The study of literature also provided the information that small molecules possessed better binding affinity that large molecules towards MAO-A receptors^11-17.

Owing to the antioxidant property of GA¹⁸ and the study of structural features it was hypothesized to design new small molecule antidepressant drugs based on GA containing piperazine/piperidine (Figure 2). The synthesized compounds would be evaluated for antidepressant activity using Forced Swim Test (FST) and Tail Suspension Test (TST).

Figure 2: Structure of the proposed derivatives

MATERIAL AND METHODS:

All the chemicals used in the study were procured from local supplier and were obtained from different sources. All the chemicals used were of laboratory grade or analytical grade and used as obtained without further purification or drying.

The melting point of each of the synthesized compounds was determined instrumentally by open capillary method and is uncorrected. Solubility was assessed qualitatively in various solvent. The structural identity of the compounds was confirmed by IR, ¹HNMR and Mass spectral study.

The target compounds were synthesized using the scheme represented in Figure 3. The scheme was designed using the reaction reported by Özkayet al¹⁹. The scheme was modified according the environmental conditions of our laboratory and optimized for maximum yield of the designed compounds.

The scheme consisted of two steps: Step 1- Synthesis of 3,4,5-trihydroxybenzoyl chloride; Step 2- Synthesis of the heterocyclic derivative (designed compounds).

Figure 3: Scheme for synthesis of the designed compounds

Synthesis of 3,4,5-trihydroxybenzoyl chloride²⁰:

The apparatus consists of a 100mL three-neck flask equipped with magnetic stirring bead and reflux condenser. The reflux condenser was connected to a wash bottle filled with 100 mL of an aqueous solution of sodium hydroxide (20%) for trapping the evolved HCl and SO₂ gases.

The reaction flask was filled with 23.8 g (14.6mL, 150 mmol) freshly distilled thionyl chloride. To this 17.1g (100mmol) GA was added in several portions while stirring. 10mL of ethanol was added to the reaction mixture to assist solubilization of the reactants. The reaction mixture was heated under reflux conditions at 80°C for 6h. After cooling, the reflux condenser was replaced by a distillation bridge and the excess of thionyl chloride was removed by distillation under reduced pressure. Dark brown crystals of 3,4,5-trihydroxybenzoyl chloride were obtained.

The progress of the reaction was monitored by thin layer chromatography using precoated aluminum backed TLC plates as the adsorbent and hexane: methanol (7:3) as the eluting solvent.

General procedure for synthesis of piperazin/piperidin-1-yl(3,4,5-trihydroxyphenyl) methanone (2a-e.)¹⁹:

3,4,5-trihydroxybenzoyl chloride (1,0.001mol, 0.188 g), potassium carbonate (0.001mol, 0.138g) and appropriate piperazine/piperidine (0.001mol) were dissolved in acetone. The solution was reﬂuxed at 40°C for 12h. Acetone was evaporated and the residue was washed with water, ﬁltered, dried and recrystallized from ethanol. The reaction was monitored by thin layer chromatography using precoated aluminum backed TLC plates as the adsorbent and hexane: methanol: glacial acetic acid (6:3:1) as the eluting solvent.

The spectral studies (NMR, Mass and IR) were conducted to confirm the structure of the synthesized compounds. The spectra were obtained for the samples and the interpretation of each spectrum was carried out to ascertain the formation of desired bonds and incorporation of the functional groups.

Piperazin-1-yl (3,4,5-trihydroxyphenyl) methanone, 2a:

IR (cm^-1): 3420 (OH), 3162 (NH), 1657 (C=O); ¹HNMR (d ppm): 5.0 (OH), 2.0 (NH), 2.8 (CH₂), 3.3 (CH₂), 6.8 (CH Ar); m/z: 240 (M⁺+2)

(4-methylpiperazin-1-yl)(3,4,5-trihydroxyphenyl) methanone, 2b:

IR (cm^-1): 3416 (OH), 3114 (NH), 1646 (C=O); ¹HNMR (d ppm): 5.0 (OH), 2.5 (NCH₃), 1.5 (CH₂), 6.8 (CH Ar); m/z: 254 (M⁺+2)

(4-ethylpiperazin-1-yl)(3,4,5-trihydroxyphenyl) methanone, 2c:

IR (cm^-1): 3410 (OH), 3121 (NH), 1654 (C=O); ¹HNMR (d ppm): 4.9 (OH), 2.4 (NCH₂), 1.2, 1.5 (CH₃), 6.8 (CH Ar); m/z: 266 (M⁺)

