Table of Contents Author Guidelines Submit a Manuscript
Journal of Chemistry
Volume 2013 (2013), Article ID 107302, 9 pages
http://dx.doi.org/10.1155/2013/107302
Research Article

Design, Synthesis, and Acetylcholinesterase Inhibition Assay of Novel 9-(1-(Substituted-benzyl)piperidin-4-yl)-2-chloro-9H-purin-6-amine Derivatives

1Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan, Shandong 250012, China
2Department of Pharmacology, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan, Shandong 250012, China

Received 22 May 2013; Revised 9 August 2013; Accepted 13 August 2013

Academic Editor: Marco Radi

Copyright © 2013 Dongwei Kang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A new series of 9-(1-(substituted-benzyl)piperidin-4-yl)-2-chloro-9H-purin-6-amine derivatives were designed, synthesized, and characterized on the basis of 1H-NMR, 13C-NMR, and mass spectra. The newly synthesized compounds were evaluated for the inhibition activity against acetylcholinesterase (AChE). Biological results revealed that four compounds among them showed moderate activities against AChE with inhibitory percentage more than 10% at 100 μM. Further pharmacology investigation towards other pathological routes in AD is currently underway.

1. Introduction

Since a cholinergic hypothesis has been postulated as a crucial element in Alzheimer’s disease (AD) symptoms etiology, catalytic acetylcholinesterase (AChE) inhibitors have constituted until today the main drugs used against AD [1]. Reversible inhibition of brain AChE by anti-AD drugs such as tacrine [2], donepezil (Figure 1) [3], and huperzine [4] increases the AChE levels and improves neurotransmission in cholinergic synapses [5]. Since AChE plays a proaggregating (noncatalytic) role to accelerating β-amyloid peptide (Aβ) aggregation and deposition into the fibrils, inhibition of AChE is still the most successful therapeutic strategy for the symptomatic treatment of AD and its progression [6]. The search for novel AChE inhibitors with improved biological profiles continues to be of great interest to medicinal chemists.

107302.fig.001
Figure 1: Structure of donepezil, 2,4-disubstituted pyrimidine AChE inhibitors, and purine derivative PU2012051296.

In the past few years, a group of 2,4-disubstituted pyrimidine derivatives were reported as cholinesterase inhibitors and agents targeting multiple pathological routes in AD. Particularly, compound PY-1 was identified as the lead candidate with a dual ChE (AChE IC50 = 9.9 μM; BuChE IC50 = 11.4 μM), Aβ-aggregation (AChE-induced = 59.3%; self-induced = 17.4% at 100 μM), and BACE-1 (34% inhibition at 10 μM) inhibitory profile along with good cell viability (81.0% neuroblastoma cell viability at 40 μM) [710]. Meanwhile, o-fluorophenyl methyl derived triazole PU2012051296, effectively suppressed Aβ-induced neurotoxicity in hippocampal slice cultures. Importantly, the neuroprotective effect of compound PU2012051296 is comparable to those of flavopiridol and roscovitine, state of the art pharmaceuticals [11].

The refinement of the central core in bioactive molecules combined with introducing privileged substituents is a common practice in medicinal chemistry to find proprietary and novel hits. Based on the considerable similarities of these lead compounds, a fragment-based design was employed to identify alternative chemotypes (Figure 2). Meanwhile, in current drug research field, substituted purines have attracted considerable attention from the medicinal chemists in recent years, owing to the high number of positive hits encountered with this heterocycle. In this paper, a novel series of 9-(1-(substituted-benzyl)piperidin-4-yl)-2-chloro-9H-purin-6-amine derivatives were designed, synthesized, and evaluated for the inhibition activity against AChE.

107302.fig.002
Figure 2: The general structure formula of newly designed 9-(1-(substituted-benzyl)piperidin-4-yl)-2-chloro-9H-purin-6-amine derivatives.

2. Results and Discussion

2.1. Chemistry

The synthetic route of the target compounds is outlined in Scheme 1. which was started from commercially available materials and completed using well established methods. Initially, N-(3,4-dimethoxybenzyl)-2-chloro-9H-purin-6-amine (2a) and N-(benzo[d] dioxol-5-ylmethyl)-2-chloro-9H-purin-6-amine (2b) intermediates were synthesized from the starting material 2,6-dichloro-9H-purine (1) by a nucleophilic aromatic substitution reaction at the C-6 position using either (3,4-dimethoxyphenyl) methanamine or benzo[d] dioxol-5-ylmethanamine in the presence of triethylamine (TEA). The reaction was run in t-BuOH at 60°C for 30 min. Intermediates 6 and 7 were obtained in good yields ranging (90.5% and 89.7%) (Scheme 1) [710]. In the meantime, tert-butyl 4-hydroxypiperidine-1-carboxylate (4) was synthesized according to the reported procedure [12]. Then treatment of intermediate 4 with compound 2a or 2b in the presence of NaH afforded tert-butyl 4-(6-(3,4-dimethoxybenzylamino)-2-chloro-9H-purin-9-yl)piperidine-1-carboxylate (5a) or tert-butyl 4-(6-(benzo[d] dioxol-5-ylmethylamino)-2-chloro-9H-purin-9-yl)piperidine-1-carboxylate (5b), respectively. The deprotection of the tert-butoxycarbonyl (t-Boc) group of 5a and 5b was accomplished using trifluoroacetic acid (TFA) in dichloromethane (DCM) to yield N-(3,4-dimethoxybenzyl)-2-chloro-9-(piperidin-4-yl)-9H-purin-6-amine (6a) and N-(benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(piperidin-4-yl)-9H-purin-6-amine (6b), respectively, in good yield (91% and 87%). In the last step, the alkylation reaction of piperidine at NH position with various substituted benzyl chloride (bromine) afforded the target compounds. The newly synthesized compounds were characterized by physicochemical and spectral means, and the MS and NMR spectral data are found in agreement with the assigned molecular structures.

107302.sch.001
Scheme 1: Reagents and Conditions. (i) t-BuOH, Et3N; (ii): methanesulfonyl chloride (MsCl), DCM, Et3N, and 4-DMAP; (iii) NaH, DMF; (iv): TFA, DCM; (v) substituted benzyl chloride (bromine, fluorine), DMF, and K2CO3.
2.2. Cholinesterase Inhibition

All the newly synthesized compounds (7a1-a13, 7b1-b13) were evaluated for their inhibitory activities toward AChE using an in vitro assay based on the reported protocol [710], in comparison with donepezil as standard drug. The anticholinesterase activities are summarized in Table 1. Preliminary results in Table 1 revealed that compound 7b9 with a 3-cyanobenzyl group in the benzo[d] dioxol-5-yl series displayed the highest AChE inhibitory activity (15.42% at 100 μM) among all the tested compounds. Besides, compounds 7a1, 7a7, and 7b10 with inhibitory percentage more than 10% at 100 μM showed similar activities with compound 7b9 against AChE. Generally, all the compounds in both 3,4-dimethoxyphenyl and benzo[d] dioxol-5-yl series demonstrated much lower inhibitory activity toward AChE than the reference drug donepezil (80.07% at 100 μM). Obviously, the activity of the compounds depends upon the nature of the substituents attached with purine ring. Even small modifications in every series can decrease and even lost their activity against AChE. Therefore, the coherent structure-activity relationship will be afforded by further pharmacology investigation, which is currently underway in our lab.

tab1
Table 1: Cholinesterase inhibition activity of 9-(1-(substituted-benzyl)piperidin-4-yl)-2-chloro-9H-purin-6-amine derivativesa.

