In-vitro Acetylcholine Esterase Inhibition activity of Chalcones with Phenothiazine Moiety Akilandapuram Velusamy Saranya and Subban Ravi* Department of Chemistry, Karpagam University, Coimbatore-21, Tamil Nadu, INDIA Available online at: www.isca.in Received 10th October 2012, revised 14th October 2012, accepted 16th October 2012 Abstract A series of chalcones (3a-g) were synthesized by Claisen-Schmidt condensation between 2-acetyl phenothizine (1) and aromatic aldehydes (2a-g). All the synthesized chalcones were characterized by their spectral data (UV, IR, 1H-NMR, 13C-NMR, MS and elemental analyses). Acetylcholine esterase inhibition activity was carried out for all the synthesized chalcones which showed an IC50 value between 1.0 to 6.4µg/ml and indicated a comparable inhibitory potency, when compared to the control neostigmine with IC50 value of 8.3µg/ml. Keywords: Chalcones, IR, NMR, MS technique, acetylcholine esterase inhibition, alzheimer's disease. Introduction For a quarter of a century, the pathogenesis of Alzheimer's disease (AD) has been linked to a deficiency in the brain neurotransmitter acetylcholine. This is based on cholinergic system abnormalities with intellectual impairment1. The cholinergic dysfunction, a role for ß-amyloid deposition, oxidative stress and inflammation has been investigated in the aetiology of AD and currently trials are underway to test modifying agents. Nevertheless, attempts to treat acetylcholine deficiency in the brain of affected individuals were first carried in form of acetylcholine esterase inhibitors (AChEIs) and however three agents' donepezil, rivastigmine and galantamine are licensed in UK. The main use of AChEIs resulted in stabilization of cognitive decline, improvement in behavioural and psychological symptoms of dementia2. The development of acetylcholine esterase (AChEI) inhibitor drugs has followed the finding that cholinergic pathways in cerebral cortex and basal forebrain are compromised in Alzheimer's disease 3 and the resultant cholinergic deficit contributes to the cognitive impairment of these patients 4. An unfortunate result of rapid rise in geriatric populations worldwide is the increasing prevalence of age related cognitive disorders5,6. Chalcones, one of the major classes of natural products with widespread occurrence in fruits, vegetables, spices, tea and soy-based food stuffs, have been recently the subject of extensive investigations due to their interesting pharmacological activities. Chemically they consist of open chain flavonoids in which the two aromatic rings are joined by three carbons a, ß-unsaturated carbonyl system7. The compounds with the backbone of chalcones have been reported to possess various biological activities such as antimicrobial, anti-inflammatory, analgesic, antiplatelet, antiulcerative, antimalarial, anticancer, antiviral, antileishmanial, antioxidant, antitubercular, antihyperglycemic, immunomodulator, inhibition of chemical mediators release, inhibition of leukotriene B4, inhibition of tyrosine, inhibition of aldose reductase activities8. From a chemical point of view an important feature of chalcones and their heteroanalogs is the ability to act as activated unsaturated systems in conjugate addition reactions of carbanions in presence of base catalysts9. 