Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 
  • Users Online: 903
  • Home
  • Print this page
  • Email this page


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 13  |  Issue : 3  |  Page : 262-272

Synthesis and evaluation of anticonvulsant activity of (Z)-4-(2-oxoindolin-3-ylideneamino)-N-phenylbenzamide derivatives in mice


1 Isfahan Pharmaceutical Sciences Research Center, Isfahan; Pharmaceutical Sciences Research Center, School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah; Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, I.R. Iran
2 Pharmaceutical Sciences Research Center, School of Pharmacy, Kermanshah University of Medical Sciences; Department of Pharmacology, Toxicology and Medical Services, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, I.R. Iran
3 Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences; Students Research Committee, Kermanshah University of Medical Sciences, Kermanshah, I.R. Iran
4 Pharmaceutical Sciences Research Center, School of Pharmacy, Kermanshah University of Medical Sciences; Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, I.R. Iran

Date of Web Publication16-Apr-2018

Correspondence Address:
Alireza Aliabadi
Pharmaceutical Sciences Research Center, School of Pharmacy, Kermanshah University of Medical Sciences; Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah
I.R. Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1735-5362.228956

Rights and Permissions
  Abstract 

Due to resistance of some epileptic patients to the current medications and the general incidence of severe side effects of these drugs, development and discovery of novel antiepileptic drugs is crucial. Isatin-based derivatives are promising compounds as antiepileptic agents. In this study a new series of isatin-containing derivatives were synthesized via the imine formation between isatin and p-aminobenzoic acid. Subsequently, the obtained acidic compound was utilized to prepare the final amidic derivatives (4a-4l) through the reaction with various aniline derivatives. Then, their anti-seizure activity was investigated using maximal electroshock seizure (MES) as well as pentylenetetrazole (PTZ) models in mice. Neurotoxicity of target compounds was also determined by rotarod test. Tested isatin-based derivatives exhibited a favorable protection in both MES and PTZ procedures with high safety levels in neurotoxicity test. The introduced derivatives have demonstrated remarkable activity in mice and could be suggested as potential anticonvulsant lead compounds. All methoxylated derivatives (4j, 4k, 4l) showed a significant anti-seizure activity in MES model. Compounds 4j (2-OCH3) and 4l (4-OCH3) also demonstrated a potent anti-seizure activity against PTZ. Compound 4k (m-OCH3) did not induce protection towards PTZ-induced convulsion.

Keywords: Synthesis; Isatin; Anticonvulsant; PTZ; MES


How to cite this article:
Khajouei MR, Mohammadi-Farani A, Moradi A, Aliabadi A. Synthesis and evaluation of anticonvulsant activity of (Z)-4-(2-oxoindolin-3-ylideneamino)-N-phenylbenzamide derivatives in mice. Res Pharma Sci 2018;13:262-72

How to cite this URL:
Khajouei MR, Mohammadi-Farani A, Moradi A, Aliabadi A. Synthesis and evaluation of anticonvulsant activity of (Z)-4-(2-oxoindolin-3-ylideneamino)-N-phenylbenzamide derivatives in mice. Res Pharma Sci [serial online] 2018 [cited 2021 Sep 21];13:262-72. Available from: https://www.rpsjournal.net/text.asp?2018/13/3/262/228956


  Introduction Top


Epilepsy is a common central nervous system (CNS) disorder that is characterized by presentation of recurrent spontaneous convulsions as well as by emergence of some sensory, motor or autonomic symptoms. According to the epidemiological surveys, approximately 1% of the world's population suffers from epilepsy [1]. The disease has a very complex nature, and diverse pathological factors can cause the disease. Epilepsy is a condition that can occur in both sexes and at all age groups [2]. Many of the currently used antiepileptic drugs have severe adverse effects that limit their use in short or long term treatment. These include CNS (ataxia, drowsiness), gastrointestinal (gingival hyperplasia, nausea, vomiting, hepatic dysfunction), hematological (megaloblastic anaemia), bone related (osteomalacia), and dermatological (rash, Stevens-Johnson syndrome) disorders. Furthermore, about 30% of epileptic patients do not show adequate response to the current drugs [3],[4],[5]. Many antiepileptic agents exert their anticonvulsant effects via a variety of mechanisms. Inhibition of ion channels such as sodium channels (phenytoin, carbamazepine), calcium channels (ethosuximide), and potassium channels (retigabine) as well as GABAA receptors (benzodiazepines, barbiturates) are common mechanisms of conventional antiepileptic drugs.

On the other hand, effect on glutamatergic receptors (lamotrigine, topiramate, felbamate) is performed by means of newer drugs.

icacy of these medications is in question for controlling and treatment of some types of epilepsy. Hence, in antiepileptic drug development research there is a deep need and demand for the development of more efficacious agents with fewer side effects [6],[7],[8],[9].

