Design, synthesis and AChE inhibitory activity of indanone and aurone derivatives

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European Journal of Medicinal Chemistry 44 (2009) 7e17 http://www.elsevier.com/locate/ejmech

Original article

Design, synthesis and AChE inhibitory activity of indanone and aurone derivatives Rong Sheng a, Yu Xu a, Chunqi Hu a, Jing Zhang b, Xiao Lin a, Jingya Li c, Bo Yang b, Qiaojun He b, Yongzhou Hu a,* a

ZJU-ENS joint laboratory of Medicinal Chemistry, School of Pharmaceutical Sciences, Zijingang Campus, Zhejiang University Hangzhou 310058, People’s Republic of China b Department of Pharmacology, School of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China c The National Center for Drug Screening, Shanghai 201203, People’s Republic of China Received 26 November 2007; received in revised form 6 March 2008; accepted 6 March 2008 Available online 16 March 2008

Abstract A new series of indanone and aurone derivatives have been synthesized and tested for in vitro AChE inhibitory activity by modified Ellman method. Most of them exhibit AChE inhibitory activities superior to rivastigmine. Further, the most potent compound 1g was selected to evaluate the effect on the acquisition and memory impairment by mice step-down passive avoidance test. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Indanone; Aurone; Synthesis; AChE inhibitory activity

1. Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and one of the most common causes of mental deterioration in the elderly. Considerable research efforts have been devoted to elucidate the molecular, biochemical, and cellular mechanisms of AD in the past decades. Several hypotheses have been proposed attempting to explain the pathogenesis of AD, including b-amyloid deposition, tau hyperphosphorylation, acetylcholine deficiency, inflammation, and oxidative stress, while the etiology of this disease is not known, and interventions able to halt or slow disease progression are as yet unproven. Up to now, AChE inhibitors are still the major and most developed class of drugs approved for AD therapy, such as donepezil, rivastigime, and galanthamine have been approved by FDA and EMEA for the symptomatic treatment of AD [1]. The X-ray crystallography of AChE (TcAChE) has ˚ gorge demonstrated that it possesses a long and narrow 20 A

* Corresponding author. Tel./fax: þ86 571 8820 8460. E-mail address: [email protected] (Y. Hu). 0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2008.03.003

with two separate ligand binding sites, a catalytic site (active site) and a peripheral anionic site (PAS) [2], the latter was found to be related with the no-catalytic function of AChE, accelerating b-amyloid peptide deposition and promoting the formation of b-amyloid fibril. It was postulated that AChE binds through its peripheral site to the nonamyloidogenic form of Ab protein acting as a chaperone protein and inducing conformational change to the amyloidogenic form with subsequent amyloid fibril formation [3e5]. Moreover, it has been shown that molecules that were able to interact with both active and peripheral sites of AChE could prevent the aggregating activity of AChE toward Ab besides the inhibitory activity [6]. All these findings stimulated a great interest in designing dual-site binding AChE inhibitors [7e9]. In our previous study, a series of 2-phenoxy-indan-1-one derivatives were proven as potent and selective AChE inhibitors in vitro (Fig. 1c), which were characterized by combining 5,6-dimethoxy-indan-1-one from donepezil (Fig. 1a) and dialkylbenzyl amine from rivastigmine (Fig. 1b), with oxygen as the linkage. The docking study reveals that these series compounds are able to bind the active and peripheral sites simultaneously [10]. It was reported that chalcone and aurone

R. Sheng et al. / European Journal of Medicinal Chemistry 44 (2009) 7e17

8

with LiAlH4eEt2NH in pentane to yield key intermediate 6aej [14]. Then, the reaction of 6aej with 5,6-dimethoxy-indan-1-one [15] or 5,6-dimethoxy-benzo-furan-3(2H)-one [16] was carried out in the presence of KOH or Al2O3, to provide 1aep [17,18]. The trans-conformation of compounds 1aep could be confirmed by the chemical shift of vinyl proton in 1 H NMR, because of the deshielding effect resulting from the carbonyl group, the vinyl protons should give a signal at a greater chemical shift than in the cis isomer, the value is higher than 7.0 [19]. Finally, 2aep were obtained through catalytic hydrogenation of 1aep with 5% Pd/C as catalyst in THF. All the newly synthesized compounds were characterized by 1H NMR, ESIeMS and IR spectroscopy.

O N

O

O

H3CO N H3CO

N

a

b O

H3CO

O N

H3CO

R1

c

R2 R3

Fig. 1.

derivatives exhibit high binding affinity to Ab aggregates in vitro [11,12], therefore, introduction of these moieties into the molecules may increase the affinity to Ab and enhance the therapeutic efficacy. Following these reasons, a series of new indanone and aurone derivatives were designed, synthesized and evaluated for ChE inhibitory activities, in which 5,6-dimethoxy-indan-1-one was remained or replaced by its bioisostere 5,6-dimethoxy-benzofuran-3(2H)-one, ]CH or eCH2 was chosen as the linkage, with different sort of aminoalkyl groups substituted at the para or meta position of the benzene. Besides study on the pharmacological activities of these derivatives, the docking program by computational modeling was also performed. 2. Results and discussion 2.1. Chemistry The target compounds 1aep and 2aep were synthesized as illustrated in Scheme 1. The readily available materials methyl 3- (or 4-) methyl benzoates 3a,b were brominated with NBS following previously reported method to give 4a,b in high yield [13]. The obtained 4a,b reacted with various secondary amines (dimethylamine, pyrrolidine, etc.) in CH2Cl2 at reflux conditions to afford 5aej, followed by selective reduction COOCH3

2.2. Biological activities All the newly synthesized indanone and aurone derivatives were tested for their inhibitory activities toward AChE and BuChE in vitro according to the modified Ellman method using rat cortex homogenate (AChE) and rat serum (BChE) with commercially available donepezil and rivastigmine as the reference standard [20]. The ChE inhibitory results are summarized in Table 1. Most of the tested compounds demonstrated higher inhibitory activities against AChE than rivastigmine and high selectivity for AChE over BuChE. Some general conclusions can be drawn from the results, as follows: (1) The variation of linkage exhibits obvious effect on the AChE inhibitory activities, compounds containing ]CH linkage (i.e. 1g, 1l, 1n, 1p) were more potent than that of containing CH2 linkage (i.e. 2g, 2l, 2n, 2p). (2) The compounds with aminomethyl at the paraposition of benzene ring (i.e. 1d, 1g, 2b, 2k) were more effective than those substituted at meta-position (1c, 1f, 2a, 2j). (3) The compounds having dimethylamine group (i.e. 1a, 2b) showed less activity than those having other groups (1g, 1o, 2k, 2p). (4) The change of Y did not show obvious effect on AChE inhibition, aurone derivatives (i.e. 1l, 2i) demonstrated similar activities to indanone derivatives (i.e. 1j, 2g). CHO

COOCH3

COOCH3

a

b

CH3

c CH2N

CH2Br

3a,b

5a-j

4a,b

R1 CH2N

R2

6a-j

O

d

Y

CH2N

1a-p

(

Y= O, CH2

R2

O

H3CO H3CO

R1

N

R1 R2

=

N

e

R1

H3CO

R2

CH3 CH3

H3CO

N

CH3 CH2CH3

N

Y

2a-p

CH2CH3 CH2CH3

CH2N

N

N

R1 R2

)

Scheme 1. (a) NBS, CCl4, hn, reflux 4 h; (b) secondary amine, CH2Cl2, reflux 2 h; (c) LiAlH4eEt2NH, pentane, r.t. 2e3 h; (d) Y]CH2, 5,6-dimethoxyindan-1one, KOH, CH3OH, r.t. 2e4 h; Y]O, 5,6-dimethoxybenzofuran-3(2H)-one, Al2O3, CH2Cl2, r.t. 2e4 h; (e) 5% Pd/C, THF, r.t. 6e8 h.

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Table 1 Structures and ChE inhibitory activities of 1aep and 2aep Compound

NR1R2

X

Y

Substituted position

Donepezil Rivastigmine N

1c 1d 1e

N

CH3 CH3

N

meta para

2.14 0.42

89.3 315

42 750

]CH

CH2

meta para para

0.49 0.27 0.70

36.8 20.1 23.6

75 74 34

meta para meta para

0.44 0.035 1.18 0.14

71 82 65.1 47.9

161 2342 55 342

1j 1k 1l 1m

N

meta para meta para

0.24 0.045 0.29 0.16

41.6 26.6 72.0 36.4

173 591 248 227

1n 1o 1p

N

meta para para

0.16 0.10 0.082

55.2 84.6 45

345 846 549

CH2CH3 CH3CH3

]CH

CH2 O

]CH

CH2 O

]CH

CH2 O

2a 2b

N

2c 2d 2e

N

CH3 CH3

eCH2

CH2

meta para

3.74 1.38

25.2 40.7

7 29

eCH2

CH2

meta para para

0.69 0.38 1.54

18.5 34.5 25.9

27 91 17

meta para meta para

1.48 0.29 1.07 0.44

32.0 11.7 38.2 16.4

22 40 36 37

meta para meta para

2.58 0.15 7.02 0.62

42.2 33.2 57.6 49.8

16 221 8 80

meta para meta

0.66 0.12 0.24

70.4 48.3 98.1

107 402 408

CH3 CH2CH3

O

2f 2g 2h 2i

N

2j 2k 2l 2m

N

2n 2o 2p

N

CH2CH3 CH2CH3

231 0.7

CH2

CH3 CH2CH3

2.77 1.41

Selectivity for AChE

]CH

O

1f 1g 1h 1i

b

IC50 for BChE (mM)b

0.012 2.07

1a 1b

a

IC50 for AChE (mM)a

eCH2

CH2 O

eCH2

CH2 O

eCH2

CH2 O

Assay performed using rat cortex homogenate. Values are means of three different experiments. Assay performed using rat serum.