3. Conclusion

In summary, a novel series of 9-(1-(substituted-benzyl)piperidin-4-yl)-2-chloro-9H-purin-6-amine derivatives were designed and synthesized, and their AChE inhibitory activities were preliminary examined. Biological assay demonstrated that four compounds showed moderate activities against AChE with inhibitory percentage more than 10% at 100 μM. Further investigation of other bioactivities associated with AD of this series is ongoing work within our group and will be reported in due course.

4. Experimental Section

4.1. Chemistry

All melting points were determined on a micromelting point apparatus and are uncorrected. 1H-NMR and 13C-NMR spectra were obtained on a Bruker Avance-400 NMR-spectrometer in the indicated solvents. Chemical shifts are expressed in δ units and TMS as internal reference. Mass spectra were taken on an LC Autosampler Device: Standard G1313A instrument. TLC was performed on Silica Gel GF254 for TLC (Merck) and spots were visualized by iodin evapours or by irradiation with UV light (λ = 254 nm). Flash column chromatography was performed on column packed with Silica Gel60 (230–400 mesh). Solvents were reagent grade and when necessary were purified and dried by standard methods. Concentration of the reaction solutions involved the use of rotary evaporator at reduced pressure.

4.1.1. General Procedure for the Preparation of 2a and 2b

2,6-Dichloro-9-H-purine (1.89 g, 10 mmol) and Et3N (1.6 mL, 11 mmol) was dissolved in t-BuOH (50 mL), and the mixed solution of 3,4-dimethoxybenzylamine (1a, 1.50 mL, 10 mmol) and t-BuOH (20 mL) or benzo[d] dioxol-5-ylmethanamine (1b, 1.27 mL, 10 mmol) and t-BuOH (20 mL) was slowly added to the previous solution in 20 min at 60°C. The resulting mixture was stirred at 60°C for 30 min. Then the reaction mixture was poured into cold H2O (100 mL), the resulting precipitate was collected by filtration underreduced pressure and washed sequentially with H2O then dried to give the corresponding product 2a as a white power with yield of 90.5% or 2b as a white power with yield of 89.7%.

4.1.2. General Procedure for the Preparation of 4

N-Boc-4-hydroxypiperidine (3, 8.05 g, and 40 mmol) was dissolved in DCM (50 mL), then methanesulfonyl chloride (3.4 mL, 44 mmol), Et3N (6.7 mL, 48 mmol) and 4-DMAP (0.05 g, 4.0 mmol) were added to the above solution slowly in turn at 0°C. The mixed solution of methanesulfonyl chloride (3.4 mL, 44 mmol) and DCM (20 mL) was slowly added to the mixed solution in 30 min at 60°C. The mixtures were stirred at 0°C overnight. Then the reaction solution was alkalized to PH = 9 with NaHCO3. The solution was washed by water (50 mL) then extracted with DCM (3 × 15 mL). Combined organic phase dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the product 4 as white solid. Yield: 94.3%.

4.1.3. General Procedure for the Preparation of 5a and 5b

2-Chloro-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine(2a, 3.20 g, 10 mmol) or N-(benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(piperidin-4-yl)-9H-purin-6-amine(2b, 3.03 g, 10 mmol) was dissolved in DMF (60 mL) and added 60% dispersion NaH (0.48 g, 12 mmol) to the solution at room temperature. Then it slowly added the intermediate 4 (3.10 g, 11 mmol) that dissolved in DMF (20 mL) to the previous mixed solution in 20 min at 60°C. The reaction mixturewas rapid stirred at 100°C for 16 h (monitored by TLC). After removal of the solvent under reduced pressure, water (30 mL) was added and extracted with ethyl acetate (3 × 10 mL). Combined organic phase was washed with brine and dried over anhydrous Na2SO4 to give the corresponding crude product, which was purified by flash column chromatography to afford compounds 5a as a pink crystal with yield of 31.5% or 5b as a pink crystal with yield of 29.7%.

4.1.4. General Procedure for the Preparation of 6a and 6b

To a solution of 5a (600 mg, 1.19 mmol) or 5b (600 mg, 1.23 mmol) in DCM (20 mL) TFA was added (3 mL) at room temperature and the solution was stirred for 5 h. Then the reaction solution was alkalized to PH = 9 with NaHCO3. The solution was washed by water (50 mL) and then extracted with DCM (3 × 15 mL). Combined organic phase dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the product 6a or 6b.

2-Chloro-N-(3,4-dimethoxbenzyl)-9-(piperidin-4-yl)-9H-purin-6-amine (6a). White crystal, yield 91%, mp: 157–160°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.73 (1H, s), 8.25(1H, s), 7.07(1H, s), 6.87 (2H, d, J = 4.04 Hz), 5.08 (1H, s, NH), 4.55 (2H, d, J = 5.36 Hz), 4.36 (1H, dt, J1 = 10.80 Hz, J2 = 5.12 Hz), 3.74 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.08 (2H, d, J = 8.88 Hz), 2.65 (2H, dt, J1 = 14.28 Hz, J2 = 3.52 Hz), 1.92–1.94 (4H, m). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 149.8, 149.0, 148.3, 140.0, 132.1, 120.1, 118.9, 112.4, 112.1, 56.0, 55.8, 53.3, 45.6 (2C), 43.5, 33.0 (2C). ESI-MS: m/z 403.5 (M + 1), 405.5 (M + 3), 525.3 (M + Na). C19H23ClN6O2 (402.16).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(piperidin-4-yl)-9H-purin-6-amine (6b). White crystal, yield 87%, mp: 205–209°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.73 (1H, s), 8.26 (1H, s), 6.93 (1H, s), 6.83 (2H, d, J = 7.68 Hz), 5.97 (2H, s, OCH2O), 5.05 (1H, s, NH), 4.54 (2H, d, J = 4.92 Hz), 4.35 (1H, dt, J1 = 10.48 Hz, J2 = 5.40 Hz), 3.05 (2H, d, J = 12.32 Hz), 2.60 (2H, dt, J1 = 14.76 Hz, J2 = 3.60 Hz), 1.96 (4H, dd, J1 = 16.64 Hz, J2 = 2.80 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 149.9, 147.6, 146.6, 140.1, 133.7, 121.1, 118.9, 101.3, 55.3, 53.4, 45.7 (2C), 43.4, 33.2 (2C). ESI-MS: m/z 387.3 (M + 1), 389.3 (M + 3), 409.4 (M + Na). C18H19ClN6O2 (386.13).