Isatin, an oxidation product of indigo, is a bright orange-colored compound with a broad range of biological activities. Presence of significant amounts of isatin in such tissues as brain, proposes that it probably has a role in CNS functions. Isatin is demonstrated to have anxiogenic as well as sedative effects. It also increases the levels of monoamines in CNS [10]. Inhibition of monoamine oxidase (MAO) enzymes especially MAOB is shown in some studies. Moreover, isatin is also formed in the tryptophan metabolic pathway [11],[12]. The chemical structure of isatin attracted the attention of many medicinal chemists for derivatization. A large number of pharmacological effects such as anticancer, antidepressant, anticonvulsant, anti-acetylcholinesterase, antibacterial, antifungal, anti-HIV, antitubercular, and anti-inflammatory properties have been observed from the compounds with isatin substructure [12],[13],[14]. Anticonvulsant activity is one of the main promising biological activities that are shown to possess some isatin-based compounds [Figure 1] [13],[14],[15],[16],[17],[18],[19]. In continuation of our previous studies on isatin-based anticonvulsant derivatvies [19], we designed a new series of anticonvulsant agents based on isatin as a substructure. As illustrated in [Figure 2], similar pharmacophoric sections could be defined for isatin derivatives which are already presented in phenytoin and carbamazepine as standard anticonvulsant agents.
Figure 1: Structures of some isatin-based compounds as anticonvulsant agents.

Click here to view
Figure 2: Pharmacophoric necessities of anticonvulsant drugs. Hydrogen bond acceptor/donor represented in green (HAD), electron donor represented in violet (ED) and aromatic ring represented in red (AR represented) for hydrophobic interaction. Target compounds were designed according to this theory.

Click here to view


The insufficient data and knowledge about the physiopathology and cellular mechanisms of epilepsy in human and the multiple pharmacological mechanisms for antiepileptic drugs makes it difficult to design and develop novel drugs [3]. Pharmacophoric studies of antiepileptic drugs have revealed that three main parts are present in chemical scaffolds of efficacious drugs for presentation of antiepileptic activity. The main requisite parts are: hydrogen bond donor/acceptor moiety, electron donor group , and hydrophobic region that is produced by aromatic rings such as phenyl residue [Figure 2] [1],[20]. Based on this information, we designed a new series of isatin-based antiepileptic agents and subsequently synthesized the designed compounds and investigated their activity using pentylenetetrazole (PTZ) and maximal electroshock seizure (MES) protocols in mice.


  Materials and Methods Top


Chemistry

All solvents as well as starting materials and corresponding reagents were purchased either from Merck or Sigma-Aldrich companies or from their branches. Aluminum based TLC sheets were applied for thin layer chromatography (TLC). Silica gel (70-230 mesh) was utilized for purification of the intermediate and final compounds. Nuclear magnetic resonance (NMR) Bruker 250 MHz apparatus was used for hydrogen-1 NMR (1HNMR) spectra acquisition. All prepared compounds were dissolved in deutrated dimethylsulfoxide (DMSO-d6) and tetramethylsilane was used as internal standard. Chemical shifts for each proton were presented as δ (ppm) related to tetramethylsilane. Potassium bromide (KBr) disk was prepared for infrared (IR) spectra in Shimadzu 470 spectrophotometer. Mass spectroscopy was performed using a Finigan TSQ-70 spectrometer (Finigan, USA) at 70 eV and mass of each fragment were provided with its frequency percentage. Melting points for final compounds was also obtained using melting point analyzer (electrothermal 9001A) with drugs nested in open capillary tubes.

Synthesis of (Z)-4-((2-oxoindolin-3-ylidene) amino)benzoic acid [3]

In a flat-bottom flask, 3 g (0.020 mmol) of isatin (MW: 147 g/mol) was mixed with 2.80 g (0.020 mmol) p-aminobenzoic acid (PABA, MW: 137 g/mol) and the resultant mixture was dissolved in absolute ethanol (40 mL). Catalytic amount (1 mL) of glacial acetic acid was added to the reaction medium and refluxing condition was applied overnight [Scheme 1].



TLC was carried out for reaction monitoring and also for determination of the reaction end. Then, ethanol was removed under reduced pressure and water/ethylacetate (50/50) was added to the residue. Organic layer was separated and washed twice by diluted sulfuric acid and consequently two times by brine. Ethylacetate was evaporated using rotary evaporator apparatus and the obtained precipitate was washed by n-hexane and diethyl ether (Et2O). Column chromatography (ethylacetate/petroleum ether 90:10) was applied for final purification [17].

General procedure for synthesis of (Z)- 4-((2-oxoindolin-3-ylidene)amino)-N-phenyl benzamide derivatives (4a-4l)

In a flat bottom flask, 0.200 g (0.75 mmol) of compound 3, were treated with 0.155 g (0.75 mmol) dicyclohexyl carbodiimide (DCC) and 0.102 g (0.75 mmol) hydroxybenzotriazole in 20 mL tetrahydrofuran (THF). Equimolar quantity of appropriate aniline derivative was also added to the reaction medium. The reaction container was put in an ice bath and stirred for 1 h. Then, stirring was continued for 24 h at room temperature.

