Design, synthesis and evaluation of difunctionalized 4-hydroxybenzaldehyde derivatives as novel cholinesterase inhibitors

Article abstract of DOI:10.1248/cpb.58.1216

A series of difunctionalized 4-hydroxybenzaldehyde derivatives were designed, synthesized and evaluated as cholinesterase (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)) inhibitors. The results demonstrated that all the compounds had more potent AChE and BChE inhibitory activities than galanthamine-HBr, one of the best cholinesterase inhibitors known so far. The inhibition mechanism revealed that the best active compound 4e displayed a mix-type mode of AChE and BChE by its dual-site interactions with the catalytic triad active center and the peripheral anionic site (PAS) of enzyme. All these data suggested that further development of such compounds may be of interest.

Full text of DOI:10.1248/cpb.58.1216

1216
Note
Chem. Pharm. Bull. 58(9) 1216—1220 (2010)
Vol. 58, No. 9
Design, Synthesis and Evaluation of Difunctionalized
4-Hydroxybenzaldehyde Derivatives as Novel Cholinesterase Inhibitors
Liang YU, Rihui CAO, Wei YI, Qin YAN, Zhiyong CHEN, Lin MA, and Huacan SONG*
School of Chemistry and Chemical Engineering, Sun Yat-sen University; 135 Xin Gang West Road, Guangzhou 510275,
China. Received February 11, 2010; accepted May 24, 2010; published online June 16, 2010
A series of difunctionalized 4-hydroxybenzaldehyde derivatives were designed, synthesized and evaluated as
cholinesterase (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)) inhibitors. The results demon-
strated that all the compounds had more potent AChE and BChE inhibitory activities than galanthamine-HBr,
one of the best cholinesterase inhibitors known so far. The inhibition mechanism revealed that the best active
compound 4e displayed a mix-type mode of AChE and BChE by its dual-site interactions with the catalytic triad
active center and the peripheral anionic site (PAS) of enzyme. All these data suggested that further development
of such compounds may be of interest.
Key words synthesis; 4-hydroxybenzaldehyde derivative; cholinesterase inhibitor; inhibition mechanism
It is well-known that two forms of cholinesterases coexist
ubiquitously throughout the body, acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE). They can catalyze the
degradation of the neurotransmitter acetylcholine, leading
to the termination of cholinergic neurotransmission.1,2)
Cholinesterases inhibitors (ChEI) represented the treatment
of choice for Alzheimer’s disease (AD).3,4) Currently, AchE
Fig. 1. Chemical Structure of Lead Compound
has a well established esterase activity, while the pharmaco-
logical role of BChE is not yet completely understood. BChE action with the peripheral anionic site (PAS) of enzyme.19—21)
may have a compensatory role in the modulation of the hy-
Recently, our group investigated the syntheses and the
drolysis of acetylcholine (ACh) in brain with degenerative inhibitory effects on AChE of a variety of 4-hydroxybenz-
changes. Consequently, BChE may be additional target for aldehyde derivatives. We found that compound 3a having a
increasing the cholinergic tone in AD patients.5,6)
piperidine moiety connected to the phenyl ring via eight CH2
Many efforts have been spent in the search for potent unit spacer (Fig. 1) was the most potent inhibitor with IC50
AChE inhibitors, and a large number of naturally occurring value lower than 92 nM and 7.3 nM against AChE and BChE,
and synthetic AChE inhibitors have already been reported.7,8) respectively. (These results will be published elsewhere.) In
Among them, four representative anti-AChE agents, tacrine, our continuing efforts for simple but efficient cholinesterase
rivastigmine, donepezil, and galanthamine, have been ap- inhibitors (ChEI), we designed and synthesized a series of
proved by FDA for the treatment of AD.9—11) Unfortunately, difunctionalized 4-hydroxybenzaldehyde derivatives by the
the potential effectiveness offered by these inhibitors is often replacement of diethylamino moieties with appropriate cyclic
limited mainly due to their some adverse effects. For exam- or dialkylamino group on the basis of the chemical structure
ple, recent clinical studies showed that tacrine had hepato- of compound 3a. These compounds were expected to exhibit
toxic liability.12) Obviously, this is still needed to search and more potent ChE inhibitory effects. To the best of our knowl-
develop novel AChE inhibitors with better activities together edge, this is the first time to report the inhibitory effects of
with lower side effects.
such compounds on the AChE and BChE.
