Synthesis of pterostilbene and resveratrol carbamate derivatives as potential dual cholinesterase inhibitors and neuroprotective agents

Article abstract of DOI:10.1007/s11164-012-1003-1

Pterostilbene and resveratrol carbamate derivatives were designed and synthesized by use of a multi-target directed drug-design strategy. Their acetylcholinesterase and butylcholinesterase inhibitory activity and neuroprotective effects against hydrogen peroxide-induced PC12 cell injury were evaluated in vitro. The results indicated that some of the compounds had dual inhibitory potency against acetylcholinesterase and butylcholinesterase, and potential neuroprotective effects, and could be considered as potential multi-target-directed agents.

Full text of DOI:10.1007/s11164-012-1003-1

Res Chem Intermed
DOI 10.1007/s11164-012-1003-1
Synthesis of pterostilbene and resveratrol carbamate
derivatives as potential dual cholinesterase inhibitors
and neuroprotective agents
•
Wen Yuan Zhipei Shang Xiaoming Qiang
•
•
•
Zhenghuai Tan Yong Deng
Received: 8 November 2012 / Accepted: 26 December 2012
Ó Springer Science+Business Media Dordrecht 2013
Abstract Pterostilbene and resveratrol carbamate derivatives were designed and
synthesized by use of a multi-target directed drug-design strategy. Their acetyl-
cholinesterase and butylcholinesterase inhibitory activity and neuroprotective
effects against hydrogen peroxide-induced PC12 cell injury were evaluated in vitro.
The results indicated that some of the compounds had dual inhibitory potency
against acetylcholinesterase and butylcholinesterase, and potential neuroprotective
effects, and could be considered as potential multi-target-directed agents.
Keywords Alzheimer’s disease ꢀ Pterostilbene carbamates ꢀ Resveratrol
carbamates ꢀ Synthesis ꢀ Cholinesterase inhibitors ꢀ Neuroprotective effects
Introduction
Alzheimer’s disease (AD), a major age-associated neurodegenerative disease, is
characterized by progressive loss of cognitive function, loss of memory, impaired
synaptic function, and massive brain cell loss that ultimately results in premature
death [1]. Although the exact cause of the disease is under intense investigation, the
prevailing hypothesis is that low levels of acetylcholine (ACh), amyloid b-protein
(Ab) deposits, and neurofibrillary tangles are important in the disease [2, 3].
Deposition of amyloid plaques causes cell death by inducing mitochondrial
dysfunction and oxidative stress. Ab deposition initiates the flavoenzyme-dependent
increase of hydrogen peroxide (H2O2) and lipid peroxides that increase free radical
W. Yuan ꢀ Z. Shang ꢀ X. Qiang ꢀ Y. Deng (&)
Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education,
West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, China
e-mail: dengyongy@sohu.com
Z. Tan
Sichuan Institute of Chinese Materia Medica, Chengdu 610041, Sichuan, China
123

W. Yuan et al.
generation [4]. In the past 10 years, much research has been conducted on the
effects of free radical formation, oxidative cell damage, and inflammation in the
pathogenesis of AD, providing promising new targets [5–7]. There is, moreover,
evidence that butylcholinesterase (BuChE) activity increases substantially in the AD
brain and this may also be involved in the etiology and progression of AD. Thus,
development of AChE and BuChE dual inhibitors may lead to improved clinical
outcome [8]. Current treatment of AD focuses on increasing cholinergic
neurotransmission in the brain by inhibiting the activity of cholinesterases with
acetylcholinesterase (AChE) inhibitors, for example tacrine, donepezil, rivastigmine
(1), and galantamine (Fig. 1). In clinical trials, however, these drugs have proved
useful for treatment of cases of mild to moderate severity only, and do not reverse or
heal the disease [9]. Because of the multifaceted etiology of AD and the insufficient
efficacy of single drugs, it is necessary to develop new anti-Alzheimer agents with
multiple potency.
Recent research has demonstrated that oxidative stress can be a risk factor in
initiation and progression of AD [10], and that oxidative damage, for example
formation of radical reactive species, precedes the appearance of the senile plaques
and neurofibrillary tangles [11]. Therefore, antioxidant compounds that specifically
scavenge oxygen radicals may have therapeutic efficacy in AD treatment. Pterostil-
bene ((E)-3,5-dimethoxy-40-hydroxystilbene, 2) and resveratrol ((E)-3,40,5-trihydr-
oxystilbene, 3) are well known natural phenolic compounds with significant
antioxidant, anti-inflammatory, and neuroprotective properties related to AD [12].
