Design, Synthesis, and Biological Evaluation of Orally Available First-Generation Dual-Target Selective Inhibitors of Acetylcholinesterase (AChE) and Phosphodiesterase 5 (PDE5) for the Treatment of Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.7b00345

Through drug discovery strategies of repurposing and redeveloping existing drugs, a series of novel tadalafil derivatives were rationally designed, synthesized, and evaluated to seek dual-target AChE/PDE5 inhibitors as good candidate drugs for Alzheimer's disease (AD). Among these derivatives, 1p and 1w exhibited excellent selective dual-target AChE/PDE5 inhibitory activities and improved blood-brain barrier (BBB) penetrability. Importantly, 1w·Cit (citrate of 1w) could reverse the cognitive dysfunction of scopolamine-induced AD mice and exhibited an excellent effect on enhancing cAMP response element-binding protein (CREB) phosphorylation in vivo, a crucial factor in memory formation and synaptic plasticity. Moreover, the molecular docking simulations of 1w with hAChE and hPDE5A confirmed that our design strategy was rational. In summary, our research provides a potential selective dual-target AChE/PDE5 inhibitor as a good candidate drug for the treatment of AD, and it could also be regarded as a small molecule probe to validate the novel AD therapeutic approach in vivo.

Full text of DOI:10.1021/acschemneuro.7b00345

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Article
Design, Synthesis, and Biological Evaluation of Orally Available First-
Generation Dual-Target Selective Inhibitors of Acetylcholinesterase (AChE)
and Phosphodiesterase 5 (PDE5) for the Treatment of Alzheimer’s Disease
Fei Mao, Huan Wang, Wei Ni, Xinyu Zheng, Manjiong Wang, Keting
Bao, Dazheng Ling, Xiaokang Li, Yixiang Xu, Haiyan Zhang, and Jian Li
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00345 • Publication Date (Web): 25 Oct 2017
Downloaded from http://pubs.acs.org on October 29, 2017
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Design, Synthesis, and Biological Evaluation of Orally Available
FirstꢀGeneration
DualꢀTarget
Selective
Inhibitors
of
Acetylcholinesterase (AChE) and Phosphodiesterase 5 (PDE5) for the
Treatment of Alzheimer’s Disease
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†,#
‡,§,#
†,#
†
†
†
Fei Mao , Huan Wang , Wei Ni , Xinyu Zheng , Manjiong Wang , Keting Bao ,
†
†
†
‡,
⊥
,
†,
Dazheng Ling , Xiaokang Li , Yixiang Xu , Haiyan Zhang *, Jian Li *
†
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China
University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
‡
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
§
University of Chinese Academy of Science, No.19A Yuquan Road, Beijing 100049,
China
⊥
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai
2
01203, China
#
These authors contributed equally to this work.
*To whom correspondence should be addressed. jianli@ecust.edu.cn,
hzhang@simm.ac.cn
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ABSTRACT
Through drug discovery strategies of repurposing and redeveloping existing
drugs, a series of novel tadalafil derivatives were rationally designed, synthesized and
evaluated to seek dualꢀtarget AChE/PDE5 inhibitors as good candidate drugs for AD.
Among these derivatives, 1p and 1w exhibited excellent selective dualꢀtarget
AChE/PDE5 inhibitory activities and improved bloodꢀbrain barrier (BBB)
penetrability. Importantly, 1w·Cit (citrate of 1w) could reverse the cognitive
dysfunction of scopolamineꢀinduced AD mice and exhibited an excellent effect on
enhancing cAMP response elementꢀbinding protein (CREB) phosphorylation in vivo,
a crucial factor in memory formation and synaptic plasticity. Moreover, the molecular
docking simulations of 1w with hAChE and hPDE5A confirmed that our design
strategy was rational. In summary, our research provides a potential selective
dualꢀtarget AChE/PDE5 inhibitor as a good candidate drug for the treatment of AD,
and it could also be regarded as a small molecule probe to validate the novel AD
therapeutic approach in vivo.
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KEYWORDS: Alzheimer’s disease, Drug repositioning, Dualꢀtarget inhibitor, AChE
inhibition, PDE5 inhibition
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INTRODUCTION
Alzheimer’s disease (AD) is a longꢀterm and incurable neuroꢀdegenerative brain
disorder clinically characterized by dysmnesia, loss of speech, and behavioral
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abnormalities. At present, there is no ideal drug for the treatment of AD, and the
search for AD drugs remain an urgent issue in the pharmaceutical community. Due to
its complex pathogenesis, a single target drug cannot cure this disease fundamentally.
Dual or multiple target drugs involved in two or more aspects of AD pathogenesis
may generate a synergistic effect and ultimately achieve an ideal therapeutic effect.
Thus, the development of multiꢀtargetꢀdirected ligands (MTDLs) for the treatment of
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ꢀ6
AD have recently attracted much attention, and some studies have confirmed the
7ꢀ9
effectiveness of MTDLs in animal models of dementia, supporting the idea that
drugs with two or more biological activities may significantly advance the field of
antiꢀAD in the future.
The current antiꢀAD therapeutic arsenal approved by the United States Food and
Drug Administration (FDA) consists of four drugs, donepezil, rivastigmine,
galantamine and memantine, which only ameliorate the symptoms of cognitive
function but do not cure the disease or reverse its progression. Among them, three are
acetylcholinesterase (AChE) inhibitors, and the other, memantine, is an
NꢀmethylꢀDꢀaspartate receptor (NMDAR) antagonist. In addition, huperzine A (Hup
A), which was approved by the Chinese Food and Drug Administration (CFDA) for
the treatment of AD, is also an efficient reversible competitive AChE inhibitor.
Although inadequate for curing AD, inhibition of AChEs remains the most successful
strategy and has been shown to be transiently capable of improving memory and
cognitive function in AD patients. Therefore, MTDLs combining AChEs inhibitors
with other active pharmacophore within a single drug would generate a more
significant reduction in AD symptoms. These compounds not only enhance
acetylcholine (ACh) levels in the brain by inhibiting acetylcholinesterase, but also
exhibit one or more other antiꢀAD biological activities such as inhibition of beta
10ꢀ12
amyloid protein (Aβ) production,
inhibition of Aβ selfꢀ or metalꢀmediated
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aggragation,
subtype
neuroprotection.
monoamine oxidase (MAO) inhibitory activity,
serotonergic
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receptor (5ꢀHT R) agonist activity,
antioxidation
and
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Phosphodiesterases (PDEs) comprise a group of enzymes that are responsible for
the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine
monophosphate (cGMP), which are intracellular second messengers that are closely
associated with cellular functions such as neurotransmitter release, neuroplasticity and
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neuroprotection.
Moreover, the elevation of cAMP and/or cGMP levels may
promote cAMP response elementꢀbinding protein (CREB) phosphorylation, which in
turn promotes synaptic plasticityꢀassociated gene transcription and influences
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longꢀterm memory formation and persistent longꢀterm potentiation (LTP).
Ultimately, the increasing levels of cAMP and/or cGMP lead to strengthening of
learning and memory ability. Moreover, different PDE inhibitor subtypes have been
confirmed to demonstrate significant memoryꢀenhancing effects in animal
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models,
especially in models of AD, such as PDE3,
PDE4, PDE5,
and
3
4
PDE9, indicating that PDE inhibitors are novel potential therapeutic approaches for
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the treatment of AD.