Piperidin-1-yl(3,4,5-trihydroxyphenyl)methanone, 2d:

IR (cm^-1): 3431 (OH), 3165 (NH), 1654 (C=O); ¹HNMR (d ppm): 5.0 (OH), 1.5 (CH₂), 3.2 (CH₂), 6.8 (CH Ar); m/z: 240 (M⁺ + 2)

(4-methylpiperidin-1-yl(3,4,5-trihydroxyphenyl) methanone, 2e:

IR (cm^-1): 3422 (OH), 3172 (NH), 1665 (C=O); ¹HNMR (d ppm): 5.0 (OH), 1.5 (CH₂), 3.3 (CH₂), 1.0 (CH₃), 6.8 (CH Ar); m/z: 253 (M⁺ + 2)

Evaluation of antidepressant action:

The in vivo antidepressant action of the synthesized compounds was carried out in male albino mice weighing between 25–30g by FST and TST method. The protocol of the present work was approved by Institutional Animal Ethical Committee (IAEC) of Technocrats Institute of Technology Pharmacy. The animal were grouped and housed in poly acrylic cages (38x23x10cm) in the animal house of the institute. Not more than four animals per cage were housed and maintained under standard laboratory conditions with natural dark and light cycle (14h light/10h dark) at 27±2°C and relative humidity (RH) 44-56% with free access to standard diet (Golden Feeds, India) and tap water ad libitum for one week for acclimatization before and during the experiments. Animal were divided into 7 groups of 6 animals each for conducting the study. Group I was administered with normal saline and served as control, group II, III, IV, V and VI were administered 40mg/kg (i.p) of the test compounds, whereas group VII served as positive control and was administered with fluoxetine, 10mg/kg (i.p).

Forced Swim Test^21,22:

The synthesized compounds and ﬂuoxetine were dissolved in DMSO and injected intraperitoneally in a standard volume of 0.05mL per 20g body weight, to each mouse 30minutes prior to the test. To determine the effect of the test compound mice were individually placed in a glass cylinder (25 cm height, 10cm diameter) filled with water (22-25°C) up to 10cm height. Each mouse was allowed to swim for 6minutes during the test, and the duration of immobility was observed and noted during the final 4minutes of the test. The time spent by the mouse floating in the water without struggling and making only those movements necessary to keep its head above water was regarded as the immobility period. The animals were dried using tower and returned back to their housing conditions.

Tail Suspension Test^21,22:

The synthesized compounds and ﬂuoxetine were dissolved in DMSO and injected intraperitoneally in a standard volume of 0.05mL per 20g body weight, to each mouse 30minutes prior to the test. To determine the effect of the test compound mice were individually suspended by tail using clamp (2cm from the tip of the tail) in a box (25 × 25 × 30cm) with the head 5cm from the bottom. Minimal background noise was maintained and the testing was carried out in dark room. All animals were suspended for total 6minutes, and the duration of immobility was observed and noted during the final 4 minutes of the test. Mice were considered immobile only when they hung passively and completely motionless.The animals were used only once for this test.

Statistical Analysis:

The results of pharmacological studies were expressed as mean±S.D. The total variations present in data were evaluated by using Graph Pad Prism 5 project software one way ANOVA (analysis of variance) followed by Dunnett’s multiple comparison Test. The result were considered statistically significant when P- value less than 0.05 (P<0.05) vs control.

RESULTS AND DISCUSSION:

RESULTS:

The target molecule 2a-2e was synthesized in two steps as shown in Figure 3. The piperazine/piperidine selected for the synthesis of final compounds is reported in Table 1.

Table 1: Piperazin/ piperidin-1-yl (3,4,5-trihydroxyphenyl) methanone derivatives

Compound
2a
2b
2c
2d
2e

The structure, yield, solubility, retention factor and melting point of all the synthesized compounds are depicted in Table 2.

The antidepressant action of the synthesized compounds was testing using two animal models (Figure 4 to 6). The immobility time was recorded and statistically analyzed using one way ANOVA followed by Dunnett’s multiple comparison test.