They can scavenge reactive oxygen species (ROS) and inhibit the formation of other
free radicals [13]. However, low bioavailability of pterostilbene or resveratrol
restricts their clinical activity as anti-Alzheimer drugs. Rivastigmine (1), marketed as
Exelon by Novartis, is a carbamate pseudo-irreversible acetylcholinesterase inhibitor
which acts selectively in the brain, specifically in the hippocampus and cortex,
interfering with the action of AChE and BuChE [14]. 1 does not reverse the
progression of AD, however, and contributes to modest improvement in cognition
only. Because it has been proved that the carbamyl group is the cholinesterase-
inhibitory pharmacophore [15], we planned to use multi-target-directed drug-design
NH2
O
MeO
MeO
N
N
Donepezil
Tacrine
Me
N
Et
N
NMe2
Me
O
Me
O
OMe
Galantamine
O
HO
Rivastigmine
Fig. 1 Chemical structures of marketed acetylcholinesterase inhibitors
123

Synthesis of pterostilbene and resveratrol carbamate derivatives
strategy to combine 2 and 3, with carbamyl groups, in a single molecule which may,
simultaneously, have cholinesterase inhibitory activity, antioxidant activity, and
neuroprotective properties.
In this article, we describe the synthesis of two novel series, including a series of
pterostilbene carbamate derivatives (6a–h) and a series of resveratrol carbamate
derivatives (7a–h). The design strategy used for the compounds is depicted in
Fig. 2. Furthermore, their acetylcholinesterase and butylcholinesterase inhibitory
activity and neuroprotective effects against hydrogen peroxide-induced PC12 cell
injury were investigated in vitro.
Results and discussion
Chemistry
Synthesis of compounds 6a–h and 7a–h was accomplished by use of the general
pathway illustrated in Scheme 1. N,N-disubstituted carbamoyl chlorides (5a–
h) were the key intermediates in synthesis of the compounds investigated. They are
usually prepared by reaction of secondary amines with phosgene [16]. This
phosgenation route has several disadvantages, however, for example low yields,
long reaction times, handling of highly corrosive materials, and highly toxic
reagents, so a non-phosgene process for preparing N,N-disubstituted carbamoyl
chlorides with good yields under extremely mild reaction conditions and short
reaction times, is highly desirable. Triphosgene (bis(trichloromethyl) carbonate,
BTC), a solid and safe surrogate of phosgene, is usually used in the synthesis of
carbamoyl chloride [17]. In the work discussed in this paper, compounds 5a–h were
prepared by reaction of secondary amines with triphosgene in the presence of
NaHCO3 in anhydrous dichloromethane; this provided the product more conve-
niently and in higher yield. Pterostilbene (2) was then reacted with 1.2 eq 5a–h in
the presence of anhydrous K2CO3 and a catalytic amount 4-dimethylaminopyridine
(4-DMAP) in CH3CN at 60–65 °C to afford the desired target compounds 6a–h in
OH
CH3
CH3
N
RO
CH3
H3C
O
N
CH3
O
Pterostilbene: R= CH3 (2)
Resveratrol: R=H (3)
OR
Rivastigmine (1)
R1
O
N
R2
R3O
O
AChEI
Antioxidant
OR3
Fig. 2 Design strategy used for the pterostilbene and resveratrol carbamate derivatives
123

W. Yuan et al.
good yield. Compounds 7a–h were obtained by reaction of resveratrol (3) with 4.5
eq 5a–h in the presence of anhydrous K2CO3 and catalytic amount 4-DMAP in
acetone at reflux. In this step acetone was chosen as solvent because resveratrol
cannot be dissolved in CH3CN. Because pterostilbene (2) and resveratrol (3) are
readily oxidized, all the reactions should be conducted under an argon atmosphere.
These target compounds have not previously been reported in the literature. Their
structures were confirmed by1H NMR spectroscopy and ESI–HRMS.
Evaluation of biological activity
Two types of novel carbamate derivative, 6a–h and 7a–h, synthesized as described
above, were evaluated for inhibitory activity against AChE from rat cortex
homogenate by the method of Ellman, with a slight modification [18]. Inhibition of
BuChE from rat serum by the same set of compounds was also evaluated to assess
the selectivity profile. Rivastigmine tartrate, a well-known AChE and BuChE dual
inhibitor, was used as reference positive drug. The neuroprotective effects of
selected carbamate derivatives (6a–c, 6g, 7a–b, 7g–h) against free radicals, which
might be involved in a self propagating cascade of neurodegenerative events, were
assessed by measuring the ability of the compounds to protect against hydrogen
O
R1
R2
(i)
R1
R2
NH
N
l
C
5a~h
4a~h
R1
N
H
O
O
R2
H
3CO
H
3CO
O
(ii)
6a~h
H
OC
H
OC
3
2
3
H
C
3
=
R1R2N
H
O
(
)
H
3 2
N
N
C
H
C
b
3
a
H
O
(
H
C
)
H
2C
3 2
N(i-Pr)2
N
N
c
d
3
H
O
R1
N
O
(iii)
N
O
e
g
f
R2
R
3O
O
N
N H
C
N
N H Ph
C
2
3
h
R
O
=
R3 R1R2NCO
7a~h
3
Scheme 1 Synthesis of carbamate derivatives of pterostilbene and resveratrol, 6a–h and 7a–h,
respectively. Reagents and conditions: (i) triphosgene, NaHCO3, CH2Cl2, -10–0 °C 6–8 h; (ii) 1.2 eq
R1R2NCOCl (5a–h), 4-DMAP, K2CO3, CH3CN, 60–65 °C, 5–10 h; (iii) 4.5 eq R1R2NCOCl (5a–h),
4-DMAP, K2CO3, acetone, reflux 12–18 h
123

Synthesis of pterostilbene and resveratrol carbamate derivatives
peroxide-induced PC12 cell injury, by use of the MTT method in accordance with a
reported procedure [19], with pterostilbene (2) and resveratrol (3) as reference
compounds. The biological profile of the tested compounds, including AChE and
BChE inhibitory potency, expressed as percentage inhibition and IC50 values, and
PC12 cell viability, expressed as a percentage, are shown in Table 1.