PDE5, a special cGMP hydrolytic enzyme, only comprises one hypotype,
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PDE5A, localized in the hippocampus, cortex and cerebellum of the brain. Research
has indicated that the level of cGMP, but not cAMP, is significantly lower in the
cerebrospinal fluid (CSF) of mild AD patients compared with nonꢀdemented controls,
3
8
owing to the upꢀregulation of PDE5 in AD brains. Therefore, PDE5 is a promising
therapeutic target in AD, and a PDE5 inhibitor may be a good therapeutic strategy for
the treatment of AD patients. Studies have shown that PDE5 inhibitors, such as
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sildenafil, tadalafil (1), and icariin (Figure 1), possess potent antiꢀAD effects in
different mouse models of AD, significantly reversing cognitive impairment and
improving learning and memory. In addition, research has indicated that the effects of
AChE and PDE5 inhibitors on object recognition memory in rats is different and
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dissociable. Therefore, AChE/PDE5 dual inhibitors could play a synergistic antiꢀAD
effect and may supply a new perspective and breakthrough for the treatment of AD.
According to the literature research, no dualꢀtarget AChE/PDE5 inhibitor has
been reported to date, and hence it is necessary and crucial to search novel dualꢀtarget
AChE/PDE5 inhibitors to further explore this synergistic therapeutic method. In this
study, we will reveal the design, synthesis and biological evaluation of a series of
novel, potent, and selective firstꢀgeneration dualꢀtarget AChE/PDE5 inhibitors with
good bloodꢀbrain barrier (BBB) penetrability and excellent improvement of memory
and cognitive function in mice with amnesia, and we anticipated finding a good lead
compound for the treatment of AD.
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O
O
H
N
O
HN
N
N
O
OH
N
N
N
N
HO
HO
O
O
O
H
O
O
O
OH
OH
O
O
S
O
O
N
OH
HO
O
HO
Sildenafil
1, Tadalafil
Icariin
Figure 1. Chemical structures of representative PDE5 inhibitors possessed potent
antiꢀAD effects in mouse models of AD.
DESIGN OF ACHE/PDE5 DUAL INHIBITORS
The drug discovery strategies of new uses for existing drugs have attracted much
attention from medicinal chemists and pharmaceutical companies because they are
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2
effective, quick and costꢀeffective. Our group is devoted to research of repurposing
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and redeveloping existing drugs and has achieved some success.
Through the
screening of our old drug library, tadalafil, a selective PDE5 inhibitor developed by
Eli Lilly and approved by the United States FDA for the treatment of erectile
dysfunction (ED) in 2003, was found to have certain AChE inhibition (inhibition rate:
6
5.43% at 40 ꢁM; IC50 = 26.159 ± 1.300 ꢁM, Figure 2). In addition, recent studies
have shown that tadalafil can reverse cognitive dysfunction in a J20 transgenic mouse
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model of AD. Based on the above findings and considering the longer halfꢀlife and
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safety of tadalafil for the chronic treatments of ED and hypertension,
it would be
an effective compound for the discovery of dualꢀtarget AChE/PDE5 inhibitors for the
treatment of AD. According to previous structureꢀactivity relationship (SAR) studies
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of tadalafil analogues,
the substituent group at the Nꢀatom of piperazineꢀ2,5ꢀdione
was replaceable for PDE5 inhibitory activity. In this context, we retained the tadalafil
parent nucleus (Figure 2) to maintain PDE5 inhibitory activity, varying only the
different substituent group at the Nꢀatom of piperazineꢀ2,5ꢀdione, such as
morpholineꢀ4ꢀethyl,
NꢀmethyꢀNꢀbenzylaminoethyl, and substituted or unsubstituted benzylpiperidine
Figure 2, Tables 1ꢀ2), expecting to obtain good dualꢀtarget AChE/PDE5 inhibitors
1ꢀbenzylpyrrolidineꢀ3ꢀyl,
N,Nꢀdimethylaminoethyl,
(
with improved BBB penetrability and excellent pharmacological activity.
Figure 2. Design strategy for the novel dualꢀtarget AChE/PDE5 inhibitors.
CHEMISTRY
The synthetic route for preparation of the key intermediates 3aꢀd (two pairs of
enantiomers 3a, 3c and 3b, 3d) is shown in Scheme 1. The PictetꢀSpengler reaction of
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commercially available Dꢀtryptophan methyl ester hydrochloride and intermediate 6
(piperonal) in isopropanol provided the main cisꢀintermediate 3a with a yield of 82%,
while it gave an almost equal amount of the cisꢀintermediate 3a (38% yield) and
transꢀintermediate 3b (40% yield) in ethanol. The PictetꢀSpengler reaction of
commercially available Lꢀtryptophan methyl ester hydrochloride and intermediate 6
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(
piperonal) in ethanol provided the cisꢀintermediate 3c (47% yield) and the
transꢀintermediate 3d (32% yield).
a
Scheme 1. Synthesis of Intermediate 3aꢀd
a
Reagents and conditions: (a) piperonal, EtOH, reflux, 24 h; (b) piperonal, iꢀPrOH,
reflux, 24 h.
Scheme 2 depicts the synthetic route of the target compounds 1aꢀw. Reaction of
3
aꢀd with 2ꢀchloroacetyl chloride yielded the key intermediates 4aꢀd. In the presence
1
of different primary amines (R NH , e.g., 2ꢀmorpholinoethanꢀ1ꢀamine,
2
2
ꢀ(1ꢀbenzylpiperidinꢀ4ꢀyl)ethanꢀ1ꢀamine, intermediates 6ꢀ13), the key intermediates
4
aꢀd proceeded through the cyclization reaction to afford the target compounds 1aꢀw.
a
Scheme 2. Synthesis of Compounds 1aꢀw
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a
3
Reagents and conditions: (a) 2ꢀchloroacetyl chloride, Et N, DCM, −10 °C to rt, 6 h;
1
(b) R NH , Et N, MeOH, reflux, 12ꢀ24 h; (c) 2ꢀ(1ꢀbenzylpiperidinꢀ4ꢀyl)ethanꢀ1ꢀamine,
2
3
Et
3
N, MeOH, reflux, overnight.
Target compounds 2aꢀq were synthesized as illustrated in Scheme 3.
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Debenzylation of compound 1q in the presence of 10% Pd/C and H at room
2
temperature and atmospheric pressure gave intermediate 5, which then reacted with
the bromoethane (bromomethyl)cyclohexane, substituted benzyl or bromide benzyl
bromideꢀgenerated target compounds 2aꢀq.
a
Scheme 3. Synthesis of Compounds 2aꢀq
O
N
Ph
O
NH
H
N
H
N
N
N
N
H
N
a
b
H
O
O
O
O
O
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N
O
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N
H
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b: R = cyclohexyl
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2
2
2
c: R = 2-fluorophenyl
2l: R = 2-cyanophenyl
N
2
2
N
H
2d: R = 3-fluorophenyl
2m: R = 3-cyanophenyl
2
2
2
2
2
2
2
e: R = 4-fluorophenyl
2n: R = 4-cyanophenyl
O
O
2
2
f: R = 3-chlorophenyl
2o: R = 4-methylphenyl
2
2
g: R = 3-bromophenyl 2p: R = 4-methoxylphenyl
2
2
h: R = 3-iodophenyl
2q:R = 3-nitrophenyl
O
2a-q
2
i: R = 2-pyridyl
a
2
Reagents and conditions: (a) Pd/C, H
2
, MeOH, rt, overnight; (b) R CH
2
Cl or
2
R CH Br, K CO , rt, 12ꢀ24 h.
2
2
3
The details of the synthetic procedures and structural characterizations of
intermediates 3ꢀ5 and target compounds 1aꢀw and 2aꢀq are described in the
experimental section, and intermediates 6ꢀ13 are shown in the Supporting Information.
The purities of all target compounds were determined by HPLC (Table S1, Supporting
Information) and were identified as nonꢀPAINS on the web of
http://fafdrugs3.mti.univparisꢀdiderot.fr/, as recommended by the editors from the
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RESULTS AND DISCUSSION
ChE Inhibitory Activity
Inhibition of these tadalafil derivatives against AChE and butyrylcholinesterase
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(BuChE) was determined by the modified Ellman method using Hup A and
donepezil as positive references. As shown in Tables 1ꢀ3, these compounds displayed
poor to excellent AChE inhibitory activities and moderate BuChE inhibitory
activities.
a
Table 1. In vitro AChE inhibitory activities of compounds 1aꢀw .
