WO2019207604A1 - Tacrine derivatives targeting nmda receptor, acetylcholine esterase, butyryl choline and beta secretase activity - Google Patents

Abstract

The present invention underlying this instant application prepares a library based on tacrine, and discloses and claims derivatives of a heterocyclic compound tacrine with the basic molecular formula C₁₃H₁₄NNH₂, and the structural formula of (Formula I):Wherein either position 2 or position 6 or both may be substituted by R1 and R2respectively, wherein R1 and R2 is selected from hydrogen, halogen or halo derivatives, phenyl, substituted benzene, benzene derivatives, heterocyclic derivates of nitrogen (N₂), furan derivatives, amide or amide derivatives, purine or pyrimidine, purine derivatives or pyrazole or pyrazole derivatives, and ester derivatives, either standalone or any combination thereof.

Description

TACRINE DERIVATIVES TARGETING NMDA RECEPTOR, ACETYLCHOLINE ESTERASE, BUTYRYL

CHOLINE AND BETA SECRETASE ACTIVITY

FIELD OF INVENTION

[0001] The present disclosure relates to novel Tacrine derivatives that target NMDA receptor, Acetylcholinesterase, Butyrylcholinesterase and Beta secretase activities.

BACKGROUND AND PRIOR ART

[0002] N-methyl-D-aspartate receptor (NMDAR) is a ligand gated ion channel, involved in several physiological and pathophysiological processes in the central nervous system. Excessive activation of NMDAR causes neuronal death by a cellular mechanism called excito toxicity. Excitotoxicity is a common mechanism of cell death in many neurological diseases. Variations in NMDAR activity can also lead to neuropsychiatric diseases. NMDA receptor activity modulators (antagonists and agonists) can be used as potential therapeutic agents in such diseases. However many NMDAR antagonists currently in clinical use, either have insufficient efficacy or have undesirable side effects. Hence new and better antagonists are necessary for treating neurological diseases.

[0003] Tacrine has been used as a drug to treat Alzheimer’s disease due to its ability to inhibit acetylcholinesterase (AChE) (Kurz A, 1998, J. Neural Transm. Suppl. Vol. 54, pp295-299) at nanomolar concentrations in vitro. Tacrine is also reported to be an antagonist of NMDAR (Hershkowitz, et al, 1991, Molecular Pharmacology, Vol. 39.5, pp592-598; Vorobjev, et al, 1994, European Journal of Pharmacology, Vol. 253.1 -ppl-8). However, it is required at high concentrations, to cause significant inhibition of NMDAR activity. The reported half maximal inhibitory concentration values (IC50) of Tacrine are 193 mM in cultured hippocampal neurons (Hershkowitz, et al, 1991, Molecular Pharmacology, Vol. 39.5, pp592-598) and 25+6 pM in hippocampal slice preparations (Vorobjev, et al, 1994, European journal of Pharmacology, Vol. 253.1 ppl-8). Hence, at the therapeutic doses of Tacrine that are enough to achieve AChE inhibition, inhibition of NMDAR would be limited. Because of this Tacrine could not be used as NMDAR inhibitor for neurological conditions such as stroke and traumatic brain injury as NMDAR inhibitor. In addition, the therapeutic dose of Tacrine has been shown to have side effects such as hepatotoxicity (Gracon, et al, 1998, Alzheimer Dis Assoc Disord., Vol. 12, rr93-101). Hence it was necessary to improve the inhibitory potency of Tacrine so that dosage could be reduced and thus hepatotoxicity could be brought within safety limits. The present invention meets this long-felt need.

OBJECTS OF THE INVENTION

[0004] An object of the present invention is to provide novel Tacrine derivatives that target NMDA receptor (NMDAR), Acetylcholinesterase (AChE), Butyrylcholinesterase (BChE) and Beta secretase activities.

[0005] Another object of the present invention is to provide a modulator for both glutamatergic and cholinergic receptor signaling pathways in the nervous system.

[0006] Yet another object of the present invention is to provide Tacrine derivatives which have NMDAR inhibitory activity with IC50 values in the range 0.20 mM to 50 pM.

[0007] A further object of the present invention is to provide Tacrine derivatives which have NMDAR inhibitory activity with maximal non cytotoxic concentration values.

[0008] A further object of the present invention is to provide Tacrine derivatives which improve memory and learning in a setting of monosodium glutamate induced neural damage. [0009] A still further object of the present invention is to provide Tacrine derivatives which show significantly lower hepatotoxicity as compared to parent Tacrine molecule.

SUMMARY OF THE INVENTION

[0010] The present invention underlying this instant application prepares a library based on tacrine, and discloses and claims derivatives of a heterocyclic compound tacrine with the basic molecular formula C13H14NNH2, and the structural formula of:

[0011] Wherein either position 2 or position 6 or both may be substituted by Rl and R2 respectively, wherein Rl and R2 is selected from hydrogen, halogen or halo derivatives, phenyl, substituted benzene, benzene derivatives, heterocyclic derivates of nitrogen (N₂), furan derivatives, amide or amide derivatives, purine or pyrimidine, purine derivatives or pyrazole or pyrazole derivatives, and ester derivatives, either standalone or any combination thereof.

[0012] The derivatives are selected from the group consisting of 6-bromo-l,2,3,4- tetrahydroacridin-9-amine designated as compound 118; 6-(l-methyl-lH-pyrazol- 4-yl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 201; 6-

(pyrimidin-5-yl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 203; 6-(lH-pyrazol-3-yl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 204; 4-(9-amino-5,6,7,8-tetrahydroacridin-3-yl)benzonitrile designated as compound 205; 6-(4-(trifluoromethoxy)phenyl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 206; 6-(2-methylpyrimidin-5-yl)-l, 2,3,4- tetrahydroacridin-9-amine designated as compound 207; 6-(2-fluorophenyl)-

1.2.3.4-tetrahydroacridin-9-amine designated as compound 208; 6-(4- fluorophenyl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 209; 6- (4-(methylthio)phenyl)- 1 ,2,3 ,4-tetrahydroacridin-9-amine designated as compound 210; 6-(4-(trifluoromethyl)phenyl)- 1 ,2,3 ,4-tetrahydroacridin-9-amine designated as compound 211; 6-(furan-3-yl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 212; 6-phenyl- 1,2, 3, 4-tetrahydroacridin-9-amine designated as compound 213; 6-(pyridin-3-yl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 214; Ethyl-9-amino-6-bromo- 1,2,3, 4-tetrahydroacridine- 2-carboxylate designated as compound 05; Ethyl-9-amino-6-(l-methyl-lH- pyrazol-4-yl)-l,2,3,4-tetrahydroacridine-2-carboxylate designated as compound 10; 9-amino-N-methyl-6-( l-methyl-lH-pyrazol-4-yl)- 1,2,3, 4-tetrahydroacridine- 2-carboxamide designated as compound 14; Ethyl-9-amino-6-(4-fluorophenyl)-

1.2.3.4-tetrahydroacridine-2-carboxylate designated as compound 107.

[0013] Further, a process of synthesis of library derivatives of a heterocyclic compound tacrine is disclosed comprising the steps of allowing reaction of the tacrine or tacrine derivatives with appropriate reagent in presence of sodium carbonate and palladium-tetrakis (triphenylphosphine), (Pd (PPh₃)₄) in presence of a mixture of l,4-dioxane and water in 8:2 to 9: 1 ratio at a definite temperature range from 50°C to l20°C, for a period from 1 to 12 hours, or allowing the similar reaction in presence of methylamine (CH3NH2), boron trifluoride (BF₃), methanol (CH₃OH), Toluene, diethyl ether at a definite temperature for a period.

[0014] Furthermore, a method for inhibiting the activity of NMDAR expressed in cell culture or for inhibiting excitotoxic cell death in primary neuronal culture by treating with an effective amount of any one of the derivatives is disclosed. [0015] Furthermore, a method for inhibiting the enzymes, acetylcholine esterase, butyryl choline esterase (BChE) or beta secretase (BACE-l) by treating with an effective amount of any one of the derivatives is disclosed.

[0016] Still further, a method for treating one or more conditions associated calcium mediated excitotoxicity has been disclosed by administering an effective amount of any one of the derivatives disclosed.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0017] The illustrated embodiments of the subject matter will be best understood by reference to the drawings, wherein like parts are designated by like numerals throughout. The following description is intended only by way of example, and simply illustrates certain selected embodiments of devices, systems, and processes that are consistent with the subject matter as claimed herein, wherein:

Fig 1: Representative diagram showing the binding mode of Tacrine at the active site of hAChE and NMDAR along with its sites of modification wherein (a) illustrates the binding mode of Tacrine to the active site of hAChE, (b) illustrates the binding mode of Tacrine to the active site of NMDAR and (c) illustrates sites of modification on the Tacrine moiety marked as Rl and R2.

Fig 2: Representative diagram showing the chemical structures of all the Tacrine derivatives developed along with their designated compound number.

Fig 3: Represents a graphical chart showing the percentage of inhibition of NMDAR activity by the Tacrine derivatives at three concentrations- 20, 50 and IOOmM.

Fig 4: Represents a graphical chart showing the IC50 values of the Tacrine derivatives towards NMDAR. Fig 5: Represents a graphical chart showing the IC50 values of the Tacrine derivatives towards AChE.

Fig 6: Represents a graphical chart showing the IC50 values of the Tacrine derivatives towards butyryl choline esterase (BChE).

Fig 7 : Represents a graphical chart showing the residual activity of beta secretase- 1 (BACE-l) on treatment with the Tacrine derivatives.

Fig 8: Represents a graphical chart showing the percent of viable cells after treatment of HepG2 cell lines with the Tacrine derivatives for 24 hours, at concentrations 10, 50, 100 and 300 mM.