4-Hydroxybenzaldehyde was originally isolated as one of
the main active constituents from Gastrodia elata, a plant
named tianma in China, which is a very important Chinese
herbal medicine used for medical treatment for a long time in
China, such as headaches, migraine and some neuralgic and
nervous disorders.13,14) On the basis of the above mentioned
information and the crystallographic structure of AChE, our
group had ever reported a series of helicid analogues and
methyl 2-[2-(4-formylphenoxy)acetamido]-2-substituted ac-
etate derivatives as potent AChE inhibitors.15,16) In addition,
some galanthamine derivatives were also found to exhibit
significant inhibitory effects on AChE,17) and these com-
pounds exhibited the potent activity mainly due to its strong
interaction with the active site gorge of AChE.18) On the
other hand, numerous investigations provided evidence that
the introduction of amine cation moiety remarkably im-
Experimental
Chemistry NMR spectra were recorded on a Varian Mercury-Plus 300
spectrometer in CDCl3 or deuteriated dimethyl sulfoxide (DMSO)-d6 at
25 °C. All chemical shifts (d) are quoted in parts per million downfield from
TMS and coupling constants (J) are given in Hertz. All reactions were moni-
tored by TLC (Merck Kieselgel 60 F254) and spots were visualized with UV
light or iodine. Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel),
butyrylcholinesterase (BChE, E.C. 3.1.1.8, from horse serum), 5,5Ј-dithio-
bis(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), butylthiocholine chlo-
ride (BTC) and acetylthiocholine chloride (ATC) were purchased from
Sigma-Aldrich (Steinheim, Germany). Galanthamine hydrobromide was ob-
tained from Sigma-Aldrich (Sintra, Portugal). All commercially available
reagents and solvents were used without further purification.
Procedure for the Synthesis of 4-(8-Bromooctyloxy)benzaldehyde (1)
A stirred suspension of 4-hydroxybenzaldehyde (20 mmol), 1,8-dibromooc-
tane (40 mmol) and K2CO3 (5.52 g, 40 mmol) in dry acetone was refluxed for
24 h. The reaction was monitored by TLC. The hot reaction mixture was
filtered and evaporated to dryness. The residue was purified by chromatogra-
proved the AChE inhibitory effects by the electrostatic inter- phy using CH2Cl2 as eluent to afford compound 1 (8.71 g, 70%) as colorless
∗ To whom correspondence should be addressed. e-mail: yjhxhc@mail.sysu.edu.cn. © 2010 Pharmaceutical Society of Japan

September 2010
1217
(C6), 26.3 (C3), 26.2 (Cpip-3,5), 24.9 (Cpip-4); ESI-MS m/z: 389 [MϩH]ϩ.
oil. 1H-NMR (CDCl3, 300 MHz) d, ppm: 9.86 (s, 1H, CHO), 7.82 (d,
Jϭ8.8 Hz, 2H, ArH), 6.99 (d, Jϭ8.7 Hz, 2H, ArH), 4.04 (t, Jϭ6.5 Hz, 2H,
H1), 3.20 (t, Jϭ7.0 Hz, 2H, H8), 1.88—1.78 (m, 4H, H7, H2), 1.42—1.30
(m, 8H, H3, H4, H5, H6).
General Procedure for the Synthesis of Compounds 2a—h Com-
pound 1 (2 mmol) and the corresponding cyclic or dialkylamine (2 mmol)
were mixed in 1,2-dichloroethane (35 ml) and then treated with sodium tri-
acetoxyborohydride (0.6 g, 14 mmol). The mixture was stirred at room tem-
perature for 1.5 h. The reaction mixture was quenched by adding aqueous
saturated NaHCO3, and the product was extracted with EtOAc. The com-
bined organic phase was dried (MgSO4), and the solvent was evaporated to
give 2a—h in 95—98% yields.
N-[4-(8-Bromooctyloxy)benzyl]-N-ethylethanamine (2a): Compound 2a
was obtained as yellow solid (0.73 g, 96%). H-NMR (CDCl3, 300 MHz) d,
ppm: 7.32 (d, Jϭ8.8 Hz, 2H, ArH), 6.85 (d, Jϭ8.8 Hz, 2H, ArH), 3.93 (t,
Jϭ6.6 Hz, 2H, H1), 3.72 (s, 2H, CH2Ph), 3.18 (t, Jϭ6.6 Hz, 2H, H8), 2.48
(q, Jϭ7.2 Hz, 4H, NCH2CH3), 1.86—1.72 (m, 4H, H7, H2), 1.42—1.30 (m,
8H, H3, H4, H5, H6), 1.01 (t, 6H, Jϭ7.1 Hz, NCH2CH3).
