Claulansine F-donepezil hybrids as anti-alzheimer’s disease agents with cholinergic, free-radical scavenging, and neuroprotective activities

Article abstract of DOI:10.3390/molecules26051303

The multifactorial nature of Alzheimer’s disease (AD) calls for the development of multitarget agents addressing key pathogenic processes. A total of 26 Claulansine F-donepezil hybrids were designed and synthesized as multitarget drugs. Among these compounds, six compounds exhibited excellent acetylcholinesterase (AChE) inhibitory activity (half maximal inhibitory concentration (IC50) 1.63-4.62 μM). Moreover, (E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol- 5-yl)-N-((1-(2-chlorobenzyl)piperidin-4-yl)methyl)acrylamide (6bd) exhibited better neuroprotective effects against OGD/R (oxygen-glucose deprivation/reoxygenation) than lead compound Claulansine F. Furthermore, 6bd could cross the blood-brain barrier in vitro. More importantly, compared to edaravone, 6bd had stronger free-radical scavenging activity. Molecular docking studies revealed that 6bd could interact with the catalytic active site of AChE. All of these outstanding in vitro results indicate 6bd as a leading structure worthy of further investigation.

Full text of DOI:10.3390/molecules26051303

molecules
Article
Claulansine F–Donepezil Hybrids as Anti-Alzheimer’s
Disease Agents with Cholinergic, Free-Radical Scavenging,
and Neuroprotective Activities
†
†
Yingda Zang , Ke Liu , Weiping Wang, Chuangjun Li, Jie Ma, Jingzhi Yang, Xinyi Chen, Xiaoliang Wang
and Dongming Zhang *
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
zydaitfj@imm.ac.cn (Y.Z.); liuke@imm.ac.cn (K.L.); wangwp@imm.ac.cn (W.W.); lichuangjun@imm.ac.cn (C.L.);
majie@imm.ac.cn (J.M.); yjzh@imm.ac.cn (J.Y.); chenxinyi@imm.ac.cn (X.C.); wangxl@imm.ac.cn (X.W.)
*
†
Correspondence: zhangdm@imm.ac.cn; Tel./Fax: +86-10-63165227
These authors contributed equally to this work.
Abstract: The multifactorial nature of Alzheimer’s disease (AD) calls for the development of multitar-
get agents addressing key pathogenic processes. A total of 26 Claulansine F–donepezil hybrids were
designed and synthesized as multitarget drugs. Among these compounds, six compounds exhibited
excellent acetylcholinesterase (AChE) inhibitory activity (half maximal inhibitory concentration
(
5
IC ) 1.63–4.62
µ
M). Moreover, (E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
50
ꢀ
ꢁꢂꢃꢄꢅꢆ
-yl)-N-((1-(2-chlorobenzyl)piperidin-4-yl)methyl)acrylamide (6bd) exhibited better neuroprotective
Citation: Zang, Y.; Liu, K.; Wang, W.;
Li, C.; Ma, J.; Yang, J.; Chen, X.; Wang,
X.; Zhang, D. Claulansine
effects against OGD/R (oxygen–glucose deprivation/reoxygenation) than lead compound Claulan-
sine F. Furthermore, 6bd could cross the blood–brain barrier in vitro. More importantly, compared to
edaravone, 6bd had stronger free-radical scavenging activity. Molecular docking studies revealed
that 6bd could interact with the catalytic active site of AChE. All of these outstanding in vitro results
indicate 6bd as a leading structure worthy of further investigation.
F–Donepezil Hybrids as
Anti-Alzheimer’s Disease Agents
with Cholinergic, Free-Radical
Scavenging, and Neuroprotective
Activities. Molecules 2021, 26, 1303.
https://doi.org/10.3390/
Keywords: Alzheimer’s disease; Claulansine F–donepezil hybrids; AChE inhibitory activity; free-
radical scavenging activity; neuroprotective effects
molecules26051303
Academic Editor: Patrizia Russo
1. Introduction
Received: 1 February 2021
Accepted: 24 February 2021
Published: 28 February 2021
Alzheimer’s disease (AD) is the most prominent form of dementia among the pop-
ulation above 65 that results in a substantial burden on human health, economy, and
society throughout the world. Considering the etiology of AD still remains difficult to
understand, strenuous efforts are being made to elucidate the underlying mechanisms of
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
the disease [1–5]. Many hypotheses including cholinesterase, β-amyloid (Aβ) deposits,
tau-protein aggregation, oxidative stress, inflammation, and dyshomeostasis of biometals
have been developed to illuminate the mechanism of AD [6,7].
At present, the treatment of AD is mainly based on cholinergic hypothesis [8]. The
hypothesis emphasizes that decreased acetylcholine can lead to cognitive and memory
impairment, and sustaining or recovering the cholinergic function is supposed to be
clinically beneficial [9]. In addition, oxidative stress is one of the earliest events in AD
Copyright: © 2021 by the authors. pathogenesis. Compelling evidence indicates that free radicals are extremely important
Licensee MDPI, Basel, Switzerland.
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distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
in causing neuronal death [10]. Antioxidants are thought to offer a good possibility of
combating neurodegeneration [11–13].
Clausena lansium, a fruit tree distributed in southern China, is used in traditional medicine.
During our continued investigation of the bioactive constituents of Clausena lansium, a con-
siderable number of carbazole alkaloids were obtained and evaluated primarily for their
neuroprotective effects via phytochemical studies and activity tracking [14]. Among these
4
.0/).
Molecules 2021, 26, 1303. https://doi.org/10.3390/molecules26051303
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1303
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carbazole alkaloids, Claulansine F (Clau F), a new pyrano[3, 2-a]carbazole alkaloid, dis-
played remarkable protection of PC12 cells against sodium nitroprusside (SNP)-induced
apoptosis [14]. Our previous pharmacological research also demonstrated that Clau F
exerts a notable effect on OH scavenging and the protection of mitochondrial integrity [15].
·
With the aim of improving the activity, our research team synthesized a series of derivatives
of Clau F. Subsequently, preliminary researches were conducted on the vitro neuropro-
tective activity of Clau F and its derivatives. Among these derivatives, CZ-7 showed the
strongest ability to scavenge free radicals and penetrate the blood–brain barrier, as well as
the strongest neuroprotective impact. More importantly, CZ-7 exposure in the brain was
4.3-fold higher than that in plasma, and preliminary acute toxicological test results showed
that CZ-7 had no toxic side effects with an oral 1 g/kg dose in ICR (Institute of Cancer
Research) mice [16]. In addition, CZ-7 has therapeutic potential for VD (vascular dementia)
by alleviating oxidative stress injury through Nrf2-mediated antioxidant responses [17].
Donepezil, a Food and Drug Administration (FDA)-approved drug, is a potent, se-
lective, uncompetitive, reversible acetylcholinesterase (AChE) inhibitor that is believed
to enhance cholinergic function by increasing ACh levels in the central nervous system
(CNS) [18]. However, clinical trials of this single-target drug on this disease have been
modest and transient because of the multifactorial nature of AD, and this has consequently
failed in the treatment of moderate to severe AD patients [19].
Considering the facts that both donepezil and Clau F exhibit a vast spectrum of biologi-
cal properties and have not been combined into one drug candidate as an anti-AD agent, we
hybridized the pharmacophores of donepezil and Clau F into one molecule (Figure 1). As
known, the benzylpiperidine fragment of donepezil that interacts with the catalytic anionic
site (CAS) of AChE has been shown as the AChE inhibitory pharmacophore. The indanone
moiety can interact with the peripheral anionic site (PAS) of AChE via aromatic stacking
interactions and inhibit Aβ aggregation. In order to introduce free-radical scavenging
activity and diversify the structures, we replaced the indanone moiety with Clau F or its
analogue CZ-7 fragments. These new derivatives may simultaneously possess dual binding
sites for AChE inhibition, free-radical scavenging activity, and neuroprotective effects.
Figure 1. Design strategy of the multifunctional Claulansine F–donepezil hybrids.
2. Results
2.1. Chemistry
The target compounds 6aa–ch were synthesized, as outlined in Scheme 1. The key
intermediates 2a,b were prepared via the reaction between 1a,b and dry malonic acid using
piperidine as a catalyst in dry pyridine. In order to afford the other key intermediates, with
an N-benzyl piperidine moiety 5a–n, two-step reactions were carried out. Firstly, commer-
cially available 4-Boc-aminopiperidine (3a), 4-(Boc-aminomethyl) piperidin (3b), and 4-(2-
Boc-aminoethyl) piperidine (3c) were reacted with different substituted benzyl bromides
to give 4a–n. Secondly, the key intermediates 5a–n were acquired via the reaction between
trifluoroacetic acid (TFA) and 4a–n in good yields without further purification. Lastly, 5a–n
reacted with 2a,b in dichloromethane (DCM) catalyzed by N-(3-(dimethylamino)propyl)-N-

