Design, synthesis and biological evaluation of rasagiline-clorgyline hybrids as novel dual inhibitors of monoamine oxidase-B and amyloid-β aggregation against Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2020.112475

A series of rasagiline-clorgyline hybrids was designed, synthesized and investigated in vitro for their inhibition of monoamine oxidase and amyloid-β aggregation. Most of compounds were found to be selective and highly potent hMAO-B inhibitors showing IC₅₀ values in the nanomolar, and exhibited a moderate inhibition of amyloid-β aggregation. 7-((5-(methyl(prop-2-yn-1-yl)amino) pentyl)oxy)chroman-4-one (6j) was the most interesting compound identified in this research, endowed with higher hMAO-B potency (IC₅₀ = 4 nM) and selectivity (SI > 25000) compared to the reference selective inhibitor rasagiline (IC₅₀ = 141 nM, SI > 355), and exhibited good inhibitory activity against Aβ_1-42 aggregation (40.78percent, 25 μM). Kinetic and molecular modeling studies revealed that 6j was a competitive reversible inhibitor for hMAO-B. Moreover, compound 6j displayed low toxicity and good neuroprotective effects in SH-SY5Y cell assay, and could penetrate the blood-brain barrier according to the parallel artificial membrane permeability assay. Pharmacokinetics assay revealed that compound 6j possessed good pharmacokinetic profiles after intravenous and oral administrations. Overall, these results highlighted that compound 6j was an effective and promising multitarget agent against Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2020.112475

Journal Pre-proof
Design, synthesis and biological evaluation of rasagiline-clorgyline hybrids as
novel dual inhibitors of monoamine oxidase-B and amyloid-β aggregation against
Alzheimer's disease
Sai-Sai Xie, Jing Liu, Chunli Tang, Chengyun Pang, Qing Li, Yuelian Qin, Xiaojie
Nong, Zhipeng Zhang, Jie Guo, Maojun Cheng, Weizhong Tang, Ningsheng Liang,
Neng Jiang
PII:
S0223-5234(20)30446-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.112475
EJMECH 112475
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 January 2020
Revised Date: 13 April 2020
Accepted Date: 15 May 2020
Please cite this article as: S.-S. Xie, J. Liu, C. Tang, C. Pang, Q. Li, Y. Qin, X. Nong, Z. Zhang, J. Guo,
M. Cheng, W. Tang, N. Liang, N. Jiang, Design, synthesis and biological evaluation of rasagiline-
clorgyline hybrids as novel dual inhibitors of monoamine oxidase-B and amyloid-β aggregation against
Alzheimer's disease, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112475.
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Graphical Abstract
Design, synthesis and biological evaluation of rasagiline-clorgyline hybrids as
novel dual inhibitors of monoamine oxidase-B and amyloid-β aggregation against
Alzheimer’s disease

Design, synthesis and biological evaluation of rasagiline-clorgyline hybrids as
novel dual inhibitors of monoamine oxidase-B and amyloid-β aggregation against
Alzheimer’s disease
Sai-Sai Xie#a,c, Jing Liu#a,d, Chunli Tanga, Chengyun Panga, Qing Lia, Yuelian Qina,
Xiaojie Nonga, Zhipeng Zhangc, Jie Guoc, Maojun Chengc, Weizhong Tangb,
Ningsheng Lianga,*, Neng Jianga,*
b
aDepartment of Pharmacy, Department of Gastrointestinal Surgery, Affiated Tumor
Hospital of Guangxi Medical University, Nanning, PR China
cNational Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal
d
Medicine, School of Pharmacy, Jiangxi University of Traditional Chinese Medicine,
Nanchang, PR China
*Corresponding authors.
E-mail addresses: Liangn01@163.com (N. Liang), cpujnh@163.com (N. Jiang)
#These authors contributed equally to this work.

Abstract
A series of rasagiline-clorgyline hybrids was designed, synthesized and investigated
in vitro for their inhibition of monoamine oxidase and amyloid-β aggregation. Most of
compounds were found to be selective and highly potent hMAO-B inhibitors showing
IC50 values in the nanomolar, and exhibited a moderate inhibition of amyloid-β
aggregation. 7-((5-(methyl(prop-2-yn-1-yl)amino) pentyl)oxy)chroman-4-one (6j)
was the most interesting compound identified in this research, endowed with higher
hMAO-B potency (IC50 = 4 nM) and selectivity (SI>25000) compared to the
reference selective inhibitor rasagiline (IC50 = 141 nM, SI>355), and exhibited good
inhibitory activity against Aβ1-42 aggregation (40.78%, 25 ꢀM). Kinetic and molecular
modeling studies revealed that 6j was a competitive reversible inhibitor for hMAO-B.
Moreover, compound 6j displayed low toxicity and good neuroprotective effects in
SH-SY5Y cell assay, and could penetrate the blood-brain barrier according to the
parallel artificial membrane permeability assay. Pharmacokinetics assay revealed that
compound 6j possessed good pharmacokinetic profiles after intravenous and oral
administrations. Overall, these results highlighted that compound 6j was an effective
and promising multitarget agent against Alzheimer’s disease.