4.1.5. General Procedure for the Target Compounds 7a and 7b

Compound 6a (or 6b) was dissolved in anhydrous DMF (10 mL) in the presence of anhydrous K2CO3 (1.2 eq), followed by addition of appropriate substituted benzyl chloride (bromine, fluorine) (1.1 eq). The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure, and then water (20 mL) was added. Extracted with ethyl acetate (3 × 10 mL), and the organic phase was washed with saturated sodium chloride (10 mL) then dried over anhydrous Na2SO4 to give the corresponding crude product, which was purified by flash column chromatography to afford compounds 7a1-13 (or 7b1-13).

2-Chloro-9-(1-(3-chlorobenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a1). Recrystallized from EA/PE as a white crystal, yield 62.3%, mp: 176–180°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30 (1H, s), 7.29–7.39 (4H, m), 7.06 (1H, s), 6.86 (2H, d, J = 4.32 Hz), 5.07 (1H, s, NH), 4.55 (2H, d, J = 7.24 Hz), 4.30 (1H, dt, J1 = 8.32 Hz, J2 = 3.27 Hz), 3.73 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.54 (2H, s), 2.92 (2H, d, J = 7.80 Hz), 2.11–2.19 (4H, m), 1.93 (2H, d, J = 8.80 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.0, 149.0, 148.3, 141.7, 140.1, 133.4, 132.2, 130.5, 128.9, 127.9, 127.4, 120.1, 118.9, 112.5, 112.2, 61.4, 56.0, 55.9, 52.9, 52.5 (2C), 43.5, 31.9 (2C). ESI-MS: m/z 527.3 (M + 1), 529.2 (M + 3), 549.3 (M + Na). C26H28Cl2N6O2 (526.17).

2-Chloro-9-(1-(3-bromobenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a2). Recrystallized from EA/PE as a white crystal, yield 57.2%, mp: 187–190°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.31 (1H, s), 7.61 (1H, dd, J1 = 7.96, J2 = 1.0 Hz), 7.52 (1H, dd, J1 = 7.64 Hz, J2 = 1.52 Hz), 7.40 (1H, dt, J1 = 7.48 Hz, J2 = 1.0 Hz), 7.23 (1H, dt, J1 = 7.76 Hz, J2 = 1.68 Hz), 7.06 (1H, s), 6.86 (2H, d, J = 8.26 Hz), 5.07 (1H, s, NH), 4.54 (2H, d, J = 5.44 Hz), 4.34 (1H, dt, J1 = 11.72 Hz, J2 = 3.84 Hz), 3.73 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.60 (2H, s), 2.96 ( 2H, d, J = 11.52 Hz), 2.27 (2H, t, J = 11.68 Hz), 2.08–2.17 (2H, m), 1.95–1.99 (2H, d, J = 7.28 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.0, 149.0, 148.3, 140.0, 138.0, 133.0, 132.2, 131.3, 129.4, 128.0, 124.4, 120.1, 119.0, 112.5, 112.2, 61.4, 56.0, 55.9, 52.8, 52.7 (2C), 43.5, 31.9 (2C). ESI-MS: m/z 573.2 (M + 3), 574.4 (M + 4), 576.2 (M + 6). C26H28BrClN6O2 (570.11).

2-Chloro-9-(1-(4-cyanobenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a3). Recrystallized from EA/PE as a white crystal, yield 66.5%, mp: 193–196°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30 (1H, s), 7.82 (2H, d, J = 4.10 Hz), 7.54 (2H, d, J = 4.08 Hz), 7.06 (1H, s), 6.87 (2H, dd, J1 = 8.64 Hz, J2 = 3.26 Hz), 5.07 (1H, s, NH), 4.54 (2H, d, J = 5.20 Hz), 4.32 (1H, dt, J1 = 8.62 Hz, J2 = 3.56 Hz), 3.74 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.62 (2H, s), 2.90 (2H, d, J = 9.28 Hz), 2.17 (4H, dd, J1 = 16.60, J2 = 8.88 Hz), 1.95 (2H, d, J = 11.56 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 149.9, 149.0, 148.3, 145.1, 140.1, 132.6 (2C), 132.1, 130.0 (2C), 120.1, 119.3, 118.9, 112.4, 112.1, 110.2, 61.6, 56.0, 55.8, 52.8, 52.6 (2C), 43.5, 31.8 (2C). ESI-MS: m/z 518.4 (M + 1), 540.4 (M + Na). C27H28ClN7O2 (517.20).

2-Chloro-9-(1-(4-nitrobenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a4). Recrystallized from EA/PE as a yellow crystal, yield: 53.4%, mp: 209–212°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.29 (1H, s), 8.21 (2H, d, J = 8.12 Hz), 7.62 (2H, d, J = 8.08 Hz), 7.06 (1H,s), 6.89 (2H, d, J = 4.68 Hz), 5.07 (1H, s, NH), 4.55 (2H, d, J = 6.32 Hz), 4.32 (1H, dt, J1 = 11.32, J2 = 3.28 Hz), 3.74 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.67 (2H, s), 2.92 (2H, t, J = 9.08 Hz), 2.14–2.25 (4H, m), 1.97 (2H, d, J = 4.58 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.0, 149.0, 148.3, 147.3, 147.1, 140.1, 132.1, 130.2 (2C), 124.0 (2C), 120.1, 118.9, 112.4, 112.1, 61.3, 56.0, 55.8, 52.8, 52.6 (2C), 43.5, 31.8 (2C). ESI-MS: m/z 538.4 (M + 1), 540.4 (M + 3), 560.3 (M + Na). C26H28ClN7O4 (537.19).

2-Chloro-9-(1-(3-fluorobenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a5). Recrystallized from EA/PE as a white crystal, yield: 49.7%, mp: 183–185°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30 (1H, s), 7.38 (1H, dt, J1 = 8.04 Hz, J2 = 6.16 Hz), 7.17 (2H, dd, J1 = 9.36 Hz, J2 = 6.28 Hz), 7.09 (2H, s), 6.87 (2H, d, J = 3.26 Hz), 5.07 (1H, s, NH), 4.55 (2H, d, J = 5.56 Hz), 4.31 (1H, dt, J1 = 10.28 Hz, J2 = 3.68 Hz), 3.74 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.55 (2H, s), 2.93 (2H, d, J = 7.16 Hz), 2.11–2.17 (4H, m), 1.93 (2H, d, J = 8.88 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 163.9, 161.5, 155.3, 153.2, 149.9, 149.0, 148.3, 142.0, 140.0, 132.1, 130.5, 125.2, 120.1, 118.9, 115.7, 114.3, 112.4, 61.5, 56.0, 55.8, 52.8, 52.5 (2C), 43.5, 31.8 (2C). ESI-MS: m/z 511.5 (M + 1), 513.4 (M + 3), 515.4 (M + 5), 533.3 (M + Na). C26H28ClFN6O2 (510.19).

