Synthesis of 5-Alkoxythieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine Derivatives and Evaluation of Their Anticonvulsant Activities

This work concerns the design and synthesis of novel, substituted 5-alkoxythieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives 5a–p prepared from 3-amino-2-thiophenecarboxylic acid methyl ester. The final compounds were screened for their in vivo anticonvulsant activity using maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests. Neurotoxicity (NT) was tested using a rotarod test. The structure-anticonvulsant activity relationship analysis revealed that the most effective structural motif involves a substituted phenol, especially when substituted with a single chlorine, fluorine or trifluoromethyl group (at the meta-position), or two chlorine atoms. These molecules possessed high activity according to the MES and scPTZ models. Quantitative assessment of the compounds after intraperitoneal administration in mice showed that the most active compound was 5-[3-(trifluoromethyl)phenoxy]thieno[2,3-e] [1,2,4]triazolo[4,3-c]pyrimidine (5o) with ED50 values of 11.5 mg/kg (MES) and 58.9 mg/kg (scPTZ). Furthermore, compound 5o was more effective in the MES and scPTZ tests than the well-known anticonvulsant drugs carbamazepine and ethosuximide.


Introduction
Epilepsy is one of the most common disorders of the human brain, affecting more than 60 million individuals worldwide [1][2][3]. It has been observed that in as many as 25% of cases currently available antiepileptic drugs (AEDs) are unable to control the seizures [4]. Additionally, in many cases the clinical use of AEDs is restricted by their side effects such as gastrointestinal disturbances, gingival hyperplasia, headaches, nausea, anorexia, ataxia, hepatotoxicity, drowsiness, attention deficit, and cognitive problems [5][6][7][8][9]. Therefore, there is a ongoing need for the discovery of new chemical entities for the development of effective and safer AEDs.
The anticonvulsant activity of these compounds was evaluated using the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests in mice, and their neurotoxicity (NT) was evaluated using the rotarod test.

Anticonvulsant Activity
The synthesized compounds were submitted to in vivo evaluation using the methods described in the Antiepileptic Drug Development Program (ADD) of the US National Institutes of Health according to previously described testing procedures [22,23]. The pharmacological evaluation was accepted by the Ethics Commission of China. Primary anticonvulsant studies involved two tests: maximal electroshock seizure (MES) and subcutaneous metrazol (scMET), in mice. It is emphasized that nearly all clinically significant AEDs are effective in at least one of these two models, making them very useful tools for initial high throughput screening of candidate anticonvulsants. The MES test employs an electrical stimulus to induce generalized tonic clonic seizures and is used to identify compounds that prevent the spread of seizures. The scMET model utilizes chemically induced myoclonic seizures and recognizes the agents that are effective because they raise the seizure threshold. In addition to the primary anticonvulsant evaluation in the MES and scMET models, the neurotoxicity was assessed using a rotarod test.
The compounds were administered intraperitoneally to mice at doses of 30, 100, and 300 mg/kg and tests were carried out 0.5 and 4 h after administration. The reference drugs, carbamazepine (for MES and rotarod test) and ethosuximide (for scPTZ test) were used as positive controls. In preliminary screening, all of the newly synthesized compounds exhibited some degree of anti-MES activity. The protection offered by these compounds was indicative of their pharmacological ability to reduce seizure spread at a certain dose level. The results obtained after investigating the anticonvulsant activity of the synthesized compounds 5a-p, 7a-d, 9a-d, and 11a-d are summarized in Tables 1 and 2. For the 5a-p series, all of the compounds were active in the MES test, which indicates their ability to prevent seizure spread. At a dose of 100 mg/kg, most of the compounds showed protection, except 5a, 5j, and 5m-n. Five compounds, 5c-d, 5f, and 5o-p showed protection against MES-induced seizures at a dose of 30 mg/kg. In these derivatives, only four compounds (5i, 5j, 5l, and 5n) did not show activity 4 h after administration. Only two compounds showed significant neurotoxicity at a dose of 100 mg/kg, and six compounds were not neurotoxic at a dose of 300 mg/kg. Compounds 5d, 5f, 5h, and 5o-p were not neurotoxic at a dose of 300 mg/kg in 4 h.  As shown in Table 2, compound 7c showed anticonvulsant activity at a low dose of 30 mg/kg, and four of the compounds showed no neurotoxicity after 0.5 h. Five compounds, 7b-c, 9c, and 11c-d showed protection against MES-induced seizures when tested 4 h after administration, and only compounds 7a, 7c, and 9c were neurotoxic at a dose of 300 mg/kg after 4 h.
The anticonvulsant activity of the compounds according to the scMET model in mice was markedly lower than that according to the MES screen. Among the compounds, five (5c, 5f-g, 5o, and 7c) were active after both 0.5 h and 4 h. Only seven compounds (5c-d, 5f-g, and 5o-p) showed considerable levels of seizure protection (after 0.5 h) at a dose of 100 mg/kg.
Based on the preliminary biological data, the most active compounds 5c, 5f, 5o, 5p, and 7c were chosen for quantification of the pharmacological parameters (ED50 and TD50) after i.p. administration to mice. The results for the new compounds along with the data for the standard AEDs (tested in the same conditions), carbamazepine and ethosuximide are shown in Table 3. Table 2. Anticonvulsant activities of compounds 7a-d, 9a-d and 11a-d in MES and scPTZ tests. The analysis of the MES quantitative data revealed lower activity of 5c, 5f, 5p, and 7c than carbamazepine, which is used as a reference antiepileptic drug in the MES model. At the same time, all of these molecules provided higher anti-MES protection in comparison with ethosuximide. The most effective compound according to the MES test was 5o with an ED50 of 11.5 mg/kg. Despite lower activity in comparison with carbamazepine, 5o was less neurotoxic and yielded a more favorable protective index (PI) of 17.7 than the reference drug's PI of 6.4. Significantly, stronger activity was observed in the PTZ-induced seizures. In this test, the most effective were 5c, 5o, and 7c with ED50 values and protective indexes much more favorable than ethosuximide (model AED for PTZ-induced seizures).
To determine the oral time of peak effect (TPE) of compounds 5c and 5o, we conducted a time-course test; compounds 5c and 5o reached the TPE at 1.5 h after oral administration. Next, we evaluated the anticonvulsant activity of compounds 5c and 5o against MES-induced seizures and neurotoxicity after oral administration to mice (Table 4), using carbamazepine as a reference. Compounds 5c and 5o showed significant oral activity against MES-induced seizures in mice, with ED50 values of 39.4 mg/kg and 28.4 mg/kg, respectively. The standard, carbamazepine, showed an ED50 value of 27.3 mg/kg and a TD50 value of 328.6 mg/kg, resulting in a PI of 12.0 under the same conditions. Thus, compounds 5c and 5o were judged safer than the anticonvulsant drug carbamazepine in MES and TOX models. In this study, we prepared four series of compounds and evaluated their preliminary anticonvulsant activities, and we established the following structure-activity relationships (SARs). Analysis of the anticonvulsant activities of compounds 5a-p resulted in the establishment of several SARs. Among the phenoxy-substituted derivatives, the position of the substituent group on the benzene ring appeared to greatly influence the anticonvulsant activity, especially with a chlorine, fluorine, trifluoromethyl group (in the meta-position), or two chlorine atoms. These molecules showed high activity in the MES and/or the scPTZ models. Changing the position of the chlorine atom to the ortho or para position as well as introduction of methyl and methoxy groups decreased the anticonvulsant activity. The same effect was observed for unsubstituted derivative 5a, which protects against MES seizures only at dose of 300 mg/kg. When the triazole ring of compounds 5c, 5f, 5o, and 5p was opened, their anticonvulsant activities were weaker, and the PIs were lower for 7a-d compared to these compounds. When the triazole ring in compounds 7a-d was replaced with other heterocycles (i.e., imidazole and pyrazole), the resultant compounds 9a-d and 11a-d had slightly decreased activity as compared to the compounds containing the triazole ring. These data indicate that separating the triazole from the thieno[3,2-d]pyrimidine, instead of incorporating the triazole into the thieno[3,2-d]pyrimidine, leads to weaker anticonvulsant activity and lower PI.