General Procedure for the Synthesis of Compounds 3a—h A mix-
ture of compounds 2a—h (2 mmol), KI (3 mmol) and piperidine (20 mmol)
in anhydrous ethanol (50 ml) was refluxed for 5—10 h. After completion of
the reaction as indicated by TLC, the solution was cooled and filtered, and
then concentrated under reduced pressure. The residue was dissolved in
CH2Cl2, and then washed with saturated NaHCO3 and brine, dried with
anhydrous Na2SO4, and solvent was removed in vacuo. The crude product
was purified by chromatography using CH2Cl2/MeOH/aqueous NH3
(50 : 1 : 0.5) as eluent to afford 3a—h in 80—95% yields.
1-{8-[4-(Pyrrolidin-1-ylmethyl)phenoxy]octyl}piperidine (3e): Compound
3e was obtained as yellow oil (0.65 g, 88%). 1H-NMR (CDCl3, 300 MHz) d,
ppm: 7.20 (d, Jϭ8.5 Hz, 2H, ArH), 6.80 (d, Jϭ8.6 Hz, 2H, ArH), 3.92 (t,
Jϭ6.6 Hz, 2H, H1), 3.53 (s, 2H, CH2Ph), 2.52—2.45 (m, 4H, Hpyr-2,5),
2.40—2.30 (m, 4H, Hpip-2,6), 2.29—2.26 (m, 2H, H8), 1.83—1.71 (m, 6H,
H2, Hpyr-3,4), 1.62—1.55 (m, 4H, Hpip-3,5), 1.51—1.40 (m, 6H, H7, Hpip-4,
H3), 1.37—1.25 (m, 6H, H6, H5, H4). 13C-NMR (CDCl3, 75 MHz) d: 158.3
(Cphenyl-1), 131.3 (Cphenyl-4), 130.3 (Cphenyl-3,5), 114.4 (Cphenyl-2,6), 68.2 (C1),
60.3 (CH2Ph), 59.9 (H8), 54.9 (Cpyr-2,5), 54.3 (Cpip-2,6), 29.9 (C2), 29.7
(C4), 29.5 (C5), 28.1 (C7), 27.2 (C6), 26.4 (C3), 26.3 (Cpip-3,5), 24.8 (Cpip
-
4), 23.7 (Cpyr-3,4); HR-MS (EI) Calcd for C24H40O1N2: 372.3135. Found:
372.3137.
1
1-{4-[8-(Piperidin-1-yl)octyloxy]benzyl}piperidine (3f): Compound 3f
1
was obtained as yellow oil (0.66 g, 86%). H-NMR (CDCl3, 300 MHz) d,
ppm: 7.19 (d, Jϭ8.8 Hz, 2H, ArH), 6.80 (d, Jϭ8.8 Hz, 2H, ArH), 3.92
(t, Jϭ6.6 Hz, 2H, H1), 3,40 (s, 2H, CH2Ph), 2.44—2.30 (m, 8H, Hpip-2,6,
HЈpip-2,6), 2.29—2.24 (m, 2H, H8), 1.81—1.71 (m, 2H, H2), 1.62—1.52 (m,
8H, Hpip-3,5 , HЈpip-3,5), 1.50—1.38 (m, 8H, H7, Hpip-4, HЈpip-4, H3), 1.37—
1.30 (m, 6H, H6, H5, H4). 13C-NMR (CDCl3, 75 MHz) d: 158.3 (Cphenyl-1),
130.6 (Cphenyl-3,5), 130.3 (Cphenyl-4), 114.2 (Cphenyl-2,6), 68.2 (H1), 63.6
(CH2Ph), 60.0 (H8), 55.0 (Cpip-2,6), 54.6 (CЈpip-2,6), 29.9 (C2), 29.7 (C4),
29.5 (C5), 28.1 (C7), 27.3 (C6), 26.4 (C3), 26.3 (Cpip-3,5 , CЈpip-3,5), 24.9
(Cpip-4), 24.8 (CЈpip-4); HR-MS (EI) Calcd for C25H42O1N2: 386.3292.
