Synthesis and Antidepressant Activity Profile of Some Novel Benzothiazole Derivatives

Within the scope of our new antidepressant drug development efforts, in this study, we synthesized eight novel benzothiazole derivatives 3a–3h. The chemical structures of the synthesized compounds were elucidated by spectroscopic methods. Test compounds were administered orally at a dose of 40 mg/kg to mice 24, 5 and 1 h before performing tail suspension, modified forced swimming, and activity cage tests. The obtained results showed that compounds 3c, 3d, 3f–3h reduced the immobility time of mice as assessed in the tail suspension test. Moreover, in the modified forced swimming tests, the same compounds significantly decreased the immobility, but increased the swimming frequencies of mice, without any alteration in the climbing frequencies. These results, similar to the results induced by the reference drug fluoxetine (20 mg/kg, po), indicated the antidepressant-like activities of the compounds 3c, 3d, 3f–3h. Owing to the fact that test compounds did not induce any significant alteration in the total number of spontaneous locomotor activities, the antidepressant-like effects of these derivatives seemed to be specific. In order to predict ADME parameters of the synthesized compounds 3a–3h, some physicochemical parameters were calculated. The ADME prediction study revealed that all synthesized compounds may possess good pharmacokinetic profiles.


Introduction
Depression is a common and serious disability-causing mental disorder with high incidence and recurrence [1,2]. It is estimated by the World Health Organization that in the future it will become the second major cause of disability after cardiovascular diseases [2]. Depression is characterized by several clinical symptoms, including sadness, loss of interest or pleasure, disturbed sleep and appetite, feelings of tiredness, low self-esteem or poor concentration [1]. Currently used antidepressant drugs alleviate the symptoms of major depression, but these treatment approaches have many limitations such as requirement of long times to produce therapeutic responses, low response rates, and various side effects [3,4], therefore, there is a strong demand to develop novel antidepressant agents with greater efficacy and fewer adverse effects.
Based on the previous papers reporting the antidepressant-like potential of the benzothiazole scaffold, in this study, we synthesized novel compounds containing this ring system and investigated their potential antidepressant-like effects.

Comp.
Comp. Structure elucidations of the final compounds were performed by FT-IR, 1 H-NMR, 13 C-NMR and HRMS spectroscopic methods. In the IR spectra, significant stretching bands due to N-H, C=O bonds at 3315-3238 cm −1 and 1705-1685 cm −1 , respectively, were observed. The stretching absorption belonging to the 1,4-disubstituted benzene unit was determined at 839-831 cm −1 . In the 1 H-NMR spectra, N-H and -CH 2 protons in the amide group observed as a singlet at 12.01-11.84 ppm and 3.38-3.01 ppm, respectively. The -CH 3 protons gave a singlet peak between 2.24-2.32 ppm. The C4 and C7 protons of the benzothiazole ring gave peaks at 7.70-7.36 and 7.53-7.20 ppm as a singlet, respectively. In the 13 C-NMR spectra carbon of C=O group was assigned to the peaks at 169.62-171.27 ppm. All measured mass and isotope scores were compatible with calculated values for the compounds 3a-3h.

Pharmacology
In this study, based on the antidepressant activity potential of benzothiazole-based compounds, we searched for potential antidepressant-like effects of the newly synthesized benzothiazole derivatives 3a-3h through TST and modified forced swimming tests (MFST). Activity cage tests were also incorporated into the experimental protocol to evaluate any possible effect of the test compounds on the spontaneous locomotor activities of mice. Test compounds were administered to the mice at a dose of 40 mg/kg, which was previously reported for benzothiazole-based compounds [33].
As represented in Figure 1, the immobility time of the mice treated with compounds 3c, 3d, 3f-3h were lower than the corresponding values of the control group in TST [F (9, 50) = 12.29; p < 0.001]. Fluoxetine, used as a reference drug, also reduced the immobility time of animals, as expected. However, compounds 3a, 3b and 3e were ineffective in TST ( Figure 1). Structure elucidations of the final compounds were performed by FT-IR, 1 H-NMR, 13 C-NMR and HRMS spectroscopic methods. In the IR spectra, significant stretching bands due to N-H, C=O bonds at 3315-3238 cm −1 and 1705-1685 cm −1 , respectively, were observed. The stretching absorption belonging to the 1,4-disubstituted benzene unit was determined at 839-831 cm −1 . In the 1 H-NMR spectra, N-H and -CH2 protons in the amide group observed as a singlet at 12.01-11.84 ppm and 3.38-3.01 ppm, respectively. The -CH3 protons gave a singlet peak between 2.24-2.32 ppm. The C4 and C7 protons of the benzothiazole ring gave peaks at 7.70-7.36 and 7.53-7.20 ppm as a singlet, respectively. In the 13 C-NMR spectra carbon of C=O group was assigned to the peaks at 169.62-171.27 ppm. All measured mass and isotope scores were compatible with calculated values for the compounds 3a-3h.