The most potent AChE inhibitory activity in vitro was observed for the compounds 1g with the IC50 values of 0.035 mM, which was chosen as the typical compound and evaluated for its pharmacological effects on cognitive enhancement in vivo. Mice step-down passive avoidance test was used to evaluate the effects of 1g on the acquisition and memory impairment induced by scopolamine [21]. Table 2 disclose that, in the acquisition memory process, scopolamine markedly reduced the escape latency on the platform and increased the times of step-down. Similar to donepezil and rivastigmine, 1g significantly ameliorate scopolamine-induced deficit at different concentrations (2, 5, 10 mg/kg, i.g.). Therefore, new indanone derivative 1g demonstrated as a potent, reversible, and highly selective AChE inhibitor and it could remarkably improve the acquisition and memory deficits. All the results suggest that indanone derivative 1g might be a promising candidate in the treatment of AD.

2.3. Molecular docking To gain insight into the molecular determinants that modulate the inhibitory activity of these compounds, molecular docking simulations for 1g to TcAChE were performed using the flexX program in Sybyl 6.9.1 software based on the Xray crystal structure of TcAChE-E2020 complex [22]. The docking and subsequent scoring were performed using the default parameters of the FlexX program, and the binding gorge of TcAChE composed of the central catalytic pocket and peripheral sites were taken as the binding site for docking. Fig. 2 demonstrated that 1g has a nice fit in the active-site gorge of AChE similar to donepezil, binding to the central subsite and the peripheral anionic site simultaneously. As shown in Fig. 3, near the bottom of the gorge (the central subsite), besides the charged nitrogen makes a cation-p interaction with Trp84, the diethylamine moiety also displays hydrophobic contacts

R. Sheng et al. / European Journal of Medicinal Chemistry 44 (2009) 7e17

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Table 2 Effect of donepezil, rivastigmine and 1g on memory deficit of acquisition process induced by scopolamine as evaluated by step-down passive avoidance test Drugs

Dose/ mg kg1

Number of mice

Memory abilities (times)

Escape latency (s)

Saline Scopolamine Scopolamine þ donepezil Scopolamine þ rivastigmine Scopolamine þ 1g

0 2.0 2.0 2.0 2.0 5.0 10.0

10 10 10 10 10 10 10

0.80  0.77 4.20  1.40a 1.90  1.52c 2.00  1.05d 2.67  1.87 2.00  1.41c 1.60  1.17d

142  52 5  6a 75  73c 73  49d 47  45b 55  56c 60  68c

a

p < 0.001 vs vehicle group. p < 0.05, cp < 0.01, dp < 0.001 vs model group.

4. Experimental protocols All solvents used were of analytical grade. Melting points were recorded on a Buchi apparatus and were not corrected, IR spectra, KBr pellets, 400e4000 cm1 were recorded on a Bruker VECTOR 22 FTIR spectrophotometer. 1H NMR and 13C NMR spectra were recorded on a Bruker AM 400 instrument (chemical shifts are expressed as d values relative to TMS as internal standard). Mass spectra (MS) and ESI (positive) were recorded on an Esquire-LC-00075 spectrometer. 4.1. Synthesis

b

with Asp72 and Phe331. In the middle of the gorge, the phenyl ring formed p-p stacking with Tyr334 and Phe330, respectively. At the top of the gorge (the PAS), the indanone ring stacks against the indole ring of Trp279 through classical pp interaction, and the oxygen of indanone makes a hydrogen bond with the nitrogen of the Phe288.

4.1.1. Procedure for preparation of 4a, 4b Methyl 3-methyl-benzoate (15.0 g, 0.1 mol) was dissolved in 150 mL carbon tetrachloride and N-bromosuccinimide (20.0 g, 0.11 mol) was added. The mixture was refluxed for 6 h accompanied with illumination and the white succinimide residue was filtered off. The solvent was evaporated under reduced pressure to give a yellow liquid, which was purified with ether at 78  C to get 4a as a yellow liquid at room temperature (21.6 g, 94.3% yield). Compound 4b was obtained from methyl 4-methyl-benzoate with the yield of 91.3%, yellow solid, m.p. 52e54  C (52e53  C [23]).

3. Conclusion In summary, on the basis of designing dual-site binding AChE inhibitors, a series of indanone and aurone derivatives were prepared and evaluated ChE inhibitory activities in vitro. Most of them demonstrated high activities against AChE, while almost no activities were observed against BChE in vitro. Compound 1g exhibited high AChE inhibitory activity (IC50 ¼ 0.035 mM) in vitro and can remarkably improve the memory deficits induced by scopolamine in mice step-down passive avoidance test. The modeling studies clearly indicated that 1g was nicely accommodated by AChE. Further seeking the AD candidates based on these results is in progress and will be reported in due course.

4.1.2. General procedure for preparation of compounds 5aej To a solution of 4a (2.29 g, 0.01 mol) in 20 mL CH2Cl2, was added 33% dimethylamine solution (3.1 mL, 0.02 mol) at r.t., the mixture was refluxed for 2 h, the solvent was removed under vacuum to nearly dryness. The residue was acidified with 20 mL of 2 mol/L hydrochloric acid. The acid solution was washed with ethyl acetate (10 mL  3), and concentrated ammonium hydroxide was added thereto up to clearly basic, the product was extracted with ethyl acetate (20 mL  3). The extract was washed with brine and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure to give crude product and then purified by silica gel column chromatography eluting with petroleum, ethyl acetate and triethylamine (20:10:1) to afford compound 5a (1.11 g, 57.5%) as oil. 4.1.2.1. Methyl 3-[(dimethylamino)methyl]-benzoate 5a. Yield: 57.5%; 1H NMR (CDCl3) d 7.92 (m, 2H, H-2 and H-6), 7.49 (d, 1H, J ¼ 7.2 Hz, H-4,), 7.37 (t, 1H, J ¼ 7.6 Hz, H-5), 3.90 (s, 3H, OCH3), 3.45 (s, 2H, benzylic-CH2), 2.23 (s, 6H, 2NCH3); MS (ESI), m/z ¼ 194 [M þ 1]; IR (KBr): 3021, 2949, 2855, 2817, 1725, 1589, 1434, 1286, 749, 694 cm1. 4.1.2.2. Methyl 4-[(dimethylamino)methyl]-benzoate 5b. Yield: 50.4%; 1H NMR (CDCl3) d 7.99 (d, 2H, J ¼ 8.0 Hz, H-2 and H-6), 7.38 (d, 2H, J ¼ 8.0 Hz, H-3 and H-5), 3.91 (s, 3H, OCH3), 3.47 (s, 2H, benzylic-CH2), 2.25 (s, 6H, 2NCH3); MS (ESI), m/z ¼ 194 [M þ 1]; IR (KBr): 3012, 2976, 2817, 1723, 1611, 1435, 1279, 865 cm1.

Fig. 2. Topography of the constricted region in the gorge of AChE.

4.1.2.3. Methyl 3-[(ethylmethylamino)methyl]-benzoate 5c. Yield: 51.7%; 1H NMR (CDCl3) d 7.97 (s, 1H, H-2), 7.90

R. Sheng et al. / European Journal of Medicinal Chemistry 44 (2009) 7e17

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Fig. 3. The binding modes of 1g in the gorge of AChE.