Molecules 2021, 26, 1303
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ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole hydrate (HOBt) to ob-
tain the target compounds 6aa–ch. All final compounds ( 6aa-ch; Supplementary Materials
1
13
Figures S1–S52) were fully characterized by H and C nuclear magnetic resonance (NMR)
and high-resolution electrospray ionization mass spectrometry (HRESIMS). The purity of
Clau F and its synthetic analogues was determined by high-performance liquid chromatog-
raphy (HPLC) analysis and was greater than 95% (Supplementary Materials Table S1).
Scheme 1. Synthetic pathways for access to compounds 6aa–ch. Reagents and conditions: (a)
◦
pyridine, piperidine, 12 h, reflux; (b) sodium hydride (NaH), tetrahydrofuran (THF), 12 h,
TFA, DCM, 0 C, 2 h; (d) HOBt, EDCI, THF, 25 C, 12 h.
−
20 C; (c)
◦
◦
2
.2. Evaluation of Cholinesterase Inhibitory Activities and Structure–Activity Relationship
(
SAR) Discussion
The inhibitory activities of the target compounds on AChE (from electric eel) and
butyrylcholinesterase (BuChE, from equine serum) were tested using the spectrophoto-
metric method of Ellman et al. with donepezil, parental Clau F, and CZ-7 as reference
compounds. The half maximal inhibitory concentration (IC ) values for eeAChE and
50
eqBuChE inhibition are shown in Table 1. Generally, the activity of cholinesterase increased
after introducing the N-benzylpiperidine moiety mimicking donepezil compared with
Clau F and CZ-7. Firstly, under the same linker condition, the AChE inhibitory activity
of the compounds with R = t-Bu were higher than those with R = OCH , for example,
3
6
aa–ad vs. 6ae–ah
;
6ba–bf vs. 6bg–bj
;
6ca–cd vs. 6ce–ch. In particular, compounds 6ba–
M) than other compounds.
bf exhibited better AChE inhibitory activity (IC50 1.63–4.62
µ

Molecules 2021, 26, 1303
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Secondly, the length of the alkyl chain between the carbazole and donepezil moiety could
significantly influence the AChE inhibitory activity. Compounds with n = 1 held relatively
better activities than n = 0 or 2. In addition, the substituent N-benzylpiperidine moiety had
no significant effect on the activity. On the other hand, compounds 6ba–bf with R = t-Bu
showed comparable potency (IC50 5.46–19.01 µM) to donepezil (4.20 µM) against BuChE.
According to the experimental results above, the compounds with R = t-Bu (6aa–ad, 6ba–bf,
and 6ca–cd) were selected as the candidate compounds for subsequent investigations.
Table 1. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities of
compounds 6aa–ch. IC , half maximal inhibitory concentration; Clau F, Claulansine F.
5
0
IC50 (µM) (a) or Inhibition Rate (%) in 100 µM
Compound
AChE
BuChE
6
6
6
6
6
6
aa
ab
ac
ad
ae
af
ag
51.92 ± 2.24
36.16 ± 0.31%
50.93 ± 2.76%
42.46 ± 6.12%
20.43 ± 7.35%
34.20 ± 10.65%
36.77 ± 1.25%
40.05 ± 1.40%
4.62 ± 0.28
22.86 ± 1.14
35.93 ± 5.13
34.26 ± 0.69
38.83 ± 2.48
69.13 ± 8.51
24.52 ± 1.94
26.38 ± 4.51
18.02 ± 2.27
12.27 ± 0.66
5.63 ± 0.31
19.01 ± 0.94
5.46 ± 0.61
6.36 ± 1.21
12.15 ± 0.76
74.86 ± 3.21
31.52 ± 3.88
32.81 ± 0.50
37.57 ± 0.56
23.37 ± 0.95
19.12 ± 2.04
33.82 ± 0.51
17.34 ± 4.82
26.39 ± 1.40
18.05 ± 3.35
26.75 ± 1.76
24.17 ± 4.78
4.20 ± 0.48
64.32 ± 7.52
59.54 ± 5.62
6
6
6
ah
ba
6
6
6
6
6
6
6
bb
bc
bd
be
bf
bg
bh
4.08 ± 0.12
4.26 ± 0.22
4.34 ± 0.46
1.69 ± 0.00
1.63 ± 0.30
27.72 ± 5.15
23.60 ± 8.31
58.36 ± 1.79
91.13 ± 4.41
9.00 ± 0.86
6
6
6
bi
bj
ca
6
6
6
6
6
6
6
cb
cc
cd
ce
cf
cg
ch
10.67 ± 0.20
14.30 ± 0.36
16.96 ± 1.51
41.70 ± 1.22
54.82 ± 7.96
72.97 ± 1.93
24.56 ± 2.10
0.17 ± 0.04
Donepezil
Clau F
28.25 ± 2.11%
32.54 ± 6.52%
CZ-7
2
.3. Neuroprotective Effects against OGD/R (Oxygen–Glucose Deprivation/Reoxygenation) Injury
on Primary Cortical Neurons
The parental compounds Clau F and CZ-7 have remarkable neuroprotective effects. To
further confirm whether the derivatives retain activity, an OGD/R model was performed
in primary cortical neurons, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assays were used to test cell viability. As shown in Figure 2, OGD/R treatment
obviously reduced the cell viability of cultured neurons to 40.4% from 100% in the control
group. The administration of most of the target compounds (10
µM) resulted in elevated
levels of cell viability. In particular, 6bb 6bd 6be 6bf, and parental comopound CZ-7
,
,
,
significantly promoted cell viability to 109%, 125.6%, 104.7%, 107.3%, and 112.7%, respec-
tively (n = 3, p < 0.01 vs. OGD/R group). According to the experimental results above,
compound 6bd was selected as the candidate compound for the subsequent investigations.

Molecules 2021, 26, 1303
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Figure 2. Neuroprotective activity screening of 6aa–ad, 6ba–bf, 6ca–cd, and CZ-7 at 10 µM in
primary cortical neurons induced by oxygen–glucose deprivation/reoxygenation (OGD/R). Neurons
were exposed to 5 mM sodium dithionite and reoxygenation. A 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay was used to test the cell viability. The absorbance of
normal cells was regarded as 100%. Error bars represent the standard deviation. ### p < 0.001 vs.
control; ** p < 0.05, *** p < 0.01 vs. model (means ± standard deviation, n = 6).
2.4. Brain Penetration
A blood–brain barrier (BBB) penetration assay in vitro is essential for anti-AD drug
development. A parallel artificial membrane permeation assay for BBB (PAMPA-BBB)
was carried out to investigate the BBB permeability of compound 6bd. According to
the obtained data on permeability in Table 2, 6bd was classified among the compounds
−
6
−1
(Pe represents
having good brain penetration with a Pe value of 4.38
±
0.05
×
10 cm
·
s
the PAMPA effective permeability coefficient). The test was performed using a good
(
corticosterone) and weak brain-penetrating reference (ofloxacin), respectively.
−
6
−1
Table 2. Permeability (Pe
×
10 cm s ) in the PAMPA-BBB assay for compound 6bd and two
·
commercial drugs used in the experiment validation.
Experiment b
Commercial Drugs
Bibliography a
Corticosterone
Ofloxacin
5.1
0.8
6.27 ± 0.32
1.32 ± 0.01
4.38 ± 0.05
6
bd
a
Taken from [20]. b Data are the means ± SD of three independent experiments.
2.5. Free-Radical Scavenging Ability of 6bd
Oxidative stress caused by reactive oxygen (for example, singlet oxygen and hy-
droxyl radical) is related to the death of neurons and the formation of intracellular A
oligomer [21 22]. Drugs designed to prevent the formation or removal of free radicals in
β
,
the brain are beneficial for AD. The main technique for detecting organic free radicals is
electron spin resonance (ESR) spectroscopy. This technology can detect radical concentra-
tions on the order of 10 nM or less under favorable conditions and has high sensitivity.
Therefore, compound 6bd was tested for its ability to scavenge active oxygen by using
ESR spectroscopy. For comparison, edaravone (Eda) was used as a reference. The singlet
−
2
oxygen scavenging of 10 mol/L compound 6bd or Eda is shown in Figure 3, with the
former having a stronger effect than the latter.

