Comparative Analysis of Anticonvulsant Activity of Trans and Cis 5,5′-Diphenylhydantoin Schiff Bases

Recently, the four 5,5′-diphenylhydantoin Schiff bases, possessing different aromatic species (SB1-Ph, SB2-Ph, SB3-Ph and SB4-Ph) were synthesized, characterized, and evaluated for anticonvulsant activity in combination with phenytoin. In the present study, the SB1-Ph and SB4-Ph compounds were selected, based on their anticonvulsant potency, and compared with their cis isomers, prepared after a one-hour exposure to the UV source, for their anticonvulsant potency in the maximal electroshock (MES) test and the kainate (KA)-induced status epilepticus (SE) test in mice. In the MES test, the cis SB1-Ph compound exhibited superior to phenytoin and trans isomer activity in the three tested doses, while the cis SB4-Ph compound entirely suppressed the electroshock-induced seizure spread at the highest dose of 40 mg/kg. Pretreatment with the cis SB1-Ph compound and the cis SB4-Ph at the doses of 40 mg/kg, respectively, for seven days, significantly attenuated the severity of KA SE compared to the matched control group pretreated with a vehicle, while phenytoin was ineffective in this test. The cis SB4-Ph but not the cis SB1-Ph demonstrated an antioxidant effect against the KA-induced SE in the hippocampus. Our results suggest that trans–cis conversion of 5,5′-diphenylhydantoin Schiff bases has potential against seizure spread in the MES test and mitigated the KA-induced SE. The antioxidant potency of cis SB4-Ph might be associated with its efficacy in mitigating the SE.


Introduction
Epilepsy is a chronic neurological disorder that affects approximately 1% of people worldwide [1].Spontaneous recurrent seizures represent the main symptom of this disease.Pharmacotherapy with anticonvulsants is the method of choice for epileptic patients.Although there is extensive funding for developing antiepileptic drugs (ASMs), about 30% of patients are resistant to treatment [2].The first generations of ASMs were characterized by severe side effects, whereas new-generation ASMs have an advantage with good tolerability and low capacity for drug interaction [3].
The first-line ASM phenytoin effectively blocks partial and tonic-clonic seizures [4].However, this ASM has low solubility and side effects during treatments.Heterocyclic systems and their Schiff bases are an essential class of compounds that have piqued the interest of researchers due to their varied range of biological functions, including anti-inflammatory, anticonvulsant, analgesic, antimicrobial, anticancer, antioxidant, anthelmintic, and antidepressant activities [5][6][7].Schiff bases, a universal pharmacophore, are studied in screening investigations [8].These compounds have an imine or azomethine (-C=N-) functional group.They are condensation products of primary amines with carbonyl compounds, which are gaining importance in medicine and pharmacy due to their ease of synthesis and isolation.A heterocyclic system, such as phenytoin, combined with an azomethine functional group would have a synergistic effect and increase biological activity.Schiff bases tend to isomerize due to the imine group (-C=N-) giving two stereoisomers cis and trans (E and Z) isomers, and the formation of these stereoisomers can be controlled kinetically or thermodynamically.These compounds have the potential to be photosensitive, undergoing rapid reverse photoisomerization from the more stable trans isomer to the less stable cis isomer.They can employ this characteristic to control, functionalize, and alter numerous biological functions.As a result, as the Schiff bases' molecular arrangement changes, the compounds' bioactivity also changes [9].
Recently, the four 5,5 -diphenylhydantoin Schiff bases, containing aromatic species (SB1-Ph, SB2-Ph, SB3-Ph and SB4-Ph), were synthesized and their structure-property characterization was carried out by X-ray, optical, and electrochemical methods [10].Furthermore, the four 3-amino-phenytoin Schiff base derivatives were explored alone and in combination with phenytoin against maximal electroshock (MES)-induced seizure spread in mice [11].Taking into consideration the potential anticonvulsant properties of 3-aminophenytoin Schiff base derivatives, in the present study, we aimed to compare the anticonvulsant activity of trans and cis 5,5 -diphenylhydantoin Schiff bases SB1-Ph and SB4-Ph against the MES-induced seizure spread.Furthermore, the potency to mitigate status epilepticus (SE) induced by kainic acid (KA) and oxidative stress in the hippocampus was evaluated after sub-chronic pretreatment with the more potent cis isomers of Schiff bases at the dose of 40 mg/kg, that was effective against tonic-clonic seizures in the MES test.