(d, 1H, J ¼ 7.6 Hz, H-6,), 7.51 (d, 1H, J ¼ 7.6 Hz, H-4,), 7.36 (t, 1H, J ¼ 7.6 Hz, H-5,), 3.90 (s, 3H, OCH3), 3.51 (s, 2H, benzylic-CH2), 2.43 (q, 2H, J ¼ 6.8 Hz, CH2CH3,), 2.17 (s, 3H, NCH3), 1.07 (t, 3H, J ¼ 6.8 Hz, CH2CH3); MS (ESI), m/ z ¼ 208 [M þ 1]; IR (KBr): 3025, 2971, 2950, 2839, 1725, 1589, 1434, 1285, 748, 693 cm1. 4.1.2.4. Methyl 4-[(ethylmethylamino)methyl]-benzoate 5d. Yield: 54.1%; 1H NMR (CDCl3) d 7.97 (d, 2H, J ¼ 8.0 Hz, H-2 and H-6), 7.38 (d, 2H, J ¼ 8.0 Hz, H-3 and H-5), 3.90 (s, 3H, OCH3), 3.53 (s, 2H, benzylic-CH2), 2.42 (q, 2H, J ¼ 7.2 Hz, CH2CH3), 2.19 (s, 3H, NCH3), 1.08 (t, 3H, J ¼ 7.2 Hz, CH2eCH3); MS (ESI), m/z ¼ 208 [M þ 1]; IR (KBr): 3016, 2971, 2842, 1725, 1611, 1435, 1278, 859 cm1. 4.1.2.5. Methyl 3-(diethylaminomethyl)-benzoate 5e. Yield: 76.0%; 1H NMR (CDCl3) d 7.99 (s, 1H, H-2), 7.90 (d, 1H, J ¼ 7.6 Hz, H-6), 7.55 (d, 1H, J ¼ 7.6 Hz, H-4), 7.36 (t, 1H, J ¼ 7.6 Hz, H-5), 3.91 (s, 3H, OCH3), 3.60 (s, 2H, benzylicCH2), 2.49 (q, 4H, J ¼ 7.6 Hz, 2CH2CH3), 1.02 (t, 6H, J ¼ 7.6 Hz, 2CH2CH3); MS (ESI), m/z ¼ 222 [M þ 1]; IR

(KBr): 3024, 2970, 2873, 1725, 1589, 1433, 1284, 745, 692 cm1. 4.1.2.6. Methyl 4-(diethylaminomethyl)-benzoate 5f. Yield: 68.7%; 1H NMR (CDCl3) d 7.97 (d, 2H, J ¼ 8.0 Hz, H-2 and H-6), 7.41 (d, 2H, J ¼ 8.0 Hz, H-3 and H-5), 3.91 (s, 3H, OCH3), 3.60 (s, 2H, benzylic-CH2), 2.49 (q, 4H, J ¼ 7.2 Hz, 2CH2eCH3), 1.02 (t, 6H, J ¼ 7.2 Hz, 2CH2-CH3); MS (ESI), m/z ¼ 222 [M þ 1]; IR (KBr): 3047, 2970, 2873, 1724, 1611, 1435, 1278, 858 cm1. 4.1.2.7. Methyl 3-[(pyrrolidin-1-yl)methyl]-benzoate 5g. Yield: 69.4%; 1H NMR (CDCl3) d 8.00 (s, 1H, H-2), 7.92 (d, 1H, J ¼ 7.6 Hz, H-6), 7.54 (d, 1H, J ¼ 8.0 Hz, H-4), 7.37 (t, 1H, J ¼ 7.6 Hz, H-5), 3.91 (s, 3H, OCH3), 3.65 (s, 2H, benzylic-CH2), 2.49 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.77 (m, 4H, pyrrolidine-CH2, H-3 and H-4); MS (ESI), m/z ¼ 220 [M þ 1]. IR (KBr): 3018, 2951, 2870, 1723, 1588, 1433, 1283, 749, 699 cm1. 4.1.2.8. Methyl 4-[(pyrrolidin-1-yl)methyl]-benzoate 5h. Yield: 59.8%; 1H NMR (CDCl3) d 7.98 (d, 2H, J ¼ 8.0 Hz,

12

R. Sheng et al. / European Journal of Medicinal Chemistry 44 (2009) 7e17

H-2 and H-6), 7.40 (d, 2H, J ¼ 8.0 Hz, H-3 and H-5), 3.91 (s, 3H, OCH3), 3.66 (s, 2H, benzylic-CH2), 2.49 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.77 (m, 4H, pyrrolidine-CH2, H-3 and H-4); MS (ESI), m/z ¼ 220 [M þ 1]; IR (KBr): 3027, 2954, 2783, 1723, 1612, 1435, 1278, 855 cm1.

J ¼ 7.6 Hz, H-4), 7.47 (t, 1H, J ¼ 7.6 Hz, H-5), 3.56 (s, 2H, benzylic-CH2), 2.45e2.50 (q, 4H, J ¼ 7.2 Hz, 2CH2-CH3), 2.20 (s, 3H, NeCH3), 1.10e1.13 (t, 3H, J ¼ 7.2 Hz, 2CH2e CH3); MS (ESI), m/z ¼ 178 [M þ 1]; IR (KBr): 3020, 2970, 2924, 2848, 2726, 1699, 1604, 1588, 1450, 784, 690 cm1.

4.1.2.9. Methyl 3-[(piperidin-1-yl)methyl]-benzoate 5i. Yield: 78.9%; 1H NMR (CDCl3) d 7.97 (s, 1H, H-2), 7.91 (d, 1H, J ¼ 7.6 Hz, H-6), 7.53 (d, 1H, H-4, J ¼ 8.0 Hz), 7.36 (t, 1H, H-5, J ¼ 7.6 Hz), 3.91 (s, 3H, OCH3), 3.50 (s, 2H, benzylicCH2), 2.37 (m, 4H, piperidine-CH2, H-20 and H-60 ), 1.54 (m, 4H, H-30 and H-50 ), 1.43 (m, 2H, H-40 ); MS (ESI), m/z ¼ 234 [M þ 1]; IR (KBr): 3026, 2935, 2853, 1725, 1589, 1434, 1285, 748, 694 cm1.

4.1.3.4. 4-[(Ethylmethylamino)methyl]-benzaldehyde 6d. Yield: 47.8%; 1H NMR (CDCl3) d 10.00 (s, 1H, CHO), 7.83 (d, 2H, J ¼ 7.6 Hz, H-2 and H-6), 7.50 (d, 2H, J ¼ 8.0 Hz, H-3 and H-5), 3.57 (s, 2H, benzylic-CH2), 2.45 (q, 2H, J ¼ 7.2 Hz, 2CH2eCH3), 2.21 (s, 3H, NeCH3), 1.10 (t, 3H, J ¼ 7.2 Hz, 2CH2eCH3); MS (ESI), m/z ¼ 178 [M þ 1]; IR (KBr): 3031, 2969, 2926, 2847, 2735, 1700, 1607, 1577, 1456, 835 cm1.

4.1.2.10. Methyl 4-[(piperidin-1-yl)methyl]-benzoate 5j. Yield: 76.8%; 1H NMR (CDCl3) d 7.97 (d, 2H, J ¼ 8.0 Hz, H-2 and H-6), 7.39 (d, 2H, J ¼ 8.0 Hz, H-3 and H-5), 3.91 (s, 3H, OCH3), 3.51 (s, 2H, benzylic-CH2), 2.37 (m, 4H, piperidine-CH2, H-2 and H-6), 1.55 (m, 4H, piperidine-CH2, H-3 and H-5), 1.43 (m, 2H, H-4); MS (ESI), m/z ¼ 234 [M þ 1]; IR (KBr): 3031, 2935, 2853, 1724, 1612, 1435, 1279, 866 cm1.

4.1.3.5. 3-[(Diethylamino)methyl]-benzaldehyde 6e. Yield: 52.8%; 1H NMR (CDCl3) d 10.02 (s, 1H, CHO), 7.86 (s, 1H, H-2), 7.75 (d, 1H, J ¼ 7.6 Hz, H-6), 7.62 (d, 1H, J ¼ 7.6 Hz, H-4), 7.45 (t, 1H, J ¼ 7.6 Hz, H-5), 3.63 (s, 2H, benzylic-CH2), 2.51 (q, 4H, J ¼ 6.8 Hz, 2CH2eCH3), 1.03 (t, 6H, J ¼ 6.8 Hz, 2CH2eCH3); MS (ESI), m/z ¼ 192 [M þ 1]; IR (KBr): 3024, 2970, 2934, 2724, 1703, 1604, 1588, 1449, 785, 689 cm1.

4.1.3. General procedure for preparation of compounds 6aej LiAlH4 (97%, 78 mg, 2.0 mmol) was dissolved in 10 mL THF, then, the THF solvent was pumped off, and diethylamine (0.41 mL, 4.0 mmol) in 5 mL of pentane was added. To this slurry was added 5a (0.39 g, 2.0 mmol) and the mixture was stirred vigorously for 2 h at room temperature. Then, the reaction mixture was cooled to 0  C, and the precipitate was filtered and washed with cold pentane several times. The separated pentane portion was washed with brine and dried over anhydrous Na2SO4. The solvent was removed under vacuum to give crude product, which was purified by silica gel column chromatography eluting with petroleum and acetone (20:1) to afford compound 6a (0.15 g, 46%) as oil.