O
H
R1
1
2a
N
6
N
N
H
O
O
O
Inhibition of AChE
I］= 40 ꢁM
1
b
Compd
R
Configuration
6R,12aR
IC50 ± SD (ꢁM)
［
c
1
a
6.26%
n.t.
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c
d
e
f
6R,12aR
6R,12aR
6S,12aR
6S,12aR
6S,12aR
6R,12aR
6R,12aR
25.22%
73.08%
16.94%
6.19%
n.t.
14.206 ± 2.137
c
n.t.
c
n.t.
c
28.54%
18.36%
59.36%
n.t.
c
1g
1h
n.t.
c
n.t.
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i
6R,12aR
6R,12aR
67.53%
26.79%
17.529 ± 0.648
c
1j
n.t.
10
11
12
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16
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1
k
6R,12aR
6R,12aR
81.17%
1.22%
6.728 ± 0.201
c
1l
n.t.
c
1
1
m
6R,12aR
6R,12aR
16.35%
11.12%
n.t.
c
n
o
n.t.
c
1
1
1
6R,12aR
6R,12aR
6R,12aR
65.35%
103.96%
98.46%
n.t.
p
q
0.036 ± 0.008
0.075 ± 0.017
c
1
1
r
s
6R,12aR
6R,12aR
12.70%
19.50%
n.t.
c
n.t.
c
1t
6R,12aR
6S,12aR
6S,12aS
13.62%
77.26%
94.73%
n.t.
1u
8.140 ± 0.400
0.851 ± 0.129
N
N
Ph
Ph
1
1
v
w
6R,12aS
6R,12aR
99.78%
65.43%
0.032 ± 0.013
26.159 ± 1.300
Tadalafil Me
Hup A
ꢀ
ꢀ
ꢀ
0.084 ± 0.017
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4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Donepezil
ꢀ
ꢀ
ꢀ
0.013 ± 0.150
a
b
c
AChE from rat cortex. Results are expressed as the mean of at least three experiments. n.t.
indicates no test.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 2. In vitro AChE inhibitory activities of compounds 2aꢀq .
Inhibition of AChE
2
b
Compd
R
IC50 ± SD (ꢁM)
［
I］= 40 ꢁM
1p
2a
2b
Ph
103.96%
96.31%
101.72%
98.89%
98.34%
101.51%
68.12%
0.036 ± 0.008
1.583 ± 0.216
0.118 ± 0.029
0.067 ± 0.013
0.060 ± 0.014
0.089 ± 0.020
10.440 ± 0.910
Me
Cy
2c
2d
2e
2f
2ꢀfluorophenyl
3ꢀfluorophenyl
4ꢀfluorophenyl
3ꢀchlorophenyl
2g
3ꢀbromophenyl
64.48%
3.390 ± 0.201
c
2
2
2
2
2
2
2
2
2
h
3ꢀiodophenyl
2ꢀpyridyl
21.60%
102.67%
90.03%
90.93%
25.34%
80.82%
95.73%
88.38%
46.98%
n.t.
i
0.093 ± 0.008
0.261 ± 0.030
0.161 ± 0.016
j
3ꢀpyridyl
k
l
4ꢀpyridyl
c
2ꢀcyanophenyl
3ꢀcyanophenyl
4ꢀcyanophenyl
4ꢀmethylphenyl
4ꢀmethoxylphenyl
n.t.
m
n
o
p
0.800 ± 0.109
0.216 ± 0.045
1.689 ± 0.146
c
n.t.
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2
q
3ꢀnitrophenyl
79.41%
0.833 ± 0.061
26.159 ± 1.300
0.084 ± 0.017
0.013 ± 0.150
Tadalafil
Hup A
Donepezil
a
ꢀ
ꢀ
ꢀ
65.43%
ꢀ
ꢀ
10
11
12
13
14
15
16
17
18
19
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22
23
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27
28
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b
c
AChE from rat cortex. Results are expressed as the mean of at least three experiments. n.t.
indicates no test.
1
The R substituent group and absolute configuration remarkably affected the
1
AChE inhibitory activities (Table 1). When R substituent groups changed from
methyl (tadalafil) into morpholineꢀ4ꢀethyl (1a), 1ꢀbenzylpyrrolidineꢀ3ꢀyl (1b, 1g),
N,Nꢀdimethylaminoethyl
(1j),
NꢀmethyꢀNꢀbenzylaminoethyl
(1m),
NꢀmethylꢀNꢀphenylaminoethyl (1l), 1Hꢀbenzo[d]imidazoleꢀ1ꢀethyl (1r), and
1
Hꢀimidazoleꢀ1ꢀethyl (1s), the AChE inhibitory activities were dramatically
1
decreased, with 1.22ꢀ28.54% inhibition at 40 ꢁM. When the R substituent groups
changed
from
methyl
(tadalafil)
into
pyrrolidineꢀ1ꢀethyl
(1c),
1
ꢀmethylpiperazineꢀ4ꢀethyl (1h), piperidineꢀ1ꢀethyl (1i), and N,Nꢀdiethylaminoethyl
(1k), the AChE inhibitory activities were reserved, with 59.36ꢀ81.17% inhibition at
40 ꢁM. Among these tested compounds with an absolute configuration of 6R,12aR,
1
1
p with the R substituent group of 1ꢀbenzylpiperidineꢀ4ꢀethyl exhibited the best
AChE inhibitory activities, with IC50 values of 36 nM. Notably, the chain length
between the tadalafil parent nucleus and 1ꢀbenzylpiperidine exerted a crucial
influence on the AChE inhibitory activities, and the two methylenes between them
was the optimal chain length. Directly tethering the 1ꢀbenzylpiperidine to the tadalafil
parent nucleus led to weak AChE inhibitory activity (1n, 11.12% inhibition at 40 ꢁM).
Changing the chain length into one methylene (1o) or three methylene (1q) led to
decreased AChE inhibitory activities (1o, 65.35% inhibition at 40 ꢁM; 1q, IC = 75
5
0
nM). Moreover, the substituent pattern of benzylpiperidine also played an important
role in AChE inhibition. Reversing the N atom position of 1ꢀbenzylpiperidineꢀ4ꢀethyl,
1
which changes the R substituent group 1ꢀbenzylpiperidineꢀ4ꢀethyl (1p) into
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2
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2
2
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6
4
ꢀbenzylpiperidineꢀ1ꢀethyl (1t), led to a dramatic decline in AChE inhibitory activity
(1t, 13.62% inhibition at 40 ꢁM).
Furthermore, the influence of stereochemistry on AChE inhibition has been
discussed. In the cisꢀisomers (6R,12aR and 6S,12aS), the enantiomer 1p (6R,12aR)
was the more potent AChE inhibitor (1p, IC50 = 0.036 ꢁM; 1v, IC50 = 0.815 ꢁM),
while in the transꢀisomers (6R,12aS and 6S,12aR), the enantiomer 1w (6R,12aS) was
the more potent AChE inhibitor (1w, IC50 = 0.032 ꢁM; 1u, IC50 = 8.140 ꢁM). The
diastereoisomers 1p and 1w showed almost the same AChE inhibitory activity, which
indicated that the stereoꢀconfiguration at position 6 of the tadalafil parent nucleus was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
key for the strong AChE inhibitory activity.
