Design, synthesis and biological evaluation of 3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives as new multipotent cholinesterase/monoamine oxidase inhibitors for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2017.03.070

A new family of multitarget molecules able to interact with acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as with monoamino oxidase (MAO) A and B, has been synthesized. Novel 3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives have been designed using a conjunctive approach that combines the JMC49 and donepezil. The most promising compound TM-33 showed potent and balance inhibitory activities toward ChE and MAO (eeAChE, eqBuChE, hMAO-A and hMAO-B with IC₅₀ values of 0.56?μM, 2.3?μM, 0.3?μM and 1.4?μM, respectively) but low selectivity. Both kinetic analysis of AChE inhibition and molecular modeling study suggested that TM-33 binds simultaneously to the catalytic active site and peripheral anionic site of AChE. Furthermore, our investigation proved that TM-33 could cross the blood-brain barrier (BBB) in vitro, and abided by Lipinski's rule of five. The results suggest that compound TM-33, an interesting multi-targeted active molecule, offers an attractive starting point for further lead optimization in the drug-discovery process against Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2017.03.070

Accepted Manuscript
Design, synthesis and biological evaluation of 3,4-dihydro-2(1H)-quinoline-O-
alkylamine derivatives as new multipotent cholinesterase/monoamine oxidase
inhibitors for the treatment of Alzheimer’s disease
Zhipei Sang, Wanli Pan, Keren Wang, Qinge Ma, Lintao Yu, Wenmin Liu
PII:
S0968-0896(17)30013-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.070
BMC 13669
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
4 January 2017
22 March 2017
Please cite this article as: Sang, Z., Pan, W., Wang, K., Ma, Q., Yu, L., Liu, W., Design, synthesis and biological
evaluation of 3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives as new multipotent cholinesterase/monoamine
oxidase inhibitors for the treatment of Alzheimer’s disease, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.03.070
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Design,
synthesis
and
biological
evaluation
of
3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives as new
multipotent cholinesterase/monoamine oxidase inhibitors for the
treatment of Alzheimer’s disease
Zhipei Sanga, *, Wanli Pana, Keren Wangb, Qinge Maa , Lintao Yua, Wenmin Liua, *.
aCollege of Chemistry and Pharmaceutical Engineering, Nanyang Normal University,
Nanyang, 473061, China
bNanyang Normal University Hospital, Nanyang Normal University, Nanyang,
473061, China
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterase; BuChE,
butyrylcholinesterase; ACh, acetylcholine; MAO, monoamine oxidase; MTDLs,
multi-target-directed ligands; EeAChE, Electrophorus electricus AChE; eqBuChE,
equine serum BuChE; SD, standard deviation; PDB, Protein Data Bank; CAS, catalytic
active site; PAS, peripheral anionic site; PAMPA-BBB, parallel artificial membrane
assay for the blood- brain barrier; TPSA, topological polar surface area; ADME,
absorption, distribution, metabolism, and excretion; HPLC, high-performance liquid
chromatography; PBL, Porcine brain lipid; ATC, acetylthiocholine chloride; BTC,
butyrylthiocholine chloride.
* Corresponding Author.
E-mail: sangzhipei@126.com (Zhipei Sang)
E-mail: liuwm1969@163.com (Wenmin Liu)
1

Abstract
A new family of multitarget molecules able to interact with acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE), as well as with monoamino oxidase
(MAO)
A
and
B,
has
been
synthesized.
Novel
3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives have been designed using a
conjunctive approach that combines the JMC49 and donepezil. The most promising
compound TM-33 showed potent and balance inhibitory activities toward ChE and
MAO (eeAChE, eqBuChE, hMAO-A and hMAO-B with IC50 values of 0.56 µM, 2.3
µM, 0.3 µM and 1.4 µM, respectively) but low selectivity. Both kinetic analysis of
AChE inhibition and molecular modeling study suggested that TM-33 binds
simultaneously to the catalytic active site and peripheral anionic site of AChE.
Furthermore, our investigation proved that TM-33 could cross the blood-brain barrier
(BBB) in vitro, and abided by Lipinski’s rule of five. The results suggest that
compound TM-33, an interesting multi-targeted active molecule, offers an attractive
starting point for further lead optimization in the drug-discovery process against
Alzheimer’s disease.
Keywords: Alzheimer’s disease; 3,4-dihydro-2(1H)-quinoline-O-alkylamines; AChE
−brain barrier
inhibitor; BuChE inhibitor; MAO-A inhibitor; MAO-B inhibitor; blood
.
2

1. Introduction
Alzheimer’s disease (AD), the most common form of adult onset dementia, is an
age-related neurodegenerative disorder characterized by a progressive memory loss, a
decline in language skills, and other cognitive impairments.1 In 2015, there was an
estimated 46 million people living with dementia worldwide, the dementia number is
estimated to increase to 131.5 million by 2050. Dementia also has a huge economic
impact, the total estimated worldwide cost of dementia is US $818 billion, and it will
become 2 trillion dollar disease by 2030.2 Current clinical treatment of AD is mainly
focused on the symptomatic aspects. The cholinergic hypothesis of memory
dysfunction in AD is the basis for treating this disease.3 In particular, the main
approach in the AD treatment is the improvement of cholinergic neurotransmission by
inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).4 By
increasing the levels of acetylcholine (ACh) in the brain through inhibition of these
enzymes, it is possible to reducing cognitive decline but cannot prevent, halt, or
reverse the progression of the disease.5
AD is a complicated disease with different molecular events, the decline in the
cholinergic function of the brain results in the loss of memory and cognitive functions
which are usually accompanied by behavioral disturbances.6 Studies showed that
serotoninergic and dopaminergic, were also thought to be responsible for the
behavioral disturbances observed in patients with AD.7-9 It suggested that inhibitors of
monoamine oxidase (IMAOs) might be also valuable for the treatment of AD. Thus,
monoamine oxidase (MAO), the enzyme that catalyzes the oxidative deamination of a
variety of biogenic and xenobiotic amines, is also an important target to be considered
for the treatment of specific features of this multifactorial disease.10 MAO exists as
two distinct enzymatic isoforms, MAO-A and MAO-B, based on their substrate and
inhibitor specificities. Selective inhibitors for MAO-A have been shown to be
effective antidepressants, whereas MAO-B inhibitors, although apparently devoid of
antidepressant action, are useful in the treatment of Parkinson’s disease.11 Moreover,
AD patients commonly present depressive symptoms that have even considered as a
risk factor for the development of the disease.12 Increased MAO-B levels due to
3

enhance astrogliosis in the brain of AD patients have also been reported.13 Overall,
these observations revealed that dual inhibition of MAO-A and MAO-B, rather than
MAO-B alone, may be of value for AD therapy.14
Given that the complexity of AD and the involvement of multi-systems in its
progression, one single drug that acts on a specific target to produce the desired
clinical effects might not be suitable for the complex nature of AD, alternatively, the
multi-target-directed ligands (MTDLs) strategy is considered as an effective way.15,16
It is well-known that many multifunctional agents have been synthesized as
therapeutics that possess simultaneously with two or more AD-relevant targets, and
the results obtained have been encouraging and convince researchers that MTDLs
might present the best pharmacological option for tackling the multifactorial nature of
AD and for halting the progression of the disease.17-19 Further studies suggested the
combination of MAO and ChE inhibition in the same molecule as a promising
strategy in the treatment of AD.20
In the development of IMAOs for the treatment of neurodegenerative diseases,
compound
JMC49
was
a
noticeable
MAO-B
inhibitor,
the
3,4-dihydro-2(1H)-quinoline scaffold was catalytic site MAO-B, and spacer was the
entrance cavity MAO-B.21,22 Moreover, the O-alkylamine side chains might increase
the MAO-A inhibitory activity.14 Donepezil is approved by the FDA as a selective
AChE inhibitor for the treatment of AD. It has been proved that the
1-benzylpiperidine fragment of donepezil is the pharmacophore. Recently, our group
has
reported
the
synthesis
of
genistein-O-alkylbenzylamines
and
scutellarein-O-alkylamines as potential multifunctional agents for the treatment of AD,
which has showed that the tertiary amino group linked lipophilic moiety binds to the
catalytic and mid-gorge sites of AChE.23,24 Taking the above mentioned into account,
based on MTDLs drug design strategy, we plan to fuse the
3,4-dihydro-2(1H)-quinoline and tertiary amino with methylene spacers of different
lengths, to test whether these novel molecules might possess more potency.
In this work, a series of 3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives
were designed based on MTDLs (Figure 1). They were also synthesized and
4