1. Introduction
Given social development, better living conditions and medical advances, the
proportion and lifespan of older people in the global population are increasing. The
aging population is a feature trends in developed and developing countries. The aging
population is closely related to the increasing incidence rate of neurodegenerative
diseases in old age and increasing government expenditure on health and social care.
Now the care costs of 35 million patients with dementia was over $ 600 billion per
year that is about one percent of global Gross Domestic Product [1]. Kuca et al. [2]
simulated prolonging the length of a person’s ‘stay’ in the Mild, Moderate, or Severe
stage, the total cost of care for all persons with dementia will increase by 2080. Kuca
et al. showed that prolonging the stay in the Mild stage of AD (by lowering the
incidence by 10%, 30%, or 50%) reduced the cost (by 4.88%, 16.78% and 32.48%,
respectively). Therefore, it is of great significance to develop drugs for the mild and
moderate stages of AD.
Alzheimer's disease (AD) is a complex neurodegenerative disease and widely
researched owing to the poor efficiency of AD treatment and brain functional damage
for daily life inabilities, leading to high familial and social burden for patient care [3,
4]. Alzheimer’s Association has reported that there are about 47 million AD patients
worldwide, and the number will rise to 100 million by 2050 [5]. Many AD studies
have shown that the loss of neurons associated with the aggregation of amyloid-β
peptide (Aβ) and hyperphosphorylation of tau protein in the brain regions [6]. Aβ
aggregation in neuronal plaques, oxidative stress produced by neuro transmitters and
many other factors are mainly causing neuronal degeneration in AD [7, 8]. A
multitarget-directed-ligand (MTDL) strategy is more promising and effective for the
treatment of AD because of the complex and multifactorial pathological mechanism
of AD [9,10].
Monoamine oxidases (MAOs) are flavin adenine dinucleotide (FAD)-containing
enzymes that catalyse the oxidative deamination of various biogenic and xenobiotic
monoamines such as serotonin, epinephrine, dopamine and xenobiotic amines from
dietary [11]. Monoamine oxidases (MAOs) are separated into two different

isoenzymes, namely MAO-A and MAO-B [12, 13]. The two isoform enzymes
encoded by different genes exhibit distinct tissue distribution, different substrate and
specific inhibition [14-18]. MAO-A is responsible for the oxidative deamination of
various neurotransmitters including norepinephrine, serotonin and epinephrine.
Clorgyline is an irreversible inhibitor of selective MAO-A, and moclobemide is a
reversible inhibitor of selective MAO-A [11]. MAO-B preferentially catalyses the
derivative of benzylamine or 2-phenethylamine. Selegiline and rasagiline are selective
and reversible inhibitors of MAO-B. MAO-A are associated with depression, whereas
MAO-B are used for the treatment of Alzheimer's disease by more and more evidence
[19-21]. With increasing age, more and more expression level of MAO-B is found in
the patients’ brain tissue and cerebral spinal fluid (CSF) [22]. High expression of
MAO-B can cause the increment of oxidative free radicals, the disorder of cholinergic
neurons and the formation of amyloid plaques [23]. Thus, high selective and
reversible inhibitors of MAO-B are considered as promising candidates for AD
treatment.
Aβ plaques plays an important role in the key pathological feature of AD. Aβ
plaques are mainly consisted of aggregation of Aβ peptide, a 39 to 43 residue long
protein degraded from the amyloid precursor protein (APP) [24-25]. Aβ in the brain
leads to the formation of oligomers, fibrils and plaques, causing neuronal toxicity,
neuronal loss and dementia [26-27]. Therefore, inhibition of Aβ aggregation is also a
promising and potential therapy for AD.
Alzheimer’s disease is a complex multifactorial neurodegenerative disease, the
initiation and progression involve multiple targets or factors [28]. The approach “one
molecule, one target” does not always lead to satisfactory efficacy. Two approaches
are often used for multi-target therapeutics. The first approach is combinations of
drugs with one target or a single active ingredient respectively, whereas the second
approach is multi-target directed ligands (MTDLs) [29]. The former approach has
some advantages, such as providing better dose flexibility by directly adjust ratio of
drugs in mixture, lower treatment cost. However, combinations of drugs often
suffered from more adverse effects such as dose-limiting toxicities, drug-drug