The ChE inhibitory activity results showed that reference compound rivastigmine
tartrate had IC50 values of 6.3 lM for AChE and 1.37 lM for BuChE. Compounds
2 and 3 had no activity toward AChE and BuChE. However, the carbamate
derivatives 6a–h and 7a–h had different amounts of AChE and BuChE inhibitory
potency. Among these, compound 7h had the best AChE and BuChE inhibitory
activity with IC50 values of 4.11 and 1.13 lM, respectively, both being higher than
those of the reference compound rivastigmine tartrate. Compounds 6g and 7g had
moderate AChE inhibitory activity—38.04 and 41.64 % inhibition at 5.0 lM and
IC50 values of 8.58 and 6.97 lM, respectively; neither had BuChE inhibitory
Table 1 Inhibition of AChE and BuChE activity by, and neuroprotective effects of, rivastigmine (1),
pterostilbene (2), resveratrol (3), and carbamate derivatives 6a–h and 7a–h
Compound
AChE
BuChE
Neuroprotective effectb
a
%
a
IC50 (lM)
%
IC50 (lM)
Cell viability (%)
1
47.5
\5.0
\5.0
\5.0
\5.0
\5.0
\5.0
\5.0
\5.0
38.04
18.59
9.00
6.3
58.68
\5.0
\5.0
\5.0
\5.0
\5.0
\5.0
\5.0
\5.0
\5.0
\5.0
9.94
1.37
N.T.c
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
[50
N.T.
N.T.
N.T.
N.T.
[50
N.T.
1.13
65.45 0.67
76.38 0.64
80.53 0.95
75.34 0.56
77.99 0.67
78.29 0.32
N.T.
2
N.T.c
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
8.58
[50
[50
[50
N.T.
N.T.
[50
[50
6.97
4.11
3
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
7g
7h
N.T.
N.T.
82.56 1.35
N.T.
75.05 1.37
75.68 0.75
N.T.
8.18
\5.0
\5.0
\5.0
\5.0
26.65
\5.0
60.48
\5.0
\5.0
5.45
N.T.
N.T.
10.27
41.64
54.29
N.T.
80.45 1.74
81.30 0.25
All experiments were performed in triplicate
a
Inhibition (%) was determined at 5.0 lM inhibitor concentration
b
Neuroprotective effect of compounds at 10 lM on cell injury induced by hydrogen peroxide (100 lM)
in PC12 cells. The cell viability in control was taken as 100 %; cell viability under hydrogen peroxide
(100 lM) conditions was 57.58 %
c
Not tested
123

W. Yuan et al.
activity. Compound 7f had weak AChE inhibitory active and moderate BuChE
inhibitory activity (26.65 % inhibition at 5.0 lM). The other carbamate derivatives
were either inactive or had only weak AChE and BuChE inhibitory activity. On the
basis of these results, the molecular structure containing the piperazine-N0-carbonyl
structure seems to be of crucial importance in AChE inhibitory activity. The reason
may be the basic piperazine tertiary amine moiety, which improved the water
solubility of the compounds; the tertiary amine group has often been important for
molecular and enzyme recognition through hydrophilic or hydrogen-bonding
interaction. It is also known that the binding pocket of BuChE is bigger than that
of AChE [20]. Therefore, compound 7h, which has a larger molecular volume than
compounds 6g and 7g, could to better fit inside the gorge of BuChE. This may
explain why compound 7h has remarkable inhibitory activity against BuChE.
In testing of neuroprotective effects against free radicals, exposure of PC12 cells to
100 lM H2O2 markedly reduced viability to 57.58 %, suggesting high sensitivity to
H2O2-induced injury. Carbamate derivatives 6a–c, 6g, 7a, b, and 7g, h at concen-
trations of 10.0 lM significantly increased the cell survival to 75.05–82.56 %. This
activity was comparable with or superior to that of the reference compounds 2
(76.38 %) and 3 (80.53 %) at the same concentration; rivastigmine tartrate had only
weak antioxidant activity (65.45 %). These results also showed that the antioxidant
activity of compounds containing the N-methylpiperazine-N0-carbonyl (6g, 7g) and
N-benzylpiperazine-N0-carbonyl (7h) structures was substantially more potent than
that of compounds which containing the N,N-dialkylcarbonyl (6a–d, 7a–d), N-
morpholinecarbonyl (6e, 7e), or N-piperidinecarbonyl (6f, 7f) structures. This may be
because of better water solubility and cell membrane permeation, mainly attributed to
the basic tertiary unit.
