NO-donating tacrine derivatives as potential butyrylcholinesterase inhibitors with vasorelaxation activity

Article abstract of DOI:10.1016/j.bmcl.2013.04.008

To search for potent anti-Alzheimer's disease (AD) agents with multifunctional effects, 12 NO-donating tacrine-flurbiprofen hybrid compounds (2a-l) were synthesized and biologically evaluated. It was found that all the new target compounds showed selective butyrylcholinesterase (BuChE) inhibitory activity in vitro comparable or higher than tacrine and the tacrine-flurbiprofen hybrid compounds 1a-c, and released moderate amount of NO in vitro. The kinetic study suggests that one of the most active and highest BuChE selective compounds 2d may not only compete with the substrate for the same catalytic active site (CAS) but also interact with a second binding site. Furthermore, 2d and 2l exhibited significant vascular relaxation effect, which is beneficial for the treatment of AD. All the results suggest that 2d and 2l might be promising lead compounds for further research.

Full text of DOI:10.1016/j.bmcl.2013.04.008

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3162–3165
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
NO-donating tacrine derivatives as potential butyrylcholinesterase
inhibitors with vasorelaxation activity
Yao Chen a,b,c, Jianfei Sun d, Zhangjian Huang a,b, Hong Liao d, , Sixun Peng a,b, Jochen Lehmann c,e,
⇑
⇑
,
Yihua Zhang a,b,
⇑
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
b Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
c Lehrstuhl für Pharmazeutische/Medizinische Chemie, Institut für Pharmazie, Friedrich-Schiller-Universität Jena, Philosophenweg 14, D-07743 Jena, Germany
d Neurobiology Lab, New Drug Screening Center, China Pharmaceutical University, Nanjing 210009, PR China
e College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
To search for potent anti-Alzheimer’s disease (AD) agents with multifunctional effects, 12 NO-donating
tacrine–flurbiprofen hybrid compounds (2a–l) were synthesized and biologically evaluated. It was found
that all the new target compounds showed selective butyrylcholinesterase (BuChE) inhibitory activity
in vitro comparable or higher than tacrine and the tacrine–flurbiprofen hybrid compounds 1a–c, and
released moderate amount of NO in vitro. The kinetic study suggests that one of the most active and high-
est BuChE selective compounds 2d may not only compete with the substrate for the same catalytic active
site (CAS) but also interact with a second binding site. Furthermore, 2d and 2l exhibited significant vas-
cular relaxation effect, which is beneficial for the treatment of AD. All the results suggest that 2d and 2l
might be promising lead compounds for further research.
Received 26 January 2013
Revised 24 March 2013
Accepted 3 April 2013
Available online 10 April 2013
Keywords:
Alzheimer’s disease
Butyrylcholinesterase inhibitors
Tacrine
Ó 2013 Elsevier Ltd. All rights reserved.
Nitric oxide
Vasodilator properties
Alzheimer’s disease (AD) is an age-related chronic, neurodegen-
erative disorder, manifested as loss of cholinergic neurons and re-
duced levels of neurotransmitter acetylcholine (ACh) in the brain.1
To improve cholinergic function, the strategy of restoring the ACh
level has been investigated.2 It is well-known that two cholinester-
ases, acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE) are responsible for ACh hydrolysis. In normal brains, AChE
accounts for the majority of cholinesterase activity and is thus ini-
tially considered as the most important target for the treatment of
AD.3,4 In fact, most currently prescribed AD drugs are AChE inhib-
itors (AChEI), such as tacrine and rivastigmine.5 However, AChEIs
can only halt the progression of AD for a while instead of prevent-
ing or reversing it.6,7 In comparison, BuChE plays a minor role in
the healthy brain, whereas its level and activity progressively
increases in the AD patient brains,8,9 which suggests BuChE should
also be taken as a crucial target. Recently, positive effects of the
administration of the selective BuChE inhibitor (BuChEI) cymserine
on learning and improving cognitive performance have already
been shown in rodents.10
In addition to the cholinergic strategy, increasing evidence
suggests that nitric oxide (NO), a free radical gas, may be beneficial
for the treatment of AD by increasing blood supply11 and regulat-
ing the cerebral circulation.12 Consequently, to search for potent
NO-donating BuChEI with multifunctional effects is of great
interest.
In this study, we conjugated nitrate, a NO donor moiety, via alk-
ylene side chain, to the tacrine–flurbiprofen hybrid compounds
NH2
COOH
F
N
tacrine
flurbiprofen
O
ONO2
n
F
F
O
O
HN m N
H
HN
N
N
m H
N
2a-l, m=2-4; n=2-4,6
⇑
Corresponding authors. Tel.: +86 25 83271043; fax: +86 25 83271142 (H.L.);
1a-c, m=2-4
tel.: +49 3641949803; fax: +49 3641949802 (J.L.); tel./fax: +86 25 83271015 (Y.Z.).
E-mail addresses: hliao@cpu.edu.cn (H. Liao), j.lehmann@uni-jena.de
(J. Lehmann), zyhtgd@163.com (Y. Zhang).
Figure 1. Structures of tacrine, flurbiprofen, compounds 1a–c, and the target
compounds 2a–l.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.008