4.1.3.6. 4-[(Diethylamino)methyl]-benzaldehyde 6f. Yield: 55.4%; 1H NMR (CDCl3) d 10.00 (s, 1H, CHO), 7.82 (d, 2H, J ¼ 8.0 Hz, H-2 and H-6), 7.52 (d, 2H, J ¼ 8.0 Hz, H-3 and H-5), 3.64 (s, 2H, benzylic-CH2), 2.51 (q, 2H, J ¼ 7.2 Hz, 2CH2eCH3), 1.03 (t, 3H, J ¼ 7.2 Hz, 2CH2e CH3); MS (ESI), m/z ¼ 192 [M þ 1]; IR (KBr): 3014, 2969, 2934, 2730, 1701, 1607, 1577, 1454, 831 cm1.

4.1.3.1. 3-[(Dimethylamino)methyl]-benzaldehyde 6a. Yield: 46.0%; 1H NMR (CDCl3) d 10.02 (s, 1H, CHO), 7.83 (s, 1H, H-2), 7.78 (d, 1H, J ¼ 7.6 Hz, H-6), 7.59 (d, 1H, J ¼ 7.6 Hz, H-4), 7.47 (t, 1H, J ¼ 7.6 Hz, H-5), 3.50 (s, 2H, CH2), 2.26 (s, 6H, 2 NeCH3); MS (ESI), m/z ¼ 164 [M þ 1]; IR (KBr): 3021, 2942, 2855, 2818, 2725, 1697, 1589, 1457, 797, 691 cm1. 4.1.3.2. 4-[(Dimethylamino)methyl]-benzaldehyde 6b. Yield: 44.4%; 1H NMR (CDCl3) d 10.03 (s, 1H, CHO), 7.84 (d, 2H, J ¼ 7.6 Hz, H-2 and H-6), 7.48 (d, 2H, J ¼ 7.6 Hz, H-3 and H-5), 3.50 (s, 2H, benzylic-CH2), 2.26 (s, 6H, 2 NCH3); MS (ESI), m/z ¼ 164 [M þ 1]; IR (KBr): 3006, 2943, 2849, 2731, 1700, 1607, 1577, 1456, 817 cm1. 4.1.3.3. 3-[(Ethylmethylamino)methyl]-benzaldehyde 6c. Yield: 44.9%; 1H NMR (CDCl3) d 10.02 (s, 1H, CHO), 7.84 (s, 1H, H-2), 7.77 (d, 1H, J ¼ 8.0 Hz, H-6), 7.61 (d, 1H,

4.1.3.7. 3-[(Pyrrolidin-1-yl)methyl]-benzaldehyde 6g. Yield: 47.6%; 1H NMR (CDCl3) d 10.02 (s, 1H, CHO), 7.86 (s, 1H, H-2), 7.77 (d, 1H, J ¼ 7.6 Hz, H-6), 7.62 (d, 1H, J ¼ 7.6 Hz, H-4), 7.46 (t, 1H, J ¼ 7.2 Hz, H-5), 3.69 (s, 2H, benzylic-CH2), 2.51 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.79 (m, 4H, H-3 and H-4); MS (ESI), m/z ¼ 190 [M þ 1]; IR (KBr): 3026, 2962, 2927, 2732, 1699, 1605, 1588, 1460, 783, 692 cm1. 4.1.3.8. 4-[(Pyrrolidin-1-yl)methyl]-benzaldehyde 6h. Yield: 54.1%; 1H NMR (CDCl3) d 10.00 (s, 1H, CHO), 7.83 (d, 2H, J ¼ 7.6 Hz, H-2 and H-6), 7.51 (d, 2H, J ¼ 7.6 Hz, H-3 and H-5), 3.70 (s, 2H, benzylic-CH2), 2.52 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.79 (m, 4H, pyrrolidineCH2, H-3 and H-4); MS (ESI), m/z ¼ 190 [M þ 1]; IR (KBr): 3024, 2964, 2875, 2733, 1699, 1607, 1577, 1461, 825 cm1. 4.1.3.9. 3-[(Piperidin-1-yl)methyl]-benzaldehyde 6i. Yield: 43.9%; 1H NMR (d, CDCl3) 10.02 (s, 1H, CHO), 7.83 (s, 1H, H-2), 7.76e7.78 (d, 1H, H-6 J ¼ 7.6 Hz), 7.61e7.63 (d, 1H, H-4, J ¼ 7.6 Hz), 7.46e7.49 (t, 1H, H-5, J ¼ 7.6 Hz), 3.54 (s, 2H, benzylic-CH2), 2.39 (m, 4H, piperidine-CH2, H-20 and H-60 ), 1.56e1.61 (m, 4H, H-30 and H-50 ), 1.44e 1.45 (m, 2H, H-40 ); MS (ESI), m/z ¼ 204 [M þ 1]; IR

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13

(KBr): 3031, 2935, 2852, 2723, 1699, 1604, 1588, 1450, 801, 691 cm1.

(KBr): 3068, 2968, 2835, 1689, 1631, 1606, 1588, 1501, 1304, 1217, 805, 695 cm1.

4.1.3.10. 4-[(Piperidin-1-yl)methyl]-benzaldehyde 6j. Yield: 48.8%; 1H NMR (CDCl3) d 10.00 (s, 1H, CHO), 7.82 (d, 2H, J ¼ 7.2 Hz, H-2 and H-6), 7.49 (d, 2H, J ¼ 7.2 Hz, H-3 and H-5), 3.55 (s, 2H, benzylic-CH2), 2.39 (m, 4H, piperidine-CH2, H-2 and H-6), 1.57 (m, 4H, piperidine-CH2, H-3 and H-5), 1.44 (m, 2H H-40 ); MS (ESI), m/z ¼ 204 [M þ 1]; IR (KBr): 3024, 2935, 2852, 2732, 1702, 1607, 1577, 863 cm1.

4.1.4.4. (E )-2-{4-[(Ethylmethylamino)methyl]benzylidene}5,6-dimethoxy-2,3-dihydroinden-1-one 1d. Yield: 82.6%; m.p. 105e107  C; 1H NMR (CDCl3) d 7.58 (m, 3H, H-7, H20 and H-60 ), 7.40 (d, 2H, J ¼ 8.4 Hz, H-30 and H-50 ), 7.34 (s, 1H, C]CH), 6.99 (s, 1H, H-4), 4.00 (s, 3H, OCH3), 3.96 (s, 2H, H-3), 3.95 (s, 3H, OCH3), 3.53 (s, 2H, benzylic-CH2), 2.45 (q, 4H, J ¼ 7.2 Hz, CH2-CH3), 2.22 (s, 3H, N-CH3), 1.10 (t, 3H, J ¼ 7.2 Hz, CH2eCH3); MS (ESI), m/z ¼ 352 [M þ 1]; IR (KBr): 3063, 2969, 2836, 1683, 1629, 1606, 1590, 1501, 837 cm1.

4.1.4. General procedure for preparation of indanone derivatives 1aed, 1f, 1g, 1j, 1k, 1n and 1o To a solution of KOH (84 mg, 1.5 mmol) in 10 mL methanol, 6a (0.16 g, 1.0 mmol) and 5,6-dimethoxy- indanone (0.21 g, 1.1 mmol) were added, the mixture was stirred for 3 h at r.t., then, the solvent was evaporated to dryness, the residue was treated with 15 mL, 2 M HCl and 15 mL Et2O, shacked, the aqueous layer was separated, alkalinized with K2CO3, then extracted with EtOAc, the organic phase was separated and washed with brine, dried over anhydrous Na2SO4, then evaporated under reduced pressure to give crude product, which was purified by silica gel column chromatography eluting with petroleum, ethyl acetate and triethylamine (20:10:1) to afford compound 1a (0.27 g, 80%) as a white solid. 4.1.4.1. (E )-2-{3-[(Dimethylamino)methyl]benzylidene}-5,6dimethoxy-2,3-dihydroinden-1-one 1a. Yield: 80.1%; m.p. 96e98  C; 1H NMR (CDCl3) d 7.56 (m, 3H, H-7, H-20 and H-40 ), 7.41 (t, 1H, H-50 , J ¼ 7.6 Hz), 7.34 (m, 2H, H-60 and C]CH), 7.00 (s, 1H, H-4), 4.00 (s, 3H, OCH3), 3.98 (s, 2H, H-3), 3.95 (s, 3H, OCH3), 3.49 (s, 2H, benzylic-CH2), 2.28 (s, 6H, 2 N-CH3); MS (ESI), m/z ¼ 338 [M þ 1]; IR (KBr): 3066, 2940, 2816, 1687, 1630, 1605, 1588, 1501, 1233, 806, 695 cm1. 4.1.4.2. (E )-2-{4-[(Dimethylamino)methyl]benzylidene}-5,6dimethoxy-2,3-dihydroinden-1-one 1b. Yield: 79.2%; m.p. 135e137  C; 1H NMR (CDCl3) d 7.60e7.63 (m, 3H, H-7, H-20 and H-60 ), 7.39 (d, 2H, J ¼ 7.6 Hz, H-30 and H-50 ), 7.36 (s, 1H, C]CH), 6.99 (s, 1H, H-4), 4.01 (s, 3H, OCH3), 3.97 (s, 2H, H-3), 3.96 (s, 3H, OCH3), 3.47 (s, 2H, benzylicCH2), 2.27 (s, 6H, 2N-CH3); MS (ESI), m/z ¼ 338 [M þ 1]; IR (KBr): 3058, 2924, 2805, 1690, 1629, 1605, 1586, 824 cm1. 4.1.4.3. (E )-2-{3-[(Ethylmethylamino)methyl]benzylidene}5,6-dimethoxy-2,3-dihydroinden-1-one 1c. Yield: 79.8%; m.p. 75e77  C; 1H NMR (d, CDCl3) 7.61 (s, 2H, H-7 and H-20 ), 7.55 (d, 1H, J ¼ 7.6 Hz, H-40 ), 7.39 (t, 1H, J ¼ 7.6 Hz, H-50 ), 7.35 (m, 2H, H-60 and C]CH), 7.00 (s, 1H, H-4), 4.00 (s, 3H, OCH3), 3.98 (s, 2H, H-3), 3.96 (s, 3H, OCH3), 3.57 (s, 2H, benzylic-CH2), 2.48 (q, 2H, J ¼ 7.2 Hz, CH2eCH3), 2.24 (s, 3H, NeCH3), 1.12 (t, 3H, J ¼ 7.2 Hz, CH2eCH3); MS (ESI), m/z ¼ 352 [M þ 1]; IR