2
The influence of the substituent group at the Nꢀatom of piperidine (R CH
2
, Table
2
) on AChE inhibitory activities was also studied, and the results are shown in Table
2. Compound 2a with an ethyl substituent at the Nꢀatom of piperidine showed lower
AChE inhibition (IC50 = 1.583 ꢁM). Compound 2b, obtained by reducing phenyl to a
sixꢀmembered nonaromatic ring, showed good AChE inhibition (IC50 = 0.118 ꢁM)
that was a little weaker than 1p (IC50 = 0.036 ꢁM). Of these compounds with a
substituted aryl methyl at the Nꢀatom of piperidine (2cꢀq), fluorophenylꢀsubstituted
compounds exhibited better AChE inhibitory activity (2cꢀe) but were still slightly
weaker than the unsubstituted compound 1p, indicating that the bulk groups at the
phenyl ring of phenylpiperidine adversely affected AChE inhibition.
Pyridylꢀsubstituted compounds (2iꢀk) exhibited nearly 2ꢀ7 times weaker AChE
inhibitory activity than 1p, with IC values of 0.093, 0.261, 0.161 ꢁM, respectively.
50
Neither the electronꢀwithdrawing (2cꢀn, 2q) nor electronꢀdonating group (2o, 2p) was
beneficial for AChE inhibition.
Taken together, diastereoisomers 1p and 1w were the most potent AChE
inhibitors, exhibiting far more potency than the hit compound tadalafil, than the
approved drug Hup A, and comparable potency to the bestꢀselling antiꢀAD drug
donepezil.
Compounds (1pꢀq, 1vꢀw, 2bꢀe, 2iꢀk, 2mꢀn, and 2q) with good AChE inhibition
(IC50 < 1 ꢁM) were further assessed for their butyrylcholinesterase (BuChE)
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inhibitory activities. The results shown in Table 3 indicated that these compounds had
weaker BuChE inhibition and good AChE selectivity, and the selectivity index values
for AChE varied from 17 to 273. Among these tested compounds, compounds with a
cyano substituent at the phenyl ring of the phenylpiperidine fragment were very weak
BuChE inhibitors, with 13.28ꢀ39.45% inhibition at 40 ꢁM. Compounds with a fluoro
or pyridyl substituent possessed moderate BuChE inhibitors, with IC50 values of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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44
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46
47
48
49
50
51
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57
58
59
60
9
1
.787ꢀ40.847 ꢁM. Interestingly, increasing the chain length between
ꢀbenzylpiperidine and the tadalafil parent nucleus led to increased BuChE inhibitory
activities (IC50 values of 1p vs 1q: 9.835 vs 1.309 ꢁM). The most potent AChE
inhibitors, 1p and 1w, exhibited weak BuChE inhibitory activity, which was 273 and
1
21 times weaker than the AChE inhibitory activity, respectively, indicating that 1p
and 1w were excellent selective AChE inhibitors over BuChE.
Table 3. In vitro BuChE inhibitory activities and selectivity for AChE of compounds
a
1pꢀq, 1vꢀw, 2bꢀe, 2iꢀk, 2mꢀn, and 2q .
IC50 ± SD (ꢁM)
IC50 ± SD (ꢁM)
2
b
c
Compd
R
n
for BuChE
SI
for AChE
(Inhibition @ 40 ꢁM)
1p
1q
Ph
1
2
ꢀ
0.036 ± 0.008
0.075 ± 0.017
0.851 ± 0.129
0.032 ± 0.013
0.118 ± 0.029
0.067 ± 0.013
0.060 ± 0.014
0.089 ± 0.020
9.835 ± 3.203
1.309 ± 0.012
16.200 ± 1.103
3.880 ± 0.275
3.977 ± 0.150
12.323 ± 0.554
13.725 ± 0.708
10.870 ± 1.454
2
1
1
1
3
1
2
73
Ph
7
1
1
2
2
2
2
v
w
b
c
ꢀ
9
ꢀ
ꢀ
21
4
Cy
1
1
1
1
2ꢀfluorophenyl
3ꢀfluorophenyl
4ꢀfluorophenyl
84
29
d
e
122
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6
2
2
2
2
2
i
2ꢀpyridyl
3ꢀpyridyl
4ꢀpyridyl
3ꢀcyanophenyl
4ꢀcyanophenyl
3ꢀnitrophenyl
ꢀ
1
1
1
1
1
1
ꢀ
0.093 ± 0.008
0.261 ± 0.030
0.161 ± 0.016
0.800 ± 0.109
0.216 ± 0.045
0.833 ± 0.061
26.159 ± 1.300
0.084 ± 0.017
0.013 ± 0.150
9.787 ± 0.430
22.080 ± 1.784
40.847 ± 3.066
> 40 (27.12%)
> 40 (13.28%)
> 40 (39.45%)
> 40(3.86%)
1
8
2
05
j
5
k
m
n
54
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
> 50
> 185
2q
>
48
Tadalafil
Hup A
> 1.53
ꢀ
ꢀ
36.400 ± 1.838
7.704 ± 0.552
4
44
93
Donepezil
ꢀ
ꢀ
5
a
b
BuChE from rat serum. Results are expressed as the mean of at least three experiments.
c
Selectivity index for AChE is defined as IC50(BuChE)/IC50(AChE).
Phosphodiesterase 5 (PDE5) Inhibitory Activity
To evaluate whether these tadalafil derivatives retained PDE5 inhibition, an
54, 55
IMAPꢀFP (immobilized metal ion affinityꢀbased fluorescence polarization) assay
was conducted using tadalafil as a reference. Compounds (1pꢀq, 1vꢀw, 2bꢀe, 2iꢀk,
2
mꢀn, and 2q) with good AChE inhibition (IC50 < 1 ꢁM) were selected to determine
the PDE5 inhibitory activity to identify good dualꢀtarget AChE/PDE5 inhibitors, and
the results are shown in Table 4. These data showed that most of the tested
compounds could retain PDE5 inhibition, with IC50 values of 0.032ꢀ23.20 ꢁM. The
chain length between 1ꢀbenzylpiperidine and the tadalafil parent nucleus was no
obvious influence on PDE5A1 inhibition (IC50 values of 1p vs 1q: 0.153 vs 0.150 ꢁM).
Compound 2b with a sixꢀmembered nonaromatic ring substituent at the Nꢀatom of
piperidine and showed weaker PDE5A1 inhibition than the sixꢀmembered aromatic
ring substituent (IC50 values of 1p vs 2b: 0.153 vs 0.335 ꢁM).
Table 4. In vitro PDE5A1 inhibitory activities of compounds 1pꢀq, 1vꢀw, 2bꢀe, 2iꢀk,
a
2mꢀn, and 2q .
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a
IC50 ± SD (ꢁM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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27
28
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2
Compd
R
n
for PDE5A1
0.153 ± 0.005
0.150 ± 0.420
23.200 ± 7.040
1.530 ± 0.004
0.335 ± 0.261
0.059 ± 0.086
0.074 ± 0.021
0.098 ± 0.021
0.054 ± 0.010
0.041 ± 0.005
0.032 ± 0.013
0.071 ± 0.006
0.097 ± 0.008
0.089 ± 0.016
0.004 ± 0.0001
> 100
1
1
p
q
Ph
1
2
ꢀ
Ph
1v
ꢀ
1
2
2
2
w
ꢀ
ꢀ
b
c
Cy
1
1
1
1
1
1
1
1
1
1
ꢀ
2ꢀfluorophenyl
3ꢀfluorophenyl
4ꢀfluorophenyl
2ꢀpyridyl
3ꢀpyridyl
4ꢀpyridyl
3ꢀcyanophenyl
4ꢀcyanophenyl
3ꢀnitrophenyl
ꢀ
d
2e
2
2
2
2
i
j
k
m
2n
2q
Tadalafil
Donepezil
ꢀ
ꢀ
a
Results are expressed as the mean of at least two experiments.