Maximal Electroshock Test
In the MES test, the cis-form of SB1-Ph but not the trans isomer showed protection against the MES-induced hindlimb tonic phase at the three tested doses (10, 20 and 40 mg/kg) (Fisher exact test: p = 0.003 compared to the control group) (Figure 1).This effect was comparable to that of the positive control phenytoin (p = 0.015 compared to control group).Similarly, the cisbut not the trans-form of SB4-Ph compound exhibited potency to suppress the MES-induced seizure spread (Fisher exact test: p = 0.003 SB4-Ph, 10 and 20 mg/kg compared to the control group).Notably, the cis isomer of SB4-Ph demonstrated 100% protection against tonic seizures at the highest dose of 40 mg/kg (p < 0.001, 40 mg/kg compared to the control group).No mortality of the cis-forms was observed except for SB4-Ph at a dose of 20 mg/kg with 16% mortality rate compared to the controls with 87% and 33% mortality rate for most of the trans isomers.

Maximal Electroshock Test
In the MES test, the cis-form of SB1-Ph but not the trans isomer showed protection against the MES-induced hindlimb tonic phase at the three tested doses (10, 20 and 40 mg/kg) (Fisher exact test: p = 0.003 compared to the control group) (Figure 1).This effect was comparable to that of the positive control phenytoin (p = 0.015 compared to control group).Similarly, the cis-but not the trans-form of SB4-Ph compound exhibited potency to suppress the MES-induced seizure spread (Fisher exact test: p = 0.003 SB4-Ph, 10 and 20 mg/kg compared to the control group).Notably, the cis isomer of SB4-Ph demonstrated 100% protection against tonic seizures at the highest dose of 40 mg/kg (p < 0.001, 40 mg/kg compared to the control group).No mortality of the cis-forms was observed except for SB4-Ph at a dose of 20 mg/kg with 16% mortality rate compared to the controls with 87% and 33% mortality rate for most of the trans isomers.

Kainate-Induced Status Epilepticus
The mice from all groups were pretreated with the positive control phenytoin (Ph group, 20 mg/kg), cis-forms of SB1-Ph and SB4-Ph, respectively (40 mg/kg), i.p. for seven days to assess the efficacy of cis isomers of novel phenytoin-related Schiff bases to mitigate seizure intensity during the KA-induced SE as well as its consequences on oxidative stress in the hippocampus.The matched control group was pretreated with a vehicle for a week in the same manner before the KA test.One hour (Ph group) or thirty minutes after the last injection the convulsant KA was i.p. applied at a dose of 30 mg/kg.The intensity of seizures was scored each hour up to 200 min.During the first 20 min of observation, the KA injection induced mild seizure behavior consisting mainly of facial automatisms and head nodding.Furthermore, at about 40th minutes, the seizure intensity progressed to forelimb clonus and rearing with occasional loss of posture (score 3-4).That behavioral reaction was sustainable until 140 min and faded out gradually till 200 min of observation in the control group (Figure 2A).No significant difference in each time interval as well as