TLC was used to determine the end of the reaction. Reaction mixture was filtered and solvent was removed using rotary evaporator. Ethylacetate/water (50/50 mL) was added to the obtained residue and aqueous layer was discarded. Organic layer was washed tree times with diluted sulfuric acid 1%, sodium bicarbonate 2% and finally two times by brine. Organic phase was dried over anhydrous sodium sulfate and then filtered. Ethylacetate was evaporated and obtained powder was washed with n-hexane and (Et2O). The obtained powder was purified by column chromatography (ethylacetate/petroleum ether 90:10) [21],[22],[23],[24],[25],[26].

Anticonvulsant and neurotoxicity identification tests

The new mentioned compounds were undertaken for the anticonvulsant screening procedure by the anticonvulsant drug development program protocol [27]. Adult male NMRI mice weighing between 20-25 g (purchased from Iran Pasteur Institute and nurtured in a temperature-controlled room under 12-h light/dark cycle) were used as experimental animals. Animals were treated in accordance with the ethical principles approved in ethical committee of Kermanshah University of Medical Sciences (ID No. 92281). The screening was performed using PTZ (Sigma Aldrich, Germany), and MES models of seizures. Drugs were dissolved in 0.5% methylcellulose and administered intraperitoneally (i.p.) in a volume of 0.01 mL/g body weight. In the preliminary screening procedure (phase I) drugs were given at doses of 30, 100, or 300 mg/kg at 0.5 h and 4 h time intervals. Neurotoxicity screening was also performed using the rotarod test. PTZ was dissolved in 0.9% saline and used at the dose of 85 mg/kg (subcutaneous). In the MES test, 60-Hz current (50 mA) was delivered for 0.2 second through corneal electrodes in each mice (the MES device was made by Borj Sanat corporation, Tehran, Iran). Phase I tests were performed according to the standard procedure provided by the antiepileptic drug development program [27].

In the MES test after the shock was delivered animals were observed to see if they show a tonic extension in their hind limbs. A tonic extension is defined as the full extension (180°) of the hind limbs with the plane of the body. Absence of this component means that the test compound was able to prevent the spreading of seizure discharge in the brain.

In the PTZ test animals were observed for 30 minutes after the PTZ injection and absence of a clonic spasm persisting for at least 5 seconds meant that the test substance has the ability to raise the seizure threshold.

In the rotorod test the speed of the rod was set at 6 rpm. If the animal was unable to maintain its balance in a 1 min challenge test the substance was considered neurotoxic.

Identification of median effective dose and median neurotoxic dose

Median effective dose (ED50) was determined for some of the most active agents in phase I tests. For ED50 determination (phase II), groups of eight mice were injected with doses of each compound around the best dose determined in the screening tests. The statistical method of Litchfield and Wilcoxon was used for determination of ED50 [28]. Median neurotoxic dose (TD50) was determined using the results of the rotorod test and the same statistical method. Briefly the best determined dose was applied to a group of animals (n = 8) and the number of responding cases were recorded. Then another dose, one half or double the initial dose, was administered to another group of animals. This procedure was repeated until we had at least two doses with 0% animal response and two doses with 100% animal response. As mentioned above the results were analyzed through the statistical log-probit method of Litchfield and Wilcoxon.


  Results Top


Chemistry

According to the [Table 1], all target compounds were prepared with an average yield. An imine derivative of isatin (compound 3, orange powder) was synthesized via the reaction of isatin with p-aminobenzoic acid in the presence of catalytic amounts of glacial acetic acid. Final compounds were obtained through the reaction of compound 3 with various derivatives of aniline bearing electron withdrawing as well as electron donating substituents. The corresponding amidation reaction was carried out using dicyclohexyl carbodiimide and hydroxybenzotriazole. Purification was done using column Purification was done using column chromatography. Melting points of all synthesized derivatives were measured by melting point analyzer and a range of 166.8-189.6 °C was observed for these series except for compounds 4c and 4e-4g that were decomposed over 200 °C. Spectroscopic methods such as 1HNMR, IR, and MS were applied for characterization of synthesized compounds. All compounds were dissolved in DMSO-d6 for NMR spectra acquisition. Wave numbers for typical functional groups were reported in IR spectra interpretation. Stretching vibrations of carbonyl groups of isatin residue and also amide bond in final derivatives were the main peaks in IR spectra. The main fragments in MS spectra were provided with related abundance. Molecular ion (M+) peak in some cases were weak and did not observe clearly.
Table 1: Properties of compounds 4a-4l.

Click here to view


(Z)-4-((2-oxoindolin-3-ylidene)amino)benzoic acid (3)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.67 (d, 2H, J = 7.5 Hz, H3,5-phenyl), 6.93 (m, 1H, H5-isatin), 7.07 (m, 1H, H6-isatin), 7.53 (m, 1H, H7-isatin), 7.76 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 8.11 (d, 1H, J = 7.5 Hz, H4-isatin), 10.90 (brs, NH-isatin), 11.10 (brs, -COOH). IR (KBr, cm-1) ῡ: 3275 (stretch, NH), 1743 (stretch, C=O, acid), 1689 (stretch, C=O, isatin). MS (m/z, %): 266 (75), 238 (100, 221 (15), 211 (12, 148 (15), 137 (15), 121 (25), 90 (15), 76 (20).