Found: 386.3294.
1-Methyl-4-{4-[8-(piperidin-1-yl)octyloxy]benzyl}piperazine (3g): Com-
pound 3g was obtained as yellow oil (0.71 g, 89%). 1H-NMR (CDCl3,
300 MHz) d, ppm: 7.18 (d, Jϭ8.5 Hz, 2H, ArH), 6.80 (d, Jϭ8.5 Hz, 2H,
ArH), 3.91 (t, Jϭ6.5 Hz, 2H, H1), 3,42 (s, 2H, CH2Ph), 2.56—2.29 (m, 12H,
Hppz-2,3,5,6, Hpip-2,6), 2.29—2.22 (m, 2H, H8), 2.26 (s, 3H, Hppz-NCH3),
1.79—1.70 (m, 2H, H2), 1.61—1.53 (m, 4H, Hpip-3,5), 1.50—1.38 (m, 6H,
H7, Hpip-4, H3), 1.37—1.25 (m, 6H, H6, H5, H4). 13C-NMR (CDCl3,
75 MHz) d: 158.4 (Cphenyl-1), 130.5 (Cphenyl-3,5), 130.1 (Cphenyl-4), 114.3
N-Ethyl-N-{4-[8-(piperidin-1-yl)octyloxy]benzyl}ethanamine (3a): Com-
pound 3a was obtained as yellow oil (0.70 g, 95%). 1H-NMR (CDCl3,
300 MHz) d, ppm: 7.20 (d, Jϭ8.8 Hz, 2H, ArH), 6.82 (d, Jϭ8.8 Hz, 2H,
ArH), 3.92 (t, Jϭ6.6 Hz, 2H, H1), 3.49 (s, 2H, CH2Ph), 2.49 (q, Jϭ6.8 Hz,
4H, NCH2CH3), 2.42—2.30 (m, 4H, Hpip-2,6), 2.26 (t, Jϭ6.4 Hz, 2H, H8),
1.80—1.71 (m, 2H, H2), 1.62—1.52 (m, 4H, Hpip-3,5), 1.50—1.40 (m, 6H,
H7, Hpip-4, H3), 1.37—1.26 (m, 6H, H6, H5, H4), 1.04 (t, 6H, Jϭ6.8 Hz,
(Cphenyl-2,6), 68.2 (H1), 62.8 (CH2Ph), 60.0 (H8), 55.4 (Cppz-3,5), 55.0 (Cpip
-
NCH2CH3). 13C-NMR (CDCl3, 75 MHz) d: 158.1 (Cphenyl-1), 131.4 (Cphenyl
-
2,6), 53.3 (Cppz-2,6), 46.4 (Cpip-NCH3), 29.9 (C2), 29.6 (C4), 29.6 (C5), 28.1
(C7), 27.3 (C6), 26.3 (C3), 26.3 (Cpip-3,5), 24.8 (Cpip-4); ESI-MS m/z: 402
[MϩH]ϩ.
4), 130.3 (Cphenyl-3,5), 114.3 (Cphenyl-2,6), 68.2 (H1), 60.0 (CH2Ph), 57.0
(H8), 55.0 (Cpip-2,6), 46.7 (2NCH2CH3), 29.9 (C2), 29.7 (C4), 29.6 (C5),
28.1 (C7), 27.2 (C6), 26.4 (C3), 26.3 (Cpip-3,5), 24.8 (Cpip-4), 12.0
(2NCH2CH3); high resolution-mass spectra (HR-MS)-electron ionization
(EI) Calcd for C24H42O1N2: 374.3292. Found: 374.3288.