interactions, complex PK/PD properties of the multiple components and poor patient
compliance [30-31]. MTDLs that are new chemical entity have some advantages,
such as the PK/PD properties be easier to formulate compared with a mixture, the
increasing therapeutic efficacy through synergies at low dosages and reduced adverse
effects enable wider therapeutic windows [32-34]. Sun [35] et al. had discussed the
advantages and disadvantages of the above two approaches.
Our group have made great efforts to find potential multitarget-directed-ligands
(MTDLs) targeting monoamine oxidases, cholinesterase, Aβ protein and oxidative
free radicals etc [36-39]. In this paper, a series of rasagiline-clorgyline hybrids was
designed, synthesized and evaluated for their biological activities, toxicity and
pharmacokinetics. In addition, the development of new drugs targeted to the CNS
requires good blood brain-barrier (BBB) permeability. A good permeability through
the BBB is essential because of the target site of AD is located in the CNS [40-41].
Drugs that can penetrate BBB is a challenging issue in AD therapy. Thus, to evaluate
the compounds whether have good brain penetration as early as possible [42-43], a
well-known high throughput screening (HTS) technique, parallel artificial membrane
permeation assay, was performed. Without consideration of P-glycoprotein (Pgp)
efflux, high plasma protein binding and low capillary permeability, this method can
quickly predict the ability of present compounds to cross the BBB [40]. Furthermore,
kinetic and molecular modeling studies were also performed to investigate the high
selective and strong affinity modes of compound 6j with hMAO-B.
2. Results and discussion
2.1. Design of rasagiline-clorgyline hybrids

Fig. 1. Design strategy for rasagiline-clorgyline hybrids
Clorgyline (hydrochloride) is an irreversible, potent and selective MAO-A inhibitor
with IC50 values of 0.0046 ꢀM for MAO-A and 62 ꢀM for MAO-B. Rasagiline is a
potent, selective and irreversible monoamine oxidase B (MAO-B) inhibitor. As shown
in Fig.1, the 2,3-dihydro-1H-indene analogues of rasagiline show the inhibitory
activity for amyloid-β aggregation. Given the similar structure between clorgyline and
rasagiline, the drug fragment of clorgyline and rasagiline are combined to design and
optimize a series of rasagiline-clorgyline hybrids, which are expected to be highly
selective and potent hMAO-B inhibitors, as well as Aβ aggregation inhibitors. In
addition, we focus the structural optimization of 2,3-dihydro-1H-indene (marked at
the blue color as shown in Fig.1.) and the length of flexible linker (marked at the red
color as shown in Fig.1.). Rasagiline-clorgyline hybrids and its analogues are under
investigation for the treatment of Alzheimer’s disease.
2.2. Chemistry

Scheme 1. Synthesis of rasagiline-clorgyline hybrids 6a-6t. Reagents and conditions:
(i) CF3SO3H, 80 °C, 30 min; (ii) 2 M NaOH (aq), 5 °C to r.t., 6 M H2SO4 (aq), 2 h; (iii)
α, ω-dibromoalkanes, K2CO3, DMF, reflx, 4 h; (iv) propargyl amine, anhydrous
K2CO3, KI, CH3CN, 65 °C, 1-2 h.
The target compounds were synthesized as shown in Scheme 1. The initial
compound 4a-4c and 4e-4h were purchased from the market. The initial compound
4d was obtained according to our previously reported experimental method [42]. The
alkylation of compound 4a-4h with corresponding α, ω-dibromoalkanes under K2CO3
o
alkaline condition at 70 C generated products 5a-h. It was noted that the reactivity
equivalent of corresponding α, ω-dibromoalkanes were ranged from 4 to 6. Finally,
the target compounds 6a-6t were obtained by nucleophilic substitution of compounds
5a-h with the appropriate propargylamine in the presence of K2CO3 and DMF at 60
oC.

2.3.MAOs inhibitory activities of all synthesized compounds 6a-6t.
The hMAO-A and hMAO-B inhibitory potencies of compounds 6a-6t were tested
by a reported fluorescence-based Amplex Red assay using clorgyline, rasagiline and
iproniazid as reference compounds [44]. The hMAO inhibition IC50 values and
corresponding selectivity indexes (SI = IC50 MAO-A/IC50 MAO-B) obtained for all
synthesized compounds 6a-6t and reference compounds were shown in Table 1. Most
of the design compounds exhibited potent the inhibition of hMAO-B enzymatic
activity in nanomolar. On the contrary, the same compounds showed no inhibition or
poor efficacy against hMAO-A, up to the highest concentration tested (100 ꢀM).
Owing to the MAO-A inhibition may cause adverse reaction in the peripheral tissues,
these high selective and potent inhibitors of MAO-B activity will be more beneficial
for AD treatment. Recently, some research groups had reported represented MAO-B
inhibitors,
such
as
hydroxypyridinonecoumarin
hybrids
[45],
(Pyrrolo-pyridin-5-yl)benzamides
[46], 1-Propargyl-4-Styrylpiperidine-Like
Analogues [47], 4H-benzopyran-4-one derivatives [48]. Most of these compounds
show similar potent and less selective than compound 6j.
In the series hybrids 6a-6t, compound 6j was the most potent and the highest
selective inhibitors of hMAO-B (IC50 = 4 nM, SI > 25000), which was more potent
and selective than the reference compounds rasagiline and iproniazid (IC50 = 141.7
nM and 7410 nM, SI >355 and = 0.89, respectively), as shown in Table 1. Due to the
linker length between aromatic ring and propargyl amine having relations with the
MAOs inhibitory potencies, compounds with the different linker length were
synthesized for evaluation of MAOs inhibition activity. A comparison of inhibitory
capabilities of the compounds 6h-6l (n = 3-8) showed that the inhibitory activity for
MAO-B was increased with the increase of carton chain length (from 3 carbon atoms
to 5 carbon atoms), but decreased with further longer carton chain (from 5 carbon
atoms to 8
Table 1. MAOs inhibitory activities of rasagiline-clorgyline hybrids 6a-6t.
X
R1
R2
R3
m
n
IC50 ± SD (nM)a
SIc
Compound

