1. Effect of synthesized compounds on PTZ – Induced Seizure in Mice

 
Compounds (Dose – 100mg/kg) (Volume – 10ml/kg) Clonic convulsions (Mean ± S.E.M) % of protection in 1 hr (mortality of animals N/F) % of protection in 24 hrs (mortality of animals N/F)
Onset of Clonic Convulsion* No. of Clonic Convulsions* (in 30 min)
Vehicle 10.00 ± 0.89 3.00 ± 0.25 2/6 = 0.33% 1/6 = 16.66%
Carbamazepine 25.50 ± 1.05 1.16 ± 0.16 6/6 = 100% 6/6 = 100%
6a 20.35 ± 1.57 1.66 ± 0.33 4/6 = 66.66% 6/6 = 100%
6b 20.32 ± 1.51 1.16 ± 0.33 6/6 = 100% 6/6 = 100%
6c 17.46 ± 0.99 2.00 ± 0.36 4/6 = 66.66% 3/6 = 50%
6d 20.71 ± 1.46 2.83 ± 0.30 4/6 = 66.66% 3/6 = 50%
6e 21.25 ± 1.15 2.00 ± 0.25 6/6 = 100% 5/6 = 83.33%
6f 17.61 ± 0.69 1.33 ± 0.21 6/6 = 100% 6/6 = 100%
6g 20.15 ± 0.92 1.16 ± 0.16 6/6 = 100% 6/6 = 100%
6h 16.97 ± 1.39 2.83 ± 0.47 3/6 = 50% 3/6 = 50%
6i 19.07 ± 1.07 1.66 ± 0.33 5/6 = 83.33% 4/6 = 66.66%
6j 16.35 ± 0.96 2.16 ± 0.47 5/6 = 83.33% 3/6 = 50%
6k 13.84 ± 1.22 1.66 ± 0.33 6/6 = 100% 6/6 = 100%
6l 17.18 ± 1.23 1.33 ± 0.21 4/6 = 66.66% 6/6 = 100%
6m 18.35 ± 1.13 1.50 ± 0.22 6/6 = 100% 6/6 = 100%
10a 8.69 ± 1.12 1.50 ± 0.22 4/6 = 66.66% 3/6 = 50%
10b 10.40 ± 0.99 1.66 ± 0.33 6/6 = 100% 4/6 = 66.66%
10c 13.91 ± 0.72 1.16 ± 0.16 5/6 = 83.33% 4/6 = 66.66%
* No of animals – 6         NUJPS – 2019 | Vol. 6 | Issue 1                                                                                                                               19  

Experimental Section

General procedure for synthesis of 1,3,4-thiadiazol derivatives:                       Scheme 1. Synthesis of 5-substituted-N-phenyl-1,3,4-thiadiazol-2-amine derivatives     To a round bottom flask, hydrazine hydrate (1), phenyl isothiocyanate (2) and acetonitrile as a solvent were mixed properly and was stirred for 20-25 min at mixture is then stirred at 25-30oC for 70-80 min. After completion of reaction, the resulted  precipitates  of   5-substituted-N- p h e n y l – 1 , 3 , 4 – t h i a d i a z o l – 2 – a m i n e   25-30 oC         to          form         4- derivatives (6a-m) were vacuum filtered.   phenylthiosemicarbazide (3). In this mixture, substituted benzaldehyde (4a-m) and thiourea (5) were added. The reaction The precipitate was further purified with methanol and results were reported. [10- 13]                                     Scheme 2. Synthesis of N-(5-substituted-1,3,4-thiadiazol-2-yl)furan-2-carboxamide derivatives 20                                                      SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF SUBSTITUTED…   To a round bottom flask, hydrazine hydrate (1), furan-2-carbonyl isothiocyanate (7) and acetonitrile as a solvent were mixed properly and was stirred for 20-25 min at 1,3,4-thiadiazol-2-yl)furan-2-carboxamide derivatives (10a-c) were vacuum filtered. The precipitate was further purified with methanol and results were reported. [10-   25-30 oC         to         form         N- 13]   (hydrazinecarbonothioyl)           furan-2- carboxamide (8). In this mixture, substituted benzaldehyde (9a-c) and thiourea (5) were added. The reaction mixture is then stirred at 25-30 oC for 70- 80 min. After completion of reaction, the resulted precipitates of N-(5-substituted- All synthesized compounds were characterized by physical characteristics like melting point and Rf. All synthesized compounds were characterized by FT-IR. Few compounds were also characterized by Mass spectral analysis.  

Table 2. Physical characteristics of synthesized compounds.

