Dual functional cholinesterase and PDE4D inhibitors for the treatment of Alzheimer's disease: Design, synthesis and evaluation of tacrine-pyrazolo[3,4-b]pyridine hybrids

Article abstract of DOI:10.1016/j.bmcl.2019.06.056

A series of tacrine-pyrazolo[3,4-b]pyridine hybrids were synthesised and evaluated as dual cholinesterase (ChE) and phosphodiesterase 4D (PDE4D) inhibitors for the treatment of Alzheimer's disease (AD). Compound 10j, which is tacrine linked with pyrazolo[

Full text of DOI:10.1016/j.bmcl.2019.06.056

Accepted Manuscript
Dual functional cholinesterase and PDE4D inhibitors for the treatment of Alz-
heimer’s disease: design, synthesis and evaluation of tacrine-pyrazolo[3,4-
b]pyridine hybrids
Tingting Pan, Shishun Xie, Yan Zhou, Jinhui Hu, Haibin Luo, Xingshu Li, Ling
Huang
PII:
S0960-894X(19)30437-8
https://doi.org/10.1016/j.bmcl.2019.06.056
BMCL 26534
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 March 2019
21 June 2019
28 June 2019
Please cite this article as: Pan, T., Xie, S., Zhou, Y., Hu, J., Luo, H., Li, X., Huang, L., Dual functional cholinesterase
and PDE4D inhibitors for the treatment of Alzheimer’s disease: design, synthesis and evaluation of tacrine-
pyrazolo[3,4-b]pyridine hybrids, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/
j.bmcl.2019.06.056
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Dual functional cholinesterase and PDE4D inhibitors for the treatment of Alzheimer’s
disease: design, synthesis and evaluation of tacrine-pyrazolo[3,4-b]pyridine hybrids
Tingting Pan, Shishun Xie , Yan Zhou , Jinhui Hu , Haibin, Luo, Xingshu Li, Ling Huang *
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
 For L. Huang: Tel.: +086-20-3994-3051; Fax: +086-20-3994-3051; e-mail: huangl72@mail.sysu.edu.cn;
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
A series of tacrine-pyrazolo[3,4-b]pyridine hybrids were synthesised and evaluated as dual
cholinesterase (ChE) and phosphodiesterase 4D (PDE4D) inhibitors for the treatment of
Alzheimer’s disease (AD). Compound 10j, which is tacrine linked with pyrazolo[3,4-b]pyridine
moiety by a six-carbon spacer, was the most potent acetylcholinesterase (AChE) with IC50 value
Accepted
Available online
of 0.125μM. Moreover, compound 10j provided a desired balance of AChE and
butylcholinesterase (BuChE) and PDE4D inhibition activities, with IC50 value of 0.449 and
0.271 μM, respectively. The above results indicated that this hybrid was a promising dual
functional agent for the treatment of AD.
Keywords:
Tacrine-pyrazolo[3,4-b]pyridine hybrids
Cholinesterase inhibitor
PDE4D inhibitors
2009 Elsevier Ltd. All rights reserved.
Alzheimer's disease
Alzheimer’s disease (AD) is a neurodegenerative illness
characterized by a progressive decline in cognitive function,
memory loss, extensive neuronal loss, decrease in cholinergic
transmission, and the presence of senile plaques. 1-2 It is the most
common single cause of dementia and is also became the world-
wide health problem with implications for an increasing number
inhibition.16-19 The newly developed PDE4D inhibitor GEBR-7b
enhanced spatial and object memory performance in mice and
rats (Fig. 1).20-21 In addition, MK-0952 (Fig. 1), another PDE4
inhibitor that enhanced cognition in preclinical studies, prompted
clinical trials in patients with mild-to-moderate AD from phase II
clinical studies.22
3
of people and countries. However, up to now, there is no
CH3
O
effective treatment is available.4 The decrease of cholinergic
transmission, caused by the loss of basal forebrain cholinergic
cells, was supposed to play an important role in the cognitive
impairment associated with AD. Studies had dedicated that
increase the levels in Ach by inhibiting acetylcholinesterase
could effectively improve the cognitive function and memory
ability.3, 5-9 Based on the cholinergic hypothesis, Cholinesterase
(ChE) became one of major targets in the current therapy of AD7
and most of the clinically approved drugs for treatment of AD are
acetylcholinesterase inhibitors (AChEIs), such as tacrine,
rivastigmine, galantamine and Donepezil (Figure 1).10-11
H
N
NH2
N
O
H
N
NH2
N
CH3
Tacrine
Rivastigmine
Galanthamine
HOOC
N
O
NH
O
O
F
O
O
N
Donepezil
MK-0952
Phosphodiesterase 4 (PDE4) is the primary cAMP-specific
hydrolase and is represented by four genes (PDE4A, B, C and D).