1, 3-diarylpropenones (Chalocnes) have been popular substrates for the generation of variety of heterocyclic, carbocyclic and flavonoids10. In the present work we report the reaction of 2-acetyl phenothiazine with different aromatic aldehydes to form chalcones (3a-g). Many reports were available for the preparation of chalcones11-14 but acetylcholine esterase activity was not reported for chalcones in literature. Molecules that possess sulfur atoms are universal and crucial in living organisms15. Phenothiazines were important kind compounds containing one sulfur and one nitrogen atom. This prompted us to synthesize chalcones containing phenothiazine moiety and to carry out the acetylcholine esterase inhibitor activity. Material and Methods Chemistry: Melting points (uncorrected) were determined using a Guna melting point apparatus. UV spectra were obtained UV 2460 shimadzu spectrophotometer. IR spectra were carried out on a Perkin-Elmer 1650 spectrophotometer. NMR spectra were recorded in CDCl3 on a Bruker AM 400 MHz spectrometer, using residual CHCl3 and TMS as an internal standard. Mass spectra were recorded on a VG-70-S instrument. Elemental analysis was carried out in a Perkin Elmer 240C model instrument. Column chromatography and TLC were carried out on silica gel 60 -120 mesh and silicagel 'G' respectively. All the chemicals are of AR grade. General procedure for the preparartion of compounds 3a-g: 2-acetyl phenothiazine 1 (0.01 mol) was dissolved in 25 ml methanol and different benzaldehyde derivatives(2a-g) (0. 01 mol) were added, heated for 6 hrs with constant stirring in a magnetic stirrer and a catalytic amount of NaOH was added in drops. The reaction was poured into ice-cold water, neutralized with con.HCl and left over night in a refrigerator. The precipitate was filtered, dried and purity of the compound was checked by TLC using chloroform as the solvent. The compound was purified by column chromatography using silica gel (60-120 mesh). (E)-3-(4-methoxyphenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one 3a: Yield 66%; m.p.: 202oC; UV ? max: 399.50, 263.50; IR (KBr) cm-1: 3344, 1666, 1590; 1H-NMR (400MHz CDCl3) d: 7.11 (d, 1H, J=1.6Hz, H-1'), 7.38 (dd, 1H, J=8Hz, 1.6Hz, H-3'), 6.88 (d, 1H, J=8Hz, H-4'), 6.98 (d, 1H, J=8Hz, H-5'), 6.76 (td, 1H, J=8, 1.6Hz, H-6'), 6.93 (dd, 1H, J=8, 1.6Hz, H-7'), 6.48 (dd, 1H, J=8, 1.6Hz, H-8'), 7.68 (d, 1H, J=16Hz, H-2), 7.23 (d, 1H, J=16Hz, H-3), 7.51 (d, 2H, J=8Hz, H-2'', 6''), 6.85 (m, 2H, H-3'', 5''), 3.79 (s, 3H, 0CH3), 8.79 (s, 1H, NH); 13C-NMR: 119.32, 137.69, 126.34), 127.64, 126.73, 114.64, 128.20, 114.46 , 140.80, 113.46, 144.62, 124.20, 189.50, 122.81, 144.23, 127.78, 130.24, 114.46, 161.00, 114.46, 130.24, 55.42; MS [M+1]+ = 361; Anal. Calcd. for C22H12O2NS: C, 77.64%; H, 3.52%; N, 4.11%; Found: C, 77.58%; H, 3.67%; N, 4.23%. (E)-3-(4-methoxyphenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one 3b: Yield 64%; m.p.: 168oC; UV ? max: 438.00, 312.00, 247.00; IR (KBr) cm-1: 3350, 1650, 1590; 1H-NMR (400MHz CDCl3) d: 7.31 (d, 1H, J=1.6Hz, H-1'), 7.46 (m, 1H, H-3'), 7.09 (d, 1H, J=7.8Hz, H-4'), 6.91 (d, 1H, J=7.8Hz, H-5'), 6.77 (t, 1H, J=7.8Hz, H-6'), 6.99 (td, 1H, J=7.8Hz, H-7'), 6.67 (d, 1H, J=7.8Hz, H-8'), 7.46 (m, 2H, H-3'',5''), 7.87 (m, 2H, H-2'', 6''), 7.63 (d, 1H, H-4''), 7.77 (d, 2H, J=16Hz, H-2,3), 8.89 (s, 1H, NH). 