Synthesis and Antiepileptic Evaluation of Some New Thiophene Incorporated 1,5-Benzothiazepine Derivatives

Chaithanya P¹, Abhishek Kumar¹*_, M Chandana Adiga¹,

Krishnapriya K R¹, Gupta Dheeraj Rajesh¹, Aravinda Pai²

¹Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences (NGSMIPS),

Nitte (Deemed to be University), Mangalore, India-575018

²Department of Pharmaceutical Chemistry, Manipal Academy of Higher Education,

Manipal College of Pharmaceutical Sciences (MCOPS), Manipal, (Karnataka) India.

*Corresponding Author E-mail: abhi12bunty@nitte.edu.in

ABSTRACT:

Epilepsy is a chronic neurological disorder affecting approximately 50 million individuals worldwide. It poses a significant challenge like osteoporosis, Stevens-Johnson syndrome, agranulocytosis, aplastic anemia, hepatic failure, pancytopenia, multiorgan hypersensitivity, and psychosis due to limited efficacy of currently available antiepileptic drugs (AEDs). The heterocyclic rings thiophene and 1,5-benzothiazepine have been studied extensively for their antiepileptic activity. This study synthesized a series of new thiophene-incorporated 1,5-benzothiazepine derivatives.  The synthetic technique involved the condensation of ketomethylene with malononitrile to form a Knoevenagel–Cope condensation product, which cyclizes with sulfur and diethyl amine to give a 2-aminothiophene derivative. This forms thienyl chalcones upon treatment with different substituted benzaldehyde in ethanol and sodium hydroxide as a base at room temperature. Upon refluxing with o-amino thiophenol, these chalcones form corresponding thiophene-incorporated benzothiazepine derivatives. The structure of these compounds was confirmed by IR, ¹H NMR, and Mass spectroscopy. Compound TBA5 was subjected to in vivo antiepileptic activity by Maximal electroshock induced seizure and Pentylenetetrazole induced seizure model. Compound TBA5, in three different dosage levels (low, medium, high), effectively decreased convulsions compared to the control. Hence, the findings highlight the potential of the newly designed compound TBA5 as a viable option for further exploration as an antiepileptic medication.

KEYWORDS: Epilepsy, Thiophene, Chalcones, Benzothiazepine, antiepileptic.

INTRODUCTION: 

Epilepsy is a most common non-communicable primary neurological disease where the activity of the nerve cells is agitated in the brain, which causes seizures. According to the World Health Organization (WHO), epilepsy is one of the oldest disorders present in humans. There are more than 50 million people worldwide who are affected by epilepsy. Epilepsy is a disorder where a person experiences intermittent seizures, usually unprovoked and stereotyped, which result from the abnormal, paroxysmal electric discharge of neurons of the cerebral cortex. Seizures may differ from muscle twitching to chronic and elongated convulsions. The frequency can vary from several months to a few days. Two groups of seizures are named partial and generalized. A hereditary disorder or a traumatic brain injury can cause epilepsy. Abnormal behavior, symptoms, sensations, and chances of unconsciousness may occur during seizures. Medication can cure convulsions, and, in some cases, surgery and dietary changes are also treatments^1,2,3. Newer antiepileptic drugs (AED) have been developed for years because there is the possibility of patients becoming resistant to AEDs. Hence, there is a need for newer AEDs without pharmacokinetic drug interactions, drugs with minimal adverse effects, and drugs with different mechanisms of action for synergistic combination therapy. The majority of existing AEDs suffer from toxicity issues. Nausea, diplopia, dizziness, headache, tiredness, and ataxia are some of the most prevalent dose-related adverse effects of AEDs^4,5,6.

Thiophene is a five-membered monocyclic heterocyclic compound with sulfur at 1st position with the molecular formula C₄H4S. It is chemically called thiacyclopentadiene. Thiophene has gained interest from early dye chemistry to modern drug design. Thiophene derivatives are extensively used due to their wide therapeutic applications. Fused heteroaromatic compounds are more widely employed than the monocyclic compound. Newer heterocyclic compounds can be synthesized by fusing thiophene and heterocyclic systems. Anti-inflammatory, anti-psychotic, anti-arrhythmic, anti-anxiety, anti-fungal, antioxidant, estrogen receptor regulating, anti-mitotic, anti-microbial, kinases inhibiting, and anti-cancer activities are found in the thiophene moiety^7,8,9. Benzothiazepines are seven-membered heterocyclic rings with nitrogen and sulfur. Benzothiazepines are an important compound because of their pharmacological properties. They have become interesting since 1,5-benzothiazepines show antifungal, antibacterial, analgesic, and anticonvulsant activity. 1,5-Benzothiazepines are well-known benzologs of 1,4-thiazepine and one of the three possible benzo-condensed derivatives, namely, 1,4-, 4,1- and1,5-benzothiazepines¹⁰. 1, 5- benzothiazepines are gaining more interest due to their broad range of chemotherapeutic applications like anticonvulsive. The first clinically used 1,5-benzothiazepine molecule is diltiazem for its cardiovascular activity, followed by clentiazem. Some of the other clinically used drugs for CNS disorders are thiazesim, clothiapine and quetiapine^11,12.