2-Chloro-9-(1-(pyridine-4-yl-methyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a6). Recrystallized from EA/PE as a yellow crystal, yield: 61.2%, mp: 158–161°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.53 (2H, dd, J1 = 4.56 Hz, J2 = 1.36 Hz), 8.30 (1H, s), 7.35 (2H, d, J = 5.80 Hz), 7.06 (1H, s), 6.86 (2H, d, J = 2.28 Hz), 5.06 (1H, s, NH), 4.53 (2H, d, J = 5.44 Hz), 4.32 (1H, dt, J1 = 7.28 Hz, J2 = 3.76 Hz), 3.73 (3H, s, OCH3), 3.70 (3H, s, OCH3), 3.57 (2H, s), 2.92 (2H, d, J = 9.40 Hz), 2.13–2.20 (4H, m), 1.94 (2H, d, J = 11.2 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.3 (2C), 149.0, 148.3, 148.0, 140.1, 132.2, 124.2 (2C), 120.2, 119.3, 118.9, 112.5, 112.2, 60.9, 56.0, 55.9, 52.7, 52.6 (2C), 43.5, 31.8 (2C). ESI-MS: m/z 494.4 (M + 1), 496.4 (M + 3), 516.5 (M + Na). C25H28ClN7O2 (493.20).

2-Chloro-9-(1-benzylpiperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a7). Recrystallized from EA/PE as a white crystal, yield: 55.7%, mp: 179–182°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.29 (1H, s), 7.33 (4H, t, J = 4.68 Hz), 7.27 (1H, t, J = 4.24 Hz), 7.06 (1H, s), 6.87 (2H, d, J = 5.06 Hz), 5.07 (1H, s, NH), 4.55 (2H, d, J = 5.16 Hz), 4.31 (1H, dt, J1 = 9.26 Hz, J2 = 4.04 Hz), 3.73 (3H, s, OCH3), 3.70 (3H, s, OCH3), 3.52 (2H, s), 2.93 (2H, d, J = 6.24 Hz), 2.12–2.14 (4H, m), 1.94 (2H, d, J = 6.43 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.0, 149.0, 148.3, 140.1, 138.9, 132.1, 129.3(2C), 128.7(2C), 127.4, 120.1, 118.9, 112.3, 112.1, 62.3, 56.0, 55.8, 52.9, 52.6 (2C), 43.5, 31.8 (2C). ESI-MS: m/z 493.4 (M + 1), 495.3 (M + 3), 515.4 (M + Na). C26H29ClN6O2 (492.20).

2-Chloro-9-(1-(4-bromobenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a8). Recrystallized from EA/PE as a white crystal, yield 52.5%, mp: 209–212°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30(1H, s), 7.52 (2H, d, J = 1.60 Hz), 7.29 (2H, d, J = 8.36 Hz), 7.07 (1H, s), 6.87 (2H, d, J = 4.38 Hz), 5.07 (1H, s, NH), 4.55 (2H, d, J = 5.76 Hz), 4.29 (1H, dt, J1 = 8.37 Hz, J2 = 5.76 Hz), 3.74 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.49 (2H, s), 2.91 (2H, d, J = 6.36 Hz), 1.99–2.14 (4H, m), 1.93 (2H, d, J = 6.24 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.0, 149.0, 148.3, 140.1, 138.4, 132.1, 131.5 (2C), 131.4 (2C), 120.4, 120.1, 118.9, 112.4, 112.1, 61.4, 56.0, 55.8, 52.8, 52.5 (2C), 43.5, 31.8 (2C). ESI-MS: m/z 571.3 (M + 1), 573.2 (M + 3), 575.3 (M + 5), 595.3 (M + Na). C26H28BrClN6O2 (570.11).

2-Chloro-9-(1-(3-cyanobenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a9). Recrystallized from EA/PE as a white crystal, yield 62.1%, mp: 209–210°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30 (1H, s), 7.77 (1H, dd, J1 = 25.48 Hz, J2 = 7.60 Hz), 7.68 (2H, d, J = 7.80 Hz), 7.56 (1H, t, J = 7.68 Hz), 7.06 (1H, s), 6.87 (2H, d, J = 9.84 Hz), 5.07 (1H, s, NH), 4.55 (2H, d, J = 5.16 Hz), 4.33 (1H, dt, J1 = 10.84, J2 = 3.26 Hz), 3.73 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.59 (2H, s), 2.93 (2H, d, J = 9.16 Hz), 2.11–2.18 (4H, m), 1.93 (2H, d, J = 12.80 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.0, 149.0, 148.3, 140.8, 140.1, 134.1, 132.6, 132.1, 131.3, 129.9, 120.1, 119.4, 118.9, 112.4, 111.7, 61.1, 56.0, 55.8, 52.7, 52.5 (2C), 43.5, 31.8 (2C). ESI-MS: m/z 518.4 (M + 1), 520.3 (M + 3), 522.6 (M + 5), 540.3 (M + Na). C27H28ClN7O2 (517.20).

2-Chloro-9-(1-(4-methoxybenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a10). Recrystallized from EA/PE as a white crystal, yield 56.3%, mp: 202–205°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.28 (1H, s), 7.24 (2H, d, J = 8.56 Hz), 7.06 (1H, s), 6.90 (2H, d, J = 8.60 Hz), 6.86 (2H, d, J = 4.56 Hz), 5.07 (1H, s, NH), 4.54 (2H, d, J = 5.48 Hz), 4.27 (1H, dt, J1 = 8.62 Hz, J2 = 3.64 Hz), 3.74 (3H, s, OCH3), 3.73 (3H, s, OCH3), 3.70 (3H, s), 3.44 (2H, s), 2.92 (2H, d, J = 6.32 Hz), 2.07–2.09 (4H, m), 1.92 (2H, s). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 158.8, 155.3, 153.2, 149.9, 149.0, 148.3, 140.1, 132.1, 130.6, 130.5 (2C), 120.1, 118.9, 114.0 (2C), 112.4, 112.1, 61.7, 56.0, 55.8, 55.5, 53.0, 52.4 (2C), 43.5, 31.8 (2C). ESI-MS: m/z 523.5 (M + 1), 525.4 (M + 3), 545.3 (M + Na). C27H31ClN6O3 (522.21).