Molecules 2021, 26, 1303
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Figure 3. The intensity of free-radical scavenging, whereby 6bd exhibits stronger free-radical scav-
enging than Eda.
2.6. Molecular Docking of 6bd on AChE
To further investigate the binding modes of these derivatives, a molecular docking
study was performed with the most promising compound 6bd using the Discovery Studio
software package. In this study, the X-ray crystal structure of the hAChE complex with
donepezil (Protein Data Bank identifier (PDB ID): 4EY7) was applied to this study [23].
Redocking simulation was performed with the native ligands to verify the docking pro-
cedures applied. The site of the grid box was on the centroid of the pre-dock ligand with
X-ray coordinates of X:
−
13.983, Y: 43.975, and Z: 27.900. As a result, donepezil highly
overlapped with the co-crystal ligand (root-mean-square deviation (RMSD) value = 0.18 Å).
After validating the docking method, compound 6bd was docked using the Discovery Stu-
dio software package. As illustrated in Figure 4, the predicted binding mode of compound
6
bd was very similar to the experimental conformation observed for the AChE–donepezil
complex, and they retained several key interactions. The phenyl moiety of 6bd was ori-
ented toward the CAS of AChE through the -stacking interaction with the phenyl ring of
Trp86. The charged nitrogen of piperidine ring could bind to the CAS by making a cation–
interaction with the aromatic ring in Tyr337 [24]. A –alkyl interaction was observed
π
π
π
between the F atom of compound 6bd and His447. Furthermore, 6bd formed a hydrogen
bond with Asp74 and Try341 of the PAS. Meanwhile, the carbazole ring of compound 6bd
formed a
bd was suitable for the active site of the enzyme, where it simultaneously interacted with
the PAS and CAS of hAChE.
π–π interaction with Trp286. All these results clearly indicated that compound
6

Molecules 2021, 26, 1303
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Figure 4.
line, pink dotted line, blue dotted line, and orange dotted line represent hydrogen bonds,
interactions, and cation– interactions, respectively.) ( ) Superimposed result of 6bd (wheat sticks) and donepezil (pink
(
A
) Three-dimensional (3D) docking models of compound 6bd (wheat stick) with hAChE (the green dotted
π
-stacking interactions, –alkyl
π
π
B
sticks) in the binding pocket.
3. Discussion
3.1. Chemistry
All commercial chemicals were used as supplied unless otherwise indicated. All
reactions were performed in oven-dried glassware. All yields reported refer to the yields
1
13
of the isolated compounds. H-NMR and C-NMR spectral data were obtained using
Bruker Avance 600 MHz and 400 MHz spectrometers at 300 K using tetramethylsilane
TMS) as an internal standard. HRESIMS was measured with an Agilent 6520 Accurate-
Mass Q-TOF LC/MS. Silica gel thin-layer chromatography (TLC) plates (Qing Dao Marine
Chemical Factory, Qingdao, China) were used to monitor the reaction progress. Column
chromatography was performed using silica gel (100–200 mesh size, Qing Dao Marine
Chemical Factory, Qingdao, China). Compounds 5a–n were synthesized as previously
described [25].
(
3.1.1. General Procedure for the Synthesis of 2a,b
A solution of 1a,b (1 equivalent (eq.)) and malonic acid (3 eq.) in a mixture of pyridine
−
1
◦
(
10 mL mmol 1a,b) and piperidine (0.01 eq.) was maintained at 40 C for 48 h. The
·
solution was concentrated by evaporation, the residue was diluted with 250 mL of water,
and the pH was adjusted to 11.0 with 2 N NaOH. The solution was extracted with four 200
mL portions of ethyl acetate to remove bases and neutral impurities. The aqueous phase
was treated with charcoal, and the filtrate was acidified to pH 1.8 by dropwise addition of
◦
6
N hydrochloric acid with stirring at 5 C. The resulting yellow precipitate was washed
well with cold water to obtain 2a,b.
E)-3-(8-Methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)acrylic acid (2a). White
(
◦
1
solid, 75%; melting point (m.p.): 182–183 C. H NMR (400 MHz, dimethyl sulfoxide
DMSO)-d6) 12.09 (s, 1H), 11.35 (s, 1H), 8.47 (s, 1H), 8.13 (d, J = 1.9 Hz, 1H), 7.96 (d,
J = 16.0 Hz, 1H), 7.40 (dd, J = 8.5, 1.9 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 6.92 (d, J = 9.8 Hz,
H), 6.62 (d, J = 16.0 Hz, 1H), 5.87 (d, J = 9.8 Hz, 1H), 1.48 (s, 6H), 1.39 (s, 9H). C-NMR
100 MHz, DMSO-d6) 168.4, 149.5, 142.0, 139.7, 138.5, 129.3, 122.9, 122.8, 119.6, 117.7, 117.4,
16.3, 115.5, 114.4, 110.3, 104.2, 76.6, 34.4, 31.8, 27.4. HRESIMS m/z = 376.19064 [M + H]+
(
δ
1
3
1
(
1
δ
(
calculated for C H O N, 376.19072).
24 26 3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)acrylic acid (2b).
◦
1
White solid, 68%; m.p.: 205–207 C. H-NMR (400 MHz, DMSO-d )
δ
12.11 (s, 1H), 11.30 (s,
H), 8.41 (s, 1H), 7.95 (d, J = 16.0 Hz, 1H), 7.70 (d, J = 2.5 Hz, 1H), 7.33 (d, J = 8.7 Hz, 1H), 6.96
dd, J = 8.7, 2.5 Hz, 1H), 6.90 (d, J = 9.8 Hz, 1H), 6.58 (d, J = 16.0 Hz, 1H), 5.86 (d, J = 9.9 Hz,
H), 3.83 (s, 3H), 1.48 (s, 6H). 13C-NMR (100 MHz, DMSO-d6)
168.4, 153.6, 149.7, 139.7,
6
1
(
1
δ

Molecules 2021, 26, 1303
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1
2
38.7, 135.0, 129.3, 123.6, 119.9, 117.6, 117.3, 115.5, 114.3, 114.0, 111.5, 104.3, 103.2, 76.7, 55.5,
+
7.4. HRESIMS m/z = 350.13959 [M + H] (calculated for C H O N, 350.13868).
21
20
4
3
.1.2. General Procedure for the Synthesis of 6aa–ch
To a solution of 2a,b (1 eq.) and 5a–n (2 eq.) in dry tetrahydrofuran (THF, 10
−
1
mL mmol 2a,b), EDCI (2.0 eq.) and HOBt (2.0 eq.) were added by stirring at room
temperature for 24 h. The solvent was removed by evaporation, and the residue was
·
diluted with EtOAc. The solution was washed with water, dried over MgSO , filtered, and
4
concentrated in vacuo. Purification by column chromatography (petroleum ether/ethyl
acetate, 3:1) gave compounds 6aa–ch.
(
E)-N-(1-Benzylpiperidin-4-yl)-3-(8-(tert-butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-
◦
1
yl)acrylamide (6aa). White solid, 58%; m.p.: 191–192 C. H-NMR (400 MHz, DMSO-d )
δ
6
1
1.31 (s, 1H), 8.16 (s, 1H), 8.01 (s, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.74 (d, J = 15.7 Hz, 1H),
7
1
2
.44–7.22 (m, 7H), 6.92 (d, J = 9.8 Hz, 1H), 6.72 (d, J = 15.8 Hz, 1H), 5.86 (d, J = 9.8 Hz,
H), 3.76–3.61 (m, 1H), 3.48 (s, 2H), 2.84–2.74 (m, 2H), 2.17–2.01 (m, 2H), 1.83–1.75 (m,
H), 1.48 (s, 6H), 1.46–1.43 (m, 2H), 1.38 (s, 9H). 13C-NMR (100 MHz, DMSO-d6)
165.0,
δ
1
1
49.3, 141.8, 138.4, 138.4, 137.8, 134.5, 129.4, 128.8, 128.2, 126.9, 122.8, 122.6, 119.7, 118.7,
17.4, 117.4, 115.6, 115.4, 110.3, 104.4, 76.5, 62.1, 51.9, 45.9, 34.4, 31.8, 31.7, 27.4. HRESIMS
+
m/z = 584.32721 [M + H] (calcd for C H O N , 548.32715).
36
42
2
3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-(1-(2-fluorobenzyl)
◦
1
piperidin-4-yl)acrylamide (6ab). White solid, 56%; m.p.: 168–169 C. H NMR (400 MHz,
DMSO-d6) 11.34 (s, 1H), 8.16 (s, 1H), 8.00 (s, 2H), 7.75 (d, J = 15.7 Hz, 1H), 7.54–7.30 (m,
H), 7.28–7.13 (m, 2H), 6.93 (d, J = 9.8 Hz, 1H), 6.72 (d, J = 15.7 Hz, 1H), 5.85 (d, J = 9.8 Hz,
H), 3.88–3.51 (m, 3H), 3.07–2.77 (m, 2H), 2.50 (s, 2H), 1.93–1.78 (m, 2H), 1.62–1.50 (m, 2H),
.48 (s, 6H), 1.38 (s, 9H). 13C NMR (100 MHz, DMSO-d6)
165.1, 160.9 (d, JC-F = 245.8 Hz),
49.3, 141.8, 138.4, 137.8, 134.6, 132.0 (d, JC-F = 4.7 Hz), 129.4, 128.7 (d, JC-F = 8.2 Hz), 124.5
δ
4
1
1
1
δ
(d, JC-F = 14.6 Hz), 124.4 (d, JC-F = 3.4 Hz), 122.8, 122.6, 119.6, 118.6, 117.5, 117.4, 115.6, 115.3,
1
15.2 (d, JC-F = 22.1 Hz), 110.4, 104.4, 76.5, 59.7, 51.5, 45.0, 34.4, 31.8, 31.7, 27.4. HRESIMS
+
m/z = 566.31696 [M + H] (calculated for C H O N F, 566.31773).
36
41
2
3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-(1-(2-methylbenzyl)
◦
1
piperidin-4-yl)acrylamide (6ac). White solid, 52%; m.p.: 201–202 C. H-NMR (400 MHz,
DMSO-d6) 11.37 (s, 1H), 8.16 (s, 1H), 8.00 (d, J = 1.8 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.74
d, J = 15.8 Hz, 1H), 7.40 (dd, J = 8.4, 1.8 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.23–7.20 (m, 1H),
δ
(
7
.16–7.11 (m, 3H), 6.93 (d, J = 9.8 Hz, 1H), 6.72 (d, J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H),
3
(
.77–3.64 (m, 1H), 3.41 (s, 2H), 2.78–2.74 (m, 2H), 2.32 (s, 3H), 2.13–2.02 (m, 2H), 1.85–1.73
m, 2H), 1.48 (s, 6H), 1.47–1.41 (m, 2H), 1.38 (s, 9H). 13C-NMR (100 MHz, DMSO-d6)
δ
165.0,
1
1
1
49.3, 141.7, 138.4, 137.8, 137.0, 136.7, 134.5, 130.0, 129.5, 129.3, 126.8, 125.4, 122.8, 122.6,
19.8, 118.7, 117.5, 117.4, 115.6, 115.4, 110.4, 104.4, 76.5, 60.3, 52.1, 46.0, 34.4, 31.9, 31.8, 27.4,
+
8.8. HRESIMS m/z = 562.34235 [M + H] (calculated for C H O N , 562.34280).
37
44
2
3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-(1-(2-chlorobenzyl)
◦
1
piperidin-4-yl)acrylamide (6ad). White solid, 62%; m.p.: 188–189 C. H-NMR (400 MHz,
DMSO-d6) 11.34 (s, 1H), 8.17 (s, 1H), 8.01 (d, J = 1.8 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.76
d, J = 15.8 Hz, 1H), 7.48 (dd, J = 7.5, 1.9 Hz, 1H), 7.43–7.25 (m, 5H), 6.93 (d, J = 9.8 Hz, 1H),
δ
(
6
.74 (d, J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.79–3.66 (m, 1H), 3.55 (s, 2H), 2.82–2.78
1
3
(
(
m, 2H), 2.21–2.10 (m, 2H), 1.85–1.75 (m, 2H), 1.55–1.43 (m, 8H), 1.38 (s, 9H). C-NMR
100 MHz, DMSO-d6) 165.1, 149.3, 141.8, 138.4, 137.8, 136.0, 134.5, 133.2, 130.7, 129.3,
29.2, 128.5, 127.0, 122.8, 122.6, 119.7, 118.7, 117.5, 117.4, 115.6, 115.4, 110.3, 104.4, 76.5,
δ
1
5
+
8.7, 52.0, 45.8, 34.4, 31.8, 31.8, 27.4.HRESIMS m/z = 582.28790 [M + H] (calculated for
C H O N Cl, 582.28818).
36
41
2
3
(
E)-N-(1-Benzylpiperidin-4-yl)-3-(8-methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-
◦
-yl)acrylamide (6ae). White solid, 57%; m.p.: 212–213 C. H-NMR (600 MHz, DMSO-d ) δ
6
1
5
11.26 (s, 1H), 8.13 (s, 1H), 7.94 (d, J = 7.4 Hz, 1H), 7.73 (d, J = 15.8 Hz, 1H), 7.58 (d, J = 2.5 Hz,
1H), 7.33 (d, J = 8.6 Hz, 1H), 7.33–7.27 (m, 4H), 7.27–7.21 (m, 1H), 6.96 (dd, J = 8.6, 2.5 Hz,
1H), 6.90 (d, J = 9.8 Hz, 1H), 6.70 (d, J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H),