Kainate-Induced Status Epilepticus
The mice from all groups were pretreated with the positive control phenytoin (Ph group, 20 mg/kg), cis-forms of SB1-Ph and SB4-Ph, respectively (40 mg/kg), i.p. for seven days to assess the efficacy of cis isomers of novel phenytoin-related Schiff bases to mitigate seizure intensity during the KA-induced SE as well as its consequences on oxidative stress in the hippocampus.The matched control group was pretreated with a vehicle for a week in the same manner before the KA test.One hour (Ph group) or thirty minutes after the last injection the convulsant KA was i.p. applied at a dose of 30 mg/kg.The intensity of seizures was scored each hour up to 200 min.During the first 20 min of observation, the KA injection induced mild seizure behavior consisting mainly of facial automatisms and head nodding.Furthermore, at about 40th minutes, the seizure intensity progressed to forelimb clonus and rearing with occasional loss of posture (score 3-4).That behavioral reaction was sustainable until 140 min and faded out gradually till 200 min of observation in the control group (Figure 2A).No significant difference in each time interval as well as total seizure intensity was detected between the Ph group and the veh group (Figure 2A,B).Significantly lower seizure scores were demonstrated in the cis-form of the SB1-Ph compound in the 80th (p < 0.05), 120th (p < 0.001), 140th (p < 0.001) and 160th min (p < 0.001), respectively, compared to veh group (Figure 2A).The cis-form of SB4-Ph compound alleviated SE at the 140th (p < 0.001) and 160th minute (p < 0.01), respectively, compared to the veh group.

Effects of Cis Isomers of SB1-Ph and SB4-Ph Derivates on the KA-Induced Oxidative St
The antioxidant capacity of the two cis isomers of SB1-Ph and SB4-Ph was asse by measurement of the level of total glutathione (GSH) and lipid peroxidation in hippocampus after the KA-induced SE in mice.A significant decrease in the total G was detected in the KA-veh group compared to the controls (p < 0.001) (Figure 3A).cis isomer of SB4-Ph significantly elevated the level of endogenous antioxidant in homogenate (p < 0.001 compared to the KA + veh group).The antioxidant activity of derivate was comparable to the effect of phenytoin (p < 0.001 compared to the KA group), while the cis isomer of SB1-Ph was ineffective (p > 0.05).
Furthermore, the KA + veh group showed elevated malondialdehyde (MDA) lev the hippocampus (p < 0.001 compared to C-veh group) suggesting an enhanced l peroxidation as a result of SE (Figure 3B).Neither the phenytoin nor the two cis isom of the new Schiff bases succeeded to suppress the KA-induced lipid peroxidation in hippocampus though the two phenytoin analogs partly mitigated this process (p < compared to KA + veh group).

Effects of Cis Isomers of SB1-Ph and SB4-Ph Derivates on the KA-Induced Oxidative Stress
The antioxidant capacity of the two cis isomers of SB1-Ph and SB4-Ph was assessed by measurement of the level of total glutathione (GSH) and lipid peroxidation in the hippocampus after the KA-induced SE in mice.A significant decrease in the total GSH was detected in the KA-veh group compared to the controls (p < 0.001) (Figure 3A).The cis isomer of SB4-Ph significantly elevated the level of endogenous antioxidant in the homogenate (p < 0.001 compared to the KA + veh group).The antioxidant activity of this derivate was comparable to the effect of phenytoin (p < 0.001 compared to the KA-veh group), while the cis isomer of SB1-Ph was ineffective (p > 0.05).

Discussion
Our findings revealed that the trans/cis conversion of 5,5′-diphenylhydantoin Schiff bases has protective activity against seizure spread in the MES test and mitigated the KAinduced SE.The antioxidant potency of cis SB4-Ph might be associated with its efficacy in reducing the severity of SE.
Two new 3-amino-5,5′-diphenylhydantoin Schiff Bases (SB1-Ph and SB4-Ph) were synthesized as described in detail in [10].The compounds SB1-Ph, and SB4-Ph were synthesized by a condensation reaction in absolute methanol between 3-amino-5,5′- Furthermore, the KA + veh group showed elevated malondialdehyde (MDA) level in the hippocampus (p < 0.001 compared to C-veh group) suggesting an enhanced lipid peroxidation as a result of SE (Figure 3B).Neither the phenytoin nor the two cis isomers of the new Schiff bases succeeded to suppress the KA-induced lipid peroxidation in the hippocampus though the two phenytoin analogs partly mitigated this process (p < 0.01 compared to KA + veh group).