1-Ethyl-4-{4-[8-(piperidin-1-yl)octyloxy]benzyl}piperazine (3h): Com-
pound 3h was obtained as yellow oil (0.72 g, 87%). 1H-NMR (CDCl3,
300 MHz) d, ppm: 7.19 (d, Jϭ8.5 Hz, 2H, ArH), 6.81 (d, Jϭ8.5 Hz, 2H,
ArH), 3.91 (t, Jϭ6.5 Hz, 2H, H1), 3,43 (s, 2H, CH2Ph), 2.69—2.30 (m, 14H,
HN1, Hppz-2,3,5,6, Hpip-2,6), 2.29—2.23 (m, 2H, H8), 1.80—1.71 (m, 2H,
H2), 1.61—1.53 (m, 4H, Hpip-3,5), 1.51—1.38 (m, 6H, H7, Hpip-4, H3),
1.37—1.24 (m, 6H, H6, H5, H4), 1.07 (t, 3H, Jϭ7.2Hz, HN2). 13C-NMR
(CDCl3, 75 MHz) d: 158.4 (Cphenyl-1), 130.6 (Cphenyl-3,5), 130.0 (Cphenyl-4),
114.3 (Cphenyl-2,6), 68.2 (H1), 62.8 (CH2Ph), 60.0 (H8), 55.0 (Cpip-2,6), 53.3
(Cppz-3,5), 53.1 (Cppz-2,6), 52,6 (CN1), 29.9 (C2), 29.7 (C4), 29.7 (C5), 28.1
(C7), 27.3 (C6), 26.4 (C3), 26.3 (Cpip-3,5), 24.9 (Cpip-4), 12.4(CN2); ESI-MS
m/z: 416 [MϩH]ϩ.
1-Methyl-1-(8-{4-[(1-methylpyrrolidinium-1-yl)methyl]phenoxy}octyl)
piperidinium iodide (4e): Compound 3e (0.40 g, 1 mmol) was dissolved in
15 ml CHCl3, and 1 ml CH3I was added to the solution. The mixture was
refluxed for 24 h. After completion of the reaction as indicated by TLC, the
solution was concentrated under reduced pressure to obtain 4e (0.61 g, 95%)
as colorless oil. 1H-NMR (DMSO-d6, 300 MHz) d, ppm: 7.17 (d, Jϭ7.8 Hz,
2H, ArH), 6.80 (d, Jϭ8.0 Hz, 2H, ArH), 4.47 (s, 2H, CH2Ph), 3.98 (t, Jϭ6.6
N-{4-[8-(Piperidin-1-yl)octyloxy]benzyl}-N-propylpropan-1-amine (3b):
Compound 3b was obtained as yellow oil (0.73 g, 91%). H-NMR (CDCl3,
1
300 MHz) d, ppm: 7.20 (d, Jϭ8.5 Hz, 2H, ArH), 6.80 (d, Jϭ8.6 Hz, 2H,
ArH), 3.93 (t, Jϭ6.5 Hz, 2H, H1), 3,48 (s, 2H, CH2Ph), 2.40—2.30 (m, 8H,
Hpip-2,6, HN1), 2.26 (t, Jϭ6.4 Hz, 2H, H8), 1.81—1.72 (m, 2H, H2), 1.62—
1.55 (m, 4H, Hpip-3,5), 1.52—1.39 (m, 10H, H7, HN2, Hpip-4, H3), 1.39—
1.23 (m, 6H, H6, H5, H4), 0.85 (t, 6H, Jϭ7.3 Hz, NCH2CH3). 13C-NMR
(CDCl3, 75 MHz) d: 158.1 (Cphenyl-1), 132.1 (Cphenyl-4), 130.1 (Cphenyl-3,5),
114.2 (Cphenyl-2,6), 68.2 (H1), 60.0 (CH2Ph), 58.2 (H8), 56.0 (Cpip-2,6), 55.0
(2HN1), 29.9 (C2), 29.7 (C4), 29.6 (C5), 28.1 (C7), 27.3 (C6), 26.4 (C3),
26.3 (Cpip3,5), 24.9 (Cpip-4), 20.5 (2HN2), 12.0 (2HN3); electrospray ioniza-
tion (ESI)-MS m/z: 403 [MϩH]ϩ.
