Total synthesis of pulmonarin B and design of brominated phenylacetic acid/tacrine hybrids: Marine pharmacophore inspired discovery of new CHE and Aβ aggregation inhibitors

Article abstract of DOI:10.3390/md16090293

A marine natural product, pulmonarin B (1), and a series of related tacrine hybrid analogues were synthesized and evaluated as cholinesterase (ChE) inhibitors. The in vitro ChE assay results revealed that 1 showed moderate dual acetylcholinesterase (AChE)/ butyrylcholinesterase (BChE) inhibitory activity, while the hybrid 12j proved to be the most potent dual inhibitor among the designed derivatives, being almost as active as tacrine. Molecular modeling studies together with kinetic analysis suggested that 12j interacted with both the catalytic active site and peripheral anionic site of AChE. Compounds 1 and 12j could also inhibit self-induced and AChE-induced Aβ aggregation. In addition, the cell-based assay against the human hepatoma cell line (HepG2) revealed that 1 and 12j did not show significant hepatotoxicity compared with tacrine and donepezil. Taken together, the present study confirmed that compound 1 was a potential anti-Alzheimer’s disease (AD) hit, and 12j could be highlighted as a multifunctional lead compound for anti-AD drug development.

Full text of DOI:10.3390/md16090293

marine drugs
Article
Total Synthesis of Pulmonarin B and Design of
Brominated Phenylacetic Acid/Tacrine Hybrids:
Marine Pharmacophore Inspired Discovery of New
ChE and Aβ Aggregation Inhibitors
Zhi-Qiang Cheng 1, Jia-Li Song 1, Kongkai Zhu 1, Juan Zhang 2, Cheng-Shi Jiang 1,
*
ID
and Hua Zhang 1,
*
ID
1
School of Biological Science and Technology, University of Jinan, Jinan 250022, China;
czq13515312897@163.com (Z.-Q.C.); 13285419800@163.com (J.-L.S.); hkhhh.k@163.com (K.Z.)
School of Biological Sciences, University of Brasília, Brasília 72220-275, Brazil; zjandzq@163.com
Correspondence: jiangchengshi-20@163.com (C.-S.J.); bio_zhangh@ujn.edu.cn (H.Z.);
Tel.: +86-531-8973-6199 (H.Z.)
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 14 July 2018; Accepted: 18 August 2018; Published: 21 August 2018
Abstract: A marine natural product, pulmonarin B (
were synthesized and evaluated as cholinesterase (ChE) inhibitors. The in vitro ChE assay results
revealed that showed moderate dual acetylcholinesterase (AChE)/ butyrylcholinesterase (BChE)
1), and a series of related tacrine hybrid analogues
1
inhibitory activity, while the hybrid 12j proved to be the most potent dual inhibitor among the
designed derivatives, being almost as active as tacrine. Molecular modeling studies together with
kinetic analysis suggested that 12j interacted with both the catalytic active site and peripheral
anionic site of AChE. Compounds
aggregation. In addition, the cell-based assay against the human hepatoma cell line (HepG2) revealed
that and 12j did not show significant hepatotoxicity compared with tacrine and donepezil. Taken
together, the present study confirmed that compound was a potential anti-Alzheimer’s disease (AD)
1 and 12j could also inhibit self-induced and AChE-induced Aβ
1
1
hit, and 12j could be highlighted as a multifunctional lead compound for anti-AD drug development.
Keywords: pulmonarin B; brominated-phenylacetic acid/tacrine hybrids; acetylcholinesterase;
butylcholinesterase; anti-amyloid aggregation; Alzheimer’s disease
1. Introduction
Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that has become the third
leading death cause behind cancer and cardiovascular diseases. AD worsens over the time with
the loss of memory, decline in language skills, deficits in cognitive functions, and severe behavioral
problems [
AD patients were diagnosed worldwide [
with the aging of the global population [4].
Although the cause of AD is still poorly understood nowadays, many pathogenic hypotheses have
been proposed over the last three decades [ ]. Among them, cholinergic hypothesis is undoubtedly
the earliest approved, which believes that increasing the level of acetylcholine (ACh) to enhance
cholinergic neurotransmission in the brain is an efficacious approach for AD treatment [ ]. In the
brain, ACh can be hydrolyzed by two types of cholinesterase (ChE): acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) [ ]. AChE is the key enzyme for the termination of neurotransmission
in cholinergic pathways via its rapid hydrolysis of ACh, almost 103-fold more active than BChE [
].
Therefore, AChE inhibition is an effective approach for the symptomatic treatment for AD. As a result,
1
]. In 2015, about 1.9 million people died of AD [
2], and in 2017, an estimated 46.8 million
3]. More seriously, the number is expected to triple by 2050
5,6
7
8
9
Mar. Drugs 2018, 16, 293; doi:10.3390/md16090293
www.mdpi.com/journal/marinedrugs

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four of five anti-AD drugs approved by the Food and Drug Administration (FDA) are AChE inhibitors
including tacrine, donepezil, galantamine, and rivastigmine (Figure 1). These drugs are used to
improve the memorial and cognitive functions of AD patients; however, none of them can stop or
slow down the course of AD. Recent studies showed that the level and activity of BChE progressively
increased in AD patients, while the activity of AChE declined [10]. BChE was found to serve as a
backup to compensate for the function of AChE. Besides cholinergic hypothesis, amyloid hypothesis
has been brought out to develop several therapeutic strategies for AD treatment. Among the factors
involved in amyloid hypothesis, the formation and accumulation of beta-amyloid (Aβ) in the brain
is considered as a primary target for developing novel anti-AD drugs, because of its ability to
trigger critical intracellular signaling pathways associated with neurotoxicity, oxidative damage,
and inflammation [11
,12]. Due to the multifactorial nature of AD, multi-target directed bioactive
molecules are believed to simultaneously modulate different targets involved in the progression of
AD [13]. Thus, the dual inhibition of ChE and Aβ aggregation could be a promising strategy for the
treatment of AD.
Figure 1. Structures of approved acetylcholinesterase (AChE) inhibitors, pulmonarin B (1), and brominated
phenylacetic acid/tacrine hybrids.
Marine natural products (MNPs) have been proven to be an extremely important source for
developing novel drugs [14 15]. As a special group of MNPs, marine halogenated metabolites possess
a wide range of biological properties, such as antibacterial, antiviral, antitumor, anti-inflammatory,
and neurological activities [16 17]. During our continued project for developing new ChE
inhibitors [18–20], pulmonarin B ( , Figure 1), a brominated phenylacetic acid derivative isolated from
,
,
1
the ascidian Synoicum pulmonaria by Seveson et al. [21], came to our line of sight. This dibrominated
compound was reported to be an AChE inhibitor (IC50 = 36 µM), and was considered as a marine hit
for further studies [21]. It is important to note that the discovery of marine-derived AChE inhibitors
was very limited according to the latest statistics of MNPs from 1984 to 2018 [22]. Thus, we recently
prepared
its anti-ChE effect. The assay results revealed that
In addition, it showed inhibitory activity against self-induced and AChE-induced Aβ1–42 aggregation
and non-hepatotoxicity against HepG2 cells. However, the terminal quaternary ammonium group in
probably makes the compound’s polarity too high to pass the blood–brain barrier. To further increase
the bioactive profiles of , molecule hybridization was applied as a powerful strategy to assemble
bioactive compounds. Tacrine as a versatile pharmacophore for the design of potent ChE inhibitors
showed excellent dual inhibition on AChE/BChE with IC50 values at the nanomolar level [23 25].
Thus, a series of brominated phenylacetic acid/tacrine hybrids were designed by the fusion of tacrine
to the terminal quaternary amine of . In the present study, we describe the total synthesis of and its
1
using a synthetic approach that was different from that previously reported, and retested
1
was a moderate dual AChE/BChE inhibitor.
1
1
–
1
1
brominated phenylacetic acid/tacrine hybrids, as well as evaluate their anti-AD potential, including
AChE/BChE inhibition, molecular docking, anti-Aβ aggregation, and cytotoxicity.

