Synthesis and characterization of pyrimidines analogues as anti-Alzheimer’s agents
Yadav Rakesh*, Maan Monika, Yadav Divya     
Department of Pharmacy, Banasthali University, Banasthali-304022 Rajasthan, India    
Abstract: Pyrimidine derivatives have been reported as neuroprotective agents useful for the treatment of various neurodegenerativedisorders. In the present study, several pyrimidine analogues have been evaluated as neuroprotective agents in Morris water maze model. It was observed that pyrimidine derivatives 817 considerably improve learning, memory, and movement deficits in animal models. Biochemical estimations of brain serum of treated animals revealed suppression of oxidative and nitrosative stress, acetylcholinesterase activity, and other parameters which leads to neurodegeneration of brain. Of all the pyrimidine derivatives, thiomorpholine derivative 8 and piperazine ethanol derivative 17 were found to be the most active neuroprotective agents and produced effects comparable to standard drug rivastigmine in terms of behavioral, biochemical, and molecular aspects.              
Keywords: Alzheimer’s disease; Neurodegeneration; Pyrimidine derivatives; Biochemical tests
CLC number: R916                Document code: A                 Article ID: 10031057(2017)1183413 

1. Introduction
Alzheimer’s disease is a neurodegenerative disorder that affects areas of the brain that controls cognition and physical awareness. The hallmarks of Alzheimer’s disease (AD) include the loss of cholinergic neural transmission and the formation of beta-amyloid plaques (Aβ-plaques) and neuro-fibrillary tangles (NFTs) of hyper-phosphorylated tau protein[1].According to the Alzheimer’s association,approximately 37 million people are living with dementia around the world today, and an estimated 67 million will be by 2030[2].
The cholinergic hypothesis is one of the most deliberated pathways and is proposed as a link betweendiminished cholinergic neural transmission and Aβ-plaque toxicity in AD pathogenesis[3].The cholinesterase (ChE) enzymes, acetylcholine esterase (AChE) and butrylcholine esterase (BuChE) are hydrolytic enzymes that act on acetylcholine (ACh) to terminate its action in the synaptic cleft upon cleavage the neurotransmitter to choline and acetate. Studies have shown that AD pathogenesis is characterized by the rapid loss of acetylcholinesterase (AChE) activity in the early stages of the disease along with the increasing ratios of BuChE to AChE as the disease progresses[1]. These findings support the need to control the activity of the ChE enzymes at different stages of AD progression.
Based on the cholinergic hypothesis, AD can treated by enhance cholinergic function to sustain or prolong the action of remaining ACh neuronsusing cholinesterase inhibitors[4].AChE and BuChE are hydrolytic cholinesterase enzymes that act on ACh to terminate its action in the synaptic cleft by cleaving the neurotransmitter to choline and acetate[5]. Recent studies have shown thatcholinesterase inhibitors have been efficacious in reducing the symptoms of early to moderate stage AD[6].
In this regard, research into the cholinergic hypothesishad led to the development of several fused heterocycles as ChE inhibitors (ChEIs) (Fig. 1). For example, the acridine derivative, tacrine (1), was one of the earliest ChEI developed to treat AD. Bis-7-tacrine (2) is a potent dual AChE and BuChE inhibitor. Furthermore, β-carbolins (3) were developed as both AChE and BuChE inhibitors. Currently marketed AD pharmacotherapies, such as donepezil (4) and galantamine (5), are multifunctional agents because they inhibit ChE, enhance nicotinic acetylcholine receptor (AChR) activity, and blockAβ-aggregation[1,7,8].As part of our program to evaluatenovel ChEIs, we report herein the synthesis and evaluation of a class of 2,4-disubsituted pyrimidine derivatives (817).

Figure 1. Heterocycles that exhibit ChE inhibition (13) and other anti-AD agents (45). 
2. Result and discussion
2.1. Chemistry
The precursor for the synthesis of targeted 2,4-disubstituted pyrimidine derivatives 817 were synthesized by the condensation of 2,4-dichloropyrimidine (6) with various substituted anilines (ag) in the presence of N,N'-diisopropylethylamine (DIPEA) and ethanol at reflux temperatures. The desired key intermediates7ag were synthesized by nucleophilic aromatic substitution at the C-4 position with good yields (80%90%).
Compounds 7agwere further condensed with secondary amines (thiomorpholine, 1-phenylpiperazine,N-hydroxyethylpiperazine) to afford the various2,4-disubstituted pyrimidine derivatives 817 byaftermicrowave irradiation at 100 °C for 15 min[7] (Scheme 1).

Scheme 1.
Reagents and conditions: (a) DIPEA/EtOH; Reflux for 46 h (b) MW at 100 ºC for 15 min.
The anti-AD activity of the newly synthesized compounds were evaluated in vivo in a mouse model through the behavioral tests and biochemical studies.  