Pharmacology
In this study, based on the antidepressant activity potential of benzothiazole-based compounds, we searched for potential antidepressant-like effects of the newly synthesized benzothiazole derivatives 3a-3h through TST and modified forced swimming tests (MFST). Activity cage tests were also incorporated into the experimental protocol to evaluate any possible effect of the test compounds on the spontaneous locomotor activities of mice. Test compounds were administered to the mice at a dose of 40 mg/kg, which was previously reported for benzothiazole-based compounds [33].
As represented in Figure 1, the immobility time of the mice treated with compounds 3c, 3d, 3f-3h were lower than the corresponding values of the control group in TST [F (9, 50) = 12.29; p < 0.001]. Fluoxetine, used as a reference drug, also reduced the immobility time of animals, as expected. However, compounds 3a, 3b and 3e were ineffective in TST ( Figure 1).            TST and MFST, rapid and validated animal tests, are widely used for screening the antidepressantlike activity [36]. In both of these tests, animals are exposed to a short-term and inescapable stress. Mice that are hanged from their tails in TST or forced to swim in a restricted space in MFST, initially carry out escape behaviors and then experience an "immobile posture" reflecting the behavioral despair phenomenon. The immobility behavior is accepted to associate with the depressive-like state of the animals and thus, agents reducing duration or frequency of the immobility are supposed to possess antidepressant-like effect [36][37][38][39].
In our study, in the TST and MFST, compounds 3c, 3d, 3f-3h induced significant decreases in the duration (Figure 1) and the frequencies (Figure 2) of immobility behaviors of mice, respectively. These findings pointed out the antidepressant-like effects of compounds 3c, 3d, 3f-3h.
MFST, superior to TST, allows to establish correlations between the "analyzed behaviors" and the "involved neurotransmitter systems". Namely, decrease in the immobility with simultaneous increase in the swimming behavior is related to the enhanced serotonergic neurotransmission.   TST and MFST, rapid and validated animal tests, are widely used for screening the antidepressantlike activity [36]. In both of these tests, animals are exposed to a short-term and inescapable stress. Mice that are hanged from their tails in TST or forced to swim in a restricted space in MFST, initially carry out escape behaviors and then experience an "immobile posture" reflecting the behavioral despair phenomenon. The immobility behavior is accepted to associate with the depressive-like state of the animals and thus, agents reducing duration or frequency of the immobility are supposed to possess antidepressant-like effect [36][37][38][39].
In our study, in the TST and MFST, compounds 3c, 3d, 3f-3h induced significant decreases in the duration (Figure 1) and the frequencies (Figure 2) of immobility behaviors of mice, respectively. These findings pointed out the antidepressant-like effects of compounds 3c, 3d, 3f-3h.
MFST, superior to TST, allows to establish correlations between the "analyzed behaviors" and the "involved neurotransmitter systems". Namely, decrease in the immobility with simultaneous increase in the swimming behavior is related to the enhanced serotonergic neurotransmission. TST and MFST, rapid and validated animal tests, are widely used for screening the antidepressant-like activity [36]. In both of these tests, animals are exposed to a short-term and inescapable stress. Mice that are hanged from their tails in TST or forced to swim in a restricted space in MFST, initially carry out escape behaviors and then experience an "immobile posture" reflecting the behavioral despair phenomenon. The immobility behavior is accepted to associate with the depressive-like state of the animals and thus, agents reducing duration or frequency of the immobility are supposed to possess antidepressant-like effect [36][37][38][39].
In our study, in the TST and MFST, compounds 3c, 3d, 3f-3h induced significant decreases in the duration (Figure 1) and the frequencies (Figure 2) of immobility behaviors of mice, respectively. These findings pointed out the antidepressant-like effects of compounds 3c, 3d, 3f-3h.