Compounds with a substituted aryl methyl at the Nꢀatom of piperidine (2cꢀe,
iꢀk, 2mꢀn, 2q) possessed better inhibitory activity than unsubstituted compound 1p,
2
indicating that the presence of bulk groups at the phenyl ring of phenylpiperidine was
beneficial for PDE5A1 inhibition. Of these tested compounds, compounds (2iꢀk) with
pyridyl substituents exhibited the best PDE5A1 inhibitory activity, with IC50 values of
0.054, 0.041, 0.032 ꢁM, respectively. The most potent AChE inhibitors, 1p and 1w,
exhibited good or moderate PDE5A1 inhibitory activity, with IC50 values of 0.153 ꢁM
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and 1.530 ꢁM, respectively, indicating that 1p and 1w were good dualꢀtarget
AChE/PDE5 inhibitors.
Noticeably, in the diastereoisomers 1p (6R,12aR) and 1w (6R,12aS), 1p
(6R,12aR) exhibited 10 times more potent PDE5A1 inhibitory activity (1p, IC50
=
0
1
2
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7
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9
0
1
2
3
4
5
6
7
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0
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5
6
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8
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2
3
4
5
6
7
8
9
0
0.153 ꢁM; 1w, IC = 1.530 ꢁM), while in the diastereoisomers 1v (6S,12aS) and 1w
50
(
6R,12aS), 1w (6R,12aS) showed 15.2 times more potent PDE5A1 inhibitory activity
1v, IC50 = 23.2 ꢁM; 1w, IC50 = 1.530 ꢁM), demonstrating both the
(
stereoꢀconfigurations at position 6 and 12 of the tadalafil parent nucleus, especially at
position 6, were vital for exerting the strong PDE5A1 inhibitory activity, and the
favored configuration was 6R, 12R.
SAR Studies
Based on the AChE and PDE5A1 inhibitory activity data shown in Tables 1ꢀ2
and 4, the noticeable SARs for compounds 1aꢀw and 2aꢀq were revealed as follows:
(1) the chain length between the tadalafil parent nucleus and 1ꢀbenzylpiperidine
exerted a large influence on the AChE inhibitory activities, with the potencies
increasing in the order of zero methylene < one methylene < three methylene < two
methylene (1n < 1o < 1q < 1p), but no influence on the PDE5A1 inhibitory activities
(1p vs 1q); (2) the substituent pattern of benzylpiperidine was pivotal for AChE
inhibition, and reversing the N atom position of 1ꢀbenzylpiperidineꢀ4ꢀethyl led to
almost no AChE inhibition (1p vs 1t); (3) the stereoꢀconfiguration at position 6 of the
tadalafil parent nucleus was key for displaying the strong AChE inhibitory activity,
and the favored configuration was 6R (1p vs 1u vs 1v vs 1w); (4) both the
stereoꢀconfigurations at position 6 and 12 of the tadalafil parent nucleus, especially at
position 6, were vital for exerting the strong PDE5A1 inhibitory activity, and the
favored configuration was 6R, 12R (1p vs 1v vs 1w); (5) replacing phenyl with an
aliphatic substituent (methyl or cyclohexyl) led to decreased inhibitions against both
AChE (2a, 2b vs 1p) and PDE5A1 (2b vs 1p); (6) bulk groups at the phenyl ring of
phenylpiperidine showed an adverse effect on the AChE inhibition (1p vs 2cꢀq) as
opposite to a favorable effect on the PDE5A1 inhibition (1p vs 2cꢀe, 2iꢀk, 2mꢀn, 2q).
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In brief, the SARs of the 40 compounds were explicit and provided important insights
into the key structural requirements for effective AChE/PDE5 inhibition.
Phosphodiesterase Selectivity Studies
10
11
12
13
14
15
16
17
18
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Furthermore, the inhibitory activities of 1p and 1w against other PDE isoforms
were also evaluated using an IMAPꢀFP assay. As shown in Table 5, 1p and 1w
possessed no inhibitory activities against PDE2A1, PDE3A, PDE4D3, PDE6C,
PDE7A and PDE9A2, demonstrating that they were highly selective PDE5 inhibitors.
Table 5. In vitro PDE2A1, PDE3A, PDE4D3, PDE6C, PDE7A and PDE9A2
inhibitory activities of compounds 1p and 1w.
a
IC50 ± SD (ꢁM)
Compd
PDE2A1
> 100
PDE3A
> 100
PDE4D3
> 100
PDE6C
> 100
PDE7A
> 100
PDE9A2
> 100
1p
1w
> 100
> 100
> 100
> 100
> 100
> 100
b
c
c
c
Trequinsin
1.062 ± 0.079 0.170 ± 0.001
0.199 ± 0.021 n.t.
n.t.
n.t.
c
c
c
b
Dipyridamole n.t.
n.t.
n.t.
n.t.
n.t.
0.943 ± 0.056 58.8 ± 3.21
n.t.
c
c
c
c
c
Zaprinast
n.t.
n.t.
n.t.
7.512 ± 0.687
a
b
c
Results are expressed as the mean of at least two experiments. unit: nM. n.t. indicates no test.
In Vitro BloodꢀBrain Barrier Permeation Assay
Good permeability of the BBB is an essential element for the development of
antiꢀAD drugs. Compounds (1p, 1q, 1w, 2bꢀe, 2i, 2k) with good dualꢀtarget
AChE/PDE5 inhibitors (AChE inhibition: IC < 0.2 ꢁM; PDE5A1 inhibition: IC <
50
50
2 ꢁM) were selected to determine their potential druggability and BBB penetrability.
The optimized compound BBB penetrabilities were evaluated by a parallel
5
6
artificial membrane permeation assay (PAMPA) as described by Di et al . First, the
permeability (P ) values of 13 commercial drugs in vitro were determined and
compared with the reported values to validate the assay. A plot of the experimental
e
e
data versus bibliographic values showed a good linear correlation, P (exp.) =
2
0
.9738P (bibl.) – 0.8157 (R = 0.9651, Figure S1, Supporting Information).
e
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According to this equation and the threshold established by Di et al. for BBB
−6
permeation, we could conclude that compounds with P values over 3.08 × 10 cm
e
−1
−6
−1
s could cross the BBB (CNS +); compounds with P
e
values from 1.13×10 cm s
−6
−1
to 3.08 × 10 cm s had uncertain BBB permeation (CNS ±); compounds with P
e
0
1
2
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7
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9
0
1
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0
1
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4
5
6
7
8
9
0
−
6
−1
values less than 1.13 × 10 cm s were identified as CNS −, with low BBB
permeation (Table S3, Supporting Information).
e
The P values of the tested compounds from the PAMPAꢀBBB assay and their
prediction of BBB penetration are presented in Table 6. All the tested compounds
exhibited good BBB permeation (CNS +), except compounds (2i, 2k) with pyridyl
e
substituents, which showed uncertain BBB permeation. The P value of the hit
compound, tadalafil, was 2.16, which indicated that tadalafil possessed poor BBB
permeation. Compounds 1p, 1q, 1w and 2bꢀe, which possess a substituted or
unsubstituted group on the phenyl ring of phenylpiperidine, have good BBB
permeation (CNS +), indicating that it is successful for improving BBB permeation by
introducing phenylpiperidine into tadalafil. Among them, compounds 1p and 1w
exhibited better BBB permeability than 1q and 2bꢀe. Therefore, compounds 1p and
1
w, the most potent AChE inhibitors with good PDE5 inhibitory activity and good
druggability, were selected as the optimal dualꢀtarget AChE/PDE5 inhibitors for
further study.
ꢀ6
ꢀ1
e
Table 6. Permeability results (P 10 cm s ) from the PAMPAꢀBBB assay for
selected compounds 1p, 1q, 1w, 2bꢀe, 2i, and 2k and their prediction of BBB
penetration.
