Synthesis, Anticonvulsant, Sedative and Anxiolytic Activities of Novel Annulated Pyrrolo[1,4]benzodiazepines

Four new pentacyclic benzodiazepine derivatives (PBDTs 13–16) were synthesized by conventional thermal heating and microwave-assisted intramolecular cyclocondensation. Their anticonvulsant, sedative and anxiolytic activities were evaluated by drug-induced convulsion models, a pentobarbital-induced hypnotic model and an elevated plus maze in mice. PBDT 13, a triazolopyrrolo[2,1-c][1,4]benzodiazepin-8-one fused with a thiadiazolone ring, exhibited the best anticonvulsant, sedative and anxiolytic effects in our tests. There was no significant difference in potency between PBDT 13 and diazepam, and we proposed that the action mechanism of PBDT 13 could be similar to that of diazepam via benzodiazepine receptors.


Biology
Benzodiazepines are common muscle relaxant, anxiolytic and anticonvulsant agents, but their side effects limit their clinical use. It is significant to develop modified benzodiazepines to minimize the side effects. Four PBDT derivatives 13-16 are synthesized from a core skeleton tetracyclic 3-amino triazolopyrrolo[2,1-c] [1,4]benzodiazepin-8-one (12), and we evaluated their anticonvulsant, sedative and anxiolytic activities by drug-induced convulsion models, a pentobarbital-induced hypnotic model, and an elevated plus maze (EPM) in mice.
Firstly, we selected two drugs (picrotoxin (10 mg/kg, sc) or strychnine (2 mg/kg, ip)) to induce convulsion to evaluate the anticonvulsant activities of PBDTs 13-16 and used diazepam as a positive control (Table 1). In the picrotoxin-induced convulsion model, only PBDT 13 could prolong the duration of clonic-tonic convulsion induced by picrotoxin or strychnine (p < 0.001). However, PBDT 14 only prolonged the duration of strychnine-induced, but not picrotoxin-induced, clonic-tonic convulsion (p < 0.001). On the contrary, PBDTs 15 and 16 only prolonged the duration of clonic-tonic convulsion induced by picrotoxin, but not strychnine (p < 0.05). Diazepam at 1 mg/kg also prolonged the latency of myoclonic jerks and the duration of clonic-tonic convulsion induced by picrotoxin or strychnine (p < 0.01, p < 0.001). Therefore, we suggested that PBDT 13 among PBDT derivatives possesses better anticonvulsant effects, and its anticonvulsant mechanism could be similar to diazepam, which mainly acts at benzodiazepine receptors.  Values are the mean ± SEM, n = 4 mice; * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the vehicle group.
Next, we evaluated the sedative effects of  Finally, we evaluated the anxiolytic effects of PBDTs 13-16 by the elevated plus maze. EPM is the most popular test of anxiety and the first-choice test for screening anxiolytic drugs [32]. The anxiolytic effects of PBDTs 13-16 and diazepam on the elevated plus maze are shown in From these above results, we suggested that PBDT 13 possessed the best anticonvulsant, sedative and anxiolytic effects among PBDT derivatives 13-16, and then, the next derivative was PBDT 15.
A new potential anxiolytic compound, PBDT 13, was found. There was no significant difference in potency between PBDT 13 and diazepam in our tests, and the action mechanism of PBDT 13 was similar to that of diazepam via the benzodiazepine receptors.

General
Melting points were recorded on a Yanaco MP-3 melting point apparatus (Yanaco Corp., Kyoto, Japan) and were not corrected. IR spectra were recorded on a Nicolet Magna FT-IR spectrophotometer (Nicolet Instrument, Inc., Madison, WI, USA). NMR spectra were recorded on Bruker AMX 500 FT-NMR spectrometers (Bruker, Karlsruhe, Germany); all chemical shifts were given in ppm from tetramethylsilane as an internal standard. Mass spectra were obtained on a VG 70-250S spectrometer by a direct inlet system (Micromass Corp., Manchester, UK).