Fig 9: Represents a graphical chart showing the amount of cell death upon glutamate treatment of primary cortical neurons in presence of Tacrine derivatives measured in terms of Glucose 6 phosphate dehydrogenase (G6PD) activity in the culture supernatant.

Fig 10: Represents a graphical chart showing percent cell viability after excitotoxicity treatment of cells with glutamate for 3 hours in presence of Tacrine derivatives by MTT assay.

Fig 11: Represents a panel of fluorescent microscopy images of primary cortical neurons treated with or without glutamate to test its neurotoxicity in presence of NMDAR inhibitor MK-801 or Tacrine derivatives or DMSO alone.

Fig 12: Represents a graphical chart showing the amount of apoptosis upon glutamate treatment in rat primary cortical neurons in presence of Tacrine derivatives, as detected by DAPI staining.

Fig 13: Represents a graphical chart showing the effect of the Tacrine derivatives on neuronal integrity, upon glutamate treatment by detection of MAP2 positive cells. Fig 14: Represents graphical charts showing parameters of behavioral studies in rats using Morris Water Maze in a mono sodium glutamate (MSG) induced cognitive impairment model in presence of Tacrine derivatives. Escape latency to reach the platform has been given from Day 1 to Day 5. Fig 15: Represents a graphical chart showing the effect of 2 and 4 g MSG per Kg body weight compared to saline, on learning and memory tasks in rats as determined by the Morris Water maze.

Fig 16: Represents a graphical chart showing the effect of Tacrine derivative compound 201 on MSG (4g/ kg body wt) induced cognitive impairment from Day 1 to Day 5 in a rat model.

Fig 17: Representative images showing day wise trajectories of rats in each experimental group to find the platform in Morris Water maze experiment on treatment with glutamate and in presence of Tacrine derivative compound 201.

DETAILED DESCRIPTION OF THE INVENTION

[0018] At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a form of this invention. However, such a form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.

[0019] Throughout the description and claims of this specification, the phrases “comprise” and“contain” and variations of them mean“including but not limited to”, and are not intended to exclude other moieties, additives, components, integers or steps. Thus, the singular encompasses the plural unless the context otherwise requires. Wherever there is an indefinite article used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0020] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with an aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification including any accompanying claims, abstract and drawings or any parts thereof, or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0021] The reader's attention is directed to all papers and documents which are filed concurrently with or before this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. Post filing patents, original peer reviewed research paper may be published.

[0022] The following descriptions of embodiments and examples are offered by way of illustration and not by way of limitation.

[0023] Unless contraindicated or noted otherwise, throughout this specification, the terms“a” and“an” mean one or more, and the term“or” means and / or.

[0024] The present invention relates to novel Tacrine derivatives. New Tacrine derivatives were synthesized by comparing the binding modes of Tacrine against AChE and NMDAR. Design of these derivatives was done using structural bioinformatics-based approach. Derivatives of Tacrine were synthesized by substituting at the benzene ring (6th position, Rl) and/or at the cyclohexyl portion (2nd position, R2) of Tacrine nucleus (Fig. 1). Initially, the binding mode of Tacrine against hAChE and NMDAR were predicted using docking methods. The crystal structure of hAChE bound with Tacrine is not available in Protein Data Bank. Hence, using the binding information of huprine W (a ligand structurally like Tacrine) from its complex with hAChE (PDB ID: 4BDT), a grid was generated and docking of Tacrine was carried out. During iterative docking studies, the importance of two water molecules (HOH2023 and HOH2040) was identified, as they play an important role in the positioning of -NH2 group of Tacrine in the crystal structure of AChE in complex with Tacrine ((PDB ID: 1ACJ). As expected, the mode of binding of Tacrine in the docked structure well agreed with that in the crystal structure of Tacrine bound to TcAChE as well as huprine W bound to hAChE. From the docking studies, it was understood that, the binding of Tacrine is mainly stabilized by stacking with enzyme residues W86 and Y337. A hydrogen bond between the ring nitrogen of Tacrine nucleus and carbonyl oxygen of H447 was also observed. The binding mode of Tacrine is shown in Fig. 1.

[0025] Binding studies using radio-labeled ligands have demonstrated that Tacrine competitively displaces [3H]N-(l-[2-thienyl]cyclohexyl)-3, 4-piperidine from the phencyclidine (PCP) binding site indicating that the Tacrine and PCP sites may be identical or at least situated near one another (Albin et ah, 1988, Neurosci. Lett. Vol. 88, pp303-307). It was also reported that the block produced by Tacrine on NMDAR mediated response was thought to be similar to that produced by Mg²⁺ (Mayer et ah, 1984, Nature, Vol. 309, p.26l; Nowak et ah, 1984, Nature, Vol. 307, p.462; Johnson and Ascher, 1990, Biophysical journal, Vol. 57, pp.1085- 1090) and anesthetics like phencyclidine (PCP), ketamine and MK-801 (Honey et ah, 1985, Neuroscience letters, Vol. 61, pp.135-139; McDonald et ah, 1991, The Journal of physiology, Vol. 432, pp.483-508; Huettner and Bean, 1988, Proceedings of the National Academy of Sciences, Vol. 85, pp.1307- 1311). These agents are known to block NMDAR responses by binding to a site within the transmembrane region of the NMDAR channel and enabling a voltage dependent blockade.

[0026] With this information, the in-silico binding studies of Tacrine against NMDAR were performed by generating a grid on the transmembrane region of the channel. Grid was set by selecting centroid of the residues A644, V640, N616, L643, N615 and A639. Interaction of Tacrine with NMDAR was found to be through hydrogen bonds between N637 of A and C chain of GluNl (Since it is a heterotetramer) and nitrogen atoms of Tacrine. N637 is located at the tip of the pore loop, causing a narrow constriction of the channel pore of NMDAR. It was observed that the Tacrine moiety is arranged in a parallel fashion to the TM helices (Fig. 1).

[0027] It was then presumed that extending the Tacrine moiety by substituting groups at C2 or C6 positions (Figure lc) may improve its binding potential towards NMDAR due to its parallel arrangement in the pore. It was also reported that the substitutions at C3 and C6 improved the AChE inhibitory activity (Reddy et al, 2017, European Journal of Medicinal Chemistry, Vol. 139, pp. 367-377). On this basis, 19 compounds were synthesized. The synthesized compounds were named as 5, 8, 10, 13, 14, 16, 17, 107, 201, 203, 204, 205, 206, 208, 209, 210, 211, 212 and 214 (Fig. 2). Of these compounds, the structures of 5, 8, 13, 16, and 17 were already reported (Reddy et al, 2017, European Journal of Medicinal

Chemistry, Vol. 139, pp. 367-377). The AChE and BChE inhibitory properties of 13 and 17 are also reported in the paper.

Computational approaches - Proteins, ligands and computational tools used for the studies

[0028] Since AD (Alzheimer’s Disease) is multi-factorial in nature, the proteins that are critically involved in disease progression were taken as the drug targets for ligand screening. Three classes of protein molecules, namely, cholinesterases (AChE and BChE), NMDA receptor and BACE (beta secretase 1) were selected in the current study.

[0029] The complex of Huprine W, a ligand structurally like tacrine, with human AChE (4BDT) was taken as the template for the docking of tacrine and its derivatives against AChE. For docking studies with NMDAR, GluNl and GluN2B containing whole receptor structure (PDB ID: 5FXJ) was used.

[0030] All the computational studies were performed using Schrodinger suite. Programs like PyMOL, ChemSketch and Desmond were used for visualization, for drawing chemical structure of the ligands and for running molecular dynamics simulations respectively.

Ligand preparation and ADME prediction

[0031] LigPrep module, implemented in the Schrodinger package (Schrodinger, LLC, New York, USA), was used for ligand preparation for in silico studies. LigPrep produces energy-minimized, three-dimensional structure of the ligand, with various ionization states, tautomeric, stereo isomeric and ring conformations, for each input structure. Epik program was used to generate the tautomeric forms and ionization states of the derivatives. Optimized potential for liquid simulations (OPLS3) force field was used for energy minimization of the ligand structure (Harder et al., 2015, J chemical theory and computation, Vol. 12, pp.281-296). Multiple conformers per ligands were generated at pH 7.0 + 2 and were used for the study. The terms molecule/ligand/compound/inhibitor are used alternatively to represent the small molecules throughout the application.

[0032] Most of the drug candidates fail during clinical trials due to poor ADME (absorption, distribution, metabolism, and excretion) profile, with a concomitant loss of time and money. Early assessment of ADME properties of the compounds, at its development stage, can generate lead candidates that are more likely to exhibit satisfactory ADME profile during clinical trials, which in turn can dramatically reduce the amount of time and resources. For this purpose, the physico-chemical parameters of the selected compounds were assessed using QikProp program running in normal mode. The compound structure was neutralized during its preparation, since the QikProp program is unable to carry out the same. QikProp predicts about 45 physically significant descriptors and pharmacologically relevant properties that are useful in determining the bioavailability and pharmacodynamics of a compound. Some of the important parameters are molecular weight (MW) and volume, CNS activity, partition coefficient (Log P), number of hydrogen bond donors and acceptors (HBD and HBA), polar surface area (PSA), number of rotatable groups (rotor), number of N and O (N and O), BBB permeability (log BB), permeability across Mandin-Darby canine kidney (MDCK) and intestinal epithelial cells (Caco-2), solvent accessible surface area (SASA) and percentage of human oral absorption (% HOA).

Protein preparation, grid generation and docking

[0033] The selected protein molecules were prepared for docking studies using protein preparation wizard that performs addition of hydrogen atoms, specifying the bond orders, incorporation of protonation states for protein residues and optimization of the hydrogen bond network to repair overlapping hydrogens.