Molecules 2021, 26, 1303
9 of 15
3
.73–3.65 (m, 1H), 3.35 (s, 2H), 2.81–2.74 (m, 2H), 2.11–2.01 (m, 2H), 1.83–1.72 (m, 2H),
1
1
1
.48 (s, 6H), 1.47–1.41 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 165.1, 162.3, 153.5, 149.5,
38.6, 137.9, 134.8, 134.7, 129.3, 128.7, 128.2, 126.8, 123.4, 119.8, 119.2, 117.1, 115.4, 113.8,
11.5, 104.4, 102.8, 76.5, 62.2, 55.5, 52.0, 46.7, 31.8, 27.4. HRESIMS m/z = 522.27576 [M + H]+
(
calculated for C H O N , 522.27512).
33 36 3 3
(
E)-N-(1-(2-Fluorobenzyl)piperidin-4-yl)-3-(8-methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-
◦
1
a] carbazol-5-yl)acrylamide (6af). White solid, 65%; m.p.: 195–196 C. H-NMR (600 MHz,
DMSO-d6) 8.13 (s, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.74 (d, J = 15.7 Hz, 1H), 7.58 (d, J = 2.5 Hz,
H), 7.42–7.38 (m, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.32–7.28 (m, 1H), 7.20–7.13 (m, 2H), 6.96
dd, J = 8.7, 2.5 Hz, 1H), 6.90 (d, J = 9.7 Hz, 1H), 6.70 (d, J = 15.8 Hz, 1H), 5.84 (d, J = 9.8 Hz,
H), 3.83 (s, 3H), 3.68 (d, J = 7.2 Hz, 1H), 3.52 (s, 2H), 2.79 (d, J = 11.2 Hz, 2H), 2.10 (t,
J = 11.2 Hz, 2H), 1.79 (dd, J = 12.8, 4.0 Hz, 2H), 1.48 (s, 6H), 1.46–1.42 (m, 2H). C-NMR
150 MHz, DMSO-d6) 165.1, 160.8 (d, JC-F = 244.4 Hz), 153.5, 149.5, 137.9, 134.8, 134.7,
31.5 (d, JC-F = 4.9 Hz), 129.3, 129.0 (d, JC-F = 8.2 Hz), 124.9 (d, JC-F = 14.6 Hz), 124.2 (d,
JC-F = 3.3 Hz), 123.4, 119.8, 119.2, 117.4, 117.2, 115.4, 115.1 (d, JC-F = 21.9 Hz), 113.8, 111.5,
04.4, 102.8, 76.5, 59.8, 55.5, 51.8, 45.9, 31.7, 27.4. HRESIMS m/z = 540.26569 [M + H]+
calculated for C H O N F, 540.26570).
δ
1
(
1
1
3
(
1
δ
1
(
33
35
3
3
(
E)-3-(8-Methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-(1-(2-methylbenzyl)
◦
1
piperidin-4-yl)acrylamide (6ag). White solid, 58%; m.p.: 215–216 C. H-NMR (600 MHz,
DMSO-d6) 8.12 (s, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 15.8 Hz, 1H), 7.57 (d, J = 2.5 Hz,
H), 7.33 (d, J = 8.7 Hz, 1H), 7.24–7.20 (m, 1H), 7.17–7.11 (m, 3H), 6.96 (dd, J = 8.7, 2.5 Hz,
H), 6.90 (d, J = 9.8 Hz, 1H), 6.70 (d, J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H),
δ
1
1
3
2
.75–3.64 (m, 1H), 3.42 (s, 2H), 2.81–2.73 (m, 2H), 2.32 (s, 3H), 2.09 (s, 2H), 1.82–1.74 (m,
H), 1.48 (s, 6H), 1.46–1.41 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ
165.1, 153.5, 149.5,
1
1
37.9, 137.0, 136.7, 134.8, 134.7, 130.1, 129.5, 129.3, 126.9, 125.4, 123.4, 119.8, 119.3, 117.4,
17.1, 115.4, 113.8, 111.5, 104.4, 102.8, 76.5, 59.8, 55.5, 52.1, 46.0, 31.8, 27.4, 18.8. HRESIMS
m/z = 536.29102 [M + H]+ (calculated for C H O N , 536.29007).
34
38
3
3
(
E)-N-(1-(2-Chlorobenzyl)piperidin-4-yl)-3-(8-methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-
◦
1
a]carbazol-5-yl)acrylamide (6ah). White solid, 68%; m.p.: 197–198 C. H-NMR (600 MHz,
DMSO-d6) 11.25 (s, 1H), 8.13 (s, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.74 (d, J = 15.8 Hz, 1H),
.58 (d, J = 2.5 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.36–7.25 (m, 3H),
.96 (dd, J = 8.7, 2.5 Hz, 1H), 6.90 (d, J = 9.8 Hz, 1H), 6.72 (d, J = 15.8 Hz, 1H), 5.85 (d,
δ
7
6
J = 9.8 Hz, 1H), 3.83 (s, 3H), 3.78–3.67 (m, 1H), 3.56 (s, 2H), 2.85–2.76 (m, 2H), 2.22–2.11 (m,
2
H), 1.84–1.76 (m, 2H), 1.54–1.43 (m, 8H). 13C-NMR (150 MHz, DMSO-d6)
δ
165.1, 153.5,
1
1
49.5, 137.9, 136.0, 134.8, 134.7, 133.3, 130.7, 129.3, 129.2, 128.5, 127.0, 123.4, 119.8, 119.2,
17.4, 117.1, 115.4, 113.8, 111.5, 104.4, 102.8, 76.5, 58.7, 55.5, 52.1, 45.9, 31.8, 27.4. HRESIMS
+
m/z = 556.23633 [M + H] (calculated for C H O N Cl, 556.23615).
33
35
3
3
(
E)-N-((1-Benzylpiperidin-4-yl)methyl)-3-(8-(tert-butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-
◦
1
a]carbazol-5-yl)acrylamide (6ba). White solid, 54%; m.p.: 163–164 C. H-NMR (400 MHz,
DMSO-d6) 11.30 (s, 1H), 8.16 (s, 1H), 8.01 (d, J = 1.8 Hz, 1H), 7.98 (d, J = 5.9 Hz, 1H), 7.74
d, J = 15.8 Hz, 1H), 7.40 (dd, J = 8.6, 1.8 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.33–7.26 (m, 4H),
δ
(
7
3
6
.26–7.21 (m, 1H), 6.91 (d, J = 9.8 Hz, 1H), 6.75 (d, J = 15.8 Hz, 1H), 5.86 (d, J = 9.8 Hz, 1H),
.45 (s, 2H), 3.12–3.06 (m, 2H), 2.86–2.77 (m, 2H), 1.91 (s, 2H), 1.69–1.59 (m, 2H), 1.48 (s,
H), 1.40–1.36 (m, 10H), 1.28–1.12 (m, 2H). 13C-NMR (100 MHz, DMSO-d6)
δ 165.9, 149.3,
1
1
41.8, 138.7, 138.4, 137.8, 134.4, 129.4, 128.8, 128.1, 126.9, 122.8, 122.6, 119.6, 118.6, 117.4,
17.4, 115.7, 115.4, 110.3, 104.4, 76.5, 62.4, 53.0, 44.3, 35.9, 34.4, 31.8, 29.7, 27.4. HRESIMS
+
m/z = 562.34210 [M + H] (calculated for C H O N , 562.34280).
37
44
2
3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-((1-(2-fluorobenzyl)
◦
1
piperidin-4-yl)methyl)acrylamide (6bb). White solid, 52%; m.p.: 142–143 C. H-NMR (400 MHz,
DMSO-d6) 11.32 (s, 1H), 8.16 (s, 1H), 8.05–7.94 (m, 2H), 7.74 (d, J = 15.7 Hz, 1H), 7.45–7.09
m, 6H), 6.92 (d, J = 9.8 Hz, 1H), 6.75 (d, J = 15.7 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.49 (s, 2H),
δ
(
3
(
(
.12–3.03 (m, 2H), 2.81 (d, J = 11.3 Hz, 2H), 1.94 (t, J = 11.3 Hz, 2H), 1.69–1.58 (m, 2H), 1.48
165.9, 160.7
d, JC-F = 244.1 Hz), 149.3, 141.8, 138.4, 137.8, 134.4, 131.4 (d, JC-F = 4.7 Hz), 129.4, 128.9(d,
s, 6H), 1.40–1.35 (m, 10H), 1.26–1.11 (m, 2H). 13C-NMR (100 MHz, DMSO-d6)
δ