N-Butyl-N-{4-[8-(piperidin-1-yl)octyloxy]benzyl}butan-1-amine
(3c):
1
Compound 3c was obtained as yellow oil (0.79 g, 92%). H-NMR (CDCl3,
300 MHz) d, ppm: 7.20 (d, Jϭ8.5 Hz, 2H, ArH), 6.82 (d, Jϭ8.5 Hz, 2H,
ArH), 3.93 (t, Jϭ6.5 Hz, 2H, H1), 3,47 (s, 2H, CH2Ph), 2.42—2.31 (m, H,
HN1, Hpip-2,6), 2.29—2.24 (m, 2H, H8), 1.81—1.72 (m, 2H, H2), 1.51—
1.39 (m, 10H, H7, H3, HN2, Hpip-3,5), 1.36—1.23 (m, 10H, H6, H5, H4,
HN3), 0.88 (t, 6H, Jϭ7.2 Hz, NCH2CH3). 13C-NMR (CDCl3, 75 MHz) d:
158.0 (Cphenyl-1), 131.4 (Cphenyl-4), 130.3 (Cphenyl-3,5), 114.3 (Cphenyl-2,6),
68.2 (H1), 60.0 (CH2Ph), 58.1 (H8), 55.0 (Cpip-2,6), 53.6 (2CN1), 30.5
(2CN2), 29.9 (C2), 29.7 (C4), 29.5 (C5), 28.1 (C7), 27.3 (C6), 26.4 (C3),
26.3 (Cpip-3,5), 24.9 (Cpip-4), 21.0 (2CN3), 14.5 (2CN4); ESI-MS m/z: 431
[MϩH]ϩ.
Hz, 2H, H1), 3.40—3.32 (m, 2H, H8), 3.32—3.21 (m, 8H, Hpyr-2,5, Hpip
-
2,6), 2.96 (s, 3H, Hpyr-NCH3), 2.85 (s, 3H, Hpip-NCH3), 2.18—2.00 (m, 4H,
Hpyr-3,4), 1.88—1.58 (m, 8H, H2, H7, Hpip-3,5), 1.57—1.46 (m, 2H, Hpip-4),
1.42—1.20 (m, 8H, H3, H4, H5, H6). 13C-NMR (DMSO-d6, 75 MHz) d:
160.5 (Cphenyl-1), 134.5 (Cphenyl-3,5), 121.2 (Cphenyl-4), 115.4 (Cphenyl-2,6),
68.4 (Cpyr-2,5), 65.6 (H1), 63.3 (CH2Ph), 63.1 (Cpip-2,6), 60.8 (H8), 48.0
(Cpip-NCH3), 47.8 (Cpyr-NCH3), 29.4 (C2), 29.4 (C4), 29.3 (C5), 26.6 (C6),
26.2 (C7), 26.2 (C3), 21.6 (Cpyr-3,4), 21.5 (Cpip-4), 20.1 (Cpip-3,5); ESI-MS
m/z: 402 [M]ϩ.
Assay of the AChE and BChE Inhibitory Activity The assay was
performed as described in the following procedure.22) Five different concen-
tration of each compound were measured at 412 nm for 1 min, each con
centration in triplicate. For buffer preparation, 0.1 M dipotassium hydrogen
phosphate was adjusted to pHϭ8.0 with 1 M potassium dihydrogen phos-
phate. Enzyme solutions were prepared to give 2.5 units/ml in 1.5 ml aliquots.
Furthermore, 0.01 M DTNB solution, 0.075 M ATC and BTC solutions,
respectively, were used. A cuvette containing 880 ml of phosphate buffer,
10 ml of the respective enzyme, 50 ml of DTNB and 20 ml of the test com-
4-{4-[8-(Piperidin-1-yl)octyloxy]benzyl}morpholine (3d): Compound 3d
was obtained as yellow oil (0.69 g, 90%). H-NMR (CDCl3, 300 MHz) d,
1
ppm: 7.20 (d, Jϭ8.4 Hz, 2H, ArH), 6.83 (d, Jϭ8.5 Hz, 2H, ArH), 3.92
(t, Jϭ6.5 Hz, 2H, H1), 3.72—3.65 (m, 4H, Hmpl3,5) 3,42 (s, 2H, CH2Ph),
2.46—2.39 (m, 4H, Hmpl2,4), 2.39—2.30 (m, 4H, Hpip-2,6), 2.29—2.24 (m,
2H, H8), 1.81—1.72 (m, 2H, H2), 1.60—1.52 (m, 4H, Hpip-3,5), 1.52—1.39
(m, 6H, H7, Hpip-4, H3), 1.37—1.23 (m, 6H, H6, H5, H4). 13C-NMR
(CDCl3, 75 MHz) d: 158.5 (Cphenyl-1), 130.5 (Cphenyl-3,5), 129.6 (Cphenyl-4),
114.4 (Cphenyl-2,6), 68.2 (H1), 67.3 (Cmpl3,5), 63.2 (CH2Ph), 60.0 (H8), 55.0
(Cpip-2,6), 53.8 (Cmpl2,4), 29.9 (C2), 29.7 (C4), 29.5 (C5), 28.1 (C7), 27.3

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Vol. 58, No. 9
Reagents and conditions: (i) Br(CH2)8Br, K2CO3 reflux, 24 h; (ii) the corresponding cyclic or dialkylamine, NaBH(OAc)3, DCE, rt 4 h; (iii) piperidine, KI, anhydrous
ethanol, reflux 15 h.
