Synthesis of benzofuran derivatives via rearrangement and their inhibitory activity on acetylcholinesterase

Article abstract of DOI:10.3390/molecules15128593

During a synthesis of coumarins to obtain new candidates for treating Alzheimer's Disease (AD), an unusual rearrangement of a benzopyran group to a benzofuran group occurred, offering a novel synthesis pathway of these benzofuran derivatives. The possible

Full text of DOI:10.3390/molecules15128593

Molecules 2010, 15, 8593-8601; doi:10.3390/molecules15128593
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Benzofuran Derivatives via Rearrangement and
Their Inhibitory Activity on Acetylcholinesterase
Xiang Zhou 1, Miao Li 2, Xiao-Bing Wang 1, Tao Wang 2 and Ling-Yi Kong 1,*
1
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang,
Nanjing 210009, China
2
Jiangsu Center for Drug Screening, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing
210009, China
* Author to whom correspondence should be addressed: E-Mail: cpu_lykong@126.com;
Tel.: +86-25-85391289; Fax: +86-25-85301528.
Received: 15 September 2010; in revised form: 18 November 2010 / Accepted: 24 November 2010 /
Published: 29 November 2010
Abstract: During a synthesis of coumarins to obtain new candidates for treating
Alzheimer’s Disease (AD), an unusual rearrangement of a benzopyran group to a
benzofuran group occurred, offering a novel synthesis pathway of these benzofuran
derivatives. The possible mechanism of the novel rearrangement was also discussed. All of
the benzofuran derivatives have weak anti-AChE activities compared with the reference
compound, donepezil.
Keywords: coumarin; benzofuran; rearrangement; anti-AChE
1. Introduction
Benzofuran derivatives are a major group of biologically active heterocycles, which are usually
important constituents of plant extracts used in medicinal chemistry for their various biological
activities [1-4]. Due to their diverse activities, much attention has been paid to synthetic strategies to
access these systems, and a number of methods have been developed [5-7], but a method involving the
rearrangement from a benzopyran group to a benzofuran group has not been reported before. More
interesting to us, studies have shown that some of benzofuran derivatives such as donepezil [8] were
able to inhibit acetylcholinesterase (AChE), or had the capability of reducing aggregated beta-amyloid

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(Aβ) in the brain [9,10]. This prompted us to continued synthesizing benzofuran derivatives through
the rearrangement of coumarins to expand our efforts on developing novel AChE inhibitors for treating
Alzheimer’s disease (AD).
As we know, AChE and toxic Aβ are the main medication targets for treating AD so far, so dual
binding AChE inhibitors [11], which can not only facilitate cholinergic transmission but also interfere
with AD pathogenesis [12,13], namely the synthesis, deposition and aggregation of toxic Aβ in brain
areas, have become the leading strategy for the development of anti-AD agents [14-17]. Hence
developing novel dual binding AChE inhibitors is our particular interest and as part of our research
program on naturally occurring biological coumarins [18-20], a series of novel coumarin derivatives
has already been designed and synthesized aiming at AChE inhibitory activity [21]. As the extension
of our efforts on developing new AChE inhibitors based on the coumarins, a novel series of derivatives
were designed (Figure 1), but surprisingly, we did not obtain the target compounds during synthesis
but rather an unusual rearrangement of the benzopyran group to a benzofuran group occurred instead,
which offered a new synthetic pathway of benzofuran derivatives not reported before. Here, the details
of the new rearrangement as well as the pharmacological characteristics of the new benzofuran
derivatives obtained through this rearrangement are discussed.
Figure 1. Design strategy of the target compounds.
OCH3
N
N
R
O
O
O
N
O
O
N
N
R
extention
O
N
N
R
O
O
N
R
N
H3C
O
O
2. Results and Discussion
2.1. Chemistry
The synthesis of the benzofuran derivatives is shown in Scheme 1. The key intermediate 4-
hydroxy-6-methylcoumarin (B) was prepared from p-cresol and malonic acid using ZnCl2 as the
catalyst in POCl3 and the mixture was stirred at 60 °C for 24 h to give B. Then B was treated with
POCl3 at reflux for 0.5 h to give 4-chloro-6-methylcoumarin (C). 4-morpholino-6-methylcoumarin (D)
was obtained through the reaction between C and morpholine under reflux for 12 h. Compound D was
brominated with NBS using BPO as the initiator to give 3-bromo-6-methyl-4-morpholinocoumarin (E)
and not the expected 6-(bromomethyl)-4-morpholinocoumarin. Finally, E reacted with the
corresponding piperazine substituents [22] and the rearranged compounds A1-A5 were obtained.

