Antidepressant/Anxiolytic and Anti-Nociceptive Effects of Novel 2-Substituted 1,4-Benzodiazepine-2-ones

Oxazepam (4a) has been used as overall starting material in the synthesis of novel 2-substituted 1,4-benzodiazepines. By reacting Oxazepam 4a with commercially available hydrazines, hydrazides, semicarbazide, aminoguanidine and N,N-dimethylamino aniline in ethanol under acetic conditions, a series of diazenyl-1,4-benzodiazepines 5a–5i and 2-amino-1,4-benzodiazepine 5k were obtained in good yields. These novel compounds served as new chemical entities (NCE) for testing in mice. The diazo-benzodiazepine 5d has shown a promising antidepressant effect in initial experiments in vivo at a dose of 5 mg/kg. The highly coloured 2-aminobenzodiazepine derivative 5k showed over a dose range from 5–50 mg/kg an analgesic effect in mice.


Introduction
Benzodiazepines are primary agents to treat anxiety and they are the most successful class of chemical compounds worldwide. Around 50 benzodiazepine derivatives have been marketed worldwide. They are all classed as anxiolytics, anticonvulsants, sedatives and muscle relaxants. They mainly act by binding to a specific regulatory site on the GABA A (γ-amino butyric acid) receptor, thus increasing the inhibitory effect of GABA [1]. Since the discovery of benzodiazepines as anxiolytics in the 1960's, the classical structures of this class of compounds have been widely varied, resulting in benzodiazepine ligands that bind to specific subtypes of the GABA A receptors.
As shown, modified benzodiazepines are uniquely fruitful in the discovery of new lead structures and drugs. Therefore, novel 2-substituted 1,4-benzodiazepines had been synthesised in high yields from 3-hydroxy-1,4-benzodiazepine (oxazepam) and have been subsequently evaluated in standard CNS animal models. The starting material towards the synthesis of 3-substituted 1,4-benzodiazepines was oxazepam [11], which had been previously prepared by various synthetic methods [12]. Oxazepam has been derived from diazepam [13], by subsequent oxidation of the 3-position [14]. The most common synthesis is the rearrangement of the N-oxide of diazepam via the acetate into oxazepam [15]. We found the most appropriate route towards the synthesis of oxazepam on a multigramme scale based on the oxime 2, which was converted into the chloride 3 and cyclised in a one pot reaction into the desired oxazepam 4a or the oxazepam salt 4b under strictly monitored reaction conditions [16] (Scheme 1).

Results and discussion
Mechanistically, the one pot reaction followed the Polonovski Rearrangement [17], in which a benzodiazocrine, an eight membered hetrocyclic ring system, was formed in situ.
A mixture without a major product was formed, when oxazepam was refluxed with amines in ethanol/acetic acid as prescribed by Kulkarmi [18]. 3-Substituted 1,4-benzodiazepines [19] were formed via the 3-chlorinated intermediates, which acted as CCK antagonists and their in vivo evaluation was recently published [20]. The reaction of 4a under reflux conditions worked well with hydrazines and hydrazides as nucleophiles and furnished the unexpected 2-diazobenzo-diazepines 5a-5g in good yields, as highly coloured crystalline compounds.
Using semicarbazide or aminoguanidine resulted in the formation of 5h or 5i (Table 1).
Dimethylaminoaniniline is the only working aniline and formed by this route the 2-amino derivative 5k. It is violet in alkaline media and turns yellow, after the addition of acid. This novel compound might be useful as an organic dye in fluorescence screening assays for analgesics ( Figure 2).  Proposed mechanism for the formation of diazo-benzodiazepines.

Animal studies
Using standard CNS assays the anxiolytic, antidepressant and analgesic properties were systematically evaluated in mice. The elevated x-maze and the light/dark box were applied to test for anxiolytic activity. Anilino-benzodiazepine 5k showed a low CCK binding activity and this was not further investigated as CCK antagonists previously developed from the 3-substituted series had a better potency [20]. The tail suspension and the forced swim test served as assays for antidepressant activity. The diazo series 5d-5f was found different from the control from 5 mg/kg onwards and the dinitro-derivative 5f displayed unwanted side effects as accessed by the rota rod and the wire mesh grasping test. These two assays were added to the screening to identify mainly unwanted muscle relaxing properties or an impairment of coordination. Considering these properties and the presence of toxicophore, a nitrogroup, the chlorinated diazo-derivative 5d was forwarded to further evaluation. The thermal tail flick test (tail immersion test) and the hot plate test were applied to investigate the analgesic effects of the 2-substituted benzodiazepines ( -- Control (5% DMSO) Desipramine (10 mg/kg)* 5d (0.05 mg/kg) 5d 0.5 mg/kg) 5d (5 mg/kg) 5d (50 mg/kg)

Fig. 5.
Dose-effect relationship of 5d on the immobility time for the forced swim test in mice.
The diazo-benzodiazepine 5d was tested further in the forced swim test for a dose range from 0.05-50 mg/kg. In presence of the standard antidepressant desimpramine (10 mg/kg) the immobility time was reduced from 180s to 120s. Dose dependant a reduction was found for 5d with a comparable potency and magnitude to desipramine ( Figure 5).
The analgesic effect of 5k was tested in the tail flick test from 5-50 mg/kg in comparison with morphine, tramadol and amitriptyline as standards and the maximum possible effects are shown in Figure 6.
It appears that the activity is opiate like and in vitro studies are ongoing. 3-Anilinobenzodiazepine based CCK antagonists [21] such as the N-methylanilino-benzodiazepine potentiated the analgesic effect of morphine, while the 2-substituted benzodiazepine 5k showed a dose dependent analgesic effect on its own.

