Design, synthesis, and biological evaluation of acetophenone derivatives as dual binding acetylcholinesterase inhibitors

Article abstract of DOI:10.1691/ph.2013.2839

As part of a project aimed at developing new agents for potential application in Alzheimer's disease, a new series of acetophenone derivatives which possess alkylamine side chains were designed, synthesized and assayed as acetylcholinesterase (AChE) inhibitors that could simultaneously bind to the peripheral and catalytic sites of the enzyme. The compounds were synthesized, and the inhibitory activities toward AChE and butyrylcholinesterase (BuChE) in vitro were determined using a modified Ellman method. Of the compounds tested, 6 derivatives were found to inhibit AChE in the micromolar range. The best compound, 2e, had an IC50 of 0.13 M. A detailed molecular modeling study was performed to explore the interaction of 2e with AChE.

Full text of DOI:10.1691/ph.2013.2839

ORIGINAL ARTICLES
College of Chemistry and Environmental Science, Anyang Institute of Technology, Anyang, China
Design, synthesis, and biological evaluation of acetophenone derivatives
as dual binding acetylcholinesterase inhibitors
Yanhong Shen, Baoli Li, Hu Xu, Guoqiang Zhang
Received October 20, 2012, accepted November 23, 2012
Yanhong Shen, College of Chemistry and Environmental Science, Anyang Institute of Technology, Anyang 455000,
China
yanhong0809@163.com
Pharmazie 68: 307–310 (2013)
doi: 10.1691/ph.2013.2839
As part of a project aimed at developing new agents for potential application in Alzheimer’s disease, a new
series of acetophenone derivatives which possess alkylamine side chains were designed, synthesized
and assayed as acetylcholinesterase (AChE) inhibitors that could simultaneously bind to the peripheral
and catalytic sites of the enzyme. The compounds were synthesized, and the inhibitory activities toward
AChE and butyrylcholinesterase (BuChE) in vitro were determined using a modified Ellman method. Of the
compounds tested, 6 derivatives were found to inhibit AChE in the micromolar range. The best compound,
2e, had an IC50 of 0.13 ␮M. A detailed molecular modeling study was performed to explore the interaction
of 2e with AChE.
1. Introduction
2. Investigations, results and discussion
Alzheimer’s disease (AD) is a chronic, neurodegenerative disor-
der, characterized by the loss of memory and cognition, severe
behavioral abnormalities, and ultimately death. Low levels of
acetylcholine (ACh) are thought to play a significant role in
the disease, therefore, the use of reversible inhibitors of acetyl-
cholinesterase (AChE) is considered to be a viable and attractive
approach to treat the disease (Bachurin 2003; Munoz-Torrero
and Camps 2006; Orhan et al. 2006). To date only acetyl-
cholinesterase (AChE) inhibitors, such as tacrine, donepezil,
rivastigmine and galantamine and a NMDA receptor antagonist,
memantine, are available for AD treatment (Giacobini 2004).
Recent studies suggest that ␤-amyloid peptide (A␤) in the brain
may be a key factor in the pathogenesis of the disease, as its
accumulation may result in a cascade of biochemical events
leading to neuronal dysfunction (Hardy and Selkoe 2002; Piazzi
et al. 2003). AChE is able to promote the aggregation of A␤ into
amyloid fibrils through residues located in the peripheral anionic
site (PAS) of the enzyme. AChE inhibitors that interact with both
the catalytic site and PAS might exert a dual pharmacological
effect by simultaneously inhibiting acetylcholine hydrolysis and
AChE-induced A␤ aggregation (Camps et al. 2008; Kwon et al.
2007; Tumiatti et al. 2010).
