Accepted Manuscript

Design, synthesis and biological evaluation of dual acetylcholinesterase and

phosphodiesterase 5A inhibitors in treatment for Alzheimer’s disease

Yao Zhu, Li-yun Zhou, Yu-ren Jiang, Xiong-jie Zhao, Dong Guo

PII: S0960-894X(17)30706-0
DOI: http://dx.doi.org/10.1016/j.bmcl.2017.07.013
Reference: BMCL 25126

To appear in: Bioorganic & Medicinal Chemistry Letters

Received Date: 23 April 2017

Revised Date: 1 July 2017
Accepted Date: 3 July 2017

Please cite this article as: Zhu, Y., Zhou, L-y., Jiang, Y-r., Zhao, X-j., Guo, D., Design, synthesis and biological
evaluation of dual acetylcholinesterase and phosphodiesterase 5A inhibitors in treatment for Alzheimer’s disease,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.013

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Design, synthesis and biological evaluation Leave this area blank for abstract info.
of dual acetylcholinesterase and
phosphodiesterase 5A inhibitors in
treatment for Alzheimer’s disease
Yao Zhu §,a, Li-yun Zhou §,a, Yu-ren Jiang a,*Xiong-jie Zhao a, Dong Guo a
 Bioorganic & Medicinal Chemistry Letters
j o ur n al h o m e p a g e : w w w . e l s e v i e r . c o m

Design, synthesis and biological evaluation of dual acetylcholinesterase and

phosphodiesterase 5A inhibitors in treatment for Alzheimer’s disease
Yao Zhu §,a, Li-yun Zhou §,a, Yu-ren Jiang a,*, Xiong-jie Zhao a, Dong Guo a
a
Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China
§
These authors contributed equally to this work

AR T IC LE IN F O A B S TR A C T

Article history: With the recent research advances in molecular biology and technology, multiple credible
Received hypotheses about the progress of Alzheimer’s disease (AD) have been proposed; multi-target
Revised drugs have emerged as an innovative therapeutic approach for AD. Current clinical therapy for
Accepted AD patients is mainly palliative treatment targeting acetylcholinesterase (AChE). Inhibition of
Available online phosphodiesterase 5A (PDE5A) has recently been validated as a potentially novel therapeutic
approach for Alzheimer’s disease (AD). In this work, series of new compounds were designed,
synthesized and evaluated as dual cholinesterase and PDE5A inhibitor. Biological results
Keywords: revealed that some of these compounds display good biological activities against AChE with
Phosphodiesterase 5A IC50 values about 44.67-169.80 nM (donepezil IC50 50.12 nM). Notably, compound 12 presented
Acetylcholinesterase potent activities against PDE5A with IC50 values about 50 µM (sildenafil IC50 12.59 µM), and
Inhibitor some of these compounds showed low cell toxicity to A549 cells in vitro.
Biological 2017 Elsevier Ltd. All rights reserved.
Alzheimer’s disease.

Alzheimer’s disease (AD) is an incurable neurodegenerative
disorder characterized by deterioration of memory, cognitive
functions in elder people. It is evaluated by World Alzheimer's
Report that more than 35 million people suffered from AD in
2015, and this number will be double by 2030 and approximately
triple to 131 million by 2050. However, limited treatment effects
for AD therapy could be achieved accompanied by a lot of side
effects using single AChE inhibitors. More attention has been
paid to the development of effective new drug. Since the precise
pathogenesis responsible for triggering neurodegenerative
disorder is controversial from the start, more and more
researchers have proposed a move from single receptor to a
strategy of developing multiple targets and multifunctional drug
decreases levels of phosphorylated Tau (pTau). Specific PDE5A
inhibitors (sildenafil, vardenafil, and tadalafil) have been shown
to improve memory performance and/or enhance synaptic
plasticity and cognitive function in different animal models of
AD.7−10
Considering the multifactor interacting property of AD
pathogenesis, the multitarget-directed ligand (MTDL) approach11
has been increasingly applied in this field by many researchers.
In order to design effective dual inhibitors targeting to AChE and
PDE5A for the treatment of AD, the MTDL approach was also
applied in this work.
O
N
O