Conclusions
By using simply refluxing oxazepam with hydrazines and hydrazides in the presence of acid novel 2-diazo-1,4-benzodiazepines were furnished, of which the diazo derivatives and 5k were highly coloured. These new chemical structures were identified as new lead structures for antidepressents, anxiolytics and analgesics. Further studies to explore the underlying biological mechanism are currently being evaluated and the colour/fluorescence of the agents may be additionally useful in pharmacology to locate the distribution of receptors.

Chemistry
The chemicals were purchased from Aldrich, UK, and Lancaster Synthesis, UK. Mass-Spectras were obtained by Atmospheric Pressure Chemical Ionisation (APCI), using a Hewlett-Packard 5989b quadrupole instrument connected to an electrospray 59987A unit, with automatic injection (Hewlett-Packard 1100 series autosampler). Samples were dissolved in HPLC grade methanol or acetonitrile. Both proton and carbon NMR spectra were obtained on a Brucker AC 250 instrument, calibrated with the solvent reference peak. Infra-red spectra were plotted from KBr discs on a Mattson 300 FTIR Spectrophotometer. Melting points were recorded from a Stuart Scientific Melting Point (SMP1). Analytical Thin Layer Chromatography was obtained using aluminium sheets, silica gel 60 F254, 250 nm and were visualized using ultraviolet light.
(A): The remaining residue was partitioned first with ether then water. The organic phase was washed with water and dried over magnesium sulphate and then concentrated to dryness. The yellow solid was heated with a minimum amount of toluene and then cooled to room temperature and allowed to crystallise.

Anxiolytic activity tests
The light/dark box: Mice were placed in the light part of the light/dark box. The box was a plexiglass cage, 25x50x20 cm, having one-third as a dark and two-third as a light compartment. A 40-W light bulb was used and positioned 10 cm above the centre of the light component. The animals could walk freely between dark and light parts through the opening. The time animals spent in light part during the 5 min interval was recorded. The mouse was considered to be in the light part when its 4 legs were in the light part. The

Nociception tests
The thermal tail-flick test: The thermal response latency was measured by the tail flick test. The animals were placed into individual restraining cages leaving the tail hanging freely. The tail was immersed into water preset at 50°C and the response time, at which the animal reacted by withdrawing its tail from water, was recorded.
The thermal response latency was measured by the tail flick test. The base line withdrawal thresholds (BT) were recorded prior to the first injection. Test thresholds (TT) were measured 60 min after the second injection. The cut off time was set to 45s. This was to avoid any tissue damage to the paw during the course of analgesia testing. The test thresholds were expressed as a percentage of Maximal Possible Effect (% MPE) using the equation: The hot plate test: Mice were placed on a hot plate that was thermostatically maintained at 50°C. A plexiglass box was used to confine the animal to the hot plate. The reaction time of each animal (either paw licking or jumping) was considered a pain response and the latency to react was recorded.

Antidepression tests
The tail suspension test: Mice were hung by their tail on the tail hanger using sticky tape for tail fixation, at approximately 1 cm from the end. The hanger was fixed in the black plastic box (20x20x45 cm) with the opening at the top front. The distance between the hanger to floor was approximately 40 cm. The mouse was suspended in the air by its tail and the immobile time was recorded during the period of 5 min. The duration of immobility was defined as the absence of all movement except for those required for respiration.
The forced swim test: The forced swim test was carried out in a glass cylinder (20 cm diameter, 30 cm height) filled with water to the height of 20 cm. The water temperature was approximately 25-28°C. Mice were gently placed into the water and the immobility time was recorded by an observer during the period of 5 min. Immobility was defined as absence of all movement and remained floating passively in the water with its head just above the water surface.

Motor activity tests
The rota-rod test: Mouse was placed on the rotating drum with the acceleration speed (Acceler. Rota-rod, Jones & Roberts, for mice 7650, Ugo Basile, Italy). The time animal spent on the rod is recorded.
The wire mesh grasping test: Mouse was placed on a wire mesh (20x30 cm). After a few seconds, the mesh was turned 180° and the time animal hold on the mesh was recorded.

Statistical methods
The data were expressed as mean ± SD and one-way analysis of variance (ANOVA) and supplementary Tukey test for pair wise comparison were tested to determine for any significant difference at p< 0.05.