hMAO-A
hMAO-B
366.3±13.2
94.8±5.8
CH2
-
-
-
1
1
1
1
2
2
2
2
2
2
2
2
-
4
5
6
6
4
5
6
3
4
5
6
8
4
6
5
4
5
4
5
6
7.93±1.03% b
31.01±4.21% b
4.42±0.97% b
11.67±1.03% b
30.88±4.97% b
26.05±2.14% b
24.99±4.50%b
32.14±11.32%b
22.78±3.26%b
6.04±0.74% b
34.06±6.25% b
28.65±3.68%b
28.61±4.11% b
37.28±5.33% b
32.33±9.46%b
21.10±2.21% b
39.52±7.52% b
29.37±3.88% b
27.13±10.11% b
37.39±7.93% b
4.58±0.35
> 273#
> 1055#
> 777#
> 1927#
> 1379#
> 2160#
> 751#
> 9#
6a
CH2
-
-
-
6b
CH2
-
-
-
128.6±9.1
51.9±5.7
6c
O
-
-
-
6d
CH2
-
-
-
72.5±7.4
6e
CH2
-
-
-
46.3±3.8
6f
CH2
O
O
O
O
O
-
-
-
-
133.1±8.5
11414±128
58.9±5.7
6g
-
-
-
6h
> 1698#
>2.5*104 #
> 855#
> 237#
> 1770#
> 4897#
> 991#
> 87#
-
-
-
-
6i
-
-
6j
4.0±0.6
-
-
-
-
117.0±8.3
422.0±15.2
56.51±4.1
20.42±2.2
100.86±7.9
1150±37
6k
-
H
-
6l
CH3CO
CH3CO
H
H
6m
-
H
H
-
6n
-
H
CH3O
H
-
6o
-
CH3O
CH3O
H
CH3CO
CH3CO
HCO
HCO
HCO
-
6p
> 15#
-
H
-
6520±24
6q
6r
-
CH3O
CH3O
CH3O
-
41.25%b
-
> 17#
-
H
-
5735±120
551.8±33
62010±870
141.70±6.34
7410 ± 340
6s
-
H
-
> 181#
0.000074
> 355#
0.89
6t
Clorgyline
Rasagiline
Iproniazid
47.91±2.31%b
6590 ± 270
a Data are expressed as mean ± S.D. from three diferent experiments; b Inhibition% at 100 µM. cSI:
d
#
hMAO-B selectivity index = IC50(hMAO-A)/ IC50(hMAO-B). Inhibition% at 50 µM. Values
obtained under the assumption that the corresponding IC50 against MAO-A was the highest
concentration tested (100 µM).
carbon atoms). Compound 6j (IC50 = 4 nM) with a five-carbon chain presented more
potent activity than the corresponding compound 6h containing three-carbon chain