2.2. Pharmacological evaluation
The present study showed the neuroprotective effects of pyrimidine analogs in scopolamine-induced amnesia.The Morris water maze was used as a behavioral assay for the assessment of learning and memory[9].Decreased escape latency in repeated trials of theMorris water maze task demonstrated intact learning and memory function. Scopolamine treated mice escape latency could not decrease significantly in repeated trials ranging from day 1 to day 5, suggesting significant impairment in the memory of scopolamine treated mice. However, upon administration of these pyrimidine analogues to the scopolamine treated mice, the time to reach the hidden platform on target quadrant in water maze task decreased significantly (Fig. 2A–B). In a memory consolidation test, the time spent in the target quadrant (Fig. 2C) was also significantly decreased in scopolamine injected mice as compared to the control group. This effect was reversed on treatment with the pyrimidine analogs. The results from the following biochemical studies were consistent with the findings of the Morris water maze test, supporting the conclusionthat these pyrimidine analogues could mitigate the brain acetylcholinesterase activity[10].

Figure 2.
Effect of pyrimidine derivatives (817) treatment on escape latency (A) path length (B) in morris water maze, time spent (C) in target quadrant in probe trial in scopolamineinjected mice. Values were expressed as mean±SEM. (*P<0.005) different from control; (#P<0.05) different from scopolamine; (**P<0.001) different from control; (##P<0.01) different from scopolamine, (***P<0.0001) different from control; (###P<0.05) different from scopolamine. Pyrimidine derivatives (2 mg/kg); SC, Scopolamine (1.5 mg/kg); RVS, Rivastigmine (2 mg/kg). 

   ACh is a neurotransmitter associated with learning and memory, and its levels are reduced in the presence of
AChE. In addition to their role in cholinergic transmission, cholinesterases may also play a role in morphogenesis and neurodegenerative diseases[11].In the present study, scopolamine, a cholinergic antagonist, was used to create animal models of amnesia. It antagonizes the muscariniccholinergic receptors and increases cholinesterase activity. Experimental results from the present study revealed that scopolamine treated mice brains showed significantly increased acetylcholinesterase activity[12]. This may lead to diminished cholinergic transmission due to a decrease in acetylcholine levels. Treatment with 2,4-disubstitudted pyrimidines analogues817 significantly inhibited acetylcholinesterase levels in the brains of scopolamine treated mice (Fig. 3A), which showed that the synthesized compounds 817could act against scopolamine-induced amnesia through cholinesterase inhibition.
Pyrimidine is a unique scaffold which is associated with many biological activities. The structure of the pyrimidine ring is similar to benzene and pyridine. In a pyrimidine ring, as the number of nitrogen atoms increase, the π electrons become less energetic and electrophilic aromatic substitution becomes more difficult, whereas the nucleophilic aromatic substitution resonance stabilization properties of pyrimidine may lead to addition and ring cleavage reactions, rather than substitutions. The findings from the previous studies suggest that pyrimidine analogs have cholinergicantagonism and antioxidant activity[13].The synthesized compounds 817 also exhibited antioxidant activity and have the ability to trap the free radicals, which are implicated in the pathogenesis of many different diseases, including neuro-inflammatory disorders.
Scopolamine-induced amnesia is associated with increased oxidative stress in particular brain structures associated with memory and learning[14]. The synthesized compounds were also found to disturb metabolism, especially for low molecular weight antioxidants such as glutathione, and therefore it increases the levels of lipid peroxidation in the brain[15]. This is due to the high abundance of polyunsaturated fatty acids, such as arachidonic acid and docosahexaenoic acid[16]. Scopolamine-induced amnesia is a very well established animal model of memory dysfunction and is widely used to test potential drugs for anti-AD properties[17]. The present study also demonstrated significant increases in the lipid peroxidation (Fig. 3B) and nitrite levels (Fig. 3C) with decreased antioxidant content (Fig. 3D–F) in the scopolamine treated group, which were reversed upon treatment with various synthesized pyrimidine analogs[18–20].

Figure 3.
Effect of treatment with pyrimidine derivatives (817) on acetylcholinesterase levels (A), lipid peroxidation (B), nitrergic stress (C) and antioxidant profile (D, E, F). Values were expressed as mean±SEM. (*P<0.005) different from control; (#P<0.05) different from scopolamine; (**P<0.001) different from control; (##P<0.01) different from scopolamine; (***P<0.0001) different from control; (###P<0.05) different from scopolamine. Pyrimidine derivatives (2 mg/kg); SC, Scopolamine (1.5 mg/kg); RVS, Rivastigmine (2 mg/kg).   
3. Conclusions
The cholinergic system plays an important role in learning and memory[21]. ACh levels are reduced by AChE, and thus inhibition of AChE would be expected to lead to enhanced ACh levels[12]. Scopolamine is an anticholinergic drug that antagonizes the muscarinic cholinergic receptors (mAChRs) (subtypes; M1 and M2) and generates deficits in learning and memory consolidation[22]. Intraperitoneal administration ofscopolamine produces an accepted and widely used model for inducing memory deficits in animal models[23]. 
The current study revealed that pyrimidine derivatives817 affect memory processes. However, obtained results may suggest that pyrimidine derivatives are able to ameliorate memory impairments caused by cholinergic dysfunction through the inhibition of AChE as well as the inhibition of oxidative stress-related processes. The present study demonstrated that administration of pyrimidine derivatives improved scopolamine-induced memory loss and consolidation impairment in the Morris water maze test in mice. Furthermore, we may also conclude that observed anti-amnestic effects of the pyrimidine derivatives may be attributed to its antioxidant activity. Currently, there is no cure for AD, and additional preclinical studies may lead to the development of effective anti-AD agents with minimal side effects. Based on the present results, we put forth that these pyrimidine derivatives may become new treatments that change the course of the disease and improve the quality of life in people with AD.
