Synthesis and Biological Activity Evaluation of Some New Coumarin Derivatives as Potent Anticonvulsant and CNS-Depressant Agents

Abstract Triazolo-thiadiazines 4a–f and triazolo-tetrazines 8g–l were prepared from reaction of 3-(4-amino-5-mercapto-4H-1,2,4-triazol-3-yl)-2H-chromen-2-one with the respective hydrazonoyl halides 2a–f and 2g–l. Moreover, the reaction of the aminomercaptotriazole derivative with phenacyl bromide and ethyl 2-chloro-3-oxobutanoate followed by the coupling reaction of the products with benzenediazonium chloride yielded the corresponding products 4f and 8g, respectively. All of the newly prepared heterocyclic derivatives were determined using spectral data, elemental analysis, and alternate methods. The in-vivo anticonvulsant and CNS-depressant activities of all the prepared derivatives were tested using the maximal electroshock method and showed excellent activities, especially triazolo[4,3-b]tetrazine derivatives 8j, 8k, and 8l. Additionally, their structure–activity relationship was proven clearly, demonstrating that electron-releasing groups increase activities while electron-withdrawing groups lower them.


Introduction
Epilepsy is a class of CNS disorders that are characterized by symptomatic cerebral dysrhythmia, which manifests as brief episodes of awareness loss or alteration, even without seizures, visual or mental symptoms. 1 Many people suffer from seizures that do not respond to medical treatment. 2 Although about 20 drugs are now on the market, only about 5 or 6 of them are frequently used in clinical trials. These drugs, however, have serious side effects such as sleepiness, megaloblastic anemia, gastrointestinal disruption, ataxia, and hepatotoxicity. 3 As consequently, there is an urgent need for antiepileptic drugs having safer and more efficient effects. The coumarin chemistry has been the subject of considerable interest due to their various biological activity, such as antiviral, anticonvulsant, anti-inflammatory, antimicrobial, antitumor, and antidiabetic [4][5][6][7][8] and therefore, they are considered as valuable scaffolds in drug research. Many coumarin compounds presented a promising anticonvulsant activity. 9,10 Furthermore, coumarin derivatives have been shown to exhibit potent effects in vitro and in vivo on numerous CNS components. 11 1,2,4-Triazoles have also gotten a lot of attention because of their anticonvulsant properties, CNS-depressant activities, and neurotoxicity activity. [12][13][14][15][16][17] It was reported as an attempt to lead optimization of anticonvulsant agents that 1,2,4-triazolylcoumarins have significant anticonvulsant activities compared to standard antiepileptic drugs (Chart 1). 18,19 The purpose of this research is to examine the effect of replacing the barbiturate ring system present in many antidepressants clinically used drugs with its closely related thiadiazine and tetrazine ring systems on the CNS-depressant and anticonvulsant activities. Also studying replacing the oxazolidinedione and hydantoins ring systems present in most clinically used anticonvulsant agents with its bioisosteric 1,2,4-triazole moiety.
Spectral (MS, 1 H-NMR, IR) and analytical data were used to identify the structure of obtained compounds (see experimental). In each case, the IR spectra revealed a band at v ¼ 3249-3274 cm À1 Chart 1. Structures of literature lead compounds and the proposed target compounds.
due to the NH group and the lack of the band assigned to the CO group. Along with the signals given to aliphatic and aromatic protons, 1 H-NMR exhibited a peculiar singlet signal near d ¼ 10.30 ppm, which was related to the NH of the hydrazone tautomer. 29 Coupling compounds 5 28 with PhN 2 Cl in pyridine at 0-5 C resulted in the synthesis of products that are identical to compound 4f in all aspects (IR spectra, mp, and mixed mp). This was used as an alternate method to confirm the structure of product 4 (Scheme 1).
As indicated in Chart 2, compounds 4 may be iminohydrazone 4A, azo-enamine 4B, or CHazo 4C. The 1 H-NMR spectra of the examined compounds exhibited one broad singlet signal at d ¼ 10.14-10.26 ppm which is assignable to the hydrazone NH, and the absence of signals assignable to the NH and CH at d ¼ 11.69 26 and 5.27 ppm, 30 associated to the azo-enamine and CHazo forms 4B and 4 C, respectively. Thus structure 4A is the actual structure of the final products according to 1 H-NMR spectra. Also, the electronic absorption spectra confirm hydrazo structure 4A. The electronic absorption spectra of compound 4a, taken as an example of the series, in various solvents are characterized by three intense maxima at v ¼ 387-378, 318-311, and 273-260 nm, which are assigned to phenylhydrazono chromophore. 31,32 Our research was expanded to investigate the behavior of compound 1 toward the other hydrazonoyl halides 2g-i and 2j-l in dioxan/TEA to obtain the triazolo[4,3-b]tetrazine derivatives 8g-l (Scheme 2).
Products 8g-l were formed via S-alkylation of thiol 1 to form thioyhdrazonate intermediate 6,33 followed by Smiles rearrangement 34 and finally removal of H 2 S from the thiohydrazide intermediate 7 (Scheme 2). MS, 1 H-NMR, IR, and elemental analyses were used to establish the identity of the obtained products. The 1 H-NMR spectrum of derivative 8g presented one singlet signal for the tetrazine NH at d 9.59 ppm 23 and its IR spectrum revealed two bands for NH and COOEt groups at t 3223 and 1737 cm À1 , respectively.
Also, product 8g was alternatively prepared via alkylation of compound 1 with 9 to yield the S-alkylated derivative 10. Coupling of 10 with PhN 2 Cl in pyridine via Japp-Klingemann cleavage of the CH 3 CO moiety yielded intermediate 11, 35 followed by the in situ rearrangement into 7 and finally removal of H 2 S (Scheme 2).