(IC50 = 11414 nM) and 6i containing four-carbon chain (IC50 = 58.9 nM). However,
the compound 6k containing six-carbon chain (IC50 = 117 nM) and 6l with
eight-carbon atom chain (IC50 = 422 nM) showed less inhibitory activity than
compound 6j containing a five-carbon chain (IC50 = 4 nM). Similar structure-activity
relationships (SARs) were also found in other compounds, such as compounds 6a-6c,
6e-6g and 6r-6t.
After exploring the SARs of chain length, the SARs of different heterocycle were
also explored. As shown in Table 1, the target compounds containing six-membered
heterocycle (6e, 6f) was about 5-fold and 2-fold more potent than the corresponding
target compounds (6a, 6b) containing five-membered heterocycle, whereas
compounds 6g containing six-membered heterocycle and compounds 6c containing
five-membered heterocycle showed similar potency. A comparison of the potency of
the four rasagiline-clorgyline hybrids (6d, 6k, 6c, 6g) revealed that the replacement of
carbon atom with oxygen atom substituent at the position 3 of benzene ring (6d vs. 6c,
6k vs. 6g) provided more hMAO-B inhibitory activity. Given the above discussion,
the chromanone moiety (compound 6k with IC50: 116 nM) produced an increment in
potency when compared to 2,3-dihydro-1H-inden-1-one (compound 6c with IC50:
128.6 nM) and 3,4-dihydronaphthalen-1(2H)-one moieties (compound 6g with IC50:
133.1 nM). Furthermore, the ring-opening of benzofuran-3(2H)-one moiety
(compound 6d) was to generate analogues 6r, 6s and 6t. The corresponding functional
group of these analogues 6r-6t were expressed as R2=HCO- and R3 = CH3O-
substituents. Surprisingly, the ring-opening analogues 6r-6t resulted in dramatic
reduction in potency towards MAO-B compared to the corresponding heterocycle 6d.
It was worth noting that, the acetyl (R2 = CH3CO-) substituent group at the position 4
of benzene ring were necessary for the potent hMAO-B inhibitory activity, as
compounds 6m and 6n showed significant MAO-B inhibition activity (IC50 = 56.51,
20.42 nM, respectively). Finally, the additional substitution with methoxy group (R1 =
CH3O-) at the position 2 of benzene ring (compounds 6p and 6q) exhibited a dramatic
loss of hMAO-B potency, compared to compounds 6m and 6n.
2.4. Docking studies of compound 6j with hMAO-B

Fig. 2. (a) 3D docking model of compound 6j with hMAO-B. Atom colors:
green-carbon atoms of 6j, gray-carbon atoms of residues of hMAO-B, yellow-carbon
atoms of FAD, dark blue-nitrogen atoms, red-oxygen atoms. The green dashed line
represents the interaction between the protein and the ligand. (b) 2D schematic
diagram of docking model of compound 6j with hMAO-B. The figure was prepared
using the ligand interactions application in MOE.
In order to rationalize the binding modes of the synthesized compounds with
hMAOs at molecular level, docking studies were carried out by the Molecular
Operating Environment (MOE 2015.10) software. The compound 6j with highly
selective and most potent toward hMAO-B was selected and researched its hMAO-B
binding modes. The binding mode of compound 6j with respect to MAO-B was
investigated based on the X-ray crystal structure of the human monoamine oxidase B
in complex with 7-(3-chlorobenzyloxy)-4-(methylamino)methyl-coumarin (PDB code
2V61), and the protein was energy minimized and 3D protonated using the structure

preparation module of MOE. It can be seen from the Fig. 2. that the propargylamine
moiety of compound 6j was oriented to the flavin adenine dinucleotide (FAD)
cofactor and establish a ‘arene-H’ interaction with Tyr 435 in the substrate cavity. The
chromanone moiety occupied the entrance cavity and interacted with leu171, Cys172,
Ile198, Ile199, Ile 316, Tyr 326, Trp 119 and Phe168 through van der waals and
hydrophobic interactions. For comparison purpose, rasagiline was also selected for
docking studies (Fig S1). The results showed that compound 6j and rasagiline showed
very similar docking poses in the substrate cavity of MAO-B; however, in comparison
to the rasagiline, compound 6j could also interact with the residues in entrance cavity
of MAO-B, thereby exhibiting higher binding affinity to MAO-B than rasagiline.
2.5. Reversibility and kinetic study of hMAO-B inhibition
Fig. 3. Recovery of enzyme inhibition activity after dilution of enzyme-compound
complex. hMAO-B were pre-incubated with compound 6j at 10 × IC50 concentration
and 100 × IC50 concentration for 30 min and then diluted to 0.1 × IC50 and 1 × IC50
concentrations, respectively.
It was well known that reversible inhibitors are more effective for the treatment of
AD than irreversible inhibitors. The most promising compound 6j, a highly selective
and potent MAO-B inhibitor, was selected for further studies. A reported enzyme
inhibition experiment [49-50] was carried to investigate whether compound 6j was
reversible or irreversible, and pargyline was used as irreversible reference compound.
Firstly, the MAO-B enzyme and compound 6j were incubated together at

concentrations of 0, 10 and 100 × IC50 for 30 min, respectively. Then, these 10 and
100×IC50 concentrations of enzyme-compound complex were diluted to
corresponding concentrations of 0, 0.1 and 1×IC50. The tested compound will be
reversible if the activity of the enzyme is restored to about 90% and 50% after
dilution to 0.1×IC50 and 1×IC50, respectively. The tested compound will be
irreversible if the activity of the enzyme is no longer restored after dilution. As shown
in Fig. 3, the activity of MAO-B enzyme was restored to about 82% and 45% after
compound 6j dilution to 0.1× IC50 and 1× IC50, respectively. The activity of MAO-B
enzyme was no longer restored (less than 10% of control) after the reference
irreversible inhibitor pargyline be diluting. These results showed that compound 6j is
a reversible MAO-B inhibitor.
Fig. 4. Kinetic study on the mechanisms of interaction between hMAO-B and
compound 6j. Overlaid Lineweaver–Burk reciprocal plots of hMAO-B at different
concentrations of compound 6j (2.5, 5 and 10 nM) using p-tyramine (0.05–3.0 mM)
as substrate.
Furthermore, an enzyme kinetic study on the mechanisms of interaction between
hMAO-B and compound 6j was carried out. As shown in Fig. 4, the
Lineweavere-Burk reciprocal plots were established by rates of MAO-B catalyzed
oxidation for p-tyramine at different concentrations of compound 6j (2.5, 5 and 10