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Scheme 1. The preparation of the target compounds.
Cl
O
OH
H3C
H3C
c
a
b
CH2(COOH)2
+
H3C
OH
O
O
B
O
C
O
N
O
BrH2C
N
O
N
H3C
O
O
O
d
O
N
H3C
O
N
N
e
O
O
R
d
A
D
H3C
Br
O
O
E
Compounds
R
Compounds
R
A1
A2
A3
H
o-CH3
p-Cl
A4
A5
p-CH3
p-OCH3
Reagents and conditions: (a) p-cresol, ZnCl2, malonic acid, POCl3, 24 h, 60 °C; (b) POCl3,
triethylamine, 0.5 h, reflux; (c) K2CO3, morpholine, acetone 12 h, reflux; (d) NBS, BPO, CCl4
under nitrogen, 8 h, reflux; (e) substituted phenylpiperazine derivatives, potassium carbonate,
15 mL acetone, 15 mL ethanol, 48 h, reflux.
1
The structures of the target compounds were elucidated by H-NMR, IR and ESI-MS. In order to
confirm their chemical structures, one of the target compounds, A1, was further identified by single
crystal X-ray diffraction (Figure 2).
Figure 2. The X-ray diffraction structure of compound A1.

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Through analyzing its chemical spectral data, the structure of D was identified as 6-methyl-4-
morpholinocoumarin and that of E as 3-bromo-6-methyl-4- morpholinocoumarin and not the expected
6-(bromomethyl)-4-morpholinocoumarin, but A1 was identified as (4-phenylpiperazin-1-yl) (5-
methyl-3-morpholino-benzofuran-2-yl) methanone, so it can be concluded that the rearrangement
should have occured in the step of synthesizing A from E. A possible mechanism of this
transformation (shown in Scheme 2) is also discussed below. As to the position of bromination, after
comparing the structure of D with the coumarin derivatives synthesized before [21], it can be noticed
that the main difference between them was the substituents on the 4-position of the coumarin group,
which was a basic morpholino group in D, while in the coumarin derivatives synthesized before it was
a methyl, methoxy or chloro group, so the substituents on the 4-position of the coumarin group should
be taken into account during the study of the position of bromination..
Scheme 2. A possible mechanism of the rearrangement.
O
N
O
N
H3C
Br
O
H3C
Br
O
O
O
N
N
N
N
R
R
O
N
O
N
H3C
Br
H3C
-Br
O
O
O
O
N
N
N
N
R
R
2.2. Anti-AChE testing
To determine the therapeutic potency of the benzofuran derivatives A1-A5 for treating AD, their
anti-AChE activities were assayed according to Ellmann’s method [23], utilizing freshly prepared
AChE from rats brain homogenate and donepezil as the reference compound. Inhibition of AChE
activities of them was shown in Table 1.
Table 1. Inhibition of AChE activities of the synthesized compounds.
AChE inhibition
(IC50, μmol/L) a
45 ± 0. 3
AChE inhibition
(IC50, μmol/L) a
11 ± 0.2
Compound
Compound
A1
A2
A3
A4
A5
donepezil
32 ± 0.1
73 ± 0. 2
21 ± 0.1
0.11 ± 0.01
a Data are means ± standard deviation of three independent experiments.