2-Chloro-9-(1-(2,6-dicholobenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a11). Recrystallized from EA/PE as a white crystal, yield 58.3%, mp: 196–198°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.28 (1H, s), 7.34 (2H, dt, J1 = 17.22 Hz, J2 = 7.20 Hz), 7.34 (1H, dd, J1 = 8.56 Hz, J2 = 7.48 Hz), 7.06 (1H, s), 6.86 (2H, d, J= 3.82 Hz), 5.06 (1H, s, NH), 4.55 (2H, d, J = 4.56 Hz), 4.29 (1H, dt, J1 = 10.26, J2 = 4.32 Hz), 3.74 (3H, s, OCH3), 3.72 (2H, s), 3.70 (3H, s, OCH3), 2.93 (2H, d, J = 11.68 Hz), 2.40 (2H, dt, J1 = 9.92 Hz, J2 = 1.80 Hz), 1.93–2.06 (4H, m). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.0, 149.0, 148.3, 140.1, 136.5(2C), 134.3, 132.0, 130.4, 129.2 (2C), 120.1, 118.9, 112.4, 112.1, 62.3, 56.3, 56.0, 55.8, 52.5 (2C), 43.2, 31.8 (2C). ESI-MS: m/z 561.2 (M + 1), 563.2 (M + 3), 565.3 (M + 5). C26H27Cl3N6O2 (560.13).

2-Chloro-9-(1-(2,4,6-trimethylbenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a12). Recrystallized from EA/PE as a white crystal, yield: 63.3%, mp: 203–206°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.27(1H, s), 7.06 (1H, s), 6.86 (2H, s), 6.80 (2H, s), 5.07 (1H, s, NH), 4.54 (2H, d, J = 5.48 Hz), 4.40 (1H, dt, J1 = 11.24 Hz, J2 = 5.48 Hz), 3.73 (3H, s, OCH3), 3.70 (3H, s, OCH3), 3.43 (2H, s), 2.86 (2H, d, J = 11.4 Hz), 2.32–2.33 (6H, m), 2.19–2.21 (5H, m), 1.92–1.94 (4H, m). 13C NMR (100 MHz, DMSO-d6, ppm) δ:155.3, 153.2, 149.9, 149.0, 148.3, 139.9, 137.9(2C), 136.0, 132.2, 132.1, 129.1 (2C), 120.1, 118.9, 112.4, 112.1, 56.0, 55.8, 55.4, 53.0, 52.3 (2C), 43.5, 32.1 (2C), 21.0, 20.2 (2C). ESI-MS: m/z 535.3 (M + 1), 537.4 (M + 3). C29H35ClN6O2 (534.25).