[0034] All the crystallographic water molecules were deleted prior to protein preparation. Protein preparation was performed using OPLS-3 force field by applying a short energy minimization with a RMSD cutoff of 0.30 A with respect to the crystal structure. Further, the grid was generated on the minimized structure by selecting the centroid of co-crystal ligands. For those proteins with no crystal ligands, the grid was prepared based on the centroid of its active site residues.

[0035] Extra precision docking (XP) method employed in the Schrodinger software was used for the docking of the ligands to AChE and NMDA receptor. XP docking results were analyzed based on visual inspection and Glide score (GScore) / docking score. GScore is calculated using the following equation. GScore = a*vdW+ b*Coul + Lipo + Hbond + Metal + BuryP + RotB + Site wherevdW denotes van der Waals energy,

Coul denotes Coulomb energy, Lipo denotes lipophilic contacts,

HBond indicates hydrogen-bonding,

Metal indicates metal-binding,

BuryP indicates penalty for buried polar groups,

RotB indicates penalty for freezing the rotatable bonds,

Site denotes polar interactions with the residues in the active site,

a (= 0.065) and b (= 0.130) are coefficients of vdW and Coul respectively.

Synthesis of Compounds

6-bromo-l,2,3,4-tetrahydroacridin-9-amine (118):

BFg Et₂O_s Toluene

CC

[0036] To a solution of 2-amino-4-bromo-benzonitrile (1) (5.0 g, 0.0253 mol, 1.0 eqiv) in anhydrous toluene was added BF₃.Et₂0 (3.76 mL, 0.0304 mol, 1.2 eqiv) slowly at room temperature. The reaction mixture was cooled to 0°C followed by the addition of cyclohexanone (2) (3.9 mL, 0.0379, 1.5 eqiv) and the reaction mixture was heated to l00°C for 4 h. After completion of reaction monitored by TLC, the reaction mixture was quenched with aq. NaOH solution upto pH= 10 and the reaction mixture was diluted with ethyl acetate. The organic layers were separated, dried over sodium sulphate and concentrated. The crude product was recrystallized from dichloromethane to obtained 6-bromo-l,2,3,4- tetrahydroacridin-9-amine (118) (5.8 g, 82%) as pale yellow solid.

*H NMR: DMSC 6 (400 MHz): d 8.13 (d, 1H, J= 8.8 Hz), 7.81 (s, 1H), 7.40-7.37 (m, 1H), 6.50 (bs, 2H), 2.82-2.79 (m, 2H), 2.55-2.52 (m, 2H), 1.82-

1.80 (m, 4H).LCMS: m/z 279.0 (M+2), Analysis calculated for Ci₃Hi₃BrN₂

6-(l-methyl-lH-pyrazol-4-yl)-l,2,3,4-tetrahydroacridin-9-amine (201):

[0037] To a solution of 6-bromo-l,2,3,4-tetrahydroacridin-9-amine (118)(l00 mg, 0.36 mmol, 1.0 eqiv) in l,4-dioxane (4 mL) and water (0.5 mL), l-methyl pyrazole 4-boronic ester (4) (90 mg, 0.434 mmol, 1.2 eqiv) and Na₂C0₃ (57 mg, 0.54 mmol, 1.5 eqiv) were added. The reaction mixture was degassed for 10 min and added Pd(PPh₃)₄ (41.0 mg, 0.036 mmol, 0.1 eqiv). The reaction mixture was heated to H0°C for 2h in sealed tube. The reaction mixture was filtered through celite and the filtrate was diluted with water (100 mL) and extracted with ethyl acetate (3x 25 mL). The organic layers separated were dried over anhydrous Na₂S0₄ and concentrated under vacuum. The crude product was purified by column chromatography using 10% methanol in dichloromethane as eluent to give 6-(l-methyl-lH-pyrazol-4-yl)-l,2,3,4-tetrahydroacridin-9-amine (201) (50 mg, 50% yield) as off white solid.

¾ NMR: NMR: MeOD (400 MHz):d 8.13-8.10 (m, 2H), 7.97 (s, 1H), 7.81 (s, 1H), 7.66 (dd, 2H, J = 8.4, 1.6 Hz), 3.98 (s, 3H), 2.97-2.94 (m, 2H), 2.65- 2.62 (m, 2H), 1.97-1.95 (m, 4H).LCMS: m/z 279.1 (M+l), Analysis calculated for C_l7H_l8N₄.

6-(pyrimidin-5-yl)-l,2,3,4-tetrahydroacridin-9-amine (203):

Synthesized following the same procedure as 201 ¾NMR: MeOD (400 MHz): d 9.18 (s, 3H), 8.22-8.20 (m, 1H), 8.00 (m, 1H), 7.69-7.66 (dd, 1H, J=8.8, 1.8 Hz), 2.97-2.94 (m, 2H), 2.66-2.63 (m, 2H), 1.96- 1.93 (m, 4H). LCMS: m/z 277.2 (M+l), Analysis calculated for C17H16N4.

6-(lH-pyrazol-3-yl)-l,2,3,4-tetrahydroacridin-9-amine (204):

Synthesized following the same procedure as 201

¾NMR: MeOD (400 MHz): d 8.10-8.05 (m, 2H), 7.82-7.79 (m, 1H), 7.71 (m, 2H), 6.8 l(d, 1H, J=2.0 Hz), 2.96-2.93 (m, 2H), 2.66-2.63 (m, 2H), 1.95-

1.90 (m, 4H).LCMS: m/z 265.2 (M+l), Analysis calculated for C16H16N4.

4-(9-amino-5,6,7,8-tetrahydroacridin-3-yl) benzonitrile (205):

Synthesized following the same procedure as 201

¾NMR: DMSO (300 MHz): d 8.29-8.26 (m, 2H), 8.19-8.16 (m, 1H), 8.00- 7.99 (m, 1H), 7.86-7.83 (m, 1H), 7.72-7.67 (m, 2H), 6.44 (bs, 2H), 2.90-2.80 (m, 2H), 2.72-2.68 (m, 2H), 1.90-1.87 (m, 4H). LCMS: m/z 300.2 (M+l), Analysis calculated for C20H17N3.

6-(4-(trifluoromethoxy)phenyl)-l,2,3,4-tetrahydroacridin-9-amine (206):

Synthesized following the same procedure as 201

¾NMR: MeOD (400 MHz):d 8.17 (d, 1H, 7= 8.8 Hz), 7.96-7.94 (m, 1H), 7.86-7.84 (dd, 2H, 7=6.4, 2.0 Hz), 7.69-7.66 (dd, 1H, 7=4.8, 2.0 Hz), 7.43-7.41 (m, 2H), 2.98-2.95 (m, 2H), 2.67-2.64 (m, 2H), 2.01-1.90 (m, 4H). LCMS: m/z 359.0 (M+l), Analysis calculated for C20H17F3N2O.

6-(2-methylpyrimidin-5-yl)-l,2,3,4-tetrahydroacridin-9-amine (207):

Synthesized following the same procedure as 201

¾NMR: MeOD (400 MHz):d 9.42 (s, 2H), 8.40-8.38 (m, 1H), 7.89-7.87 (m, 1H), 7.60 (s, 1H), 3.10-3.03 (m, 2H), 2.68-2.64 (m, 2H), 2.57 (s, 3H), 2.08- 1.95 (m, 4H).LCMS: m/z 291.0 (M+l), Analysis calculated for C18H18N4.

6-(2-fluorophenyl)-l,2,3,4-tetrahydroacridin-9-amine (208):

Synthesized following the same procedure as 201

¾NMR: MeOD (400 MHz):d 8.42 (d, 1H, J= 8.8 Hz), 7.94 (s, 1H), 7.94-7.80 (m, 1H), 7.67-7.72 (m, 1H), 7.60-.7.49 (m, 1H), 7.40-7.37 (m, 1H), 7.37-7.25

(m, 1H), 3.05-3.02 (m, 2H), 2.68-2.65 (m, 2H), 2.03-1.98 (m, 4H).LCMS: m/z 293.2 (M+l), Analysis calculated for C19H17FN2.

6-(4-fluorophenyl)-l,2,3,4-tetrahydroacridin-9-amine (209) :

Synthesized following the same procedure as 201

¾NMR: MeOD (400 MHz):d 8.37 (d, 1H, /= 8.4 Hz), 7.87-7.81 (m, 2H), 7.79-7.77 (m, 2H), 7.70-7.61 (m, 1H), 7.58-7.55 (m, 1H), 7.29-7.24 (m, 2H), 3.03-3.02 (m, 2H), 2.63-2.61 (m, 2H), 2.05-1.90 (m, 4H). LCMS: m/z 293.2

(M+l), Analysis calculated for C19H17FN2.

6-(4-(methylthio) phenyl)-l,2,3,4-tetrahydroacridin-9-amine (210):

Synthesized following the same procedure as 201

¾NMR: MeOD (400 MHz):68.39 (d, 1H, J= 8.0 Hz), 7.91 (m, 2H), 7.43 (d, 2H, J=8.8), 7.42 (d, 2H, J=8.8), 3.05- 3.02(m, 2H), 2.67-2.64 (m, 2H), 2.56 (s, 3H), 2.01-2.00 (m, 4H). LCMS: m/z 321.2 (M+l), Analysis calculated for C20H20N2S.

6-(4-(trifluoromethyl) phenyl)-l,2,3,4-tetrahydroacridin-9-amine (211):

Synthesized following the same procedure as 201

¾NMR: MeOD (400 MHz): 68.47 (d, 1H, J= 8.8 Hz), 8.01-7.92 (m, 4H), 7.88-7.86 (m, 2H), 3.10-3.06 (m, 2H), 2.72-2.65 (m, 2H), 2.10-1.99 (m, 4H). LCMS: m/z 343.0 (M+l), Analysis calculated for C20H17F3N2.