Figure 4: Effect of test compounds 2a-2e (40mg/kg) and fluoxetine (10mg/kg) on immobility time of mice in TST. *p<0.05, **p<0.01, ***p<0.001, Values are represented as mean ± SD, (n = 6)

Figure 5: Effect of test compounds 2a-2e (40mg/kg) and fluoxetine (10mg/kg) on immobility time of mice in FST. **p<0.05, ***p<0.001, ns-not significant, Values are represented as mean ± SD, (n = 6)

Figure 6: Effect of test compounds 2a-2e (40mg/kg) and fluoxetine (10mg/kg) on swimming frequency of mice in TST. *p<0.05, **p<0.01, ***p<0.001, Values are represented as mean ± SD, (n = 6)

DISCUSSION:

Chemistry:

Derivatives of GA have been widely investigated for their antioxidant potential and other related pharmacological actions thereof^23-26. In the present work the target compounds were synthesized in two steps as shown in Figure 3. Initially 3,4,5-trihydroxybenzoyl chloride, 1 was prepared by the reaction of GA and thionyl chloride. The completion of reaction was monitored by TLC and could also be considered as the end of formation of HCl gas bubbles. In the second step, compound 1 was reacted with substituted piperazine (2a-2c) and substituted piperidine (2d, 2e) in acetone to obtain the target compounds in good yields. A decrease in the reaction time affected the yield of the compounds significantly.

The structure elucidation of the compounds was performed by IR, ¹HNMR and mass spectroscopy^27-30. The IR spectra of all the compounds exhibited the stretching vibration peaks due to O-H, C=O, C-N at 3400-3700 cm^-1, 1640-1690 cm^-1 and 1120-1350 cm^-1 (medium) respectively. The stretching absorption of N-H was obtained in 2a at 3100-3200. The other vibrations that appeared in the spectra included those from aromatic C=C and C-H, C-H alkane.The ¹HNMR spectra obtained displayed the peaks of CH₂, OH and aromatic CH at 2-3.3, 5.0 and 6.7-7.2ppm respectively. The mass spectra displayed the molecular ion peak and the isotopic peaks as calculated.

Pharmacology:

Owing to the antidepressant potential displayed by GA and various piperazine and piperidine compounds, the antidepressant effects of the synthesized compounds 2a-2e were evaluated by TST and FST. The test compounds were administered at a dose of 40mg/kg body weight intraperitoneally as GA has been studied at the similar dose in earlier study³². As it can be seen from Figure 4 that the immobility time for the compounds 2b, 2c and 2e was much lower than the control group and was comparable to that of fluoxetine at a dose on 10mg/kg. However the results obtained by compounds 2a and 2d were not as effective as it was expected (p<0.05) signifying the presence of substitution on the piperazine nitrogen for antidepressant effect.

As it can be seen that the reference drug fluoxetine and the test compounds 2b, 2c and 2e decreased the immobility of mice in FST whereas the swimming frequency was increased significantly. An activity profile similar to TST was seen and the compounds 2a and 2d did not give the expected results. Both TST and FST are very commonly used behavioral distress models employing rodent to predict the antidepressant potential of drugs and molecules. These models are based on the concept that mice when hanged by tails or forced to swim initially try to perform the escape behavior activity and when they experience helplessness they exhibit the immobile posture. The immobility behavior has been accepted to be associated to depression and the reduction in the immobility is used as a measure of antidepressant effect^33,34.

Though the synthesized compounds were soluble in water instead of organic solvent the excellent antidepressant like effect exhibited by the compounds may be attributed to the antioxidant property of gallic acid and also it may be possible that the compounds were able to target serotonin transporter or inhibit the enzyme MAO-A in the neurons. A further investigation of the possible mechanism by which the gallic acid analogues elicit their antidepressant effect is planned to be studied in the next part of the study.

CONCLUSION:

The objective of the present work was to design and synthesize newer gallic acid derivatives that would be able to treat depression. Owing to the antioxidant potential of gallic acid it was foreseen that the compounds would have lesser adverse effects as compared to the already available antidepressants. The results led to the conclusion that gallic acid conjugated to piperazine or piperidine could be a potential lead for designing of novel antidepressant molecules that may elicit their action by virtue of their capability to combat oxidative stress. A further investigation of the possible mechanism by which the gallic acid analogues elicit their antidepressant effect is planned to be studied in the next part of the study.

CONFLICT OF INTERESTS:

The authors declare no conflict of interests.

REFERENCES:

1. National Institute of Mental Health, 2016. Depression Basics. NIH Publication No. 19-MH-8079

2. Rajagopal K, Leethyal V. Prevalence of postnatal depression among postnatal mothers. Asian Journal of Nursing Education and Research. 2012;2(1):33-36.

3. Udapi G. A Study to Assess the Knowledge Regarding Depression and Suicidal Behavior in Patients among the Nurses Working at the Selected Tertiary Care Hospital in the Belgaum City, Karnataka. Asian Journal of Nursing Education and Research. 2014; 4(3): 314-316.