                 
Comp. No. Molecular Formula Substitution % Yield Rf * M.P. (in oC)
6a C14H10N3SCl 4-chlorophenyl 61.02 0.78 176-178
6b C14H10N3SF 4-fluorophenyl 57.04 0.64 158-160
6c C14H11ON3S 3-hydroxyphenyl 70.53 0.77 169-172
6d C16H15O2N3S 3,4-dimethoxy phenyl 64.66 0.69 186-188
6e C14H10N3SBr 3-bromophenyl 40.22 0.79 178-180
6f C14H10 O2N4S 2-nitrophenyl 55.33 0.81 158-160
6g C14H10N3SCl 2-chlorophenyl 58.71 0.71 113-115
6h C15H13ON3S 4-methoxyphenyl 42.81 0.75 170-171
6i C14H10 O2N4S 3-nitrophenyl 30.31 0.70 166-168
  NUJPS – 2019 | Vol. 6 | Issue 1                                                                                                                               21  
6j C16H16N4S 4-dimethylamino phenyl 52.62 0.65 164-166
6k C14H11ON3S 4-hydroxyphenyl 47.86 0.80 170-172
6l C19H14N4S Nicotinaldehyde 74.50 0.83 170-172
6m C18H13ON3S 4-furan 66.04 0.69 170-172
10a C13H8O2N3SF 4-fluorophenyl 58.91 0.79 158-160
10b C15H13O4N3S 3,4-dimethoxy phenyl 51.43 0.84 181-183
10c C13H8O2N3SCl 4-chlorophenyl 48.94 0.77 169-170
* n-Hexane : Ethyl acetate :: 4:1

Table 3. Spectral characteristics of synthesized compounds.

 
Comp. No. Substitution FT-IR (cm-1) Mass
6a 4-chlorophenyl 3309, 3137, 2550, 1670, 1500, 1083 288 (M+ + 1)
6b 4-fluorophenyl 3330, 3145, 2510, 1690, 1480, 1089 273 (M+ + 1)
6c 3-hydroxyphenyl 3100, 3145, 2530, 1680, 1520, 1090 272 (M+ + 2)
6d 3,4-dimethoxy phenyl 3310, 3158, 2490, 1695, 1495, 1097 314 (M+)
6e 3-bromophenyl 3280, 3133, 2525, 1650, 1470, 1098 333 (M+ + 1)
6f 2-nitrophenyl 3230, 3110, 2470, 1650, 1474, 1091 301 (M+ + 2)
6g 2-chlorophenyl 3309, 3137, 2550, 1670, 1500, 1083 289 (M+ + 2)
6h 4-methoxyphenyl 3290, 3100, 2515, 1678, 1514, 1075 284 (M+ + 1)
6i 3-nitrophenyl 3230, 3110, 2470, 1650, 1474, 1091 299 (M+)
6j 4-dimethylamino phenyl 3330, 3210, 2570, 1490, 1091 298 (M+ + 1)
6k 4-hydroxyphenyl 3100, 3145, 2530, 1680, 1520, 1090 271 (M+ + 1)
  22                                                      SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF SUBSTITUTED…  
6l Nicotinaldehyde 3225, 3090, 2512 1650, 1515, 1070 333 (M+ + 2)
6m 4-furan 3269, 3110, 2550, 1690, 1550, 1089 318 (M+ + 1)
10a 4-Fluorophenyl 3310, 3108, 2495, 1705, 1510, 1090 257 (M+ + 1)
10b 3,4-dimethoxy phenyl 3290, 3150, 2480, 1653, 1515, 1100 300 (M+ + 1)
10c 4-chlorophenyl 3230, 3105, 2525, 1690, 1510, 1110 274 (M+ + 2)
   

Pharmacological Evaluation

The pharmacological screening protocol was approved by the Institutional Animal Ethics Committee.

Pentylenetetrazole (Metrazol) induced convulsions

The assay is used primarily to evaluate antiepileptic drugs.
Procedure
Mice of either sex with a body weight between 18 and 22 g were used. The test compounds or the reference drug were injected s.c. or i.p. or given orally to groups of 6 mice. Another group of 6 mice served as control. Fifteen min after sc.-injection or 30 min after i.p.-injection or 60 min after oral administration, 60 mg/kg MTZ (Metrazol) was injected, subcutaneously. Each animal was placed into an individual plastic cage for observation lasting for 1 h. Seizures and tonic-clonic convulsions were recorded. It is noted that at least 80% of the animals in the control group showed convulsions [14].