ꢀ6
ꢀ1 a
b
Compd
P
e
(10 cm s )
Prediction
1
1
p
q
7.67 ± 1.21
6.98 ± 0.95
9.25 ± 0.93
7.92 ± 0.76
5.10 ± 0.05
5.39 ± 0.44
CNS +
CNS +
CNS +
CNS +
CNS +
CNS +
1w
2
2
b
c
2d
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5.69 ± 0.41
3.06 ± 0.67
2.74 ± 0.60
2.16 ± 0.48
8.78 ± 2.24
CNS +
2i
CNS ±
CNS ±
CNS ±
CNS +
2
k
Tadalafil
Donepezil
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a
PBS/EtOH (70:30) was used as the solvent.Values are expressed as the mean ± SD of at least
b
−6
−1
three independent experiments. CNS + : Compounds with P
e
> 3.08 × 10 cm s could cross
−
6
−1
the BBB by passive diffusion; CNS−: Compounds with P
< 1.13 × 10 cm s could not cross the
e
−
6
−1
−6
−1
BBB; CNS±: Compounds with 1.13 × 10 cm s < P < 3.08 × 10 cm s show uncertain BBB
e
permeation.
Passageway Water Maze Test
To evaluate the in vivo effect of compounds 1p and 1w on cognitive
improvement, the passageway water maze experiment was performed on a
scopolamine (Scop)ꢀinduced cognitive deficit mouse model, a classical AD model to
57
evaluate anticholinergic drug candidates, with donepezil (Don) as a positive control
(
Ctrl).
As shown in Figure 3, the Scop group (model group) exhibited a longer escape
#
##
latency and more frequent errors than the Ctrl group (negative group, p < 0.001).
The groups treated with 10 mg/kg 1w·Cit (citrate of 1w) demonstrated a shorter
escape latency and less frequent errors than the Scop group (*p < 0.05), confirming
that 1w·Cit could relieve the Scopꢀinduced learning and memory deficits at a dosage
of 10 mg/kg, which was comparable to donepezil (no significant difference, p > 0.05).
The groups treated with 10 mg/kg 1p·Cit (citrate of 1p) also demonstrated a shorter
escape latency and less frequent errors than the Scop group, but this difference was
not significant. Therefore, compound 1w·Cit demonstrated the most potential as an
AChE/PDE5 dualꢀtarget antiꢀAD lead compound due to its comparable effectivity
with donepezil in the cognitive ability improvement of AD mouse model.
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0
Figure 3. Effects of compounds 1p·Cit and 1w·Cit (10 mg/kg) on
scopolamineꢀinduced cognitive deficits in mice. Donepezil (10 mg/kg) as a reference.
(A) The escape latency of mice in the passageway water maze. (B) The number of
errors of mice in the passageway water maze. Data are shown as the mean ± SEM,
#
##
*
p < 0.001 vs control group, p < 0.05 vs Scop group; N = 12. Ctrl group: solvent +
saline; Scop group: solvent + scopolamine.
Western Blot Analysis
The inhibition of PDE5 led to an increase in cGMP levels, which promoted
CREB phosphorylation (pCREB), a crucial factor in memory formation and synaptic
24
plasticity. Furthermore, PDE5 inhibition of 1p·Cit and 1w·Cit in vivo were
confirmed by examining the level of pCREBꢀSer133 (Figure 4). As expected, after
administration of the PDE5 inhibitors 1p·Cit and 1w·Cit at 10 mg/kg, the levels of
CREB phosphorylation were increased significantly, indicating that both optimal
compounds could inhibit PDE5 in vivo and enhance CREB phosphorylation.
Moreover, although PDE5 inhibition by 1p in vitro was 10 times better than by 1w,
their levels of CREB phosphorylation were almost equivalent, 1.9 times and 1.8 times
greater than the vehicle control (Ctrl), respectively. Taken together, the more effective
compound (1w·Cit) for improving the cognitive dysfunction of Scopꢀinduced
cognitive deficit mice also exhibited an excellent effect on enhancing CREB
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phosphorylation in vivo, which may ameliorate the cognitive impairment and restore
synaptic function in AD.
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Figure 4. Effects of compounds 1p·Cit and 1w·Cit (10 mg/kg) on pCREBꢀSer133 in
vivo. (A) Representative bands in the western blot are shown. (B) The histogram
shows the quantification of immunochemically reactive bands in the western blot.
Data are the mean ± SEM, *p < 0.05 vs scopolamine group; N = 3. Ctrl group: solvent
+ saline; Scop group: solvent + scopolamine.
Molecular Docking Studies of 1w with AChE and PDE5
To explore the interaction mode of the optimal compound 1w with AChE and
PDE5, molecular docking simulation was performed using TriposSybyl 2.0 software
5
8
based on the crystal structure of hAChE complexed with donepezil (PDB ID 4EY7 )
59
and hPDE5A complexed tadalafil (PDB ID 2V60 ), respectively.
As shown in Figure 5, compound 1w could interact with both the catalytic active
site (CAS) and peripheral anionic site (PAS) of hAChE. The benzylpiperidine
fragment of compound 1w bound to the bottom of the hAChE gorge (Figure 5A) and
interacted with CAS via two cationꢀπ stacking interactions and πꢀπ stacking
interactions. Specifically, the phenyl of the benzylpiperidine fragment interacted with
Trp86 (4.0 Å) via faceꢀtoꢀface πꢀπ stacking interactions, and the protonated N atom of
the benzylpiperidine fragment interacted with Trp86 (4.9 Å) and Trp337 (5.6 Å)
through cationꢀπ stacking interactions. Importantly, in the midꢀgorge site of hAChE,
the carbonyl of the piperazinedione fragment participated in a hydrogen bond
interaction with Phe295 (2.0 Å), while in the PAS of hAChE, the indole ring of 1w
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interacted with Trp286 (4.8 Å) via faceꢀtoꢀface πꢀπ stacking interactions and the
phenyl ring of benzo[d][1,3]dioxole fragment interacts with Trp341 (5.0 Å) via
edgeꢀtoꢀface
vertical
πꢀπ
stacking
interactions.
In
addition,
the
hexahydropyrazino[1',2':1,6]pyrido[3,4ꢀb]indoleꢀ1,4ꢀdione moiety bounds to the
middle and external of the hAChE gorge, forming hydrophobic interactions with
Trp286, Phe295 and Leu289 (Figure 5B).
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9
0
Figure 5. Predicted binding mode of compound 1w with hAChE. (A) Surface
representations of the binding mode of compound 1w (carbon in green, oxygen in red,
nitrogen in blue) within the active site pockets of hAChE. (B) 3D binding mode of
compound 1w within the active site pocket of hAChE. Hydrogen bonds are
represented by dashed lines. The figures were generated using Pymol.
Additionally, the predicted binding mode of compound 1w within the active site
pockets of hPDE5A is presented in Figure 6. In general, compound 1w contacts with
Gln817, Phe820, Met816, Leu804 and Val782 of hPDE5A via a hydrogen bond and
hydrophobic interactions. In detail, the benzo[d][1,3]dioxole fragment of 1w occupies
the hydrophobic pocket of hPDE5A, forming hydrophobic interactions with Met816,
Leu804 and Val782. Furthermore, the indole fragment of 1w interacts with the phenyl
ring of Phe820 through faceꢀtoꢀface πꢀπ stacking interactions (4.7 Å) and the NH of
indole fragment interacts with Gln817 (2.1 Å) via hydrogen bond interactions.
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Figure 6. Predicted binding mode of compound 1w with hPDE5A. (A) Surface
representations of the binding mode of compound 1w (carbon in green, oxygen in red,
nitrogen in blue) within the active site pockets of hPDE5A. (B) 3D binding mode of
compound 1w within the active site pocket of hPDE5A. Hydrogen bonds are
represented by dashed lines. The figures were generated using Pymol.