4. https://www.mentalhealth.org.uk/a-to-z/d/depression; assessed on 08/04/2022

5. World Health Organization, 2017. “Depression: let’s talk” says WHO, as depression tops list of causes of ill health [press release]. https://www.who.int/news-room/detail/30-03-2017--depression-let-s-talk-says-who-as-depression-tops-list-of-causes-of-ill-health assessed on 08/04/2022

6. RhoadsJ, Murphy PJM. Nurses’ Clinical Consult to Psychopharmacology 2012, NY: Springer Publishing Company

7. Uppala PK, Murali Krishna B., Atchuta Kumar K, Ramji V. Experimental Evaluation of Antidepressant activity of Aqueous and Methanolic Leaf and Shoot Extracts of Ageratum conyzoides Linn in Mice. Asian Journal of Pharmaceutical Research. 2016; 6(3): 153-158.DOI: 10.5958/2231-5691.2016.00022.8

8. Jadhav SS, Salunke VR, Magdum CS. Daily Consumption of Antioxidants:-Prevention of Disease is better than Cure. Asian Journal of Pharmaceutical Research. 2013;3(1): 33-39.

9. Xu Y, WangC, Klabni JJ, O’Donnell JM. Novel Therapeutic Targets in Depression and Anxiety: Antioxidants as a Candidate Treatment. Current Neuropharmacology. 2014;12: 108-119. doi: 10.2174/1570159X11666131120231448

10. Gautam M, Agrawal M, Gautam M, Sharma P, Gautam AS, Gautam S. Role of antioxidants in generalized anxiety disorder and depression. Indian Journal of Psychiatry. 2012;54(3): 244-247. doi: 10.4103/0019-5545.102424.

11. Mishra N, Jain P, Mishra B.Derivatization of Gallic Acid with amino acids for accentuation of its antioxidant potential. Journal of Pharmacology and Biomedicine. 2017;1(3): 94-102

12. Fatahala SS, Nofal S, Mahmoud E, Abd El-Hameed RH.Pyrrolopyrazoles: Synthesis, evaluation and pharmacological screening as antidepressant agents. Medicinal Chemistry. 2019;15(8): 911-922. doi: 10.2174/1573406414666181108090321

13. Wen H, Qin W, Yang G, Guo Y.Design and synthesis of arylamidine derivatives as serotonin/norepinephrine dual reuptake inhibitors. Molecules. 2019; 24: 497-511. doi: 10.3390/molecules24030497

14. Wang S, Liu H, Wang X, Lei K, Li G, Quan Z. Synthesis and evaluation of antidepressant activities of 5-Aryl-4,5-dihydrotetrazolo [1,5-a]thieno[2,3-e]pyridine derivatives. Molecules. 2019;24: 1857-1869. doi: 10.3390/molecules24101857

15. Gu Z-S, Zhou A-N, Xiao Y, Zhang Q-W, Li J-Q.Synthesis and antidepressant-like activity of novel aralkylpiperazine derivatives targeting SSRI/5-HT1A/5-HT7. European Journal of Medicinal Chemistry. 2018; 144: 701-715. doi: 10.1016/j.ejmech.2017.12.063.

16. Wang J, Song Q, Xu A, Bao Y, Xu Y, Zhu Q.Design, synthesis and biological evaluation of aminobenzyloxyarylamide derivatives as selective κ opioid receptor antagonists. European Journal of Medicinal Chemistry. 2017; DOI: 10.1016/j.ejmech.2017.02.029

17. Song MX, Rao BQ, Cheng BB, Wu Y, Zeng H, Luo Y-G, Deng X-Q.Design, synthesis and evaluation of the antidepressant and anticonvulsant activities of triazole-containing benzo[d]oxazoles. CNS and Neurological Disorders - Drug Targets. 2017;: 187-198. doi: 10.2174/1871527315666160822112501

18. KuamwatRS, Mruthunjaya K, Gupta MK. Hepatoprotective effect of Gallic acid and Gallic acid Phytosome against Carbon Tetrachloride induced damage in albino rats. Research Journal of Pharmacy and Technology. 2012; 5(5): 677-681

19. Özkay ÜD, Kaya C, Çevik UA, Can OD. Synthesis and antidepressant activity proﬁle of some novel benzothiazole derivatives. Molecules. 2017; 22: 1490-1503. doi: 10.3390/molecules22091490

20. https://www.oc-praktikum.de/nop/en/instructions/pdf/2013_en.pdf; assessed on 11/02/2022

21. Guan LP, Zhao D-H, Chang Y,Wen Z-S, Tang L-M, Huang F-F.Synthesis of 2,4-dihydroxychalcone derivatives as potential antidepressant eﬀect. Drug Research. 2013; 63: 46-51. doi: 10.1055/s-0032-1333229.