molecules.1-3
NH H3 CO
N

N H3 CO

Cholinergic hypothesis is one of the classical hypotheses of OH N

AD. The AChE accounts for approximately 80% Ach hydrolysis

A B

in healthy brain.4 The crystallographic structure of human AChE O

R
O
R
O

indicates that the active pocket of AChE is a gulch having a N N

N R

length of 20Å (1Å = 0.1nm) by the enzyme surface to internal. 5 It

N R N O
H H O

is composed of two separate ligand binding sites, a peripheral C D E

cationic site (PAS) at the entrance and a catalytic active site

(CAS) at the bottom. Inhibitors binding to these sites can O

interdict combination with ACh. R

O R R

+ O
PDEs, extensively distributed in the brain, hydrolyze the
O NH
N

second messengers cyclic guanosine monophosphate (cGMP) R O

and cyclic adenosine monophosphate (cAMP).6 In fact, PDE5A, a F G

cGMP-specific phosphodiesterase, is up-regulated in the brains Figure 1 Desi gn of t arget compounds

of AD patients compared with that in age-matched healthy
Recently, it was found that alkaloids fraction showed potential
control subjects. Moreover, by favoring the inactive form of
inhibitory effects on the AChE activity. The camel Artemisia
GSK3β (phosphorylated at GSK3β-Ser9), PDE5A inhibition
alkaloid, consisted of beta-carboline and vasicinone and extracted
 Scheme 1. Reagents and conditions: (i) chloral hydrate, hydroxylamine hydrochloride, 80-90 °C; (ii) 98% H2SO4 , 80 °C, 15 min; (iii) (CH3 )2 NCOCl, pyridine,
0 °C; DMF; NaH or substituted benzoyl chloride, pyridine, 0 °C; (iv) NBS, AIBN, ClCH2CH2Cl, reflux; amines, K2CO3 , 0 °C (v) amines, K2CO3, reflux; (vi)
N2 H4-H2 O, KOH, HOCH2CH2 OH, 120 °C; (vii)amines, NBS, 10% AIBN, ClCH2CH2 Cl, 50 °C, K2CO3 . (viii) CH3 OH or benzoyl chloride, reflux, 1 h.