CONCLUSIONS
AChEs inhibitors remains the most successful means of improving the memory
and cognitive function of AD patients, and PDE5 upꢀregulation in AD brains is an
underlying magnetic AD therapeutic target. Therefore, AChE/PDE5 dual inhibitors
may provide new perspectives and breakthroughs for the treatment of AD. Through
screening of our old drug library, tadalafil, a selective PDE5 inhibitor, was found to
have certain AChE inhibition.
To obtain good dualꢀtarget AChE/PDE5 inhibitors with improved AChE
inhibitory activity and BBB penetrability, a series of tadalafil derivatives were
rationally designed, synthesized and evaluated. The SAR studies revealed that the
stereoꢀconfiguration at position 6 of the tadalafil parent nucleus was vital to display
strong AChE inhibitory activity, and the favored configuration was 6R, while
stereoꢀconfiguration at both position 6 and 12 of the tadalafil parent nucleus,
especially position 6, was vital to exert strong PDE5A1 inhibitory activity, and the
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favored configuration was 6R, 12R. Research has confirmed that 1p and 1w are
excellent selective dualꢀtarget AChE/PDE5 inhibitors with improved BBB
penetrability. Remarkably, 1w·Cit demonstrated comparable effectivity with
donepezil in cognitive ability improvement in the AD mouse model at a dosage of 10
mg/kg, and excellent effect on increasing the level of CREB phosphorylation in vivo,
a crucial factor in ameliorating the cognitive impairment and restoring the synaptic
function of AD. As CREB phosphorylation is a downstream effect of PDE5 inhibition,
more studies will be needed to further determine the PDE inhibitory effect of 1w Cit
in vivo. Moreover, the molecular docking simulations of 1w with hAChE and
hPDE5A also confirmed the rationality of our design strategy.
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In conclusion, a series of novel tadalafil derivatives with excellent selective
dualꢀtarget AChE/PDE5 inhibitory activities and improved BBB penetrability were
discovered through drug discovery strategies of repurposing and redeveloping
existing drugs. The optimal compound 1w·Cit could improve cognitive dysfunction
in an AD mouse model and increase the level of CREB phosphorylation in vivo. Our
research presents a potential selective dualꢀtarget AChE/PDE5 inhibitor as a good
candidate drug for the treatment of AD, and they can also be regarded as an excellent
molecule probe to explore the novel AD therapeutic approach in vivo.
EXPERIMENTAL SECTION
Chemistry. General Method. Reagents and solvents were purchased from
Adamasꢀbeta, Energy Chemical, J&K, TCI, and Target Molecule, which were used
without further purification. Analytical thinꢀlayer chromatography (TLC) was
performed using HSGF 254 (150ꢀ200 ꢁm thickness; Yantai Huiyou Co., China), and
spots were visualized with UV light. Melting points were measured in capillary tubes
on a SGW Xꢀ4 melting point apparatus without correction, and yields were not
optimized. Nuclear magnetic resonance (NMR) spectroscopy was performed on a
Bruker AMXꢀ400 NMR. Chemical shifts were reported in parts per million (ppm, δ)
downfield from tetramethylsilane. Proton coupling patterns were described as singlet
(s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Highꢀresolution
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mass spectra (HRMS) were obtained by electric ionization (EI) using a Waters GCT
Premie and Waters LCT. HPLC data analysis of compounds 1aꢀt and 2aꢀq were
performed on an Agilent 1100 with a quaternary pump and diodeꢀarray detector
(DAD). The peak purity was verified by UV spectra. All analogs were confirmed to
be ≥95% pure (Table S1, supporting information).
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General procedure for the synthesis of target compounds 1aꢀ1w. A solution of
intermediate 4 (0.213 g, 0.5 mmol) and amine (1.2 equiv) in methanol (10 mL) with
trimethylamine (1.5 equiv) was refluxed for 12ꢀ24 h and was monitored by TLC.
After the reaction was complete, methanol was removed by evaporation. The residue
was dissolved in dichloromethane. The organic layer was washed with saturated
sodium bicarbonate solution and brine and dried over anhydrous sodium sulfate.
Evaporation of the solvent gave a residue that was purified by chromatography with
dichloromethane/methanol (40:1 – 10:1) as the eluent to afford the target compound.
(6
R
,12a
R
)ꢀ6ꢀ(benzo[
d
][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ(2ꢀmorpholinoethyl)ꢀ2,3,6,7,12,12aꢀhexa
]indoleꢀ1,4ꢀdione (1a). The intermediate and
hydropyrazino[1',2':1,6]pyrido[3,4ꢀ
b
amine were 4a and 2ꢀmorpholinoethanꢀ1ꢀamine, respectively. White solid, 62% yield,
1
mp 248–249 °C. H NMR (400 MHz, CDCl
3
) δ 7.93 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H),
7
6
1
.33 (d, J = 8.1 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 6.98 (s, 1H),
.82 (s, 1H), 6.73 (s, 2H), 5.95 (s, 2H), 4.33 (t, J = 12.8 Hz, 2H), 4.06 (d, J = 17.5 Hz,
H), 3.91 (s, 1H), 3.71 (s, 4H), 3.52 (dd, J = 15.3, 3.5 Hz, 1H), 3.24 (s, 1H), 2.99 (s,
+
1H), 2.60 (s, 6H). HRMS (EI) m/z calcd for C27
H
28
N
4
O
5
(M ) 488.2060, found
488.2061.
(6
R
,12a
R
)ꢀ6ꢀ(benzo[
d
][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ((
R
)ꢀ1ꢀbenzylpyrrolidinꢀ3ꢀyl)ꢀ2,3,6,7,12,
]indoleꢀ1,4ꢀdione (1b).
1
2aꢀhexahydropyrazino[1',2':1,6]pyrido[3,4ꢀ
b
The intermediate and amine were 4a and (R)ꢀ1ꢀbenzylpyrrolidinꢀ3ꢀamine, respectively.
1
White solid, 56% yield, mp 135–136 °C. H NMR (400 MHz, CDCl
3
) δ 8.13 (s, 1H),
7
.62 – 7.09 (m, 4H), 7.05 – 6.61 (m, 4H), 5.91 (s, 2H), 5.16 (s, 1H), 4.27 (d, J = 20.2
Hz, 2H), 4.12 (d, J = 17.3 Hz, 1H), 3.73 – 3.37 (m, 3H), 3.12 – 2.42 (m, 3H), 2.26 (s,
+
2
H), 1.82 (s, 2H). HRMS (EI) m/z calcd for C32
H
30
N
4
O
4
(M ) 534.2267, found
534.2268.
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d
][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ(2ꢀ(pyrrolidinꢀ1ꢀyl)ethyl)ꢀ2,3,6,7,12,12aꢀ
hexahydropyrazino[1',2':1,6]pyrido[3,4ꢀb]indoleꢀ1,4ꢀdione (1c). The intermediate
and amine were 4a and 2ꢀ(pyrrolidinꢀ1ꢀyl)ethanꢀ1ꢀamine. White solid, 53% yield, mp
1
278–280 °C. H NMR (400 MHz, CDCl
3
) δ 8.12 (s, 1H), 7.53 (d, J = 7.7 Hz, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.31 (d, J = 8.0 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H), 7.15 (t, J = 7.4 Hz, 1H), 6.97 (s, 1H),
6
1
1
.80 (s, 1H), 6.70 (s, 2H), 5.92 (d, J = 3.5 Hz, 2H), 4.39 – 4.24 (m, 2H), 4.10 (d, J =
7.7 Hz, 1H), 3.84 – 3.69 (m, 1H), 3.52 (dd, J = 15.4, 4.0 Hz, 1H), 3.42 – 3.27 (m,
H), 3.03 – 2.90 (m, 1H), 2.73 (d, J = 4.6 Hz, 2H), 2.59 (s, 4H), 1.79 (s, 4H). HRMS
+
(EI) m/z calcd for C27
H
28
N
4
O
4
(M ) 472.2111, found 472.2164.