4.1.4.5. (E )-2-{3-[(Diethylamino)methyl]benzylidene}-5,6dimethoxy-2,3-dihydroinden-1-one 1f. Yield: 79.4%; m.p. 78e80  C; 1H NMR (CDCl3) d 7.64 (s, 1H, H-7), 7.61 (s, 1H, H-20 ), 7.53 (d, 1H, J ¼ 6.8 Hz, H-40 ), 7.36 (m, 3H, H-50 , H-60 and C]CH), 6.99 (s, 1H, H-4), 4.00 (s, 3H, OCH3), 3.98 (s, 2H, H-3), 3.95 (s, 3H, OCH3), 3.63 (s, 2H, benzylic-CH2), 2.53 (q, 4H, J ¼ 7.2 Hz, 2CH2eCH3), 1.06 (t, 6H, J ¼ 7.2 Hz, 2CH2eCH3); MS (ESI), m/z ¼ 366 [M þ 1]; IR (KBr): 3071, 2967, 2932, 1681, 1630, 1605, 1588, 1501, 1305, 1233, 804, 691 cm1. 4.1.4.6. (E )-2-{4-[(Diethylamino)methyl]benzylidene}-5,6dimethoxy-2,3-dihydroinden-1-one 1g. Yield: 76.7%, m.p. 116e118  C; 1H NMR (CDCl3) d 7.60 (m, 3H, H-7, H-20 and H-60 ), 7.42 (d, 2H, J ¼ 7.6 Hz, H-30 and H-50 ), 7.36 (s, 1H, C]CH), 6.99 (s, 1H, H-4), 4.00 (s, 3H, OCH3), 3.97 (s, 2H, H-3), 3.96 (s, 3H, OCH3), 3.61 (s, 2H, benzylic-CH2), 2.52 (q, 4H, J ¼ 7.2 Hz, 2CH2eCH3,), 1.05 (t, 6H, J ¼ 7.2 Hz, 2CH2eCH3); 13C NMR (CDCl3, 100 MHz) d 192.9, 155.1, 149.4, 144.7, 141.8, 134.6, 133.8, 132.1, 130.9, 130.28, 129.1, 107.0, 104.8, 57.1, 56.1, 55.9, 46.7, 32.0, 11.6; MS (ESI), m/z ¼ 366 [M þ 1]; IR (KBr): 3061, 2967, 2932, 1690, 1629, 1606, 1500, 1223, 832 cm1. Anal. Calcd for C23H27NO3: C 75.59, H 7.45, N 3.83. Found: C 75.29, H 7.16, N 3.60. 4.1.4.7. (E )-5,6-Dimethoxy-2-{3-[(pyrrolidin-1-yl)methyl]benzylidene}-2,3-dihydroinden-1-one 1j. Yield: 82.6%, m.p. 112e113  C; 1H NMR (CDCl3) d 7.61 (s, 2H, H-7 and H20 ), 7.55 (d, 1H, J ¼ 7.2 Hz, H-40 ), 7.36 (m, 3H, H-50 , H-60 and C]CH), 7.00 (s, 1H, H-4), 4.01 (s, 3H, OCH3), 3.99 (s, 2H, H-3), 3.97 (s, 3H, OCH3), 3.69 (s, 2H, benzylic-CH2), 2.56 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.82 (m, 4H, pyrrolidine-CH2, H-3 and H-4); MS (ESI), m/z ¼ 364 [M þ 1]; IR (KBr): 3064, 2960, 2825, 1689, 1630, 1601, 1585, 1234, 809, 688, cm1. 4.1.4.8. (E )-5,6-Dimethoxy-2-{4-[(pyrrolidin-1-yl)methyl]benzylidene}-2,3-dihydroinden-1-one 1k. Yield: 77.1%, m.p. 138e140  C; 1H NMR (CDCl3) d 7.60 (m, 3H, H-7, H-20 and H-60 ), 7.42 (d, 2H, J ¼ 8.0 Hz, H-30 and H-50 ), 7.35 (s, 1H, C]CH), 6.99 (s, 1H, H-4), 4.00 (s, 3H, OCH3), 3.97 (s, 2H, H-3), 3.96 (s, 3H, OCH3), 3.66 (s, 2H, benzylic-CH2),

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2.54 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.81 (m, 4H, pyrrolidine-CH2, H-3 and H-4); MS (ESI), m/z ¼ 364 [M þ 1]; IR (KBr): 3066, 2969, 2928, 1682, 1628, 1606, 1590, 1224, 826, cm1. 4.1.4.9. (E )-5,6-Dimethoxy-2-{3-[(piperidin-1-yl)methyl]benzylidene}-2,3-dihydroinden-1-one 1n. Yield: 84.9%, m.p. 127e129  C; 1H NMR (CDCl3) d 7.61 (s, 2H, H-7 and H20 ), 7.54 (d, 1H, J ¼ 6.8 Hz, H-40 ), 7.35 (m, 3H, H-50 , H-60 and C]CH), 6.99 (s, 1H, H-4), 4.01 (s, 3H, OCH3), 3.98 (s, 2H, H-3), 3.95 (s, 3H, OCH3), 3.52 (s, 2H, benzylic-CH2), 2.41 (m, 4H, piperidine-CH2, H-2 and H-6), 1.57 (m, 4H, piperidine-CH2, H-3 and H-5), 1.45 (m, 2H, piperidine-CH2, H-4); MS (ESI), m/z ¼ 378 [M þ 1]; IR (KBr): 3068, 2934, 2851, 1686, 1628, 1601, 1584, 1234, 809, 687 cm1. 4.1.4.10. (E )-5,6-Dimethoxy-2-{4-[(piperidin-1-yl)methyl]benzylidene}-2,3-dihydroinden-1-one 1o. Yield: 82.2%, m.p. 124e126  C; 1H NMR (CDCl3) d 7.59 (m, 3H, H-7, H-20 and H-60 ), 7.40 (d, 2H, J ¼ 7.6 Hz, H-30 and H-50 ), 7.34 (s, 1H, C]CH), 6.98 (s, 1H, H-4), 4.00 (s, 3H, OCH3), 3.95 (s, 5H, H-3 and OCH3), 3.52 (s, 2H, benzylic-CH2), 2.38 (m, 4H, piperidine-CH2, H-2 and H-6), 1.57e1.61 (m, 4H, piperidine-CH2, H-3 and H-5), 1.44 (m, 2H, piperidine-CH2, H4); MS (ESI), m/z ¼ 378 [M þ 1]; IR (KBr): 3064, 2969, 2933, 2852, 1689, 1633, 1606, 1589, 1222, 863 cm1. 4.1.5. General procedure for preparation of aurone derivatives 1e, 1h, 1i, 1l, 1m and 1p 5,6-Dimethoxybenzofuran-3(2H)-one (0.12 g, 0.62 mmol) and compound 6c (0.104 g, 0.59 mmol) were dissolved in 5 mL CH2Cl2, then 0.3 g Al2O3 (neutral) was added, the mixture was stirred for 3 h at r.t. filtered to remove Al2O3, washed with 15 mL CH2Cl2, the organic phase was separated and washed with brine, dried over anhydrous Na2SO4, then evaporated under reduced pressure to dryness, the residue was purified by silica gel column chromatography eluting with petroleum, ethyl acetate and triethylamine (10:10:1) to afford compound 1e (0.16 g, 76.9%) as a yellow solid. 4.1.5.1. (Z )-2-{3-[(Ethylmethylamino)methyl]benzylidene}-5,6dimethoxy-benzofuran-3(2H)-one 1e. M.p. 138e141  C; 1H NMR (CDCl3) d 7.83 (d, 2H, J ¼ 8.0 Hz, H-20 and H-60 ), 7.39 (d, 2H, J ¼ 8.0 Hz, H-30 and H-50 ), 7.18 (s, 1H, C]CH), 6.83 (s, 1H, H-4), 6.81 (s, 1H, H-7), 4.02 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.53 (s, 2H, benzylic-CH2), 2.44 (q, 2H, J ¼ 7.2 Hz, CH2eCH3), 2.22 (s, 3H, NeCH3), 1.09 (t, 3H, J ¼ 7.2 Hz, CH2eCH3); MS (ESI), m/z ¼ 354 [M þ 1]; IR (KBr): 3063, 2971, 2838, 1695, 1656, 1607, 1501, 1280, 816 cm1. 4.1.5.2. (Z )-2-{3-[(Diethylamino)methyl]benzylidene}-5,6dimethoxybenzofuran-3(2H)-one 1h. Yield: 73.1%, m.p. 95e 98  C; 1H NMR (CDCl3) d 7.81 (m, 1H, H-60 ), 7.78 (s, 1H, H-20 ), 7.38 (m, 2H, H-40 and H-50 ), 7.18 (s, 1H, C]CH), 6.82 (s, 1H, H-4), 6.81 (s, 1H, H-7), 4.02 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.62 (s, 2H, benzylic-CH2), 2.53 (q, 4H,