6-(furan-3-yl)-l,2,3,4-tetrahydroacridin-9-amine (212):

Synthesized following the same procedure as 201

¾NMR: MeOD (400 MHz): d 8.33 (d, 1H, 7=8.8 Hz), 8.21 (s, 1H), 7.87-7.81 (m, 2H), 7.65-7.55 (m, 2H), 3.04-3.01 (m, 2H), 2.66-2.63 (m, 2H), 2.03-1.99 (m, 4H). LCMS: m/z 265.2 (M+l), Analysis calculated for C17H16N2O.

6-phenyl-l,2,3,4-tetrahydroacridin-9-amine (213):

Synthesized following the same procedure as 201

¾NMR: MeOD (400 MHz):d 8.39 (d, 1H, J= 8.8 Hz), 7.92-7.87 (m, 2H), 7.78-7.76 (m, 2H), 7.56-7.52 (m, 2H), 7.49-7.45 (m, 1H), 3.04-3.01 (m, 2H), 2.65-2.62 (m, 2H), 2.00-1.98 (m, 4H). LCMS: m/z 275.2 (M+l), Analysis calculated for C₁₉H₁₈N₂.

6-(pyridin-3-yl)-l,2,3,4-tetrahydroacridin-9-amine (214):

Synthesized following the same procedure as 201

*H NMR: MeOD (400 Hz): 9.44 (s, 1H), 9.14 (d, 1H, J= 8.0 Hz), 9.01 (d, 1H, J= 6.4 Hz), 8.62 (d, 1H, J= 8.8 Hz), 8.31 (m, 1H), 8.24 (s, 1H), 8.09 (d, 1H, J= 8.8 Hz), 3.11-3.09 (m, 2H), 2.70-2.67 (m, 2H), 2.03-2.02 (m, 4H); LCMS: m/z 276.0 (M+l) calculated for C18H17N3.

Synthesis of 5, 10 & 14:

Ethyl 9-amino-6-bromo-l,2,3,4-tetrahydroacridine-2-carboxylate (05):

[0038] To a solution of 2-amino-4-cyano-benzonitrile 1 (5.0 g, 0.0253 mol, 1.0 eqiv) in anhydrous toluene was added BF₃.Et₂0 (3.76 mL, 0.0304 mol, 1.2 eqiv) slowly at room temperature. The reaction mixture was cooled to 0°C followed by the addition of ethyl-4-oxocyclohexanecarboxylate (6.4 mL, 0.0379, 1.5 eqiv) and the reaction mixture was heated to l00°C for 4 h. After completion, the reaction was monitored by TLC, the reaction mixture was quenched with aq. NaOH solution up to pH= 10 and the reaction mixture was diluted with ethyl acetate. The organic layers were separated, dried over sodium sulphate and concentrated. The crude product was recrystallized from dichloromethane to obtain ethyl 9-amino-6- bromo-l,2,3,4-tetrahydroacridine-2-carboxylate (05) (7.0 g, 85%) as off white solid.

*H NMR (400 MHz, DMSO-d6): d 8.13 (d, lH,/= 8.96 Hz), 7.79 (s, 1H), 7.40 (dd, 1H, J= 8.92, 8.92 Hz), 6.61 (s, 2H), 4.19-4.07 (m, 2H), 2.94-2.85 (m, 4H), 2.68-2.61 (m, 1H), 2.l5-2.l l(m, 1H), 1.89-1.80 (m, 1H), 1.24 (t, 3H, /= 7.12 Hz). LCMS: 351.7 (M+2), m/z calculated for Ci6H₁₇BrN₂0₂.

Ethyl-9-amino-6-(l-methyl-lH-pyrazol-4-yl)-l,2,3,4-tetrahydroacridine-2- carboxylate (10):

[0039] To a solution of 05 (1.0 g, 2.87 mmol) in l,4-dioxane (10 mL) and water (1 mL), l-methyl pyrazole 4-boronic ester (0.9 g, 4.31 mmol) and Na2C03 (0.6 g, 5.74 mmol) were added. The reaction mixture was degassed for 10 min and added Pd(PPh3)4 (0.33 g, 0.287 mmol). The reaction mixture was heated to H0°C for 2h in sealed tube. The reaction mixture was filtered through celite and the filtrate was diluted with water (100 mL) and extracted with ethyl acetate (3x100 mL). The organic layers separated was dried over anhydrousNa2S04 and concentrated under vacuum. The residue obtained was recrystallized in dichloromethane to afford compoundlO (0.52 g, 52%) as white solid.

¾ NMR (400 MHz, DMSO-rf6):6 8.26(s, 1H), 8.16 (d, J= 8.0 Hz, 1H), 7.99 (m, 1H), 7.82 (m, lH),7.55 (m, 1H), 6.57 (bs, 2H), 4.15 (m, 2H), 3.89 (s, 3H), 2.88(m, 4H), 2.69 (m, 1H), 2.16 (m, 1H), 1.90 (m, 1H), 1.26 (t, J = 7.12, 3H). LCMS: 351.4 (M+H) m/z calculated for C20H22N4O2. 9-amino-N-methyl-6-(l-methyl-lH-pyrazol-4-yl)-l,2,3,4-tetrahydroacridine- 2-carboxamide (14):

[0040] To a solution of compound 10 (100 mg, 0.284 mmol, 1.0 eqiv) in methanol (10 mL) was added 50% aqueous methyl amine (3 mL) and the reaction mixture was heated to 60°C for 4 h. The reaction mixture was concentrated and co-distilled with toluene three times. The crude product was recrystallized from dichloromethane to give 9-amino-N-methyl-6-(l-methyl- lH-pyrazol-4-yl)-l,2,3,4-tetrahydroacridine-2-carboxamide (50 mg, 92 % yield) as a white solid.

¾ NMR (400 MHz, DMSO-rf6):6 8.37 (s, 1H), 8.16 (d, 7= 8.4 Hz, 1H), 7.99 (s, 1H), 7.95 (m, 1H), 7.81 (s, 1H), 7.56 (d, 7= 8.0 Hz, lH),6.67 (bs, 2H), 3.89 (s, 3H), 2.89 (m, 2H), 2.60 (m, 4H), 2.33 (m, 1H), 2.15 (m, 1H), 1.91 (m, 1H), 1.81 (s, 1H). LCMS: 336.2 (M+H) m/z calculated for C19H21N5O.

Synthesis of ethyl 9-amino-6-(4-fluorophenyl)-l,2,3,4-tetrahydroacridine- 2-carboxylate 107:

Synthesized following the same procedure as 10

¾ NMR (400 MHz, MeOD):6 8.14 (d, 7=8.8 Hz, 1H), 7.91 (m, 1H), 7.80-7.75 (m, 2H), 7.58 (dd, 7=8.0, 2.0 Hz, 1H), 7.24 (m, 2H), 4.22 (m, 2H), 3.05-3.01 (m, 2H), 2.99-2.90 (m, 2H), 2.89-2.82 (m, 1H), 2.33-2.29 (m, 1H), 2.6-1.98 (m, 1H),

1.32 (t, 7=7.2 Hz, 3H);LCMS: 365.2 (M+H) m/z calculated for C22H21FN2O2. In vitro cholinesterase (AChE and BChE) inhibition assays

[0041] In order to confirm the binding potential of compounds towards ChEs, in vitro enzyme inhibition studies were conducted. The assays for AChE were carried out in a 96-well plate using AMPLITE™ AChE assay kit (AAT Bioquest, Inc., Sunnyvale, CA). Electric eel AChE (0.3EG) was used for the study, due to its structural similarity with the nerve and muscle AChE of vertebrates (Sussman et ah, 1991; Bon et al, 1979). The assay buffer (pH 7.4), 5,5-dithiobis-(2- nitrobenzoic acid) (DTNB, known as Ellman’s reagent) and the substrate acetylthiocholine (AChT) is included in the assay kit. The assay system works based on Ellman's method (Ellman et al, 1961). Thiocholine, produced by the action of AChE on acetylthiocholine, forms a yellow colored product with 5,5’- dithiobis-(2-nitrobenzoic acid), DTNB. The intensity of the colored product, measured at 410 ± 5 nm, is proportionate to enzyme activity. 100 pL reaction mixture was prepared by mixing the enzyme, 500 pM AChT solution in double distilled water (ddH₂0) and 500 pM DTNB in assay buffer.

[0042] Enzyme activity was determined by measuring the increase in absorbance as a result of enzyme substrate reaction at 405 nm for 2 minutes interval at 37°C. The same experiment was repeated in the presence of all tested compounds. All the compounds were dissolved in DMSO and were pre-incubated at room temperature for 20 minutes prior to the addition of reaction mixture containing AChT and DTNB. The optical density of enzyme in the presence and absence of compounds was plotted against time. The relative activity of enzyme in the presence of compounds was calculated with respect to native enzyme activity. The half maximal inhibitory concentration ( IC50 ) was determined for all the compounds by performing the assay at different inhibitor concentrations.

[0043] The inhibitory effect of all the compounds against BChE was also determined in a similar way. BChE from equine serum (0.3EG), butyrylthiocholine (500 pM) and DTNB (500 pM) in PBS (pH 7.4) were used for the inhibition assays. The IC50 value presented is the mean ± standard deviation of at least three separate experiments.