(Z)-4-((2-oxoindolin-3-ylidene)amino)-N-phenylbenzamide (4a)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.80 (m, 5H, aromatic), 6.92 (d, 2H, J = 7.5 Hz, H3,5-phenyl), 7.07 (t, 1H, H5-isatin), 7.33 (t, 1H, H6-isatin), 7.49 (d, 1H, J = 7.5 Hz, H7-isatin), 7.69 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 7.78 (d, 1H, J = 7.5 Hz, H4-isatin), 9.44 (brs, NH-isatin), 10.97 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3251 (stretch, NH), 1747 (C=O, isatin), 1647 (C=O, amide).

(Z)-N-(2-chlorophenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4b)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.86 (d, 2H, J = 8 Hz, H3,5-phenyl), 6.86-7.08 (m, 4H, 2-chlorophenyl), 7.38 (d, 1H, J = 7.5 Hz, H4-isatin), 7.48 (t, 1H, J = 7.5 Hz, H5-isatin), 7.69 (d, 1H, J = 7.5 Hz, H8-isatin), 7.95 (d, 1H, J = 7.5 Hz, H7-isatin), 7.97 (d, 2H, J = 8 Hz, H2,6-phenyl), 9.44 (brs, NH-isatin), 10.97 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3325 (stretch, NH), 1735 (C=O, isatin), 1624 (C=O, amide).

(Z)-N-(3-chlorophenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4c)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.61 (s, 1H, H2-3-chlorophenyl), 6.70 (d, 2H, J = 7.5 Hz, H3,5-phenyl), 6.82 (d, 1H, H6-2-chlorophenyl),7.23 (m, 2H, 2-chlorophenyl), 7.38 (t, 1H, J = 7.5 Hz, H5-isatin), 7.51 (t, 1H, J = 7.5 Hz, H6-isatin), 7.68 (d, 1H, J = 7.5 Hz, H7-isatin), 7.71 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 7.95 (d, 1H, J = 7.5 Hz, H4-isatin), 10.23 (brs, NH-isatin), 10.51 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3325 (stretch, NH), 1735 (C=O, isatin), 1624 (C=O, amide).

(Z)-N-(4-chlorophenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4d)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.73 (d, 2H, J = 7.5 Hz, H3,5-phenyl), 7.04-7.18 (m, 2H, aromatic), 7.29-7.49 (m, 4H, aromatic), 7.59 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 7.80-7.96 (m, 2H, aromatic), 10.45 (brs, NH-isatin), 11.02 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3325 (stretch, NH), 1735 (C=O, isatin), 1624 (C=O, amide). MS (m/z, %): 357 (M+, 2), 246 (45), 218 (20), 146 (20), 136 (40), 127 (100), 120 (90), 92 (75), 65 (70).

(Z)-N-(2-fluorophenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4e)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.75 (d, 2H, J = 7.5 Hz, H3,5-phenyl), 6.90 (t, 1H, H6-4-fluorophenyl), 7.05 (t, 1H, J = 7.5 Hz, H5-isatin), 7.41 (t, 1H, J = 7.5 Hz, H6-isatin), 7.46-7.66 (m, 3H, 4-fluorophenyl), 7.56 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 7.88-8.07 (m, 4H, aromatic), 10.10 (brs, NH-isatin), 10.95 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3325 (stretch, NH), 1732 (C=O, isatin), 1624 (C=O, amide).

(Z)-N-(3-fluorophenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4f)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.80 (d, 2H, H3,5-phenyl), 6.95 (m, 1H, aromatic), 7.10 (t, 1H, H4-3-fluorophenyl), 7.26 (t, 1H, H6-isatin), 7.27 (t, 1H, 3-fluorophenyl), 7.50 (dd, 1H, 3-fluorophenyl), 7.63 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 7.73-7.95 (m, 2H, aromatic), 9.85 (brs, NH-isatin), 10.95 (brs, NH). IR (KBr, cm-1) ῡ: 3325 (stretch, NH), 1732 (C=O, isatin), 1651 (C=O, amide).

(Z)-N-(4-Fluorophenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4g)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.92 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 7.06 (t, 2H, J = 7.5 Hz, H2,6-4-fluorophenyl), 7.39 (t, 1H, H6-isatin), 7.47-7.58 (m, 4H, aromatic), 7.65 (d, 1H, J = 7.5 Hz, H7-isatin), 7.96 (d, 1H, J = 7.5 Hz, H4-isatin), 10.11 (brs, NH-isatin), 10.16 (brs, NH-isatin), 10.97 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3325 (stretch, NH), 1732 (C=O, stretch, isatin), 1620 (C=O, stretch, amide). MS (m/z, %): 359 (M+, weak), 224 (20), 147 (40), 119 (100), 110 (45), 91 (50), 76 (30).