J ¼ 7.2 Hz, 2  CH2), 1.05 (t, 6H, J ¼ 7.2 Hz, 2  CH3); MS (ESI), m/z ¼ 368 [M þ 1]; IR (KBr): 3071, 2967, 2793, 1690, 1648, 1605, 1482, 1280, 1139, 804, 698 cm1. 4.1.5.3. (Z )-2-{4-[(Diethylamino)methyl]benzylidene}-5,6dimethoxybenzofuran-3(2H)-one 1i. Yield: 82.9%, m.p. 117e 121  C; 1H NMR (CDCl3) d 7.84 (d, 2H, J ¼ 7.6 Hz, H-20 and H-60 ), 7.43 (d, 2H, J ¼ 8.0 Hz, H-30 and H-50 ), 7.20 (s, 1H, C]CH), 6.84 (s, 1H, H-4), 6.82 (s, 1H, H-7), 4.04 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.62 (s, 2H, benzylic-CH2), 2.53 (q, 4H, J ¼ 7.6 Hz, 2  CH2), 1.05 (t, 6H, J ¼ 7.6 Hz, 2  CH3); MS (ESI), m/z ¼ 368 [M þ 1]; IR (KBr): 3061, 2966, 2934, 1693, 1652, 1606, 1498, 1276, 814 cm1. 4.1.5.4. (Z )-5,6-Dimethoxy-2-{3-[(pyrrolidin-1-yl)methyl]benzylidene}-benzofuran-3(2H)-one 1l. Yield: 63.4%, m.p. 125e130  C; 1H NMR (CDCl3) d 7.83 (m, 1H, H-60 ), 7.78 (s, 1H, H-20 ), 7.39 (m, 2H, H-40 and H-50 ), 7.18 (s, 1H, C]CH), 6.83 (s, 1H, H-4), 6.82 (s, 1H, H-7), 4.03 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.68 (s, 2H, benzylic-CH2), 2.53 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.79 (m, 4H, pyrrolidine-CH2, H-3 and H-4); MS (ESI), m/z ¼ 366 [M þ 1]; IR (KBr): 3064, 2964, 2783, 1691, 1653, 1608, 1499, 1281,1135, 818, 697 cm1. 4.1.5.5. (Z )-5,6-Dimethoxy-2-{4-[(pyrrolidin-1-yl)methyl]benzylidene}-benzofuran-3(2H)-one 1m. Yield: 71.9%, m.p. 149e152  C; 1H NMR (CDCl3) d 7.83 (d, 2H, J ¼ 8.0 Hz, H-20 and H-60 ), 7.40 (d, 2H, J ¼ 8.0 Hz, H-30 and H-50 ), 7.18 (s, 1H, C]CH), 6.83 (s, 1H, H-4), 6.82 (s, 1H, H-7), 4.02 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.66 (s, 2H, benzylicCH2), 2.53 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.80 (m, 4H, pyrrolidine-CH2, H-3 and H-4); MS (ESI), m/z ¼ 366 [M þ 1]; IR (KBr): 3066, 2955, 2786, 1690, 1650, 1605, 1499, 1277, 818 cm1. 4.1.5.6. (Z )-5,6-Dimethoxy-2-{3-[(piperidin-1-yl)methyl]benzylidene}-benzofuran-3(2H)-one 1p. Yield: 87.2%, m.p. 153e156  C; 1H NMR (CDCl3) d 7.82 (d, 2H, J ¼ 8.4 Hz, H-20 and H-60 ), 7.39 (d, 2H, J ¼ 8.0 Hz, H-30 and H-50 ), 7.18 (s, 1H, C]CH), 6.83 (s, 1H, H-4), 6.82 (s, 1H, H-7), 4.02 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.51 (s, 2H, benzylic-CH2), 2.39 (m, 4H, piperidine-CH2, H-2 and H-6), 1.56 (m, 4H, piperidine-CH2, H-3 and H-5), 1.44 (m, 2H, piperidine-CH2, H-4); MS (ESI), m/z ¼ 380 [M þ 1]; IR (KBr): 3064, 2935, 2793, 2756, 1690, 1649, 1601, 1498, 1276, 824 cm1. 4.1.6. General procedure for preparation of compounds 2aep A mixture of compound 1a (0.17 g, 0.5 mmol) in THF (20 mL) and 5% palladium on carbon (10 mg) was stirred under hydrogen (balloon) for 6 h, The mixture was then filtered through diatomaceous earth, the filter cake was washed with methanol, and the combined filtrates were concentrated to give a slight yellow solid. Purification by silica gel column chromatography eluting with petroleum ether, ethyl acetate

R. Sheng et al. / European Journal of Medicinal Chemistry 44 (2009) 7e17

and triethylamine (10:10:1) afforded compound 2a as a white solid. 4.1.6.1. 2-{3-[(Dimethylamino)methyl]benzyl}-5,6-dimethoxy2,3-dihydroinden-1-one 2a. Yield: 70.6%, m.p. 76e78  C; 1 H NMR (CDCl3) d 7.19 (m, 3H, H-7, H-20 and H-50 ), 7.14 (d, 2H, J ¼ 8.0 Hz, H-40 and H-60 ), 6.81 (s, 1H, H-4), 3.94 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.35e3.45 (m, 3H, PhCH2N and PheCH2-a), 3.03 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 7.6 Hz, H-3a), 2.97 (m, 1H, H-2), 2.75 (d, 1H, J ¼ 16.4 Hz, H-3b), 2.62 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.0 Hz, PheCH2b), 2.23 (s, 6H, 2 NeCH3); MS (ESI), m/z ¼ 340 [M þ 1]; IR (KBr): 3073, 2927, 2852, 1681, 1606, 1591, 1501, 1455, 780, 706 cm1. 4.1.6.2. 2-{4-[(Dimethylamino)methyl]benzyl}-5,6-dimethoxy2,3-dihydroinden-1-one 2b. Yield: 76.6%, m.p. 99e101  C; 1 H NMR (CDCl3) d 7.18 (m, 5H, H-7, H-20 , H-30 , H-50 and H-60 ), 6.81 (s, 1H, H-4), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.41 (s, 2H, PhCH2N), 3.34 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 4.0 Hz, PheCH2-a), 3.04 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 7.6 Hz, H-3a), 2.96e3.01 (m, 1H, H-2), 2.74 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 3.2 Hz, H-3b), 2.61 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.0 Hz, PheCH2-b), 2.25 (s, 6H, 2 N-CH3); MS (ESI), m/z ¼ 340 [M þ 1]; IR (KBr): 3073, 2928, 2853, 1681, 1605, 1592, 1502, 1472, 1454, 805 cm1. 4.1.6.3. 2-{3-[(Ethylmethylamino)methyl]benzyl}-5,6-dimethoxy-2,3-dihydroinden-1-one 2c. Yield: 74.1%, m.p. 68e 69  C; 1H NMR (CDCl3) d 7.22 (d, 1H, J ¼ 7.6 Hz, H-40 ), 7.20 (m, 2H, H-7 and H-20 ), 7.12 (m, 2H, H-60 and H-50 ), 6.81 (s, 1H, H-4), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.57 (s, 2H, PhCH2N), 3.34 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 4.0 Hz, PheCH2-a), 3.04 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 7.2 Hz, H-3a), 2.96 (m, 1H, H-2), 2.75 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 2.8 Hz, H-3b), 2.62 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.0 Hz, PheCH2-b), 2.42 (q, 2H, J ¼ 7.2 Hz, CH2eCH3), 2.18 (s, 3H, NeCH3), 1.09 (t, 3H, J ¼ 7.2 Hz, CH2eCH3); MS (ESI), m/z ¼ 354 [M þ 1]; IR (KBr): 3056, 2970, 2931, 2837, 1681, 1591, 1502, 1443, 774, 706 cm1. 4.1.6.4. 2-{4-[(Ethylmethylamino)methyl]benzyl}-5,6-dimethoxy-2,3-dihydroinden-1-one 2d. Yield: 79.3%, m.p. 92e 94  C; 1H NMR (CDCl3) d 7.18 (m, 5H, H-7, H-20 , H-30 , H50 and H-60 ), 6.81 (s, 1H, H-4), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.50 (s, 2H, PhCH2N), 3.34 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 4.0 Hz, PheCH2-a), 3.04 (dd, 1H, J1 ¼ 16.8 Hz, J2 ¼ 7.6 Hz, H-3a), 2.96 (m, 1H, H-2), 2.75 (dd, 1H, J1 ¼ 16.8 Hz, J2 ¼ 2.4 Hz, H-3b), 2.61 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.0 Hz, PheCH2-b), 2.44 (q, 4H, J ¼ 7.2 Hz, 2CH2e CH3), 2.20 (s, 3H, NeCH3), 1.09 (t, 3H, J ¼ 7.2 Hz, 2CH2e CH3); MS (ESI), m/z ¼ 354 [M þ 1]; IR (KBr): 3072, 2963, 2926, 2853, 1682, 1606, 1592, 1501, 1455, 820 cm1. 4.1.6.5. 2-{3-[(Ethylmethylamino)methyl]benzyl}-5,6-dimethoxy-benzofuran-3(2H)-one 2e. Yield: 29.8%; oil; 1H NMR (CDCl3) d 7.25 (m, 4H, H-20 , H-30 , H-50 and H-60 ), 6.96