MFST, superior to TST, allows to establish correlations between the "analyzed behaviors" and the "involved neurotransmitter systems". Namely, decrease in the immobility with simultaneous increase in the swimming behavior is related to the enhanced serotonergic neurotransmission. On the other hand, agents acting on the noradrenergic system reduce immobility with a corresponding increase in the climbing behavior [36,40,41]. In this study, compounds 3c, 3d, 3f-3h, similar to serotonergic agent fluoxetine, induces significant decrease in the immobility and increase in the swimming frequencies of mice (Figures 2 and 3). These findings pointed out that serotonergic rather than noradrenergic system plays a significant role in the antidepressant-like effects of compounds 3c, 3d, 3f-3h.
It is known that agents that alter locomotor activity counts of animals may induce false positive or false negative results in TST and MFST [42,43]. In the present study, in order to clarify whether the antidepressant-like effects of the tested compounds are associated with a possible change in the locomotor activities of mice, we performed activity cage tests. Obtained results revealed that test compounds did not induce any alteration in the total number of horizontal or vertical locomotor activities of the mice (Figures 5 and 6). These data indicate that observed antidepressant-like effects of the test compounds were specific.
Most of new drug candidates fail in clinical trials owing to their toxicological profiles and reduced ADME (absorption, distribution, metabolism, and excretion) properties. The late-stage failures significantly cause to growing cost of new drug development. The capability to identify problematic issues early can intensely reduce the amount of missed time and funds, and rationalize the overall development progression. Therefore, pharmacokinetic properties of new drug candidates are extremely vital and should be assessed as early as possible in the drug development process. In this context, ADME prediction can be used to focus lead optimization to improve the preferred features of a compound [44].
In this study, predictions of ADME parameters of synthesized compounds 3a-3h were carried out using the QikProp 4.8 software [45]. This software calculates the violations of Lipinski's rule of five [46] and Jorgensen's rule of three [47], which evaluate the ADME properties of new drug candidates, and is essential for the optimization of a biologically active compound. The theoretical calculations about the CNS score; number of rotatable bonds (RB) molecular weight (MW), molecular volume (MV), number of hydrogen donors (DHB), number of hydrogen acceptors (AHB), octanol/water partition coefficient (logP), aqueous solubility (logS), apparent Caco-2 cell permeability (PCaco), number of likely primer metabolic reactions (PM), percent of human oral absorption (%HOA), polar surface area (PSA) are presented in Table 1 along with the violations of rules of three (VRT) and five (VRF). According to Lipinski's rule of five, all compounds 3a-3h abide to the rules causing no violation. Moreover, these compounds meet Jorgensen's rule of three with no more than one violation. Besides, it can be seen that all results of the rules of three and five are within the recommended ranges. CNS scores (1-2) of the compounds (Table 1) indicate that all compounds are able to cross through CNS which is essential for antidepressant drugs. However, the compounds 3a and 3b, carrying piperidine moiety, were inactive. This finding suggests that piperazine ring system has more contribution to antidepressant activity than piperidine moiety.
Among the piperazine bearing compounds 3c-3h, compound 3e was ineffective. Comparing the structural distance of these compounds, it was thought that extended side chain on the 4th position of the piperazine may cause an activity decrease in compound 3e. On the other hand, due to the preliminary pharmacological activity screening, a single dose (40 mg/kg) of the compounds was administered to animals and a dose-response relationship was not established. Therefore, investigating potential effect of different doses of compound 3e may be beneficial in further studies.
Antidepressant-like potency of compounds 3c, 3d, 3f-3h also different from each other (Figures 1 and 2). Although the difference did not reach the statistical significance, compounds 3f, 3g and 3h, carrying phenyl or benzyl substituents on 4th position of piperazine, seemed to be more active than compounds 3c and 3d. This may be caused by the better penetrance of compounds 3f, 3g and 3h to the CNS; since these compounds have higher scores of logP, a predictive indicator of lipophilicity and membrane penetration, than 3c and 3d.