nM). All lines were orthogonal on the Y-axis, which suggested that compound 6j was
a competitive MAO-B inhibitor.
2.6. Aβ1-42 self-aggregation inhibitory activity of all synthesized compounds 6a-6t
All synthesized compounds tested for their MAOs inhibitory activity were also
evaluated for their capabilities against Aβ1-42 self-induced aggregation by thioflavin-T
Table 2. Inhibitions of Aβ1-42 self-induced aggregation of rasagiline-clorgyline
hybrids 6a-6t.
Inhibition rate (%)a
31.27 ± 3.01%
11.37 ± 1.10%
0.68 ± 0.22%
9.76 ± 1.53%
13.39 ± 1.41%
10.28 ± 2.34%
18.15 ± 2.17%
7.77 ± 2.19%
43.9 ± 4.85%
40.78 ± 6.27%
Inhibition rate (%)a
0.66 ± 0.26%
0.34 ± 0.08%
6.18 ± 1.97%
12.72 ± 2.04%
22.23 ± 4.55%
21.79 ± 2.74%
5.74 ± 0.31%
0.86 ± 0.14%
0.82 ± 0.20%
0.99 ± 0.33%
Compound
Compound
6a
6k
6b
6l
6c
6m
6d
6n
6e
6o
6f
6p
6g
6q
6h
6r
6i
6j
6s
6t
46.1 ± 6.71%
Curcumin
a
Inhibition of self-induced Aβ1-42 aggregation (means ± SD of three experiments). The
thioflavin-T fluorescence method was used, and the measurements were carried out in the
presence of 25 ꢀM inhibitor.
based fluorescence assay [51-53]. Curcumin (Cur) were used as reference compounds,
and Aβ1-42 self-aggregation inhibitory activity data of all synthesized compound were
summarized in Table 2. As can be seen from the Table 2, most of compounds showed
weak-to-moderate potencies compared to the reference compound curcumin (46.1 ±
6.71%, at 25 ꢀM). Compounds 6i, 6j which exhibited remarkable inhibitory activities
against MAO-B, had good inhibition property of Aβ self-induce aggregation (43.9 ±
4.85%, 40.78 ± 6.27% at 25 ꢀM). It was hard to draw the structure-activity
relationships, due to the vague outline between these compounds and inhibitory
activities of Aβ self-aggregation.

To further analyze the effect of compounds on inhibition of Aβ aggregation,
compound 6j with potent inhibitory activity was selected for the TEM assay. The
results indicated that the TEM image analysis is consistent with ThT binding assay
(Fig S2 and S3). More well-defined Aβ1-42 aggregates were observed in the presence
of Aβ alone, and fewer Aβ1-42 aggregates were observed when compound 6j and
curcumin were added to the samples.
2.7. In vitro blood-brain barrier permeation assay
The blood-brain barrier (BBB) was the main obstacle of the entrance of the
anti-AD agents for central nerval system into the brain. Hence, it was necessary to
assess the blood-brain barrier permeability of target compounds. The blood-brain
barrier
Table 3. Permeability Pe (×10-6) in the PAMPA-BBB assay for 9 commercial drugs in
the experiment validation.
Bibliographya
Experimentb
16.06 ± 0.53
12.45 ± 0.46
9.26 ± 0.34
7.12 ± 0.17
2.17 ± 0.08
0.76 ± 0.02
1.54 ± 0.05
1.66 ± 0.11
0.69 ± 0.03
Commercial drugs
Testosterone
Estradiol
17.0
12.0
9.3
Progesterone
Chlorpromazine
Corticosterone
Hydrocortisone
Caffeine
6.5
5.1
1.9
1.3
1.02
0.1
Atenolol
Theophylline
a Taken from Ref [54].
b Experimental data are expressed as mean ± SD from three independent experiments, using PBS :
EtOH (70:30) as solvent.