Pharmacological screening
Based on a strong biological rational of the structural similarity of the aforementioned antiepileptic and anticonvulsant agents and newly synthesized compounds, in this study, some of the prepared derivatives were subjected to pharmacological screening as anticonvulsant and CNSdepressant agents. As demonstrated in Tables 1 and 2, the outcomes showed that the majority of the investigated derivatives had a wide range of action when compared to the reference drug. Most derivatives showed potent CNS-depressant and anticonvulsant activity. The ascending order of activities for both properties of the prepared derivatives was as follows: 4b, 4d, 4a, 4e, 4c, 4f, 8i, 8g, 8h, 8l, 8j, and 8k.

Structural activity relationship (SAR)
Careful examination of the anticonvulsant and CNS-depressant activities of the examined derivatives and their structures leads to the following SAR assumptions.  For the triazolo-thiadiazines 4: the methoxy and methyl substituents with þ M effects provide more activities than the hydrogen ones and the latter more active than the electron withdrawing -I effects bromine and chlorine substituent (OMe > Me > H > Br > Cl) . For the triazolo-tetrazines 8: CONHPh provide more activities than COOEt and on fixing CONHPh or COOEt, the methyl substituent with þ M effects provide more activities than the hydrogen ones and the latter more active than the electron withdrawing -I effects chlorine substituent (Me > H > Cl).

General methods
A Shimadzu 470 spectrometer was employed to collect FT-IR data of some synthesized compounds. The uncorrected melting point of the fabricated compounds was measured using an electrothermal melting point device. 1 H (400 MHz) and 13 C NMR (100 MHz) spectra were detected by tetramethylsilane as an inner standard on a Bruker 400 DRXAvance NMR spectrometer. Finnigan MATSSQ-7000 mass spectrometer was also utilized to measure the mass spectra. Elemental analyses were performed using various elemental at microanalytical entity, central Services laboratory, NRC, Cairo, Egypt, and were obtained to be around ± 0.5% of the hypothetical quantities. Compounds 2 were synthesized according to prior reports in the literature. 36

Synthesis of compounds 4a-f and 8g-l
General procedure: A mixture of an equimolar amounts of aminomercaptotriazole 1 (1 mmol, 0.260 g) and compounds 2a-l (1 mmol) in dioxan (20 mL) containing TEA (0.1 g) was heated under reflux for 4-8 h, and then the resulting solid was crystallized from EtOH or dioxan or mixture of them to give the respective triazolo-thiadiazines 4a-f and triazolo-tetrazines 8g-l. The analytical data for the products are presented below. The 1-chloro-2-phenyldiazene solution was portionwise added to a stirred cold solution of thiadiazine derivative 5 (1 mmol, 0.360 g) in 10 mL of pyridine then the reaction mixture was left overnight, filtered, and finally recrystallized from dioxan to yield triazolo-thiadizine derivative 4f.

Coupling of 10
Compound 8g was obtained by repeating the alternative synthesis of compound 4f but replacing compound 10 with compound 5.

Anticonvulsant activity
The maximal electroshock method was used to evaluate the anticonvulsant activity. 37 Details were inserted in supplementary data.

CNS-depressant activity
The sleep deprivation method was used to evaluate the acute toxicity. 38 Details were inserted in supplementary data.

Statistical analysis
The difference between the control and tested compounds was compared statistically using oneway ANOVA and Duncan's multiple comparison test Ã p < 0.05.

Conclusions
Regardless of the type of hydrazonoyl halides, aminomercaptotriazole's remarkable reactivity produced triazolothiadiazines or triazolotetrazines. The elemental analyses, spectral data, and alternative synthetic methods were used to elucidate the newly prepared compounds. Moreover, the anticonvulsant and CNS-depressant activities of the products were determined and the results revealed potent activities. The study of the SAR showed that the activities of these compounds appear to be closely related to type of the substituent on the ring. It showed that the triazolotetrazine derivatives 8g-l is more active than the triazolo-thiadiazine derivatives 4a-f and electronreleasing groups (e.g. Me and OMe groups) enhance activities while electron-withdrawing groups (e.g. Cl and Br groups) decrease activities.