O
N
NH
O
O
O
O
O
H
12
PDE4D, which is relatively high expression in the frontal
GEBR-7b
N
O
O
Rolipram
cortex13, has been demonstrated to be the main subtype involved
in the process of memory consolidation and LTP.14 Rolipram
(Fig. 1), a typical PDE4D inhibitor, has beneficial effects on
hippocampal dependent memory tasks, but PDE4D-deficient
mice did not show altered memory with the administration of
Fig. 1 Structure of tacrine, rivastigmine, galantamine, donepezil, GEBR-
7b, MK-0952 and rolipram.
Tacrine, the first inhibitor of both AChE and
butylcholinesterase (BuChE) approved by the FDA for the
treatment of AD, was withdrawn from the market due to its side
rolipram, which further demonstrated the crucial role of PDE4D
15
in memory consolidation.
Recently, several strategies were
proposed to enhance cerebral cyclic adenosine monophosphate
(cAMP) for the treatment of neurological disorders by PDEs
23
effects such as hepatotoxicity.
Nevertheless, medicinal

chemists remain interested in researching tacrine analogues or
related new candidates as the excellent inhibition activity.
were stirred for 12 hours under room temperature to give the
target compound 10a-l.
24-27
Pyrazolo[3,4-b]pyridine, a novel scaffold reported as potent and
selective PDE4 inhibitors, may could be bind to peripheral
anionic binding site (PAS) of AChE.28 Taking into account the
importance of PDE4 and AChE inhibition in AD therapeutics, we
designed, synthesized and evaluated a series of new tacrine-
pyrazolo[3,4-b]pyridine hybrids hybrids as dual functional
ligands for the treatment of Alzheimer’s disease (Fig. 2). In this
paper, We variated the length of the linker chain between the
tacrine and pyrazolo[3,4-b]pyridine moiety to obtain a hybrid
with the desired balance of AChE and BuChE and PDE4D
inhibition activities.
Cl
N
NH(CH2)nNH2
COOH
a
b
R
NH2
R
R
N
7
8
9a-9l
NH
O
H
10a:
10b:
10c:
10d:
10e:
10f:
R = H, n = 2
R = H, n = 4
R = H. n = 5
R = H. n = 6
R = H, n = 7
R = H, n = 8
10g:
10h:
10i:
10j:
10k:
10l:
R = Cl, n = 2
R = Cl, n = 4
R = Cl, n = 5
R = Cl, n = 6
R = Cl, n = 7
R = Cl, n = 8
c
N
N
R
N
N
H
n
N
N
10
Scheme 2. Synthesis of 10a-l. Reagents and conditions: (a)
cyclohexanone, POCl3, 0ꢀ°C-reflux; (b) diamine, KI, 1-pentanol, 160ꢀ°C; (c)
6, CH2Cl2, TEA, rt, 12h. 69% for 10a, 68% for 10b, 73% for 10c, 65% for
10d, 68% for 10e, 71% for 10f, 78% for 10g, 75% for 10h, 70% for 10i, 65%
for 10j, 72% for 10k, 75% for 10l.
NH2
Target to PDE4 and peripheral anionic
site (PAS) of AChE
N
Tacrine, AChE inhibitor
NH
N
O
Combine
H
N
N
R
N
N
H
n
N
The AChE and BuChE inhibitory effects of the hybrids were
determined by the spectroscopic method described by Ellman et
al. using tacrine as the standard.31 AChE (E.C.3.1.1.7) and
BuChE (E.C. 3.1.1.8) were obtained from electric eel and equine
serum, respectively. The IC50 values for AChE and BuChE
inhibition are summarized in Table 1. Most of the hybrids
demonstrated potent inhibitory activity against AChE and BuChE
with IC50 values in the micromolar to sub-micromolar range. The
results of eeAChE inhibition of compounds showed that the
AChE inhibitory potency was closely related to the length of the
alkylene chain. It appears that a 6-carbon spacer seems to be the
proper length for a linker between the two pharmacophore groups
in both series. Compounds 10d and 10j gave the most active
AChE inhibitory activities with IC50 value of 0.149 and 0.125μM,
respectively. Increasing or decreasing the length of carbon linker
resulted a slightly decreasing the inhibitory activities (10c, n = 5,
IC50 = 0.358 μM；10e, n = 7, IC50 = 0.316μM； 10i, n = 5, IC50 =
0.306μM； 10k, n = 5, IC50 = 0.0.391μM). However, the same
trend was not observed in BuChE inhibition. Almost all the
compounds showed potent activity against BuChE. Additionally,
some compounds exhibited almost equal inhibitory activity with
AChE. Based on these results, these hybrids should be good dual
AChE/BChE inhibitors to treat AD.