BACE-1 Inhibition assays

[0044] BACE assay kit (PanVera) utilizes fluorescence resonance energy transfer (FRET) technology to identify BACE-l inhibitor. BACE-l assay was carried out according to the protocol given by the manufacturer (Invitrogen, USA). (http://tools.invitrogen.com/content/sfs/manuals/L0724.pdf). An APP-based peptide substrate (Rhodamine (Rh)-EVNLDAEFK-Quencher) containing rhodamine as a fluorescence donor at one end and a quencher at the other end was used. The distance between the two fluorophores is very crucial. Upon light excitation, the donor fluorescence energy is significantly quenched by the acceptor via a quantum mechanical phenomenon known as resonance energy transfer. During enzymatic cleavage, the fluorophore separates from the quencher and the substrate becomes highly fluorescent. Enzyme activity is linearly proportional to increase in fluorescence. Assays were performed using 1U BACE- 1 enzyme and 750 nM substrate in 50 mM sodium acetate buffer at pH 4.5. The substrate solution was prepared fresh every time. The protocol is as follows: lOpL of BACE-l substrate is added to lOpL assay buffer, mixed gently and then lOpL BACE-l enzyme is added to start the reaction. After incubating the reaction mixture for 60 minutes at 25 °C, under dark conditions, it was stopped using 2.5 M sodium acetate. Fluorescence was measured at 545 nm excitation and 585 nm emission using Infinite® 200 (TECAN). The inhibitor concentration used was 50mM and DMSO concentration was 0.03%. The obtained values are the mean values of three different experiments.

Cell viability assay

[0045] Cytotoxicity of the compounds were tested using 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This is a colorimetric assay system which measures the reduction of MTT (pale yellow) to dark purple formazan crystals by the action of mitochondrial succinate dehydrogenase. After incubation, the cells were solubilized by DMSO addition and was measured on spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells, the level of activity is directly proportional to the number of viable cells. Two cell lines namely human embryonic kidney (HEK-293) and HepG2 were used. Cytotoxicity of the derivatives were checked in HEK-293 cells. Hepatotoxicity of the synthesized compounds based on tacrine was studied in HepG2 cell line. HEK-293 and HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 10,000 units/mL penicillin, 10 mg/mL streptomycin and 25 pg/mL amphotericin- B in T-25 flasks. Cells were trypsinized using 0.25% trypsin and seeded onto 96- well plates (-15000 cells/well). After 24 hrs, the medium was aspirated and replaced with varying concentrations of the test compounds. Following 24 hrs of compound treatment, 5 mg/mL MTT solution was added to the cells. After 2 hrs of MTT treatment, the formazan crystals formed were dissolved in lOOpL DMSO. Absorbance was measured at 570 nm using EnSpire multimode plate reader (PerkinElmer) or TECAN. Cell viability is expressed as the percentage of viable cells compared to untreated cells. The concentrations of the compounds used for testing cell viability is described in the respective chapters.

Preparation of primary cortical neurons from E18 rat embryos

[0046] Primary cultures of cortical neurons were prepared from El 8 old Wistar rat embryos. All animal studies were carried out at Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, according to the Institutional Animal Ethics Committee guidelines.

[0047] The pregnant female rat was sacrificed, and uterus was carefully removed, with the embryos, and placed in a 90 mm petri dish containing ice cold PBS. Embryos were removed from the uterus and the brain was dissected from each embryo. The cortical hemispheres were separated, and the meninges were removed with the help of a stereotaxic microscope. Cortical tissues were minced gently in Hank’s balanced salt solution (HBSS) and washed with HBSS at 1500 rpm for 3 minutes. The tissue was then subjected to dissociation using 0.05% Trypsin-EDTA and DNase I for 10 minutes. Trypsin action was inactivated by addition of 10% FBS, followed by wash with HBSS at 1500 rpm for 3 minutes. The tissues were triturated to a single cell suspension using DMEM + DNase I and washed twice with DMEM at 1500 rpm for 5 minutes. Live/dead cell count was carried out using trypan blue dye exclusion method. The dead cells stain blue, while the live cells stain white. The cells were then seeded onto 24-well plates and 96-well plates, pre-coated with poly-D-lysine (100 pg/mL) and laminin (1 pg/mL), at a density of 1 x 10⁵ cells/well and 20 x 10³ cells/well respectively, in neurobasal media (NBM) supplemented with IX GlutaMAX, IX antibiotic/antimycotic solution, IX B27 supplement and ciprofloxacin (10 pg/mL). The plates were incubated at 37°C, 5% C0₂. The cells were maintained in culture, by changing the media at regular intervals, till the day of experiment.

Induction of excitotoxicity in primary cortical neurons using glutamate

[0048] Glutamate-induced excitotoxicity was performed on nine-th day in vitro (DIV9) in rat primary cortical neurons. The cells were initially washed with solution I (HBSS, lOmM HEPES and 0.2 mM EGTA) for 10 minutes, followed by pretreatment with solution II (HBSS, lOmM HEPES) for 10 minutes. The cells were then treated with solution III (HBSS, 10 mM HEPES, lOOpM glutamate, lOpM glycine, 1.2 mM CaCl₂) for 60 minutes for induction of excitotoxicity. The tested compounds were added to the cells in both solution II and III. MK-801 (20 pM) was used as a positive control. After 60 minutes of glutamate treatment, the treatment solutions were replaced with NBM for another 24 hours. The spent media was used for biochemical estimation of glucose 6-phosphate dehydrogenase (G6PD) release. The cells were fixed using 4% paraformaldehyde and analyzed by immunocytochemistry and DAPI staining. In 96-well plates, MTT assay was also carried out after 24 hrs of excitotoxic treatment.

Glucose 6-phosphate dehydrogenase assay for measuring cell death

[0049] Cell death of cortical neurons, after glutamate treatment, was measured using Vybrant™ cytotoxicity assay kit (Molecular Probes). This kit monitors the release of the cytosolic enzyme, glucose 6-phosphate dehydrogenase (G6PD), from damaged cells into the surrounding medium, through a two-step enzymatic process. The oxidation of glucose-6-phosphate by G6PD results in the generation of NADPH. The released NADPH is coupled to the reduction reaction of resazurin to red fluorescent resorufin (Abs/Em: 563/587nm) by the action of diaphorase enzyme. The resulting fluorescence intensity is proportional to the amount of G6PD released into the medium which correlates with cell death.

[0050] The kit includes the following reagents: resazurin (75pg), dimethylsulphoxide (DMSO), reaction mixture (lyophilized mixture of diaphorase, glucose 6-phosphate and NADP⁺), 5X reaction buffer (0.5M Tris, pH 7.5) and 100X lysis buffer. The stock solution of resazurin (4mM) was prepared by adding 75pL DMSO. The 5X reaction buffer was diluted to IX with deionized water (dH₂0) and the lyophilized reaction mixture was dissolved in 400pL of IX reaction buffer. A working concentration of 2X resazurin/reaction mixture was prepared by combining 75pL of 4mM resazurin stock solution, 400pL of reaction mixture solution and 9.52 mL of IX reaction buffer.

[0051] After 24 hrs of excitotoxicity treatment, the spent media of neurons were subjected to G6PD assay for monitoring cell death. 50pL of spent media was mixed with 50pL of 2X resazurin/reaction mixture in a 96-well plate, incubated at 37°C for 40 minutes and fluorescence emission was measured at 587 nm. All necessary experimental controls and samples were assayed in duplicates. Immuno and DAPI staining of primary cortical neurons

[0052] Immuno staining was carried out to visualize the distribution and localization of the protein of interest during excitotoxic conditions and DAPI was used as a nuclear counterstain. The experimental procedure is as follows: paraformaldehyde-fixed cells after excitotoxicity experiment were washed thrice with PBS (Phosphate -buffered saline containing 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen orthophosphate, 137 mM sodium chloride, 2.7 mM potassium chloride, pH 7.4) and the cells were permeabilized/blocked simultaneously with PBS containing 0.2% triton X-100 and 3% BSA for 1 hour at room temperature. After removing the blocking solution, the cells were incubated with the respective primary antibody (MAP2 - 1:1000) diluted in PBS containing 2% BSA and 0.3% triton X-100 for overnight at 4°C. The primary antibody was removed and washed thrice with PBS followed by incubation with secondary antibodies conjugated to either Cy3 (in case of MAP2) or FITC (for b tubulin), at a dilution of 1:500, for 1-2 hrs at room temperature. The cells were washed thrice with PBS before being stained with DAPI (0.5pg/mL) for 20 minutes. The cells were again washed thrice with PBS and the coverslips were mounted using Fluoromount and sealed with DPX. The slides were viewed using fluorescence microscope (Leica) and the images were captured at 40X magnification.

Effect of compounds on NMDA receptor activity

[0053] In order to check the effect of compounds on NMDA receptor (NMDAR) activity, a cell-basedassay system was used. The assay system works based on protein-protein interaction between NMDAR and CaMKII, which was developed by Dr. R. V. Omkumar at Molecular Neurobiology Division, RGCB (US Patent Nos. 8, 304, 198; Indian Patent No. 260367; European Patent No. 2162742). The plasmids coding for NMDAR subunits, GluNl and GluN2B, and CaMKII tagged with GFP (GFP-CaMKII) was co-transfected into HEK-293 cells and the activity of GluN2B containing NMDAR was detected. In order to perform the assay, following procedures were done.