evaluated for their multifunctional biological activities, including inhibition of ChE
(AChE and BuChE) and MAO (MAO-A and MAO-B), the kinetics of enzyme
inhibition, and the ability of cross blood−brain barrier in vitro,. Finally, molecular
modeling studies were also performed to afford insight into the binding mode and the
structure-activity
relationships
of
the
novel
3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives.
<Insert Figure 1>
2. Results and discussion
2.1. Chemistry
The synthetic of 3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives were
depicted via the general pathway in Scheme 1, in which
7-hydroxy-3,4-dihydro-2(1H)-quinoline (1) was used as the starting material.
Alkyation of with excessive amounts of 1,3-dibromopropane (2a),
1
1,4-dibromobutane (2b), 1,5 –dibromopentane (2c) or 1,6-dibromohexane (2d) in the
presence of K2CO3 in CH3CN at 60–65°C resulted in the intermediates 3a-d. In
sequence,these intermediates were reacted with the corresponding secondary amines
4a-k in the presence of K2CO3 in CH3CN at 60–65°C to obtain the
final3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives TM1~33. All new
compounds were purified by chromatography, and the analytical and spectroscopic
data confirmed their structures, as detailed in the experimental section.
<Insert Scheme 1>
2.2. Pharmacology
2.2.1. Inhibition studies on AChE and BuChE
To
study
the
multipotent
profile
of
the
3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives, they were first evaluated as
inhibitors of AChE and BuChE. The inhibitory activities of target compounds
TM-1~TM-33 against eeAChE (from electric eel) and eqBuChE (from equine serum)
were measured according to the Ellman assay.23 Donepezil, a well-known
cholinesterase inhibitor, was used as reference standards, the precursor compound
5

7-hydroxy-3,4-dihydro-2(1H)-quinoline (1) was also evaluated for comparative
purpose. Furthermore, all the compounds were reevaluated using ratAChE and rat
BuChE. The results are listed in Table 1, expressed as IC50 values. Compared with
7-hydroxy-3,4-dihydro-2(1H)-quinoline (1) and donepezil, the tested target
compounds showed moderate to good potent regarding the inhibition of EeAChE with
IC50 values ranging from 0.56 to 23.5 µM (Table 1). Moreover, they exhibited similar
potencies against eqBuChE, as IC50 values range from 0.87 to 39.6 µM. Most target
compounds displayed slightly selective AChE inhibitors (the ratio
IC50(eqBuChE)/IC50(EeAChE) varies from 1.3 to 14.9), except compounds TM-7,
TM-12, TM-14, TM-15, TM-19, TM-21, TM-22, TM-23, TM-26, TM-28, TM-29,
TM-30 and TM-32. Accordingly, the length of the linker and the terminal groups
NR1R2 seem to be a significant factor for the inhibitory potency against AChE and
BuChE. The most potent compound was compound TM-33, which are characterized
by IC50 values of 0.56 µM against AChE and IC50 values of 2.3 µM against BuChE.
For EeAChE inhibition, the structure-activity relationship is from Table 1,
although the precursor compound 7-hydroxy-3,4-dihydro-2(1H)-quinoline (1) did not
show significant inhibitory activity, introduction of the O-alkylamines observably
increased the AChE inhibitory capacity. The results revealed that the terminal groups
NR1R2 of side chain caused significant effects on AChE inhibitory activities. To be
more specific, to start with, compounds TM-1 ~ TM-30 were synthesized, the data
showed that the piperazine substituent (4e, 4d, 4f) showed better inhibitory activity
than the others (4a, 4b, 4e, 4g, 4h). The methylene chain length also was an important
factor, with the same terminal groups NR1R2, AChE inhibitory activity increased with
an increase in methylene chain length, except compounds TM-9 and TM-21, where
the optimal chain length was four and five, respectively. However, inhibitory activity
decreased with an increase of methylene chain length when the terminal groups
NR1R2 was 1-(2-pyridyl)piperazine and N-benzylpiperazine, for example compounds
(TM-4, TM-12, TM-19, TM-26) and compounds (TM-6, TM-13, TM-20, TM-28).
It might be that the methylene chain length significantly affect the conformation,
further enhance the affinity for AChE via intermolecular hydrogen bond, π-π, σ-π
6

interaction with important amine acid residues, such as TM-3-TcAChE complex
(Figure S1A), TM-11-TcAChE (Figure S1B), TM-18-TcAChE complex (Figure
S1C) and TM-25-TcAChE (Figure S1D). Among of these derivatives, compound
TM-25, with isopropylpyrazine group and six methylene chain, exhibited the best
AChE inhibitory activity (IC50 = 0.76 µM). In addition, compounds TM-31~TM-33
was synthesized and evaluated, revealed good inhibitory activity. Among of which,
compound TM-33, with 4-benzylpiperidine, indicated the higher AChE inhibitory
activity (IC50 = 0.56 µM) than that of TM-25. Furthermore, the optimal compound
TM-33 was re-evaluated using huAChE, and showed a good inhibitory activity with
IC50 value was 1.02 0.01 µM .
For eqBuChE inhibition, generally, the compounds with NR1R2 substituent
groups 4c, 4g, 4i, 4j and 4k displayed better inhibitory potency than that with others.
In the same conditions, BuChE inhibitory activity increased with an increase in
methylene chain length, except compounds TM-10 and TM-21, where the optimal
chain length was four and five, respectively. Compound TM-32, with pyrrole,
appeared the best BuChE inhibitory activity (IC50 = 0.87 µM). Compound TM-33
also indicated good BuChE inhibitory with IC50 value of 2.3 µM. From the
re-evaluated results by additional testing using ratAChE and ratBuChE, it is notable
that most of the tested compounds were selective AChE inhibitors and exhibited
weaker inhibitory activity towards ChE (ratAChE and ratBuChE) than towards ChE
(EeAChE and eqBuChE), expect compounds TM-3, TM-7, TM-10, TM-14, TM-17,
TM-18, TM-24, TM-25 and TM-31, which were selective BuChE inhibitors.
Compound TM-33 presented the highest inhibitory activity toward ratAChE and
ratBuChE with IC50 value of 1.9 µM and 3.6 µM, respectively.
<Insert Table 1>
2.2.2. Kinetic studies of AChE inhibition
To investigate the AChE inhibitory mechanism for this class of
3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives, a kinetic study was carried
out with a representative compound TM-33 using EeAChE (Figure 2).25 The
Lineweaver–Burk plots exhibited that both inhibitions had rising slopes and
7

increasing intercepts at higher concentration, which indicated a mixed-type inhibition.
The result demonstrated that compound TM-33 could bind to both CAS and PAS of
AChE.
<Insert Figure 2>
2.2.3. Molecular modeling studies
In order to explore possible binding mechanism to AChE and BuChE, a further
computational study was performed for compound TM-33 using the docking program,
AutoDock 4.2 package with Discovery Studio 2.5, based on the X-ray crystal
structure of TcAChE (PDB code: 1EVE) and huBuChE (PDB code: 4TPK).23,25 As
shown in Figure 3, the results indicated that TM-33 occupied the entire enzymatic
CAS, the mid-gorge sites and the PAS, and could simultaneously bind to both the
catalytic site and the peripheral site thus providing an explanation for its highly potent
inhibitory activity against AChE. In the TM-33-TcAChE complex, the
4-benzylpiperidine of moiety of TM-33 was observed to bind to the CAS via a
sigma-Pi interaction with TRY334. The long chain of methylene could fold into a
conformation in the gorge that allowed them to interact with PHE330 and TYR121
through hydrophobic interaction. In addition, the carbonyl group and NH at the
3,4-dihydro-2(1H)-quinoline nucleus could simultaneously bind to the SER124 and
TRP84 via two intermolecular hydrogen bonds. The results of the docking in Figure 4
also showed that compound TM-33 bind with BuChE via multiple sites. The carbonyl
group and NH at the 3,4-dihydro-2(1H)-quinoline nucleus could simultaneously bind
to the TYR128 via two intermolecular hydrogen bonds. The benzene ring of
3,4-dihydro-2(1H)-quinoline nucleus via two Pi-Pi interaction with TRP82. Moreover,
hydrophobic interactions could be observed between the ligand and GLY116, GLY117,
ALA328, PHE329 and HIS438. Based on these results above, the strong affinity of
compound TM-33 bind to amino acid residues may be partially explained the high
potent in inhibiting AChE and BuChE.
<Insert Figure 3>
<Insert Figure 4>
2.2.4. Recombinant human MAO-A and-B inhibition studies
8