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In general, the data in Table 1 clearly shows that all of these compounds exhibited moderate
inhibition activities toward the cholinesterase. Among them compound A4 (4-(p-tolylpiperazin-1-yl)
(5-methyl-3-morpholinobenzofuran-2-yl) methanone showed the best AChE inhibitory activity with
the IC50 value of 11 μmol/L.
3. Conclusions
By linking a substituted phenylpiperazine moiety to the benzofuran backbone conformationally
restricted derivatives that are as potential inhibitors of AChE for treating AD were synthesized. An
unusual rearrangement from a benzopyran group to a benzofuran group occurred. The rearrangement
ocurred under moderate conditions and offers a facile and practical preparation of biologically active
benzofuran derivatives, so it may be worthwhile for developing potential AChE inhibitors through the
rearrangement of benzopyran rings to benzofuran derivatives under mild conditions.
4. Experimental
4.1. General
Reaction progress was monitored using thin layer chromatography (TLC) on precoated Merck silica
gel Kiesegel 60 F254 plates and the spots were detected under UV light (254 nm). Flash
chromatography was performed with 230–400 mesh silica gel. The IR spectra were measured on a
Jasco FT/IR-430 spectrophotometer. Melting points (mp) were obtained on a B-540 Buchi melting-
1
point apparatus and are uncorrected. The H-NMR spectra were recorded on a 500 MHz Bruker
spectrometer. The chemical shifts are reported downfield in ppm relative to internal TMS, and
coupling constants are reported in Hertz (Hz). Mass spectra were run on a HP 5989A electrospray
ionization mass spectrometer spectrometer. The mass spectra analysis is reported as m/z values.
4.2. 4-Chloro-6-methylcoumarin (C)
Compound B (0.005 mol) and triethylamine (0.005 mol) were added to POCl3 (5 mL). The mixture
was refluxed for 30 min, and poured into water. Then the solution was extracted with methylene
chloride. The methylene chloride layer was dried with anhydrous Na2SO4 and evaporated under
reduced pressure. Flash chromatography (cyclohexane-acetone = 10:1) was performed to give C 0.8 g
1
(yield 82.3%); H-NMR (CDCl3) δ: 7.56 (d, 1H, H-5, J = 2.1 Hz), 7.26 (dd, 1H, H-7, J = 2.1 Hz,
8.7 Hz), 7.28 (d, 1H, H-8, J = 8.7 Hz), 6.53 (s, 1H, H-3), 2.39 (s, 3H, 6-CH3).
4.3. 6-Methyl-4-morpholinocoumarin (D)
C (0.01 mol), K2CO3 (0.02 mol) and morpholine (0.01 mol) were dissolved in acetone (50 mL),
then the mixture was refluxed for 12 h., after which the reaction mixture was filtered and the filtrate
was evaporated under reduced pressure. Flash chromatography (cyclohexane-acetone = 10:1) was
1
performed to give D 0.97 g (yield 75.6%); H-NMR (CDCl3) δ: 7.36 (d, 1H, H-5, J = 1.9 Hz), 7.31
(dd, 1H, H-7, J = 1.9 Hz, 8.5 Hz), 7.24 (d, 1H, H-8, J = 8.5 Hz), 5.73 (s, 1H, H-3), 3.24 [m, 4H,
N1(CH2)2], 3.94 [m, 4H, (CH2)2O], 2.41 (s, 3H, 6-CH3).

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4.4. 3-Bromo-6-methyl-4-morpholinocoumarin (E)
D (0.003 mol) were added to anhydrous CCl4 (34 mL) with the reaction system protected by N2.
The mixture was refluxed for 0.5 h, then 1-bromopyrrolidine- 2, 5- dione (NBS, 0.0028 mol) and
methyl benzoperoxoate (BPO, 100 mg) were added to the reaction mixture. After the reaction refluxed
for 5 h, additional BPO (100 mg) was added again, and the reaction continued for 3 h. Then the
reaction mixture was cooled down to room temperature and filtered. The filtrate was evaporated under
reduced pressure. Flash chromatography (cyclohexane-acetone = 5:1) was performed to give E 0.32 g
(yield 59.6%); 1H-NMR (CDCl3) δ: 7.36 (m, 1H, H-9), 7.34 (dd, 1H, H-7, J = 1.9 Hz, 8.4 Hz), 7.24 (d,
1H, H - 6, J = 8.4 Hz), 3.54 [m, 4H, N1(CH2)2], 3.93 [m, 4H, (CH2)2O], 2.44 (s, 3H, 10-CH3). 13C-
NMR (CDCl3): 169.3 (C-3); 157.2 (C-1); 149.8 (C-8); 136.2 (C-5); 132.3 (C-6); 127.7 (C-4); 117.3
(C-7); 114.9 (C-9); 75.8 (C-2); 67.7(C-11 and C-12); 48.9 (C-10 and C-13); 22.3 (CH3). ESI-MS:
325.3 ([M+H]+).
4.5. General synthetic procedure for target compounds A
E (1 mmol) was added to a mixture of acetone (15 mL) and ethanol (15 mL) containing a
substituted piperazine derivative (1 mmol) and potassium carbonate (2 mmol). The mixture was
refluxed for 48 h and the reaction mixture was evaporated. Flash chromatography was performed to
afford the target compounds that were further purified by recrystallization from ethanol.
(5-Methyl-3-morpholinobenzofuran-2-yl)-(4-phenylpiperazin-1-yl) methanone (A1) Yield 49%;
colorless powder; mp 131–133 °C; IR (KBr) ν cm−1: 3449, 2826, 1620, 1598, 1464, 1338, 1260, 1200,
1
1021, 902. H-NMR (CDCl3) δ: 7.58 (s, 1H, H-9), 7.44 (m, 1H, H-7), 7.22~7.28 (m, 3H, Ph, H - 6),
6.82~7.00 (m, 3H, Ph), 3.61~3.84 (m, 8H, CH2-15, CH2-18, O(CH2)2), 3.15~3.20 (m, 8H, CH2-11,
CH2-14, CH2-16, CH2-17), 2.42 (s, 3H, CH3-10); ESI-MS: 406.2 ([M+H]+). CCDC number 740474
contains the supplementary crystallographic data for this compound. These data can be obtained free
of
charge
from
The
Cambridge
Crystallographic
Data
Centre
via
www.ccdc.cam.ac.uk/data_request/cif. Empirical formula, C24H27N3O3; molecular weight, 405.49;
crystal dimensions, 0.40  0.34  0.25 mm; Orthorhombic, Pbca; a = 15.385 (2) Å, b = 7.301 (2) Å, c
= 37.579 (3) Å; α = 90.00°, β = 90.00°, γ = 90.00°; V = 4221.3 (14) Å3; Z = 8; Dx = 1.276 Mg· m-3;
R1 = 0.0546; ωR2 = 0.1205; GOOF = 1.055;  = 1.71-25.01°;  = 0.085 mm-1; T = 298(2)K.
(4-o-Tolylpiperazin-1-yl)-(5-methyl-3-morpholinobenzofuran-2-yl) methanone (A2) Yield 43%;
colorless powder; mp 122–125 °C; IR (KBr) ν cm−1: 3424, 2954, 1617, 1518, 1385, 1259, 1158, 802;
1H-NMR (CDCl3) δ: 7.42 (s, 1H, H-9), 7.37 (m, 1H, H-7), 7.17 (m, 1H, H-6), 7.08~7.10 (m, 2H, Ph),
6.85~6.87 (m, 2H, Ph), 3.86~3.95 [m, 8H, CH2-15, CH2-18, O(CH2)2], 3.19~3.29 (m, 8H, CH2-11,
CH2-14, CH2-16, CH2-17), 2.42 (s, 3H, CH3-10), 2.42 (s, 3H, CH3). ESI-MS: 420.2 ([M+H]+).
(4-(4-Chlorophenyl)piperazin-1-yl)-(5-methyl-3-morpholinobenzofuran-2-yl) methanone (A3) Yield
42%; colorless powder; mp 128–130 °C; IR (KBr) ν cm−1: 2955, 2862, 1620, 1593, 1499, 1387, 1259,
1
1231, 813. H-NMR (CDCl3) δ: 7.43 (s, 1H, H-9), 7.29 (m, 1H, H-7), 7.19~7.24 (m, 3H, Ph, H-6),