Conclusion
To investigate the efficacy of novel drugs for treatment of Alzheimer’s disease
(AD), sixteen pterostilbene and resveratrol carbamate derivatives were designed on
the basis of a multi-target-directed drug-design strategy. The results indicated that
compounds 6g and 7g had moderate inhibiting activity and better selectivity toward
AChE than rivastigmine. Compound 7h had dual inhibitory potency toward AChE
and BuChE, and potential neuroprotective effects, and could be considered as a
potential multi-target directed agent. Further studies to evaluate 7h in vivo are in
progress in our laboratory and will be reported in due course.
Experimental
Melting points were recorded on a YRT-3 melting-point apparatus (China) and are
uncorrected. 1H NMR spectra were recorded on a Varian INOVA 400 NMR
spectrometer with CDCl3 as solvent and TMS as internal standard. Proton chemical
shifts are expressed in parts per million (ppm) and coupling constants in hertz. Mass
spectra were recorded on a Agilent-6210 TOF LC–MS spectrometer. All
123

Synthesis of pterostilbene and resveratrol carbamate derivatives
commercially available reagents and anhydrous solvents were of the highest quality
and were used without further purification. Pterostilbene and resveratrol were
commercially available and purchased from Hangzhou Great Forest Biomedical.
Reactions were monitored by TLC on silica gel GF254 plates from Qingdao Haiyang
Chemical (China); detection was by means of a UV lamp (254 nm). Where
appropriate, crude products were purified by column chromatography on silica gel
(230–400 mesh) from Qingdao Haiyang Chemical.
General procedure for synthesis of carbamyl chlorides (5a–h)
A solution of the secondary amine (4a–h) (30.00 mmol) in dichloromethane (5 ml)
was added dropwise, over half of an hour, to a stirred slurry of sodium bicarbonate
(5.04 g, 60 mmol) and triphosgene (5.94 g, 20 mmol) in dichloromethane (30 ml)
at -10 °C. The reaction mixture was left to warm to room temperature and stirred
for 6–8 h. The mixture was then filtered and the filtrate was evaporated under
reduced pressure to give the carbamyl chloride crude product. The crude product
was distilled under reduced pressure or purified by chromatography on silica,
with petroleum ether–dichloromethane as mobile phase, to give the desired product
(5a–h).
N, N-Dimethylcarbamyl chloride (5a)
Colorless oil, yield 50.4 %, bp 69.0–72.0 °C at 3.4 kPa. (lit., [21] 167–168 °C at
775 mm Hg).
N-Ethyl-N-Methylcarbamyl chloride (5b)
Colorless oil, yield 73.9 %, bp 94.0–96.0 °C at 6.0 kPa. (lit., [21] 88.0–89.0 °C at
40 mm Hg).
N, N-Diethylcarbamyl chloride (5c)
Colorless oil, yield 73.2 %, bp 70.0–72.5 °C at 3.4 kPa. (lit., [21] 60.0–62.0 °C at
15 mm Hg).
N, N-Diisopropylcarbamyl chloride (5d)
White solid, yield 45.6 %, bp 120.0–122.5 °C at 2.0 kPa, mp 52.0–54.0 °C. (lit.,
[22] mp 55.0 °C).
N-Morpholinecarbonyl chloride (5e)
Colorless oil, yield 75.3 %, bp 114.0–116.0 °C at 2.0 kPa. (lit., [21] 101–103 °C at
8 mm Hg).
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W. Yuan et al.
N-Piperidinecarbonyl chloride (5f)
Colorless oil, yield 50.5 %, bp 118.0–120.0 °C at 2.0 kPa. (lit., [21] 111–113 °C at
13 mm Hg).
N-Methyl-N0-piperazinecarbonyl chloride (5g)
Colorless oil, yield 73.8 %, bp 122.0–124.0 °C at 2.0 kPa. (lit., [22] 84–85 °C at
0.2 mm Hg).
N-Benzyl-N0-piperazinecarbonyl (5h)
White solid, yield 51.2 %, mp 90.0–92.0 °C. (lit., [23] mp 92 °C).
General procedure for preparation of pterostilbene carbamate derivatives 6a–h
Carbamyl chloride 5a–h (0.585 mmol) was added to a mixture of anhydrous K2CO3
(92 mg, 0.663 mmol), 4-DMAP (5.0 mg, 0.039 mmol), and pterostilbene (100 mg,
0.390 mmol) in 5 ml MeCN. The reaction mixture was warmed to 60–65 °C and
stirred for 5–10 h under an argon atmosphere. The reaction was monitored by TLC.
On completion of the reaction the solvent was evaporated under reduced pressure.
Water (20 ml) was added to the residue and the mixture was extracted with
dichloromethane (2 9 30 ml). The combined organic phases were washed sequen-
tially with 5 % NaOH solution (20 ml), 5 % HCl solution (20 ml), and saturated
aqueous sodium chloride (25 mL), dried over anhydrous Na2SO4, and filtered. The
solvent was evaporated under reduced pressure and the residue was purified by
silica gel flash column chromatography (mobile phase 2:1 CH2Cl2–EtOAc) to give
compounds 6a–h.