Molecules 2021, 26, 1303
10 of 15
JC-F = 8.2 Hz), 125.0 (d, JC-F = 14.5 Hz), 124.1(d, JC-F = 3.4 Hz), 122.8, 122.6, 119.6, 118.6, 117.5,
1
2
17.3, 115.6, 115.4, 115.1 (d, JC-F = 22.0 Hz), 110.3, 104.4, 76.5, 54.9, 52.9, 44.2, 35.8, 34.4, 31.8,
+
9.7, 27.4. HRESIMS m/z = 580.33258 [M + H] (calculated for C H O N F, 580.233338).
37
43
2
3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-((1-(2-methylbenzyl)
◦
1
piperidin-4-yl)methyl)acrylamide (6bc). White solid, 54%; m.p.: 168–169 C. H-NMR (400 MHz,
DMSO-d6) 11.33 (s, 1H), 8.16 (s, 1H), 8.05–7.96 (m, 2H), 7.74 (d, J = 15.8 Hz, 1H), 7.46–7.32
m, 2H), 7.27–7.07 (m, 4H), 6.92 (d, J = 9.8 Hz, 1H), 6.75 (d, J = 15.8 Hz, 1H), 5.85 (d,
δ
(
J = 9.8 Hz, 1H), 3.38 (s, 2H), 3.13–3.05 (m, 2H), 2.79 (d, J = 11.4 Hz, 2H), 2.30 (s, 3H), 1.93 (t,
J = 11.4 Hz, 2H), 1.70–1.59 (m, 2H), 1.48 (s, 6H), 1.48–1.41 (m, 1H), 1.38 (s, 9H), 1.25–1.06
(
1
1
m, 2H). 13C-NMR (151 MHz, DMSO)
δ
165.9, 153.5, 149.5, 137.9, 136.9, 136.7, 134.8, 134.6,
30.0, 129.4, 129.3, 126.7, 125.3, 123.4, 119.6, 119.2, 117.4, 117.1, 115.3, 113.8, 111.5, 104.4,
02.8, 76.5, 60.5, 55.5, 53.2, 44.3, 36.0, 29.9, 27.4, 18.8. HRESIMS m/z = 576.35785 [M + H]+
(
calculated for C H O N , 576.35785).
38 46 2 3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-((1-(2-chlorobenzyl)
◦
1
piperidin-4-yl)methyl)acrylamide (6bd). White solid, 66%; m.p.: 175–176 C. H-NMR (400 MHz,
DMSO-d6) 11.31 (s, 1H), 8.16 (s, 1H), 8.05–7.95 (m, 2H), 7.74 (d, J = 15.7 Hz, 1H), 7.51–7.23
m, 6H), 6.92 (d, J = 9.8 Hz, 1H), 6.75 (d, J = 15.7 Hz, 1H), 5.86 (d, J = 9.8 Hz, 1H), 3.52 (s,
δ
(
2
H), 3.13–3.04 (m, 2H), 2.83 (d, J = 11.3 Hz, 2H), 2.01 (t, J = 11.3 Hz, 2H), 1.71–1.62 (m, 2H),
1
1
1
.48 (s, 6H), 1.40–1.35 (m, 10H), 1.26–1.15 (m, 2H). 13C-NMR (101 MHz, DMSO)
δ
165.9,
49.3, 141.8, 138.4, 137.8, 136.1, 134.4, 133.2, 130.6, 129.4, 129.2, 128.4, 127.0, 122.8, 122.6,
19.6, 118.6, 117.4, 117.4, 115.7, 115.4, 110.3, 104.4, 76.5, 58.9, 53.1, 44.3, 35.9, 34.4, 31.8, 29.8,
+
2
7.4. HRESIMS m/z = 596.30298 [M + H] (calculated for C H O N Cl, 596.30383).
37
43
2
3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-((1-(3-chlorobenzyl)
◦
1
piperidin-4-yl)methyl)acrylamide (6be). White solid, 59%; m.p.: 171–172 C. H-NMR (400 MHz,
DMSO-d6) 11.30 (s, 1H), 8.16 (s, 1H), 8.07–7.93 (m, 2H), 7.74 (d, J = 15.7 Hz, 1H), 7.40 (dd,
δ
J = 8.5, 1.9 Hz, 1H), 7.37–7.32 (m, 3H), 7.32–7.26 (m, 2H), 6.91 (d, J = 9.8 Hz, 1H), 6.75 (d,
J = 15.7 Hz, 1H), 5.86 (d, J = 9.8 Hz, 1H), 3.46 (s, 2H), 3.14–3.05 (m, 2H), 2.83–2.74 (m, 2H),
1
3
1
.97–1.85 (m, 2H), 1.69–1.61 (m, 2H), 1.48 (s, 6H), 1.38 (s, 10H), 1.25–1.16 (m, 2H). C-NMR
(
100 MHz, DMSO-d6) 165.9, 149.3, 141.8, 138.4, 137.8, 136.1, 134.4, 132.9, 130.0, 129.4,
δ
1
5
28.2, 127.3, 126.8, 122.8, 122.6, 119.6, 118.6, 117.4, 117.4, 115.7, 115.4, 110.3, 104.4, 76.5, 61.5,
3.0, 44.3, 35.9, 34.4, 31.8, 29.8, 27.4. HRESIMS m/z = 596.30365 [M + H] (calculated for
+
C H O N Cl, 596.30383).
37
43
2
3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-((1-(4-chlorobenzyl)
◦
1
piperidin-4-yl)methyl)acrylamide (6bf). White solid, 62%; m.p.: 158–159 C. H-NMR (400 MHz,
DMSO-d6) 11.30 (s, 1H), 8.16 (s, 1H), 8.01 (d, J = 1.8 Hz, 1H), 7.98 (d, J = 5.9 Hz, 1H), 7.74
d, J = 15.8 Hz, 1H), 7.40 (dd, J = 8.5, 1.9 Hz, 1H), 7.40–7.32 (m, 3H), 7.31 (d, J = 8.5 Hz,
δ
(
2H), 6.92 (d, J = 9.8 Hz, 1H), 6.75 (d, J = 15.8 Hz, 1H), 5.86 (d, J = 9.8 Hz, 1H), 3.42 (s, 2H),
3
.13–3.04 (m, 2H), 2.82–2.72 (m, 2H), 1.96–1.84 (m, 2H), 1.68–1.58 (m, 2H), 1.48 (s, 6H),
1
1
1
.38 (s, 9H), 1.26–1.11 (m, 3H). 13C-NMR (100 MHz, DMSO-d6)
δ 165.9, 149.3, 141.8, 138.4,
37.8, 134.4, 131.2, 130.4, 129.4, 128.1, 122.8, 122.6, 119.6, 118.6, 117.4, 117.4, 115.7, 115.4,
10.3, 104.4, 76.5, 61.5, 52.9, 48.6, 44.3, 35.9, 34.4, 31.8, 29.8, 27.4. HRESIMS m/z = 596.30365
+
[
M + H] (calculated for C H O N Cl, 596.30383).
37
43
2
3
(
E)-N-((1-Benzylpiperidin-4-yl)methyl)-3-(8-methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-
◦
1
a]carbazol-5-yl)acrylamide (6bg). White solid, 64%; m.p.: 182–183 C. H-NMR (600 MHz,
DMSO-d6) 11.31 (s, 1H), 8.24 (s, 1H), 8.14 (s, 1H), 7.75 (d, J = 15.8 Hz, 1H), 7.58 (d,
J = 2.5 Hz, 1H), 7.38–7.22 (m, 6H), 6.96 (dd, J = 8.6, 2.5 Hz, 1H), 6.91 (d, J = 9.8 Hz, 1H),
δ
6
.75 (d, J = 15.8 Hz, 1H), 5.84 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 3.58 (s, 2H), 3.13–3.07 (m,
2
(
H), 2.93–2.84 (m, 2H), 2.14–2.05 (m, 2H), 1.72–1.62 (m, 2H), 1.51–1.44 (m, 7H), 1.30–1.20
m, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ
165.9, 153.5, 149.5, 138.0, 137.0, 134.9, 134.7,
1
7
29.2, 128.2, 127.3, 123.4, 123.4, 119.5, 119.2, 117.4, 117.2, 115.3, 113.8, 111.5, 104.4, 102.8,
6.5, 61.7, 55.5, 52.5, 44.0, 35.5, 29.1, 27.4. HRESIMS m/z = 536.29095 [M + H] (calculated
+
for C H O N , 536.29077).
34
38
3
3
(
E)-N-((1-(2-Fluorobenzyl)piperidin-4-yl)methyl)-3-(8-methoxy-3,3-dimethyl-3,11-dihydropyrano
◦
1
[
3,2-a]carbazol-5-yl)acrylamide (6bh). White solid, 56%; m.p.: 162–163 C. H-NMR (600 MHz,