In our previous study, a series of 2-phenoxy-indane-1-one
derivatives (compound 1) which possess alkylamine side chains
have been designed and synthesized. Most of them demonstrated
potent AChE inhibitory activity, and a docking study revealed
that compound 1 bound with the active and peripheral sites
simultaneously (Shen et al. 2008; Sheng et al. 2005). In this
paper, 5,6-dimethoxy-indan-1-one in compound 1 was replaced
by 3,4-dimethoxyacetophenone to result in compound 2 with a
more flexible structure that may lead to a higher affinity or better
selectivity for acetylcholinesterase. This provided an opportu-
nity to explore the relationship between structure flexibility and
inhibitory activity (Fig. 1).
2.1. Chemistry
The target compounds 2a–g were synthesized as illus-
trated in the Scheme. The readily available material
3,4-dimethoxyacetophenone 3 was brominated with CuBr2 to
obtain 2-bromo-1-(3,4-dimethoxyphenyl) ethanone 4 (Belluti
et al. 2005). For the synthesis of intermediates 5a–g, different
routeswerefollowedaccordingtopreviouslypublishedmethods
(Grove et al. 2002; Tramontini 1973). Finally, the target com-
pounds were obtained by refluxing 5a–g with 4 in acetonitrile
in the presence of K2CO3.
2.2. Biological activity
The synthetic compounds 2a–g were then assayed for their
AChE (rat cortex homogenate) and BuChE (rat serum) inhi-
bition potency using the modified Ellman method (Cheng and
Tang 1998; Ellman et al. 1961), with donepezil as the reference
standard. The ChE inhibitory results were summarized in the
Table.
Most of the tested compounds demonstrated moderate or high
inhibitory activities against AChE. Among them, compounds
2a, 2e and 2 g showed sub-micromolar inhibition of AChE.
Compound 2e possessed the most potent activity with IC50 of
0.13 ␮M. Based on the previously published data on compound
1, the replacement of the indanone moiety of compound 1 by
3,4-dimethoxyacetophenone resulted in a decrease in the AChE
inhibitory activity of compound 2 compared to compound 1
(Shen et al. 2008). This indicates that a more flexible struc-
ture may not lead to a higher affinity for the peripheral sites
of AChE than the rigid indanone moiety. Comparing series 2a–
d with 2e–g, it appeared that the compounds with a carbonyl
group in the linker between the benzene ring and tertiary amino
group generally possessed higher inhibitory potency (i.e. 2e and
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ORIGINAL ARTICLES
Fig. 1: Chemical structure of compound 1 and acetophenone derivatives 2a–g
Scheme: Synthesis of 2a–g. Reagents and conditions: (i): CuBr2, CHCl3, AcOEt, reflux (ii) K2CO3, CH3CN, reflux
2 g). Finally, mostofthesyntheticcompoundsshowedinhibitory
selectivity for AChE over BuChE. The most potent compound
2e was 206-fold more active in inhibiting AChE than BuChE,
but less selective than donepezil.
of the optimal compound 2e was performed using the Tripos
FlexiDock program and visualized using PyMOL program. As
seen in Fig. 2, compound 2e extended well into the gorge of
the enzyme. Fig. 3 illustrates that compound 2e makes several
interactions with both the catalytic site and the PAS. Near the
bottom of the gorge, the protonated nitrogen of the ligand made
a cation–␲ interaction with Trp84. At the midgorge recognition
site, the ethereal oxygen atom was hydrogen-bonded with
the OH group of Tyr121 with a bond length of 2.41 Å, and
the carbonyl group in the COCH2CH2 linker formed direct
hydrogen bonds with the OH group in Ser200. At the peripheral
site, the methoxy groups interacted with residues Phe288 and
Arg289 through hydrophobic interactions. Therefore, when
5,6-dimethoxy-indan-1-one in compound 1 was replaced by
3,4-dimethoxyacetophenone, compound 2e assumed a slightly
different orientation compared to compound 1. Especially at
the PAS, compound 2e could not make a close contact with the
key residue Trp279, which may have resulted in the decreased
AChE inhibitory activity.
2.3. Molecular modeling
To explore the possible binding conformation and
protein–ligand interactions, a molecular modeling study
Table: AChE and BuChE inhibition data (IC50, ␮M) of pre-
pared compoundsa
b
Compd.