(E)-4-(3,5-Dimethoxystyryl)phenyl N,N-dimethylcarbamate (6a)
White needle crystals, yield 84.2 %, mp 124.0–125.0 °C. 1H NMR (400 MHz,
CDCl3) d: 7.49 (d, 2H, J = 8.8 Hz, H-20, H-60), 7.11 (d, 2H, J = 8.8 Hz, H-30,
H-50), 7.07 (d, J = 16.0 Hz, 1H, =CH–), 6.97 (d, 1H, J = 16.0 Hz, =CH–), 6.66
(d, 2H, J = 2.0 Hz, H-2, H-6), 6.39 (t, 1H, J = 2.0 Hz, H-4), 3.83 (s, 6H, 3-OCH3,
5-OCH3), 3.11 (s, 3H, NCH3), 3.02 (s, 3H, NCH3); HRMS (ESI) calcd for
C19H22NO4 [M ? H]? m/z: 328.1549, found 328.1557.
(E)-4-(3,5-Dimethoxystyryl)phenyl N-ethyl-N-methylcarbamate (6b)
1
White crystals, yield 79.3 %, mp 66.6–67.6 °C. H NMR (400 MHz, CDCl3) d:
7.49 (d, 2H, J = 8.4 Hz, H-20, H-60), 7.11 (d, 2H, J = 8.4 Hz, H-30, H-50), 7.07
(d, 1H, J = 16.4 Hz, =CH–), 6.97 (d, 1H, J = 16.4 Hz, =CH–), 6.66 (d, 2H,
J = 2.0 Hz, H-2, H-6), 6.39 (t, 1H, J = 2.0 Hz, H-4), 3.84 (s, 6H, 3-OCH3,
5-OCH3), 3.49–3.41 (m, 2H, NCH2), 3.08 (s, 3/2H, 1/29 NCH3), 3.00 (s, 3/2H,
123

Synthesis of pterostilbene and resveratrol carbamate derivatives
1/29 NCH3), 1.27–1.18 (m, 3H, CH3); HRMS (ESI) calcd for C20H24NO4
[M ? H]? m/z: 342.1705, found 342.1701.
(E)-4-(3,5-Dimethoxystyryl)phenyl N,N-diethylcarbamate (6c)
1
White crystals, yield 82.7 %, mp 53.5–54.5 °C. H NMR (400 MHz, CDCl3) d:
7.49 (d, 2H, J = 8.4 Hz, H-20, H-60), 7.12 (d, 2H, J = 8.4 Hz, H-30, H-50), 7.07
(d, 1H, J = 16.0 Hz, =CH–), 6.97 (d, 1H, J = 16.0 Hz, =CH–), 6.66 (d, 2H,
J = 2.0 Hz, H-2, H-6), 6.39 (t, 1H, J = 2.0 Hz, H-4), 3.83 (s, 6H, 3-OCH3,
5-OCH3), 3.45–3.39 (m, 4H, 29 NCH2), 1.28–1.19 (m, 6H, 29 CH3); HRMS (ESI)
calcd for C21H26NO4 [M ? H]? m/z: 356.1862, found 356.1855.
(E)-4-(3,5-Dimethoxystyryl)phenyl N,N-diisopropylcarbamate (6d)
1
White crystals, yield 75.6 %, mp 106.4–107.3 °C. H NMR (400 MHz, CDCl3) d:
7.49 (d, 2H, J = 8.4 Hz, H-20, H-60), 7.12 (d, 2H, J = 8.4 Hz, H-30, H-50), 7.07
(d, 1H, J = 16.4 Hz, =CH–), 6.97 (d, 1H, J = 16.4 Hz, =CH–), 6.66 (d, 2H,
J = 2.0 Hz, H-2, H-6), 6.39 (t, 1H, J = 2.0 Hz, H-4), 4.11 (brs, 1H, NCH), 3.95
(brs, 1H, NCH) 3.83 (s, 6H, 3-OCH3, 5-OCH3), 1.33–1.24 (m, 12H, 49 CH3);
HRMS (ESI) calcd for C23H30NO4 [M ? H]? m/z: 384.2175, found 384.2172.
(E)-4-(3,5-Dimethoxystyryl)phenyl morpholine-N-carboxylate (6e)
White needle crystals, yield 83.3 %, mp 156.5–156.9 °C. 1H NMR (400 MHz,
CDCl3) d: 7.50 (d, 2H, J = 8.8 Hz, H-20, H-60), 7.11 (d, 2H, J = 8.8 Hz, H-30,
H-50), 7.07 (d, 1H, J = 16.4 Hz, =CH–), 6.98 (d, 1H, J = 16.4 Hz, =CH–), 6.66
(d, 2H, J = 2.0 Hz, H-2, H-6), 6.40 (t, 1H, J = 2.0 Hz, H-4), 3.83 (s, 6H, 3-OCH3,
5-OCH3), 3.76 (t, 4H, J = 4.8 Hz, 29 NCH2), 3.69 (brs, 2H, OCH2), 3.58 (brs, 2H,
OCH2); HRMS (ESI) calcd for C21H24NO5 [M ? H]? m/z: 370.1654, found
370.1646.