Discussion
Our findings revealed that the trans/cis conversion of 5,5 -diphenylhydantoin Schiff bases has protective activity against seizure spread in the MES test and mitigated the KA-induced SE.The antioxidant potency of cis SB4-Ph might be associated with its efficacy in reducing the severity of SE.

Discussion
Our findings revealed that the trans/cis conversion of 5,5′-diphenylhydantoin Schiff bases has protective activity against seizure spread in the MES test and mitigated the KAinduced SE.The antioxidant potency of cis SB4-Ph might be associated with its efficacy in reducing the severity of SE.
Two new 3-amino-5,5′-diphenylhydantoin Schiff Bases (SB1-Ph and SB4-Ph) were synthesized as described in detail in [10].The compounds SB1-Ph, and SB4-Ph were synthesized by a condensation reaction in absolute methanol between 3-amino-5,5′diphenylimidazolidine-2,4-dione (1) and the corresponding aromatic aldehyde: thiophene-2-carbaldehyde (2) or pyridine-2-carbaldehyde (3) in a 1:1 molar ratio in the presence of catalytic quantities of glacial acetic acid (Scheme 1).Heterocycles thienyl, respectively pyridyl moiety in the SB1-Ph and SB4-Ph give the electron-donating properties of the molecules.Recently [10] have been studying ground state DFT calculations as trans-isomers (SB1-Ph and SB4-Ph), supported by X-ray investigation, which has revealed a near planar shape around the -CH=N-bond.The cis-isomers are distinguished by their twisting shape Recently [10] have been studying ground state DFT calculations as trans-isomers (SB1-Ph and SB4-Ph), supported by X-ray investigation, which has revealed a near planar shape around the -CH=N-bond.The cis-isomers are distinguished by their twisting shape and the creation of a weak noncovalent interaction.Azomethine aromatic compounds that can make weak noncovalent interactions with hydantoin rings in polar solvents play a critical function.Therefore, it was important to study and compare the anticonvulsant activity of both isomers transand cisand to show that the stereoisomeric and conformation states of the molecules play an important role and possess different activity.
We found that compared to the trans isomers, the cis isomers of the two phenytoin Schiff Bases SB1-Ph and SB4-Ph exerted higher potency to suppress seizure spread in the MES test, which is consistent with earlier bioactivity investigations [11][12][13][14].Furthermore, unlike phenytoin, the sub-chronic pretreatment with the two cis isomers of these Schiff Bases mitigated the severity of the KA-induced SE in mice.The detected potency of the cis-form of the SB4-Ph analog to elevate the total level of GSH and partly to reduce the lipid peroxidation in the hippocampus, suggest that the potency of this drug to minimize seizure severity during SE is closely related to its antioxidant activity in the hippocampus.However, the seizure-suppressing effect of SB1-Ph analog during SE was not accompanied by mitigation of oxidative stress, suggesting a difference from the SB1-Ph mechanism of its anticonvulsant effect.
Azomethine aromatic compounds SB1-Ph and SB4-Ph have a donor thiophene/pyridine ring that can give favorable lipophylic interactions with the corresponding receptors.SB1-Ph contains the large S atom's sterical hindrance in the molecule's variable ring part, as opposed to SB4-Ph which contains an N atom.The enhanced biological activity of the cis-form in comparison to the trans-form, on the other hand, may also be due to the better conformational states and matches with the target receptors.We hypothesize that the underlying mechanism of the anticonvulsant activity of the two 5,5 -diphenylhydantoin Schiff bases, which are structurally similar to phenytoin, is different [11].The two heterocyclic substituents, in particular, can be the molecule's key pharmacophore.
cis-form of the SB4-Ph analog to elevate the total level of GSH and partly to reduce the lipid peroxidation in the hippocampus, suggest that the potency of this drug to minimize seizure severity during SE is closely related to its antioxidant activity in the hippocampus.However, the seizure-suppressing effect of SB1-Ph analog during SE was not accompanied by mitigation of oxidative stress, suggesting a difference from the SB1-Ph mechanism of its anticonvulsant effect.
Azomethine aromatic compounds SB1-Ph and SB4-Ph have a donor thiophene/pyridine ring that can give favorable lipophylic interactions with the corresponding receptors.SB1-Ph contains the large S atom's sterical hindrance in the molecule's variable ring part, as opposed to SB4-Ph which contains an N atom.The enhanced biological activity of the cis-form in comparison to the trans-form, on the other hand, may also be due to the better conformational states and matches with the target receptors.We hypothesize that the underlying mechanism of the anticonvulsant activity of the two 5,5′-diphenylhydantoin Schiff bases, which are structurally similar to phenytoin, is different [11].The two heterocyclic substituents, in particular, can be the molecule's key pharmacophore.