Chart 1. Synthesis of Compounds 3a—h
Table 1. Chemical Structure and Inhibitory Activity against Isolated
AChEa) and BChEb) of Compounds 3a—h and 4e, and Resulting Selectivi-
ties Expressed as the Ratio of IC50 Values
Reagents and conditions: (i) CH3I, CHCl3, 24 h.
Chart 2. Synthesis of Compound 4e
ChE inhibition
Selectivity
IC50 (mM)
pound solution was allowed to pre-incubate for 15 min at 37 °C, and the
reaction was started by addition of 40 ml of the substrate solution
(ATC/BTC). The reaction was monitored by measuring the absorbance at
412 nm. For the reference value, 20 ml of dimethyl sulfoxide (DMSO) re-
placed the test compound solution. For determining the blank value, addi-
tionally 10 ml of buffer replaced the enzyme solution.
Assay of Kinetic Characterization of AChE and BChE Inhibition
Kinetic characterization of AChE was performed using a reported method.23)
Seven different concentrations of AChE and inhibitors were mixed in the
assay buffer (pHϭ8.0), containing 40 mM of 5,5Ј-dithio-bis(2-nitrobenzoic
acid), 0.035 units/ml AChE. Test compound was added into the assay solu-
tion and pre-incubated with the enzyme at 37 °C for 15 min, followed by the
addition of substrate with various concentrations. Kinetic characterization of
the hydrolysis of acetylthiocholine catalyzed by AChE was done spectromet-
rically at 412 nm. A parallel control with no inhibitor in the mixture, allowed
adjusting activities to be measured at various times.
Yield
(%)
Compd.
R1
R2
AChEc) BChEc) (AChE/BChE)
0.092 0.0073
3a
3b
3c
3d
3e
3f
95
12.50
0.54
2.05
0.33
5.45
3.52
1.05
2.44
91
92
90
88
86
89
87
93
0.21
0.39
0.47
0.39
0.19
1.42
0.048 0.0088
0.081 0.023
Results and Discussion
Synthetic Chemistry The synthetic routes of com-
pounds 3a—h were outlined in Chart 1. The 4-hydroxybenz-
aldehyde was treated with 1,8-dibromooctane in the presence
of K2CO3 to afford compound 1.24) Compound 1 was
reacted with the selected cyclic or dialkylamines in 1,2-
dichloroethane (DCE), the resulting product was reduced
with NaBH(OAc)3 to afford compounds 2a—h.25) Com-
pounds 2a—h were reacted with piperidine to give the final
compounds 3a—h. The synthetic routes of compound 4e was
outlined in Chart 2. The compound 3e was treated with
iodomethane in CHCl3 to generate the compound 4e. The
chemical structures of all the synthesized compounds were
characterized by 1H- and 13C-NMR spectra.
3g
3h
4e
0.20
1.17
0.19
0.48
0.022 0.0086
2.56
Galanthamine-HBrd)
0.67
1.52
0.044
a) AChE, E.C. 3.1.1.7, from electric eel. b) BChE, E.C. 3.1.1.8, from horse
serum. c) IC50 values are means of three different experiments. d) IC50 values of
AChE reported in the literature: 0.3—0.8 mM.
Inhibitory Activity The inhibitory activities of the
newly synthesized compounds against AChE and BChE were
investigated by determining the rate of hydrolysis of acetyl-
thiocholine (ATCh) and butyrylthiocholine (BuTCh) in com- tively, demonstrated more potent anti-AChE activities than
parison with reference compound galanthamine-HBr (one of lead compound 3a (Fig. 1). However, replacement of the
the best cholinesterase inhibitors known so far), using the diethylamino moiety of compound 3a with the dipropyl-
slightly modified method of Ellmann et al.22) The IC50 value amino, dibutylamino, morpholino, 1-methylpiperazino and
of all compounds investigated was summarized in Table 1.