MATERIAL AND METHODS:

All reactions were carried out under the specified laboratory conditions. All the chemicals and solvents employed in the synthesis process were of the analytical and laboratory standard. Recrystallization of the products was done using a suitable solvent. A Digital Point device was used to calculate melting points.

Synthesis of 1,5-benzothiazepine derivatives:

The synthesis of thiophene-incorporated benzothiazepine  involved the following three steps.

Synthesis of 2-aminothiophene:

An equimolar mixture (0.1 M) of ketomethylene and malononitrile with 0.11 M of sulphur was taken in a flask. 10-30 mL of ethanol as a solvent and 10 mL of diethylamine as a base were added. This reaction mixture was stirred in a water bath for about 3h at 45-50 ⁰C. The temperature of the reaction should be maintained between 60⁰C. The solid obtained at the end of the reaction was filtered, washed, and dried. It was recrystallized using ethanol to get orange color solids^13,14.

Synthesis of thienyl chalcones:

0.1 M of 2-aminothiophene and 0.1 M of different substituted benzaldehyde were dissolved in 20 mL of ethanol. The mixture was stirred for 24hr in the presence of 3-4 mL of 20% NaOH solution. TLC confirmed the completion of the reaction. The solution was poured into the crushed and acidified with dilute HCl. The chalcones obtained were filtered, washed, dried, and recrystallized using suitable solvent^15,16.

Synthesis of thiophene incorporated benzothiazepine:

20mmol of the chalcone was dissolved in 100ml anhydrous methanol, which was then acidified to pH 2 with a few drops of concentrated hydrochloric acid. To the above acidified solution, 20mmol of o-aminothiophenol was added. This mixture was refluxed until the solid was precipitated out. The solid obtained was cooled, washed, dried, and recrystallized using ethanol to form needle-shaped crystals¹⁷.

Pentylenetetrazole (PTZ) induced convulsions in mice:

The animals were separated into five groups consisting of six animals each. The control group was given an intraperitoneal injection of 0.1% CMC, while the standard group received an intraperitoneal injection of 0.5mg/kg of Diazepam. The remaining three groups received different doses of the test compound, categorized as low, medium, and high, and were administered orally 30 minutes before being injected with PTZ (80 mg/kg). The presence of tonic seizures and clonus was observed, and the animals were monitored for protection at 0.5 hours and 24 hours after administration^18,19.

Maximal electroshock (MES) induced convulsion in mice:

The mice selected for the experiments were divided into 5 groups of 6 animals each, control, standard and test (low, medium and high dose). The animals in the control group received 0.1% of CMC i.p. the standard received 25mg/kg of Phenytoin i.p. the test group received 1.04mg/kg, 3mg/kg and 8.04 mg/kg of compound CBT-05 orally. After 60 min of the drug administration an electric shock of 150 mA, 60Hz for 0.2 sec were applied through the ear clip electrodes by Orchid scientific digital electroconvulsiometer (model EC02) ²⁰. These animals were observed for different stages of MES-induced seizures like flexion, extensor, stupor, and tonic-clonic. The duration of each phase is compared with standard and control. The mortality of each group was observed after 24 hours.

Scheme

RESULTS:

Spectral Characterization of Synthesized Compounds

2-amino-5-(4-(4-hydroxy-3-methoxyphenyl)benzo[b] [1,4]thiazepin-2-yl)-4-methylthiophene-3-carbonitrile: (TBA4) Yield:47 %, m.p:203, IR (cm^-1): 2975 (Ar C-H str), 2893 (Aliphatic.C-H str),1541(C=C str),3564(NH₂ str), 3359 (O-H str), 2210(C≡N),1650 (C=N str), 697(C-S str). ¹H NMR(400 MHz, DMSO-d₆): δ7.315-8.324 (m, 8H, Ar-H), 7.315-7.733 (m, 5H, benzothiazepine), 5.207 (s, 2H, NH₂), 3.318 (s, 3H, OCH₃). MS ( M⁺): 418