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2. Results and Discussion
2.1. Chemistry
Given the particularly low yield of the key intermediate 3,5-(dibromo-4-methoxyphenyl)acetic
acid in the protocol reported by Seveson et al. and the high price of the starting material
5-(dimethylamino) amylamine [21], the total synthetic route of pulmonarin B (
successfully achieved with the agents in hand. As illustrated in Scheme 1, methyl 2-(4-methoxyphenyl)
acetate ( ) was prepared from the esterification of starting material 2-(4-methoxyphenyl) acetic acid
). Ester was dibrominated with NBS/FeCl3 in acetonitrile to give compound , which was then
hydrolyzed to yield acid . The yield from to is 43%, which was twofold higher than that (21%)
reported by Severson et al. Next, amide was prepared by a coupling reaction of with tert-butyl
(5-aminopentyl)carbamate. The deprotection of the N-Boc group of in trifluoroacetic acid directly
gave amine . Then, compound was generated by the reductive amination of . Finally, N-methylation
of with CH3I enabled the full synthesis of pulmonarin B ( ). The total yield of was increased to 3.5%,
which was over twofold what was reported in the literature (1.6%) [21]. Also, the present synthetic
1) was redesigned and
3
(2
3
4
5
2
5
6
5
6
7
8
7
8
1
1
route afforded more pulmonarin B analogues by modifying the terminal amino group of intermediate 7.
The 1H, 13C NMR and MS spectrascopic data of the synthetic
1 were in agreement with those of the
natural 1 [21].
Scheme 1. Synthesis of pulmonarin B (1). (a) Thionyl chloride, CH3OH, reflux; (b) N-bromosuccinimide
(NBS), FeCl3, CH3CN, room temperature (r.t.); (c) CH3OH, H2O, NaOH, reflux; (d) 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI), hydroxybenzotriazole (HOBT), Et3N, CH2Cl2, tert-butyl
(5-aminopentyl)carbamate, r.t., 8 h; (e) trifluoroacetic acid, CH2Cl2; (f) 37% formaldehyde solution,
NaCNBH3, CH3OH, HCl (aq); (g) CH3OH, CH3I, K2CO3.
The synthetic routes for two series of compounds 10a
–
10h and 12a
–
12l were shown in Scheme 2.
10h
11l afforded the
Briefly, intermediate was reacted with appropriate amines to give the corresponding hybrids 10a
4
–
(Table 1). Similarly, the coupling reaction of 9f with different phenylacetic acids 11a
corresponding 12a–12l (Table 2).
–
Scheme 2. Synthesis of 10a–10h and 12a–12l. (a) EDCI, HOBT, Et3N, CH2Cl2, r.t., 8 h.

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2.2. In Vitro Inhibition of AChE and BChE, and Structure–Activity Relationship (SAR) Analysis
At first, the in vitro AChE and BChE inhibitory activities of
1
were tested. From the results in
showed comparable AChE inhibitory activity (IC50 = 37.02 2.11 M) to the
M [21]. In addition, it was also found to be a BChE inhibitor with an IC50 value of
was much weaker than that of the positive
M; BChE, IC50 = 0.046 0.002 M). Aiming to improve the
, brominated phenylacetic acid/tacrine hybrids 10a 10h were prepared based on molecule
hybridization. Compared with , all of these hybrids showed more potent ChE inhibitory activity,
Table 1, compound
reported value of 36
1
µ
±
µ
30.70
control tacrine (AChE, IC50 = 0.159
activity of
±
1.44
µ
M. However, the ChE inhibitory activity of
1
±
0.007
µ
±
µ
1
–
1
and clearly this improvement could be attributed to the introduction of tacrine moiety. In both enzyme
bioassays, compound 10f with 7-carbon aliphatic spacer in the linker showed the best activity (AChE,
IC50 = 0.314
±
0.010
µ
M; BChE, IC50 = 0.053
±
0.007 µM). The preliminary SAR study indicated that
the ChE inhibitory activities of these compounds increased with the elongation of the diamine linker
(from 10a to 10f) and then decreased (from 10f to 10h), and the 7-carbon diamino linkage seemed to be
the optimal distance between the brominated phenylacetyl and 1,2,3,4-tetrahydroacridine moieties.
Table 1. In vitro inhibition of AChE and butyrylcholinesterase (BChE) presented as IC50 (µM) 1.
Compound
EeAChE 2
eqBChE 3
1
10a
10b
10c
10d
10e
10f
10g
10h
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
37.02 ± 2.11
0.880 ± 0.074
0.771 ± 0.027
1.045 ± 0.161
0.764 ± 0.016
0.686 ± 0.045
0.314 ± 0.010
0.427 ± 0.016
0.638 ± 0.026
0.324 ± 0.033
0.750 ± 0.054
0.607 ± 0.045
0.304 ± 0.004
0.724 ± 0.003
0.597 ± 0.028
0.696 ± 0.052
0.729 ± 0.021
0.748 ± 0.017
0.182 ± 0.006
0.383 ± 0.054
0.760 ± 0.092
0.159 ± 0.007
30.70 ± 1.44
0.230 ± 0.010
0.233 ± 0.013
0.195 ± 0.011
0.100 ± 0.014
0.054 ± 0.006
0.053 ± 0.007
0.325 ± 0.004
0.516 ± 0.037
0.140 ± 0.004
0.694 ± 0.003
0.152 ± 0.006
0.091 ± 0.017
0.149 ± 0.021
0.214 ± 0.004
0.062 ± 0.006
0.204 ± 0.011
0.049 ± 0.004
0.064 ± 0.006
0.104 ± 0.001
0.680 ± 0.054
0.046 ± 0.002
1
2
3
4
5
6
7
9
R1 = 2-Br, R2 = 4-OMe
R1 = H, R2 = 4-OMe
R1 = 3-Br, R2 = 4-OMe
R1 = 2-Br, R2 = H
R1 = 3-Br, R2 = H
R1 = 4-Br, R2 = H
R1 = 2-Br, R2 = 5-Br
R1 = 3-Br, R2 = 5-Br
R1 = 3-Br, R2 = 4-F
R1 = 5-Br, R2 = 2-F
R1 = 2-Br, R2 = 5-Cl
R1 = R2 = H
Tacrine
1
3
2
Results were expressed as mean
eqBChE: BChE from equine serum.
±
SD from three separate experiments. EeAChE: AChE from electric eel.
Next, the substituent effect of benzene was investigated, and compound 10f was selected as a model
compound for further structural modification. Consequently, 12 analogues (12a 12l) were prepared,
and their bioassay results were shown in Table 1. Among these analogues, compound 12j showed the
best ChE inhibitory activity (AChE, IC50 = 0.182 0.006 M; BChE, IC50 = 0.064 0.006 M), which was
–
±
µ
±
µ
almost equal to that of tacrine. From the bioassay results, it was clear that different substituents had
obvious impact on the AChE and BChE inhibitory activities. For example, compound 12j with 5-Br and
2-F showed increased inhibitory activity toward AChE by fourfold compared with 12a, and toward
BChE by fourfold over 12f. Compounds 12b and 12l without Br substitution showed low anti-ChE
activity in comparison with 12j.

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2.3. Kinetic Study of AChE and BChE Inhibition
Herein, compound 12j was selected as a representative prototype for kinetic assay to obtain
information on the mode of action and binding site of this series of analogues. The mechanisms of
AChE and BChE inhibitions were analyzed by recording substrate concentration enzyme velocity
−
curves in the presence of different concentrations of 12j.
Graphical analyses revealed both increasing slopes (decreasing Vmax) and increasing intercepts
(Kmax) at rising concentrations of 12j (Figures 2A and 3A). The equilibrium constant for binding with
the free enzyme (Ki) was obtained from the slope against inhibitor concentration [26]. The estimated
Ki value of 12j was 97.50 nM for AChE (Figure 2B), and that for BChE was 9.91 nM (Figure 3B). These
patterns revealed compound 12j as a linear mixed-type ChE inhibitor, indicating that 12j may not
only bind to the catalytic active site (CAS), but also interact with the peripheral anionic site (PAS) of
both enzymes.
Figure 2. Kinetic assay on AChE inhibition displayed by 12j. (A) Lineweaver–Burk reciprocal plots of
initial velocity and increasing substrate concentrations (0.078–0.625 mM); (B) Secondary plot of slopes
versus 12j concentrations.
Figure 3. Kinetic assay on BChE inhibition displayed by 12j. (A) Lineweaver–Burk reciprocal plots of
initial velocity and increasing substrate concentrations (0.078–0.625 mM); (B) Secondary plot of slopes
versus 12j concentrations.
2.4. Docking Study
As compound 12j showed the best inhibition toward AChE, it was selected for docking simulation.
First, the ligand alkylene-linked bis-tacrine that was derived from the original X-ray structure of AChE
(PDB ID: 5EI5) was redocked to the protein to validate the molecular modeling methodology, as shown
in Figure S1 (Supporting Information). The docking result could well reproduce the crystal structure,
which indicated that the method was suitable for a docking study of AChE. Since the redocking of the
ligand tacrine that originated from BChE (PDB ID: 4BDS) to its protein could not well reproduce the
crystal structure, the binding mode of 12j with BChE was not discussed in this study. As shown in
Figure 4, 12j was obviously able to bind to the active pocket of AChE. Here, AChE (PDB: 5EI5) [27
was used in the docking analysis because of its high resolution. The docking score for 12j/AChE was
17.50. The tacrine moiety of 12j could be spatially located in CAS (Figure 4A) and showed stacking
]
−