NH
N
O
N
N
H
Target to catalytic active
site (CAS) of AChE
N
Tacrine-pyrazolo[3,4-b]pyridine hybrids
PDE4 inhibitor
Fig. 2. Design strategy of tacrine-pyrazolo[3,4-b]pyridine hybrids
Routes for the synthesis of the key intermediate 6, target
compounds 10a-l are summarized in Scheme 1 and Scheme 2.
Commercially available compound 1 was refluxed with diethyl
ethoxymethylenemalonate (EMME) at high temperature for 12
hours to get compound 2, and it was refluxed with phosphorus
oxychloride, now intermediate
3
has been obtained.
Triethylamine was added to a mixed solution of intermediate 3
and cyclohexylamine, and stirred at room temperature for 10
hours to obtain 4. After hydrolysis, it was reacted with
phosphorus oxychloride to obtain an acid chloride intermediate 6.
OH
Cl
CO2Et
CO2Et
b
a
N
N
N
NH2
N
N
N
N
N
3
2
1
As PDE4D is one of the key subtypes participating in LTP,
the inhibition of PDE4D2 by the tacrine-pyrazolo[3,4-b]pyridine
hybrids were determined using a previously described method,
with rolipram as the reference compounds. 32 The results in Table
1 indicated that most of the hybrids exhibited more potent or
similar inhibitory activities against PDE4D2 compared to
rolipram. The results of PDE4D2 inhibition of compounds
showed that 6-chlorin substituted group in tacrine was beneficial
to the inhibitory activities of PDE4D2, for example, compound
10f (IC50 = 0.025 μM) was more potent than 10a (IC50 = 0.041
μM) by 1.5-fold improvement. Similar to the SAR of AChE
inhibitory activities, a structure-activity relationship analysis
showed that the PDE4D2 inhibitory potency was closely related
to the length of the alkylene chain. 2-carbon spacer was more
favorable for the activity than the other positions. For example，
hybrids 10a and 10g demonstrated the most potent PDE4D2
inhibitory activities with IC50 value of 0.041 and 0.0.025μM,
respectively, which are about 10-fold more active than rolipram.
When the length of the alkyl chain was changed from 2 to 8, the
activity against PDE4D2 decreased slightly. The hybrids 10b,
10c, 10d, 10e and 10f, with four, five, six, seven, and eight
carbon spacers between tacrine and tacrine-pyrazolo[3,4-
b]pyridine hybrids unit (10b, n = 4, IC50 = 0.108 μM；10c, n = 6,
IC50 = 0.477μM； 10d, n = 7, IC50 = 0.501μM；10e, n = 8, IC50
NH
HN
d
c
CO2Et
COOH
N
N
N
N
N
N
4
5
HN
e
COCl
N
N
N
6
Scheme 1. Synthesis of intermediate 6. Reagents and conditions: (a)
EMME, reflux 12h; (b) POCl3, 120ꢀ°C, reflux 12h; (c) cyclohexylamine,
TEA, rt, 10h; (d) NaOH, CH3CH2OH, rt, 10h; (e) POCl3, reflux 2h.
The synthesis route of the target compounds 10a-l are
summarized in Scheme 2. Intermediate 9a-l was synthesized
according to the reported procedure. 29-30 Firstly, using anthranilic
acid or 2-amino-4-chlorobenzoic acid as the starting materials,
POCl3 was added dropwise to the mixed solution of the starting
materials and cyclohexanone at 0°C, then recrystallized to give
compound 8. Dissolving intermediate 8, KI and diamines of
different carbon chains in n-pentanol. They were refluxed at
160°C for 12 hours to give 9a-l. Finally, in the presence of
anhydrous dichloromethane, compound 9a-l and intermediate 6

= 1.071μM) had slightly weak inhibitory activities than 10a . The
same tread was also observed in 6- chlorinetacrine-pyrazolo[3,4-
b]pyridine series compounds. Considering the results in
inhibiting AChE and BuChE, compound 10j provided a desired
balance of AChE and BuChE and PDE4D inhibition activities,
with IC50 value of 0.125, 0.449 and 0.271 μM, respectively.