2-amino-5-(4-(4-hydroxyphenyl)benzo[b][1,4]thiazepin-2-yl)-4-methylthiophene-3-carbonitrile: (TBA5) Yield: 88 %, m.p: 202,IR (cm^-1): 3437(O-H str), 3353 (NH₂ str), 3248 (Ar. C-H str), 2214(C≡N str), 1646 (C=N str), 1449(C=C str),635(C-S str). ¹H NMR(400 MHz, DMSO-d₆): δ 7.315-8.324 (m, 9H, Ar-H), 7.315-7.733 (m, 5H, benzothiazepine), 7.795-8.324 (m,4H, Ar-H),5.207 (s, 2H, NH₂), 7.115 (s, 1H, OH). MS ( M⁺): 388

2-amino-5-(4-(4-hydroxy-3-methoxyphenyl)benzo [b][1,4]thiazepin-2-yl)-4-phenylthiophene-3-carbonitrile: (TBB4) Yield: 48 %, m.p: 235, IR (cm^-1): 3061(Ar C-H str), 1417 (C=C str),3413(O-H str), 3313 (NH₂ str), 1622 (C=N str), 637(C-S str). ¹H NMR (400 MHz, DMSO-d₆): δ7.195-8.376 (m, 13H Ar-H), 7.195-7.740 (d, 5H, benzothiazepine), 7.059(s, 1H,OH). MS (M⁺) : 480

Table 1: Physical data of thiophene incorporated 1,5-benzothiazepine Derivatives

Compound code	R	R^I	Mol. formula	Mol. wt	m.p (^0C)	% yield
TBA1	CH3	4-Cl	C21H14ClN3S2	407.93	188	60 %
TBA2	CH3	2-Cl	C21H14ClN3S2	407.93	173	72 %
TBA4	CH3	3-OMe, 4-OH	C22H17N3O2S2	418.51	203	47 %
TBA5	CH3	4-OH	C21H15N3OS2	388.48	202	88 %
TBA6	CH3	2-OMe	C22H17N3OS2	403.51	201	70 %
TBA9	CH3	3-NO2	C21H14N4O2S2	400.45	198	52 %
TBA10	CH3	4-F	C21H14FN3S2	373.44	193	50 %
TBA11	CH3	2,4-Cl	C21H13Cl2N3S2	442.34	186	58 %
TBA13	CH3	3-Cl	C21H14ClN3S2	407.93	173	60 %
TBB4	C6H5	3-OMe, 4-OH	C27H19N3O2S2	480.58	235	48 %

Table 2: Effect of compound TBA5 on PTZ model

Groups	Treatment	Dose (mg/kg)	Latency (Sec/0.5hr)	Tonic convulsion (Sec/0.5hr)	Status of animal after 0.5hrs		Status of animal after 24hrs
					No of animal alive	% Protection	No of animal alive	% Protection
I	control	-	48.26±1.25	98.52±3.56	0/6	0	0/6	0
II	STD (Diazepam)	5	178.9±2.38*	127.2±2.38*	6/6	100	5/6	83.3
III	Low	8.75	67.85±1.58**	179.5±2.58*	4/6	66.7	4/6	66.7
IV	Medium	17.5	118.5±2.89*	142.5±1.85**	4/6	66.7	4/6	66.7
IV	High	35	129.5±1.39*	125.6±3.25*	4/6	66.7	4/6	66.7

Values are expressed as mean ± SD for 6 animals; ***p < 0.001, *p < 0.001 and *p < 0.05 Vs control.

Figure 1: Effect of compound TBA5 on PTZ model

Figure 2: Different phases of convulsion in PTZ inducedmodel

Table 3: Effect of compound TBA5 on MES model

Groups	Treatment	Flexion	Extensor	Clonus	Stupor	HLE	% protection
I	control	2.6± 1.2	22.31± 3.6	39.56± 3.5	86.25± 2.5	101.3± 3.5	0
II	STD (Phenytoin)	1.2± 0.89**	9.35± 1.20*	15.25± 2.50**	26.52± 2.60*	56.25± 1.89*	100
III	Low	2.3± 0.85**	12.52± 3.58*	29.56± 2.53*	56.25± 3.89*	86.52± 2.52**	66.7
IV	Medium	3.6± 1.25*	10.85± 1.89**	30.24± 2.30^ns	45.13± 0.65**	66.52± 0.98*	66.7
IV	High	2.9± 0.89*	13.56± 1.25*	26.52± 1.25*	41.28± 2.85*	63.25± 0.89*	66.7

Values are expressed as mean ± SD for 6 animals; ***p < 0.01, *p < 0.001 and *p < 0.05 Vs control.