Mar. Drugs 2018, 16, 293
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interactions with Trp84 and Phe330 (Figure 4B). Furthermore, the protonated nitrogen atom in the
tacrine ring underwent cation interaction with residues Phe330 and Trp84, and also formed an H-bond
with the key residue His440. The brominated benzene moiety was oriented in the PAS by forming
stacking interactions with Try70, Try121, and Trp279. The H atom of the amide group serves as an
H-bond donor for interacting with Tyr121. In addition, hydrophobic interactions between 12j and key
residues also contributed to the affinity of 12j to AChE.
Figure 4.
(A) The proposed orientation of 12j (carbon in green) in the active pocket of AChE;
(B) The interactions of 12j with key amino acid residues in the active site of AChE.
2.5. Inhibition of Self-Induced and AChE-Induced Aβ Aggregation
Studies showed that ChE inhibitors not only increased the levels of ACh in the brain, they also
reduced and prevented the formation of Aβ aggregation [28]. Compounds 1 and 12j were assessed
for their ability to inhibit self-induced and AChE-induced Aβ1–42 aggregation using the thioflavin
T (ThT) fluorescence method. Tacrine and donepezil were used as positive reference compounds,
and galantamine was included as a negative control. The results are shown in Table 2. The inhibitions
of compound
32.37 0.62%, respectively, which were higher than that of donepezil (17.95
compounds 1 and 12j also exhibited a significant inhibitory effect on AChE-induced Aβ aggregation,
with inhibition ratios of 27.60 1.96% and 47.73 4.35%, respectively. As for galantamine,
no significant inhibition was observed in self-induced and AChE-induced Aβ1–42 aggregation assay.
1
and 12j at 10
µ
M against self-induced Aβ1–42 aggregation were 29.78
±
1.45% and
±
±
0.77%). In addition,
±
±
Table 2. Inhibition of self-induced and AChE-induced Aβ aggregation by compounds 1 and 12j in
comparison with tacrine, donepezil, and galantamine.
Inhibition of Aβ Aggregation (%)
Compounds
Self-Induced 1
AChE-Induced 2
1
12j
29.78 ± 1.45
32.37 ± 0.62
NT 3
27.60 ± 1.96
47.73 ± 4.35
21.84 ± 1.60
22.42 ± 2.56
3.58 ± 1.42
Tacrine
Donepezil
Galantamine
17.95 ± 0.77
1.25 ± 0.46
1
Inhibition of self-induced Aβ1–42 aggregation (2.5
µM) produced by the tested compounds at 10 µM concentration.
2
Values are expressed as the mean SD of three separate experiments. Co-aggregation inhibition of Aβ1–42
aggregation (2.5
as the mean ± SD of three separate experiments. Not tested.
±
µ
M) and AChE (0.2 U/mL) by the tested compounds at 8
µM concentration. Values are expressed
3
2.6. In Vitro Cytotoxicity toward HepG2 Cells
The main reason for the withdrawal of tacrine from the market is its hepatotoxicity. Thus, to verify
the hepatotoxicity of and 12j, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay [29 30] on the human hepatoma cell line (HepG2) was carried out. Tacrine and donepezil were
used as control drugs, and the IC50 values of all of the tested compounds toward HepG2 cells were
1
,

Mar. Drugs 2018, 16, 293
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summarized in Table 3. The results indicated that compounds
1
and 12j did not show significant
cytotoxicity against HepG2 cell (cell viability > 50% up to 80 µM) compared with tacrine and donepezil.
Table 3. Results of in vitro cytotoxicity toward human hepatoma cell line (HepG2) cells.
1
Compound
IC50
>80 2
>80
1
12j
Tacrine
Donepezil
38.87 ± 0.53
37.92 ± 1.46
1 Values are expressed as the mean
±
SD of three separate experiments. 2 Cell viability >50% of compounds at 80
µM.
3. Materials and Methods
3.1. General Information
Melting points were measured by a Melting Point YRY-3 apparatus (Tianjin Precision Apparatus
Factory, Tianjin, China). Commercially available reagents were used without further purification.
Organic solvents were evaporated with reduced pressure using Büchi R-100 evaporators. Reactions
were monitored by thin-layer chromatography (TLC) using Yantai JingYou (Yantai, China) GF254
silica gel plates. Silica gel column chromatography was performed on an Isolera One system (Biotage,
Uppsala, Sweden) with and silica gel (200–300 mesh) from Qingdao Hailang Inc. (Qingdao, China).
NMR spectra were measured on a BrukerAvance III 600 spectrometer (Bruker, Fällanden, Switzerland).
Chemical shifts were expressed in
δ (ppm) and coupling constants (J) in Hz, with residual solvent
signals as internal standards (CDCl3, δH 7.26 ppm and δC 77.0 ppm; DMSO-d6, δH 2.50 ppm and δC
39.5 ppm). Electron spray ionization (ESI)-MS analyses were performed on an Agilent 1260-6460 Triple
Quard LC-MS instrument and HR-ESI-MS data were acquired on an Agilent Q-TOF 6520 spectrometer
(Agilent, Waldbronn, Germany).
3.2. Chemistry
Methyl 2-(4-methoxyphenyl) acetate (3). Thionyl chloride (2.5 mg, 0.021 mmol, 0.003 equiv.) was added
to a solution of 2-(4-methoxyphenyl) acetic acid (1156 mg, 7 mmol, 1 equiv.) in methanol (5 mL) at
0 ◦C. Then, the reaction solution was refluxed for 4 h. The solution was concentrated under reduced
pressure to give 3, which was used in the next step without further purification. Colorless oil. Yield:
1
1215 mg, 96.4%. H NMR (600 MHz, CDCl3)
δ 7.20 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 3.79
(s, 3H), 3.68 (s, 3H), 3.57 (s, 2H). 13C NMR (150 MHz, CDCl3)
δ 172.5, 158.8, 130.4, 126.2, 114.1, 55.4,
52.1, 40.4. ESI-MS m/z 203.0 [M + Na]+.
2-(3,5-Dibromo-4-methoxyphenyl)acetate (4). Ferric chloride (1097 mg, 6.75 mmol, 1 equiv.) and NBS
(2403 mg, 13.5 mmol, 2 equiv.) were added to a solution of
CH3CN at 0 C. The mixture was stirred for 5 h at room temperature. After purification by flash
3 (1215 mg, 6.75 mmol, 1 equiv.) in
◦
chromatography, product
1H NMR (600 MHz, CDCl3)
CDCl3) δ 171.1, 153.5, 133.6, 132.5, 118.2, 60.8, 52.5, 39.7. ESI-MS m/z 335.9 [M + H]+.
4
was obtained in moderate yield. Colorless oil. Yield: 1540 mg, 67.5%.
δ
7.43 (s, 2H), 3.87 (s, 3H), 3.71 (s, 3H), 3.53 (s, 2H). 13C NMR (150 MHz,
2-(3,5-Dibromo-4-methoxyphenyl)acetic acid ( ). A solution of (1540 mg, 4.5 mmol, 1 equiv.) and
5
4
NaOH (540 mg, 13.5 mmol, 3 equiv.) in CH3OH-H2O (v:v = 9:1, 10 mL) was refluxed for 24 h. Then,
the reaction mixture was acidified with 1 M HCl and filtered. The obtained filter was washed three
times with water to give compound
5
, which was used for the next step without further purification.
12.51 (s, 1H,
172.1, 152.0, 134.6,
White solid. Yield: 956 mg, 66.2%. Mp. 125.2–126.6 ◦C. 1H NMR (600 MHz, DMSO-d6)
δ
COOH), 7.57 (s, 2H), 3.78 (s, 3H), 3.60 (s, 2H). 13C NMR (150 MHz, DMSO-d6)
133.9, 116.9, 60.4, 38.6. ESI-MS m/z 322.8 [M + H]+.
δ