22. de Oliveira KN, Costa P, Santin JR, Mazzambani L, Burger C, Mora C, Nunes RJ, et al.Synthesis and antidepressant-like activity evaluation of sulphonamides and sulphonyl-hydrazones. Bioorganic and Medicinal Chemistry. 2012;19: 4295-4306. doi: 10.1016/j.bmc.2011.05.056.

23. Lone SH, Shakeel-U-Rehman, Bhat KA.Synthesis of Gallic-Acid-1-phenyl-1H-[1,2,3]triazol-4-yl methyl esters as effective antioxidants. Drug Research. 2016;http://dx.doi.org/10.1055/s-0042-118860

24. da Silva MM, Comin M, Duarte TS, Foglio MA, De Carvalho JE, Vieira MDC, Formagio ASN.Synthesis, antiproliferative activity and molecular properties predictions of galloyl derivatives. Molecules.2015; 20: 5360-5373. doi: 10.3390/molecules20045360

25. Hejchman E, Taciak P, Kowalski S, Maciejewska D, Czajkowska A, Borowska J, Sladowski D, et al.Synthesis and anticancer activity of 7-hydroxycoumarinyl gallates. Pharmacological Reports. 2014;http://dx.doi.org/10.1016/j.pharep.2014.09.008

26. Fei X, Je I-G, Shin T-Y, Kim S-H, Seo S-Y.Synthesis of gallic acid analogs as histamine and pro-inﬂammatory cytokine inhibitors for treatment of mast cell-mediated allergic inﬂammation. Molecules. 2017; 22: 898-911. doi: 10.3390/molecules22060898.

27. Patel RC, Patel CN, PanigrahiBB, Bhaskar VH.Synthesis and SAR Study of Some New BenzhydrylPiperazine Sulfonamide and Carboxamide as Antimicrobial Agents. Asian Journal of Research in Chemistry. 2009;2(4): 448-451.

28. Khobragade CN, Bobade RG,Manwar AV. (2010) Synthesis and Antioxidant Activity of Some Flavone Derivatives. Asian Journal of Research in Chemistry. 2010; 3(1): 139-141.

29. Divekar K, Hardik J, Priyadarshini SB. Synthesis and Biological Evaluation of Some Novel Pyrimidine Derivatives. Asian Journal of Research in Chemistry. 2011; 4(1): 64-67.

30. Uddandam A, Sreerama UR, Aruna Devi M, Rajitha G. Synthesis and Evaluation of Substituted Imidazolones for Antibacterial and Antioxidant Activities. Asian Journal of Research in Chemistry, 2011; 4(2): 257-259

31. Pereira MM, de Morais H, dos Santos Silva E, Corso CR, Adami ER, Acco A, Zanoveli JM.The antioxidant gallic acid induces anxiolytic, but not antidepressant-like effect, in streptozotocin-induced diabetes. Metabolic Brain Disease. 2018; https://doi.org/10.1007/s11011-018-0264-9

32. Oliveira CE, Sari MH, Zborowski VA,Araujo PSO, Nogueira CW, Zeni G. p,p′-Methoxyl-diphenyldiselenide elicits an antidepressant-like effect in mice without discontinuation anxiety phenotype. Pharmacology Biochemistry and Behavior. 2017; 154: 31–38. https://doi.org/10.1016/j.pbb.2017.02.002

33. Cryan JF, MombereauC, Vassout A.The tail suspension test for assessing antidepressant activity: Review of pharmacological and genetic studies in mice. Neuroscience and Biobehavioral Reviews. 2005; 29: 571–625.doi: 10.1016/j.neubiorev.2005.03.009.

Received on 05.10.2022 Modified on 20.01.2023

Research J. Pharm. and Tech 2023; 16(11):5051-5057.

DOI: 10.52711/0974-360X.2023.00818

Compound	Structure	Yield (%)	Solubility	Melting Point (°C)	Retention factor (R_f value)
2a		74	Soluble in water and DMSO	252-254	0.62
2b		71	Soluble in water and DMSO	230-232	0.73
2c		79	Soluble in water and DMSO	237-239	0.57
2d		81	Soluble in water and DMSO	211-213	0.69
2e		80	Soluble in water and DMSO	223-225	0.61