13C-NMR: 121.90, 141.10, 126.24, 127.94, 126.11, 114.57, 128.75, 112.92, 142.82, 115.19, 143.72, 123.58, 188.02, 122.52, 143.72, 136.85, 122.08, 128.90, 130.57, 134.66, 126.40. MS [M+1]+ = 329; Anal. Calcd. for C21H15ONS: C, 76.59%; H, 4.55%; N, 4.25%; Found: C, 76.67%; H, 4.44%; N, 4.34%. (E)-3-(4-chlorophenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one 3c: Yield 65%; m.p.: 212oC. UV ? max: 342.00, 282.00; IR (KBr) cm-1: 3380, 1650, 1590; 1H-NMR (400MHz CDCl3) d: 7.30 (d, 1H, J=1.6Hz H-2'), 7.63 (dd, 1H, J=8,1.6Hz, H-3'), 7.09 (d, 1H, J=8Hz, H-4'), 6.93 (dd, 1H, J=8Hz, H-5'), 6.78 (dt, 1H, J=8, 1.6Hz, H-6'), 7.00 (td, 1H, J=8, 1.6Hz, H-7'), 6.52 (dd, 1H, J=8, 1.6Hz, H-8'), 7.71 (d, 1H, J=16Hz, H-2), 7.83 (d, 1H, J=16Hz, H-2), 7.91 (d, 2H, J=8Hz, H-2'',6''), 7.53 (d, 2H, H-3'',5''), 8.79 (s, 1H, NH); 13C-NMR: 115.16, 141.07, 126.34, 127.98, 126.10, 114.58, 128.94, 112.86, 136.75, 113.75, 142.13, 122.09, 187.92, 122.09, 142.24, 135.04, 127.98, 128.94, 133.65. MS [M+1]+ = 362; Anal. Calcd. for C21H14ONSCl: C, 69.34%; H, 3.85%; N, 3.85%; Found: C, 69.29%; H, 3.80%; N, 3.91%. (E)-1-(10H-phenothiazin-2-yl)-3-p-tolylprop-2-en-1-one 3d: Yield 62%; m.p.: 170oC. UV ? max: 432.50, 315.50, 248.50; IR (KBr) cm-1: 3380, 1620, 1590; 1H-NMR (400MHz CDCl3) d: 7.30 (d, 1H, J=1.6Hz, H-1'), 7.61 (dd,1H, J=8, 1.6Hz, H-3'), 7.08 (d, 1H, J=8Hz, H-4'), 6.91 (dd, 1H, J=8, 1.6Hz, H-5'), 6.76 (td, 1H, J=8, 1.6Hz, H-6'), 6.92 (td, 1H, J=8, 1.6Hz, H-7'), 6.65 (dd, 1H, J=8, 1.6Hz, H-8'), 7.71 (d, 1H, J=16Hz, H-2), 7.75 (d, 1H, J=16Hz, H-3), 7.71 (d, 2H, H-2'',6''), 7.28 (d, 1H, J=8Hz, H-3'',5''), 2.35 (s, 3H, CH3), 8.77 (s, 1H, NH); 13C-NMR: 115.23, 141.12, 126.23, 127.98, 126.08, 114.57, 128.76, 112.93, 136.97, 123.44, 143.81, 122.41, 187.98, 122.07, 142.11, 131.93, 129.53, 131.93, 140.66, 21.05. MS [M+1]+ = 345; Anal. Calcd. for C22H17ONS: C, 76.96%; H, 4.95%; N, 4.07%; Found: C, 76.93%; H, 4.84%; N, 4.23%. (E)-3-(3-nitrophenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one 3e: Yield 61%; m.p.: 198oC.; UV ? max: 432.50, 315.50, 248.50; IR (KBr) cm-1: 3336, 1658, 1593; 1H-NMR (400MHz CDCl3) d: 7.31 (d, 1H, J=1.6Hz, H-1'), 7.68 (dd, 1H, J=8, 1.6Hz, H-3'), 7.08 (d, 1H, J=8Hz, H-4'), 6.93 (dd, 1H, J=8, 1.6Hz, H-5'), 6.79 (td, 1H, J=8, 1.6Hz, H-6'), 6.99 (td, 1H, J=8, 1.6Hz, H-7'), 6.67 (d, J=8, 1.6Hz, 1H, H-8'), 7.80 (d, 1H, J=16Hz, H-2), 8.02 (d, 1H, J=16Hz, H-3), 8.74 (m, 1H, H-2''), 8.26 (dd, 1H, J=8, 1.6Hz, H-4''), 7.74 (t, 1H, J=8Hz, H-5''), 8.28 (m, 1H, H-6''), 8.75 (s, 1H, NH); 13C-NMR: 112.93, 141.03, 126.21, 122.82, 126.07, 114.58, 127.97, 112.86, 136.57, 124.62, 141.11, 124.57, 187.85, 124.04, 142.12, 136.53, 148.39, 130.31, 134.92, 122.08, 122.82; MS [M+1]+ = 374; Anal. Calcd. for C21H14O3N2S: C, 67.38%; H, 3.74%; N, 7.48%; Found: C, 67.48%; H, 3.65%; N, 7.54%. (E)-3-(4-bromophenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one 3f: Yield 65%; m.p.: 208oC. UV ? max: 448.50, 319.50, 247.50; IR (KBr) cm-1: 3348, 1651, 1581; 1H-NMR (400MHz CDCl3) d: 7.30 (d, 1H, J=8Hz, H-1'), 7.61 (dd, 1H, J=8Hz, H-3'), 7.09 (d, 1H, J=8Hz, H-4'), 6.91(dd, 1H, J=8, 1.6Hz, H-5'), 6.77 (td, 1H, J=8, 1.6Hz, H-6'), 7.00 (td, 1H, J=8, 1.6Hz, H-7'), 6.68 (dd, 1H, J=8, 1.6Hz, H-8'), 7.67 (m, 1H, J=16Hz, H-2), 7.83 (m, 1H, J=16Hz, H-3), 7.80 (m, 2H, H-2'',6''), 7.67 (m, 2H, H-3'',5''), 8.78 (s, 1H, NH); 13C-NMR: 115.17, 141.06, 126.23, 127.97, 126.10, 114.58, 123.90, 112.87, 136.75, 123.76, 142.13, 122.70, 187.94, 122.09, 142.32, 133.97, 130.64, 131.87, 122.59; MS [M+1]+ = 407; Anal. Calcd. for C21H14ONSBr: C, 61.78%; H, 3.43%; N, 3.43%; Found: C, 61.84%; H, 3.51%; N, 3.45%. (E)-4-(3-oxo-3-(10H-phenothiazin-2-yl)prop-1-enyl)benzaldehyde 3g: Yield 64%; m.p.: 191oC; UV ? max: 455.00, 304.50, 249.50; IR (KBr) cm-1: 3344, 1666, 1590; 1H-NMR(400MHz CDCl3) d: 7.31 (d, 1H, J=1.6Hz, H-1'), 7.61 (dd, 1H, J=8, 1.6Hz, H-3'), 7.09 (d, 1H, J=8Hz, H-4'), 6.91 (dd, 1H, J=8, 1.6Hz, H-5'), 6.78 (td,1H, J=8, 1.6Hz, H-6'), 7.00 (td, 1H, J=8, 1.6Hz, H-7'), 6.67 (d, 1H, J=8, 1.6Hz, H-8'), 7.90 (m, 2H, H-2'',6''), 8.09 (m, 2H, H-3'',5''), 7.78 (d, 1H, J=16Hz, H-2), 7.91 (d, 2H, J=16Hz, H-3), 8.79 (s, 1H, NH), 10.05 (s, 1H, CHO); 13C-NMR: 115.14, 141.03, 126.23, 127.99, 126.12, 114.59, 124.02, 112.87 , 136.99, 124.82, 142.16, 122.70, 187.94, 124.02, 142.00, 136.60, 129.85, 129.26, 140.30, 192.59; MS [M+1]+ = 357; Anal. Calcd. for C22H15O2NS: C, 73.39%; H, 4.20%; N, 3.91%; Found: C, 73.47%; H, 4.25%; N, 4.31%. In- vitro acetylcholine esterase inhibition activity: Acetylcholine esterase activity16 was carried out for all the synthesized compounds 3a-g as shown in table 1. Spectrophotometric assay was used to determine the inhibitory potential of the compounds against acetylcholine esterase enzyme isolated from red blood cells. Acetyl thiocholine iodide was used as a substrate. 2.81ml of phosphate buffer of pH 8 was taken in each test tube. The test sample solutions of different concentrations of 2µg, 4µg, 6µg, 8µg, 10µg were added and 30µl of enzyme were added. The mixture was allowed standing for 10min. The colouring reagent DTNB (dithiobisnitro benzoic acid) was added which produces the yellow anion of 5-thio-2-nitro benzoic acid and then substrate 30µl followed by incubation for 20 min. The absorbance was measured at 412nm. The percentage inhibition in enzyme activity can be calculated as follows: % inhibition = Absorbance (control) - Absorbance (test) / Absobance (control) × 100 Table-1 AChEI assay with IC50 value of the compounds 3a-g S.NoCompoundIC50 value in (µg/ml)13a4.023b6.433c1.043d2.953e2.863f3.373g4.08Neostigmine8.3 Results and Discussion Spectral values of chalcones 3a-g: Compounds (3a-g) were synthesized by the reaction between 2-acetyl phenothiazine with different aromatic aldehydes by Claisen- Schmidt condensation reaction as shown in scheme 1. For the compounds 3a-g, IR spectra showed characteristic absorption bands to show the presence of carbonyl group at 1651cm-1, C=C at 1600cm-1, NH stretching at 3336.85 cm-1. For all the synthesized compounds, the signals for the aromatic carbons and protons were assigned using known effects of substituents, position, multiplicities and integral values. In 1H-NMR spectra for the compound (3a-g) H-2 and H-3 are found to be trans protons where d value appears between d 7.30 and 7.77 and the coupling constant J value is 16Hz. NH proton appeared as a singlet at d 8.79. In compound 3a, OCH3 proton appeared as a singlet in the range d 1.18 showing the presence of three protons, similarly in 3d, CH3 appeared at d 2.35. In 3g, the singlet at d 10.05 is due to CHO group and all the aromatic protons appeared between d 6.50-8.28. The 13C -NMR signals were assigned based on their positions and intensities. The 13C-NMR spectrum of chalcone were recorded in CDCl3 and spectral signals were in good agreement with the proposed structures; C-1 (i.e) C=O group shows the presence at d 187.92. In compound 3a, the methoxy carbon appeared at d 55.42 and in compound 3d, CH3 carbon appeared at d 21.05. For 3g, the aldehyde carbon appeared at d 192.59 and all the aromatic carbon or unsaturated C=C appeared between 100-160. Characteristic molecular ion peaks were observed in the mass spectra of the chalcone and shown in experimental section. In-vitro acetylcholine esterase