4. Experimental section
4.1. General
Infrared (FT-IR) spectra were recorded on an Agilent Technology Cary 600 series Fourier Transform-Infrared spectrophotometer using potassium bromide pellets (νmax in cm–1). 1HNMR spectra were recorded on Bruker advance II 400 MHzspectrometer (CDCl3–DMSO as solvent). Melting points (m.p.) were determined by using glass capillary tubes on a Veego melting point apparatus and are uncorrected. Compounds (817) showed a single spot on thin-layer chromatography (TLC) using a solvent system of chloroformmethanol (9.5:0.5, v/v). Spots were visualized using an iodine chamber. All reagents were of AR grade.
4.2.General procedure for the synthesis of 4-substituted-2-chloropyrimidine-4-amine (7ag)
Equimolar concentrations of 2,4-dichloropyrimidine (6) and substituted anilines (ag) were dissolved in ethanol (25 mL) with continuous stirring at 0 °C (ice bath) in the presence of a catalytic amount of N,N-diisopropylethylamine (DIPEA).The reaction mixture was allowed to stir in the water bath for 5 min and then refluxed at 70–80 °C for 6 h[1].The reaction was monitored by TLC. After completion, excess solvent was removed under reduced pressure to obtain a solid residue, which was washed with cold water to afford the desired products 7ag.
4.2.1. 2-Chloro-N-phenylpyrimidin-4-amine (7a)
Yield: 97%. Mp: 180182 ºC. FT-IR vmax. (KBr) cm–1: 3239 (-NH), 1647 (C=N), 1458 (C=C), 1204 (C-N), 750 (C-Cl). 1H NMR (400 MHz, DMSO) δ: 10.04(s, 1H, NH), 8.16 (d, 2H, ArH, J 5.6 Hz), 7.59 (d, 2H, ArH), 7.37 (t, 2H, ArH), 7.09 (t, 1H, ArH), 6.76 (d, 2H, ArH, J 6 Hz).
4.2.2. 2-Chloro-N-(3-chlorophenyl)-pyrimidin-4-amine(7b)
Yield: 76.66%. Mp: 150154 ºC. FT-IR vmax. (KBr) cm–1:3296 (-NH), 1624 (C=N), 1473(C=C), 1206 (C-N), 721 (C-Cl). 1H NMR (400 MHz, DMSO) δ: 10.25 (s, 1H, NH), 8.22 (d, 1H, ArH, J 5.6 Hz), 7.82 (s, 1H, ArH), 7.52 (d, 1H, ArH), 7.39 (t, 1H, ArH), 7.14 (d, 1H, ArH), 6.81 (d, 1H, ArH, J 5.6 Hz).
4.2.3. 2-Chloro-N-(4-chlorophenyl)-pyrimidin-4-amine (7c)
Yield: 77.14%. Mp: 180184 ºC. FT-IR vmax. (KBr) cm1:3083 (-NH), 1615 (C=N), 1489 (C=C), 1210 (C-N), 747 (C-Cl). 1H NMR (400 MHz, DMSO) δ: 10.94 (s, 1H, NH), 8.03 (d, 1H, ArH, J 7.2 Hz), 7.65 (t, 2H, ArH), 7.42 (m, 2H, ArH), 6.59 (d, 1H, ArH, J 7.2 Hz). 
4.2.4. 2-Chloro-N-(4-methoxyphenyl)-pyrimidin-4-amine (7d)
Yield: 67.64%. Mp: 160162 ºC. FT-IR vmax. (KBr) cm1:3195 (-NH), 2828 (OCH3), 1581 (C=N), 1453 (C=C), 1246 (C-N), 731 (C-Cl). 1H NMR (400 MHz, DMSO) δ: 8.06 (d, 1H, ArH, J 5.6 Hz), 7.21 (d, 2H, ArH), 6.99 (s, 1H, NH), 6.93 (m, 2H, ArH), 6.41 (d, 1H, ArH, J 6 Hz), 3.83 (s, 3H, -OCH3).
4.2.5. 2-Chloro-N-p-tolyl-pyrimidin-4-amine (7e)
Yield: 75.60%. Mp: 208212 ºC. FT-IR vmax. (KBr) cm–1:3219 (-NH), 2904 (CH3), 1608 (C=N), 1421 (C=C), 1243 (C-N), 748 (C-Cl). 1H NMR (400 MHz, DMSO) δ: 8.09 (d, 1H, ArH, J 6 Hz), 7.22 (d, 2H, ArH), 7.17 (d, 1H, ArH), 6.96 (s, 1H, NH), 6.53 (d, 1H, ArH, J 5.6 Hz), 2.36 (s, 3H, -CH3).
4.2.6. 2-Chloro-N-(4-fluorophenyl)-pyrimidin-4-amine (7f)
Yield: 89.61%. Mp: 162164 ºC. FT-IR vmax. (KBr) cm–1:3195 (-NH), 1613 (C=N), 1423(C=C), 1219 (C-N), 1094 (C-F). 1H NMR (400 MHz, DMSO) δ: 10.09 (s, 1H, NH), 7.89 (d, 1H, ArH, J 5.6 Hz), 7.57 (m, 2H, ArH), 7.31 (m, 2H, ArH), 6.77 (d, 1H, ArH, J 5.6 Hz).