(Z)-N-(3-Nitrophenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4h)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.76 (d, 2H, J = 7.5 Hz, H3,5-phenyl), 6.92 (d, 1H, J = 7.5 Hz, H7-isatin), 7.05 (t, 1H, J = 7.5 Hz, H5-isatin), 7.25 (t, 1H, J = 7.5 Hz, H6-isatin), 7.43-7.58 (m, 3H, aromatic), 7.69 (s, 1H, H2-3-nitrophenyl), 7.96 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 8.05 (d, 1H, H4-3-nitrophenyl), 10.12 (brs, NH-isatin), 10.92 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3344 (stretch, NH), 1716 (C=O, stretch, isatin), 1608 (C=O, stretch, amide), 1523 (asymmetric stretch, NO2), 1338 (symmetric stretch, NO2).

(Z)-N-(4-Nitrophenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4i)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 6.63 (d, 2H, J = 7.5 Hz, H3,5-phenyl), 6.74 (d, 1H, J = 7.5 Hz, H7-isatin), 6.92 (d, 2H, J = 7.5 Hz, H2,6-4-nitrophenyl), 7.05 (t, 1H, J = 7.5 Hz, H5-isatin), 7.40 (t, 1H, J = 7.5 Hz, H6-isatin), 7.52 (d, 2H, J = 7.5 Hz, H3,5-4-nitrophenyl), 7.63 (d, 1H, J = 7.5 Hz, H4-isatin), 7.94 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 9.91 (brs, NH-isatin), 10.95 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3360 (stretch, NH), 1735 (C=O, stretch, isatin), 1627 (C=O, stretch, amide), 1500 (asymmetric stretch, NO2), 1303 (symmetric stretch, NO2). MS (m/z, %): 386 (M+, weak), 267 (25), 239 (30), 224 (45), 145 (30), 138 (100), 120 (75), 120 (70), 110 (60), 92 (45), 76 (20).

(Z)-N-(2-Methoxyphenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4j)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 3.81 (s, 3H, -OCH3), 6.68 (d, 1H, H3-2-methoxyphenyl), 6.87 (d, 1H, J = 7.5 Hz, H2,6-phenyl), 7.02 (t, 1H, H5-2-methoxyphenyl), 7.32 (t, 1H, H4-2-methoxyphenyl), 7.42 (t, 1H, H5-isatin), 7.46 (d, 1H, H6-isatin), 7.54 (t, 1H, H4-isatin), 7.60 (d, 1H, J = 7.5 Hz, H3,5-phenyl), 7.88 (d, 1H, J = 7.5 Hz, H7-isatin), 7.99 (d, 1H, J = 7.5 Hz, H6-2-methoxyphenyl), 9.89 (brs,, NH-isatin), 11.04 (brs, NH). IR (KBr, cm-1) ῡ: 3325 (stretch, NH), 1728 (C=O, stretch, isatin), 1624 (C=O, stretch, amide).

(Z)-N-(3-Methoxyphenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4k)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 3.69 (s, 3H, -OCH3), 6.86 (d, 2H, J = 7.5 Hz, H3,5-phenyl), 7.03 (t, 1H, J = 7.5 Hz, H5-3-methoxyphenyl), 7.36 (t, 1H, J = 7.5 Hz, H5-isatin), 7.46 (d, 2H, J = 7.5 Hz, H2,6-phenyl), 7.50 (t, 1H, J = 7.5 Hz, H6-isatin), 7.54 (m, 7.50 (t, 1H, J = 7.5 Hz, H6-isatin), 7.54 (m, 2H, J = 7.5 Hz, 3-methoxyphenyl), 7.66 (d, 1H, J = 7.5 Hz, H7-isatin), 7.93 (d, 1H, J = 7.5 Hz, H4-isatin), 10.07 (brs, NH-isatin), 10.99 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3329 (stretch, NH), 1732 (C=O, isatin), 1624 (C=O, amide).

(Z)-N-(4-Methoxyphenyl)-4-((2-oxoindolin-3-ylidene)amino)benzamide (4l)

1HNMR (250 MHz, DMSO-d6) δ (ppm): 3.80 (s, 3H, -OCH3), 6.82 (d, 2H, J = 7.5 Hz, H3,5-4-methoxyphenyl), 6.86 (d, 2H, H3,5-phenyl), 6.90 (d, 2H, J = 7.5 Hz, H2,6-4-methoxyphenyl), 7.05 (t, 1H, H5-isatin), 7.53 (t, 3H, H6-isatin), 7.64 (d, 1H, J = 7.5 Hz, H7-isatin), 7.73 (d, 2H, H2,6-phenyl), 7.82 (d, 1H, J = 7.5 Hz, H7-isatin), 9.45 (brs, NH-isatin), 10.95 (brs, NH-amide). IR (KBr, cm-1) ῡ: 3325 (stretch, NH), 1732 (C=O, stretch, isatin), 1624 (C=O, stretch, amide). MS (m/z, %): 371 (M+, weak), 329 (20), 224 (45), 165 (20), 143 (30), 123 (95), 99 (35), 56 (100).