The synthesized derivatives were tested in vitro for their MAO-A and –B
inhibitory activities using clorgyline, rasagiline and iproniazid as reference drugs.
Recombinant human MAO-A and –B were employed as enzyme sources, and their
activities were determined by a fluorimetric method with kynuramine as a common
substrate.14 As shown in Table 2, the synthesized target compounds were significantly
selective hMAO-A inhibitors, except compounds TM-1, TM-8, TM-9, TM-10, TM-11, TM-17,
TM-22 and TM-28 were selective hMAO-B inhibitors. All the derivatives displayed
moderate to good MAO-A inhibition (IC50 values range from 0.3 to 14.3 µM) and
significant MAO-B inhibition (IC50 values range from 1.4 to 31.7 µM). Among of
which, compound TM-33, with 4-benzylpiperidine, exhibited the most potent
compound toward both isoforms (IC50 of 0.3 0.001 µM and 1.4 0.01 µM for
MAO-A and MAO-B, respectively). The data appeared that MAO-A inhibitory
activity increased with an increase in methylene chain length, except compounds
TM-20, where the optimal chain length was five. However, inhibitory activity
decreased with an increase of methylene chain length when substituent group NR1R2
was 1-(2-pyridyl)piperazine, for example compounds (TM-4, TM-12, TM-19,
TM-26). The O-alkylamines side chain also had significant effects on MAO-B
inhibition. Generally, with the same terminal groups NR1R2, MAO-B inhibitory
activity increased with an increase in methylene chain length, except compounds
TM-10, TM-11 and TM-21, where the optimal chain length was four. However,
inhibitory activity decreased with an increase of methylene chain length when the
terminal groups NR1R2 was piperidine, for example compounds (TM-1, TM-9,
TM-16, TM-23). The terminal groups NR1R2 (4f, 4g, 4h, 4i, 4j and 4k) showed good
inhibitory potency toward MAO-B inhibition. In particular, compound TM-33, with
4-benzylpiperidine, indicated the highest inhibitory activity with IC50 values of 1.4
µM.
<Insert Table 2>
2.2.5. Cytotoxic effects
To examine the cytotoxicity of the representative compound TM-33, PC12 cells were
9

exposed to the test compound with increasing concentrations (1, 10 and 100 µM) for
24 h, and the cell viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium (MTT) assays. As shown in Figure 5, TM-33 did not show
modified cell viability up to the concentration of 10 µM. With increased concentration
to 100 µM, TM-33 induced a decrease of cell viability (87.3%). The result showed
that the target compound TM-33 had a wide therapeutic safety range on PC12 cells.
<Insert Figure 5>
2.2.6. In vitro blood−brain barrier permeation assay
Brain penetration and acute toxicity are major requirements for successful CNS
drugs. To evaluate the brain penetration of the compound TM-33, we used a parallel
artificial membrane assay for the blood- brain barrier (PAMPA-BBB), as described by
Di et al.27,28 We compared the permeability of 11 commercial drugs with reported
values to validate the assay (Table S1). A plot of the experimental data versus the
reported values produced a good linear correlation, Pe (exp) = 0.9163Pe (bibl.)
−0.2247 (r2 = 0.9558) (Figure S1). From this equation, and considering the limit
established by Di et al. for blood−brain barrier permeation, we determined that
compounds with permeabilities above 3.44×10−6 cm/s could cross the blood−brain
barrier (Table S2). According to the measured permeability (Table 3), as expected,
TM-33 could cross the BBB and reach the biological targets located in the CNS.
<Insert Table 3>
2.2.7. Theoretical evaluation of ADME properties.
To correlate better the druglike properties of ferulic acid-donepezil hybrids, the
lipophilicity, expressed as the octanol/water partition coefficient and herein called log
P, as well as other theoretical calculations such as the topological polar surface area
(TPSA), the number of hydrogen-bond acceptors, and the number of hydrogen-bond
donors were calculated using the Molinspiration property program.23 The theoretical
prediction of the absorption, distribution, metabolism, and excretion (ADME)
properties of compound TM-33 is summarized in Table 4.
<Insert Table 4>
10

From the data obtained for the theoretical evaluation of the ADME properties,
compound TM-33 do not break any point of the Lipinski’s rule of five, making them
promising leads for drug candidates. TPSA and log P values are compatible with those
described as a predictive indicator of a drug’s capacity for membrane penetration.
3. Conclusion
In conclusion, this article involved the synthesis of a new series of
3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives acted as multi-target-directed
ligands for the treatment of Alzheimer’s disease. Among the synthesized compounds,
compound TM-33 demonstrated the highest AChE and BuChE inhibitory activities
with IC50 values of 0.56 µM and 2.3 µM, and exhibited favourable hMAO-A (IC50 =
0.3
0.001 µM) and hMAO-B (IC50 = 1.4
0.01 µM) inhibitory potency. The
molecular modeling might play a key role in the structure-activity relationship. The
kinetic analysis suggested that TM-33 showed mixed-type inhibition, and could bind
to both CAS and PAS of AChE, which was consistent with the molecular modeling
study. More interestingly, our study proved that TM-33 could cross the blood-brain
barrier (BBB) in vitro, and complied with Lipinski’s rule of five. The present study
indicated that TM-33 could be considered as an interesting multi-targeted lead
compound for the further study in the treatment of advanced AD. Further
investigations to evaluate compound TM-33 in vivo and to develop structural
refinements are in progress and will be reported in due course.
4. Experimental section
4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers
and used without further purification. The purity of all final compounds was
determined by high-performance liquid chromatography (HPLC) analysis to be over
97%. HPLC analysis was carried out on a Waters600-2487 plus system with the use of
1
a Kromasil C18 column (4.6 mm × 250 mm, 5um). H and 13C NMR spectra were
recorded using TMS as the internal standard in CDCl3 with a Varian INOVA
spectrometer at 400 and 100 MHz, respectively. Coupling constants were given in Hz.
11

Multiplicities are given as s (singlet), d (doublet), dd (double-doublet), t (triplet), q
(quadruplet), m (multiplet), and br (broad signal). MS spectra data were obtained on
an Agilent-6210 TOF LC-MS spectrometer. All the reactions were monitored by
thin-layer chromatography (TLC) using silica gel GF254 plates from Qingdao Haiyang
Chemical Co. Ltd. (China) with an UV lamp (254nm). Column chromatography using
silica gel (230–400 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd.
(China).
4.1.1. General procedure for the synthesis of intermediates 3a-3d. The appropriate
dibromoalkane derivative 2a-2d (4.4 mmol) was added to a mixture of the starting
material 7-hydroxy-3,4-dihydro-2(1H)-quinoline (1) (2.0 mmol), anhydrous K2CO3
(290 mg, 2.1 mmol) in CH3CN (8 mL). The reaction mixture was heated to 60-65 °C
and stirred for 8-10 h under an argon atmosphere. After complete reaction, the solvent
was evaporated under reduced pressure. Water (30 mL) was added to the residue and
the mixture was extracted with dichloromethane (30 mL × 3). The combined organic
phases were washed with saturated aqueous sodium chloride, dried over sodium
sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure.
The residue was purified on
a
silica gel chromatography using
dichloromethane/acetone (50:1) as eluent to give the intermediates 3a-3d.
7-(3-Bromopropoxy)-3,4-dihydroquinolin-2(1H)-one (3a). Colorless oil, 65.3%
1
yield. H NMR (400 MHz, CDCl3) δ 8.15 (s, 1H, CONH ), 7.06 (d, J = 8.0 Hz, 1H,
Ar-H), 6.54 (d, J = 8.0 Hz, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 4.08 (t, J = 5.6 Hz, 2H,
OCH2), 3.60 (t, J = 6.0 Hz, 2H, BrCH2), 2.90 (t, J = 7.2 Hz, 2H, phCH2), 2.62 (t, J =
7.2 Hz, COCH2), 2.33-2.27 (m, 2H, CH2).
7-(4-Bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (3b). Colorless oil, 68.7% yield.
1H NMR (400 MHz, CDCl3) δ 8.55 (s, 1H, CONH ), 7.04 (d, J = 8.0 Hz, 1H, Ar-H),
6.51 (d, J = 8.0 Hz, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 3.97 (t, J = 5.6 Hz, 2H, OCH2),
3.49 (t, J = 6.4 Hz, 2H, BrCH2), 2.89 (t, J = 7.2 Hz, 2H, phCH2), 2.62 (t, J = 7.2 Hz,
COCH2), 2.09-2.02 (m, 2H, CH2), 1.96-1.89 (m, 2H, CH2).
7-((5-Bromopentyl)oxy)-3,4-dihydroquinolin-2(1H)-one (3c). Colorless oil, 60.2%
1
yield. H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H, CONH ), 7.06 (d, J = 8.0 Hz, 1H,
12