(E)-4-(3,5-Dimethoxystyryl)phenyl piperidine-N-carboxylate (6f)
Colorless oil, yield 82.5 %. 1H NMR (400 MHz, CDCl3) d: 7.49 (d, 2H,
J = 8.4 Hz, H-20, H-60), 7.10 (d, 2H, J = 8.4 Hz, H-30, H-50), 7.07 (d, 1H,
J = 16.0 Hz, =CH–), 6.97 (d, 1H, J = 16.0 Hz, =CH–), 6.66 (d, 2H, J = 1.6 Hz,
H-2, H-6), 6.39 (t, 1H, J = 1.6 Hz, H-4), 3.84 (s, 6H, 3-OCH3, 5-OCH3), 3.56 (brs,
4H, 29 NCH2), 1.65 (s, 6H, 39 CH2); HRMS (ESI) calcd for C22H26NO4
[M ? H]? m/z: 368.1862, found 368.1868.
(E)-4-(3,5-Dimethoxystyryl)phenyl N-methylpiperazine-N0-carboxylate (6g)
Colorless oil, yield 84.0 %. 1H NMR (400 MHz, CDCl3) d: 7.49 (d, 2H, J = 8.8 Hz,
H-20, H-60), 7.11 (d, 2H, J = 8.8 Hz, H-30, H-50), 7.07 (d, 1H, J = 16.4 Hz, =CH–),
6.97 (d, 1H, J = 16.4 Hz, =CH–), 6.66 (d, 2H, J = 2.4 Hz, H-2, H-6), 6.40 (t, 1H,
J = 2.4 Hz, H-4), 3.83 (s, 6H, 3-OCH3, 5-OCH3), 3.74 (brs, 2H, CONCH2), 3.64
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W. Yuan et al.
(brs, 2H, CONCH2), 2.52 (brs, 4H, 29 NCH2), 2.39 (s, 3H, NCH3); HRMS (ESI)
calcd for C22H27N2O4 [M ? H]? m/z: 383.1971, found 383.1980.
(E)-4-(3,5-dimethoxystyryl)phenyl N-benzylpiperazine-N0-carboxylate (6h)
White solid, yield 88.0 %, mp 147.0–147.3 °C. 1H NMR (400 MHz, CDCl3) d: 7.49
(d, 2H, J = 8.4 Hz, H-20, H-60), 7.32 (m, 5H, Ar–H) 7.10 (d, 2H, J = 8.4 Hz, H-30,
H-50), 7.07 (d, 1H, J = 16.0 Hz, =CH–), 6.97 (d, 1H, J = 16.0 Hz, =CH–), 6.66
(d, 2H, J = 2.0 Hz, H-2, H-6), 6.39 (t, 1H, J = 2.0 Hz, H-4), 3.83 (s, 6H, 3-OCH3,
5-OCH3), 3.69 (s, 2H, PhCH2), 3.58 (s, 4H, 29 CONCH2); 2.51 (s, 4H, 29 NCH2);
HRMS (ESI) calcd for C28H31N2O4 [M ? H]? m/z: 459.2284, found 459.2280.
General procedure for preparation of resveratrol carbamate derivatives 7a–h
Carbamyl chloride 5a–h (1.58 mmol) was added to a mixture of anhydrous K2CO3
(233 mg, 1.69 mmol), 4-DMAP (5.0 mg, 0.039 mmol), and resveratrol (80 mg,
0.35 mmol) in 10 ml acetone. The reaction mixture was heated under reflux for
12–18 h under an argon atmosphere until reaction was complete (monitored by
TLC). The mixture was then cooled to room temperature and the solvent was then
evaporated under reduced pressure. The residue was dissolved in dichloromethane
(60 ml) and the solution was washed sequentially with water (2 9 25 mL), 5 %
NaOH solution (25 ml), 5 % HCl solution (25 ml), and saturated aqueous sodium
chloride (25 mL) before being dried over anhydrous Na2SO4. The solvent was
evaporated in vacuo and the residue was purified by silica gel flash column
chromatography (mobile phase 30:1 CH2Cl2–CH3OH) to give compounds 7a–h.
(E)-3,5,40-Tri[(N, N-dimethylcarbamoyl)oxy]-stilbene (7a)
White needle crystals, yield 66.8 %, mp 143.0-143.4 °C. 1H NMR (400 MHz,
CDCl3) d: 7.45 (d, 2H, J = 8.4 Hz, H-20, H-60), 7.11–7.08 (m, 4H, H-30, H-50, H-2,
H-6), 7.06 (d, 1H, J = 16.0 Hz, =CH–), 6.95 (d, 1H, J = 16.0 Hz, =CH–), 6.86
(s, 1H, H-4), 3.09 (brs, 9H, 39 NCH3), 3.06–2.98 (m, 9H, 39 NCH3); HRMS (ESI)
calcd for C23H28N3O6 [M ? H]? m/z: 442.1978, found 442.1975.