Molecules 2021, 26, 1303
11 of 15
DMSO-d6)
δ
11.25 (s, 1H), 8.13 (s, 1H), 8.01 (d, J = 6.1 Hz, 1H), 7.73 (d, J = 15.8 Hz, 1H),
7
7
.58 (d, J = 2.5 Hz, 1H), 7.42–7.36 (m, 1H), 7.33 (d, J = 8.6 Hz, 1H), 7.33–7.26 (m, 1H),
.19–7.11 (m, 2H), 6.95 (dd, J = 8.6, 2.5 Hz, 1H), 6.90 (d, J = 9.8 Hz, 1H), 6.73 (d, J = 15.8 Hz,
1
2
H), 5.85 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 3.48 (s, 2H), 3.12–3.04 (m, 2H), 2.84–2.75 (m,
1
3
H), 2.00–1.89 (m, 2H), 1.67–1.59 (m, 2H), 1.50–1.45 (m, 7H), 1.24–1.13 (m, 2H). C-NMR
165.9, 160.8(d, JC-F = 244.1 Hz), 153.5, 149.5, 137.9, 134.8, 134.6,
31.4 (d, JC-F = 4.6 Hz), 129.3, 128.9 (d, JC-F = 8.3 Hz), 125.0 (d, JC-F = 14.6 Hz), 124.1 (d,
(150 MHz, DMSO-d6) δ
1
JC-F = 3.2 Hz), 123.4, 119.6, 119.2, 117.4, 117.1, 115.3, 115.1 (d, JC-F = 22.1 Hz), 113.8, 111.5,
04.4, 102.8, 76.5, 59.8, 55.5, 52.8, 44.3, 35.8, 29.7, 27.4. HRESIMS m/z = 554.28119 [M + H]+
calculated for C H O N F, 554.28135).
1
(
34
37
3
3
(
E)-3-(8-Methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-((1-(2-methylbenzyl)
◦
1
piperidin-4-yl)methyl)acrylamide (6bi). White solid, 61%; m.p.: 181–182 C. H-NMR (600 MHz,
DMSO-d6) 8.14 (s, 1H), 8.02 (t, J = 5.8 Hz, 1H), 7.75 (d, J = 15.8 Hz, 1H), 7.58 (d, J = 2.4 Hz,
H), 7.34 (d, J = 8.6 Hz, 1H), 7.22–7.18 (m, 1H), 7.16–7.07 (m, 3H), 6.96 (dd, J = 8.6, 2.4 Hz,
H), 6.90 (d, J = 9.8 Hz, 1H), 6.74 (d, J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H),
.37 (s, 2H), 3.12–3.06 (m, 2H), 2.82–2.71 (m, 2H), 2.29 (s, 3H), 1.97–1.88 (m, 2H), 1.68–1.60
m, 2H), 1.52–1.42 (m, 7H), 1.22–1.10 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
165.9, 153.5,
δ
1
1
3
(
δ
1
1
49.5, 137.9, 136.9, 136.7, 134.8, 134.6, 130.0, 129.4, 129.3, 126.7, 125.3, 123.4, 119.6, 119.2,
17.4, 117.1, 115.3, 113.8, 111.5, 104.4, 102.8, 76.5, 60.5, 55.5, 53.2, 44.3, 36.0, 29.9, 27.4, 18.8.
+
HRESIMS m/z = 550.30609 [M + H] (calculated for C H O N , 550.30642).
35
40
3
3
(
E)-N-((1-(2-Chlorobenzyl)piperidin-4-yl)methyl)-3-(8-methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-
◦
1
a]carbazol-5-yl)acrylamide (6bj). White solid, 63%; m.p.: 165–166 C. H-NMR (600 MHz,
DMSO-d6) 8.13 (s, 1H), 8.03 (d, J = 6.0 Hz, 1H), 7.73 (d, J = 15.8 Hz, 1H), 7.58 (d, J = 2.5 Hz,
H), 7.52–7.23 (m, 5H), 6.95 (dd, J = 8.7, 2.5 Hz, 1H), 6.90 (d, J = 9.8 Hz, 1H), 6.73 (d,
J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 3.53 (s, 2H), 3.13–3.05 (m, 2H),
δ
1
2
.87–2.78 (m, 2H), 2.05–1.96 (m, 2H), 1.69–1.62 (m, 2H), 1.51–1.43 (m, 7H), 1.26–1.15 (m, 2H).
1
3
C-NMR (150 MHz, DMSO-d6)
δ 165.8, 153.5, 149.5, 137.9, 136.0, 134.8, 134.6, 133.2, 130.7,
1
7
29.3, 129.2, 128.5, 127.0, 123.4, 119.6, 119.2, 117.4, 117.1, 115.3, 113.8, 111.5, 104.4, 102.8,
6.5, 58.9, 55.5, 53.1, 44.1, 35.8, 29.8, 27.4. HRESIMS m/z = 570.25189 [M + H] (calculated
+
for C H O N Cl, 570.25180).
34
37
3
3
(
E)-N-(2-(1-Benzylpiperidin-4-yl)ethyl)-3-(8-(tert-butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-
◦
a]carbazol-5-yl)acrylamide (6ca). White solid, 68%. White solid, 65%; m.p.:144–145 C.
H-NMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H), 8.17 (s, 1H), 8.01 (d, J = 1.8 Hz, 1H), 7.97 (t,
1
J = 5.6 Hz, 1H), 7.74 (d, J = 15.8 Hz, 1H), 7.40 (dd, J = 8.5, 1.9 Hz, 1H), 7.37–7.31 (m, 5H),
7
.30–7.26 (m, 1H), 6.93 (d, J = 9.8 Hz, 1H), 6.71 (d, J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H),
3
.60 (s, 2H), 3.27–3.14 (m, 2H), 2.89 (s, 2H), 2.26–1.98 (m, 2H), 1.75–1.64 (m, 2H), 1.48 (s, 6H),
1
1
1
.45–1.40 (m, 2H), 1.38 (s, 9H), 1.29–1.13 (m, 3H). 13C-NMR (100 MHz, DMSO-d6)
δ 165.7,
49.3, 143.9, 141.7, 138.4, 137.8, 137.7, 134.4, 129.3, 128.9, 128.2, 122.8, 122.6, 119.6, 118.7,
17.5, 117.3, 115.7, 115.3, 110.3, 104.4, 76.5, 59.8, 52.8, 36.1, 35.9, 34.4, 32.3, 31.8, 31.2, 27.4.
+
HRESIMS m/z = 576.35858 [M + H] (calculated for C H O N , 576.35845).
38
46
2
3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-(2-(1-(2-fluorobenzyl)
◦
1
piperidin-4-yl)ethyl)acrylamide (6cb). White solid, 57%; m.p.: 122–123 C. H-NMR (400 MHz,
DMSO-d6) 11.35 (s, 1H), 8.17 (s, 1H), 8.01 (s, 1H), 7.96 (t, J = 5.7 Hz, 1H), 7.73 (d, J = 15.8 Hz,
H), 7.40 (dd, J = 8.4, 1.8 Hz, 1H), 7.38–7.26 (m, 3H), 7.19–7.11 (m, 2H), 6.93 (d, J = 9.8 Hz,
H), 6.71 (d, J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.49 (s, 2H), 3.27–3.15 (m, 2H),
.83–2.76 (m, 2H), 2.02–1.88 (m, 2H), 1.71–1.59 (m, 2H), 1.48 (s, 6H), 1.45–1.39 (m, 2H), 1.38
δ
1
1
2
13
(
s, 10H), 1.21–1.08 (m, 2H). C-NMR (100 MHz, DMSO-d )
δ
165.7, 160.8 (d, JC-F = 244.5
6
Hz), 149.3, 141.8, 138.4, 137.8, 134.3, 131.5 (d, JC-F = 5.0 Hz), 129.3, 128.9 (d, JC-F = 8.3 Hz),
124.9 (d, JC-F = 14.6 Hz), 124.1 (d, JC-F = 3.2 Hz), 122.8, 122.6, 119.6, 118.7, 117.5, 117.4, 115.7,
1
2
15.3, 115.1 (d, JC-F = 22.0 Hz), 110.3, 104.4, 76.5, 54.9, 53.1, 36.2, 36.0, 35.0, 34.4, 32.7, 31.8,
+
7.4. HRESIMS m/z = 594.34808 [M + H] (calculated for C H O N F, 594.34903).
38
45
2
3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-(2-(1-(2-methylbenzyl)
◦
1
piperidin-4-yl)ethyl)acrylamide (6cc). White solid, 48%; m.p.: 158–159 C. H-NMR (400 MHz,
DMSO-d6) 11.34 (s, 1H), 8.17 (s, 1H), 8.02 (d, J = 1.8 Hz, 1H), 7.96 (t, J = 5.6 Hz, 1H), 7.75
δ