IC AChE (␮M)
50
IC BuChE (␮M)
50
Selectivity for AChE
2a
2b
2c
2d
2e
2f
2g
0.28
32.8
3.40
7.46
0.13
7.2
76.3
89.4
156.7
42.6
26.9
46.3
28.6
6.72
272.5
2.73
46.1
29.2
206.9
6.43
0.73
0.011
39.17
610.9
Donepezil
2.4. Conclusion
a
In conclusion, a novel class of acetophenone derivatives were
designed, synthesized and tested for their biological activity, and
Values are means of three different experiments.
Selectivity for AChE is defined as IC (BuChE)/IC (AChE)
50 50
b
308
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ORIGINAL ARTICLES
Fig. 2: Compound 2e in the gorge of AChE
Fig. 3: Interaction between compound 2e and AChE
most of the tested compounds demonstrated moderate or high
inhibitory activities against AChE. The optimum inhibitor 2e
showed potent inhibitory activity with an IC50 value of 0.13 ␮M.
The molecular modeling results showed that it likely interacts
with both the catalytic site and the PAS of the enzyme, but with-
out close contact with the key residue Trp279, which may result
in decreased of AChE inhibitory activity.
3.2.1. 2-(4-((Diethylamino)methyl)phenoxy)-1-(3,4-
dimethoxyphenyl)ethanone (2a)
Yield 51%, pale yellow syrup. MS(ESI): 358(M + 1). 1H NMR (CDCl3): ␦
7.64(d, 1H, J = 8.4 Hz, H-6), 7.57(s, 1H, H-2), 7.27(d, 2H, J = 8 Hz, H-3’, H-
5’), 6.90(m, 3H, H-5, H-2’, H-6’), 5.21(s, 2H, COCH2), 3.96 (s, 3H, OCH3),
3.93(s, 3H, OCH3),3.62(s, 2H, benzylic CH2), 2.60 (q, 4H, NCH2CH3,
J = 7.6 Hz), 1.08 (t, 6H, NCH2CH3, J = 7.6 Hz); IR (KBr), ␯ (cm−1): 2967,
1689, 1593, 1510, 1266, 1162, 760. Anal. calcd for C21H27NO4: C, 70.56;
H, 7.61; N, 3.92; found: C, 70.40; H, 7.53; N, 3.98.
3. Experimental
3.1. Chemistry
3.2.2. 1-(3,4-Dimethoxyphenyl)-2-(4-(pyrrolidin-1-
ylmethyl)phenoxy)ethanone (2b)
Melting points were obtained on a B-540 Buchi melting-point apparatus and
uncorrected. ESI-MS were recorded on an Esquire-LC-00075 mass spec-
trometer (Bruker, USA). 1H NMR and 13C NMR spectra were recorded
on a Brucker Advance DMX 400 MHz spectrometer (chemical shifts are
expressed as ␦ values relative to TMS as internal standard). Infrared spec-
tra were recorded on a JASCO FT/IR-4100 spectrophotometer. Elemental
analyses were performed on a Flash EA 1112 elemental analyzer.
2-Bromo-1-(3,4-dimethoxyphenyl)ethanone 4 and key intermediates 5a–g
were synthesized according to published methods (Belluti et al. 2005; Grove
et al. 2002; Tramontini 1973).
Yield 46%, pale yellow syrup. MS(ESI): 356(M + 1). 1H NMR (CDCl3):
␦ 7.65 (d, 1H, J = 8.4 Hz, H-6), 7.59 (s, 1H, H-2), 7.26 (d, 2H, J = 8 Hz,
H-3’, H-5’), 6.88-6.94 (m, 3H, H-5, H-2’, H-6’), 5.22 (s, 2H, COCH2),
3.96 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.63(s, 2H, benzylic-CH2),
2.55 (m, 4H, pyrrolidine-CH2, H-2”, H-5”), 1.81 (m, 4H, H-3”, H-4”); IR
(KBr), ␯ (cm−1): 2876, 1683, 1592, 1511, 1467, 1163, 773. Anal. calcd for
C
21H25NO4: C, 70.96; H, 7.09; N, 3.94; found: C, 71.15; H, 7.02; N, 3.90.