(E)-3,5,40-Tri[(N-ethyl-N-methylcarbamoyl)oxy]-stilbene (7b)
1
White solid, yield 66.0 %, mp 80.5–82.2 °C. H NMR (400 MHz, CDCl3) d: 7.46
(d, 2H, J = 8.8 Hz, H-20, H-60), 7.11–7.08 (m, 4H, H-30, H-50, H-2, H-6), 7.07 (d, 1H,
J = 16.4 Hz, =CH–), 6.96 (d, 1H, J = 16.4 Hz, =CH–), 6.78 (s, 1H, H-4), 3.49–3.41
(m, 6H, 39 NCH2), 3.07–3.00 (m, 9H, 39 NCH3), 1.25–1.18 (m, 9H, 39 CH3);
HRMS (ESI) calcd for C26H34N3O6 [M ? H]? m/z: 484.2448, found 484.2456.
(E)-3,5,40-Tri[(N, N-diethylcarbamoyl)oxy]-stilbene (7c)
White needle crystals, yield 74.6 %, mp 106.0–106.5 °C. 1H NMR (400 MHz,
CDCl3) d: 7.46 (d, 2H, J = 8.4 Hz, H-20, H-60), 7.14–7.09 (m, 4H, H-30, H-50, H-2,
123

Synthesis of pterostilbene and resveratrol carbamate derivatives
H-6), 7.08 (d, 1H, J = 16.4 Hz, =CH–), 6.97 (d, 1H, J = 16.4 Hz, =CH–), 6.89
(s, 1H, H-4), 3.44–3.39 (m, 12H, 69 NCH2), 1.25–1.19 (m, 18H, 69 CH3); HRMS
(ESI) calcd for C29H40N3O6 [M ? H]? m/z: 526.2917, found 526.2916.
(E)-3,5,40-Tri[(N, N-diisopropylcarbamoyl)oxy]-stilbene (7d)
1
White solid, yield 80.0 %, mp 82.0–84.0 °C. H NMR (400 MHz, CDCl3) d: 7.47
(d, 2H, J = 8.8 Hz, H-20, H-60), 7.12–7.09 (m, 4H, H-30, H-50, H-2, H-6), 7.08
(d, 1H, J = 16.4 Hz, =CH–), 6.97 (d, 1H, J = 16.4 Hz, =CH–), 6.88 (t, 1H,
J = 2.0 Hz, H-4), 4.11 (brs, 3H, 39 NCH), 3.95 (brs, 3H, 39 NCH), 1.33–1.30 (m,
36H, 129 CH3); HRMS (ESI) calcd for C35H52N3O6 [M ? H]? m/z: 610.3856,
found 610.3860.
(E)-3,5,40-Tri[(morpholine-N-carbonyl)oxy]-stilbene (7e)
White needle crystals, yield 79.9 %, mp 193.0–193.7 °C. 1H NMR (400 MHz,
CDCl3) d: 7.47 (d, 2H, J = 8.8 Hz, H-20, H-60), 7.13–7.10 (m, 4H, H-30, H-50, H-2,
H-6), 7.08 (d, 1H, J = 16.4 Hz, =CH–), 6.96 (d, 1H, J = 16.4 Hz, =CH–), 6.89
(t, 1H, J = 2.0 Hz, H-4), 3.77–3.71 (m, 12H, 69 NCH2), 3.67 (s, 6H, 39 OCH2),
3.58 (s, 6H, 39 OCH2); HRMS (ESI) calcd for C29H34N3O9 [M ? H]? m/z:
568.2295, found 568.2299.
(E)-3,5,40-Tri[(piperidine-N-carbonyl)oxy]-stilbene (7f)
White solid, yield 99.3 %, mp 191.0–192.5 °C. 1H NMR (400 MHz, CDCl3) d: 7.45
(d, 2H, J = 8.4 Hz, H-20, H-60), 7.10–7.08 (m, 4H, H-30, H-50, H-2, H-6), 7.06
(d, 1H, J = 16.4 Hz, =CH–), 6.95 (d, 1H, J = 16.4 Hz, =CH–), 6.86 (brs, 1H, H-4),
3.59–3.52 (m, 12H, 69 NCH2), 1.64 (s, 18H, 99 CH2); HRMS (ESI) calcd for
C32H40N3O6 [M ? H]? m/z: 562.2917, found 562.2908.
(E)-3,5,40-Tri[(N-methylpiperazine-N0-carbonyl)oxy]-stilbene (7g)
Colorless oil, yield 70.8 %. 1H NMR (400 MHz, CDCl3) d: 7.46 (d, 2H,
J = 8.4 Hz, H-20, H-60), 7.12–7.09 (m, 4H, H-30, H-50, H-2, H-6), 7.07 (d, 1H,
J = 16.4 Hz, =CH–), 6.96 (d, 1H, J = 16.4 Hz, =CH–), 6.87 (t, 1H, J = 2.0 Hz,
H-4), 3.69 (brs, 6H, 39 CONCH2), 3.60 (brs, 6H, 39 CONCH2), 2.46 (t, 12H,
J = 4.8 Hz, 69 NCH2), 2.35 (s, 9H, 39 NCH3); HRMS (ESI) calcd for
C32H43N6O6 [M ? H]? m/z: 607.3244, found 607.3248.