1-ethylpiperazino, respectively, to provide compounds 3b—d
As shown in Table 1, most of compounds displayed more and 3g—h, which caused the decreasing anti-AChE and anti-
potent activities against AChE and BchE than the reference BChE activities. Of compounds 3a—h, compound 3e having
inhibitor galanthamine-HBr. Particularly, compounds 3e and pyrrolidino connected with the benzyl group exhibited the
3f, bearing pyrrolidino and piperidino substituents, respec- most potent anti-AChE activity with IC50 value of 0.048 mM.

September 2010
1219
Fig. 2. Lineweaver–Burk Plots Resulting from Substrate–Velocity Curves
of AChE Activity with Different Substrate Concentrations (100—400 mM) in
the Absence and Presence of Compound 4e with Concentration of 10 and
20 nM
Fig. 3. Lineweaver–Burk Plots Resulting from Substrate–Velocity Curves
of BChE Activity with Different Substrate Concentrations (100—400 mM) in
the Absence and Presence of Compound 4e with Concentration of 2 and
5 nM
Interestingly, compound 4e, a cationic compound with qua- pound 4e bound with the catalytic triad active centre of
ternary nitrogen derived from compound 3e, showed two- enzyme and the piperidine moiety interacted with the PAS of
fold anti-AChE activities higher than compound 3e. These enzyme. Therefore, we concluded that compound 4e caused
results suggested that (1) the nature and size of dialkylamino a mix-type of inhibition, that is, compound 4e could interact
moiety connected with benzyl group significantly influenced with the active site and the second distal site of enzyme at
the cholinesterase inhibitory potencies, and the small size the same time.
amine moieties were more preferable to their inhibitory
activities; (2) the quaternary ammonium salt derivative facili- Conclusion
tated their inhibitory effects on AChE.
In conclusion, a series of difunctionalized 4-hydroxybenz-
We also investigated the inhibitory effect and the selectiv- aldehyde derivatives bearing various cyclic or dialkylamino
ity of various dialkylamino moieties on AChE and BChE. As moiety connected with benzyl group described in this paper
shown in Table 1, compound 3a presented the best BChE were proved to have significant inhibitory activity against
inhibitory potencies with an IC50 value of 0.0073 mM and AChE and BChE. Structure–activity relationships analysis
showed a surprising selectivity to BChE. Other compounds indicated that the nature and size of dialkylamine moiety
also showed moderate to strong BChE inhibiting property, which connects with benzyl group obviously influenced the
but with decreased selectivity.
cholinesterase inhibitory potency, and the small size amine
Inhibitory Mechanism Analysis Compound 4e was moiety was more favorable. In addition, the quaternary am-
selected for kinetic measurements because it showed the monium salt derivative showed higher inhibitory effects on
highest inhibitory activity against AChE and BChE. The AChE, but not obviously on BChE. The inhibition kinetics
mechanism of inhibition was analyzed by recording sub- analyzed by Lineweaver–Burk plots revealed that such com-
strate–velocity curves in the absence and the presence of pounds were mix-type inhibitors. All these results suggested
compound 4e at different concentrations. Substrate concen- that such compounds might be utilized for the development
tration was varied from 100 to 400 mM. For AChE, 0 nM, of new candidates for treatment of Alzheimer’s disease.
10 nM and 20 nM concentrations of compound 4e were ap-
Acknowledgment This work was supported by the Science and Tech-
nology Project of Guangdong Province, China (2009B060700048) and
the Natural Science Foundation of Guangdong Province, China
plied. For BChE, 0 nM, 2 nM and 5 nM concentrations of com-
pound 4e were used. Figure 2 showed the Lineweaver–Burk
plots, which are reciprocal rates versus reciprocal substrate
concentrations for the different inhibitor concentrations
resulting from the substrate–velocity curves for AChE. The
results showed that the plots of 1/V versus 1/[S] gave a fam-
ily of straight lines with different slopes but they intersected
one another in the third quadrant. Similar results were ob-
tained for BChE (Fig. 3). The inhibitory behavior of com-
pound 4e, as deduced from Fig. 2, is virtually the same as
that of some reported compounds which could bind simulta-
neously at the catalytic site and at the PAS of AChE and
could be characterized by a linear mix-type of enzyme inhi-
bition.26) The reason for the unexpected inhibitory potency of
compound 4e might be that, to a certain extent, the interac-
tion between compound 4e and enzyme was formed, and the
pyrrolidine moiety connected with benzyl group of com-
(2004B30101007).
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