Experimental Design
A description of experimental groups and consequent procedural steps is described Figure 5.In short, the mice were allocated to two main experimental protocols.In Experiment#1, sixteen groups were used as follows: C group (control group, injected with a vehicle) (n = 16); Ph group (positive control, injected with phenytoin in 20 mg/kg) (n = 12); three groups, injected with trans-form of SB1-Ph in doses of 10, 20 and 40 mg/kg, respectively) (12 × 3); three groups, injected with cis-form of SB1-Ph in doses of 10, 20 and 40 mg/kg, respectively) (12 × 3); three groups, injected with trans-form of SB4-Ph in doses of 10, 20 and 40 mg/kg, respectively) (12 × 3); three groups, injected with cis-form of SB4-Ph in doses of 10, 20 and 40 mg/kg, respectively) (12 × 3).In Experiment#2, five groups were used as follows: C-veh group (control group, treated with a vehicle for 7 days) (n = 8); KA + Ph group (positive control, treated with phenytoin at a dose of 20 mg/kg for 7 days) (n = 8); KA + SB1-Ph groups (experimental group treated with cis-form of SB1-Ph at a dose of 40 mg/kg) (8); KA + SB4-Ph groups (experimental group treated with cis-form of SB4-Ph at a dose of 40 mg/kg) (8).
and with EC Directive 2010/63/EU for animal experiments.The experimental procedures were approved by the Bulgarian Food Safety Agency (License No: 354/2023).

Experimental Design
A description of experimental groups and consequent procedural steps is described Figure 5.In short, the mice were allocated to two main experimental protocols.In Experiment#1, sixteen groups were used as follows: C group (control group, injected with a vehicle) (n = 16); Ph group (positive control, injected with phenytoin in 20 mg/kg) (n = 12); three groups, injected with trans-form of SB1-Ph in doses of 10, 20 and 40 mg/kg, respectively) (12 × 3); three groups, injected with cis-form of SB1-Ph in doses of 10, 20 and 40 mg/kg, respectively) (12 × 3); three groups, injected with trans-form of SB4-Ph in doses of 10, 20 and 40 mg/kg, respectively) (12 × 3); three groups, injected with cis-form of SB4-Ph in doses of 10, 20 and 40 mg/kg, respectively) (12 × 3).In Experiment#2, five groups were used as follows: C-veh group (control group, treated with a vehicle for 7 days) (n = 8); KA + Ph group (positive control, treated with phenytoin at a dose of 20 mg/kg for 7 days) (n = 8); KA + SB1-Ph groups (experimental group treated with cis-form of SB1-Ph at a dose of 40 mg/kg) (8); KA + SB4-Ph groups (experimental group treated with cis-form of SB4-Ph at a dose of 40 mg/kg) (8).