Table 1.The structure of the compounds

Compound R1 R2 R3 R4 R5 Y

4 -CH3 - - - - CO

5 -CH3 -H -H - - CO

6 -CH3 -NO2 -H - - CH2

7 -CH3 -H -Br - - CH2

8 -Br -H -Br - - CH2

9 -Br -NO2 -H - - CH2

10 - -H -H -NH-p-PhOCH3 - CO

11 - -H -H -NH-p-PhBr - CO

12 - -H -H -piperidine - CO

13 - -H -H -N-methyl piperazine - CO

14 - -H -H -N(CH2CH3)2 -N(CH2 CH3)2 CO

15 - -H -H CO

16 -CH3 -H -H - - CO

17 - -H -H -NHCO(CH2)2 COOH - CO

18 -CH3 -H -H - -OCH3 CO

and separated from Chinese herb medicine, is chosen as a lead inhibitor binding pocket in 3D structure of AChE, we opened the
compound.12, 13 In our previous work, based on the research of ring in camel Artemisia alkaloid (Figure 1A), and designed the
key intermediate 2-substituted-6-amino-4 (3H) quinazolinone
(Figure 1C) and quinazoline-2, 4(1H, 3H)-dione (Figure 1D).
Donepezil (Figure 1B), a potent AChE inhibitor and commercial
available drug in treatment with AD, has been widely used in
both clinic and academic field. We also chose it as a lead
compound, and designed key intermediate isoindoline-1, 3-dione
(Figure 1E). Such derivatives with three parts of aromatics
moieties or others can interact with catalytic site, peripheral site
and central channel active site of acetylcholinesterase. Ultimately
on the basis of virtual screening, we synthesized
2-substituted-6-amino-4 (3H) quinazolinone (Figure 1C)
derivatives, quinazoline-2, 4-(1H, 3H)-dione (Figure 1D)
derivatives, and isoindoline-1, 3-dione (Figure 1E) derivatives,
most of these compounds exerted excellent activities against
AChE. On the basis of our previous studies, in this work we
designed and synthesized a series of compounds with structures
based on indoline-2, 3-dione derivatives (Figure 1F). It is well
reported that isatin and its derivatives can protect critical proteins
in the cells and inhibit Aβ aggregation which may prevent the
progression of AD. It has also found that quinazolinone
derivatives have a certain inhibitory activity for PDE5A. In
conclusion, it is meaningful to design more effective anti-AD
candidates based on indoline-2, 3-dione derivatives in present
work. In order to investigate the relationships between the
structural flexibility and enzyme inhibition activity, the ring
opening derivatives of indoline-2, 3-dione was also designed and
synthesized. (Figure 1G)
The synthetic route used to generate the target compounds is
summarized in Scheme 1. The structures of the compounds were
shown in Table 1. Majorities of compounds were characterized
by melting point (MP), High Performance Liquid
Chromatography (HPLC) and 1HNMR, and compound 17 was
also characterized by MS. The inhibitory activity against AChE
and PDE5A of synthesized compounds were shown in Table 2.
The pharmacokinetic parameters predicted of compound 4, 12
and 14 at https://preadmet.bmdrc.kr/toxicity/ were shown in
Table 3.
All the synthetic compounds were tested for the
acetylcholinesterase inhibitory activities in vitro by employing
the method of Ellman method.14 Donepezil, a well-known AChE
inhibitor, acted as positive control (Table 2). Preliminary
investigation demonstrated that some of derivatives have good
inhibiting effect against AChE. Most of indoline derivatives and
aldoketones derivatives were potent inhibitors for AChE with
IC50 values ranging from nanomolar to micromolar. 4, 8, 10, 12,
13 and 14 revealed the most potent inhibition for AChE (IC50 =
144.50 nM, IC50 = 89.13 nM, IC50 = 169.80 nM, IC50 = 79.43 nM

Table 2. Molecular formula, molecular weight, purity and AChE and PDE5A inhibition activity for synthesized compounds ( - means no activity).

Compound molecular formula molecular weight Purity (HPLC) a AChE IC50 (nM)b PDE5A IC50 (µM)b

4 C12 H12N2O3 232.24 98.43% 144.50 100.00

5 C16H11 NO3 265.27 99.24% >1000 746.45
6 C16 H12N2O4 296.28 96.05% 199.50 200.00
7 C16H12 BrNO2 330.18 95.60% 204.17 -
8 C15H9Br2 NO2 395.05 90.75% 89.13 130.62
9 C15H9BrN2O4 361.15 98.36% 263.00 -
10 C23 H18N2O4 386.41 98.35% 169.80 100.00
11 C22 H15BrN2O3 435.28 99.66% 398.10 -
12 C21 H20N2O3 348.4 97.99% 79.43 50.00
13 C21 H21N3O3 363.42 95.04% 47.86 >1000
14 C24 H31N3O3 409.53 98.59% 44.67 157.51
15 C24 H23N3O6 449.46 96.17% 229.08 -
16 C16H13 NO2 251.29 91.69% >1000 91.20
17 C21H19 NO5 365.39 99.15% >1000 17.60
18 C17H15 NO4 297.31 95.59% >1000 -