Evaluation

The number of protected animals in the treated groups is calculated as percentage of NUJPS – 2019 | Vol. 6 | Issue 1 affected animals in the control group. Furthermore, the time interval between MTZ-injection and occurrence of seizures was measured. The delay of onset was calculated in comparison with the control group.

Maximal electroshock seizure (MES) in mice

Purpose and Rationale
The electroshock assay in mice is used primarily as an indication for compounds which are effective in grand mal epilepsy. Tonic hind limb extensions are evoked by electric stimuli which are suppressed by anti-epileptics but also by other centrally active drugs.
Procedure
Groups of 6 male NMRI mice (20–25 g) were used. The test was started 30 min after i.p. injection or 60 min after oral treatment with the test compound or the vehicle. An apparatus with corneal or ear electrodes was used to deliver the stimuli. The intensity of stimulus 12 mA, 50 Hz for 0.2 s was used for screening.     23  

Evaluation

The animals were observed closely for 2 min. Disappearance of the hind leg extensor tonic convulsion were used as positive criterion. Percent of inhibition of seizures relative to controls were calculated.

Measurement of the Activity

Carbamazepine (100 mg/kg i.p.) and the dose of Pentylenetetrazole which induced convulsions in 97% of animals (CD97: 60 mg/kg s.c mice) were used. A suspension of 5% sodium carboxyl methyl cellulose (CMC) at a dose of 10 ml/kg was used as control. All synthesized derivatives were administered as a suspension of 5% sodium CMC. Swiss albino mice of either sex (5-6- weeks-old) weighing 30-35g were housed under standard laboratory conditions (relative humidity 55-56 %, room  temperature 23.0 ± 2.0 oC and 12 h light:dark cycle).The animals were fed with standard diet and water ad libitum. They were fasted overnight prior to experiment. [15] The mice were divided into groups of 6 animals each. One group was served as control which received 5% sodium CMC (10 ml/kg i.p.) 30 min before administration of PTZ (60 mg/kg s.c.). PTZ were administered by injecting into a loose fold of skin in the midline of the neck. Second group received carbamazepine (100 mg/kg i.p.) dissolved in a suspension of 5% sodium CMC as a standard reference drug. Remaining groups received test compounds, at a dose of 100 mg/kg i.p. dissolved in suspension of 5% sodium CMC respectively 30 min before the administration of PTZ (60 mg/kg s.c.). Each animal was placed into an individual plastic cage for observation lasting 0.5 h. The time taken before the onset of clonic convulsions, the duration of clonic convulsions and percentage of mortality protection were recorded

Conclusion

E p i l e p s y i s a d i s e a s e d c o n d i t i o n characterized by recurrent seizures of cerebral origin. It is associated with episodes o f s e n s o r y, m o t o r o r a u t o n o m i c phenomenon  with  or   without   loss   of c o n s c i o u s n e s s . C u r r e n t l y u s e d anticonvulsant agents for treatment of epilepsy have certain disadvantages and a clear need for safer and more effective antiepileptic drugs is of urgent need. Therefore, the development of new antiepileptic drugs with greater effects is an important challenge for scientists working in the field of drug discovery. In present work, for getting synergistic response of mentioned moiety, series of potential anticonvulsant agents  belonging to s u b s t i t u t e d 1 , 3 , 4 – t h i a d i a z o l e w e r e synthesized. Reaction monitoring was done by TLC, using precoated Sillica gel G plates. T h e s y n t h e s i z e d c o m p o u n d s w e r e characterized for physical constants like melting point. Structure elucidation of synthesized compounds was done by FTIR and Mass spectroscopy. The anticonvulsant activity of the final derivatives was evaluated by using PTZ model (60mg/kg) and carbamazepine taking as a reference standard (100 g/kg). All synthesized compounds showed no sedation side effect   24                                                      SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF SUBSTITUTED…   as compared to carbamazepine. From all synthesized compounds, few compounds showed significant protection against pentylenetetrazole induced convulsions as well as mortality within 24 h in mice and   a l s o d e c r e a s e d n u m b e r o f c l o n i c convulsions (P<0.01) and increased onset time for clonic convulsion (P<0.05) which was statistically significant in comparison to control. Among these, few compounds gave anticonvulsant activity comparable to the standard carbamazepine and emerged as a lead compounds in the  series  when they w e r e s u b j e c t e d t o p r e l i m i n a r y anticonvulsant screenings. They  also showed 100% protection against mortality. These can be regarded as strong candidates for future investigations.

Acknowledgement

The authors are thankful to Nirma University, Ahmedabad, India for providing necessary facilities to carry out the research work.

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