4.2.7. N-(4-Bromophenyl)-2-chloropyrimidin-4-amine (7g)
Yield: 76.06%. Mp: 204210 ºC. FT-IR vmax. (KBr) cm1: 3215 (-NH), 1649 (C=N), 1418 (C=C), 1242 (C-N), 636 (C-Br). 1H NMR (400 MHz, DMSO) δ: 10.14 (s, 1H, NH), 8.19 (d, 1H, ArH, J 5.6 Hz), 7.57 (m, 4H, ArH), 6.77 (d, 1H, ArH, J 5.6 Hz).
4.3. General procedure for the synthesis of 2,4-disubstituted pyrimidine-4-amine derivatives (817)[1,7]
Compounds 7ag were condensed with their respective secondary amines (IIII) under microwave irradiation for 15 min at 100 °C (40 W). The residue wasthen washed with methanol/water to obtain desired compounds 817.
4.3.1. N-Phenyl-2-thiomorpholinopyrimidin-4-amine (8)
Yield: 62.5%. Mp: 148151 ºC. FT-IR vmax. (KBr) cm–1:3273 (-NH), 1563 (C=N), 1428 (C=C), 1216 (C-N), 583 (C-S). 1H NMR (400 MHz, DMSO-d6) δ: 7.99 (d, 1H, ArH, J 5.72 Hz), 7.34 (m, 4H, ArH), 7.12 (m, 1H, ArH), 6.52 (s, 1H, NH), 6.01(d, 1H, ArH, J 5.76 Hz), 4.12 (t, 4H, 2×N-CH2, J1 2.55 Hz, J2 2.65 Hz). 13C NMR (400 MHz, CDCl3) δ:27.0 (2S-CH2), 47.12 (2N-CH2), 97.47 (ArC), 116.3 (2ArC), 118.12 (ArC), 128.6 (2ArC),140.31 (ArC), 156.57 (ArC), 160.02 (ArC), 161.62 (ArC).Calcd. for (C14H16N4S):C, 61.74%; H, 5.92%; N, 20.57%. Found:C, 60.75%; H, 5.55%; N, 19.93%.
4.3.2.N-Phenyl-2-(4-phenylpiperazin-1-yl)-pyrimidin-4-amine (9)
Yield: 25.80%. Mp: 197198 ºC. FT-IR vmax. (KBr) cm–1:3264 (-NH), 1642 (C=N), 1458 (C=C), 1370 (C-N). 1H NMR (400 MHz, DMSO-d6) δ:7.43 (t, 1H, ArH), 7.23 (m, 5H, ArH), 7.11 (d, 1H, ArH), 6.96 (d, 4H, 3×ArH, 1×NH), 6.84 (t, 2H, ArH), 3.48 (t, 4H, 2×N-CH2), 3.37 (t, 4H, 2×N-CH2). 13C NMR (100 MHz, DMSO-d6) δ: 49.6 (4N-CH2), 96.6 (ArC), 114.0 (2ArC), 116.8 (2ArC),118.2 (2ArC), 128.7 (4 ArC), 130.1 (ArC), 139.5 (ArC),150.6 (ArC), 155.2 (ArC), 160.5 (ArC). Calcd. for (C20H21N5):C, 72.48%; H, 6.39%; N, 21.13%. Found:C, 63.95%; H, 6.66%; N, 17.09%.
4.3.3. 2-(4-(4-(Phenylamino)-pyrimidin-2-yl)-piperazin-1-yl)-ethanol (10)
Yield: 52%. Mp: 116120 ºC. FT-IR vmax. (KBr) cm–1:3678 (OH), 3437 (-NH), 1648 (C=N), 1436 (C=C), 1249 (C-N). 1H NMR (400 MHz, DMSO-d6) δ: 8.02 (d, 1H, ArH, J 5.68 Hz), 7.36 (d, 4H, ArH), 7.12 (t, 1H, ArH), 6.64 (s, 1H, NH), 6.04 (d, 1H, ArH, J 5.68 Hz), 3.83 (m, 4H, 2×N-CH2), 3.69 (t, 2H, CH2), 2.93 (s, 1H, OH), 2.59 (m, 6H, 3×N-CH2). 13C NMR (100 MHz, DMSO-d6) δ: 39.51 (2N-CH2), 48.24 (2N-CH2), 53.51 (N-CH2), 55.35 (O-CH2), 89.12 (ArC), 117.73 (2ArC), 120.19 (ArC), 129.6 (2ArC), 132.62 (ArC), 152.35 (ArC), 156.09 (ArC), 158.25 (ArC). Calcd. for (C16H21N5O):C, 64.19%; H, 7.07%; N, 23.39%. Found:C, 64.19%; H, 7.07%; N, 23.39%.