donepezil C24H29 NO3 379.49 > 98% 50.12 -

sildenafil C22 H30N6O4 474.58 > 98% - 12.59

a
All the synthetic compounds were tested for the purity by High Performance Liquid Chromatography (HPLC)
b
The in vitro test compound concentration required to produce 50% inhibition of AChE and PDE5A
.
IC50 = 47.86 nM, IC50 = 44.67 nM, donepezil IC50 = 50.12 nM). level kit assay, sildenafil acted as positive control. As reported in
We can find that the inhibitory activities of compounds based on Table 2, compound 4, 8, 10, 12, 14, 16 and 17 worth further
indole-2, 3-dione are more significant than the compounds based studying owing to its satisfactory activity (IC50 = 100.00 µM,
on 2- indolinone. IC50 = 130.62 µM, IC50 = 100.00 µM, IC50 = 50.00 µM, IC50 =
157.51 µM, IC50 = 91.20 µM, IC50 = 17.60 µM, sildenafil IC50 =
Some synthetic compounds were tested for the
12.59 µM).
phosphodiesterases inhibitory activities in vitro by human PDE5A
 Considering the inhibitory activity of compounds for two The predicted pharmacokinetic parameters were computed
enzymes was comprehensive, the effect of compound 4, 12 and by using PreADMET at https://preadmet.bmdrc.kr/adme/. The
14 on the viability of A549 cells was studied by the CCK-8 value ( Table 3) indicated that compound 4, may have a better
assay. The compounds were added to the cells in 50 µg/mL blood-brain barrier permeability and chemical stability than
respectively and incubated for 24 h at 37 °C. The results were compound 12 and 14.
demonstrated in Figure 2. The values indicated that there are not
Table 3. The predicted pharmacokinetic parameters of 4, 12 and 14.
great cytotoxicity differences among 4, 12 and 14. Hence, these
results demonstrate that all these tested compounds do not show ID 4 (value) 12 (Value) 14 (Value)
obvious toxicity in the CCK-8 assay, suggesting that 4, 12 and 14
BBB 0.855 0.082 0.100
lack general toxicity.
Buffer solubility/mg_L-1(pH=7.4) 216.76 2.70 40.16
Caco2 21.26 24.51 35.41
CYP 2C19 inhibition Non Non Non
CYP 2C9 inhibition Non Non Non
CYP 2D6 inhibition Non Inhibitor Inhibitor
CYP 2D6 substrate Non Substrate Weakly
CYP 3A4 inhibition Non Non Non
CYP 3A4 substrate Substrate Weakly Substrate
MDCK 26.08 58.21 0.13
Pgp inhibition 71.03 Non Non
Plasma Protein Binding 1.63 79.69 69.22
SKlogD value 1.63 2.04 2.07
SKlogP value 3.03 3.61 3.63
Figure 2. Compound (4, 12 and 14) with a concentration of 50 µg/mL on
CYP (cytochrome P450); Pgp (p-glycoprotein)
the A549 cell viability. Data is expressed as means ± SD.

To further explore the interactions between the inhibitor and
AChE or PDE5A, molecular docking study was employed for
compound 12 by software package MOE 2008.1015. Before
starting the docking process, the 3D coordinates of hAChE and
hPDE5A were retrieved from the Protein Data Bank
((http://www.rcsb.org/pdb/home/home.do). PDB code 4EY716 is
for the crystallographic structure of hAChE complex with
donepezil, and PDB code 1TBF17 stands for the crystallographic
structure of hPDE5A complex with sildenafil. Specifically the
residues within a radius of 6.5 Å around the substrate or
inhibitors in the crystal structures were selected as active site.
In this work, on the basis of our previous studies, we had
designed a series of isatin derivatives and the ring opening
derivatives, which related to AChE and PDE5A inhibition
respectively. Several synthesized compounds (Table 2) exhibit
good AChE and PDE5A inhibitory activity. In addition, the
cytotoxicity assay of compound 4, 12 and 14 showed low cell
toxicity to A549 cells in vitro. Among all these synthesized
compounds, 4, 8, 10, 12, 13 and 14 exhibits potent AChE
inhibition and 4, 8, 10, 12, 14, 16 and 17 exhibits potent PDE5A
inhibition. In conclusion, 4, 12 and 14 should be considered as
promising anti-AD candidates.
Acknowledgments

The financial support from the National Natural Science

Foundation of China (No. 20876180) is gratefully acknowledged.
The authors also would like to thank School of Pharmaceutical
Sciences of Central South University for support of MOE for this
research project.