Y. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3162–3165
3163
(1a–c, Fig. 1), which were previously prepared by our group as po-
tential BuChEIs.13 Herein we report the synthesis and biological
evaluation of the target compounds (2a–l).
subsequent treatment of AgNO3 in dry CH3CN offered nitrates
11a–d. The nitrates were deprotected under basic conditions to
yield acids 12a–d, which were coupled with 4a–c in the presence
of DCC/DMAP to provide the target compounds 2a–l (Scheme 2).
Target compounds 2a–l were tested for in vitro inhibition of
AChE from electrophorus electricus (eeAChE) and BuChE from
equine serum, respectively, using the Ellman assay15 (Table 1). It
was observed that all the compounds showed comparable or better
BuChE inhibitory activity (IC50s range from 3.9–13.9 nM) than
tacrine and 1a–c. Interestingly, it was found that the length of lin-
ker connecting the phenol and nitrate moieties influenced the
selectivity of compounds. Generally, when the length of the linker
increased, the AChE inhibitory activity decreased, leading to
enhanced BuChE selectivity. The best results were obtained with
2d and 2l, which contain a six-carbon chain linker, and have IC50
values of 4309.5 and 1456.4 nM against AChE and IC50 ratios are
567 and 373, respectively.
The synthesis of 2a–l is outlined in Schemes 1 and 2. The inter-
mediate acridine 3 was synthesized starting from anthranilic acid
and cyclohexanone according to a previously reported protocol.14
Subsequently, various alkylenediamines were introduced to 3 by
substitution giving the aminoalkylamino acridines 4a–c (Scheme
1). To synthesize the other key intermediates 12a–d, the carboxyl
group of flurbiprofen was methylated to generate ester 5, followed
by the Friedel–Crafts acylation and the Baeyer–Villiger oxidation
rearrangement to yield methyl ester 7, which was hydrolyzed
and acetylated to produce 40-OH flurbiprofen 8. Methylation of 8
resulted in ester 9, which was treated with the corresponding dib-
romoalkanes to give the halogenated intermediates 10a–d, and
To gain insights into the binding mode of this new family of
compounds on BuChE, a kinetic study was carried out with one
of the most promising compound 2d. The mechanism was
C
O
COOH
NH2
a
N
3
b
Table 1
HN m NH2
Inhibition of AChE and BuChE (IC50 values), selectivity ratios and NO release
(presented as nitrite)
N
Compd
IC50 (nM) SEMa
Selectivity ratiob Nitritec
(lg/ml)
4a,
4b
, m=3
m=2;
AChE
BuChE
4c, m=4
1a
1b
1c
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
Tacrine
ISMN
714.9 251.7
344.8 45.5
193.1 8.5
182.7 25.2
340.9 89.1
1345.4 990.3
4309.5 1801.0
112.4 7.8
188.6 71.9
356.5 40.3
1010.8 315.1
227.3 25.3
261.2 59.0
641.2 128.2
1456.4 226.9
69.8 11.1
58.7 7.3
13.9 3.4
11.0 1.6
9.9 0.7
10.0 0.4
10.1 0.8 133.2
7.6 0.5 567.0
8.1 1.2
8.6 1.1
7.9 1.0
12.2
24.8
17.6
18.5
34.1
NDd
ND
ND
Scheme 1. Synthetic route of 4a–c. Reagents and conditions: (a) POCl3, reflux, 3 h;
(b) pentanol, NH2(CH2)mNH2, reflux, 18 h.
0.209 0.012
0.302 0.039
0.456 0.014
0.424 0.039
0.219 0.009
0.308 0.051
0.366 0.051
0.353 0.058
0.268 0.017
0.347 0.045
0.321 0.039
0.321 0.058
0.011 0.004
0.412 0.013
COOCH3
COOH
13.9
21.9
45.1
a
b
F
F
5
8.1 1.6 124.8
13.9 1.5
10.6 0.6
7.7 0.7
3.9 0.3 373.4
10.6 1.1
ND
16.4
24.6
83.3
COOCH3
c
COOCH3
d
O
F
F
O
6
O
7
6.6
ND
ND
a
b
c
COOCH3
Data is the mean of at least three determinations.
Selectivity ratio = (IC50 of AChE)/(IC50 of BuChE).
All values are the mean SEM.
COOH
e
f
F
F
HO
d
HO
Br
ND means not determined.
9
8
COOCH3
g
COOCH3
h
Lineweaver-Burk plot BuChE inhibition by compound 2d
F
F
O
O2NO
O
n
n
10a
10b
n=3
n=2;
15
11a n=2; 11b n=3
10c n=4; 10d n=6
11c
11d
n=4;
n=6
O
ONO2
n
F
10
O
COOH
i
m NH
[2d] = 10 nM
HN
N
F
O2NO
O
5
n
[2d] = 4 nM
[2d] = 2 nM
[2d] = 1 nM
[2d] = 0 nM
2a-l
12a n=2; 12b n=3
12c 12d
n=4;
n=6
2a, m=2, n=2; 2b, m=2, n=3; 2c, m=2, n=4
2d, m=2, n=6; 2e, m=3, n=2; 2f, m=3, n=3
2g, m=3, n=4; 2h, m=3, n=6; 2i, m=4, n=2
2j, m=4, n=3; 2k, m=4, n=4; 2l, m=4, n=6
-0.03 -0.02
0.01
0.02
0.03
0.04
0.05
1/[S] in ¦ÌM-1
Scheme 2. Synthetic route of 2a–l. Reagents an conditions: (a) CH3OH, H2SO4,
reflux, 3 h; (b) CH3COCl, AlCl3, anhydrous CH2Cl2, reflux, 12 h; (c) mCPBA, CH2Cl2,
room temperature, 48 h; (d) NaOH, THF/CH3OH/H2O, room temperature, 6 h; (e)
CH3OH, H2SO4, reflux, 3 h; (f) Br(CH2)nBr, DMF, K2CO3, 65 °C, 6 h; (g) AgNO3, CH3CN,
60 °C, 6 h; (h) LiOH, THF/CH3OH/H2O, room temperature, 24 h; (i) DCC, DMAP,
anhydrous CH2Cl2, room temperature, 24 h.
-5
Figure 2. Linewear–Burk plots resulting from sub-velocity curves of BuChE activity
with different substrate concentrations (25–450 lM) in the absence and presence
of 2d (1, 2, 4, 10 nM).