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Tert-butyl 5-(2-(3,5-dibromo-4-methoxyphenyl)acetamido)pentylcarbamate (
6). A 10-mL flask was charged
with 5 (162 mg, 0.5 mmol, 1 equiv.), Et3N (151.5 mg, 4.5 mmol, 3 equiv.), HOBT (33.7 mg, 0.25 mmol,
0.5 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (143.2 mg, 0.75 mmol, 1.5 equiv.)
and tert-butyl (5-aminopentyl)carbamate (101 mg, 0.5 mmol, 1.0 equiv.) under nitrogen atmosphere.
After dissolution in 5 mL of anhydrous dichloromethane (DCM), the mixture was stirred at room
temperature for 8 h. The organic phase was washed three times with water and dried over anhydrous
MgSO4. The residue was purified by flash chromatography (petroleum ether:EtOAc = 1:1) to give
1
product
6
. Colorless oil. Yield: 133 mg, 52.3%. H NMR (600 MHz, CDCl3)
δ
7.43 (s, 2H), 5.80 (brs,
1H, NH), 3.86 (s, 3H), 3.43 (s, 2H), 3.24–3.21 (m, 2H), 3.12–3.08 (m, 2H), 1.52–1.47 (m, 4H), 1.43 (s, 9H),
1.33–1.29 (m, 2H). 13C NMR (150 MHz, CDCl3)
δ 169.7, 156.3, 153.4, 133.9, 133.5, 118.4, 79.3, 60.7, 42.2,
40.3, 39.8, 29.9, 29.1, 28.6, 24.0. ESI-MS m/z 540.9 [M + Cl]–.
Tert-butyl 5-(2-(3,5-dibromo-4-methoxyphenyl)acetamido)pentylcarbamate (8). Compound 6 (127 mg,
0.25 mmo◦l, 1 equiv.) was treated with trifluoroacetic acid (285 mg, 2.5 mmol, 10 equiv.) in 5 mL
DCM at 0 C to produce 7, which was used in the next reaction without further purification. A solution
of 7, 37% formaldehyde solution (16.5 mg, 0.55 mmol, 2.5 equiv.), NaCNBH3 (41.4 mg, 0.66 mmol,
3 equiv.) and 12 M HCl(aq) (0.055 mL, 0.66 mmol, 3 equiv.) was stirred in CH3OH (5 mL) for 12 h. Then,
the solution was concentrated, and the residue was purified by flash chromatography (CH2Cl2:CH3OH
1
= 9:1) to give product
8
. Yellow oil. Yield: 90.3 mg, 94.2%. H NMR (600 MHz, CDCl3)
δ
7.42 (s, 2H),
3.86 (s, 3H), 3.42 (s, 2H), 3.25–3.21 (m, 2H), 2.28–2.25 (m, 2H), 2.22 (s, 6H), 1.51–1.45 (m, 4H), 1.33–1.28
(m, 2H). 13C NMR (150 MHz, CDCl3)
169.7, 153.4, 133.9, 133.5, 118.4, 60.8, 59.4, 45.4, 42.3, 39.9, 29.2,
27.1, 24.6. ESI-MS m/z 435.0 [M + H]+.
δ
Pulmonarin B ( ). An amount of (90 mg, 0.2 mmol, 1 equiv.) was dissolved in 5 mL of CH3OH, and then
1
8
K2CO3 (57 mg, 0.4 mmol, 2 equiv.) was added. The resulting mixture was stirred at room temperature
for 15 min before methyl iodide (31 mg, 0.22 mmol, 1.1 equiv.) was added. The reaction was stirred
overnight, and then all of the volatiles were evaporated. A final purification of 10 mg of crude solid
material gave purified
= 1:1:0.4
1
by using semipreparative reversed-phase HPLC (CH3OH/H2O/CH3COOH
1
×
10−4). White solid. Yield: 1.5 mg, 16.6%. H NMR (600 MHz, DMSO-d6)
δ
8.49 (t, J = 5.9 Hz,
1H, NH), 7.56 (s, 2H), 3.77 (s, 3H), 3.43 (s, 2H), 3.28–3.24 (m, 2H), 3.05 (s, 9H), 3.05–3.03 (m, 2H),
1.69–1.66 (m, 2H), 1.48–1.42 (m, 2H), 1.28–1.24 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 169.3, 151.9,
136.2, 133.3, 116.9, 65.2, 65.2, 60.4, 52.1, 40.5, 38.3, 28.4, 23.2, 21.7. ESI-MS m/z 449.0 [M]+. HR-ESIMS:
[M]+ calculated for C17H27Br2N2O2+ 449.0434, found 449.0439.
General Procedures for the Synthesis of 10a–10h and 12a–12l
For the synthesis of 10a
, 80 mg, 0.25 mmol, 1 equiv.), hydroxybenzotriazole (HOBT) (19 mg, 0.125 mmol, 0.5 equiv.), EDCI
(58 mg, 0.375 mmol, 1.5 equiv.), triethylamine (75 mg, 0.75 mmol, 3 equiv.), and corresponding amines
9a 9h (0.25 mmol, 1 equiv.) in 5 mL of CH2Cl2 were stirred at room temperature overnight. For the
synthesis of 12a 12l, the mixtures of corresponding phenylacetic acids 11a 11l (0.26 mmol, 1 equiv.),
–10h, the mixtures of 2-(3,5-dibromo-4-methoxyphenyl)acetic acid
(4
–
–
–
HOBT (20 mg, 0.13 mmol, 0.5 equiv.), EDCI (60 mg, 0.39 mmol, 1.5 equiv.), triethylamine (79 mg,
0.78 mmol, 3 equiv.), and N1-(1,2,3,4-tetrahydroacridin-9-yl)heptane-1,7-diamine (80 mg, 0.26 mmol,
1 equiv.) in 5 mL of CH2Cl2 were stirred at room temperature overnight. The above solutions were
evaporated, and the residues were purified by flash chromatography using CH2Cl2:MeOH = 10:1 as
an eluent to give the target compounds 10a–10h and 12a–12l.
2-(3,5-Dibromo-4-methoxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)acetamide (10a). White
solid. Yield: 57.8 mg, 42.3%. Mp. 97.4–100.0 ◦C. 1H NMR (600 MHz, CDCl3)
δ 9.11 (brs, 1H, NH), 8.19
(d, J = 8.4 Hz, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.52 (s, 2H), 7.45 (dd, J = 8.7, 7.5 Hz, 1H), 7.40 (brs, 1H,
NH), 7.26 (dd, J = 8.4, 7.5 Hz,1H), 4.11–4.06 (m, 2H), 3.78 (s, 3H), 3.76–3.71 (m, 2H), 3.66 (s, 2H), 3.11
(t, J = 6.2 Hz, 2H), 2.48 (t, J = 6.1 Hz, 2H), 1.84–1.79 (m, 2H), 1.78–1.73 (m, 2H). 13C NMR (150 MHz,
CDCl3) δ 173.4, 156.0, 153.1, 150.7, 138.6, 134.2, 133.5, 132.0, 124.9, 124.7, 120.2, 118.1, 115.7, 111.4, 60.7,