3.2. General procedure for the synthesis of 2a–g
3.2.3. 2-(4-(2-(Diethylamino)ethyl)phenoxy)-1-(3,4-
dimethoxyphenyl)ethanone (2c)
A solution of 2-bromo-1-(3,4-dimethoxyphenyl) ethanone (0.124 g, 0.48
mmol) in CH3CN (3 mL) was added dropwise to a mixture of compound 5
(0.4 mmol), K2CO3 (0.066 g, 0.048 mmol) in CH3CN (3 mL) and the mix-
ture was refluxed for 1 h. After cooling to room temperature, the reaction
mixture was filtered and the solvent was removed in vacuo to give syrups
that were dissolved in dichloromethane (50 mL) and washed with water
and brine. The organic phase was dried and evaporated under reduced pres-
sure and the residue was purified by a silica-gel column chromatography
(CH2Cl2/MeOH/TEA = 100: 5:1)
Yield 37%, white solid, mp 82–84 ◦C. MS(ESI): 372(M + 1). 1H NMR
(CDCl3): ␦7.67(d, 1H, J = 8.4 Hz, H-6), 7.57 (s, 1H, H-2), 7.16 (d,
2H, J = 8 Hz, H-3’, H-5’), 6.88-6.94 (m, 3H, H-5, H-2’, H-6’), 5.24 (s,
2H, COCH2), 3.98 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.03 (m, 6H,
PhCH2CH2N, NCH2CH3), 2.19 (m, 2H, PhCH2CH2N), 1.32 (m, 6H,
CH2CH3). IR (KBr), ␯ (cm−1): 2969, 1689, 1591, 1512, 1267, 1162, 1018,
758. Anal. calcd for C22H29NO4: C, 71.13; H, 7.87; N, 3.77; found: C,
71.36; H, 7.79; N, 3.81.
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ORIGINAL ARTICLES
3.2.4. 1-(3,4-Dimethoxyphenyl)-2-(4-(2-(pyrrolidin-1-
molecules and ligands were removed from the complexes, and hydrogen
yl)ethyl)phenoxy)ethanone (2d)
atoms were subsequently added. The structures of the ligands were pre-
pared in MOL2 format using the sketcher module, and Gasteiger-Huckel
charges were assigned to the ligand atoms. The minimization was run until
Yield 41%, white solid, mp 116–118 ◦C. MS(ESI): 370(M + 1). 1H NMR
(CDCl3): ␦ 7.63 (d, 1H, J = 8.4 Hz, H-6), 7.52 (s, 1H, H-2), 7.14 (d, 2H,
J = 8 Hz, H-3’, H-5’), 6.84-6.91 (m, 3H, H-5, H-2’, H-6’), 5.22 (s, 2H,
COCH2), 3.94 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.22 (m, 6H, pyrrolidine-
CH2, H-2”, H-5”, CH2CH2N), 2.09 (m, 2H), 1.39 (m, 4H). IR (KBr), ␯
(cm−1): 2940, 1685, 1595, 1514, 1465, 1264, 1162, 771. Anal. calcd for
it converged to a maximum derivative of 0.001 kcal mol−1
Å
−1. The dock-
ing and subsequent scoring were performed using the default parameters of
the FlexX program in the SYBYL.
C22H27NO4: C, 71.52; H, 7.37; N, 3.79; found: C, 71.66; H, 7.39; N, 3.63.