Ar-H), 6.53 (dd, J1 = 6.4 Hz, J2 = 2.0 Hz, 1H, Ar-H), 6.39 (d, J = 2.0 Hz, 1H, Ar-H),
3.96 (t, J = 6.4 Hz, 2H, OCH2), 3.46 (t, J = 6.4 Hz, 2H, BrCH2), 2.91 (t, J = 7.2 Hz,
2H, phCH2), 2.64 (t, J = 7.2 Hz, COCH2), 1.99-1.91 (m, 2H, CH2), 1.85-1.77 (m, 2H,
CH2), 1.65-1.61 (m, 2H, CH2).
7-((6-Bromohexyl)oxy)-3,4-dihydroquinolin-2(1H)-one (3d). Colorless oil, 56.8%
1
yield. H NMR (400 MHz, CDCl3) δ 9.37 (s, 1H, CONH ), 7.04 (d, J = 8.0 Hz, 1H,
Ar-H), 6.52 (dd, J1 = 6.0 Hz, J2 = 2.0 Hz, 1H, Ar-H), 6.43 (d, J = 1.6 Hz, 1H, Ar-H),
3.94 (t, J = 6.4 Hz, 2H, OCH2), 3.43 (t, J = 6.8 Hz, 2H, BrCH2), 2.90 (t, J = 7.6 Hz,
2H, phCH2), 2.63 (t, J = 8.0 Hz, COCH2), 1.92-1.87 (m, 2H, CH2), 1.80-1.76 (m, 2H,
CH2), 1.52-1.49 (m, 4H, 2 × CH2).
4.1.2.
General
procedure
for
the
synthesis
of
3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives TM1~TM31. To a mixture
of the corresponding secondary amines 4a~4k (0.6 mmol), anhydrous K2CO3 (89.7
mg, 0.65 mmol) in anhydrous CH3CN (7 mL) were added the appropriate
intermediates 10–13 (0.5 mmol). The reaction mixture was warmed to 60-65 °C and
stirred for 6–10 h under an argon atmosphere. After complete reaction, the solvent
was evaporated under reduced pressure. The residue was dissolved in water (30 mL)
and the mixture was extracted with dichloromethane (20 mL × 3). The combined
organic phases were washed with saturated aqueous sodium chloride (30 mL), dried
over sodium sulfate, and filtered. The solvent was evaporated to dryness under
reduced pressure. The residue was purified on a silica gel chromatography using
mixtures of dichloromethane/acetone (30:1) as eluent to afford the corresponding
3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives TM1~TM31.
7-(3-(Piperidin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one (TM-1). Colorless
1
oil, 75.2% yield. 98.2% HPLC purity. H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H),
7.03 (d, J = 8.4 Hz, 1H), 6.52 (dd, J1 = 6.4 Hz, J2 = 1.6 Hz, 1H), 6.36 (s, 1H), 3.98 (t,
J = 6.4 Hz, 2H), 2.89 (t, J = 7.6 Hz, 2H), 2.62 (t, J = 7.6 Hz, 2H), 2.51 (t, J = 7.6 Hz,
2H), 2.46-2.41 (m, 4H), 1.64-1.59 (m, 4H), 1.48-1.42 (m, 2H). MS (ESI) m/z: 289.2
[M + H] +.
7-(3-(Diethylamino)propoxy)-3,4-dihydroquinolin-2(1H)-one (TM-2). Colorless
13

1
oil, 80.6% yield. 98.6% HPLC purity. H NMR (400 MHz, CDCl3) δ 9.23 (s, 1H),
6.95 (d, J = 8.4 Hz, 1H), 6.44 (dd, J1 = 6.0 Hz, J2 = 2.4Hz, 1H), 6.36 (d, J = 2.4 Hz,
1H), 3.90 (t, J = 6.0 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H),
2.57-2.51 (m, 4H), 1.91-1.84 (m, 2H), 1.00 (t, J = 7.2 Hz, 6H). MS (ESI) m/z: 277.2
[M + H] +.
7-(3-(4-Isopropylpiperazin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one (TM-3).
1
Light yellow oil, 69.4% yield. 98.0% HPLC purity. H NMR (400 MHz, CDCl3) δ
8.48 (s, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.52 (dd, J1 = 6.0 Hz, J2 = 2.4 Hz, 1H), 6.35 (d,
J = 2.0 Hz, 1H), 3.97 (t, J = 6.4 Hz, 2H), 2.89 (t, J = 7.6 Hz, 2H), 2.71-2.65 (q, J = 6.4
Hz, 1H), 2.61 (t, J = 8.0 Hz, 2H), 2.58-2.50 (m, 10H), 1.99-1.92 (m, 2H), 1.06 (d, J =
6.4 Hz, 6H). MS (ESI) m/z: 332.2 [M + H] +.
7-(3-(4-(Pyridin-2-yl)piperazin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one
1
(TM-4). Light yellow oil, 76.8% yield. 98.5% HPLC purity. H NMR (400 MHz,
CDCl3) δ 8.49 (s, 1H), 8.19 (d, J = 3.6 Hz, 1H), 7.48 (t, J = 7.2 Hz, 1H), 7.04 (d, J =
8.0 Hz, 1H), 6.67-6.61 (m, 2H), 6.53 (dd, J1 = 6.0 Hz, J2 = 2.4 Hz, 1H), 6.37 (d, J =
2.0 Hz, 1H), 4.02 (t, J = 6.0 Hz, 2H), 3.59 (t, J = 8.4 Hz, 4H), 2.89 (t, J = 7.2 Hz, 2H),
2.63-2.59 (m, 8H), 2.06-1.99 (m, 2H). MS (ESI) m/z: 367.2 [M + H] +.
7-(3-(4-(Pyrrolidin-1-yl)piperidin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one
1
(TM-5). Light yellow oil, 84.1% yield. 98.0% HPLC purity. H NMR (400 MHz,
CDCl3) δ 8.98 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.67-6.61 (m, 2H), 6.51 (dd, J1 = 6.0
Hz, J2 = 2.0 Hz, 1H), 6.42 (d, J = 2.0 Hz, 1H), 3.98 (t, J = 6.0 Hz, 2H), 2.98 (d, J =
11.2 Hz, 2H), 2.88 (t, J = 8.0 Hz, 2H), 2.80-2.76 (m, 4H), 2.61 (t, J = 8.0 Hz, 2H),
2.51 (t, J = 7.2 Hz, 2H), 2.30 (t, J = 6.8 Hz, 1H), 2.03 (t, J = 10.8 Hz, 2H), 1.98-1.86
(m, 8H), 1.76-1.68 (m, 2H). MS (ESI) m/z: 358.2 [M + H] +.
7-(3-(4-Benzylpiperazin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one
(TM-6).
1
Light yellow oil, 63.9% yield. 97.8% HPLC purity. H NMR (400 MHz, CDCl3) δ
9.39 (s, 1H), 7.33-7.24 (m, 5H), 7.03 (d, J = 8.4 Hz, 1H), 6.52 (dd, J1 = 5.6 Hz, J2 =
2.4 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 3.97 (t, J = 6.0 Hz, 2H), 3.53 (s, 2H), 2.89 (t, J
= 8.0 Hz, 2H), 2.62 (t, J = 8.0 Hz, 2H), 2.58-2.46 (m, 10H), 1.99-1.92 (m, 2H). MS
(ESI) m/z: 380.2 [M + H] +.
14