(6
S,12a )ꢀ6ꢀ(benzo[
R
d
][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ(2ꢀmorpholinoethyl)ꢀ2,3,6,7,12,12aꢀhexa
]indoleꢀ1,4ꢀdione (1d). The intermediate and
hydropyrazino[1',2':1,6]pyrido[3,4ꢀ
b
amine were 4b and 2ꢀmorpholinoethanꢀ1ꢀamine, respectively. White solid, 58% yield,
1
mp 147–150 °C. H NMR (400 MHz, CDCl
3
) δ 7.87 (s, 1H), 7.60 (d, J = 7.3 Hz, 1H),
7.28 (d, J = 7.6 Hz, 1H), 7.23 – 7.11 (m, 2H), 6.86 (d, J = 8.0 Hz, 1H), 6.74 (s, 1H),
6
4
1
.69 (d, J = 8.0 Hz, 1H), 6.20 (s, 1H), 5.87 (d, J = 8.5 Hz, 2H), 4.45 – 4.14 (m, 2H),
.05 – 3.89 (m, 1H), 3.88 – 3.48 (m, 6H), 3.41 (s, 1H), 3.22 (dd, J = 15.7, 11.7 Hz,
+
H), 2.76 – 2.31 (m, 6H). HRMS (EI) m/z calcd for C27
H
28
N
4
O
5
(M ) 488.2060,
found 488.2059.
,12a )ꢀ6ꢀ(benzo[
2aꢀhexahydropyrazino[1',2':1,6]pyrido[3,4ꢀ
(6
S
R
d
][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ((
R
)ꢀ1ꢀbenzylpyrrolidinꢀ3ꢀyl)ꢀ2,3,6,7,12,
]indoleꢀ1,4ꢀdione (1e). The
1
b
intermediate and amine were 4b and (R)ꢀ1ꢀbenzylpyrrolidinꢀ3ꢀamine, respectively.
1
White solid, 60% yield, mp 138–140 °C. H NMR (400 MHz, CDCl ) δ 7.88 (s, 1H),
3
7.60 (d, J = 7.4 Hz, 1H), 7.35 – 7.27 (m, 1H), 7.22 – 7.12 (m, 2H), 6.84 (d, J = 7.6 Hz,
1
H), 6.75 – 6.66 (m, 2H), 6.21 (s, 1H), 5.87 (d, J = 8.5 Hz, 2H), 5.21 (s, 1H), 4.45 (d,
J = 17.2 Hz, 1H), 4.24 (dd, J = 11.4, 4.6 Hz, 1H), 3.97 (d, J = 17.7 Hz, 1H), 3.69 (dd,
J = 15.9, 4.8 Hz, 2H), 3.49 (s, 1H), 3.22 (dd, J = 15.9, 11.5 Hz, 1H), 3.00 (s, 1H), 2.72
+
(
s, 1H), 2.51 – 2.03 (m, 4H). HRMS (EI) m/z calcd for C32
found 534.2269.
,12a )ꢀ6ꢀ(benzo[
hexahydropyrazino[1',2':1,6]pyrido[3,4ꢀ
H
30
N
4
O
4
(M ) 534.2267,
(6
S
R
d
][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ(2ꢀ(pyrrolidinꢀ1ꢀyl)ethyl)ꢀ2,3,6,7,12,12aꢀ
]indoleꢀ1,4ꢀdione (1f). The intermediate
b
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1
2
3
4
5
6
7
8
9
and amine were 4b and 2ꢀ(pyrrolidinꢀ1ꢀyl)ethanꢀ1ꢀamine, respectively. White solid,
1
5
2% yield, mp 140–142 °C. H NMR (400 MHz, CDCl ) δ 7.93 (s, 1H), 7.54 (d, J =
3
7
.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H),
6.98 (s, 1H), 6.82 (s, 1H), 6.73 (s, 2H), 5.95 (s, 2H), 4.33 (t, J = 12.8 Hz, 2H), 4.06 (d,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
J = 17.5 Hz, 1H), 3.91 (s, 1H), 3.71 (s, 4H), 3.52 (dd, J = 15.3, 3.5 Hz, 1H), 3.24 (s,
+
1
H), 2.99 (s, 1H), 2.60 (s, 6H). HRMS (EI) m/z calcd for C27
found 472.2155.
,12a )ꢀ6ꢀ(benzo[
hexahydropyrazino[1',2':1,6]pyrido[3,4ꢀ
28
H N
4
O
4
(M ) 472.2111,
(6
R
R
d
][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ(1ꢀbenzylpyrrolidinꢀ3ꢀyl)ꢀ2,3,6,7,12,12aꢀ
]indoleꢀ1,4ꢀdione (1g). The intermediate
b
and amine were 4a and 1ꢀbenzylpyrrolidinꢀ3ꢀamine, respectively. White solid, 59%
1
yield, mp 129–131 °C. H NMR (400 MHz, CDCl
3
) δ 8.00 (s, 1H), 7.59 (d, J = 7.4 Hz,
1
H), 7.29 (d, J = 7.6 Hz, 1H), 7.22 – 7.10 (m, 2H), 6.83 (d, J = 7.8 Hz, 1H), 6.71 (s,
1H), 6.68 (d, J = 7.8 Hz, 1H), 6.21 (s, 1H), 5.86 (d, J = 7.2 Hz, 2H), 5.16 (s, 1H), 4.37
(s, 1H), 4.25 (d, J = 6.4 Hz, 1H), 4.01 (d, J = 17.0 Hz, 1H), 3.68 (d, J = 15.9 Hz, 2H),
3
.31 – 3.12 (m, 1H), 2.97 (d, J = 83.1 Hz, 2H), 2.32 (s, 2H), 1.93 – 1.66 (s, 3H).
+
HRMS (EI) m/z calcd for C32
,12a )ꢀ6ꢀ(benzo[
12,12aꢀhexahydropyrazino[1',2':1,6]pyrido[3,4ꢀ
H
30
N
4
O
4
(M ) 534.2267, found 534.2272.
(
6
R
R
d
][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ(2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ethyl)ꢀ2,3,6,7
]indoleꢀ1,4ꢀdione (1h). The
,
b
intermediate and amine were 4a and 2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ethanꢀ1ꢀamine,
1
respectively. White solid, 56% yield, mp 189–190 °C. H NMR (400 MHz, CDCl
3
) δ
7.88 (s, 1H), 7.61 (d, J = 7.3 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.25 – 7.12 (m, 2H),
6.86 (d, J = 8.1 Hz, 1H), 6.72 (dd, J = 17.6, 4.7 Hz, 2H), 6.21 (s, 1H), 5.88 (d, J =
11.9 Hz, 2H), 4.34 (d, J = 6.3 Hz, 1H), 4.22 (d, J = 17.0 Hz, 1H), 3.94 (d, J = 17.1 Hz,
1
2
5
H), 3.72 (dd, J = 16.1, 4.9 Hz, 1H), 3.67 (s, 2H), 3.24 (dd, J = 15.9, 11.8 Hz, 1H),
+
.75 – 2.35 (m, 10H). HRMS (EI) m/z calcd for C H N O (M ) 501.2376, found
28
31
5
4
01.2375.
,12a )ꢀ6ꢀ(benzo[
exahydropyrazino[1',2':1,6]pyrido[3,4ꢀ
(6
R
R
d
][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ(2ꢀ(piperidinꢀ1ꢀyl)ethyl)ꢀ2,3,6,7,12,12aꢀh
]indoleꢀ1,4ꢀdione (1i). The intermediate
b
and amine were 4a and 2ꢀ(piperidinꢀ1ꢀyl)ethanꢀ1ꢀamine, respectively. White solid, 48%
1
yield, mp 130–131 °C. H NMR (400 MHz, CDCl
3
) δ 7.87 (s, 1H), 7.60 (d, J = 6.8 Hz,
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