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Y. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3162–3165
Table 2
analyzed by Lineweaver–Burk reciprocal plots, which were reci-
procal rates versus reciprocal substrate concentrations for different
concentrations of 2d. The results indicated both increased slopes
(decreased Vmax) and intercepts (higher Km) at increasing con-
centration of 2d (Fig. 2), suggesting a mixed-type inhibition of
2d, which may not only compete with the substrate for the same
catalytic active site (CAS), but also interact with a second binding
site.
Relaxation activity (%) of tacrine, 2d and 2l at 100 lM
Compounds
Relaxation activitya (%)
Tacrine
2d
2l
3.4 3.7
21.9 7.0
31.3 5.7
a
All values are the mean SEM.
To investigate the location of the second binding site for the
enzyme and compound 2d, a molecular docking study using Libdock
within Discovery Studio (DS, Accelrys) was performed. The results
(Fig. 3) showed that the tacrine scaffold of 2d could insert into the
CAS surrounded by residue His438, Tyr440, Ser198 and Ile69. At
the mouth of the gorge, the benzene ring of flurbiprofen moiety
that NO release from the compounds might be closely associated
with their vascular relaxation activity, leading to potential
anti-AD activity.
In conclusion, a series of NO-donating tacrine–flurbiprofen
hybrid compounds have been designed and synthesized as novel
potential BuChEIs. All of the compounds showed comparable or
higher BuChE inhibitory activity compared to tacrine and the par-
ent compounds 1a–c. The inhibitory mechanism of one of the most
active and highest selective compound 2d was analyzed by
Lineweaver–Burk reciprocal plots, suggesting a dual site binding
mode for BuChE. All the compounds could release moderate
amount of NO in vitro, and compounds 2d and 2l displayed signif-
icant vascular relaxation activity as compared to tacrine, which is
beneficial for the treatment of AD. All the results suggest that the
novel NO-donating tacrine–flurbiprofen hybrids 2d and 2l might
be promising lead compounds for further research.
in 2d showed strong parallel p–p stacking against the benzene ring
of Tyr332, which was considered as the key residue in the second
binding site. Additionally, the nitrate group in 2d could form
hydrogen bonds with Ala277, Glu276 and Asn68, which can
enhance the intermolecular recognition between 2d and BuChE.
The ability of compounds 2a–l to release NO was measured
using the Griess reaction16 by quantifying the nitrite produced
from the oxidative reaction of NO, oxygen and water (Table 1).
The results showed that all the target compounds generated higher
levels of nitrite than tacrine and similar levels (0.209–0.456
compared to the positive control isosorbide mononitrate (ISMN,
0.412 g/ml).
lg/ml)
l
To evaluate the vasorelaxation effect, the two active compounds
2d and 2l were tested in an ex vivo organ bath (coronar arteries
from rat) using vascular relaxation assay, in which tacrine was
employed as the negative control. As shown in Table 2, both 2d
(21.9%) and 2l (31.3%) exhibited significant blood vessel relaxation
activity in comparison with tacrine (3.4%). These results suggest
Acknowledgments
The authors thank the financial support from National Science
Foundation of China (NSFC, No. 30973608) and the Natural Science
Foundation of Jiangsu Province of China (No. BK2009296).
Figure 3. Representation of the binding mode of compound 2d with BuChE (PDB id: 1POM), compound 2d and the key residues of BuChE are shown as stick form.

Y. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3162–3165
3165
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Supplementary data
Supplementary data (syntheses and spectra data of intermedi-
ates and all target compounds 2a–l, and protocols of pharmacolog-
ical assays) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.008.
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