7-(3-(3,4-Dihydroisoquinolin-2(1H)-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one
(TM-7). Colorless oil, 67.6% yield. 98.9% HPLC purity. 1H NMR (400 MHz, CDCl3)
δ 9.40 (s, 1H), 7.12-6.98 (m, 5H), 6.52 (dd, J1 = 6.0 Hz, J2 = 2.4 Hz, 1H), 6.41 (d, J =
2.0 Hz, 1H), 4.00 (t, J = 6.0 Hz, 2H), 3.65 (s, 2H), 2.90 (t, J = 5.6 Hz, 2H), 2.85 (t, J =
8.0 Hz, 2H), 2.75 (t, J = 5.6 Hz, 2H), 2.68 (t, J = 7.2 Hz, 2H), 2.59 (t, J = 7.6 Hz, 2H),
13
2.08-2.02 (m, 2H). C NMR (100 MHz, CDCl3) 172.53, 158.69, 138.35, 134.74,
134.32, 128.69, 128.53, 126.61, 126.18, 125.66, 115.69, 108.80, 102.43, 66.43, 56.20,
54.93, 51.03, 31.10, 29.11, 27.06, 24.58. MS (ESI) m/z: 337.2 [M + H] +.
7-(3-(Benzyl(prop-2-yn-1-yl)amino)propoxy)-3,4-dihydroquinolin-2(1H)-one
(TM-8). Colorless oil, 66.7% yield. 98.5% HPLC purity. 1H NMR (400 MHz, CDCl3)
δ 7.68 (s, 1H), 7.36-7.27 (m, 5H), 7.06 (d, J = 8.4 Hz, 1H), 6.54 (dd, J1 = 6.4 Hz, J2 =
1.6 Hz, 1H), 6.29 (d, J = 1.6 Hz, 1H), 4.02 (t, J = 6.4 Hz, 2H), 3.68 (s, 2H), 3.36 (d, J
= 1.6 Hz, 2H), 2.92 (t, J = 7.2 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H), 2.64 (t, J = 7.2 Hz,
2H), 2.25 (s, 1H), 2.01-1.94 (m, 2H). MS (ESI) m/z: 349.2 [M + H] +.
7-(4-(Piperidin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (TM-9). Light yellow
1
oil, 68.7% yield. 98.1% HPLC purity. H NMR (400 MHz, CDCl3) δ 9.19 (s, 1H),
7.02 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 7.6 Hz, 1H), 6.40 (s, 1H), 3.94 (t, J = 6.0 Hz,
2H), 2.89 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 7.6 Hz, 2H), 2.43-2.33 (m, 6H), 1.79-1.72
(m, 2H), 1.69-1.64 (m, 2H), 1.60-1.57 (m, 4H), 1.46-1.42 (m, 2H, CH2). MS (ESI)
m/z: 312.2 [M + H] +.
7-(4-(Diethylamino)butoxy)-3,4-dihydroquinolin-2(1H)-one (TM-10). Colorless
oil, 83.5% yield. 98.6% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 8.55 (s, 1H),
6.96 (d, J = 8.4 Hz, 1H), 6.44 (dd, J1 = 6.0 Hz, J2 = 2.4 Hz, 1H), 6.32 (d, J = 2.4 Hz,
1H), 3.89 (t, J = 5.6 Hz, 2H), 2.82 (t, J = 8.0 Hz, 2H), 2.71 (q, J = 7.2 Hz, 4H), 2.65 (t,
J = 7.6 Hz, 2H), 2.54 (t, J = 8.0 Hz, 2H), 1.75-1.68 (m, 4H), 1.10 (t, J = 7.6 Hz, 2H).
13C NMR (100 MHz, CDCl3) 172.00, 158.07, 138.56, 128.43, 115.89, 108.74, 102.50,
67.07, 51.05, 46.64, 31.01, 26.32, 24.48, 20.47, 8.68. MS (ESI) m/z: 291.2 [M + H] +.
7-(4-(4-Isopropylpiperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (TM-11).
1
Light yellow oil, 67.1% yield. 98.2% HPLC purity. H NMR (400 MHz, CDCl3) δ
7.95 (s, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.51 (dd, J1 = 6.0 Hz, J2 = 2.4 Hz, 1H), 6.30 (d,
15

J = 2.4 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 2.90 (t, J = 7.6 Hz, 2H), 2.74-2.52 (m, 11H),
2.42 (t, J = 7.6 Hz, 2H), 1.82-1.75 (m, 2H), 1.71-1.63 (m, 2H), 1.09 (d, J = 6.4 Hz,
6H). 13C NMR (100 MHz, CDCl3) 172.14, 158.41, 138.46, 128.41, 115.64, 108.76,
102.38, 67.54, 57.12, 56.27, 50.61, 47.40, 31.04, 26.84, 24.50, 22.62, 17.36. MS (ESI)
m/z: 346.2 [M + H] +.
7-(4-(4-(Pyridin-2-yl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one
1
(TM-12). Light yellow oil, 60.8% yield. 98.0% HPLC purity. H NMR (400 MHz,
CDCl3) δ 9.07 (s, 1H), 8.19 (d, J = 2.4 Hz, 1H), 7.50-7.45 (m, 1H), 7.03 (t, J = 8.0 Hz,
1H), 6.66-6.60 (m, 2H), 6.52 (dd, J1 = 5.6 Hz, J2 = 2.4 Hz, 1H), 6.37 (d, J = 13.6 Hz,
1H), 3.96 (t, J = 5.6 Hz, 2H), 3.57-3.53 (m, 4H), 2.91-2.86 (m, 2H), 2.64-2.57 (m,
6H), 2.48-2.43 (m, 2H), 1.85-1.78 (m, 2H), 1.75-1.71 (m, 2H). MS (ESI) m/z: 381.2
[M + H] +.
7-(4-(4-Benzylpiperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one
(TM-13).
1
Light yellow oil, 68.7% yield. 97.8% HPLC purity. H NMR (400 MHz, CDCl3) δ
8.28 (s, 1H), 7.34-7.25 (m, 5H), 7.05 (d, J = 8.4 Hz, 1H), 6.52 (dd, J1 = 6.0 Hz, J2 =
2.0 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 3.95 (t, J = 6.4 Hz, 2H), 3.53 (s, 2H), 2.91 (t, J
= 7.6 Hz, 2H), 2.63 (t, J = 8.0 Hz, 2H), 2.56-2.46 (m, 8H), 2.42 (t, J = 7.6 Hz, 2H),
1.81-1.75 (m, 2H), 1.71-1.64 (m, 2H). MS (ESI) m/z: 394.2 [M + H] +.
7-(4-(3,4-Dihydroisoquinolin-2(1H)-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one
1
(TM-14). Light yellow oil, 73.5% yield. 98.3% HPLC purity. H NMR (400 MHz,
CDCl3) δ 8.52 (s, 1H), 7.15-7.10 (m, 3H), 7.06-7.03 (m, 2H), 6.54 (dd, J1 = 6.4 Hz, J2
= 2.0 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 3.99 (t, J = 6.0 Hz, 2H), 3.67 (s, 2H),
2.95-2.88 (m, 4H), 2.77 (t, J = 6.0 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.60 (t, J = 6.0
Hz, 2H), 1.90-1.76 (m, 4H). 13C NMR (100 MHz, CDCl3) 171.97, 158.70, 138.18,
134.75, 134.31, 128.66, 128.62, 126.60, 126.14, 125.61, 115.68, 108.78, 102.26,
67.94, 57.93, 56.14, 50.92, 31.11, 29.07, 27.24, 24.59, 23.68. MS (ESI) m/z: 351.2 [M
+ H] +.
7-(4-(Benzyl(prop-2-yn-1-yl)amino)butoxy)-3,4-dihydroquinolin-2(1H)-one
1
(TM-15). Light yellow oil, 69.3% yield. 97.6% HPLC purity. H NMR (400 MHz,
CDCl3) δ 8.96 (s, 1H), 7.38-7.27 (m, 5H), 7.05 (d, J = 8.0 Hz, 1H), 6.53 (dd, J1 = 6.8
16

Hz, J2 = 1.6 Hz, 1H), 6.40 (d, J = 1.6 Hz, 1H), 3.95 (t, J = 6.4 Hz, 2H), 3.66 (s, 2H),
3.36 (s, 2H), 2.91 (t, J = 7.6 Hz, 2H), 2.64 (t, J = 6.8 Hz, 4H), 2.27 (s, 1H), 1.88-1.81
(m, 2H), 1.74-1.66 (m, 2H). MS (ESI) m/z: 363.2 [M + H] +.
7-((5-(Piperidin-1-yl)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-one
(TM-16).
Colorless oil, 88.9% yield. 99.2% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 8.83 (s,
1H), 7.03 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 8.4 Hz, 1H), 6.38 (s, 1H), 3.92 (t, J = 6.4
Hz, 2H), 2.89 (t, J = 7.6 Hz, 2H), 2.62 (t, J = 7.6 Hz, 2H), 2.48-2.41 (m, 4H), 2.36 (t,
J = 7.2 Hz, 2H), 1.81-1.74 (m, 2H), 1.64-1.55 (m, 6H), 1.49-1.41 (m, 4H). MS (ESI)
m/z: 317.2 [M + H] +.
7-((5-(Diethylamino)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-one (TM-17). Light
yellow oil, 87.6% yield. 98.6% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 9.18 (s,
1H), 6.94 (d, J = 8.0 Hz, 1H), 6.43-6.40 (m, 2H), 3.86 (t, J = 6.0 Hz, 2H), 2.85 (q, J =
7.2 Hz, 4H), 2.80 (t, J = 7.6 Hz, 2H), 2.73 (t, J = 8.0 Hz, 2H), 2.52 (t, J = 8.0 Hz, 2H),
1.75-1.64 (m, 4H), 1.18 (t, J = 7.6 Hz, 6H), 1.47-1.39 (m, 2H). 13C NMR (100 MHz,
CDCl3) 172.04, 158.35, 138.47, 128.43, 115.68, 108.82, 102.43, 67.51, 51.32, 46.58,
31.05, 28.43, 24.50, 23.49, 23.15, 8.63. MS (ESI) m/z: 305.2 [M + H] +.
7-((5-(4-Isopropylpiperazin-1-yl)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-one
1
(TM-18). Light yellow oil, 83.9% yield. 98.4% HPLC purity. H NMR (400 MHz,
CDCl3) δ 8.45 (s, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 8.0 Hz, 1H), 6.35 (s, 1H),
3.92 (t, J = 6.4 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.68-2.45 (m, 11H), 2.37 (t, J = 8.0
Hz, 2H), 1.82-1.74 (m, 2H), 1.61-1.53 (m, 2H), 1.50-1.42 (m, 2H), 1.06 (d, J = 6.4 Hz,
6H). MS (ESI) m/z: 360.3 [M + H] +.
7-((5-(4-(Pyridin-2-yl)piperazin-1-yl)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-one
1
(TM-19). Light yellow oil, 82.6% yield. 98.0% HPLC purity. H NMR (400 MHz,
CDCl3) δ 8.60 (s, 1H), 8.20 (d, J = 7.2 Hz, 1H), 7.49 (t, J = 8.8 Hz, 1H), 7.04 (d, J =
8.4 Hz, 1H), 6.67-6.62 (m, 2H), 6.52 (dd, J1 = 6.0 Hz, J2 = 2.4 Hz, 1H), 6.38 (d, J =
2.4 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 3.61-3.56 (m, 4H), 2.90 (t, J = 8.0 Hz, 2H),
2.64-2.58 (m, 6H), 2.45 (t, J = 8.0 Hz, 2H), 1.84-1.77 (m, 2H), 1.69-1.59 (m, 2H),
1.54-1.47 (m, 2H). MS (ESI) m/z: 395.2 [M + H] +.
7-((5-(4-Benzylpiperazin-1-yl)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-one
17