Molecules 2021, 26, 1303
12 of 15
(d, J = 15.7 Hz, 1H), 7.40 (dd, J = 8.6, 1.8 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.22–7.16 (m, 1H),
7
.14–7.08 (m, 3H), 6.93 (d, J = 9.8 Hz, 1H), 6.72 (d, J = 15.7 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H),
3
.36 (s, 2H), 3.26–3.17 (m, 2H), 2.81–2.71 (m, 2H), 2.29 (s, 3H), 1.96–1.86 (m, 2H), 1.69–1.60
1
3
(
(
m, 2H), 1.48 (s, 6H), 1.45–1.39 (m, 2H), 1.39–1.37 (m, 10H), 1.19–1.05 (m, 2H). C-NMR
100 MHz, DMSO-d6) 165.7, 149.3, 141.8, 138.4, 137.8, 136.9, 136.8, 134.4, 130.0, 129.4,
29.3, 126.7, 125.3, 122.8, 122.6, 119.6, 118.7, 117.5, 117.4, 115.7, 115.4, 110.3, 104.4, 76.5,
δ
1
6
0.6, 53.5, 36.3, 36.1, 34.4, 32.9, 32.0, 31.8, 27.4, 18.8. HRESIMS m/z = 590.37402 [M + H]+
(
calculated for C H O N , 590.37410).
39 48 2 3
(
E)-3-(8-(tert-Butyl)-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-(2-(1-(2-chlorobenzyl)
◦
1
piperidin-4-yl)ethyl)acrylamide (6cd). White solid, 56%; m.p.: 160–161 C. H-NMR (400 MHz,
DMSO-d6) 11.31 (s, 1H), 8.17 (s, 1H), 8.02 (d, J = 1.8 Hz, 1H), 7.96 (t, J = 5.6 Hz, 1H), 7.75
d, J = 15.8 Hz, 1H), 7.49–7.23 (m, 6H), 6.92 (d, J = 9.8 Hz, 1H), 6.72 (d, J = 15.8 Hz, 1H),
δ
(
5
.85 (d, J = 9.8 Hz, 1H), 3.51 (s, 2H), 3.26–3.17 (m, 2H), 2.86–2.71 (m, 2H), 2.08–1.92 (m, 2H),
1
3
1.72–1.58 (m, 2H), 1.48 (s, 6H), 1.45–1.40 (m, 2H), 1.38 (s, 10H), 1.27–1.05 (m, 2H). C-NMR
(
100 MHz, DMSO-d6) 165.7, 149.3, 141.8, 138.4, 137.8, 136.1, 134.4, 133.2, 130.7, 129.3,
δ
1
5
29.2, 128.4, 126.9, 122.8, 122.6, 119.6, 118.7, 117.5, 117.4, 115.7, 115.4, 110.3, 104.4, 76.4, 59.0,
3.4, 36.9, 36.2, 34.9, 34.4, 32.7, 31.8, 27.4. HRESIMS m/z = 610.31873 [M + H] (calculated
+
for C H O N Cl, 610.31948).
38
45
2
3
(
E)-N-(2-(1-Benzylpiperidin-4-yl)ethyl)-3-(8-methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-
◦
1
a]carbazol-5-yl)acrylamide (6ce). White solid, 47%; m.p.:152–153 C. H-NMR (600 MHz,
DMSO-d6) 8.14 (s, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.73 (d, J = 15.8 Hz, 1H), 7.59 (d, J = 2.5 Hz,
H), 7.38–7.18 (m, 6H), 6.96 (dd, J = 8.7, 2.5 Hz, 1H), 6.90 (d, J = 9.8 Hz, 1H), 6.69 (d,
J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 3.44 (s, 2H), 3.25–3.16 (m, 2H), 2.86–
.73 (m, 2H), 1.98–1.84 (m, 2H), 1.71–1.58 (m, 2H), 1.48 (s, 6H), 1.44–1.37 (m, 2H), 1.36–1.25
m, 1H), 1.22–1.09 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
165.7, 153.5, 149.5, 138.1,
δ
1
2
(
δ
1
1
37.9, 134.8, 134.6, 129.3, 128.8, 128.1, 126.8, 123.4, 119.7, 119.2, 117.4, 117.1, 115.3, 113.8,
11.5, 104.4, 102.8, 76.5, 62.4, 55.5, 53.2, 36.3, 36.0, 32.8, 31.8, 27.4. HRESIMS m/z = 550.30670
+
[
M + H] (calculated for C H O N , 550.30642).
35
40
3
3
(
E)-N-(2-(1-(2-Fluorobenzyl)piperidin-4-yl)ethyl)-3-(8-methoxy-3,3-dimethyl-3,11-dihydropyrano
◦
1
[
3,2-a]carbazol-5-yl)acrylamide (6cf). White solid, 58%; m.p.: 141–142 C. H-NMR (400 MHz,
DMSO-d6) 11.26 (s, 1H), 8.13 (s, 1H), 7.96 (t, J = 5.6 Hz, 1H), 7.72 (d, J = 15.8 Hz, 1H), 7.58
d, J = 2.5 Hz, 1H), 7.41–7.36 (m, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.31–7.26 (m, 1H), 7.19–7.11
δ
(
(
(
(
2
m, 2H), 6.95 (dd, J = 8.7, 2.5 Hz, 1H), 6.90 (d, J = 9.9 Hz, 1H), 6.68 (d, J = 15.8 Hz, 1H), 5.85
d, J = 9.9 Hz, 1H), 3.83 (s, 3H), 3.48 (s, 2H), 3.24–3.15 (m, 2H), 2.84–2.72 (m, 2H), 1.98–1.88
m, 2H), 1.70–1.59 (m, 2H), 1.48 (s, 6H), 1.44–1.35 (m, 2H), 1.25–1.21 (m, 1H), 1.20–1.08 (m,
1
3
H). C-NMR (100 MHz, DMSO-d6)
δ 165.7, 160.8 (d, JC-F = 244.2 Hz), 153.5, 149.4, 138.0,
1
1
1
34.9, 134.5, 131.5 (d, JC-F = 4.5 Hz), 129.3, 128.9 (d, JC-F = 8.1 Hz), 124.9 (d, JC-F = 14.7 Hz),
24.1(d, JC-F = 3.5 Hz), 123.4, 119.7, 119.2, 117.4, 117.2, 115.3, 115.1(d, JC-F = 22.0 Hz), 113.8,
11.5, 104.4, 102.8, 76.5, 55.5, 54.9, 53.1, 36.2, 36.0, 32.7, 31.8, 27.4. HRESIMS m/z = 568.29767
+
[
M + H] (calculated for C H O N F, 568.29809).
35
39
3
3
(
E)-3-(8-Methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazol-5-yl)-N-(2-(1-(2-methylbenzyl)
◦
1
piperidin-4-yl)ethyl)acrylamide (6cg). White solid, 51%; m.p.: 161–162 C. H-NMR (600 MHz,
DMSO-d6) 8.14 (s, 1H), 7.98 (t, J = 5.7 Hz, 1H), 7.74 (d, J = 15.8 Hz, 1H), 7.59 (d, J = 2.5 Hz,
H), 7.34 (d, J = 8.6 Hz, 1H), 7.22–7.07 (m, 4H), 6.96 (dd, J = 8.6, 2.5 Hz, 1H), 6.90 (d,
J = 9.8 Hz, 1H), 6.70 (d, J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 3.37 (s, 2H),
.26–3.18 (m, 2H), 2.80–2.71 (m, 2H), 2.29 (s, 3H), 1.95–1.85 (m, 2H), 1.68–1.60 (m, 2H),
.48 (s, 6H), 1.42–1.37 (m, 2H), 1.35–1.27 (m, 1H), 1.20–1.06 (m, 2H). 13C-NMR (150 MHz,
DMSO-d6) 165.7, 153.5, 149.5, 137.9, 136.9, 136.7, 134.8, 134.6, 130.0, 129.4, 129.3, 126.7,
25.3, 123.4, 119.7, 119.2, 117.4, 117.1, 115.3, 113.8, 111.5, 104.4, 102.8, 76.5, 60.5, 55.5,
δ
1
3
1
δ
1
5
+
3.5, 36.3, 36.0, 33.0, 32.0, 27.4, 18.8. HRESIMS m/z = 564.32147 [M + H] (calculated for
C H O N ,564.32207).
36
42
3
3
(
E)-N-(2-(1-(2-Chlorobenzyl)piperidin-4-yl)ethyl)-3-(8-methoxy-3,3-dimethyl-3,11-dihydropyrano
◦
1
[
3,2-a]carbazol-5-yl)acrylamide (6ch). White solid, 57%; m.p.: 145–146 C. H-NMR (400 MHz,
DMSO-d6) 11.24 (s, 1H), 8.13 (s, 1H), 7.97 (t, J = 5.6 Hz, 1H), 7.72 (d, J = 15.8 Hz, 1H), 7.59
δ