References
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3.2.5. 1-(4-(2-(3,4-Dimethoxyphenyl)-2-oxoethoxy)phenyl)-3-
(piperidin-1-yl)propan-1-one (2e)
Yield 45%, white solid, mp 62–64 ◦C. MS(ESI): 412(M + 1). 1H NMR
(CDCl3): 7.96 (d, 2H, H-2’, H-6’), 7.65 (d, 1H, J = 8.4 Hz, H-6), 7.56 (s,
1H, H-2), 6.92-6.98 (m, 3H, H-5, H-3’, H-5’), 5.34 (s, 2H, COCH2), 3.98 (s,
3H, OCH3), 3.95 (s, 3H, OCH3), 3.27 (t, 2H, J = 7.6 Hz, COCH2CH2), 2.93
(t, 2H, J = 7.6 Hz, CH2CH2N), 2.58 (m, 4H, piperidine-CH2, H-2”, H-6”),
1.70 (m, 4H, H-3”, H-5”), 1.49 (m, 2H, H-4”). IR (KBr), ␯ (cm−1): 2932,
1682, 1595, 1512, 1264, 1162, 1014, 763. Anal. calcd for C24H29NO5: C,
70.05; H, 7.10; N, 3.40; found: C, 70.18; H, 7.10; N, 3.27.
3.2.6. 1-(4-(2-(3,4-Dimethoxyphenyl)-2-oxoethoxy)phenyl)-3-
(pyrrolidin-1-yl)propan-1-one (2f)
Yield 42%, white solid, mp 55–57 ◦C. MS(ESI): 398(M + 1). 1H NMR
(CDCl3): 7.97 (d, 2H, H-2’, H-6’), 7.66 (d, 1H, J = 8.4 Hz, H-6), 7.57 (s,
1H, H-2), 6.93-6.96 (m, 3H, H-5, H-3’, H-5’), 5.34 (s, 2H, COCH2), 3.98
(s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.23 (t, 2H, J = 7.6 Hz, COCH2CH2),
2.97 (t, 2H, J = 7.6 Hz, COCH2CH2), 2.65 (m, 4H, pyrrolidine-CH2, H-2”,
H-5”), 1.84(m, 4H, H-3”, H-4”). 13C NMR (␦, CDCl3): 197.2, 192.1, 161.9,
154.1, 149.3, 130.5, 130.4, 127.4, 122.7, 114.5, 110.1, 70.2, 56.1, 56.0, 54.2,
50.9, 37.1, 23.4. IR (KBr), ␯ (cm−1): 2960, 1680, 1596, 1512, 1419, 1266,
1163, 1017, 763. Anal. calcd for C23H27NO5: C, 69.50; H, 6.85; N, 3.52;
found: C, 69.47; H, 6.80; N, 3.41.
3.2.7. 3-(Diethylamino)-1-(4-(2-(3,4-dimethoxyphenyl)-2-
oxoethoxy)phenyl)propan-1-one (2 g)
Yield 35%, pale yellow syrup. MS(ESI): 400(M + 1). 1H NMR (CDCl3):
7.91 (d, 2H, H-2’, H-6’), 7.61 (d, 1H, J = 8.4 Hz, H-6), 7.51 (s, 1H, H-2),
6.88-6.94 (m, 3H, H-5, H-3’, H-5’), 5.32 (s, 2H, COCH2), 3.93 (s, 3H,
OCH3), 3.90 (s, 3H, OCH3), 3.26 (t, 2H, J = 7.6 Hz, COCH2CH2), 3.09
(t, 2H, J = 7.6 Hz, CH2CH2N), 2.78 (m, 4H, NCH2CH3), 2.14 (m, 6H,
NCH2CH3). IR (KBr), ␯ (cm−1): 2967, 1681, 1596, 1512, 1266, 1163,
1016, 764. Anal. calcd for C23H29NO5: C, 69.15; H, 7.32; N, 3.51; found:
C, 69.32; H, 7.18; N, 3.53.
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pyrophosphoramido (isoOMPA) (0.04 mmol/L), a selective inhibitor of
BuChE. To assess AChE or BuChE activity, a reaction mixture contain-
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buffer (pH 7.4), homogenate or serum, and different concentrations of the
tested compounds was incubated at 37 ◦C for 15 min. The reaction was ter-
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3.4. Molecular modeling of compound 2e
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310
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