Preparation of GluNl, GluN2B and GFP-CaMKII plasmids

[0054] The glycerol stocks of bacterial cells, containing the respective plasmids, were inoculated in Luria Bertani (LB) broth containing antibiotics (ampicillin in the case of GluNl and GluN2B and kanamycin for GFP-CaMKII) and the bacterial cultures were incubated at 37°C, overnight with rigorous shaking (-200 rpm). The bacterial cells in culture was pelleted by centrifugation at 6000 rpm for 15 min at 4°C. The plasmids for mammalian expression were prepared using QIAGEN Midi Kit. The bacterial pellet was resuspended in 4 mL of resuspension buffer, Pl, containing 50 mMTris-Cl, pH 8.0, 10 mM EDTA and l00pg/mL RNase A for approximately lhr. To this solution, 4 mL of lysis buffer, P2, containing 200 mMNaOH with 1% SDS was added, mixed thoroughly by inverting 4-6 times and incubated at room temperature for 5 mins. Next, 4mL of neutralization buffer, P3, containing 3.0 M potassium acetate, pH 5.0 was added, mixed gently by inverting 4-6 times, and incubated on ice for 15 minutes. After incubation, the solution was centrifuged at 14,000 rpm for 30 min at 4°C. The supernatant containing the plasmid DNA was removed carefully and centrifuged at 14,000 rpm for 15 min at 4°C to get a clear solution of plasmid DNA. An anion exchange resin, DEAE coupled column (QIAGEN-tip-lOO), was equilibrated using 4 mL equilibration buffer QBT (750 mMNaCl, 50 mM MOPS, pH 7.0, 15% isopropanol, 0.15% Triton X-100) and the column was allowed to empty by gravity flow. The supernatant, obtained after centrifugation, was allowed to flow through QIAGEN-tip and to the resin by gravity flow. Following binding, QIAGEN-tip was washed with 2X 10 mL wash buffer QC (1.0 M NaCl, 50 mM MOPS, pH 7.0, 15% isopropanol). The bound plasmid was eluted with 5 mL elution buffer QF (1.25 M NaCl, 50 mMTris-Cl, pH 8.5, 15% isopropanol). The plasmid DNA, present in the elute, was precipitated using 3.5 mL isopropanol at room temperature. This solution was mixed and centrifuged at 14,000 rpm for 30 minutes at 4°C. The supernatant was carefully decanted. The pellet was washed with 2 mL 70% ethanol at room temperature at 14,000 rpm for 10 min. The pellet was air dried and dissolved in a suitable volume of water. The concentration of the plasmids was quantified using NanoDrop 2000 and its purity was checked on 1% agarose gel.

Transfection of plasmids into HEK-293 cells

[0055] To detect the activity of GluN2B containing NMDAR, HEK-293 cells were co-transfected with plasmids coding for GFP-a-CaMKII and GluNl and GluN2B subunits of NMDAR. Co-transfection was carried out using lipofectamine (Invitrogen) according to manufacturer's protocol. For transfection, HEK-293 cells were seeded on sterile 12 mm coverslips placed in 24-well plates (-1.5 x 10⁴ cells/well). After 18 hrs of seeding, the cells were co-transfected with the plasmids.

The following solutions were prepared for transfection:

[0056] Solution A: For each transfected well, 0.15 pg of plasmid DNA for GFP- a-CaMKII and 0.35 pg each of plasmid DNA encoding subunits GluNl and GluN2B was added to 62.5 pF Opti-MEM (Invitrogen) and mixed well.

Solution B : For each transfected well, 2 pF lipofectamine reagent was added to 62.5 pF Opti-MEM and mixed well.

[0057] Solution A was added to Solution B, mixed gently by pipetting and incubated at room temperature for 45 minutes. After incubation, 125 pF Opti- MEM was added to the tube containing lipid-DNA complexes. The media in 24- well plate was replaced with 250pF of prepared solution. Cells were incubated for 5 hours at 37°C in a C0₂ incubator. After 5 hours, 250 pF of Opti-MEM medium containing 20% FBS and 20pM MK-801 was added to stop transfection. The addition of MK-801, an NMDAR antagonist, can prevent cell death in transfected cells due to activation of NMDAR. The transfection solution was aspirated after 12 hours and 500 pL of fresh DMEM containing 10% FBS and 20 mM MK-801 was added. The cells were further incubated at 37°C for 24 hours.

NMDAR dependent translocation of GFP-a-CaMKII to GluN2B

[0058] About 24 hours after transfection, cells were washed twice, first with HBSS containing lmM HEPES and 0.5 mM EGTA (Solution I) and then with HBSS containing lmM HEPES (Solution II). NMDAR was activated with its agonists, glutamate (lOOpM) and glycine (lOpM), along with Ca²⁺ (2mM) (Solution III). After 5 minutes of activation, cells were fixed with 4% paraformaldehyde for 10-15 minutes and washed thrice with PBS. The cover slips containing cells were mounted on a clean glass slide using glycerol: PBS (1:1) and were observed using fluorescence microscope. The activity of GluN2B- NMDAR was detected based on the activation of GFP-CaMKII by Ca²⁺ influx through NMDAR and its subsequent translocation to GluN2B subunit. Interaction between CaMKII and GluN2B is observed as localization spots (punctate appearance) in the endoplasmic reticulum and plasma membrane. To check the effect of compounds on NMDAR activity, the compounds at respective concentrations were added to cells with both solution II (preincubation for 5 min) and solution III. In order to determine the localization efficiency, the cells were counted using an inverted fluorescence microscope (Leica) at 40X. The number of green fluorescent cells with punctate appearance and the total number of green fluorescent cells were counted and the percentage of green fluorescent cells having punctate pattern was calculated as “(number of punctate cells/ total number of green fluorescent cells) X 100”. This number was taken as the efficiency of punctae formation or punctate cell count. For each slide, 5 or more fields were randomly selected. Mean ± SD of the percentage of cells showing GFP-CaMKII translocation, obtained from three independent experiments, is presented as values in respective graphs. Effect of compounds on GFP-CaMKII and GluN2B interaction

[0059] In order to study the effect of compounds on GFP-CaMKII and GluN2B interaction, HEK-293 cells stably expressing GFP-a-CaMKII and MLS-NR2B (a binding motif of GluN2B which binds to CaMKII, tagged with mitochondrial localization signal) was used. For activating GFP-a-CaMKII, a Ca²⁺ ionophore, ionomycin, was used which can form calcium channels on the membrane. Cells were first washed with HBSS containing 1 mM HEPES and 0.5 mM EGTA for 5 minutes. Following this, the cells were incubated with 250 pL HBSS containing 1 mM HEPES and 15 pM ionomycin for 5 minutes. After incubation, 200pL of solution was removed and the cells were treated with HBSS containing 1 mM HEPES, 2 mM Ca²⁺ and 3 pM ionomycin for 5 minutes. The cells were fixed using 4% paraformaldehyde for 10 minutes and washed twice with PBS for 5 minutes each. The coverslips were mounted onto glass slides and used for imaging. Ca²⁺ influx into the cell results in translocation of GFP-a-CaMKII to MLS-NR2B, which is observed as bright perinuclear granules under a fluorescence microscope.

In vivo experiments

Morris water maze test

[0060] Based on the neuroprotective activity of the tacrine derivatives obtained from primary cortical neurons, four of the derivatives were tested for their in vivo neuro-cognitive effect using Morris water maze test. The behavioral study was carried out using 4-6 weeks old, male Wistar rats (~l00g) for 15 days. Monosodium glutamate (MSG), a flavor enhancer in processed foods, was used for inducing excitotoxicity in the animals. It has been reported that excitotoxic glutamate exposure can reduce memory retention and Y-maze learning abilities (Ma et ah, 2007, Neuroscience bulletin, Vol. 23, pp.209-214). All the solutions were administered, intra-peritoneally (IP), for 15 days. Water maze was performed on I I^th day of injection, as described by Morris (Morris, 1984, Journal of Neuroscience Methods, N ol. 11, pp.47-60), with a slight modification. The water maze apparatus consists of a large circular pool (183 cm diameter, 64 cm height), with an escape platform (10 cm diameter, 35cm height) and filled with milk water, just above the platform. The rat was placed in the pool, facing the wall (starting point) and allowed to search for the platform for 60s (maximum trial time) based on the visual cues present in the pool. The time taken by each rat to reach the platform (escape latency) was recorded using a video camera connected to EthoVision XT (Noldus Information Technology). If the rat could find the platform before 60s, it was allowed to stay on the platform for 5-l0s and then returned to its home cage. If it was unable to find the platform, it was placed on the platform for lOs and then returned to its home cage. Each animal was given five trials per day for 5 days with ~ 10- l5s interval between each trial. The pool was divided into four equal quadrants and parameters such as path length and escape latency were analyzed using the software.

[0061] For the in vivo screening of compounds, the rats were divided into the following groups, with three rats in each group (n=3): (i) Vehicle control (0.03% DMSO in saline), (ii) MSG (2g/Kg body weight), (iii) compound 201 control, (iv) compound 201 + MSG, (v) compound 208 control, (vi) compound 208 + MSG, (vii) compound 211 control, (viii) compound 211 + MSG, (ix) compound 212 control, (x) compound 212 + MSG, (xi) tacrine control and (xi) tacrine + MSG. After 5th day of trial, the animals were sacrificed by cervical dislocation and the brain and liver were dissected. The cortex, hippocampus and cerebellum of each dissected brain was separated and stored at -80 °C. Following the initial screening, compound 201 was also checked for its cognitive enhancement property in the presence of 4 g/Kg body weight MSG in a similar manner.

[0062] The activity and efficacy of the Tacrine derivatives underlying the present invention has been elaborated in the following sections with examples:

Example 1 Effect of the derivative(s) on NMDAR activity:

[0063] The derivatives were experimentally tested for their effect on NMDAR activity using previously patented calcium sensing technology (US patent No. 8,304,198; Indian patent No. 260367; European patent No. 2162742). HEK-293 cells transfected with NMDAR (GluNl and GluN2B) subunits were used for identifying the antagonistic potential of the compounds. This system provides direct data on the activity of compounds against an NMDAR subtype with a specific subunit composition (GluNl-GluN2B), unlike in the case of neurons wherein such specificity towards receptor subtypes is difficult to achieve. It was found that the 14 derivatives had NMDAR inhibitory activity (Fig. 3) and some of them were significantly more potent as evident from the IC50 values that were more than lOO-fold lesser (Fig. 4).