Chemistry
All chemicals were purchased either from Sigma-Aldrich ( (1) 3,4-Dimethylaniline (0.04 mol, 4.84 g) and KSCN (0.048 mol, 4.66 g) in glacial acetic acid (10 mL) were cooled in an ice bath. Bromine (0.048 mol, 2.47 mL) in glacial acetic acid (10 mL) was added dropwise at 0 • C. The mixture was allowed to stir at room temperature overnight. The iced-water (50 mL) was added in the mixture and then pH was adjusted to 11 using sodium hydroxide pellets. The precipitate was filtered, dried and recrystallized from ethanol [27]. in THF (50 mL) were allowed to stir on an ice bath. Chloroacetyl chloride (0.032 mol, 3.58 g) in THF (10 mL) was added drop by drop. After this stage, the content was stirred for 1 h at room temperature. THF was evaporated and the product was recrystallized from ethanol [27,48].

Prediction of ADME Parameters
In order to predict ADME parameters of synthesized compounds (3a-3h), some physicochemical parameters were calculated by using the QikProp 4.8 software (Schrödinger, LLC, New York, NY, USA) [45].

Animals
Experiments were realized with adult BALB/c mice (Source: Anadolu University, Center for Animal Experiments and Research, Eskişehir, Turkey, 30-35 g). All animals were maintained under controlled environmental conditions (temperature 25 ± 1 • C, relative humidity 65% and 12 h light-dark cycle-lights on at 8:00 AM). Animals were acclimatized to laboratory conditions 24 h before the onset of experiment. The animals were maintained and treated during the experiments in accordance with the instructions of the Local Ethical Committee on Animal Experimentation of Anadolu University, Eskişehir, Turkey, which approved this study. All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments.

Drugs and Treatments
Test compounds were administered to the mice at a dose of 40 mg/kg [33]. As test compounds were dissolved in sunflower oil, control groups received the same volume of it. In order to validate the experiments, fluoxetine (20 mg/kg, Sigma-Aldrich Chemical Company), a classical antidepressant drug, was used as a positive control. Test compounds, fluoxetine and vehicle were administrated to animals in a constant 10 mL/kg volume of body weight, through an oral gavage three times 24, 5 and 1 h before the experiments [43].

Behavioral Experiments
Tail Suspension Test The TST was realized according to method described previously [39]. Acoustically and visually isolated mice were individually suspended 30 cm above the floor by their tail using adhesive tape (placed approximately 1 cm from the tip of the tail). After initial escape-oriented movements, mice hung passively and become completely motionless which is defined as immobile posture. The total duration of immobility (s) was recorded during the last 4 of 6 min test duration [49].

Modified Forced Swimming Test
The MFST was performed as described previously [40,50]. Mice were placed individually in a transparent glass cylinder (12 cm in diameter, 30 cm height), which was filled with water to a height of 20 cm. The temperature of the water was adjusted to 25 ± 1 • C. Two swimming sessions were performed: an initial 15-min pretest period and 24 h afterwards a 5-min test period. During the 5-min test, one of the following behavioral categories over a 5-s interval were scored [40,51]:

1.
Immobility: Mouse was in an upright position on the surface with its front paws together and making only those movements required to keep the head above the water.

2.
Swimming: Mouse was moving in horizontally throughout the swim chamber and crossing into another quadrant. 3.
Climbing: Mouse was making vertical movements with its forepaws along the side of the swim chamber.
The water in the cylinder was changed after the test was performed for each mouse to avoid the influence of alarm substances. After both swimming sessions, the animals were removed from the cylinders, dried with towels, and returned to their home cages.

Activity Cage Test
The locomotor activities were evaluated individually for each animal with activity cage device (Ugo Basile, No. 7420, Varese, Italy). Parts located on two opposite sides of the device produce infrared (IR) beams. Horizontal and vertical movements of the animals were disrupted the IR light beams to the photocells and these interruptions were recorded automatically for 4 min by apparatus. The floor of the apparatus was cleaned with ethanol solution between tests [52,53].

Statistical Analyses
All analyses were performed using the GraphPad Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA). The data used in statistical analyses were acquired from 6 animals for each group. Statistical analysis of the behavioral tests was performed by analysis of variance (ANOVA), which was followed by Tukey's post hoc comparison test. All experimental results are presented as mean (s) ± standard error of the mean (S.E.M). Probability values less than 0.05 (p < 0.05) were accepted as significant.

Conclusions
In this study, the antidepressant-like activities of some novel benzothiazole derivative compounds were examined using some in vivo methods. The obtained results indicated that compounds 3c, 3d, and 3f-3h possess antidepressant-like effects in the TST and MFST, with no accompanying effect on locomotor activities of the animals. Moreover, theoretical prediction of ADME properties of the synthesized compounds indicated that all compounds seem to have good pharmacokinetic profiles. In addition to the potent intrinsic pharmacological activities of the tested benzothiazole derivatives, the good pharmacokinetic profiles of these compounds make them interesting drug candidates. However, detailed studies are required to clarify the pharmacological mechanisms underlying the observed antidepressant-like action. Moreover, comprehensive preclinical, and also clinical studies are needed to validate antidepressant efficacy of these compounds.