Rotarod Test
The motor coordination was assessed as previously described [12].The inability to keep position on a rotating rod (3.2 cm in diameter, at a speed of 10 rpm) for at least of one minute out of three trials was accepted as a criterion for neurotoxicity (>half of mice per group with lost balance).

Kainate-Induced Status Epilepticus
The mice were assigned in groups of 8 mice.On the day of the 7th i.p. injection of the drug/vehicle, each animal was i.p. administered by 30 m/kg KA (FOT, Bulgaria).The convulsant was dissolved in saline (10 mL/kg of body weight).The observation of seizure onset and its intensity was scored according to the scale [15] as follows: stage 1 (facial clonus), stage 2 (nodding), stage 3 (forelimb clonus), stage 4 (forelimb clonus with rearing), and stage 5 (rearing and lost posture).The SE was characterized by continuous clonic seizures of stage 4 or 5.The seizures of the highest score detected during every 20 min up to 200 min were evaluated.Immediately after a 3 h period of observation, the mice were decapitated, brains were dissected rapidly on ice and the two hippocampi were isolated, quickly frozen in liquid nitrogen and stored at −20 • C until ELISA analysis.

Measurement of Glutathione (GSH) and Malondialdehyde (MDA) in the Hippocampus
The isolated hippocampi were kept on ice, weighed, and preserved at 20 • C until homogenization in cold PBS buffer (pH 7.4) containing 1 mM EGTA, 50 mM NaF, 1 mM 270 EDTA and 1 mM PMSF.After centrifugation of the tissue homogenate at 5000× g, 4 • C for 271 10 min, GSH and MDA were measured in duplicates using an ELISA kit (Elabscience cat.272 No E-EL-0060 and E-EL-0026) according to the manufacturer's instructions.The results (273) were expressed in µg/mg protein (GSH) and ng/mg protein (MDA).

Data Analysis
The data are expressed as means ± SEM.The results from the MES test and rotarod test 315 were analyzed by Fisher's exact test.Data from the grip strength test, KA test and bio-316 chemistry were assessed by the one-way analysis of variance (ANOVA) followed by the 317 post hoc Bonferroni's test.p < 0.05 was considered statistically significant.

Conclusions
In conclusion, the cis isomers of 3-amino-5,5 -diphenylhydantoin Schiff Bases (SB1-Ph and SB4-Ph) exhibited higher potency than their trans-forms to suppress seizure spread and tonic seizures in mice.The anticonvulsant activity of the cis isomer SB4-Ph against the neurotoxin KA might be associated with the antioxidant potency in the hippocampus during SE.

4. 2 .
Experimental Rodents Male albino ICR mice (23-26 g), delivered by the vivarium of the Institute of Neurobiology-BAS, were left undisturbed for seven days before experimental procedures.The rodents were kept in transparent cages (10 in groups), with standard pellets and tap water ad libitum and in an artificial light-dark cycle regime (12:12; light on at 07:00 a.m.), T o = 21 ± 1 • C and humidity: 40 ± 5%).The experiments were conducted in the morning (10:00-11:00 p.m.).All performed manipulations were consistent with the Declaration of Helsinki Guiding Principles on Care and Use of Animals (DHEW Publication, NHI 80-23) and with EC Directive 2010/63/EU for animal experiments.The experimental procedures were approved by the Bulgarian Food Safety Agency (License No: 354/2023).

Table 1 .
Effects of trans and cis 5,5 -diphenylhydantoin Schiff bases-SB1-Ph and SB4-Ph on neuromuscular tone in the grip strength test and motor coordination in the rotarod test in mice.

/Treatment Dose (mg/kg). i.p. Neuromuscular Strength (N) Rotarod Test N/F
Data are presented as mean muscle strength (in Newtons ± S.D. of 3 determinations) in mice subjected to the grip strength test.The Schiff bases were injected i.p. 0.5 h before the tests at different doses (10, 20 and 40 mg/kg) as shown above.One-way ANOVA was used for the grip strength test and Fisher's exact test for statistical analysis of the rotarod test.