Oleanolic acid acrylate elicits antidepressant-like effect mediated by 5-HT1A receptor

The development of new drugs for the treatment of depression is strategic to achieving clinical needs of patients. This study evaluates antidepressant-like effect and neural mechanisms of four oleanolic acid derivatives i.e. acrylate (D1), methacrylate (D2), methyl fumarate (D3) and ethyl fumarate (D4). All derivatives were obtained by simple one-step esterification of oleanolic acid prior to pharmacological screening in the forced swimming (FS) and open field (OF) tests. Pharmacological tools like α-methyl-p-tyrosine (AMPT, catecholamine depletor), p-chlorophenylalanine (serotonin depletor), prazosin (PRAZ, selective α1-receptor antagonist), WAY-100635 (selective serotonin 5-HT1A receptor antagonist) as well as monoamine oxidase (MAO) and functional binding assays were conducted to investigate possible neural mechanisms. In the FS test, D1 showed the most promising antidepressant-like effect without eliciting locomotor incoordination. Unlike group of mice pretreated with AMPT 100 mg/kg, PCPA 100 mg/kg or PRAZ 1 mg/kg, the effect of D1 was attenuated by WAY-100635 0.3 mg/kg pretreatment. D1 demonstrated moderate inhibition of MAO-A (IC50 = 48.848 ± 1.935 μM), potency (pEC50 = 6.1 ± 0.1) and intrinsic activity (Emax = 26 ± 2.0%) on 5-HT1A receptor. In conclusion, our findings showed antidepressant-like effect of D1 and possible involvement of 5-HT1A receptor.


General pharmacological test of D1 effect on mice.
In the general pharmacological test, D1 elicited a reduction in exploratory activity at the dose of 250 mg/kg after 30 minutes of subcutaneous (s.c) administration (Table 2). A reduction in exploratory activity and sedative effect were observed following intraperitoneal (i.p) and s.c administration of D1 at doses 10, 50 and 250 mg/kg between 30 minute and 1hour. Oral (p.o) administration of D1 elicited increase in exploratory activity starting from 30 minute to 1 hour at the doses of 10, 50 and 250 mg/kg ( Table 2). All the behavioural manifestations disappeared within 4 hours of D1 administration without any record of toxicity or death.