4.3.4. 2-(4-(4-(3-Chlorophenylamino)-pyrimidin-2-yl)-piperazin-1-yl)-ethanol (11)
Yield: 17.86%. Mp: 82100 ºC. FT-IR vmax. (KBr) cm–1: 3403 (OH), 3286 (-NH), 1612 (C=N), 1465 (C=C), 1271 (C-N), 771 (C-Cl).1H NMR (400 MHz, DMSO-d6) δ: 8.03 (d, 1H, ArH, J 5.6 Hz), 7.60 (s, 1H, ArH), 7.25 (t, 1H, ArH), 7.12 (d, 1H, ArH), 6.99 (d, 1H, ArH), 6.49 (s, 1H, NH), 5.98 (d, 1H, ArH, J 5.6 Hz), 3.82 (t, 4H, 2×N-CH2), 3.67 (t, 2H, CH2), 2.81 (s, 1H, OH), 2.59(m, 6H, 3×N-CH2). 13C NMR (100 MHz, DMSO-d6) δ: 36.12 (2N-CH2), 47.61 (2N-CH2), 52.0 (N-CH2), 54.7 (O-CH2), 90.6 (ArC), 114.4 (ArC), 116.7 (ArC), 118.9 (ArC), 128.8 (ArC), 131.6 (ArC), 144.5 (ArC), 151.8 (ArC), 158.0 (ArC), 160.6 (ArC). Calcd. for (C16H20ClN5O): C, 57.57%; H, 6.04%; N, 20.98%. Found:C, 54.9%; H, 5.67%; N, 18.56%.
4.3.5. N-(4-Chlorophenyl)-2-thiomorpholinopyrimidin-4-amine (12)
Yield: 32.5%. Mp: 168170 ºC. FT-IR vmax. (KBr) cm1:3213 (-NH), 1616 (C=N), 1497 (C=C), 1274 (C-N), 775 (C-Cl), 692 (C-S). 1H NMR (400 MHz, DMSO-d6) δ:9.41 (s, 1H, NH), 7.96 (d, 1H, ArH, J 5.6 Hz), 7.63 (m, 2H, ArH), 7.35 (m, 2H, ArH), 6.04 (d, 1H, ArH, J 5.6 Hz), 4.01 (m, 4H, 2×N-CH2), 2.59 (m, 4H, 2×S-CH2). 13C NMR(100 MHz, DMSO-d6) δ: 26.02 (2S-CH2), 46.14 (2N-CH2), 96.67 (ArC), 113.12 (ArC), 120.99 (2ArC),130.97 (2ArC), 139.37 (ArC), 155.77 (ArC), 160.12 (ArC), 160.40 (ArC). Calcd. for (C14H15ClN4S):C, 54.81%; H, 4.93%; N, 18.26%. Found:C, 53.52%; H, 4.76%; N, 17.49%.
4.3.6. 2-(4-(4-(4-Chlorophenylamino)-pyrimidin-2-yl)-piperazin-1-yl)-ethanol (13)
Yield: 56.75%. Mp: 120122 ºC. FT-IR vmax. (KBr) cm–1:3742 (OH), 3148 (-NH), 1612 (C=N), 1497 (C=C), 1224 (C-N), 793 (C-Cl).1H NMR (400 MHz, DMSO-d6) δ: 8.02 (d, 1H, ArH, J 5.6 Hz), 7.31 (m, 4H, ArH), 6.42 (s, 1H, NH), 5.96 (d, 1H, ArH, J 5.6 Hz), 3.81 (t, 4H, -NCH2-), 3.68 (t, 2H, -CH2-), 2.81 (s, 1H, OH), 2.59 (m, 4H, -2×N-CH2-), 1.61 (s, 4H, -2×N-CH2-).13C NMR(100 MHz, DMSO-d6) δ: 38.19 (2N-CH2), 49.24 (2N-CH2), 52.52 (N-CH2), 55.46 (O-CH2), 90.6 (ArC), 113.49 (ArC), 118.5 (2ArC), 129.52 (2ArC), 131.73 (ArC), 152.76 (ArC), 155.5 (ArC), 156.7 (ArC). Calcd. for (C16H20ClN5O):C, 57.57%; H, 6.04%; N, 20.98%. Found:C, 56.55%; H, 5.94%; N, 20.2%.
4.3.7. 2-(4-(4-(4-Methoxyphenylamino)-pyrimidin-2-yl)-piperazin-1-yl)-ethanol (14)
Yield: 56.25%. Mp: 130134 ºC. FT-IR vmax. (KBr) cm–1:3743 (OH), 3308 (-NH), 2819 (OCH3), 1577 (C=N), 1447 (C=C), 1240 (C-N). 1H NMR (400 MHz, DMSO-d6) δ: 7.97 (d, 1H, ArH, J 5.7 Hz), 7.26 (m, 2H, ArH), 7.16 (m, 2H, ArH), 6.46 (s, 1H, NH), 5.97 (d, 1H, ArH,J 5.7 Hz), 3.82 (m, 4H, 2×NCH2), 3.68 (t, 2H, -CH2-), 3.43 (s, 3H, -OCH3) 2.87 (s, 1H, -OH), 2.58 (m, 6H, 3×N-CH2). 13C NMR (100 MHz, DMSO-d6) δ: 37.95 (2N-CH2), 49.4 (2N-CH2), 50.0 (O-CH3), 52.1 (N-CH2), 53.42 (O-CH2), 92.6 (ArC), 115.3 (2ArC), 120.41 (2ArC), 134.62 (ArC), 150.52 (ArC), 152.49 (ArC), 158.22 (ArC),160.43 (ArC). Calcd. for (C17H23N5O2):C, 61.99%; H, 7.04%; N, 21.26%. Found:C, 61.26%; H, 7.03%; N, 20.41%.