[0064] There was also improvement in AChE inhibitory activity of some derivatives (Fig. 5). All derivatives showed inhibition of butyrylcholineesterase (BChE) activity also (Fig. 6). In addition, some derivatives also showed beta secretase inhibitory activity while Tacrine did not show beta secretase inhibitory activity at the concentration tested (Fig. 7). Thus, the derivatives were active against multiple therapeutic targets for neuroprotection. Hepatotoxicity of the derivatives as seen in HepG2 cell line was similar or lesser compared to Tacrine (Fig. 8).

[0065] Compounds with substitutions either at both positions and at the benzene ring alone were effective in antagonizing NMDAR activity at the concentrations tried. However, Tacrine did not show significant inhibition at these concentrations (Table 1), consistent with the studies done by Fi et al, in 2005 (Fi et al, 2005, J Biol Chem., Vol. 280, rr18179-88). The compounds that are having substitutions only at cyclohexyl portion also showed reduced inhibitory effect. It was found that some of the compounds with substitutions at the benzene ring alone displayed the lowest IC50 values. Particularly, the compounds such as 208, 210 and 211 showed the maximum antagonistic activity against NMDAR and were significantly more potent than the parent compound. The list of compounds with their cytotoxicity profiles and NMDAR inhibitory profiles are given in Table 1, Fig. 3 and Fig. 4.

[0066] Tacrine is reported to attenuate NMD A receptor mediated neurotoxicity in murine cortical cultures with an IC50 value of 500mM (Cynthia et al, 1988, European Journal of Pharmacology, Vol. 154, pp73-78). The Tacrine derivatives such as 210, 208 and 211 can protect rat primary cortical neurons from glutamate induced excitotoxicity at significantly reduced concentrations (0.5 mM or less).

Table 1

*More than 80% of cell viability may be considered as noncytotoxic in nature. Derivatives 201 and 203 showed only 72% and 67% viability at 20mM respectively.

Example 2

In silico ADME properties predicted for the tacrine derivatives:

[0067] Computational studies were also conducted to understand the mechanism of binding and absorption, distribution, metabolism and excretion (ADME) profile of the newly synthesized compounds. The structure-activity relationship guided study suggested that many of these novel series of tacrine derivatives might be useful as neuroprotective agents by antagonizing the activity of NMDAR.

[0068] The drug like behaviour of the 14 tacrine derivatives were assessed using in silico methods. All derivatives satisfied the Lipinski's rule of five. Some important parameters including MW, Volume, CNS activity, log P, HBD and HBA, PSA, rotor, N and O, log BB, permeability across MDCK and Caco-2 cells and % HO A that are crucial for CNS activity were identified and are displayed in Table 2 and 3. All compounds showed general drug like properties and exhibited preferred CNS activities except 10, 17, 8, 13, 14, 204 and 205. These compounds showed a negative parameter in the CNS active scale indicating that they might be CNS inactive. The lipophilicity in terms of LogP value of 211 is found to be slightly higher. It has been already reported that CNS acting agents may have slightly higher log P value (Lenz, G.R., 1999. Technical problems in getting results. From data to drugs: strategies for benefiting from the new drug discovery technologies (Haberman AB, Lenz GR, Vaccaro DE, eds.), rr.95-114). Caco-2 and MDCK permeability and % HO A of 17 and 8 is found to be low as compared to other ligands. QikProp program in the Schrodinger was used for the study.

Table 2

Example 3

In vitro inhibition assay against cholinesterase:

[0069] ChEs inhibition assays of the synthesized compounds were carried out using Ellman's method. IC50 values were determined by assaying AChE and BChE at different concentrations of the compounds (Fig. 5 and Fig. 6). Substitution at C2 position was found to improve the inhibitory potency, while substitution at both C2 and C6 position together decreased the enzyme inhibitory activity compared to tacrine. The activity of 13 and 17 towards ChEs has already been reported. (Reddy et al., 2017, European journal of medicinal chemistry, Vol. 139, pp.367-377). [0070] The IC50 value of tacrine against BChE is 14.26 ± 1.07 nM showing comparatively higher potency than against AChE (94.69+4.88 nM) which indicates the selectivity of tacrine towards BChE inhibition (Hu et al., 2002). The IC50 values of all the synthesized compounds against BChE were in the mM range while, those against AChE were in the nM range. Hence, the new compounds exhibited high selectivity profile for AChE over BChE.

Example 4

Effect of tacrine derivatives on NMDAR activity:

[0071] The effect of tacrine derivatives on NMDAR was further checked using a cell-based assay system which is based on protein-protein interaction between NMDA receptor and a-CaMKII. The NMDAR activity was observed as localization spots in the presence of NMDAR agonists and Ca²⁺. The reduction in localization spots observed in the presence of compounds is due to the block of Ca²⁺ influx through NMDAR. Initially, two different inhibitor concentrations (50 and 100 mM) of the compounds were used for testing the effect on NMDAR activity. All compounds except tacrine, 16, 5, 17, 8 and 13 showed more than 60% inhibition at 50 pM, amongst which 208, 209, 210 and 211 showed almost 100% of inhibition. Hence, their percentage of inhibition at 20 pM was also checked (Fig. 3). The concentration dependent antagonistic potential of the ligands on NMDAR activity was carried out and their IC50 values were determined (Fig. 4). A hundred-fold difference in potency (i.e., ranging from 30 pM to 0.3 pM) was observed among these compounds. From this, it was assumed that substitutions at C6 position alone might be enough to antagonize NMDAR activity.

Example 5

Effect of compounds on BACE-1 activity: [0072] To check the inhibitory effect of tacrine derivatives on BACE-l activity, a FRET based enzyme inhibition assay was performed. Four compounds namely 10, 204, 208 and 210 caused significant reduction (>60%) in the enzyme activity at 50 mM (Figure 7). The inhibition on BACE-l activity is an additional benefit to the ligand as it can reduce the production of Ab peptides.

Example 6

Hepatotoxicity of the tacrine derivatives:

[0073] As mentioned earlier, tacrine was withdrawn as a drug due to its hepatotoxicity. The cytotoxicity of the tacrine derivatives were tested on HepG2 cell line. HepG2 is one of the in vitro systems used to test the hepatotoxicity of compounds (Thabrew et al., 1977, J. Pharm. Pharmacol. Vol. 11, (1997) H32el l35; Sassa et al., 1987, Biochem. Biophys. Res. Commun. Vol. 143, pp52e57). As depicted in the graph (Fig. 8), tacrine was safe only up to 50 pM and cell viability started decreasing at 100 pM. At 300 pM, significant reduction in the cell viability was observed. Among the tested tacrine derivatives, all compounds were found to be less toxic compared to tacrine.

Example 7

Effect of tacrine derivatives on glutamate induced excitotoxicity:

[0074] As two of the tacrine derivatives (210, 211 and 208) were found to be potent blockers of NMDAR, their effect on glutamate induced excitotoxicity was checked on primary cortical neurons prepared from the rat embryos. The neuroprotective property of compounds (14, 201 and 212) that showed moderate affinity to NMDA receptor were also checked. Neuronal cultures at DIV9 were used for inducing excitotoxicity. Here, the MTT assay was also performed to understand the neuroprotective effect. All compounds were dissolved in DMSO and it has been shown that DMSO does not have any protective effect against glutamate toxicity. DMSO-plus/minus glutamate is used as the control for all experiments.

Example 8

Neuroprotective effect of tacrine derivatives quantified by G6PD assay:

[0075] G6PD assay was performed for measuring the extent of cell death. Compounds 14, 211, 210 and 208 were checked. G6PD assay results show that the compounds can protect against glutamate toxicity at their IC50 (for NMDAR) concentration or above (Fig. 9).

[0076] Compounds 201, 212, 211, 210 and 208 were selected based on their NMDAR inhibitory effect and were treated along with glutamate for 3 hours after pre-treatment with compounds for 10 minutes as mentioned earlier. Compared to the control without glutamate, the glutamate treated sample showed a significant reduction in neuronal viability, as quantified by MTT assay, and it was found to be prevented when treated with compounds (Fig. 10).

Example 9

Immuno and DAPI staining for detecting neuronal viability:

[0077] Based on the results obtained from G6PD assay and MTT assay, compounds 14, 201, 211, 210 and 208 were checked for neuronal protection by DAPI staining and immuno staining for MAP2 protein. DAPI staining of cortical neurons after glutamate treatment in presence of compounds 14, 201, 211, 210 and 208 showed a clear decrease in the condensed nuclei as compared to glutamate treatment alone indicating prevention of cell death.

[0078] Immuno staining against MAP2 protein also showed increased number of MAP2 positive neurons in presence of the compounds compared to glutamate treatment indicating protection of neurons from excitotoxic cell death in presence of compounds. The representative images of the cortical neurons stained for DAPI and MAP2 are given in Fig. 11 and the quantitative data for DAPI and MAP2 staining are given in Fig. 12 and 13 respectively.