Discussion
Chemical modification remains an important strategy for new drug synthesis and development from natural products 35 . The rationale for modification of oleanolic acid structure by esterification with acrylic and fumaric acid chlorides was to introduce electrophilic centre through the formation of Michael acceptor-type OAD that could interact with nucleophilic residues of binding site of the receptor.
In order to investigate antidepressant-like effects, mice were treated orally with OAD and subjected to the FS test. The FS test is one of the most used behavioural tests for screening antidepressant-like activity of new compounds 36,37 . Oral administration of D1 or D2 increased the swimming time. In contrary, D4 reduced this parameter while D3 did not show any significant effect on this parameter. An increase in swimming time by D1 or D2 administration that was accompanied by a significant reduction in immobility time is a valid measure of antidepressant-like effect in this model.
The sensitivity of FS test to acute antidepressant treatment, stimulants, sedatives, myorelaxants or motor-impairing compounds can influence animal performance in this model and lead to false-negative or false-positive results 38 . Hence, it is imperative to assess locomotor activity of the mice in the OF test in order to ascertain that the antidepressant-like effect of OAD was not masked by other general pharmacological activity. An open field test provides simultaneous measurement of locomotion, exploratory and anxiolytic activities 39 . The effects of D4 in the FS (reduction of swimming time) and OF (increase in freezing time, decrease in total crossing and number of grooming activities) could be associated to a sedative or myorelaxant effect of this derivative. In contrary, a dose dependent increase in swimming time and reduction in immobility time in the FS test by D1 without any alteration in total crossing, grooming activity and freezing time in the OF test reinforced specificity of antidepressant-like effect of D1. The increase in total crossing (an effect that could suggests stimulation) by D2 20 mg/kg (a dose that induced increase in swimming time and reduction in immobility time) makes further investigation of D1 more promising. In addition to antidepressant-like effect, an increase in crossing and time spent at the centre of OF suggests anxiolytic-like effect of D1 at the dose of 10 mg/kg. Anti-anxiety drugs like diazepam (benzodiazepine receptor agonist) or buspirone (partial agonist of 5-HT 1A receptor) are known to alter these parameters 40 . The choice of reference drugs and their respective doses in the FS and OF tests was based on previous study 7 . The IMI 15 mg/kg elicited antidepressant-like effect in FS test without   Having demonstrated antidepressant-like effect of D1 at the dose of 5 mg/kg, pharmacological tools like AMPT and PCPA (tyrosine and tryptophan hydroxylase enzyme inhibitors, respectively), PRAZ and WAY-100635 (selective antagonist of α 1-adrenoceptor and 5HT 1A receptors, respectively) were used to investigate possible mechanism of antidepressant-like effect of D1. The modulation of monoamine neurotransmission, transport and/or inhibition of their oxidative deamination by MAO have been associated with the mechanism of action of many clinically available antidepressant drugs 41 . In the present study, depletion of serotonin with PCPA or catecholamine with AMPT did not attenuate the antidepressant-like effect of D1. Unlike PRAZ, WAY-100635 pretreatment blocked the effect of D1, thereby suggesting involvement of 5HT 1A receptor. Unlike previous study that showed the blockade of antidepressant-like effect of OA by PCPA, AMPT, NAN, PRAZ and WAY100635 7 , the effects of D1 seems to be devoid of plural mechanisms.
The inhibition and/or activation of metabolic enzymes that are involved in the synthesis or breakdown of monoamine are critical targets of some antidepressant drugs. Although selective inhibitors of MAO-B are clinically use for the treatment of neurological disorders such as Parkinson's disease and Alzheimer's disease, the isoenzyme MAO-A inhibitors are currently in use for the treatment of depression 42 . MAO is an outer mitochondrial membrane bound flavin adenine dinucleotide-linked enzyme involved in the oxidative deamination of biogenic amines and xenobiotic arylalkylamines to aldehydes 43 . Serotonin (5-HT) and norepinephrine are preferentially metabolized by MAO-A isoform whereas MAO-B isoform deaminates benzylamine as a substrate. Previous study showed that antidepressant-like effect of OA was independent of MAO activity and that the levels of 4-hydroxy-3-methoxyphenylglycol and 3,4-dihydroxyphenylacetic acid in the brain hippocampus and cortex remained unaltered after administration of OA 7,25 . Consistent with these results, D1 showed only moderate inhibitory effect on MAO-A and no effect on MAO B. Unlike D1, clorgyline and deprenyl showed significant inhibition of MAO isoenzymes. Clorgyline and (R)-deprenyl have been reported to be potent and selective inhibitors of MAO-A and -B, repectively 42,44 .   Although the antidepressant-like effect of D1 could be associated to the inhibition of MAO A, the preservation of antidepressant-like effect of D1 despite AMPT and PCPA pretreatments suggests that the enzymes that are involved in the synthesis of monoamines (tyrosine and tryptophan hydroxylase, respectively) are possibly not critical to the effect of D1. Hence, we hypothesized that the antidepressant-like effect of the acute oral administration of this new oleanolic acid derivative is independent of monoamine synthesis. The inhibition of MAO A by D1 could increase synaptic concentration of 5-HT, bioavailability, prolongation of its interaction with 5-HT 1A receptor and subsequent potentiation of serotonergic transmission. The blockade of D1 effect by WAY-100635, which seems to be consistent with serotonergic mechanism, is not sufficient to discriminate between direct activity of D1 on the 5-HT 1A receptor or indirect effect through inhibition of MAO A to facilitate high level of 5-HT.
The interaction of 5-HT with diverse serotonergic receptors has been hypothesized in the regulation of neuronal activity 45 . As 5-HT 1A receptor seems to be involved in the effect of D1, a functional characterization of D1 at this serotonergic receptor subtype in vitro became necessary. Our findings showed similar potency and intrinsic activity of D1 and buspirone. The variations in the intrinsic activity could differentiate among full agonists, partial agonists, and antagonists, based on their high, intermediate, and zero intrinsic activity, respectively 46 . Unlike (+ )8-OH-DPAT which is full agonist of 5-HT 1A receptors, D1 could be considered as a partial agonist of this receptor on the basis of its the intrinsic activity. The 5-HT 1A receptors are relevant to the clinical response to antidepressant drugs. They are located presynaptically in the raphe nuclei (where they act as cell body autoreceptors to inhibit serotonergic transmision) and postsynaptically in limbic and cortical regions to attenuate firing activity 47 . The azapirones are full agonists at 5-HT 1A autoreceptors and are generally partial agonists at postsynaptic 5-HT 1A receptors (e.g buspirone).
In addition to the promising therapeutic value of D1, the data on general pharmacological test showed that this OAD did not elicit any sign of toxicity or behavioural alterations that could constitute harm to the mice even at the highest dose of 250 mg/kg. Except for the mild sedation that was observed within 4 hours of the administration at the highest dose, there was no record of weight gain or animal's death during the 7 days of observation. These observations demonstrate that the chemical modification of OA did not make the derivative unsafe for administration.
In conclusion, four new Michael acceptor-type oleanolic acid derivatives esterified on C-3 were synthesized and their antidepressant-like activity investigated. It is important to reiterate that the therapeutic application of OA or its derivatives is still very limited due to dearth of pharmacological data. The overall findings showed promising antidepressant-like property of D1. The mechanism of antidepressant-like effect of this compound suggested the involvement of 5HT 1A receptor. General Experimental Procedures:. All commercially available reagents were used without further purification. All these reactions were performed under Argon atmosphere and anhydrous dichloromethane was used as solvent, which was purchased from Sigma-Aldrich. The 1 H NMR spectra were recorded on a Bruker Avance-400 spectrometer using CDCl 3 as solvent, δ values in ppm and coupling constant J (Hz) assignments of 1 H resonance coupling. Thin-layer chromatography (TLC) was performed on 250 μ m layer plates Whatmann PE SIL G/UV silica gel (backing polyester) plates using n-hexane/ EtOAc, 1:1 solvent system. Spots on TLC visualized with anisaldehyde / H 2 SO 4 in methanol. Column chromatography was performed with silica gel (230 × 400 mesh) from GA, USA. Analytical HPLC was carried out on Waters 2487 with duel λ Absorbance detector system on Phenomenex Luna-C 18 column Scientific RepoRts | 5:11582 | DOi: 10.1038/srep11582 (4.6 × 250 mm, 5 μ m) with elution at a flow rate 1.0 mL/min. All biologically evaluated compounds were found to possess more than 95% purity by HPLC.