4.3.8. 2-(4-(4-(p-Tolylamino)-pyrimidin-2-yl)-piperazin-1-yl)-ethanol (15)
Yield: 55.55%. Mp: 7880 ºC. FT-IR vmax. (KBr) cm–1:3742 (OH), 3302 (-NH), 2816 (CH3), 1569 (C=N), 1441 (C=C), 1224 (C-N). 1H NMR (400 MHz, DMSO-d6) δ: 7.97 (d, 1H, ArH, J 5.7 Hz), 7.26 (m, 2H, ArH), 7.16 (m, 2H, ArH), 6.46 (s, 1H, NH), 5.97 (d, 1H, ArH, J 5.7 Hz), 3.82 (m, 4H, 2×NCH2), 3.68 (t, 2H, -CH2-), 2.87 (s, 1H, OH), 2.58 (m, 6H, 3×NCH2), 2.34 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ: 15.63 (CH3), 38.59 (2N-CH2), 47.64 (2N-CH2), 52.51 (N-CH2), 54.20 (O-CH2), 89.10 (ArC), 116.93 (2ArC), 124.49 (ArC), 128.61 (2ArC), 130.86 (ArC), 151.76 (ArC), 156.09 (ArC), 156.47 (ArC). Calcd. for (C17H23N5O): C, 65.15%; H, 7.4%; N, 22.35%. Found:C, 64.91%; H, 7.38%; N, 21.9%.
4.3.9. 2-(4-(4-(4-Fluorophenylamino)-pyrimidin-2-yl)-piperazin-1-yl)-ethanol (16)
Yield: 33.33%. Mp: 128130 ºC. FT-IR vmax. (KBr) cm–1:3744 (OH), 3302 (-NH), 1578 (C=N), 1447 (C=C), 1335 (C-N), 1219 (C-F). 1H NMR (400 MHz, DMSO-d6) δ: 7.99 (d, 1H, ArH, J 5.64 Hz), 7.31(m, 2H, ArH), 7.05 (m, 2H, ArH), 6.42 (s, 1H, NH), 5.91(d, 1H, ArH, J 5.68 Hz), 3.81(t, 4H, 2×NCH2), 3.67 (t, 2H, -CH2-), 2.81 (s, 1H, OH), 2.58 (m, 6H, 3×NCH2). 13C NMR (100 MHz, DMSO-d6) δ: 37.45 (2N-CH2), 48.6 (2N-CH2),53.7 (N-CH2), 55.16 (O-CH2), 88.0 (ArC), 115.7 (2ArC),120.71 (2ArC), 124.9 (ArC), 131.6 (ArC), 150.7 (ArC),157.0 (ArC), 157.7 (ArC). Calcd. for (C16H20N5FO):C, 60.55%; H, 6.35%; N, 22.07%. Found:C, 60.15%; H, 6.28%; N, 21.34%.
4.3.10. 2-(4-(4-(4-Bromophenylamino)-pyrimidin-2-yl)-piperazin-1-yl)-ethanol (17)
Yield: 56.25%. Mp: 137140 ºC. FT-IR vmax. (KBr) cm–1:3310 (OH), 3150 (-NH), 1603 (C=N), 1488 (C=C), 1227 (C-N), 678 (C-Br). 1H NMR (400 MHz, DMSO-d6) δ: 8.01 (d, 1H, ArH, J 3.6 Hz), 7.45 (m, 2H, ArH), 7.30 (m, 2H, ArH), 6.56 (s, 1H, NH), 5.96 (d, 1H, ArH,J 3.2 Hz), 3.80 (t, 4H, -2×NCH2-), 3.67 (t, 2H, -CH2-), 2.74 (s, 1H, OH), 2.57 (m, 6H, 3×NCH2-). 13C NMR (100 MHz, DMSO-d6) δ: 39.19 (2N-CH2), 49.24 (2N-CH2), 53.52 (N-CH2), 55.46 (O-CH2), 91.6 (ArC), 113.49 (ArC), 118.5 (2ArC), 129.52 (2ArC), 131.73 (ArC), 152.76 (ArC), 155.5 (ArC), 156.7 (ArC). Calcd. for (C16H20BrN5O):C, 50.8%; H, 5.33%; N, 18.51%. Found:C, 50.21%; H, 5.23%; N, 17.85%.
5. Pharmacological methods
5.1. Material and methods
Adult Swiss albino mice male bred in Central Research Institute Kasauli, H.P. were used in this study. They had free access to food (Ashirwad Industries, Mohali, India) and water. The animals were maintained under standard laboratory conditions with alternating light/darkcycles. The animals were acclimatized for at least 2 d to the laboratory conditions prior to allbehavioral studies. The protocol was approved by the Institutional Animal Ethics Committee, Banasthali Vidyapith, Banasthali.
5.1.2. Drugs and treatment
Scopolamine and rivastigmine were purchased from Sigma Aldrich, St. Louis, MO, USA. All chemicals used for biochemical tests were of analytical grade. All drug solutions were freshly prepared on a daily basis and administered in a constant volume of 10 mL/kgbody weight. Scopolamine (1.5 mg/kg) was prepared bydissolving in double distilled water and was administeredby intraperitoneal injection 30 min prior drug treatment.Pyrimidine derivatives (817, 2 mg/kg) and rivastigmine (2 mg/kg) were prepared in 0.25% CMC solution and orally administered daily for 5 d (Fig. 1).