Anticonvulsant assay

As seen in [Table 2] the molecules are divided into four classes based on their results in MES test. [1] anti-seizure activity at 100 mg/kg or less, [2] anti-seizure effects at doses higher than 100 mg/kg, [3] compound having no activity at any doses up to 300 mg/kg, and [4] agents inactive at 300 mg/kg and toxic at 30 mg/kg or less [27]. Although most agents are classified as group 1 compounds the screening results show that compounds 4a, 4b, and 4h have anti-seizure effects at the minimum dose of 30 mg/kg and compounds 4g and 4l have anti-seizure activity at the minimum dose of 100 mg/kg. These are actually the most active compounds which were selected for further evaluation in the phase II tests. The results of phase II tests show [Table 3] that all the selected compounds have a better protective index (PI) than diazepam (PI = 0.07). PI = TD50/ED50 was calculated for the MES test. When there was a solubility limitation the results were reported as being greater than the last meaningful PI [Table 3].
Table 2: Anti-seizure activities after intraperitoneal administration of compounds 4a-4l in mice.

Click here to view
Table 3: Median effective dose (ED50) and median neurotoxic dose (TD50) of selected compounds.

Click here to view



  Discussion Top


Anti-seizure activities of all synthesized compounds 4a-4l were explored using MES and PTZ [Table 1]. Compounds 4a, 4b, and 4h were chosen in phase I of the pharmacologic studies based on the fact that they could prevent seizure in most animals used in the screening procedure. All tested doses of these derivatives could not prevent the convulsing effects of MES as well as PTZ. It hypothesized that ortho positioning of chlorine atom and also meta positioning of nitro moiety on the phenyl residue have beneficial impact on anti-seizure activity. Comparison of compound 4b (2-Cl) with compound 4e (2-F) imply that electron withdrawing effect of the corresponding moieties is an interrupting factor, because the enhancement of the electron receiving effect in fluorine atom attenuated the activity. Chlorine has larger size than fluorine and therefore more lipophilicity and steric effect. It could be imagined that steric effect and lipophilicity that associated with chlorine are the fortifying factors. But, according to the potent activity of compound 4a without any moiety on the phenyl residue, it could be concluded that the presence of chlorine for anti-seizure potency is not a requisite at ortho position. Compound 4j (2-OCH3) with methoxy substituent at ortho position also demonstrated a remarkable activity in dose 300 mg/kg. Compound 4c (3-Cl) exhibited an acceptable onset and duration of action at dose 300 mg/kg after 0.5 and 4 h in both models. Fluorine substituent was effective in some positions of the phenyl residue. The electron withdrawing effect, hydrogen bond capability and also low steric impact are the featured properties of the fluorine moiety. The fluorine caused an acceptable anti-seizure effect especially at ortho and para positions (4e) (4g). Compounds 4e and 4g exerted their anti-seizure activity at 100 as well as 300 mg/kg doses in both MES and PTZ models. The onset action for compound 4e was slower than 4g in MES model. Compound 4f (3-F) exhibited its anti-seizure activity solely in 300 mg/kg in MES model. Nitro group rendered a better anti-seizure activity while substituted at meta position compared to para position. Nitro group is a strong electron withdrawing as well as hydrophilic moiety. As observed in compounds 4c (3-Cl) and 4f (3-F) electron receiving effect is a beneficial effect at meta position. However, it is probable that hydrophilic property of nitro moiety at para position reduces the anti-seizure effect as seen in compound 4h. It is interesting that nitro moiety caused a favorable anti-seizure activity when substituted at meta position. Chlorine and fluorine groups with lipophilic property showed a superior activity than nitro at para position. More hydrophilic characteristic of the nitro group compared to chlorine and fluorine substituents maybe a logical reason for abolishment of the anti-seizure effect. All methoxylated derivatives (4j, 4k, 4l) showed a significant anti-seizure activity in MES model especially at 100 and 300 mg/kg doses. Compound 4k (3-OCH3) was the most active methoxylated derivative in PTZ model. This compound displayed its anti-seizure effect in all tested doses in PTZ model. But, compounds 4j exhibited its anti-seizure activity in 300 mg/kg dose and compound 4l showed this effect at both 100 and 300 mg/kg. Comparison of the anticonvulsant activity of newly synthesized compounds with our previously reported results [19] demonstrated that using p-aminobenzoic acid as the linker in these series did not cause an increase in anticonvulsant activity rather than compounds without this linker. p-aminobenzoic acid attenuated the anticonvulsant potency in both of utilized methods namely PTZ and MES.

Neurotoxicity of tested compounds was investigated using rotarod protocol. All tested derivatives demonstrated a high safety in tested dose 30 mg/kg except compounds 4e (2-F) and 4l (4-OCH3) [Table 2]. Compound 4e did not render any toxicity after 0.5 h of injection of tested compounds but the toxicity was observed after 4 h at all tested dose levels. Compound 4l displayed the most toxicity among the tested derivatives. Namely, 0.5 h after injection of compound 4l the neurotoxicity was induced in mice. Overall, tested compounds showed a high safety. Neurotoxicity was observed merely at dose levels higher than 30 mg/kg in some cases.


  Conclusion Top


A new series of isatin-based anti-seizure derivatives were designed, synthesized and their activity was investigated by MES and PTZ models in mice. These compounds displayed a remarkable protective activity in both utilized convulsing models. A low level of toxicity was also observed for all tested derivatives in rotarod protocol. All methoxylated derivatives (4j, 4k, 4l) showed a significant anti-seizure activity in MES model. Compounds 4j (2-OCH3) and 4l (4-OCH3) also demonstrated a potent anti-seizure activity against PTZ. Compound 4k (m-OCH3) did not cause protection towards PTZ induced convulsion.