Mar. Drugs 2018, 16, 293
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50.9, 41.3, 39.8, 28.5, 24.3, 22.1, 20.8. ESI-MS m/z 546.2 [M + H]+. HR-ESI-MS: [M + H]+ calculated for
C24H26Br2N3O2+ 546.0386, found 546.0393.
2-(3,5-Dibromo-4-methoxyphenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)acetamide (10b). White
solid. Yield: 51 mg, 36.4%. Mp. 72.3–73.1 ◦C. 1H NMR (600 MHz, CDCl3)
δ 7.92 (d, J = 8.3 Hz, 1H),
7.88 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 8.4, 8.0 Hz, 1H), 7.39 (s, 2H), 7.34 (dd, J = 8.3, 8.0 Hz, 1H), 6.23 (brs,
1H, NH), 3.78 (s, 3H), 3.45 (s, 2H), 3.45–3.39 (m, 4H), 3.04 (brs, 2H), 2.71 (brs, 2H), 1.92–1.88 (m, 4H),
1.82–1.76 (m, 2H). 13C NMR (150 MHz, CDCl3)
δ 170.5, 158.5, 153.5, 150.7, 147.1, 133.5, 133.5, 128.6,
128.5, 124.2, 122.6, 120.4, 118.5, 116.9, 60.7, 46.0, 42.2, 37.5, 33.9, 31.3, 25.1, 23.1, 22.8. ESI-MS m/z 560.0
[M + H]+. HR-ESI-MS: [M + H]+ calculated for C25H28Br2N3O2+ 560.0543, found 560.0594.
2-(3,5-Dibromo-4-methoxyphenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)acetamide (10c). White
solid. Yield: 58.2 mg, 40.5%. Mp. 70.3–72.1 ◦C. 1H NMR (600 MHz, CDCl3)
δ 8.25 (d, J = 8.1 Hz, 1H),
8.24 (d, J = 7.9 Hz, 1H), 8.09 (t, J = 5.5 Hz, 1H, NH), 7.59 (dd, J = 7.9, 7.5 Hz 1H), 7.47 (s, 2H), 7.39 (dd,
J = 8.1, 7.5 Hz, 1H), 6.73 (brs, 1H, NH), 3.97–3.92 (m, 2H), 3.76 (s, 3H), 3.58 (s, 2H), 3.36–3.31 (m, 2H),
3.12 (t, J = 6.3 Hz, 2H), 2.66 (t, J = 6.2 Hz, 2H), 1.99–1.93 (m, 2H), 1.87–1.82 (m, 2H), 1.81–1.77 (m, 2H),
1.73–1.67 (m, 2H). 13C NMR (150 MHz, CDCl3)
δ 170.8, 156.0, 152.8, 150.8, 138.7, 134.9, 133.5, 132.4,
125.3, 124.8, 120.2, 117.9, 115.9, 111.2, 60.7, 47.9, 41.8, 38.7, 28.6, 27.9, 26.3, 24.2, 22.1, 20.8. ESI-MS m/z
574.0 [M + H]+. HR-ESI-MS: [M + H]+ calculated for C26H30Br2N3O2+ 574.0699, found 574.0714.
2-(3,5-Dibromo-4-methoxyphenyl)-N-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)acetamide (10d). White
solid. Yield: 84.1 mg, 57.1%. Mp. 74.6–76.0 ◦C. 1H NMR (600 MHz, CDCl3)
δ 7.93 (d, J = 8.4 Hz, 1H),
7.89 (d, J = 8.3 Hz, 1H), 7.54 (dd, J = 8.3, 7.1 Hz, 1H), 7.39 (s, 2H), 7.34 (dd, J = 8.4, 7.1 Hz, 1H), 5.69 (brs,
1H, NH), 3.85 (s, 3H), 3.45 (t, J = 7.2 Hz, 2H), 3.40 (s, 2H), 3.26–3.21 (m, 2H), 3.04 (brs, 2H), 2.69 (brs,
2H), 1.94–1.89 (m, 4H), 1.70–1.62 (m, 2H), 1.54–1.47 (m, 2H), 1.41–1.34 (m, 2H). 13C NMR (150 MHz,
CDCl3) δ 169.7, 158.5, 153.4, 150.8, 147.4, 133.7, 133.5, 128.7, 128.5, 123.8, 122.9, 120.3, 118.5, 116.1, 60.8,
49.4, 42.2, 39.7, 34.1, 31.4, 29.5, 25.0, 24.3, 23.2, 22.9. ESI-MS m/z 588.1 [M + H]+. HR-ESI-MS: [M + H]+
calculated for C27H32Br2N3O2+ 588.0856, found 588.0853.
2-(3,5-Dibromo-4-methoxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)acetamide (10e). White
solid. Yield: 98.6 mg, 65.4%. Mp. 72.1–74.0 ◦C. 1H NMR (600 MHz, CDCl3)
δ 7.93 (d, J = 8.5 Hz, 1H),
7.88 (d, J = 8.2 Hz, 1H), 7.54 (dd, J = 8.2, 7.0 Hz, 1H), 7.39 (s, 2H), 7.33 (dd, J =8.5, 7.0 Hz, 1H), 5.73 (brs,
1H, NH), 3.85 (s, 3H), 3.45 (t, J = 7.2 Hz, 2H), 3.39 (s, 2H), 3.23–3.18 (m, 2H), 3.04 (brs, 2H), 2.69 (brs,
2H), 1.93–1.89 (m, 4H), 1.65–1.59 (m, 2H), 1.49–1.43 (m, 2H), 1.41–1.34 (m, 2H), 1.32–1.26 (m, 2H). 13
C
NMR (150 MHz, CDCl3) δ 169.7, 158.6, 153.4, 150.8, 147.6, 133.8, 133.5, 128.8, 128.4, 123.7, 123.0, 120.3,
118.4, 116.1, 60.7, 49.4, 42.2, 39.7, 34.2, 31.8, 29.5, 26.7, 26.6, 24.9, 23.2, 22.9. ESI-MS m/z 602.1 [M + H]+.
HR-ESI-MS: [M + H]+ calculated for C28H34Br2N3O2+ 602.1012, found 602.1018.
2-(3,5-Dibromo-4-methoxyphenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (10f). White
solid. Yield: 119 mg, 77.4%. Mp. 54.6–56.8 ◦C. 1H NMR (600 MHz, CDCl3)
δ 8.34 (d, J = 8.6 Hz, 1H),
8.22 (d, J = 8.7 Hz, 1H), 7.62 (dd, J = 8.7, 7.4 Hz, 1H), 7.44 (s, 2H), 7.41 (dd, J = 8.6, 7.4 Hz, 1H), 7.21 (brs,
1H, NH), 3.87 (t, J = 7.2 Hz, 2H), 3.78 (s, 3H), 3.55 (s, 2H), 3.23–3.20 (m, 2H), 3.19 (t, J = 6.2 Hz, 2H), 2.65
(t, J = 6.1 Hz, 2H), 1.91–1.86 (m, 2H), 1.84–1.77 (m, 4H), 1.50–1.45 (m, 2H), 1.41–1.35 (m, 2H), 1.32–1.28
(m, 4H). 13C NMR (150 MHz, CDCl3)
δ 170.3, 155.6, 152.9, 151.4, 139.4, 134.8, 133.5, 132.2, 125.1, 124.6,
121.0, 118.0, 116.2, 111.3, 60.7, 48.4, 41.9, 39.6, 30.7, 29.2, 28.9, 28.4, 26.4, 26.4, 24.1, 22.1, 20.9. ESI-MS
m/z 616.1 [M + H]+. HR-ESI-MS: [M + H]+ calculated for C29H36Br2N3O2+ 616.1169, found 616.1180.
2-(3,5-Dibromo-4-methoxyphenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)acetamide (10g). White
solid. Yield: 80.7 mg, 51.2%. Mp. 60.2–61.4 ◦C. 1H NMR (600 MHz, CDCl3)
δ 7.96 (d, J = 8.4 Hz, 1H),
7.92 (d, J = 8.4 Hz, 1H), 7.55 (dd, J = 8.4, 7.4 Hz, 1H), 7.42 (s, 2H), 7.35 (dd, J = 8.4, 7.4 Hz, 1H), 5.59 (brs,
1H, NH), 3.86 (s, 3H), 3.50 (t, J = 7.2 Hz, 2H), 3.43 (s, 2H), 3.23–3.18 (m, 2H), 3.07 (brs, 2H), 2.70 (brs,
2H), 1.94–1.90 (m, 4H), 1.68–1.62 (m, 2H), 1.46–1.40 (m, 2H), 1.38–1.33 (m, 2H), 1.31– 1.23 (m, 6H). 13
C
NMR (150 MHz, CDCl3) δ 169.5, 158.1, 153.3, 151.1, 147.1, 133.7, 133.4, 128.5, 128.4, 123.7, 122.9, 120.0,