15

(s, 1H, H-4), 6.54 (s, 1H, H-7), 4.74 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 3.2 Hz, H-2), 3.93 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.51 (s, 2H, PhCH2N), 3.32 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 3.2 Hz, PheCH2-a), 2.91 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 8.4 Hz, PheCH2-b), 2.44 (q, 2H, J ¼ 7.2 Hz, CH2eCH3), 2.20 (s, 3H, NeCH3), 1.09 (t, 3H, J ¼ 7.2 Hz, CH2eCH3); MS (ESI), m/z ¼ 356 [M þ 1]; IR (KBr): 3072, 2970, 2938, 2838, 1698, 1616, 1486, 1440, 1270, 826 cm1. 4.1.6.6. 2-{3-[(Diethylamino)methyl]benzyl}-5,6-dimethoxy2,3-dihydroinden-1-one 2f. Yield: 76.3%, m.p. 75e77  C, 1H NMR (CDCl3) d 7.17 (m, 4H, H-7, H-20 , H-40 and H-50 ), 7.18 (d, 1H, J ¼ 7.6 Hz, H-60 ), 6.80 (s, 1H, H-4), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.55 (s, 2H, PhCH2N), 3.35 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 4.0 Hz, PheCH2-a), 3.03 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 7.2 Hz, H-3a), 2.97 (m, 1H, H-2), 2.75 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 2.8 Hz, H-3e), 2.61 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.0 Hz, PheCH2-b), 2.48 (q, 4H, J ¼ 6.8 Hz, 2CH2eCH3), 1.02 (t, 6H, J ¼ 6.8 Hz, 2CH2e CH3); 13C NMR (CDCl3) d 206.5, 155.4, 149.3, 148.9, 140.0, 139.5, 129.3, 129.2, 128.2, 127.2, 126.8, 107.3, 104.3, 57.4, 56.1, 56.0, 49.0, 46.6, 37.2, 31.8, 11.6; MS (ESI), m/z ¼ 368 [M þ 1]; IR (KBr): 3023, 2967, 2930, 2871, 1680, 1605, 1590, 1502, 1477, 763, 704 cm1. 4.1.6.7. 2-{4-[(Diethylamino)methyl]benzyl}-5,6-dimethoxy 2,3-dihydroinden-1-one 2g. Yield: 70.8%, m.p. 98e100  C 1 H NMR (CDCl3) d 7.24 (d, 2H, J ¼ 8.0 Hz, H-2 and H-6), 7.17 (m, 3H, H-7, H-3 and H-5), 6.81 (s, 1H, H-4), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.54 (s, 2H, PhCH2N), 3.34 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 4.0 Hz, PheCH2-a), 3.04 (dd, 1H, J1 ¼ 16.8 Hz, J2 ¼ 7.6 Hz, H-3a), 2.96 (m, 1H, H2), 2.75 (dd, 1H, J1 ¼ 16.8 Hz, J2 ¼ 2.8 Hz, H-3b), 2.60 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.0 Hz, PheCH2-b), 2.49 (q, 4H, J ¼ 7.2 Hz, 2CH2eCH3), 1.02 (t, 6H, J ¼ 7.2 Hz, 2CH2e CH3); 13C NMR (CDCl3) d 206.5, 155.4, 149.3, 148.9, 138.0, 137.7, 129.2, 129.0, 128.6, 107.3, 104.3, 57.0, 56.1, 56.0, 49.1, 46.5, 36.9, 31.8, 11.6; MS (ESI), m/z ¼ 368 [M þ 1]; IR (KBr): 3073, 2931, 2874, 1683, 1606, 1593, 1505, 1474, 821 cm1. 4.1.6.8. 2-{3-[(Diethylamino)methyl]benzyl}-5,6-dimethoxy benzofuran-3(2H)-one 2h. Yield: 36.9%; oil; 1H NMR (CDCl3) d 7.18 (m, 4H, H-20 , H-40 , H-50 and H-60 ), 6.96 (s, 1H, H-4), 6.53 (s, 1H, H-7), 4.76 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 4.0 Hz, H-2), 3.93 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.53 (s, 2H, PhCH2N), 3.33 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 4.0 Hz, PheCH2-a), 2.92 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 8.8 Hz, PheCH2-b), 2.46 (q, 4H, J ¼ 7.2 Hz, 2  CH2), 1.01 (t, 6H, J ¼ 7.2 Hz, 2  CH3); MS (ESI), m/z ¼ 370 [M þ 1]; IR (KBr): 3023, 2969, 2935, 2836, 1697, 1616, 1485, 1440, 1270, 782, 712 cm1. 4.1.6.9. 2-{4-[(Diethylamino)methyl]benzyl}-5,6-dimethoxy benzofuran-3(2H)-one 2i. Yield: 32.8%; oil; 1H NMR (CDCl3) d 7.23 (m, 4H, H-20 , H-30 , H-50 and H-60 ), 6.95 (s, 1H, H-4), 6.53 (s, 1H, H-7), 4.74 (dd, 1H, J1 ¼ 8.4 Hz,