2-Chloro-9-(1-(2-fluorobenzyl)piperidin-4-yl)-N-(3,4-dimethoxbenzyl)-9H-purin-6-amine (7a13). Recrystallized from EA/PE as a white crystal, yield 54.3%, mp: 188–192°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.27 (1H, s), 7.44 (1H, dt, J1 = 8.04 Hz, J2 = 1.80 Hz), 7.34 (1H, dt, J1 = 5.48 Hz, J2 = 1.72 Hz), 7.15–7.22 (2H, m), 7.06 (1H, s), 6.86 (2H, d, J = 4.32 Hz), 5.07 (1H, s, NH), 4.55 (2H, d, J = 4.96 Hz), 4.29 (1H, dt, J1 = 11.26 Hz, J2 = 7.40 Hz), 3.73 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.58 (2H, s), 2.95 ( 2H, d, J = 10.52 Hz), 2.09–2.22 (4H, m), 1.93 (2H, d, J = 8.28 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 162.5, 160.1, 155.3, 153.2, 149.9, 149.0, 148.3, 140.1, 132.1, 129.6, 125.1, 124.6, 120.1, 118.9, 115.7, 115.5, 112.3, 56.0, 55.8, 54.9, 52.8, 52.4 (2C), 43.5, 31.8 (2C). ESI-MS: m/z 511.4 (M + 1), 513.4 (M + 3), 533.3 (M + Na). C26H28ClFN6O2 (510.19).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(3-chlorobenzyl)piperidin-4-yl)-9H-purin-6-amine (7b1). Recrystallized from EA/PE as a white crystal, yield 55.9%, mp: 146–150°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.32 (1H, s), 7.38 (1H, d, J = 10.44 Hz), 7.33 (3H, dd, J = 9.96 Hz, 8.28 Hz), 6.93 (1H, s), 6.83 (2H, dd, J1 = 8.38 Hz, J2 = 2.83 Hz), 5.96 (2H, s, OCH2O), 5.04 (1H, s, NH), 4.53 (2H, d, J = 5.20 Hz), 4.32 (1H, dt, J1 = 9.24, J2 = 3.64 Hz), 3.54 (2H, s), 2.92 (2H, d, J = 7.92 Hz), 2.15 (4H, t, J = 9.72 Hz), 1.96 (2H, t, J = 8.88 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.0, 147.6, 146.6, 141.5, 140.0, 133.6, 133.4, 130.5, 128.9, 127.9, 127.4, 126.7, 121.1, 118.9, 108.5, 101.3, 61.4, 52.8, 52.5 (2C), 43.4, 31.8 (2C). ESI-MS: m/z 511.3 (M + 1), 513.4 (M + 3), 533.2 (M + Na). C25H24Cl2N6O2 (510.13).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(3-bromobenzyl)piperidin-4-yl)-9H-purin-6-amine (7b2). Recrystallized from EA/PE as a white crystal, yield 62.1%, mp: 174–178°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.31 (1H, s), 7.61 (1H, d, J = 7.92 Hz), 7.52 (1H, d, J = 7.48 Hz), 7.39 (1H, t, J = 7.36 Hz), 7.21 (1H, t, J = 7.44 Hz), 6.92 (1H, s), 6.83 (2H, dd, J1 = 12.16, J2 = 4.28 Hz), 5.97 (2H, s, OCH2O), 5.04 (1H, s, NH), 4.53 (2H, d, J = 5.28 Hz), 4.32 (1H, dt, J1 = 7.92 Hz, J2 = 4.48 Hz), 3.61 (2H, s), 2.96 (2H, d, J = 11.40 Hz), 2.27 (2H, t, J = 11.44 Hz), 2.14 (2H, dd, J1 = 21.84 Hz, J2 = 11.72 Hz), 1.96 (2H, d, J = 10.24 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.2, 153.2, 150.0, 147.6, 146.6, 140.1, 137.9, 133.6, 133.0, 131.3, 129.4, 128.1, 124.4, 121.1, 120.3, 118.9, 108.5, 101.3, 61.4, 55.3, 52.6 (2C), 43.4, 31.8 (2C). ESI-MS: m/z 555.2 (M + 1), 558.2 (M + 4), 579.1 (M + Na). C25H24BrClN6O2 (554.08).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(4-cyanobenzyl)piperidin-4-yl)-9H-purin-6-amine (7b3). Recrystallized from EA/PE as a white crystal, yield 58.4%, mp: 221–225°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30 (1H, s), 7.80 (2H, d, J = 8.04 Hz), 7.54 (2H, d, J = 8.06 Hz), 6.83 (2H, dd, J1 = 10.88 Hz, J2 = 7.84 Hz), 6.93 (1H, s), 5.97 (2H, s, –OCH2O–), 5.03 (1H, s, NH), 4.53 (2H, d, J = 4.68 Hz), 4.32 (1H, dt, J1 = 10.88 Hz, J2 = 4.26 Hz), 3.62 (2H, s), 2.92 (2H, d, J = 9.20 Hz), 2.17 (4H, t, J = 9.44 Hz), 1.97 (2H, t, J = 11.60 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.3, 153.2, 150.0, 147.6, 146.6, 145.1, 143.2, 140.1, 133.6 (2C), 132.6, 130.0 (2C), 128.4, 121.1, 119.4, 110.2, 108.5, 101.3, 65.9, 61.6, 52.5 (2C), 43.3, 31.8 (2C). ESI-MS: m/z 502.3 (M + 1), 504.3 (M + 3), 506.3 (M + 5), 524.4 (M + Na). C26H24ClN7O2 (501.17).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(4-nitrobenzyl)piperidin-4-yl)-9H-purin-6-amine (7b4). Recrystallized from EA/PE as a yellow crystal, yield 49.5%, mp: 137–141°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30 (1H, s), 8.22 (2H, dd, J1 = 7.08 Hz, J2 = 1.68 Hz), 7.62 (2H, d, J = 8.68 Hz), 6.92 (1H, s), 6.83 (2H, dd, J1 = 8.38 Hz, J2 = 2.83 Hz), 5.96 (2H, s, –OCH2O–), 5.04 (1H, s, NH), 4.53 (2H, d, J = 5.20 Hz), 4.32 (1H, dt, J1 = 11.40, J2 = 3.88 Hz), 3.67 (2H, s), 2.93 (2H, d, J = 10.44 Hz), 2.17 (4H, t, J = 10.44 Hz), 1.96 (2H, t, J = 3.84 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.2, 153.2, 149.7, 147.6, 147.3, 147.1, 146.6, 140.2, 133.6, 130.2 (2C), 128.6, 124.1 (2C), 121.1, 118.9, 108.5, 101.3, 61.3, 52.8, 52.6 (2C), 43.3, 31.8 (2C). ESI-MS: m/z 522.3 (M + 1), 524.4 (M + 3), 544.4 (M + Na). C25H24ClN7O4 (521.16).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(3-fluorobenzyl)piperidin-4-yl)-9H-purin-6-amine (7b5). Recrystallized from EA/PE as a white crystal, yield 64.1%, mp: 144–148°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.31 (1H, s), 7.38 (1H, dt, J1 = 8.04 Hz, J2 = 6.48 Hz), 7.17 (2H, dt, J1 = 7.96 Hz, J2 = 2.12 Hz), 7.08 (1H, dt, J1 = 8.64 Hz, J2 = 2.23 Hz), 6.93 (1H, s), 6.83 (2H, t, J = 7.72 Hz), 5.97 (2H, s, –OCH2O–), 5.05 (1H, s, NH), 4.54 (2H, d, J = 5.72 Hz), 4.32 (1H, dt, J1 = 7.24 Hz, J2 = 4.26 Hz), 3.55 (2H, s), 2.93 (2H, d, J = 6.92 Hz), 2.16 (4H, t, J = 8.96 Hz), 1.94 (2H, d, J = 7.72 Hz). 13C NMR (400 MHz, DMSO-d6, ppm) δ: 163.9, 161.5, 155.3, 153.2, 150.0, 147.6, 146.6, 142.1, 140.1, 133.6, 130.6, 125.1, 121.1, 118.9, 115.7, 114.2, 108.5, 101.2, 61.5, 52.8, 52.5 (2C), 43.4, 31.8 (2C). ESI-MS: m/z 495.3 (M + 1), 497.4 (M + 3), 517.4 (M + Na). C25H24ClFN6O2 (494.16).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(pyridine-4-yl-methyl)piperidin-4-yl)-9H-purin-6-amine (7b6). Recrystallized from EA/PE as a yellow crystal, yield: 62.3%, mp: 154–157°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.53 (2H, dd, J1 = 4.48 Hz, J2 = 1.44 Hz), 8.31 (1H, s), 7.35 (2H, d, J = 5.84 Hz), 6.92 (1H, s), 6.83 (2H, dd, J1 = 10.12 Hz, J2 = 7.80 Hz), 5.97 (2H, s, –OCH2O–), 5.04 (1H, s, NH), 4.53 (2H, d, J = 5.76 Hz), 4.32 (1H, dt, J1 = 10.48 Hz, J2 = 3.62 Hz), 3.57 (2H, s), 2.93 (2H, d, J = 9.20 Hz), 2.17 (4H, t, J = 12.16 Hz), 1.95 (2H, d, J = 10.64 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.2, 153.2, 150.0 (2C), 148.0, 147.6, 147.0, 146.6, 140.1, 133.6, 124.2 (2C), 121.1, 120.3, 118.9, 108.5, 101.3, 60.9, 52.6, 49.1 (2C), 43.4, 31.8 (2C). ESI-MS: m/z 478.3 (M + 1), 480.3 (M + 3), 482.4 (M + 5), 500.2 (M + Na). C24H24ClN7O2 (477.17).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-benzylpiperidin-4-yl)-9H-purin-6-amine (7b7). Recrystallized from EA/PE as a white crystal, yield 58.5%, mp: 169–172°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30 (1H, s), 7.33 (4H, t, J = 4.60 Hz), 7.25 (1H, dd, J1 = 8.52 Hz, J2 = 4.28 Hz), 6.92 (1H, s), 6.83 (2H, dd, J1 = 10.0 Hz, J2 = 7.80 Hz), 5.96 (2H, s, –OCH2O–), 5.04 (1H, s, NH), 4.52 (2H, d, J = 5.68 Hz), 4.29 (1H, dt, J1 = 7.81, J2 = 3.88 Hz), 3.52 (2H, s), 2.92 (2H, d, J = 6.36 Hz), 2.13 (4H, dt, J1 = 11.36, J2 = 7. 40 Hz), 1.94 (2H, s). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.2, 153.1, 150.0, 147.6, 146.6, 140.2, 138.9, 133.6, 129.3 (2C), 128.6 (2C), 127.4, 121.1, 120.3, 118.9, 108.5, 101.3, 62.3, 52.9, 52.6 (2C), 43.4, 31.8 (2C). ESI-MS: m/z 477.3 (M + 1), 479.3 (M + 3), 581.4 (M + 5), 599.3 (M + Na). C25H25ClN6O2 (476.17).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(4-bromobenzyl)piperidin-4-yl)-9H-purin-6-amine (7b8). Recrystallized from EA/PE as a white crystal, yield 54.3%, mp: 204–208°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30 (1H, s), 7.54 (2H, d, J = 1.60 Hz), 7.28 (2H, d, J = 8.32 Hz), 6.93 (1H, s), 6.83 (2H, dd, J1 = 7.26 Hz, J2 = 1.84 Hz), 5.97 (2H, s, –OCH2O–), 5.04 (1H, s, NH), 4.54 (2H, d, J = 5.80 Hz), 4.30 (1H, dt, J1 = 7.28 Hz, J2 = 4.28 Hz), 3.50 (2H, s), 2.92 (2H, d, J = 6.6 Hz), 2.20 (4H, dt, J1 = 12.36, J2 = 8.60 Hz), 1.94 (2H, s). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.2, 153.2, 150.0, 147.6, 146.7, 140.2, 138.4, 133.6, 131.5 (2C), 131.4 (2C), 121.1, 120.9, 120.4, 118.9, 108.5, 101.2, 61.4, 52.9, 52.5 (2C), 43.4, 31.8 (2C). ESI-MS: m/z 557.1 (M + 3), 559.1 (M + 5). C25H24BrClN6O2 (554.08).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(3-cyanobenzyl)piperidin-4-yl)-9H-purin-6-amine (7b9). Recrystallized from EA/PE as a white crystal, yield 48.4%, mp: 200–204°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.31 (1H, s), 7.76 (1H, dd, J1 = 10.84 Hz, J2 = 7.64 Hz), 7.69 (2H, d, J = 7.84 Hz), 6.57 (1H, t, J = 7.68 Hz), 6.93 (1H, s), 6.83 (2H, dd, J1 = 9.76, J2 = 7.80 Hz), 5.97 (2H, s, –OCH2O–), 5.04 (1H, s, NH), 4.53 (2H, d, J = 5.40 Hz), 4.31 (1H, dt, J1 = 8.12, J2 = 3.84 Hz), 3.60 (2H, s), 2.93 (2H, d, J = 9.28 Hz), 2.17 (4H, dt, J1 = 12.04 Hz, J2 = 9.68 Hz), 1.94 (2H, d, J = 11.04 Hz). 13C NMR (400 MHz, DMSO-d6, ppm) δ: 155.2, 153.2, 150.0, 147.6, 146.6, 140.8, 140.1, 134.1, 133.6, 132.5, 131.3, 129.9, 121.1, 119.4, 118.8, 111.7, 108.5, 101.3, 61.1, 55.4, 52.7, 52.5 (2C), 43.4, 31.8 (2C). ESI-MS: m/z 502.3 (M + 1), 504.3 (M+3), 506.3 (M + 5), 524.4 (M + Na). C26H24ClN7O2 (501.17).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(4-methoxybenzyl)piperidin-4-yl)-9H-purin-6-amine (7b10). Recrystallized from EA/PE as a white crystal, yield 59.4%, mp: 182–185°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.30 (1H, s), 7.24 (2H, d, J = 8.52 Hz), 6.90 (3H, t, J = 6.92 Hz), 6.83 (2H, dd, J1 = 7.52 Hz, J2 = 3.68 Hz), 5.97 (2H, s, –OCH2O–), 5.04 (1H, s, NH), 4.54 (2H, d, J = 5.92 Hz), 4.28 (1H, dt, J1 = 8.25, J2 = 3.32 Hz), 3.74 (3H, s), 3.44 (2H, s), 2.92 (2H, d, J = 6.24 Hz), 2.07 (4H, t, J = 7.88 Hz), 1.93 (2H, s). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 158.8, 155.3, 153.2, 150.0, 147.6, 146.6, 140.1, 134.5, 133.6, 130.7, 130.5 (2C), 121.1, 118.9, 114.0 (2C), 108.5, 101.3, 61.7, 55.5, 53.0, 52.4 (2C), 43.4, 31.8 (2C). ESI-MS: m/z 507.3 (M + 1), 509.3 (M + 3), 529.3 (M + Na). C26H27ClN6O3 (506.18).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(2,6-dicholobenzyl)piperidin-4-yl)-9H-purin-6-amine (7b11). Recrystallized from EA/PE as a white crystal, yield: 56.8%, mp: 199–203°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.28(1H, s), 7.48 (2H, dt, J1 = 8.40 Hz, J2 = 0.76 Hz), 7.34 (1H, dd, J1 = 7.54 Hz, J1 = 8.62 Hz), 6.92 (1H, s), 6.83 (2H, dd, J1 = 11.74, J1 = 7.9 Hz), 5.97 (2H, s, –OCH2O–), 5.04 (1H, s, NH), 4.54 (2H, d, J = 5.76 Hz), 4.54 (2H, d, J = 6.04 Hz), 4.32 (1H, dt, J1 = 7.82 Hz, J2 = 4.26 Hz), 3.74 (2H, s), 2.95 (2H, d, J = 11.6 Hz), 2.39 (2H, t, J = 1.6 Hz), 1.99 (4H, dt, J1 = 11.51 Hz, J2 = 9.62 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.2, 153.2, 150.0, 147.6, 146.6, 140.1, 136.6 (2C), 134.3, 133.6, 131.9, 130.4, 129.1 (2C), 121.1, 118.9, 108.5, 101.3, 62.3, 56.3, 52.7 (2C), 43.3, 31.8 (2C). ESI-MS: m/z 547.2 (M + 3), 549.2 (M + 5), 551.2 (M + 7). C25H23Cl3N6O2 (544.09).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(2,4,6-trimethylbenzyl)piperidin-4-yl)-9H-purin-6-amine (7b12). Recrystallized from EA/PE as a white crystal, yield 63.4%, mp: 164–167°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.28 (1H, s), 6.92 (1H, s), 6.80–6.85 (4H, m), 5.96 (2H, s, –OCH2O–), 5.03 (1H, s, NH), 4.53 (2H, d, J = 5.20 Hz), 4.29 (1H, dt, J1 = 10.28 Hz, J2 = 4.80 Hz), 3.44 (2H, s), 2.86 (2H, d, J = 11.40 Hz), 2.32 (6H, s), 2.19–2.21 (5H, m), 1.94–1.96 (4H, m). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 155.2, 153.2, 150.0, 147.6, 146.6, 140.0, 138.1, 137.9, 136.0, 133.6, 132.2, 129.1 (2C), 121.0, 118.8, 108.5, 101.2, 61.9, 55.4, 52.3 (2C), 43.3, 32.1 (2C), 21.0, 20.2, 19.7. ESI-MS: m/z 519.4 (M + 1), 521.3 (M + 3), 523.5 (M + 5), 541.4 (M + Na). C28H31ClN6O2 (518.22).