Fig. 5. Lineal correlation between experimental and reported permeability of
commercial drugs using the PAMPA-BBB assay. Pe (exp.) = 0.9707 Pe (bibl.) - 0.1043
(R2 = 0.9573).
Table 4. Permeability Pe (×106 cm/s) in the PAMPA-BBB assay for the target
compounds and their predicted penetration into CNS.
Pe (×106 cm/s)a
9.61 ± 0.88
10.91 ± 1.02
12.85 ± 0.91
8.30 ± 0.54
8.70 ± 0.65
10.63 ± 0.73
8.58 ± 0.43
9.21 ± 0.32
13.66 ± 1.01
10.25 ± 0.68
8.54 ± 0.42
8.91 ± 0.61
10.63 ± 0.40
8.69 ± 0.52
10.54 ± 0.39
Predictionb
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
Compound
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o

12.53 ± 0.87
12.94 ± 0.94
13.14 ± 1.12
14.04 ± 0.64
11.18 ± 0.39
CNS+
CNS+
CNS+
CNS+
CNS+
6p
6q
6r
6s
6t
a
Permeability Pe (×106 cm/s) values were expressed as mean±SD from three independent
experiments, using PBS: EtOH (70:30) as solvent.
b CNS+ was predicted as high BBB permeation with Pe (× 106 cm/s) > 3.78.
(BBB) was detected by the parallel artificial membrane permeation assay [54]. As
shown in Table 3, 9 commercial drugs endowed with reported values were used as
reference compounds. Described Pe were regarded as dependent variables and
experimental Pe as independent variables when we performed simple linear regression
analysis: Pe (exp.) = 0.9707 Pe (bibl.) - 0.1043 (R2 = 0.9573) (Fig. 5). Based on this
equation and the limit established by Di et al. for BBB permeation, the value of
permeability was as follow: Pe (× 106 cm/s) > 3.78 represented high BBB permeation
(CNS+). From Table 4, the Pe values of all compounds were higher than 3.78, which
indicated all compounds had high permeability of blood-brain barrier.
2.8. SH-SY5Y neuroblastoma cell toxicity
Fig. 6. SH-SY5Y cell viability after treatment of various concentrations of compound
6j by MTT assay. Data were shown as mean ± SD of three independent experiments
(*P <0.05, **P <0.01, compared to control group).

To evaluate the biological safety of these potent inhibitors for MAO-B and
amyloid-β aggregation, compound 6j was selected for cytotoxicity test in human
neuroblastoma cell line (SH-SY5Y). SH-SY5Y cells were incubated with different
concentrations of compound 6j for 24h, and then the cell viability was tested by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) experiment [55]. As
shown in Fig. 6, compound 6j showed non-toxic at the concentrations of 25 ꢀM. Due
to the high inhibitory potency of compound 6j on MAO-B, it would be safe at
therapeutic concentration.
2.9. Neuroprotection assays in SH-SY5Y cells
The neuroprotection was extraordinary significant for AD treatment, so the
potential protective effects against neurotoxins-induced damage was investigated in
SH-SY5Y cells using the method described by zheng et al [56]. Compound 6j was
selected to
Fig. 7. Neuroprotective effects of compound 6j against 6-OHDA-induced toxicity in
SH-SY5Y cells. Rasagiline was the reference compound. Results were shown as cell
viability. All data were the means ± SEM of three independent experiments (*P <0.05,
**P <0.01, ***P <0.01, compared to 6-OHDA group).
verify the neuroprotective effect. The bar chart of Fig. 7 showed that cell viability rate
of 6-hydroxydopamine-treated SH-SY5Y cell group significantly decreased when
compared to that of control and DMSO group. Different concentrations of compound

6j were respectively incubated with 6-OHDA (200 ꢀM) in SH-SY5Y cells for 24h,
and the corresponding cell viability rates were tested by MTT assay. Cell survival
rates were gradually increased when 6-OHDA-treated SH-SY5Y cells were
respectively incubated with 1.563, 3.125, 6.25, 12.5, 25 ꢀM (the increasing
concentrations) of compound 6j. A similar phenomenon of reference compound
rasagiline was also observed under the same conditions. The results indicated that
compound 6j had neuroprotective capability against neurodegeneration disease.
2.10. Pharmacokinetic evaluation of compound 6j
Compound 6j with potent inhibitory activities for MAO-B and Aβ1-42
self-aggregation was selected as representatives for further pharmacokinetic studies
following intravenous (iv) and oral (po) dosing in SD rats. The key pharmacokinetic
parameters of compound 6j were shown in Table 5. Compound 6j showed a high
maximal concentration (Cmax = 639.29 and 142.17 ꢀg/L), appropriate half-life (t1/2
=
1.02 and 1.33 h), and good oral bioavailability (36.1%). These results suggested that
compound 6j would have acceptable pharmacokinetic properties.
Table 5. Pharmacokinetic parameters of compound 6j after po and i.v. administration
Parameters a
i.v. (3 mg/kg)
po (10 mg/kg)
T1/2 (h)
Tmax(h)
Cmax(ꢀg/L)
639.29±89.06
142.17±72.21
AUC0-inf (ꢀg/L*h)
247.74±11.48
Cl(L/h/kg)
3.33±0.15
-
F%
-
1.02±0.17
-
1.33±0.16
0.3
268.49±69.72
36.10%
a Compounds were dosed to equal number of male Sprague-Dawley rats in po (10 mg/kg) and i.v.
(3 mg/kg) administration (n = 3).
3. Conclusion
To seek effective drugs for Alzheimer disease, a series of new dual inhibitors 6a-6t
by hybridization of rasagiline and clorgyline were designed, synthesized and
evaluated. All the target compounds were investigated for their ability to inhibit the
monoamine oxidases and amyloid-β aggregation. We were surprised to find that all
compounds were selective hMAO-B inhibitors with IC50 values ranging from 5.7 ꢀM
to 4 nM, and could penetrate the blood-brain barrier. Among these compounds,
compound 6j exhibited higher hMAO-B potency and selectivity (IC50 = 4 nM,
SI>25000) than the reference inhibitor rasagiline (IC50=141 nM, SI > 355), as well as