Table 1. In vitro inhibition of AChE, BuChE and PDE4D activities of tacrine-pyrazolo[3,4-b]pyridine hybrids 10a-l.
NH
O
H
N
N
R
N
N
H
n
N
N
10a-l
IC50(µM)±SDa
AChEc
SIe
IC50(µM)±SD
PDE4D2b
compounds
n
R
BuChEd
10a
10b
10c
10d
10e
10f
10g
10h
10i
2
4
5
6
7
8
2
4
5
6
H
0.243 ± 0.023
0.333 ± 0.015
0.358 ± 0.018
0.149 ± 0.009
0.316 ± 0.015
0.392 ± 0.020
0.412 ± 0.013
0.335 ± 0.030
0.306 ± 0.023
0.125 ± 0.010
0.891 ± 0.084
0.889 ± 0.010
0.264 ± 0.028
0.332 ± 0.009
0.324 ± 0.009
0.320 ± 0.003
0.713 ± 0.020
1.283 ± 0.020
0.434 ± 0.053
0.449 ± 0.062
3.67
2.67
0.73
2.23
1.02
0.82
6.54
3.82
1.42
3.60
0.041 ± 0.007
0.108 ± 0.003
0.477 ± 0.015
0.501 ± 0.012
1.071 ± 0.081
1.307 ± 0.020
0.025 ± 0.001
0.087 ± 0.009
0.308 ± 0.003
H
H
H
H
H
Cl
Cl
Cl
Cl
10j
0.271 ± 0.011
0.934 ± 0.021
1.077 ± 0.034
/
10k
7
8
-
Cl
Cl
-
0.391 ± 0.019
0.812 ± 0.003
0.110 ± 0.007
/
0.245 ± 0.004
0.314 ± 0.065
0.216 ± 0.018
/
0.63
4.30
1.96
/
10l
Tacrine
Rolipram
/
/
0.671 ± 0.03
aResult are the mean of three independent experiments(n=3) ± SD
bResult are the mean of three independent experiments(n=3) ± SD
CAChE from electriceel was used
dBuChE from equine serum was used.
eSelectivity ratio: IC50 (BuChE)/ IC50 (AChE)
In summary, this study involved the design, synthesis and
biological evaluation of a novel series of tacrine-pyrazolo[3,4-
b]pyridine hybrids as dual cholinesterase (ChE) and
phosphodiesterase 4D (PDE4D) inhibitors for the treatment of
Alzheimer’s disease (AD). Most of the target compounds
displayed excellent inhibition of AChE and BuChE and PDE4D
activity . The results indicated that both the AChE and PDE4D2
inhibitory potency were closely related to the length of the
alkylene chain. Anomy them, compound 10j, which is tacrine
linked with pyrazolo[3,4-b]pyridine moiety by a six-carbon
spacer, was the most potent acetylcholinesterase (AChE) with
IC50 value of 0.125μM. Moreover, compound 10j provided a
desired balance of AChE and butylcholinesterase (BuChE) and
PDE4D inhibition activities, with IC50 value of 0.449 and 0.271
μM, respectively. The above results indicated that this hybrid was
a promising dual functional agent for the treatment of AD.
We thank the National Natural Science Foundation of China
(21772241), Guangdong High-level personnel of special support
program -Young top-notch talent project (2015TQ01R244),
Guangzhou Pearl River New Star Fund Science And Technology
Planning Project (201610010111) and Guangdong Provincial
Key
Laboratory
of
Construction
Foundation
(No.
2017B030314030) for financial support of this study.
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Graphical Abstract
Dual functional cholinesterase and PDE4D inhibitors for the treatment of Alzheimer’s disease:
design, synthesis and evaluation of tacrine-pyrazolo[3,4-b]pyridine hybrids
Tingting Pan, Shishun Xie , Yan Zhou , Jinhui Hu , Haibin, Luo, Xingshu Li, Ling Huang *
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China