1
(TM-20). Light yellow oil, 80.7% yield. 98.8% HPLC purity. H NMR (400 MHz,
CDCl3) δ 8.34 (s, 1H), 7.33 (d, J = 8.4 Hz, 4H), 7.28-7.24 (m, 1H), 7.05 (d, J = 8.4 Hz,
1H), 6.52 (dd, J1 = 5.6 Hz, J2 = 2.4 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 3.93 (t, J = 6.4
Hz, 2H), 3.53 (s, 2H), 2.91 (t, J = 8.0 Hz, 2H), 2.63 (t, J = 8.0 Hz, 2H), 2.54-2.42 (m,
8H), 2.38 (t, J = 8.0 Hz, 2H), 1.83-1.75 (m, 2H), 1.61-1.54 (m, 2H), 1.51-1.43 (m,
2H). MS (ESI) m/z: 408.3 [M + H] +.
7-((5-(3,4-Dihydroisoquinolin-2(1H)-yl)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-on
e (TM-21). 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.33 (d, J = 8.4 Hz, 4H),
7.28-7.24 (m, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.52 (dd, J1 = 5.6 Hz, J2 = 2.4 Hz, 1H),
6.34 (d, J = 2.4 Hz, 1H), 3.93 (t, J = 6.4 Hz, 2H), 3.53 (s, 2H), 2.91 (t, J = 8.0 Hz, 2H),
2.63 (t, J = 8.0 Hz, 2H), 2.54-2.42 (m, 8H), 2.38 (t, J = 8.0 Hz, 2H), 1.83-1.75 (m,
2H), 1.61-1.54 (m, 2H), 1.51-1.43 (m, 2H). 13C NMR (100 MHz, CDCl3) 172.17,
158.74, 138.23, 134.77, 134.31, 128.65, 128.57, 126.60, 126.11, 125.59, 115.62,
108.76, 102.30, 68.02, 58.29, 56.18, 51.00, 31.10, 29.18, 29.04, 26.91, 24.58, 24.08.
MS (ESI) m/z: 365.2 [M + H] +.
7-((5-(Benzyl(prop-2-yn-1-yl)amino)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-one
(TM-22). Light yellow oil, 82.6% yield. 98.2% HPLC purity. 1H NMR (400 MHz,
CDCl3) δ 9.08 (s, 1H), 7.38-7.27 (m, 5H), 7.05 (d, J = 8.0 Hz, 1H), 6.54 (d, J = 8.0 Hz,
1H), 6.41 (s, 1H), 3.94 (t, J = 6.4 Hz, 2H), 3.65 (s, 2H), 3.34 (s, 2H), 2.91 (t, J = 7.6
Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H), 2.26 (s, 1H), 1.83-1.76 (m,
2H), 1.64-1.56 (m, 2H), 1.55-1.48 (m, 2H). MS (ESI) m/z: 377.2 [M + H] +.
7-((6-(Piperidin-1-yl)hexyl)oxy)-3,4-dihydroquinolin-2(1H)-one
(TM-23).
Colorless oil, 87.5% yield. 98.7% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 8.44 (s,
1H), 7.03 (d, J = 8.0 Hz, 1H), 6.51 (d, J = 8.4 Hz, 1H), 6.36 (s, 1H), 3.91 (t, J = 6.4
Hz, 2H), 2.89 (t, J = 8.0 Hz, 2H), 2.61 (t, J = 8.0 Hz, 2H), 2.58-2.51 (m, 4H), 2.45 (t,
J = 8.0 Hz, 2H), 1.78-1.66 (m, 6H), 1.65-1.58 (m, 2H), 1.51-1.44 (m, 4H), 1.40-1.33
(m, 2H). MS (ESI) m/z: 331.2 [M + H] +.
7-((6-(Diethylamino)hexyl)oxy)-3,4-dihydroquinolin-2(1H)-one (TM-24). Light
1
yellow oil, 82.9% yield. 98.7% HPLC purity. H NMR (400 MHz, CDCl3) δ 9.19 (s,
1H), 6.96 (d, J = 8.0 Hz, 1H), 6.44 (dd, J1 = 6.0 Hz, J1 = 2.4 Hz, 1H), 6.34 (d, J = 2.4
18

Hz, 1H), 3.84 (t, J = 6.4 Hz, 2H), 2.81 (t, J = 7.6 Hz, 2H), 2.54 (t, J = 8.0 Hz, 2H),
2.47 (q, J = 7.2 Hz, 4H), 2.36 (d, J = 8.0 Hz, 2H), 1.72-1.64 (m, 2H), 1.45-1.35 (m,
4H), 1.30-1.22 (m, 2H), 0.96 (t, J = 7.6 Hz, 6H). MS (ESI) m/z: 319.2 [M + H] +.
7-((6-(4-Isopropylpiperazin-1-yl)hexyl)oxy)-3,4-dihydroquinolin-2(1H)-one
1
(TM-25). Light yellow oil, 79.5% yield. 98.1% HPLC purity. H NMR (400 MHz,
CDCl3) δ 9.59 (s, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.42 (d, J = 8.0 Hz, 1H), 6.37 (s, 1H),
3.82 (t, J = 6.0 Hz, 2H), 2.79 (t, J = 6.4 Hz, 2H), 2.68-2.44 (m, 11H), 2.28 (t, J = 5.6
Hz, 2H), 1.72-1.65 (m, 2H), 1.48-1.42 (m, 2H), 1.40-1.35 (m, 2H), 1.32-1.25 (m, 2H),
0.99 (d, J = 6.0 Hz, 6H). 13C NMR (100 MHz, CDCl3) 172.42, 158.67, 138.33, 128.41,
115.48, 108.71, 102.30, 67.94, 58.53, 54.58, 53.19, 48.43, 31.05, 29.11, 27.28, 26.63,
25.92, 24.53, 18.47. MS (ESI) m/z: 374.3 [M + H] +.
7-((6-(4-(Pyridin-2-yl)piperazin-1-yl)hexyl)oxy)-3,4-dihydroquinolin-2(1H)-one
(TM-26). Light yellow oil, 80.2% yield. 98.8% HPLC purity. 1H NMR (400 MHz,
CDCl3) δ 8.13 (d, J = 4.0 Hz, 1H), 8.02 (s, 1H), 7.44 (t, J = 7.6 Hz, 1H), 6.97 (d, J =
8.4 Hz, 1H), 6.60 (t, J = 8.4 Hz, 2H), 6.44 (d, J = 8.0 Hz, 1H), 6.28 (s, 1H), 3.85 (t, J
= 6.0 Hz, 2H), 3.72-3.69 (m, 4H), 2.84-2.74 (m, 6H), 2.59-2.52 (m, 4H), 1.73-1.66 (m,
4H), 1.45-1.40 (m, 2H), 1.38-1.32 (m, 2H). MS (ESI) m/z: 409.3 [M + H] +.
7-((6-(4-(Pyrrolidin-1-yl)piperidin-1-yl)hexyl)oxy)-3,4-dihydroquinolin-2(1H)-on
e (TM-27). Light yellow oil, 79.5% yield. 98.4% HPLC purity. 1H NMR (400 MHz,
CDCl3) δ 8.22 (s, 1H), 7.44 (t, J = 7.6 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.44 (dd, J1 =
6.0 Hz, J1 = 2.0 Hz, 1H), 6.27 (s, 1H), 3.84 (t, J = 6.4 Hz, 2H), 2.89-2.85 (m, 2H),
2.82 (t, J = 7.6 Hz, 2H), 2.57-2.52 (m, 6H), 2.25 (t, J = 7.6 Hz, 2H), 2.04-1.98 (m,
1H), 1.90 (t, J = 7.6 Hz, 2H), 1.83 (d, J = 12.8 Hz, 2H), 1.75-1.65 (m, 6H), 1.59-1.51
(m, 2H), 1.49-1.42 (m, 2H), 1.41-1.35 (m, 2H), 1.32-1.26 (m, 2H). MS (ESI) m/z:
400.3 [M + H] +.
7-((6-(4-Benzylpiperazin-1-yl)hexyl)oxy)-3,4-dihydroquinolin-2(1H)-one (TM-28).
Light yellow oil, 83.6% yield. 98.0% HPLC purity. 1H NMR (400 MHz, CDCl3) δ
8.62 (s, 1H), 7.34 (d, J = 4.4 Hz, 4H), 7.29-7.24 (m, 1H), 7.05 (d, J = 8.4 Hz, 1H),
6.53 (dd, J1 = 6.0 Hz, J2 = 2.4 Hz, 1H), 6.35 (dd, J1 = 9.2 Hz, J2 = 2.0 Hz, 1H), 3.92 (t,
J = 6.4Hz, 2H), 3.53 (s, 2H), 2.91 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 8.0 Hz, 2H),
19