N-(Benzo[d] dioxol-5-ylmethyl)-2-chloro-9-(1-(2-fluorobenzyl)piperidin-4-yl)-9H-purin-6-amine (7b13). Recrystallized from EA/PE as a white crystal, yield 64.8%, mp: 192–195°C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.29 (1H, s), 7.44 (1H, dt, J1 = 8.0, J2 = 1.56 Hz), 7.33 (1H, dt, J1 = 5.64, J2 = 1.92 Hz), 7.18 (2H, dd, J1 = 14.2 Hz, J2 = 6.4 Hz), 6.92 (1H, s), 6.85 (2H, t, J = 7.8 Hz), 5.97 (2H, s, –OCH2O–), 5.04 (1H, s, NH), 4.54 (2H, d, J = 5.76 Hz), 4.30 (1H, dt, J1 = 11.52 Hz, J2 = 4.16 Hz), 3.59 (2H, s), 2.95 (2H, d, J = 10.2 Hz), 2.15 (4H, dt, J1 = 23.2 Hz, J2 = 11.72 Hz), 1.94 (2H, t, J = 12.88 Hz). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 162.4, 160.0, 155.2, 153.2, 149.9, 147.6, 146.7, 140.2, 133.6, 132.1, 129.6, 125.2, 124.6, 121.1, 118.9, 115.7, 108.5, 101.3, 54.9, 52.8, 52.4 (2C), 43.4, 31.8 (2C). ESI-MS: m/z 495.3 (M + 1), 497.4 (M + 3), 517.4 (M + Na). C25H24ClFN6O2 (494.16).