Figure 3: Effect of compound TBA5 on MES model

Figure 4: Different phases of convulsion in MES model

Synthesis of thiophene incorporated 1,5-benzothiazepine derivative

The in-silico studies of the designed molecules were published²⁰. Based on the in-silico studies of the designed molecules best ten compounds were selected for synthesis. The technique involves the condensation of ketomethylene with malononitrile to form a Knoevenagel–Cope condensation product which cyclizes with sulfur and diethyl amine to give 2-aminothiophene derivative. Different thienyl chalcone derivatives were prepared by reacting 2-aminothiophene derivative with different substituted benzaldehyde in ethanol and sodium hydroxide as a base. These compounds were characterized by TLC, melting point, and IR spectra showing characteristic absorption bands at 1650-1685 (C=O str) for chalcones. The title compounds thiophene incorporated benzothiazepine derivatives were prepared by cyclization of thienyl chalcones with o-amino thiophenol in glacial acetic acid as given in the scheme. The IR spectra of benzothiazepine derivatives showed characteristic absorption bands at 1590-1610 cm^-1 corresponding to C=N str of benzothiazepine, which was absent in the intermediate thienyl chalcones. Similarly the ¹H NMR of the synthesized benzothiazepine derivatives showed one characteristic signal at δ 7.315-7.733 (m, 5H, benzothiazepine) which was absent in the ¹H NMR spectra of thienyl chalcones. Hence, the formation of the title compounds benzodiazepines was confirmed and further established by mass spectra in accordance with molecular formula.

The PTZ induced seizures model aimed to evaluate the effects of different treatments on latency to tonic convulsions and the status of animals after 0.5 hours and 24 hours. The treatments administered were a control group, a standard treatment group (STD) receiving Diazepam, and three experimental groups receiving different doses of compound TBA5 (Low, Medium, and High doses). The results indicated a significant decrease in convulsion duration across the three doses of the compound and in comparison to both the control and standard values, as given in Table 2 and Figure 1. The STD group (administered with Diazepam) showed a significantly longer latency to tonic convulsions (178.9 seconds) and a shorter duration of convulsions (127.2 seconds) compared to the control group. Among the experimental groups, the high and medium doses showed similar efficacy in increasing latency to convulsions and reducing the duration of convulsions when compared to the control group. The status of animals after 0.5 hours and 24 hours demonstrated that the STD group had the highest percentage of animal survival (100% after 0.5 hours and 83.3% after 24 hours), followed by the experimental groups receiving low, medium, and high doses (all with 66.7% survival after 0.5 hours and 24 hours). Overall, the experimental groups receiving different doses of the compound TBA5 also showed some protective effects as standard. Compound TBA5 was evaluated for its effects on MES-induced seizures at three doses, as shown in Table 3 and Figure 3. The convulsive phases, including Flexion, extensor, clonus, stupor, HLE, and percentage protection, were compared to the control group. In the control group (I), the mean values for all parameters are significantly higher compared to the treated groups, indicating a higher severity of induced seizures in the control animals. The STD (Phenytoin) treatment (group II) significantly reduces all seizure parameters compared to the control group, as indicated by the lower mean values. The low treatment (group III) also demonstrates a significant reduction in seizure parameters compared to the control group, similar to the STD treatment group. The medium treatment (group IV) shows a significant reduction in flexion, extensor, clonus, and HLE scores compared to the control group, but the stupor score is not significantly different. The high treatment (group V) exhibits a significant reduction in flexion, extensor, clonus, stupor, and HLE scores compared to the control group. Compared to the control animals, the test compound exhibited HLE durations of 86.52±2.52**, 66.52±0.98*, and 63.25±0.89* for the low, medium, and high doses, respectively. There was a significant reduction in convulsion duration for all parameters across all doses compared to the standard and control groups. Therefore, it is possible to propose that electron-donating groups such as hydroxyl in the phenyl ring and methyl and amino in thiophene significantly reduced seizures.

CONCLUSION

In this study, the synthesis of novel thiophene incorporated benzothiazepine derivatives was successful by conventional method with moderate yields in the 47 to 88% range. Compound TBA5 was subjected for in vivo antiepileptic study using MES and PTZ induced methods at three different doses taking phenytoin and Diazepam as standards respectively. TBA5 was successful in reducing the seizures induced by MES and PTZ models to a certain extent, similar to the seizures induced by standard compounds phenytoin and Diazepam.

ACKNOWLEDGMENT

The authors are thankful to Nitte (Deemed to be University) for providing the necessary facilities to carry out this research.

CONFLICT OF INTEREST:

The authors declare that there was no conflict of interest.

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Received on 08.11.2024      Revised on 04.03.2025

Accepted on 10.05.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4363-4368.

DOI: 10.52711/0974-360X.2025.00625

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