2.59-2.39 (m, 8H), 2.35 (t, J = 8.0 Hz, 2H), 1.81-1.74 (m, 2H), 1.57-1.44 (m, 4H),
1.40-1.32 (m, 2H). MS (ESI) m/z: 422.3 [M + H] +.
7-((6-(3,4-Dihydroisoquinolin-2(1H)-yl)hexyl)oxy)-3,4-dihydroquinolin-2(1H)-on
1
e (TM-29). Light yellow oil, 76.1% yield. 98.1% HPLC purity. H NMR (400 MHz,
CDCl3) δ 8.96 (s, 1H), 7.12-7.08 (m, 3H), 7.03-7.01 (m, 2H), 6.51 (dd, J1 = 6.4 Hz, J2
= 2.0 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 3.92 (t, J = 6.4 Hz, 2H), 3.64 (s, 2H),
2.92-2.86 (m, 4H), 2.74 (t, J = 2.0 Hz, 2H), 2.61 (t, J = 8.0 Hz, 2H), 2.52 (t, J = 8.0
Hz, 2H), 1.81-1.74 (m, 2H), 1.68-1.60 (m, 2H), 1.54-1.46 (m, 2H), 1.45-1.38 (m, 2H).
13C NMR (100 MHz, CDCl3) 172.16, 158.77, 138.22, 134.74, 134.31, 128.65, 128.57,
126.61, 126.12, 125.60, 115.61, 108.76, 102.29, 68.09, 58.36, 56.17, 50.98, 31.11,
29.20, 29.01, 27.34, 27.09, 26.03, 24.59. MS (ESI) m/z: 379.2 [M + H] +.
7-((6-(Benzyl(prop-2-yn-1-yl)amino)hexyl)oxy)-3,4-dihydroquinolin-2(1H)-one
(TM-30). Light yellow oil, 76.8% yield. 98.3% HPLC purity. 1H NMR (400 MHz,
CDCl3) δ 9.21 (s, 1H), 7.38-7.31 (m, 4H), 7.29-7.25 (m, 1H), 7.05 (d, J = 8.4 Hz, 1H),
6.54 (dd, J1 = 6.0 Hz, J2 = 2.0 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 3.94 (t, J = 6.4 Hz,
2H), 3.65 (s, 2H), 3.35 (d, J = 1.6 Hz, 2H), 2.91 (t, J = 7.6 Hz, 2H), 2.64 (t, J = 7.2 Hz,
2H), 2.58 (t, J = 7.2 Hz, 2H), 2.26 (s, 1H), 1.82-1.75 (m, 2H), 1.60-1.53 (m, 2H),
1.51-1.40 (m, 4H). MS (ESI) m/z: 391.2 [M + H] +.
7-((5-(Benzyl(ethyl)amino)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-one (TM-31).
Light yellow oil, 87.2% yield. 98.9% HPLC purity. 1H NMR (400 MHz, CDCl3) δ
9.72 (s, 1H), 7.35 (d, J = 7.2 Hz, 2H), 7.29 (t, J = 7.2 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H),
6.99 (d, J = 8.0 Hz, 1H), 6.47 (t, J = 8.4 Hz, 2H), 3.88 (t, J = 6.0 Hz, 2H), 3.59 (s, 2H),
2.85 (t, J = 7.2 Hz, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.54 (q, J = 7.2 Hz, 2H), 2.47 (t, J =
7.2 Hz, 2H), 1.75-1.68 (m, 2H), 1.58-1.51 (m, 2H), 1.46-1.39 (m, 2H), 1.05 (t, J = 7.2
Hz, 3H). 13C NMR (100 MHz, CDCl3) 172.68, 158.78, 139.30, 138.39, 129.03,
128.49, 128.22, 126.93, 115.54, 108.86, 102.43, 68.05, 57.99, 52.96, 47.29, 31.10,
29.73, 29.14, 26.61, 24.58, 23.91, 11.58. MS (ESI) m/z: 367.2 [M + H] +.
7-((5-(Pyrrolidin-1-yl)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-one (TM-32). Light
yellow oil, 88.1% yield. 99.2% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 9.35 (s,
1H), 6.93 (d, J = 8.0 Hz, 1H), 6.42-6.39 (m, 2H), 3.84 (t, J = 6.0 Hz, 2H), 2.79 (t, J =
20

7.6 Hz, 2H), 2.74-2.68 (m, 4H), 2.59 (t, J = 8.0 Hz, 2H), 2.52 (t, J = 7.6 Hz, 2H),
1.84-1.79 (m, 4H), 1.72-1.66 (m, 2H), 1.65-1.58 (m, 2H), 1.45-1.38 (m, 2H). 13C
NMR (100 MHz, CDCl3) 172.28, 158.59, 138.37, 128.45, 115.58, 108.75, 102.35,
67.81, 56.05, 53.97, 31.07, 29.65, 28.89, 27.56, 24.54, 23.90, 23.34.MS (ESI) m/z:
303.2 [M + H] +.
7-((6-(4-Benzylpiperidin-1-yl)hexyl)oxy)-3,4-dihydroquinolin-2(1H)-one (TM-33).
Light yellow oil, 89.0% yield. 98.2% HPLC purity. 1H NMR (400 MHz, CDCl3) δ
9.78 (s, 1H), 7.27-7.22 (m, 2H), 7.18-7.09 (m, 3H), 6.99 (t, J = 7.6 Hz, 1H), 6.50-6.43
(m, 2H), 3.87 (t, J = 6.0 Hz, 2H), 2.96 (d, J = 10.0 Hz, 2H), 2.83 (t, J = 7.2 Hz, 2H),
2.61-2.55 (m, 2H), 2.52 (t, J = 6.0 Hz, 2H), 2.37-2.33 (m, 2H), 1.90 (t, J = 11.2 Hz,
2H), 1.79-1.69 (m, 2H), 1.63 (d, J = 12.4 Hz, 2H), 1.56-1.50 (m, 3H), 1.46-1.40 (m,
2H), 1.38-1.30 (m, 4H). 13C NMR (100 MHz, CDCl3) 172.56, 158.74, 140.56, 138.41,
129.11, 128.47, 128.18, 125.81, 115.51, 108.80, 102.40, 68.00, 58.87, 53.85, 43.10,
37.82, 31.82, 31.10, 29.71, 29.16, 27.39, 26.63, 25.96, 24.57. MS (ESI) m/z: 421.3
[M + H] +.
4.2. Biological assay.
4.2.1 Inhibition experiments of AChE and BuChE
Acetylcholinesterase (eeAChE, from the electric eel; huAChE, from human
erythrocytes AChE), butyrylcholinesterase (eqBuChE, from equine serum; huBuChE,
human serum BuChE), 5,5’-dithiobis-2-nitrobenzoic acid (Ellman’s reagent, DTNB),
acetylthiocholine chloride (ATC), and butyrylthiocholine chloride (BTC) were
purchased form Sigma Aldrich. ratAChE and rat BuChE was from Institute of
Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan Academy of
Chinese Medicine Sciences.
The determination of AChE and BuChE inhibitory capacity of the compounds
were tested by Ellman assay with slight modification.23 For AChE inhibition assays, a
reaction mixture (100 µL) containing ATC (1 mmol/L, 30 µL), phosphate-buffered
solution (0.1 M KH2PO4/K2HPO4, PH = 8.0, 40 µL), 10 µL enzyme (EeAChE or
HuAChE 0.45 U/mL, rat AChE 5% homogenate) and different concentrations (DMSO
< 1%) of test compounds (20 µL) was incubated at 37°C for 15 min. Then
21