inhibition activity: In the literature, the structure activity relation (SAR) of many nitrogen containing AChE inhibitors such as tacrine, physostigmine, benylamines, benzyl piperidine, benziooxazoles and huperzine A has been reported. All of them gave an overall conclusion that these drugs bind to acetylcholine esterase through the nitrogen containing heterocyclic part of the molecule. It was also reported that quarternary ammonium salts act as strong acetylcholine esterase inhibitors. In previous reports regarding the SAR of AChE inhibitors, it was concluded that the substitution in the benzene ring enhanced the activity of the molecule16. In present study, in-vitro acetylcholine esterase inhibition activity was carried out for all the synthesized chalcones from 3a-g as shown in table 1. However, the synthesized chalcone contains phenothiazine moiety with one nitrogen and sulphur are present in a heterocyclic moiety. The substitution on aromatic ring was found to markedly improve AChE activity. All the derivatives showed greater affinity and potency when compared to the control neostigmine. The potency of the molecules follows the order 3c > 3e > 3d > 3f > 3g, 3a > 3b which was based on IC50 value from 1.0 to 6.4µg/ml whereas for the control neostigmine IC50 value was found to be 8.3µg/ml. The most potent compound was 3c where the aromatic ring having the substituent Cl yielded excellent activity. However other electronegative groups like NO2, Cl, Br, and CHO substituted in aromatic ring enhanced the activity than the control. Compound 3a and 3d having methoxy and methyl substituent in aromatic and the unsubstituted benzene ring 3b also showed good activity but it was less when compared to the other molecule. Conclusion In conclusion, a series of chalcones (3a-g) were synthesized by Claisen-Schmidt condensation reaction. The in vitro acetylcholine esterase inhibition activity was evaluated for all synthesized compounds showed a good inhibitory potency with an IC50 value between 1.0 to 6.4µg/ml, when compared to the control neostigmine with IC50 value of 8.3µg/ml. Acknowledgements We are thankful to the Management, Vice-chacellor, Registrar and HOD, Dept. of chemistry, Karpagam University, Coimbatore, Tamil Nadu, India for providing the facilities to carry out the research work. And also I'm thankful to my beloved parents (Father: - A.D. Velusamy, Mother: - V. Selvam) for their encouragement and I thank my friend (S. Venkatachalapathi) for his great support in all my efforts. Scheme-1 Synthesis of Chalcones 3a-g References 1. 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Durre Shahwar, Muhammad Asam Raza and Tania Khan, Synthesis and evaluation of enzyme inhibitory potential of some derivatives of scopolamine, Turk J Chem., 36, 257-266 (2012) Research Journal of Chemical Sciences _______________________________________________________ ISSN 2231-606X Vol. 1(5), 1-7, Aug. (2011) Res.J.Chem.Sci. International Science Congress Association 2 Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 1(12), 40-43, December (2012) Res.J.Recent Sci. International Science Congress Association 40 Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502 Vol. 1(12), 40-43, December (2012) Res. J. Recent Sci. International Science Congress Association 42 Research Journal of Chemical Sciences________________________________________________ ISSN 2231-606X Vol. 1(5), 1-7, Aug. (2011) Res.J.Chem.Sci International Science Congress Association 5