Molecules 2021, 26, 1303
13 of 15
(
(
d, J = 2.5 Hz, 1H), 7.51–7.38 (m, 2H), 7.36–7.23 (m, 3H), 6.95 (dd, J = 8.7, 2.5 Hz, 1H), 6.90
d, J = 9.8 Hz, 1H), 6.69 (d, J = 15.8 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 3.53 (s,
2
1
H), 3.27–3.17 (m, 2H), 2.91–2.74 (m, 2H), 2.07–1.93 (m, 2H), 1.72–1.62 (m, 2H), 1.48 (s, 6H),
.45–1.37 (m, 2H), 1.38–1.26 (m, 1H), 1.23–1.11 (m, 2H). 13C-NMR (100 MHz, DMSO-d6)
165.7, 153.5, 149.4, 138.0, 136.1, 134.9, 134.5, 133.2, 130.7, 129.3, 129.2, 128.5, 127.0, 123.4,
δ
1
3
19.7, 119.2, 117.4, 117.2, 115.3, 113.8, 111.5, 104.4, 102.8, 76.5, 59.0, 55.5, 53.4, 36.2, 36.0, 32.7,
+
1.9, 27.4. HRESIMS m/z = 584.26843 [M + H] (calculated for C H O N Cl, 584.26745).
35
39
3
3
3.2. Biological Evaluation
3.2.1. Anti-Cholinesterase Activity Assays
The activity of AChE was tested using an acetylcholinesterase activity detection kit
A024-1-1, Jian Cheng, Nanjing). AChE is a kind of serine hydrolase, which exists widely
(
in the tissue and serum of animals. AChE acts as a key role in nervous conduction. It can
hydrolyze acetyl choline into acetyl coenzyme A and choline. The activity of BuChE was
tested using a butyrylcholinesterase activity detection kit (A025-1-1, Jian Cheng, Nanjing).
It can hydrolyze butyryl choline into acetic acid and choline. Choline can interact with
disulfide p-nitrobenzoic acid (DTNB) to produce 5-mercapto-nitrobenzoic acid (TNB),
which has an absorption peak at 412 nm. Thus, we could analyze the increasing rate of
412 nm absorption to calculate the activity of AChE and BuChE.
The specific analysis method was as follows: a standard solution of AChE (Sigma-
Aldrich) was prepared in 50 mM Tris-HCl, pH = 8.0, 0.1% w/v, bovine serum albumin
BSA) into 0.22 U/mL, and a standard solution of BuChE (Sigma-Aldrich) was prepared
in 50 mM Tris-HCl, pH = 8.0, 0.1% w/v, BSA into 0.12 U/mL. Next, 50 L of the above
standard solution and 10 L of different concentrations of drug solution (0, 20 M, 40 M,
M, 80 M, and 100 M) were used to test the change in activity. The procedure was
processed as described by the kit instructions.
(
µ
µ
µ
µ
6
0
µ
µ
µ
3.2.2. Primary Cortical Neuron Culture and Treatment
Rat cortical neurons were prepared from newborn Sprague-Dawley (SD) rats. All
cell suspensions were grown in neurobasal/B27 medium with 100-U/mL penicillin and
1
00-
µ
g/mL streptomycin. The cells were plated in poly-L-lysine (PLL, 0.1 mg/mL)-coated
◦
multiwell plates or chamber slides and maintained at 37 C in a humidified atmosphere of
5
% CO and 95% air. Seven day cultures were used for treatment. The treatment methods
2
were similar to those used for PC12, but the concentrations of H O and sodium dithionite
2
2
were 100 µM and 2 mM, respectively.
3.2.3. Cell Viability
Cell viability was determined by MTT assay. Cells were incubated with 5 mg/mL
◦
MTT (10
µ
L) at 37 C for 4 h. The formazan crystals were dissolved, and absorbance was
determined at 570 nm using a Microplate Reader (Thermo, Germany). The cell viability
was expressed as a percentage of the optical density (OD) value of the control cultures.
3.2.4. Parallel Artificial Membrane Permeability Assay
The compounds’ blood–brain barrier penetration was evaluated by using the parallel
artificial membrane permeation assay (PAMPA) described by Di et al. [20]. The two
commercial drugs were purchased from Aladdin (China). Porcine brain lipid was obtained
from Sigma (China). The donor microplate (polyvinylidene fluoride (PVDF) membrane,
pore size 0.45 µm) and acceptor microplate were both from Millipore (China). The 96-well
ultraviolet (UV) plate (COSTAR) was from Corning Incorporated (USA). The detailed
procedure was described in [26,27].
3.2.5. Free-Radical Scavenging
ESR signals were recorded with a 10 mW incident microwave and 10,000 MHz
field modulation of 1 G. The scan width was 150 G for 2, 2, 6, 6-tetramethylpiperidine

Molecules 2021, 26, 1303
14 of 15
(
5
TEMP) experiments. All measurements were performed at room temperature (RT). Briefly,
× 10 mol/L TEMP and 10 mol/L 6bd or Eda were mixed in DMSO, and ESR signals
−
2
−2
were obtained after exposure to UVA light for 1 min.
4. Conclusions
In our previous investigations, we identified several carbazole alkaloids with neuro-
protective effects, such as Clau F and CZ-7. In this study, a series of Claulansine F–donepezil
hybrids were designed and synthesized as multitarget drugs and their cholinesterase in-
hibitory activities were evaluated. On this basis, we discussed the SAR. Among the
tested compounds, the compounds with R= t-Bu exhibited the better potency against
cholinesterase. Compound 6bd showed the strongest neuroprotective effects in vitro. Fur-
thermore, 6bd could cross the blood–brain barrier in vitro. Most importantly, 6bd showed
stronger free-radical scavenging capacity than Eda. Therefore, 6bd may be valuable for
intervention against Alzheimer’s disease. We are interested in further investigating the
mechanisms underlying the inhibition of Alzheimer’s disease by 6bd.
Supplementary Materials: The following are available online.
Author Contributions: D.Z. and X.W. designed the experiments and revised the paper; Y.Z., K.L.,
W.W., C.L., J.M., J.Y., and X.C. performed the experiments, analyzed the data, and wrote the paper.
All authors read and approved the final manuscript.
Funding: This work was supported financially by the National Natural Science Foundation of China
(No. 81730093), the National Key R&D Program of China (2019YFC1711000), the Drug Innovation
Major Project (No. 2018ZX09711001-002-010, 2018ZX09735006), and the CAMS Innovation Fund for
Medical Sciences (No. 2017-I2M-3-010).
Institutional Review Board Statement: “Not applicable” for studies not involving humans or animals.
Informed Consent Statement: “Not applicable” for studies not involving humans.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
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