Mar. Drugs 2018, 16, 293
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118.3, 115.6, 60.6, 49.3, 42.2, 39.8, 33.8, 31.7, 29.4, 29.1, 29.0, 26.7, 26.6, 24.7, 23.0, 22.7. ESI-MS m/z 630.1
[M + H]+. HR-ESI-MS: [M + H]+ calculated for C30H38Br2N3O2+ 630.1325, found 630.1330.
2-(3,5-Dibromo-4-methoxyphenyl)-N-(10-((1,2,3,4-tetrahydroacridin-9-yl)amino)decyl)acetamide (10h). White
solid. Yield: 72.6 mg, 44.1%. Mp. 74.6–76.1 ◦C. 1H NMR (600 MHz, CDCl3)
δ 7.96 (d, J = 8.4 Hz, 1H),
7.89 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 8.4, 7.4 Hz, 1H), 7.40 (s, 2H), 7.33 (dd, J = 8.4, 7.4 Hz, 1H), 6.01 (brs,
1H, NH), 3.84 (s, 3H), 3.49 (t, J = 7.1 Hz, 2H), 3.42 (s, 2H), 3.32–3.18 (m, 2H), 3.04 (brs, 2H), 2.69 (brs,
2H), 1.92–1.88 (m, 4H), 1.66–1.60 (m, 2H), 1.47–1.41 (m, 2H), 1.38–1.31 (m, 2H), 1.29–1.20 (m, 10H). 13
C
NMR (150 MHz, CDCl3) δ 169.6, 158.3, 153.3, 151.1, 147.3, 134.0, 133.5, 128.5, 128.5, 123.7, 123.1, 120.1,
118.3, 115.7, 60.7, 49.5, 42.2, 39.9, 33.9, 31.8, 29.5, 29.4, 29.4, 29.3, 29.2, 26.9, 26.9, 24.8, 23.1, 22.8. ESI-MS
m/z 658.2 [M + H]+. HR-ESI-MS: [M + H]+ calculated for C32H42Br2N3O2+ 658.1638, found 658.1637.
2-(2-Bromo-4-methoxyphenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12a). Yellow oil.
1
Yield: 81.4 mg, 58.2% yield. H NMR (600 MHz, CDCl3)
δ
8.41 (d, J = 8.5 Hz, 1H), 8.21 (d, J = 8.7 Hz,
1H), 7.63 (dd, J = 8.7, 7.6 Hz, 1H), 7.41 (dd, J = 8.5, 7.6 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 2.6 Hz,
1H), 6.80 (dd, J = 8.5, 2.6 Hz, 1H), 5.90 (brs, 1H, NH), 3.87 (t, J = 7.1 Hz, 2H), 3.75 (s, 3H), 3.63 (s, 2H),
3.23 (t, J = 6.2 Hz, 2H), 3.21–3.16 (m, 2H), 2.63 (t, J = 6.1 Hz, 2H), 1.91–1.86 (m, 2H), 1.85–1.77 (m,
2H), 1.47–1.42 (m, 2H), 1.41–1.36 (m, 2H), 1.35–1.31 (m, 2H). 1.31–1.25 (m, 4H). 13C NMR (150 MHz,
CDCl3)
δ 170.2, 159.4, 155.0, 151.6, 139.4, 132.2, 129.8, 127.1, 125.3, 125.1, 124.5, 121.3, 118.4, 116.1, 114.0,
111.1, 55.7, 48.4, 43.1, 39.5, 30.9, 29.8, 29.4, 28.6, 26.5, 26.5, 24.0, 22.1, 20.9. ESI-MS m/z 538.2 [M + H]+.
HR-ESI-MS: [M + H]+ calculated for C29H37BrN3O2 538.2064, found 538.2060.
+
2-(4-Methoxyphenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12b). Yellow oil. Yield:
1
43.4 mg, 36.3%. H NMR (600 MHz, CDCl3)
δ 7.94 (d, J = 7.9 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.54
(dd, J = 8.4, 7.0 Hz, 1H), 7.33 (dd, J = 7.9, 7.0 Hz, 1H), 7.14 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H),
5.43 (brs, 1H, NH), 3.78 (s, 3H), 3.49 (s, 2H), 3.45 (t, J = 7.2 Hz, 2H), 3.18–3.14 (m, 2H), 3.05 (brs, 2H),
2.69 (brs, 2H), 1.93–1.88 (m, 4H), 1.64–1.58 (m, 2H), 1.41–1.36 (m, 2H), 1.35–1.31 (m, 2H), 1.30–1.25
(m, 2H), 1.24–1.18 (m, 2H). 13C NMR (150 MHz, CDCl3)
δ 171.4, 158.9, 158.5, 150.9, 147.5, 130.6, 128.8,
128.4, 127.1, 123.7, 123.0, 120.3, 116.0, 114.5, 55.4, 49.5, 43.6, 39.6, 34.1, 31.8, 29.5, 29.0, 26.9, 26.7, 24.9,
23.1, 22.8. ESI-MS m/z 460.2 [M + H]+. HR-ESI-MS: [M + H]+ calculated for C29H38N3O2+ 460.2959,
found 460.2950.
2-(3-Bromo-4-methoxyphenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12c). Yellow oil.
1
Yield: 50.8 mg, 36.3%. H NMR (600 MHz, CDCl3)
δ
8.49 (d, J = 7.4 Hz, 1H), 8.16 (d, J = 8.7 Hz, 1H), 7.68
(dd, J = 8.7, 7.3 Hz, 1H), 7.43 (dd, J = 7.4, 7.3 Hz, 1H), 7.44 (d, J = 2.1 Hz, 1H), 7.18 (dd, J = 8.4, 2.1 Hz,
1H), 6.84 (d, J = 8.4 Hz, 1H), 5.86 (brs, 1H, NH), 3.87 (brs, 2H), 3.86 (s, 3H), 3.49 (s, 2H), 3.29 (t, J = 6.2 Hz,
2H), 3.23–3.18 (m, 2H), 2.60 (t, J = 6.0 Hz, 2H), 1.96–1.90 (m, 2H), 1.90–1.84 (m, 3H), 1.82–1.76 (m, 2H),
1.48–1.39 (m, 4H), 1.37–1.32 (m, 2H), 1.31–1.26 (m, 2H). 13C NMR (150 MHz, CDCl3)
δ 171.1, 155.4,
154.9, 151.6, 139.6, 134.0, 132.0, 129.6, 129.3, 125.1, 124.5, 121.3, 116.3, 112.1, 111.7, 111.4, 56.4, 48.4, 42.3,
39.5, 30.8, 29.3, 29.0, 28.5, 26.5, 26.4, 24.1, 22.1, 20.9. ESI-MS m/z 538.1 [M + H]+. HR-ESI-MS: [M + H]+
calculated for C29H37BrN3O2+ 538.2064, found 538.2070.
2-(2-Bromophenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12d). Yellow oil. Yield:
1
90.4 mg, 68.4%. H NMR (600 MHz, CDCl3)
δ
8.21 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.60 (dd,
J = 8.5, 7.3 Hz, 1H), 7.54 (dd, J = 8.0, 1.0 Hz, 1H), 7.38 (dd, J = 8.1, 7.3 Hz, 1H), 7.33 (dd, J = 7.6, 1.7Hz,
1H), 7.28 (ddd, J = 7.8, 7.6, 1.0 Hz, 1H), 7.13 (ddd, J = 8.0, 7.8, 1.7 Hz, 1H), 5.67 (brs, 1H, NH), 3.69
(s, 2H), 3.69–3.66 (m, 4H), 3.23–3.18 (m, 2H), 3.17 (t, J = 6.1 Hz, 2H), 2.64 (t, J = 6.0 Hz, 2H), 1.93–1.84
(m, 4H), 1.75–1.68 (m, 2H), 1.47–1.41 (m, 2H), 1.40–1.34 (m, 2H), 1.34–1.29 (m, 2H), 1.29–1.25 (m, 2H).
13C NMR (150 MHz, CDCl3)
δ 169.7, 154.5, 153.5, 142.8, 135.2, 133.1, 131.8, 130.5, 129.1, 128.0, 127.7,
125.0, 124.5, 123.8, 117.9, 113.2, 48.9, 44.1, 39.6, 31.3, 31.0, 29.4, 28.7, 26.7, 26.6, 24.3, 22.5, 21.7. ESI-MS
m/z 508.1 [M + H]+. HR-ESI-MS: [M + H]+ calculated for C28H35BrN3O+ 508.1958, found 508.1963.
2-(3-Bromophenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12e). Yellow oil. Yield:
1
56.7 m, 42.9%. H NMR (600 MHz, CDCl3)
δ
7.94 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.54 (dd,