Animals
Male ICR mice, weighing 20-25 g, were used for the anticonvulsant, hypnotic and anxiolytic assays. All mice were used in accordance to the Guiding Principles and the experimental protocol (No. 102-250-B) was approved on 20 July 2013 by the Institutional Animal Care and Use Committee (IACUC) of the China Medical University. They were housed for at least 1 week before starting the experiment with free access to standard food pellets (supplied and designed by Fwusow Industry Co. LTD., Taiwan) and tap water and housed in a regulated environment (23 ± 1 °C temperature and 60% humidity), wherein a 12:12 h light/dark cycle (light phase: 08:00-20:00 h) was maintained. PBDT derivatives were administered, and the anticonvulsant, hypnotic and anxiolytic assays were performed using the double-blind method.

Picrotoxin-or Strychnine-Induced Convulsion in Mice
In brief, clonic-tonic convulsion was induced by a subcutaneous (sc) injection of picrotoxin or intraperitoneal (ip) injection of strychnine. The mice were pretreated with PBDT derivatives (1 mg/kg, ip), diazepam (1 mg/kg, ip) or vehicle, 15 min before the injection of picrotoxin (10 mg/kg, sc) or strychnine (2 mg/kg, ip). After the picrotoxin or strychnine injection, mice were placed in the testing chamber. The latencies to myoclonic jerks and the duration from clonic to tonic convulsion were recorded [33].

Pentobarbital-Induced Hypnotic Model in Mice
In brief, the hypnotic model was induced by an intraperitoneal injection of sodium pentobarbital. The mice were pretreated with PBDT derivatives (1 mg/kg, ip), diazepam (1 mg/kg, ip) or vehicle, 15 min before the injection of sodium pentobarbital (30 mg/kg, ip). After the sodium pentobarbital injection, mice were placed in the testing chamber. The latency to the loss of righting reflex (induction time in seconds) and the time required to recover righting reflex or awakening (sleeping time in minutes) were recorded [33].

Elevated Plus Maze in Mice
The elevated plus maze is comprised of two open arms (30 × 5 × 0.25 cm) and two closed arms (30 × 5 × 15 cm) that extended from a common central platform (5 × 5 cm) that was elevated to a height of 50 cm above the floor level. Mice were given PBDT derivatives (1 mg/kg, ip), diazepam (1 mg/kg, ip) or vehicle 15 min before their placement on the elevated plus maze. In the experimental period, every precaution was taken to ensure that no external stimuli could evoke anxiety in the mice.
After each test, the maze was carefully cleaned up with a wet tissue paper (10% ethanol solution) to eliminate the interference of the olfactory cues on the next mice. All tests were recorded by a video camera and an automated video tracking system device equipped with Etho Vision XT software (Noldus Information Technology, Leesburg, VA, USA). The number of entries and the time spent in the open and closed arms were recorded during a 5-min test period. The percentage of arm entries in each arm (open or closed arm entry × 100/total entries) and the percentage of time spent in each arm (time spent in open or closed × 100/time spent in both arms) were calculated for each mouse [33].

Statistical Analysis
All data were expressed as the mean ± SEM for each experimental group. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's test. When the probability (p) was less than 0.05, the difference was considered significant. IBM SPSS Statistics 12.0 (SPSS Inc., Chicago, IL, USA) was used in this study.

Conclusions
We have synthesized four pentacyclic benzodiazepine derivatives (PBDTs 13-16) from 3-amino triazolopyrrolo[2,1-c] [1,4]benzodiazepin-8-one (12) via conventional thermal heating and microwave-assisted intramolecular cyclocondensation. The biological evaluation of these compounds revealed that PBDT 13 possessed the best anticonvulsant, sedative and anxiolytic effects. There was no significant difference in potency between PBDT 13 and diazepam in our tests, and the action mechanism of PBDT 13 could be similar to that of diazepam via the benzodiazepine receptors.