Molecules 2010, 15
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6.85~6.87 (m, 2H, Ph), 3.73~3.88 [m, 8H, CH2-15, CH2-18, O(CH2)2], 3.21~3.29 (m, 8H, CH2-11,
CH2-14, CH2-16, CH2-17), 2.42 (m, 3H, CH3). ESI-MS: 440.1 ([M+H]+).
(4-p-Tolylpiperazin-1-yl)-(5-methyl-3-morpholinobenzofuran-2-yl) methanone (A4) Yield 43%;
colorless powder; mp 122–125 °C; IR (KBr) ν cm−1: 2968, 2832, 1623, 1597, 1494, 1270, 1200, 1024,
1
917, 768; H-NMR (CDCl3) δ: 7.42 (s, 1H, H-9), 7.30 (m, 1H, H-7), 7.16~7.21 (m, 3H, Ph, H-6),
7.01~7.04 (m, 2H, Ph), 3.70~3.90 [m, 8H, CH2-15, CH2-18, O(CH2)2], 2.96~3.31 (m, 8H, CH2-11,
CH2-14, CH2-16, CH2-17), 2.45 (s, 3H, CH3-10), 2.34 (s, 3H, CH3). ESI-MS: 420.2 ([M+H]+).
(4-(4-Methoxyphenyl)piperazin-1-yl) (5-methyl-3-morpholinobenzofuran-2-yl) methanone (A5) Yield
39%; colorless powder; mp161–163 °C; IR (KBr) ν cm−1: 2914, 2828, 1619, 1514, 1461, 1228, 1157,
1
816. H-NMR (CDCl3) δ: 7.42 (s, 1H, H-9), 7.29 (m, 1H, H-7), 7.17 (m, 1H, H-6), 6.91 (m, 2H, Ph),
6.85~6.87 (m, 2H, Ph), 3.86~3.95 [m, 8H, CH2- 15, CH2-18, O(CH2)2], 3.12~3.29 (m, 8H, CH2-11,
CH2-14, CH2-16, CH2-17), 3.77 (s, 3H, OCH3), 2.42 (m, 3H, CH3-10). ESI-MS: 436.2 ([M+H]+).
4.6. In vitro AChE inhibition assay
AChE activity was measured in duplicate by the spectrophotometric method reported by
Ellman et al. [23] with some modifications, mainly involving the source of the enzyme and the
reference compound. Rat brain homogenate was used as the enzyme source. The whole brain except
for the cerebellum was homogenized in nine volumes of 100 mM sodium phosphate buffer (pH 7.0).
The test compounds were dissolved in dimethyl sulphoxide (DMSO). The AChE activity was
expressed as a change in OD at 412 nm.
Acknowledgements
This research work was supported by the Chang Jiang Scholar Program of Ministry of Education, P.
R. China (for L.-Y. Kong).
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