Supplementary data

a b
Supplementary data associated with this article can be found,
in the online version, at
Figure 3. The docking picture of compound 12 to AChE (a) and PDE5A (b)
References

We could find that, compound 12 has interactions with the
residues Trp 86, Tyr 337, Tyr 341 and Trp 286 of AchE (Figure
3) Also it has interactions with the residues Phe820, Asp654,
Asp764 and Tyr612 of PDE5A. The carbonyl of indole-2,
3-dione plays a key role in the interactions with the active site of
the enzymes, so the ring opening derivatives and indolin-2-one
derivatives may be difficult to generate high inhibitory activity to
AChE and PDE5A simultaneously. The experiment data (Table
2) of enzyme inhibitory activity in vitro shows a satisfactory
agreement with the molecular docking study result.
1. Bolognesi M. L.; Matera, R.; Minarini, A.; Rosini, M.;
Melchiorre, C. Curr. Opin. Chem. Biol. 2009, 13, 303.
2. Munoz-Torrero, D.; Camps, P.; Curr. Med. Chem. 2006, 13, 399.
3. Van der Schyf, C. J.; Mandel, S.; Geldenhuys, W. J.; Amit, T.;
Avramovich, Y.; Zheng, H.; Fridkin, M.; Gal, S.; Weinreb, O.;
Bar Am, O.; Sagi, Y.; Youdim, M. B.; Curr. Alzheimer Res.
2007, 4, 522.
4. Greg, N. H.; Utsuki, T.; Yu, Q.; Zhu, X.; Holloway, H. W.; Perry,
T.; Lee, B.; Ingram, D. K.; Curr. Med. Res. Opin. 2001, 17, 159.
5. Bourne, Y.; Taylor, P.; Radic, Z.; Marchot, P.; Embo. J. 2003, 22
6. Maurice, D. H.; Ke, H.; Ahmad, F.; Wang, Y.; Chung, J.;
Manganiello, V. C. Nat. Rev. Drug Discovery 2014, 13, 290−314.
7. Cuadrado-Tejedor, M.; Hervias, I.; Ricobaraza, A.; Puerta,
E.;Perez-Rolda ́ n, J. M.; García-Barroso, C.; Franco, R.; Aguirre,
N.; García- ́Osta, A. Br. J. Pharmacol. 2011, 164, 2029−2041.
8. García-Barroso, C.; Ricobaraza, A.; Pascual-Lucas, M.; Unceta,
N.;Rico, A. J.; Goicolea, M. A.; Salles, J.; Lanciego, J. L.;
Oyarzabal, J.; F
́ ranco, R.; Cuadrado-Tejedor, M.; García-Osta, A.
Neuropharmacology 2013, 64, 114−123.
9. Reneerkens, O. A. H.; Rutten, K.; Akkerman, S.; Blokland, A.;
Shaffer, C. L.; Menniti, F. S.; Steinbusch, H. W. M.; Prickaerts, J.
Neurobiol. Learn Mem. 2012, 97, 370−379.
10. Rabal O, Sánchezarias J A, Cuadradotejedor M, et al. 2016,
59(19).
11. Melchiorre, C., M.L. Bolognesi, A. Minarini, M. Rosini and V.
Tumiatti, J. Med. Chem. 2010. 53(16): 5906-5914.
12. Zheng, X. Y.; Zhang, Z. J.; Chou, G. X.; Wu, T.; Cheng, X. M.;
Wang, C. H.; Wang, Z. T.; Arch. Pharmacal Res. 2009, 32,1245.
13. Zhao, T.; Ding, K. M.; Zhang, L.; Cheng, X. M.; Wang, C. H.;
Wang, Z. T.; J. Chemistry. 2013.
14. Ellman, G.L., K.D. Courtney, V. Andres and R.M. Featherstone.
Biochemical pharmacology, 1961 7(2): 88-95.
15. Molecular Operating Environment (MOE) 2008.10. Chemical
Computing Group, Montreal, Quebec, Canada.
16. Cheung, J.; Rudolph, M. J.; Burshteyn, F.; J. Med. Chem., 2012,
55(22): 10282-10286.
17. Zhang K Y J, Card G L, Suzuki Y, et al. Molecular cell, 2004,
15(2): 279-286.