(E)-3,5,40-Tri[(N-benzylpiperazine-N0-carbonyl)oxy]-stilbene (7h)
Pale yellow oil, yield 88.4 %. 1H NMR (400 MHz, CDCl3) d: 7.45 (d, 2H,
J = 8.8 Hz, H-20, H-60), 7.36–7.29 (m, 15H, 159 Ar–H), 7.10–7.08 (m, 4H, H-30,
H-50, H-2, H-6), 7.06 (d, 1H, J = 16.0 Hz, =CH–), 6.94 (d, 1H, J = 16.0 Hz, =CH–
), 6.86 (t, 1H, J = 2.0 Hz, H-4), 3.68 (s, 6H, 39 PhCH2), 3.54 (s, 12H, 69
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W. Yuan et al.
CONCH2), 2.52 (s, 12H, 69 NCH2); HRMS (ESI) calcd for C50H55N6O6 [M ? H]?
m/z: 835.4183, found 835.4170.
AChE and BuChE inhibition assays
Materials
Acetylthiocholine iodide (lot no. R10L15) and 5,50-dithiobis-2-nitrobenzoic acid
(DTNB; lot no. LV10M21) were purchased from J&K Scientific. Butyrylthiocholine
iodide (lot no. 28IFN-KC) was purchased from TCI (Shanghai) Development.
Tetraisopropyl pyrophosphoramide (lot no. 089K1178V) was purchased from Sigma.
All other chemicals were of analytical grade or the highest chromatographic purity
available. Acetylcholinesterase and butyrylcholinesterase were obtained from SPF
male Sprague–Dawley rats (200–300 g), which were supplied by the Center of
Experimental Animals of Sichuan Academy of Chinese Medicine Sciences (eligi-
bility certification no. SCXK-Sichuan 2008-19).
Assay methods
To assess the inhibitory activity of the compounds toward AChE or BuChE, we
followed the spectrophotometric method of Ellman, using 5 % rat cortex
homogenate and 25 % rat serum as the source of AChE and BuChE. Brain
homogenate was preincubated for 5 min with tetraisopropyl pyrophosphoramide
(iso-OMPA, selective inhibitor of BuChE, 4.0 mmol/L). For assay of AChE or
BuChE activity, the reaction mixture (100 lL) containing acetylthiocholine iodide
(1.0 mmol/L, 30 lL) or butyrylthiocholine iodide (1.0 mmol/L, 30 lL), phosphate-
buffered solution (0.1 mmol/L, 40 lL, pH 7.4), 5 % homogenate or 25 % serum
(10 lL), and different concentrations of the test compound (20 lL) was incubated at
37 °C for 15 min. 5,50-Dithiobis-2-nitrobenzoic acid (DTNB; 0.2 %, 30 lL) was
then added to produce the yellow anion of 5-thio-2-nitrobenzoic acid. Changes in
absorbance were detected at 405 nm by use of a Wellscan MK2 spectrophotometric
plate reader (Denley Dragon). Compounds inhibiting AChE or BuChE activity
reduced color generation. Thus, IC50 values were calculated as the compound
concentrations causing 50 % inhibition of AChE or BuChE activity. Rivastigmine
hydrogen tartrate (purity[99.5 %) was used as positive reference drug. All samples
were assayed in triplicate.
Neuroprotective effect assay
Materials
DMEM medium and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
were purchased from Sigma. Newborn calf serum was purchased from Hangzhou
123

Synthesis of pterostilbene and resveratrol carbamate derivatives
Sijiqing Biological Engineering Materials. All other chemicals were of analytical
grade or the highest chromatographic purity available.
Assay methods
PC12 cells were seeded into 96-well plates at a density of 1 9 105 cells per mL in
DMEM medium supplemented with 10 % heat-inactivated bovine calf serum. After
overnight incubation, test compound at 10.0 lmoL/L was added and incubated with
the cells. After pretreatment with the compounds for 2 h, H2O2 was added to the
PC12 cell cultures for 30 min, then replaced with fresh DMEM medium without
calf serum and phenol red. Cell-viability assays were performed 24 h after culture in
fresh medium. At the end of the experiments, the cells were incubated with 0.5 mg/
mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 2 h at
37 °C. The supernatant layer was then carefully removed, 100 lL DMSO was
added into each well, and the absorbance at 490 nm of the MTT formazan was
determined by use of an Elx-800 microplate recorder (Bio-Tek Instruments).
Resveratrol and pterostilbene (purity [99.0 %) was used as positive reference
drugs. Results, as a percentage of control data, are expressed as mean SD.
Acknowledgments This work was financially supported by the Chinese National Natural Science
Foundation (20672077, 20872099), the Research Fund for the Doctoral Program of Higher Education
(20110181110079), and the National Science and Technology Major Project on ‘‘Key New Drug Creation
and Manufacturing Program’’ (2013ZX09301304-002).
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