16

R. Sheng et al. / European Journal of Medicinal Chemistry 44 (2009) 7e17

J2 ¼ 3.6 Hz, H-2), 3.92 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.52 (s, 2H, PhCH2N), 3.31 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 3.6 Hz, PheCH2-a), 2.90 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 8.4 Hz, PheCH2-b), 2.45 (q, 4H, J ¼ 7.2 Hz, 2  CH2), 0.99 (t, 6H, J ¼ 6.8 Hz, 2  CH3); MS (ESI), m/z ¼ 370 [M þ 1]; IR (KBr): 3073, 2968, 2931, 1699, 1616, 1485, 1440, 1270, 825 cm1. 4.1.6.10. 5,6-Dimethoxy-2-{3-[(pyrrolidin-1-yl)methyl]-benzyl}2,3-dihydroinden-1-one 2j. Yield: 76.7%, m.p. 79e81  C; 1H NMR (CDCl3) d 7.19 (m, 4H, H-7, H-20 , H-40 and H-50 ), 7.13 (d, 1H, J ¼ 7.6 Hz, H-60 ), 6.80 (s, 1H, H-4), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.60 (s, 2H, PhCH2N), 3.33 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 4.0 Hz, PheCH2-a), 3.04 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 7.2 Hz, H-3a), 2.97 (m, 1H, H-2), 2.75 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 2.8 Hz, H-3b), 2.65 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.0 Hz, PheCH2-b), 2.54 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.80 (m, 4H, pyrrolidineCH2, H-3 and H-4); MS (ESI), m/z ¼ 366 [M þ 1]; IR (KBr): 3075, 2968, 2927, 1681, 1606, 1591, 1502, 1472, 778, 704 cm1. 4.1.6.11. 5,6-Dimethoxy-2-{4-[(pyrrolidin-1-yl)methyl]-benzyl}-2,3-dihydroinden-1-one 2k. Yield: 82.2%, m.p. 122e 124  C; 1H NMR (CDCl3) d 7.26 (d, 2H, J ¼ 8.0 Hz, H-2 and H-6), 7.18 (m, 3H, H-7, H-3 and H-5), 6.81 (s, 1H, H4), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.65 (s, 2H, PhCH2N), 3.34(dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 4.0 Hz, PheCH2a), 3.04 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 7.2 Hz, H-3a), 2.95 (m, 1H, H-2), 2.74 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 3.2 Hz, H-3b), 2.61 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.0 Hz, PheCH2-b), 2.58 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.82 (m, 4H, pyrrolidine-CH2, H-3 and H-4); MS (ESI), m/z ¼ 366 [M þ 1]; IR (KBr): 3072, 2926, 2852, 1682, 1605, 1592, 1502, 1471, 1454, 863 cm1. 4.1.6.12. 5,6-Dimethoxy-2-{3-[(pyrrolidin-1-yl)methyl]-benzyl}-benzofuran-3(2H)-one 2l. Yield: 48.8%; oil; 1H NMR (CDCl3) d 7.16e7.26 (m, 4H, H-20 , H-40 , H-50 and H-60 ), 6.95 (s, 1H, H-4), 6.53 (s, 1H, H-7), 4.76 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 3.6 Hz, H-2), 3.92 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.57 (s, 2H, PhCH2N), 3.32(dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 3.2 Hz, PheCH2-a), 2.93 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 8.4 Hz, PheCH2-b), 2.44 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.75e1.78 (m, 4H, pyrrolidine-CH2, H-3 and H-4); MS (ESI), m/z ¼ 368 [M þ 1]; IR (KBr): 3075, 2928, 2785, 1695, 1614, 1485, 1438, 1269, 778, 710 cm1. 4.1.6.13. 5,6-Dimethoxy-2-{4-[(pyrrolidin-1-yl)methyl] benzyl}-benzofuran-3(2H)-one 2m. Yield: 50.7%; oil; 1H NMR (CDCl3) d 7.24 (m, 4H, H-20 , H-30 , H-50 and H-60 ), 6.96 (s, 1H, H-4), 6.54 (s, 1H, H-7), 4.73 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 3.6 Hz, H-2), 3.93 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.59 (s, 2H, PhCH2N), 3.31 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 3.2 Hz, PheCH2-a), 2.90 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 8.4 Hz, PheCH2-b), 2.49 (m, 4H, pyrrolidine-CH2, H-2 and H-5), 1.75 (m, 4H, pyrrolidine-CH2, H-3 and H-4); MS (ESI),

m/z ¼ 368 [M þ 1]; IR (KBr): 3072, 2930, 2784, 1697, 1616, 1487, 1440, 1270, 824 cm1. 4.1.6.14. 5,6-Dimethoxy-2-{3-[(piperidin-1-yl)methyl]-benzylidene}-2,3-dihydroinden-1-one 2n. Yield: 84.4%, m.p. 81e 83  C; 1H NMR (CDCl3) d 7.16 (m, 4H, H-7, H-20 , H-40 and H-50 ), 7.11 (d, 1H, H-60 , J ¼ 7.6 Hz), 6.80 (s, 1H, H-4), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.49 (s, 2H, PhCH2N), 3.34 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 3.2 Hz, PheCH2-a), 3.04 (dd, 1H, J1 ¼ 16.0 Hz, J2 ¼ 7.2 Hz, H-3a), 2.97 (m, 1H, H2), 2.75 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 2.4 Hz, H-3b), 2.63 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.0 Hz, PheCH2-b), 2.35 (m, 4H, piperidine-CH2, H-2 and H-6), 1.56 (m, 4H, piperidine-CH2, H3 and H-5), 1.42 (m, 2H, piperidine-CH2, H-4); MS(ESI), m/ z ¼ 380 [M þ 1]; IR (KBr1): 3073, 2927, 2853, 1682, 1605, 1591, 1502, 1472, 1455, 773, 702 cm1. 4.1.6.15. 5,6-Dimethoxy-2-{4-[(piperidin-1-yl)methyl]-benzyl}2,3-dihydroinden-1-one 2o. Yield: 73.9%, m.p. 92e94  C; 1H NMR (d, CDCl3): 7.22e7.26 (d, 2H, J ¼ 8.0 Hz, H-2 and H6), 7.17 (m, 3H, H-7, H-3 and H-5), 6.81 (s, 1H, H-4), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.65 (s, 2H, PhCH2N), 3.34 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 4.0 Hz, PheCH2-a), 3.03 (dd, 1H, J1 ¼ 16.8 Hz, J2 ¼ 7.2 Hz, H-3a), 2.96 (m, 1H, H2), 2.75 (dd, 1H, J1 ¼ 16.4 Hz, J2 ¼ 2.8 Hz, H-3b), 2.59 (dd, 1H, J1 ¼ 14.0 Hz, J2 ¼ 10.4 Hz, PheCH2-b), 2.36 (m, 4H, piperidine-CH2, H-2 and H-6), 1.54 (m, 4H, piperidine-CH2, H3 and H-5), 1.42 (m, 2H, piperidine-CH2, H-4); MS (ESI), m/ z ¼ 380 [M þ 1]; IR (KBr): 3072, 2936, 2853, 1681, 1605, 1591, 1502, 1472, 1454, 866 cm1. 4.1.6.16. 5,6-Dimethoxy-2-{3-[(piperidin-1-yl)methyl]-benzyl}benzofuran-3(2H)-one 2p. Yield: 40.8%; oil; 1H NMR (CDCl3) d 7.23 (m, 4H, H-20 , H-30 , H-50 and H-60 ), 6.96 (s, 1H, H-4), 6.54 (s, 1H, H-7), 4.74 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 3.6 Hz, H-2), 3.93 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.43 (s, 2H, PhCH2N), 3.31 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 3.6 Hz, PheCH2-a), 2.90 (dd, 1H, J1 ¼ 14.8 Hz, J2 ¼ 8.8 Hz, PheCH2-b), 2.31 (m, 4H, piperidine-CH2, H-2 and H-6), 1.52-(m, 4H, piperidine-CH2, H-3 and H-5), 1.41e1.42 (m, 2H, piperidine-CH2, H-4); MS (ESI), m/ z ¼ 382 [M þ 1]; IR (KBr): 3072, 2928, 2852, 1695, 1614, 1483, 1268, 822 cm1. 4.2. Pharmacology 4.2.1. ChE assay AChE and BuChE activities were measured by the spectrophotometric method with slight modification with rat cortex homogenate and rat serum was used as the resource of AChE and BuChE, respectively. The brain homogenate was preincubated for 5 min with tetraisopropyl pyrophosphoramide (isoOMPA) (0.04 mmol/L), a selective inhibitor of BuChE. For assay of AChE or BuChE activity, a reaction mixture of 200 ml containing acetylthiocholine iodide 0.3 mmol/L or butyrylthiocholine iodide 0.4 mmol/L, sodium phosphate buffer (0.1 mmol/L, pH 7.4) 100 ml, homogenate or serum

R. Sheng et al. / European Journal of Medicinal Chemistry 44 (2009) 7e17

20 ml and different concentrations of test compounds 20 ml were incubated at 37  C for 15 min. The reaction was terminated by adding 50 ml 3% sodium lauryl sulfate, then, 50 ml 0.2% 5,50 -dithio-bis(2-nitrobenzoic acid) was added to produce the yellow anion of 5-thio-2-nitrobenzoic acid. The rate of color production was measured spectrophotometrically at 440 nm. Assays were performed with at least seven concentrations of compounds and IC50 (nM drug concentration that inhibits 50% AChE activity) was calculated according to the inhibition curve. Donepezil and rivastigmine were applied as positive drugs. All samples were assayed in duplicate.

4.2.2. Step-down passive avoidance test The apparatus was a 60  32  12 cm plastic box of which consisted of parallel stainless steel bars (0.3 cm diameter spaced 1 cm apart). The apparatus is divided into 5 cells and a milky plastic platform (4  4  4 cm) was placed on the same corner of each cell. In the training session, animals were placed on the platform and their latency to step down on the grid with all four paws was measured. Immediately after stepping down on the grid, animals received electric shocks. Training session lasted 3 min and the electric shocks received during these 3 min would form the memory of passive avoidance. Retention test session was carried out 24 h after training and was procedurally identical to training. Step-down latency was used as a measure of memory retention.

Acknowledgments This study was financially supported by the National Natural Science Foundation of China (30572239), the Specialized Research Fund for the Doctoral Program of Higher Education (20050335045), the Science and Technology Department of Zhejiang Province of China (2005C23008) and Health Bureau of Zhejiang Province Foundation (No.WKJ2007-016).

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