Anticonvulsant Effects in the MES Test
Seizures were induced in mice with a 60 Hz alternating current of 50 mA intensity. The current was applied via corneal electrodes for 0.2 s. Effective protection against the spread of MES-induced seizures was defined as the prevention of the hind leg and tonic maximal extension component of the seizure. 0.5 h and 4 h after the administration of the compounds, the activities were evaluated using an MES test [22,23].

Neurotoxicity (NT) Screening
The neurotoxicity of the compounds was measured in mice using the rotarod test. The mice were trained to stay on an accelerating rotarod of diameter 3.2 cm that rotates at 10 rpm. The trained animals were given intraperitoneal (i.p) injection of the test compounds. Neurotoxicity was indicated by the inability of the animals to maintain equilibrium on the rod for at least 1 min in each of the trials [22,23].

The Subcutaneous Metrazol Seizure Test (scMET)
This screen utilizes a dose of pentylenetetrazole (85 mg/kg) that produces clonic seizures lasting for a period of at least five seconds in 97% (CD97) of animals tested. At the anticipated time of testing, the convulsant is administered subcutaneously. The test compound was administered intraperitoneally in mice, and the animals were observed over a 30 min period. Mice were tested at least two different time points (15 min, 30 min, 1 h, or 4 h) following i.p administration of 100 and 300 mg/kg of test compound. Absence of clonic spasms indicated a compound's ability to counteract the effect of pentylenetetrazole on seizure threshold [24].

Pharmacological Evaluation of Compounds 5c and 5o Administered Orally to Mice
The time-course effect of compounds 5c and 5o in the MES test was determined. A suspension of compounds 5c and 5o (50 mg/kg) in 0.5% methylcellulose was injected into mice by oral administration (p.o.). The mice were divided into six groups (n = 10). Subsequently, the animals were subjected to the MES test at various times: 0.5, 1, 1.5, 2, 2.5, and 3 h. The time of peak effect (TPE) was 1.5 h after the p.o. injection. Then, compounds 5c and 5o were evaluated for anticonvulsant activity against MES-induced seizures and oral neurotoxicity at its TPE when administered orally. This test involved the same procedures for determining ED50 and TD50 as used in the MES test and the TOX test screening, except that the test drug was administered orally to mice.