Mar. Drugs 2018, 16, 293
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J = 8.4, 7.1 Hz, 1H), 7.41–7.36 (m, 2H), 7.33 (dd, J = 7.8, 7.1 Hz, 1H), 7.19–7.16 (m, 2H), 5.64 (brs, 1H,
NH), 3.49 (s, 2H), 3.46(t, J = 7.2 Hz, 2H), 3.20–3.15 (m, 2H), 3.04 (brs, 2H), 2.69 (brs, 2H), 1.93–1.88
(m, 4H), 1.65–1.58 (m, 2H), 1.43–1.37 (m, 2H), 1.37–1.30 (m, 2H), 1.30–1.26 (m, 2H), 1.24–1.20 (m, 2H).
13C NMR (150 MHz, CDCl3)
δ 170.1, 158.4, 151.0, 147.4, 137.5, 132.4, 130.5, 130.5, 128.6, 128.5, 128.1,
123.7, 123.0, 123.0, 120.2, 115.9, 49.5, 43.4, 39.7, 34.0, 31.7, 29.4, 29.0, 26.9, 26.7, 24.9, 23.1, 22.8. ESI-MS
m/z 508.1 [M + H]+. HR-ESI-MS: [M + H]+ calculated for C28H35BrN3O+ 508.1958, found 508.1949.
2-(4-Bromophenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12f). Yellow oil. Yield:
1
51.8 mg, 39.2%. H NMR (600 MHz, CDCl3)
δ 7.93 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.53
(dd, J = 8.4, 7.0 Hz, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.33 (dd, J = 8.5, 7.0 Hz, 1H), 7.11 (d, J = 8.3 Hz, 2H),
5.57 (brs, 1H, NH), 3.47 (s, 2H), 3.45 (t, J = 7.3 Hz, 2H), 3.19–3.14 (m, 2H), 3.04 (brs, 2H), 2.69 (brs, 2H),
1.93–1.87 (m, 4H), 1.64–1.57 (m, 2H), 1.42–1.35 (m, 2H), 1.34–1.30 (m, 2H), 1.29–1.25 (m, 2H), 1.23–1.19
(m, 2H). 13C NMR (150 MHz, CDCl3)
δ 170.3, 158.6, 150.8, 147.6, 134.2, 132.1, 131.1, 128.9, 128.4, 123.7,
122.9, 121.3, 120.4, 116.0, 49.5, 43.2, 39.7, 34.2, 31.8, 29.5, 29.0, 26.9, 26.7, 24.9, 23.2, 22.9. ESI-MS m/z
508.1 [M + H]+. HR-ESI-MS: [M + H]+ calculated for C28H35BrN3O+ 508.1958, found 508.1948.
2-(2,5-Dibromophenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12g). Yellow oil, Yield:
1
54.5 mg, 35.7%. H NMR (600 MHz, CDCl3)
δ 7.94 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.54
(dd, J = 8.3, 7.0 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.34 (dd, J = 8.4, 7.0 Hz, 1H),
7.26 (dd, J = 8.5, 2.4 Hz,1H), 5.53 (brs, 1H, NH), 3.62 (s, 2H), 3.46 (t, J = 7.2 Hz, 2H), 3.24–3.18 (m,
2H), 3.05 (brs, 2H), 2.70 (brs, 2H), 1.94–1.89 (m, 4H), 1.65–1.59 (m, 2H), 1.46–1.41 (m, 2H), 1.38–1.33
(m, 2H), 1.32–1.27 (m, 2H), 1.27–1.23 (m, 2H). 13C NMR (150 MHz, CDCl3)
δ 168.7, 158.6, 150.9, 147.6,
137.2, 134.5, 134.4, 132.2, 128.9, 128.4, 123.7, 123.6, 123.0, 121.8, 120.3, 116.0, 49.6, 43.9, 39.8, 34.2, 31.8,
29.5, 29.0, 26.9, 26.8, 24.9, 23.2, 22.9. ESI-MS m/z 586.0 [M + H]+. HR-ESI-MS: [M + H]+ calculated for
C28H34Br2N3O+ 586.1063, found 586.1067.
2-(3,5-Dibromophenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12h). Yellow oil. Yield:
1
56.6 mg, 37.1%. H NMR (600 MHz, CDCl3)
δ
7.95 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.57 (t,
J = 1.7 Hz, 1H), 7.55 (dd, J = 8.4, 7.1 Hz, 1H), 7.35 (d, J = 1.7 Hz, 2H), 7.34 (dd, J = 8.5, 7.1 Hz, 1H), 5.64
(brs, 1H, NH), 3.47 (d, J = 7.1 Hz, 2H), 3.45 (s, 2H), 3.22–3.17 (m, 2H), 3.05 (brs, 2H), 2.70 (brs, 2H),
1.93–1.90 (m, 4H), 1.65–1.60 (m, 2H), 1.44–1.39 (m, 2H), 1.38–1.33 (m, 2H), 1.32–1.28 (m, 2H), 1.27–1.24
(m, 2H). 13C NMR (150 MHz, CDCl3)
δ 169.4, 158.5, 151.0, 147.5 139.0, 133.0, 131.2, 128.7, 128.5, 123.8,
123.3, 123.0, 120.3, 116.0, 49.5, 42.9, 39.9, 34.1, 31.8, 29.4, 29.0, 26.9, 26.8, 24.9, 23.2, 22.9. ESI-MS m/z
586.0 [M + H]+. HR-ESI-MS: [M + H]+ calculated for C28H34Br2N3O+ 586.1063, found 586.1065.
2-(3-Bromo-4-fluorophenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12i). Yellow oil.
1
Yield: 55.7 mg, 40.7%. H NMR (600 MHz, DMSO-d6) δ 8.39 (d, J = 8.7 Hz, 1H), 8.16 (t, J = 5.5 Hz, 1H,
NH), 7.95 (d, J = 8.1 Hz, 1H), 7.84 (dd, J = 8.1, 7.5 Hz, 1H), 7.78 (brs, 1H, NH), 7.59–7.56 (m, 1H), 7.55
(dd, J = 8.7, 7.5 Hz, 1H), 7.28 (brs, 1H), 7.26 (d, J = 1.0 Hz, 1H), 3.85–3.79 (m, 2H), 3.40 (s, 2H), 3.04–2.97
(m, 4H), 2.65 (brs, 2H), 1.85–1.79 (m, 4H), 1.73–1.66 (m, 2H), 1.39–1.33 (m, 2H), 1.31–1.18 (m, 6H). 13
C
NMR (150 MHz, DMSO-d6) δ 169.3, 157.8, 156.2, 155.6, 150.6, 138.0, 134.8 (d, J = 3.57 Hz), 133.6, 132.5,
130.2 (d, J = 7.24 Hz), 125.0, 119.2, 116.3 (d, J = 21.93 Hz), 115.5, 111.1, 107.4 (d, J = 20.86 Hz), 47.2, 40.9,
38.5, 29.8, 28.9, 28.2, 28.0, 26.2, 26.0, 24.0, 21.5, 20.3. ESI-MS m/z 526.1 [M + H]+. HR-ESI-MS: [M + H]+
calculated for C28H34BrFN3O+ 526.1864, found 526.1868.
2-(5-Bromo-2-fluorophenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12j). Yellow oil.
1
Yield: 47.9 mg, 35.0%. H NMR (600 MHz, CDCl3)
δ
8.03–7.96 (m, 2H), 7.56 (dd, J = 8.1, 7.1 Hz, 1H), 7.44
(dd, J = 6.6, 2.5 Hz, 1H), 7.37–7.32 (m, 2H), 6.93 (t, J = 9.0 Hz, 1H), 5.75 (brs, 1H, NH), 3.55 (t, J = 7.1 Hz,
2H), 3.51 (s, 2H), 3.23–3.19 (m, 2H), 3.08 (brs, 2H), 2.67 (brs, 2H), 1.92–1.88 (m, 4H), 1.69–1.62 (m, 2H),
1.47–1.41 (m, 2H), 1.39–1.34 (m, 2H), 1.33–1.29 (m, 2H), 1.29–1.26 (m, 2H). 13C NMR (150 MHz, CDCl3)
δ
169.1, 160.9, 159.3, 151.9, 145.7, 134.4 (d, J = 4.03 Hz), 132.1 (d, J = 8.21 Hz), 129.3, 127.2, 124.8 (d,
J = 17.42 Hz), 124.0, 123.3, 119.4, 117.3 (d, J = 23.63 Hz), 117.0 (d, J = 3.36 Hz), 114.9, 49.3, 39.8, 36.7, 32.9,

Mar. Drugs 2018, 16, 293
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31.6, 29.4, 28.9, 26.8, 26.6, 24.7, 22.9, 22.5. ESI-MS m/z 526.1 [M + H]+. HR-ESI-MS: [M + H]+ calculated
for C28H34BrFN3O+ 526.1864, found 526.1867.
2-(2-Bromo-5-chlorophenyl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12k). Yellow oil.
1
Yield: 70.4 mg, 49.9%. H NMR (600 MHz, CDCl3)
δ
8.37 (d, J = 8.5 Hz, 1H), 8.23 (d, J = 8.7 Hz, 1H), 7.63
(dd, J = 8.7, 7.6 Hz, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.41 (dd, J = 8.5, 7.6 Hz, 1H), 7.25 (d, J = 8.2 Hz, 1H),
7.18 (dd, J = 8.2, 2.0 Hz, 1H), 6.50 (brs, 1H, NH), 3.93–3.86 (m, 2H), 3.69 (s, 2H), 3.25–3.17 (m, 4H), 2.63
(brs, 2H), 1.90–1.85 (m, 2H), 1.84–1.77 (m, 4H), 1.50–1.43 (m, 2H), 1.42–1.36 (m, 2H), 1.35–1.26 (m, 4H).
13C NMR (150 MHz, CDCl3)
δ 169.5, 155.8, 151.0, 138.9, 134.2, 133.7, 132.4, 132.4, 132.4, 128.0, 125.3,
125.2, 124.7, 120.7, 115.9, 111.0, 53.6, 48.4, 43.2, 39.6, 30.8, 29.3, 28.5, 28.5, 26.5, 24.0, 22.1, 20.8. ESI-MS
m/z 542.1 [M + H]+. HR-ESI-MS: [M + H]+ calculated for C28H34BrClN3O+ 542.1568, found 542.1561.
2-Phenyl-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)acetamide (12l). Yellow oil. Yield: 52.7 mg,
1
47.2%. H NMR (600 MHz, CDCl3)
δ
7.93 (d, J = 8.5 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.53 (dd, J = 8.4,
7.0 Hz, 1H), 7.34–7.30 (m, 3H), 7.26 (dd, J = 8.5, 7.0 Hz, 1H), 7.24–7.21 (m, 2H), 5.48 (brs, 1H, NH),
3.54 (s, 2H), 3.44 (t, J = 7.2 Hz, 2H), 3.19–3.14 (m, 2H), 3.05 (brs, 2H), 2.69 (brs, 2H), 1.94–1.87 (m, 4H),
1.64–1.57 (m, 2H), 1.40–1.35 (m, 2H), 1.34–1.29 (m, 2H), 1.29–1.25 (m, 2H), 1.23–1.17 (m, 2H). 13C NMR
(150 MHz, CDCl3) δ 171.0, 158.6, 150.8, 147.6, 135.1, 129.5, 129.1, 128.8, 128.3, 127.4, 123.7, 122.9, 120.3,
116.0, 49.5, 44.0, 39.6, 34.2, 31.8, 29.5, 29.0, 26.9, 26.7, 24.9, 23.1, 22.9. ESI-MS m/z 430.2 [M + H]+.
HR-ESI-MS: [M + H]+ calculated for C28H36N3O+ 430.2853, found 430.2847.
3.3. AChE/BChE Inhibitory Assay
The AChE and BChE inhibitory activities of compounds were determined by using a slightly
0
modified Ellman’s method [30
,
31]. Electric eel AChE, equine serum BChE, 5,5 -dithiobis(2-nitrobenzoic
acid) (DTNB), phosphate buffer solution (PBS, pH 8.0), acetylthiocholine (ATC) iodide,
and butyrylthiocholine (BUC) iodide were purchased from Sigma-Aldrich (Steinheim, Germany).
Tacrine was used as positive control. Enzyme solutions were prepared at 2.0 U/mL in 2-mL aliquots.
The assay medium consisted of 10
µL of enzyme, 40 µL of PBS, 20 µL of 0.01 M DTNB, and 10 µL of
tested compound. Assayed solutions of tested compounds were pre-incubated with corresponding
ChE for 5 min. The reaction was initiated by the addition of 20 µL of 0.01 M substrate (ATC or BUC).
The activity was determined by measuring the increase in absorbance at 410 nm at 37 ◦C in 2-min
intervals using Tecan Spark multimode microplate reader (Mannedorf, Switzerland). The percentage
of inhibition (I) was calculated from the measured data as follows: I = (Ac
Ai and Ac represent the change in the absorbance in the presence of an inhibitor and without an
inhibitor, respectively.
−
Ai)/Ac
×
100%, where
3.4. Kinetic Assay
Kinetic studies of inhibition on AChE and BChE were performed by using Ellman’s method
as described above. The concentrations of used substrates were 0.07813
and 0.625 M. Linear regression was used for calculation of Lineweaver
calculations were performed using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA).
µ
M, 0.1563
Burk plots, and all of the
µM, 0.3125 µM,
µ
−
3.5. Molecular Docking
Molecular docking studies were performed using the Autodock 4.2 program (The Scripps Research
Institute, La Jolla, CA, USA) [32,33]. The crystal structure of AChE (PDB ID: 5EI5) was obtained
from the Protein Data Bank after eliminating the inhibitor and water molecules. The 3D structure
of the ligand was built, and performed MMFF94 minimization by using ChemBio3D Ultra 12.0
(CambridgeSoft Corporation, Cambridge, MA, USA). Using Autodock Tools 1.5.6, the preparation
of the receptor was made by the addition of hydrogen atoms and Gasteiger charges, and finally an
assignment of atomic types as AD4 type, and then autotorsion was used to define the rotatable bonds
in the ligand preparation. The resulting enzyme structure was used as an input for the Autogrid