  Acknowledgement Top


The content of this paper is extracted from the Pharm. D thesis (No. 92281) submitted by Aref Moradi which was financially supported by research council of Kermanshah University of Medical Sciences, Kermanshah, I.R. Iran.

 
  References Top

1.
Prakash CR, Raja S. Design, synthesis and antiepileptic properties of novel 1-(substituted benzylidene)-3-(1-(morpholino/piperidino methyl)-2,3-dioxoindolin-5-yl)urea derivatives. Eur J Med Chem. 2011;46(12):6057-6065.   Back to cited text no. 1
    
2.
Verma M, Pandeya SN, Singh KN, Stables JP. Anticonvulsant activity of Schiff bases of isatin derivatives. Acta Pharm. 2004;54:49-56.  Back to cited text no. 2
    
3.
Aliabadi A, Gholamine B, Karimi T. Synthesis and antiseizure evaluation of isoindoline-1,3-dionederivatives in mice. Med Chem Res. 2014;23(6):2736-2743.  Back to cited text no. 3
    
4.
Rajak H, Deshmukh R, Aggarwal N, Kashaw S, Kharya MD, Mishra P. Synthesis of novel 2,5-disubstituted 1,3,4-thiadiazoles for their potential anticonvulsant activity: pharmacophoric model studies. Arch Pharm (Weinheim). 2009;342(8):453-461.  Back to cited text no. 4
    
5.
Deng XQ, Dong ZQ, Song MX, Shu B, Wang SB, Qua ZS. Synthesis and anticonvulsant activities of some triazolothiadiazole derivatives. Arch Pharm (Weinheim). 2012;345(7):565-573.  Back to cited text no. 5
    
6.
Kwan P, Brodie MJ. Early identification of refractory epilepsy. New Engl J Med. 2000;342(5):314-319.  Back to cited text no. 6
    
7.
Kwan P, Brodie MJ. Drug treatment of epilepsy: when does it fail and how to optimize its use? CNS Spectr. 2004;9(2):110-119.  Back to cited text no. 7
    
8.
Leppik IE. Antiepileptic drugs in development: prospects for the near future. Epilepsia. 1994;35(suppl 4):S29‐S40.  Back to cited text no. 8
    
9.
Meldrum BS, Rogawski MA. Molecular targets for antiepileptic drug development. Neurotherapeutics. 2007;4(1):18-61.  Back to cited text no. 9
    
10.
Medvedev AE, Clow A, Sandler M, Glover V. Isatin: a link between natriuretic peptides and monoamines? Biochem Pharmacol. 1996;52(3): 385-391.  Back to cited text no. 10
    
11.
Han K, Zhou Y, Liu F, Guo Q, Wang P, Yang Y, et al. Design, synthesis and in vitro cytotoxicity evaluation of 5-(2-carboxyethenyl)isatin derivatives as anticancer agents. Bioorg Med Chem Lett. 2014;24(2):591-594.  Back to cited text no. 11
    
12.
Lashgari N, Mohammadi Ziarani G. Synthesis of heterocyclic compounds based on isatin through 1,3-dipolar cycloaddition reactions. Arkivoc. 2012;2012:277-320.  Back to cited text no. 12
    
13.
Sridhar SK, Pandeya SN, Stables JP, Ramesh A. Anticonvulsant activity of hydrazones, Schiff and Mannich bases of isatin derivatives. Eur J Pharm Sci. 2002;16:129-132.  Back to cited text no. 13
    
14.
Rahmani-Khajouei M, Mohammadi-Farani A, Ghorbani H, Aliabadi A. Synthesis and acetylcholinesterase inhibitory assessment of 3-(2-(4-benzoylpiperazin-1-yl)ethylimino)indolin-2-one with potential anti-Alzheimer effects. J Rep Pharm Sci. 2015;4(2):148-157.   Back to cited text no. 14
    
15.
Bhrigu B, Pathak D, Siddiqui N, Alam MS, Ahsan W, Search for biological active isatins: a short review. Int J Pharm Sci Drug Res. 2010;2(4):229-235.  Back to cited text no. 15
    
16.
Smitha S, Pandeya SN, Stables JP, Ganapathy S. Anticonvulsant and sedative-hypnotic activities of N-acetyl/methyl isatin derivatives. Sci Pharm. 2008;76(4):621-636.  Back to cited text no. 16
    
17.
Pandeya SN, Raja AS. Stables JP. Synthesis of isatin semicarbazones as novel anticonvulsants-role of hydrogen bonding. J Pharm Pharm Sci. 2002;5(3):266-271.  Back to cited text no. 17
    
18.
Eggadi V, Kulandaivelu U, Sharvanabhava BS, Jupally VR. Screening of the anticonvulsant activity of some isatin derivatives in experimental seizure models and its effect on brain GABA levels in mice. Am J Pharmacol Sci. 2013;1(3):42-46.  Back to cited text no. 18
    