5,5’-dithiobis-2-nitrobenzoic acid (DTNB, 0.2%, 30 µL) was added. The activities
were determined by using a Varioskan Flash Multimode Reader at 412 nm. Each
concentration was assayed in triplicate. The inhibition percent was calculated by the
following expression: (1−Ai/Ac) × 100, where Ai and Ac are the absorbance obtained
for AChE in the presence and absence of inhibitors, respectively. In vitro BuChE
assay was performed using a method similar as described above.
4.2.2. Kinetic Characterization of AChE Inhibition
Kinetic characterization of AChE inhibition was performed based on a reported
method using purified AChE from Electrophorus electricus (EeAChE).24 The assay
solution (100 µL) consists of 0.1 M phosphate buffer (pH 8.0), with the addition of 30
µL of 0.2% DTNB, 10 µL of 0.5 units/mL EeAChE, and 20 µL of substrate
(acetylthiocholine iodide). Three different concentrations of inhibitors were added to
the assay solution and pre-incubated for 15 min at 37°C with the EeAChE followed
by the addition of substrate in different concentrations. Kinetic characterization of the
hydrolysis of substrate catalyzed by EeAChE was done spectrometrically at 412 nm.
The parallel control experiments were performed without inhibitor in the assay. The
plots were assessed by a weighted least square analysis that assumed the variance of v
to be a constant percentage of v for the entire data set. Slopes of these reciprocal plots
were then plotted against the concentration of TM-33 in a weighted analysis, and Ki
was determined as the intercept on the negative x-axis.
4.2.3. Molecular modeling studies
Dock was employed to identify the potential binding of compound TM-33 to
BuChE and AChE. The crystal structure of huBuChE (PDB ID: 4tpk) and TcAChE
(PDB ID: 1EVE) were obtained from the Protein Data Bank after removing the
original inhibitors and water molecules.23 The 3D Structure of TM-33 was built and
performed geometry optimization by molecular mechanics. Docking studies were
performed using the AUTODOCK 4.2.6 program. By using Autodock Tools (ADT;
version 1.5.6), polar hydrogen atoms were added to amino acid residues, and
Gasteiger charges were assigned to all atoms of the enzyme. The resulting enzyme
structure was used as an input for the AUTOGRID program. AUTOGRID performed
22

a pre-calculated atomic affinity grid maps for each atom type in the ligand, plus an
electrostatics map and a separate desolvation map presented in the substrate molecule.
Flexible ligand docking was performed for the compounds. Each docked system was
performed by 100 runs of the AUTODOCK search by the Lamarckian genetic
algorithm (LGA). Other than the referred parameters above, the other parameters
were accepted as default. A cluster analysis was performed on the docking results
using a root mean square (RMS) tolerance of 1.0 and the lowest energy conformation
of the highest populated cluster was selected for analysis. Graphic manipulations and
visualizations were done by Autodock Tools or Discovery Studio 2.5 software.
4.2.4. Recombinant human MAO-A and MAO-B inhibition studies.
Recombinant human MAO-A and MAO-B were purchased from Sigma-Aldrich
and stored at -80 °C. Tested compounds were prepared in DMSO (2.5 mM) and
diluted with potassium phosphate buffer (100 mM, pH 7.40, containing KCl 20.2 mM)
to a final volume of 500 µL containing various concentrations of test compounds
(0-100 µM) and kynuramine (45 µM for MAO-A and 30 µM for MAO-B). The
reactions were initiated by the addition of the enzyme (7.5 µg/mL) and then incubated
for 30 min at 37. Then 400 µL NaOH (2N) and 1000 µL water were added to
terminated the enzymatic reactions and the mistures were centrifuged at 16000g for
10 min.25 The concentrations of the generated 4-hydroxyquinoline were determined
by measuring the fluorescence of the supernatant on a Varioskan Flash Multimode
Reader (PerkinElmer) with excitation and emission wavelengths at 310 nm and 400
nm, respectively. IC50 values were calculated from sigmoidal dose-response curves
(graphs of the initial rate of kynuramine oxidation versus the logarithm of inhibitor
concentration). Each sigmoidal curve was constructed form six different compound
concentrations spanning at least three orders of magnitude. Data analyses were carried
out with GraphRad Prism 5 employing the one site competition model. IC50 values
were determined in triplicate and expressed as mean SD.
4.2.5. In vitro blood−brain barrier permeation assay
The blood-brain barrier penetration of compounds was evaluated using the parallel
artificial membrane permeation assay (PAMPA) described by Di et al.28 Commercial
23

drugs were purchased from Sigma and Alfa Aesar. Porcine brain lipid (PBL) was
obtained from Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size
0.45 mm) and acceptor microplate were both from Millipore. The 96-well UV plate
(COSTAR) was from Corning Incorporated. The acceptor 96-well microplate was
filled with 350 µL of PBS/EtOH (70:30), and the filter membrane was impregnated
with 4 µL of PBL in dodecane (20 mg/mL). Compounds were dissolved in DMSO at
5 mg/mL and diluted 50-fold in PBS/EtOH (70:30) to a final concentration of 100
µg/mL. Then 200 µL of the solution was added to the donor wells. The acceptor filter
plate was carefully placed on the donor plate to form a sandwich, which was left
undisturbed for 18 h at 25 °C. After incubation, the donor plate was carefully removed,
and the concentration of compounds in the acceptor wells was determined using the
Varioskan Flash Multimode Reader (Thermo Scientific). Every sample was analyzed
at ten wavelengths in four wells and in at least three independent runs. Pe was
−
calculated using the following expression: Pe = {−VdVa/[(Vd + Va)At]}ln(1
drugacceptor/drugequilibrium), where Vd is the volume of donor well, Va is the volume in the
acceptor well, A is the filter area, t is the permeation time, drug acceptor is the
absorbance obtained in the acceptor well, and drug equilibrium is the theoretical
equilibrium absorbance. The results are given as the mean standard deviation. In the
experiment, 11 quality control standards of known BBB permeability were included
to validate the analysis set. A plot of the experimental data versus literature values
gave a strong linear correlation, Pe(exp) = 0.9163Pe(bibl.) −0.2247 (R2 = 0.9558).
From this equation and the limit established by Di et al. (Pe (bibl.) = 4.0 × 10−6 cm/s)
for blood−brain barrier permeation, we concluded that compounds with a permeability
greater than 3.44 ×10−6 cm/s could cross the blood−brain barrier.
Acknowledgments
This work was supported in part by the Scientific and Technological Project of Henan
Provincial (162300410103), the Key Scientific Research Project of Colleges and
Universities in Henan Province (17B350002), the Special Project of Nanyang Normal
University (ZX2016017) and Nanyang Normal University (STP2016009).
24

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Figures, Schemes, and Tables Legends
Figure 1. Design strategy for 3,4-dihydro-2(1H)-quinoline-O-alkylamine derivatives
26

Figure 2. Steady state inhibition by compound TM-33 of the AChE hydrolysis of
ACh. The plots show mixed-type AChE inhibition for compound TM-33.
Figure 3 (A) Representation of compound TM-33 (green stick) interacting with
residues in the binding site of TcAChE (PDB code: 1EVE). (B) 3D docking model of
compound TM-33 (green stick) with TcAChE.
Figure 4 (A) Representation of compound TM-33 (green stick) interacting with
residues in the binding site of huBuChE (PDB code: 4tpk). (B) 3D docking model of
compound TM-33 (green stick) with huBuChE.
Figure 5. Toxicity effects of TM-33 on cell viability in human PC12 cells. Data are
mean values SEM of three independent experiments.
27

O
H
O
N
O
Br
N
MeO
MeO
JMC49 (MAO-BI)
hMAO-B IC50 = 2.9 nM
SI > 2751
Donepezil (AChEI)
AChE IC50 = 0.016
BuChE IC50 = 8.2
MTDLs
Entrance cavity MAO-B
Catalytic site MAO-B
Enchance MAO-A
inhibitory activity
R1
Spacer
N
O
N
H
O
R2
PAS AChE
CAS AChE
3,4-Dihydro-2(1 )-quinoline- -alkylamine derivatives
H
O
Figure 1.
28

Figure 2.
29