Synthesis of OAD.
General procedure for the synthesis of OAD (D1, D2, D3 and D4): Oleanolic acid (100 mg, 0.05 mmol) and a catalytic amount of trimethylamine were dissolved in dichloromethane (15 mL). An appropriate acid chloride (0.125 mmol) was added, and the reaction mixture was stirred for 4 h at room temperature. After TLC indicated completion of the reaction, the reaction mixture was quenched with water and the organic layer separated. The organic phase was washed with dilute aqueous HCl (0.01 mol/L, 2 mL) followed by saturated NaHCO 3 (2 mL). The organic layer was dried over anhydrous MgSO 4 , evaporated, and the residue was purified by column chromatography (SiO 2 ; eluent: CHCl 3 / MeOH) to obtain the target products with 75-90% yield. The synthetic scheme for the designed compounds D1-D4 is shown in Fig. 6 with (a) reagents and conditions -appropriate acid chloride, Et 3 N, dry.

FS test.
In the FS model, mice were randomly divided into 5 groups [control (vehicle), 3 different doses of OAD and IMI]. Mice were exposed to a modified version of FS test 48 after oral administration of vehicle, OAD (5, 10 or 20 mg/kg) or IMI 15 mg/kg. FS apparatus is a cylindrical water container (42 cm in height, 18 cm in diameter) filled with water up to 30 cm (total volume ( r h 2 π ) of 7635.06 cm 3 at 24 ± 2 °C). In this model, the initial escape-directed behaviour like swimming and climbing by mouse are generally followed with a relatively passive and immobile posture. The container was cleaned with 10% ethanol solution prior to the exposure of naïve mouse. This test session (6-min duration) was videotaped and the swimming and immobility time was later scored and analyzed. AMPT 100 mg/kg (catecholamine depletor) 4 h prior to the oral administration of vehicle 10 mL/kg or D1 5 mg/kg. It was demonstrated that AMPT (tyrosine hydroxylase inhibitor) reduced 57% of dopamine and 53% of noradrenaline levels in mice without affecting the levels of serotonin 50 . In a separate experiment, pretreatment (i.p) of mice with saline solution or PCPA 100 mg/kg (serotonin depletor) for four consecutive days prior to oral administration of vehicle or D1 5 mg/kg was used to investigate effect of serotonin depletion on antidepressant-like activity of this OAD. The regimen of PCPA in this study has been reported to deplete about 60% of endogenous serotonin content without altering the noradrenaline or dopamine levels [51][52][53] . Two separate experiment were also conducted where mice were pretreated with PRAZ (α 1-adrenoceptor antagonist) 1 mg/kg or WAY-100635 -WAY (a selective antagonist of 5-HT 1A ) 0.3 mg/kg prior to the oral administration of vehicle 10 mL/kg or D1 5 mg/kg (30 minutes interval between i.p. pretreatment and p.o. treatment). In all the four experiments, oral administration of vehicle or D1 5 mg/kg was followed by FS test (60 min interval between p.o. treatment and behavioural testing). Inhibition assay using recombinant human MAO-A and -B. In order to investigate the effect of OAD on the activities of recombinant human MAO-A and -B, the kynuramine deamination assay was performed in 96-well plates as described earlier 54 . The assays were performed at a fixed concentration of the substrate (kynuramine) and varying inhibitor concentrations to determine the IC 50 values. The substrate concentration of 80 and 50 μ M kynuramine was chosen for MAO-A and -B, respectively, based on the previously reported apparent K m values (the substrate concentration at half V max ) for substrate binding 55 . The assay was performed with the addition of inhibitor. Inhibition was calculated as percent of product formation compared to the corresponding control (enzyme-substrate reaction) without the inhibitors. The reactions were carried out in 0.1 M potassium phosphate buffer at pH 7.4. Incubation mixtures contained 5 μ g/mL of MAO-A (50 μ L in buffer) or 12.5 μ g/mL of MAO-B (50 μ L in buffer). The compounds were dissolved in DMSO. The total reaction volume was 200 μ L yielding a final DMSO concentration of 1.0% in the reaction mixture. The reaction mixtures were pre-incubated for 10 min at 37 °C followed by the addition of MAO-A or MAO-B to initiate the enzymatic reactions. The assay plates were incubated for 20 min at 37 °C and enzymatic reactions were stopped by the addition of 75 μ L of 2N NaOH. The formation of 4-hydroxyquinoline was determined fluorometrically by SpectraMax M5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with an excitation and emission wavelength of 320 nm and 380 nm, respectively, using the Soft Max Pro program.