Mar. Drugs 2018, 16, 293
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program. Autogrid performed pre-calculated atomic affinity grid maps for each atom type in the
ligand, plus an electrostatics map and a separate de-solvation map presented in the substrate molecule.
The dimensions of the box were set to 60
×
60
×
60 with grid spacing of 0.375 Å. Rigid ligand docking
was performed for the compounds. Docking calculations were carried out using the Lamarckian
genetic algorithm (LGA). The proposed docking complex image was created by Pymol 1.5 (DeLano
Scientific LLC, San Carlos, California, USA).
3.6. Determination of the Inhibitory Potency on Self-Aβ1–42 Aggregation
In order to investigate the inhibition of compounds on Aβ1–42 self-aggregation, a ThT-based
fluorometric assay was performed [34]. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) pre-treated Aβ1–42
(GL Biochem Ltd., Shanghai, China) was dissolved in DMSO to make a 200 µM stock solution.
The stock solution was centrifuged at the speed of 13,500 rpm for 10 min. The above supernatant was
used for experiments. The tested compounds were dissolved in DMSO at concentrations of 0.8 mM.
A screening assay for the tested compounds to inhibit A aggregation was performed by measuring
ThT fluorescence emission. Compounds (2
of phosphate-buffered saline (PBS at pH 7.4) in a 96-well plate. After incubation for 24 h at room
temperature, 80 L of 5 M of ThT solution (in 50 mM of glycine-NaOH at pH 8.5) was added to the
µL) and 2 µL of 200 µM Aβ1–42 were added into 76 µL
µ
µ
reaction solution. Fluorescence emission was measured at 490 nm with an excitation wavelength of
450 nm on a Tecan Spark multimode microplate reader. Identical spectra were recorded by performing
the independent experiments thrice. The fluorescence intensities were compared, and the % inhibition
was calculated by the following equation: 100
−
[(Fi
−
Fb)/(Fo
−
Fb)
×
100], where Fi, Fo and Fb are
the fluorescence intensities obtained for A aggregation in the presence of inhibitors Aβ1–42 and ThT;
β
in the presence of Aβ1–42 and THT but no inhibitors; and the blanks containing ThT only.
3.7. Determination of the Inhibitory Potency on Aβ1–42 Aggregation Induced by AChE
The co-incubation experiment of Aβ1–42 with AChE was performed by ThT bioassay according to
a reported protocol [35]. HFIP pre-treated Aβ1–42 (GL Biochem Ltd., shanghai, China) was dissolved in
DMSO to make a 200
for 10 min. The above supernatant was used for experiments. The tested compounds were dissolved in
DMSO at concentrations of 10 mM, and diluted to 640 M by phosphate-buffered saline (PBS at pH 7.4).
A screening assay for tested compounds to inhibit A aggregation was performed by measuring ThT
fluorescence emission. Compounds (2 L) and 2 L of 200 M Aβ1–42 and 20 L AchE (2 /mL, in PBS
at pH 8.0) were added into 76 L of PBS (pH 8.0) in a 96-well plate. After incubation for 24 h at room
temperature, 100 L of 5 M ThT solution (in 50 mM of glycine-NaOH at pH 8.5) was added to the
µM stock solution. The stock solution was centrifuged at the speed of 13,500 rpm
µ
µ
µ
µ
µ
µ
µ
µ
µ
reaction solution. Fluorescence emission was measured at 490 nm with an excitation wavelength of
450 nm on a Tecan Spark multimode microplate reader. Identical spectra were recorded by performing
the independent experiments thrice. The fluorescence intensities were compared, and the % inhibition
was calculated by the following equation: 100
−
[(Fi
−
Fb)/(Fo
−
Fb)
×
100], where Fi, Fo and Fb are
the fluorescence intensities obtained for A aggregation in the presence of inhibitors Aβ1–42, AChE,
β
and ThT; in the presence of Aβ1–42, AChE, and ThT but no inhibitors; and the blanks containing
ThT only.
3.8. Cytotoxicity Bioassay
The cytotoxicity of selected compounds was evaluated on HepG2 cells. Cells were inoculated
into 96-well plates. After incubation for 24 h, the cells were treated with different concentrations of
tested compounds for 24 h, and then were incubated with 10
diphenyltetrazolium bromide (MTT) at 37 C for 2 h. The formazan dye product was measured by the
absorbance at 490 nm on a Tecan Spark multimode microplate reader.
µ
L of 3-(4,5-dimethylthiazol-2-yl)-2,5-
◦

Mar. Drugs 2018, 16, 293
14 of 16
4. Conclusions
In summary, pulmonarin B (
10h and 12a
found to be a moderate dual AChE/BChE inhibitor and also showed inhibition on self-induced
and AChE-induced A aggregation, which established as an interesting marine hit for further
anti-AD study. Among these hybrids, the best result was obtained for compound 12j with IC50 of
0.182 ± 0.006 µM for AChE and 0.064 0.006 M for BChE. The kinetic and molecular docking studies
well confirmed 12j as a mixed-type AChE inhibitor. Moreover, 12j displayed the highest inhibitory
activity against self-induced and AChE-induced A aggregation. In addition, compound 12j did
1) and a series of brominated phenylacetic acid/tacrine hybrids
10a
–
–12l were synthesized and evaluated for their anti-AD potential. Compound 1 was
β
1
±
µ
β
not show obvious hepatotoxicity in comparison with tacrine and donepezil. In the present study,
the biological evaluation together with computational analyses demonstrated these newly designed
brominated phenylacetic acid/tacrine hybrids as attractive lead compounds toward the discovery of
multifunctional anti-AD drugs.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/16/9/293/s1,
Supplementary materials including 1H, 13C NMR and HR-ESIMS spectra for compounds
1, 10a–10h and 12a–12l.
Author Contributions: Chemical synthesis and bioassay, Z.-Q.C., J.Z. and J.-L.S.; Molecular docking, K.Z.;
Writing-Original Draft Preparation, C.-S.J.; Writing-Review, H.Z.; Supervision, C.-S.J. and H.Z.
Funding: This research was funded by the National Natural Science Foundation of China [No. 21672082], Shandong
Key Development Project [No. 2016GSF201209], the Young Taishan Scholars Program [No. tsqn20161037], Natural
Science Foundation of Shandong Province [Nos. ZR2017BH038, JQ201721], and Shandong Talents Team
Cultivation Plan of University Preponderant Discipline [No. 10027].
Acknowledgments: Authors are grateful to Jin-Tong Song (School of Biological Science and Technology, University
of Jinan) for NMR analytical support.
Conflicts of Interest: The authors declare no conflict of interest.
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