4.2. Acetylcholinesterase Inhibition Assay

The ability of derivatives 7a1-7a13 and 7b1-7b13 to inhibit human acetylcholinesterase (hAChE) (Sigma-Aldrich, USA) was examined using the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) method (to generate a yellow chromophore (5-mercapto-2-nitrobenzoic acid) detectable at the 405–412 nm range) with the acetylcholinesterase assay kit (Keygen, China). The assay was carried out as a previously described protocol [10] using donepezil as controls ( ). Stock solutions of test compounds were dissolved in a minimum volume of DMSO (1%) and were diluted using saline. In 96-well plates, 50 μL of AChE (0.22 U/mL diluted using saline) was incubated with 10 μL of various concentrations of test compounds (1.0, 10.0, 100 μM) at room temperature for 10 min followed by the addition of relative agents according to the kit instruction. The absorbance was measured at a wavelength of 405 nm with Thermo MK3 microplate reader. Percent inhibition was calculated by the comparison of compound-treated to various control incubations.

Conflict of Interests

The authors declare no conflict of interests.

Acknowledgments

The financial support from the National Natural Science Foundation of China (NSFC no. 81102320, no. 81273354), Key Project of NSFC for International Cooperation (no. 30910103908), the Natural Science Foundation of Shandong Province (ZR2009CM016), and China Postdoctoral Science Foundation funded project (no. 20100481282, 2012T50584) is gratefully acknowledged.

References

  1. A. Enz, R. Amstutz, H. Boddeke, G. Gmelin, and J. Malanowski, “Brain selective inhibition of acetylcholinesterase: a novel approach to therapy for Alzheimer's disease,” Progress in Brain Research, vol. 98, pp. 431–438, 1993. View at Google Scholar · View at Scopus
  2. G. R. Proctor and A. L. Harvey, “Synthesis of tacrine analogues and their structure-activity relationships,” Current Medicinal Chemistry, vol. 7, no. 3, pp. 295–302, 2000. View at Google Scholar · View at Scopus
  3. H. Sugimoto, Y. Iimura, Y. Yamanishi, and K. Yamatsu, “Synthesis and structure-activity relationships of acetylcholinesterase inhibitors: 1-benzyl-4-[(5,6-dimethoxy-1-oxoindan-2-yl)methyl]piperidine hydrochloride and related compounds,” Journal of Medicinal Chemistry, vol. 38, no. 24, pp. 4821–4829, 1995. View at Publisher · View at Google Scholar · View at Scopus
  4. D. L. Bai, X. C. Tang, and X. C. He, “Huperzine A, a potential therapeutic agent for treatment of Alzheimer's disease,” Current Medicinal Chemistry, vol. 7, no. 3, pp. 355–374, 2000. View at Google Scholar · View at Scopus
  5. J. Winkler, L. J. Thal, F. H. Gage, and L. J. Fisher, “Cholinergic strategies for Alzheimer's disease,” Journal of Molecular Medicine, vol. 76, no. 8, pp. 555–567, 1998. View at Publisher · View at Google Scholar · View at Scopus
  6. A. Musiał, M. Bajda, and B. Malawska, “Recent developments in cholinesterases inhibitors for Alzheimer's disease treatment,” Current Medicinal Chemistry, vol. 14, no. 25, pp. 2654–2679, 2007. View at Publisher · View at Google Scholar · View at Scopus
  7. T. Mohamed and P. P. Rao, “Design, synthesis and evaluation of 2,4-disubstituted pyrimidines as cholinesterase inhibitors,” Bioorganic & Medicinal Chemistry Letters, vol. 20, no. 12, pp. 3606–3609, 2010. View at Google Scholar
  8. T. Mohamed, X. Zhao, L. K. Habib, J. Yang, and P. P. Rao, “Design, synthesis and structure-activity relationship (SAR) studies of 2,4-disubstituted pyrimidine derivatives: dual activity as cholinesterase and Aβ-aggregationinhibitors,” Bioorganic & Medicinal Chemistry Letters, vol. 19, no. 7, pp. 2269–2281, 2011. View at Google Scholar
  9. T. Mohamed, J. C. Yeung, and P. P. Rao, “Development of 2-substituted-N-(naphth-1-ylmethyl) and N-benzhydrylpyrimidin-4-amines as dual cholinesterase and Aβ-aggregation inhibitors: synthesis and biological evaluation,” Bioorganic & Medicinal Chemistry Letters, vol. 21, pp. 5881–5887, 2011. View at Google Scholar
  10. T. Mohamed, J. C. Yeung, M. S. Vasefi, M. A. Beazely, and P. P. Rao, “Development and evaluation of multifunctional agents for potential treatment of Alzheimer's disease: application to a pyrimidine-2,4-diamine template,” Bioorganic & Medicinal Chemistry Letters, vol. 22, no. 14, pp. 4707–4712, 2012. View at Google Scholar
  11. P. V. Reddy, N. G. Nair, M. A. Smith, and W. Kudo, “Purine-based triazoles,” WO2012051296A2.
  12. J. J. Cui, M. Tran-Dubé, H. Shen et al., “Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK),” Journal of Medicinal Chemistry, vol. 54, no. 18, pp. 6342–6363, 2011. View at Publisher · View at Google Scholar · View at Scopus