5.2. Morris water maze (EthoVision software)
Swiss albino mice were evaluated using a spatial version of the Morris water maze model for 5 d. The Morris water maze for mice consisted of a circular pool (150 cm in diameter, 45 cm in height) filled to a depth of 25 cm with water maintained at 25±2 °C. The tank was located in a dark room and water was made opaque with a nontoxic black dye. The tank was divided into four equal quadrants with two threads fixed at a right angle to each other on the rim of the pool. A submerged platform (with top surface 4×4 cm2) was placed inside the target quadrants (Q4 in present study) of this pool 2 cm below the surface of the water, and the position of platform was not altered throughout the training session. The mice received four consecutive daily training trials (day 1 to 4), with each trial having a total limit of 90 s and a rest interval of approximately 30 s. During the training sessions, the mice wereplaced into the water with their heads facing the wall at one of four starting positions (selected randomly)and given 90 s to locate the submerged platform. If the mouse failed to find the platform within 90 s, it was guided gently onto the platform and allowed to remain there for 20 s. On the fifth day, escape latency, the time taken by the animal to move from the starting quadrant to find the hidden platform in the targetquadrant, was measured (EthoVision software, Noldus Information Technology, Wageningen, Netherlands) alongwith mean time spent in the target quadrant (TSTQ), which was taken as an indicator of retrieval or memory.
5.3. Biochemical estimation
5.3.1. Brain tissue homogenate preparation
The brain tissue homogenates were prepared on day 5 for mice. The mice (n = 6) were sacrificed under deep anesthesia, and their brains were rapidly removed. Brain tissue was rinsed with ice cold normal saline (0.9% sodium chloride), and homogenate was prepared in chilled 0.1 M phosphate buffer (pH 7.4) using a homogenizer. The homogenate was further centrifuged at 11 000 r/min for 20 min at 4 °C to obtain the postmitochondrial supernatant (PMS), which wasused for biochemical studies, such as assaying foracetylcholinesterase activity, lipid peroxidation, reducedglutathione, nitrite level, catalase, and superoxide dismutaseactivity.
5.3.2. Estimation of acetylcholinesterase (AChE assay)
Cholinergic dysfunction was assessed by measuring acetylcholinesterase (AChE) levels in PMS. The results were calculated using the molar extinction coefficient of the chromophore (1.36×104 M−1?cm−1) and expressed as percentage of control group.
5.3.3. Estimation of lipid peroxidation (LPO assay)
The malondialdehyde (MDA) content, a measure of lipid peroxidation, was assayed in the form of thiobarbituricacid-reactive substances. The post-mitochondrial supernatant (PMS, 0.5 mL) and 0.5 mL of Tris-HCl were incubated at 37 °C for 2 h. After incubation, 1 mL of 10% trichloro acetic acid was added, and the mixture was centrifuged at 1000 ×g for 10 min. Then 1 mL 0.67% thiobarbituric acid was added to 1 mL of supernatant and tubes were kept in boiling water for 10 min. After cooling, 1 mL of double distilled water was added and the absorbance was measured at 532 nm. Thiobarbituric acid-reactive substances were quantified using an extinction coefficient of 1.56×105 M1?cm−1 and expressed as moles of malondialdehyde per mg of protein. Tissue protein was estimated using the Biuret method, and the brain malondialdehyde content was expressed as moles of MDA per milligram of protein.
5.3.4. Estimation of reduced glutathione (GSH assay)
Reduced glutathione was assayed by precipitating PMS (1.0 mL) with 1.0 mL of sulphosalicylic acid (4%). The samples were kept at 4 °C for at least 1 h and then subjected to centrifugation at 1200g for 15 min at 4 °C. The assay mixture contained 0.1 mL of supernatant, 2.7 mL of phosphate buffer (0.1 M, pH 7.4), and 0.2 mLof 5,5-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent, 0.1 mM, pH 8.0) in a total volume of 3.0 mL. The yellow color developed was read immediatelyat 412 nm, and the reduced glutathione levels were expressed as nmol of GSH per milligram of protein. 
5.3.5. Estimation of superoxide dismutase (SOD assay)
The assay system consisted of 0.1 mM EDTA, 50 mMsodium carbonate, and 96 mM of nitro-blue tetrazolium (NBT). In a cuvette, 2 mL of the above mixture was taken, and 0.05 mL of PMS and 0.05 mL of hydroxylamine hydrochloride (adjusted to pH 6.0 with NaOH) were added to it. The auto-oxidation of hydroxylamine was observed by measuring the change in optical density at 560 nm for 2 min at 30 or 60 s intervals.
5.3.6. Estimation of catalase (Catalase assay)
Briefly, the assay mixture consisted of 1.95 mL of phosphate buffer (0.05 M, pH 7.0), 1.0 mL of hydrogen peroxide (0.019 M), and 0.05 mL of PMS in a final volume of 3.0 mL. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated and expressed as micromoles of H2O2 decomposed per minute per milligram of protein.