Example 10

Effect of tacrine derivatives on monosodium glutamate (MSG) induced cognitive impairment evaluated by Morris water maze test:

[0079] Although, different types of neurodegenerative diseases are caused by different mechanisms, neuronal injury and subsequent neuronal death are common to all neurodegenerative conditions, which is thought to occur via excessive activation of NMD A receptor. Monosodium glutamate (MSG) was used to establish an excitotoxic model in the in vivo condition because over the years, MSG has been shown to induce a series of behavioral disorders and brain lesions by stimulating glutamate receptors in various experiments (Park et al., 2000, Toxicology letters, Vol. 115, pp.117-125; Olney, 1969, Science, Vol. 164, pp.7l9- 721; Gonzalez-Burgos et al., 2001, Neuroscience letters, Vol. 297, pp.69-72; James and Yetunde, 2011, Journal of Neuroscience and Behavioral Health, Vol. 3, pp.51-56; Ma et al., 2007, Neuroscience bulletin, Vol. 23, pp.209-214). Using this information, MSG has been used to build a chronic excitotoxic condition in the animal model to study the learning impairment caused by neuronal damage. Enhancement of cognitive ability is another important property of an anti-AD (Alzheimer’s Disease) drug candidate. To induce excitotoxic conditions, MSG (2g/Kg body wt) was given intraperitoneally (i.p) for 15 days and saline was used as the control. Four compounds 201, 212, 211, 208 and tacrine were also injected i.p along with MSG or Saline for 15 consecutive days. The test included 5 days of learning and memory training from I I^th day of injection onwards. The mean escape latency values of all the groups on each day was calculated and are shown in Fig. 14. Compared to saline group, MSG treated animals showed significant delay in latency to reach the platform from 3rd day of the trials (p value <0.005). The neuroprotective effect of the compounds was analyzed on the basis of 3 days of trial. The compound 201 (p value <0.005) and 208 (p value <0.05) when administered along with MSG showed a significant improvement in the escape latency to reach the platform. All the compounds when administered with MSG showed improvement in the learning ability compared to MSG alone group.

Example 11

Effect of 201 on high dose of MSG:

[0080] Since the effect of 2g MSG on day 4 and 5 of trial was not significant compared to saline control, the dose required to induce the cognitive impairment was increased to 4g/ Kg body weight. The escape latency to reach the platform for 4g MSG treated animals were checked. Compared to the control group, MSG significantly prolonged the latency to platform from day 3 of trial indicating that the cognitive impairment model was successfully designed (Fig. 15). As compound 201 turned to be a potential candidate from the initial screening experiment, it's effect on higher dose of MSG was tested. Treatment with 201 (3 mg/Kg body weight) significantly reduced the latency to target, signifying that 201 could ameliorate the memory impairment caused by MSG (Fig. 16). The representative trajectories of the trial are given in the Fig. 17. Treatment with 201 along with saline also showed improvement in its learning ability compared to saline alone control.

[0081] The work described mainly focuses on the identification of compounds that can modulate both cholinergic and glutamatergic signaling pathways. For this, tacrine, was modified into different compounds and their inhibitory potency towards cholinesterase and NMDAR activities were tested. Results of in vitro studies showed that the antagonistic potential towards NMDAR was enhanced around lOO-fold upon derivatization. Derivatization also improved AChE inhibitory potential as seen in the case of compounds 201, 212, 209, 208 and 203. The compounds 10, 107, 204 and 208 caused significant reduction in BACE-l activity which was studied using a FRET based assay system. In silico analysis of ADME parameters showed that most of the predicted properties of the compounds were in the allowed range. Preliminary assessment of hepatotoxicity of tacrine derivatives on HepG2 cell lines showed that derivatization lowered the toxic nature of the compounds compared to the parent compound, tacrine.

[0082] Compounds 201, 212, 211, 210 and 208 could reverse the toxicity induced by glutamate in primary cortical neurons obtained from embryos of rat. Further in vivo effect of these compounds on MSG induced excitotoxic model was assessed by Morris water maze test. From preliminary screening, only 201 and 208 showed significant improvement in the learning skills compared to MSG treated animals. The neuroprotective effect of 201 was further studied using higher dose of MSG and it could significantly reduce the latency to target. Reduction in the latency to target in presence of compound 201 compared to saline treated animals indicates that normal NMDAR activities are not affected in the presence of compounds as it improved the learning ability of the rats. From these results it can be assumed that compound 201 might be a potential drug candidate against AD, as it can influence both cholinergic and glutamatergic system, which is currently one of the most opted treatment strategies towards AD.

[0083] Now, the crux of the invention is claimed implicitly and explicitly through the following claims.

Claims

We Claim:

1. Derivatives of a heterocyclic compound tacrine with the basic molecular formula C13H14NNH2, and the structural formula of:

Wherein either position 2 or position 6 or both may be substituted by Rl and R2 respectively, wherein Rl and R2 is selected from hydrogen, halogen or halo derivatives, phenyl, substituted benzene, benzene derivatives, heterocyclic derivates of nitrogen (N₂), furan derivatives, amide or amide derivatives, purine or pyrimidine, purine derivatives or pyrazoleorpyrazole derivatives, and ester derivatives, either standalone or any combination thereof.

2. The heterocyclic derivatives as claimed in claim 1, wherein Rl is selected from hydrogen, halogen or halo derivatives, phenyl substituted benzene, benzene derivatives, heterocyclic derivates of nitrogen (N₂), furan derivatives, and purine or pyrimidine, purine derivatives or pyrazole or pyrazole derivatives, either standalone or any combination thereof.

3. The heterocyclic derivatives as claimed in claim 1, wherein R2 is selected from amide or amide derivatives or ester or ester derivatives, either standalone or any combination thereof.

4. The derivatives as claimed in claiml, selected from the group consisting of-

- 6-bromo-l,2,3,4-tetrahydroacridin-9-amine designated as compound 118;

-6-( 1 -methyl- 1 H-pyrazol-4-yl)- 1 ,2,3 ,4-tetrahydroacridin-9-amine designated as compound 201 ;

-6-(pyrimidin-5-yl)-l,2,3,4-tetrahydroacridin-9-aminedesignated as compound 203;

-6-(lH-pyrazol-3-yl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 204;

-4-(9-amino-5,6,7,8-tetrahydroacridin-3-yl)benzonitrile designated as compound 205;

-6-(4-(trifluoromethoxy)phenyl)- 1 ,2,3,4-tetrahydroacridin-9-amine designated as compound 206;

-6-(2-methylpyrimidin-5-yl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 207 ;

-6-(2-fluorophenyl)- 1 ,2,3 ,4-tetrahydroacridin-9-amine designated as compound 208;

-6-(4-fluorophenyl)- 1 ,2,3 ,4-tetrahydroacridin-9-amine designated as compound 209;

-6-(4-(methylthio)phenyl)- 1 ,2,3,4-tetrahydroacridin-9-amine designated as compound 210;

-6-(4-(trifluoromethyl)phenyl)- 1 ,2,3,4-tetrahydroacridin-9-amine designated as compound 211;

-6-(furan-3-yl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound

212;

-6-phenyl- 1,2, 3, 4-tetrahydroacridin-9-amine designated as compound 213; -6-(pyridin-3-yl)-l,2,3,4-tetrahydroacridin-9-amine designated as compound 214;

-Ethyl-9-amino-6-( 1 -methyl- lH-pyrazol-4-yl)- 1 ,2,3,4-tetrahydroacridine-2- carboxylate designated as compound 10;

-9-amino-N-methyl-6-( 1 -methyl- lH-pyrazol-4-yl)- 1 ,2,3,4-tetrahydroacridine- 2-carboxamide designated as compound 14;

-Ethyl-9-amino-6-(4-fluorophenyl)-l,2,3,4-tetrahydroacridine-2-carboxylate designated as compound 107.

5. The derivatives as claimed in claim 1 and claim 4, wherein one or more of them can inhibit the activity of NMDAR.

6. The derivatives as claimed in claiml and claim 4, wherein one or more of them show NMDAR inhibition at a concentration ranging from 20 mM to 100 mM.

7. The derivatives as claimed in claim 1 and claim 4, wherein one or more of them show maximal non cytotoxic concentration values in the range 10 mM to 50 mM.

8. The derivatives as claimed in claim 1 and claim 4, wherein one or more of them show IC50 values in the range 0.2 mM to 50 mM for inhibiting NMDAR.

9. The derivatives as claimed in claim 1 and claim 4, wherein one or more of them can inhibit glutamatergic and cholinergic signaling in neural cells.

10. The derivatives as claimed in claim 1 and claim 4, wherein one or more of them can inhibit glutamate-induced excitotoxic cell death in neuronal cells.

11. The derivatives as claimed in claim 1 and claim 4, wherein one or more of them are capable of inhibiting the activity of beta secretase.

12. The derivatives as claimed in claim 1 and claim 4, wherein one or more of them are capable of inhibiting BChE.

13. The derivatives as claimed in claim 12, wherein one or more of them show IC₅o values in the range 0.5 m M to 20 mM for inhibition of BChE.

14. The derivatives as claimed in claim 1 and claim 4, wherein one or more of them are capable of inhibiting AChE.

15. The derivatives as claimed in claim 9, wherein one or more of them show IC50 values in the range 10 nM to 200 nM for inhibiting AChE.

16. The derivatives as claimed in claim 1 and claim 4, wherein one or more of them show reduced hepatotoxicity compared to Tacrine.

17. A process of synthesis of library derivatives of a heterocyclic compound tacrine comprising the steps of allowing reaction of the tacrine or tacrine derivatives with appropriate reagent in presence of sodium carbonate and palladium-tetrakis (triphenylphosphine), (Pd(PPh₃)4) in presence of a mixture of 1 ,4-dioxane and water at a definite temperature for a period, or allowing the similar reaction in presence of methylamine (CH3NH2), boron trifluoride (BF3), methanol (CH3OH), Toluene, diethyl ether at a definite temperature for a period.

18. The process as claimed in claim 17, wherein the time required is 1 to 12 hours, and the temperature required ranged from 50°C-l20°C.

19. The process as claimed in claim 17, wherein the ratio of l,4-dioxane and water ranges from 8:2 to 9: 1.

20. A method for treating one or more conditions associated with plaque accumulation and calcium mediated excitotoxicity by administering an effective amount of any one of the derivatives claimed in claim 1 and 4.

21. The method as claimed in claim 20, wherein administering the derivative(s) improves learning and memory in a setting of monosodium glutamate induced neural damage and cognitive impairment.

22. The method claimed in claim 20, wherein administering the compound reduces neurofibrillary tangle formation.