19.
Rahmani-Khajouei M, Mohammadi-Farani A, Mirzaei D, Aliabadi A. Isatin-based anticonvulsant agents: Synthesis and antiseizure evaluation in mice. J Rep Pharm Sci. 2017;6:13-22.  Back to cited text no. 19
    
20.
Siddiqui N, Alam MS, Stables JP. Synthesis and anticonvulsant properties of 1-(amino-N-arylmethanethio)-3-(1-substituted benzyl-2, 3-dioxoindolin-5-yl)urea derivatives. Eur J Med Chem. 2011;46(6):2236-2242.  Back to cited text no. 20
    
21.
Mohammadi-Farani A, Heidarian N, Aliabadi A. N-(5-Mercapto-1,3,4-thiadiazol-2-yl)-2-phenylacetamide derivatives: Synthesis and in vitro cytotoxcity evaluation as potential anticancer agents. Iran J Pharm Res. 2014;13(2):487-492.  Back to cited text no. 21
    
22.
Mohammadi-Farani A, Foroumadi A, Rezvani Kashani M, Aliabadi A. N-Phenyl-2-p-tolylthiazole-4-carboxamide derivatives: Synthesis and cytotoxicity evaluation as anticancer agents. Iran J Basic Med Sci. 2014;17(7):502-508.  Back to cited text no. 22
    
23.
Aliabadi A, Hasanvand Z, Kiani A, Mirabdali SS. Synthesis and in vitro cytotoxicity assessment of N-(5-(Benzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide with potential anticancer activity. Iran J Pharm Res. 2013;12(4):687-693.  Back to cited text no. 23
    
24.
Aliabadi A, Andisheh S, Tyarani-Najaran Z, Tayarani-Najaran M. 2-(4-Fluorophenyl)-N-phenylacetamide derivatives as anticancer agents: synthesis and in vitro cytotoxicity evaluation. Iran J Pharm Res. 2013;12(3):267-271.  Back to cited text no. 24
    
25.
Nazaritarhan H, Hosseinzadeh L, Aliabadi A, Babak Gholamine, Foroumadi A. Cytotoxic and apoptogenic properties of 2-phenylthiazole-4-carboxamide derivatives in human carcinoma cell lines. Res Pharm Sci. 2012;7(5):S129.  Back to cited text no. 25
    
26.
Agamennone M, Campestre C, Preziuso S, Consalvi V, Crucianelli M, Mazza F, et al. Synthesis and evaluation of new tripeptide phosphonate inhibitors of MMP-8 and MMP-2. Eur J Med Chem. 2005;40(3):271-279.  Back to cited text no. 26
    
27.
Porter RJ, Cereghino JJ, Gladding GD, Hessei BJ, Kupferberg HJ, Scoville B. Antiepileptic drug development program. Cleve Clin Q. 1984;51(2):293-305.  Back to cited text no. 27
    
28.
Litchfield JT, Wilcoxon F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther. 1949;96(2):99-113.  Back to cited text no. 28
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


This article has been cited by
1 Design, molecular docking, synthesis and evaluation of new 5-aminoisatin derivatives as tyrosine kinase inhibitors
Noor Waleed Ibrahim,Monther Faisal Mahdi,Ayad M.R. Raauf
Materials Today: Proceedings. 2021;
[Pubmed] | [DOI]
2 Synthesis, in silico, in vitro and in vivo evaluations of isatin aroylhydrazones as highly potent anticonvulsant agents
Saeed Emami,Mehdi Valipour,Fatemeh Kazemi Komishani,Fatemehsadat Sadati-Ashrafi,Maria Rasoulian,Majid Ghasemian,Mahmood Tajbakhsh,Patrick Honarchian Masihi,Aidin Shakiba,Hamid Irannejad,Nematollah Ahangar
Bioorganic Chemistry. 2021; 112: 104943
[Pubmed] | [DOI]
3 Synthesis and Evaluation of Anti-Epileptic Properties of New Phthalimide-4,5-Dihydrothiazole-Amide Derivatives
Leila Sadat Tabatabaei Rafiei,Mehdi Asadi,Faezeh Sadat Hosseini,Arash Amanlou,Mahmood Biglar,Massoud Amanlou
Polycyclic Aromatic Compounds. 2020; : 1
[Pubmed] | [DOI]
4 Isatin Containing Heterocycles for different biological activities: Analysis of Structure activity relationship
Rajarshi Nath,Shelly Pathania,Gourav Grover,Md Jawaid Akhtar
Journal of Molecular Structure. 2020; : 128900
[Pubmed] | [DOI]
5 A review on the synthesis and applications of molecules as anticonvulsant drug agent candidates
Oguz Özbek,Meliha Burcu Gürdere
Medicinal Chemistry Research. 2020;
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
   Abstract
  Introduction
   Materials and Me...
  Results
  Discussion
  Conclusion
  Acknowledgement
   References
   Article Figures
   Article Tables

 Article Access Statistics
    Viewed1983    
    Printed41    
    Emailed0    
    PDF Downloaded213    
    Comments [Add]    
    Cited by others 5    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]