5.3.7. Estimation of nitro-dative stress (Nitrite assay)
Nitrite was estimated in the PMS using the Greiss reagent and served as an indicator of nitric oxide production. 500 mL of Greiss reagent (1:1 solution of 1% sulfanilamide in 5% phosphoric acid and 0.1% napthaylaminediamine dihydrochloric acid in water) was added to 100 mL of postmitochondrial supernatant, and absorbance was measured at 546 nm. The nitrite concentration was calculated using a standard curve for sodium nitrite, and nitrite levels are expressed as mg/mL. It is important to note that the Griess spectrophotometric assay is an indirect measure of nitric oxide content.
5.4. Statistical analysis 
The results were expressed as mean±SEM and inter-group variation was measured by one-way analysisof variance (ANOVA) followed by Tukey’s test. Two-way ANOVA followed by Tukey’s test was employed to discover the inter-group variation in escape latency and path length data of Morris water maze by considering day of testing and treatment as two independent variables.Difference of P<0.05 was considered significant. The statistical analysis was done using the GraphPad Prism® statistical software version 5.01.
6. Results
6.1. Behavioral tests
6.1.1. Effect of pyrimidine derivatives on escape latency of scopolamine administered mice in Morris water maze task
Cognitive function was assessed in the Morris water maze test. The mice received four consecutive daily training trials in the following 5 d, i.e. each mice received 20 replicates during experimental period. Two-way ANOVA followed by Tukey’s test was employed to reveal the inter-group variation in escape latency of Morris water maze by considering day of testing and treatment as two independent variables. The mean escape latency did not differ between any of the groups on the first day of testing in Morris water maze but from second day onwards, there was significant difference in transfer latency. Scopolamine treated mice showed a reduced ability to find the platform and discover its location in the 5th day of training (Fig. 2A). This poor performance was significantly (P<0.05) mitigated by the treatment with pyrimidine derivatives or rivastigmine and resulted decreased latency to find the platform from the 2nd day of training. However, compounds 8, 13, 15, 16 and 17 showed decreased escape latency as compared to rivastigmine.
6.1.2.Effect of pyrimidine derivatives on total distance travelled to reach the hidden platform (path length)
Progressive decreases in path length to reach the hidden platform on subsequent days in water maze task is associated with the intact memory in animals. During four consecutive daily training trials for 5 d, the total distance travelled to reach the hidden platform did not differ between any of the groups on the first day of testing in Morris water maze, but from second day onwards, there was significant difference in path length of scopolamine mice as compared to that of control animals (Fig. 2B). Pyrimidine derivatives (817) and rivastigmine treatment significantly (P<0.05) decreased the total distance travelled to reach the platform in scopolamine group, suggesting improvements in memory.
6.1.3.Effect of pyrimidine derivatives on time spent in target quadrant in scopolamine administered mice
At the end of 5thday, the extent of memory consolidation was assessed by a probe trial, which measures how well the animals had learned and consolidated the platform location during the training period. The intergroup variation was measured by one-way analysis of variance (ANOVA) followed by Tukey’s test. The mice treated with scopolamine showed significantly less time spent in the target quadrant as compared to the control group. The mice treated with pyrimidine derivatives or rivastigmine along with scopolamine spent more time in the target quadrant than the scopolamine group in the probe test (P<0.05) (Fig. 2C).
6.2. Biochemical and molecular observations
6.2.1. Effect of pyrimidine derivatives on brain acetylcholinesterase activity
The acetylcholinesterase levels in PMS of mice were found to be elevated in the animals treated with scopolaminethan in the control group. Pyrimidine derivatives (817) treatment significantly reduced acetylcholinesterase levels in the scopolamine administeredmice brains. However, compounds 8, 9, 12, 13, 16 and 17 showed comparative reductionsin AChE levels as compared to the standard drug (rivastigmine) treatment (Fig. 3A).
6.2.2. Effect of pyrimidine derivatives on lipid peroxidation
Scopolamine administered mice PMS showed increased levels of malondialdehyde (MDA) levels as compared to the control group. Pyrimidine derivatives (817) or rivastigmine treated mice brain produced significant (P<0.05) reductions in MDA levels in scopolamine administrated mice (Fig. 3B).
6.2.3. Effect of pyrimidine derivatives on nitrergic stress
Nitrite levels were significantly elevated in scopolamineadministered animals as compared to the control group. Treatment with pyrimidine derivatives (817) or rivastigmine significantly inhibited the rise of brain nitrite levels (Fig. 3C). Moreover, the compounds 8,13 and 17 mitigate these levels to a normal extent.
6.2.4. Effect of pyrimidine derivatives on antioxidant profile
The enzymatic activities of catalase and superoxide dismutase, and the levels of glutathione (Fig. 3D, 3E, 3F) were significantly decreased in scopolamine administered mice PMS as compared to control group. These changes were significantly (P<0.05) reversed by treatment with the pyrimidine derivatives (817).
The authors are thankful to Vice-chancellor, BanasthaliUniversity for providing the necessary research facilities. Authors are thankful to DST-CURIE for financialsupport. One of the authors is also thankful to UGC-NRC, Panjab University, Chandigarh for providing the necessarytraining during the course.  
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Received: 2017-05-17, Revised: 2017-06-19, Accepted: 2017-09-28.
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