good inhibition of Aβ1-42 aggregation. In addition, kinetic and molecular modeling
studies suggested compound 6j was a competitive and reversible inhibitor for
hMAO-B. Meanwhile, compound 6j showed low cytotoxicity and neuroprotective
effects according to cell viability and neuroprotection activity assay. The further
pharmacokinetics studies showed that compound 6j had good pharmacokinetic
characteristics after intravenous and oral administrations. These properties highlighted
that compound 6j could serve as an effective and promising candidate for AD therapy.
4. Experimental section
4.1. Chemistry
Chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (China).
Thin layer chromatography (TLC, GF254 Qingdao Haiyang Chemical, QingDao,
China) was used for checking reaction, and components were visualized using UV
light. The compounds were purified by column chromatography (silica gel, 90-150
mm; Qingdao Marine Chemical Inc.). The synthesized compounds were characterized
by melting points apparatus, 1H NMR, 13C NMR and a Mariner ESI-TOF
spectrometer (HRESIMS) respectively. 1H NMR and 13C NMR spectra were recorded
on a Bruker AM-600 spectrometer, using CDCl3 or DMSO-d6 as the solvent.
4.2. 3-Chloro-1-(2,4-dihydroxyphenyl)propan-1-one (3)
A mixture of resorcinol (5.5 g) and 3-chloropropionic acid (5.4 g) were added
trifloromethanesulfonic acid (15 g). The reaction was stirred at 80 oC for 30 min. The
completed reaction solution was cool to room temperature and then poured into ice
water (120 ml). The water solution was extracted with Cl2CH2. The Cl2CH2 layer was
concentrated under vacuum to afford crude product used for the next reaction, without
further purification. It is the same method as our group reported reference [42].
4.3. 7-Hydroxychroman-4-one (4d)
To a solution of 2 N aqueous, crude compound 3 was added and stirred at room
temperature for 2h. After the reaction completed, the solution was acidified with 6 M
H2SO4 to PH = 2, and then was extracted with ethyl acetate. The product 4d was
purified by silica gel chromatography. Structural identification data of compound 4d
as shown in our reported reference [42].

4.4. General procedures for the preparation of compounds 6a-6t
A mixture of compound 4a-4h (4 mmol), suitable α, ω-dibromoalkanes (40 mmol)
and powdered K2CO3 (8 mmol) in acetone was stirred at reflux for 4h. The obtained
residue was purified by silica gel chromatography with PE/EA (6:1) as elution solvent
to give compounds 5a-h [42]. To a solution of compounds 5a-5h (1 mmol) and
K2CO3 (2 mmol) in DMF was added propargyl amine (1.4 mmol). The reaction was
stirred at 60 oC for 6-12 h. After completion, the residue was added ethyl acetate, and
the organic phase was washed with water three times. The combined organic phase,
dried over anhydrous Na2SO4, concentrated on vacuum, and was purified by silica gel
chromatography with PE/EA (4:1) as eluent to obtain the target compounds 6a-6t.
4.4.1. 5-(4-(methyl(prop-2-yn-1-yl)amino)butoxy)-2,3-dihydro-1H-inden-1-one (6a)
Yield 83%; pale yellow oil; 1H NMR (500 MHz, CDCl3) δ 7.68–7.63 (m, 1H), 6.87 (d,
J = 5.4 Hz, 2H), 4.04 (t, J = 6.3 Hz, 2H), 3.36 (d, J = 2.2 Hz, 2H), 3.12–3.02 (m, 2H),
2.69–2.61 (m, 2H), 2.54–2.47 (m, 2H), 2.32 (s, 3H), 2.22 (t, J = 2.2 Hz, 1H), 1.89–
13
1.80 (m, 2H), 1.72–1.61 (m, 2H). C NMR (126 MHz, CDCl3) δ 205.45 (s), 164.83
(s), 158.27 (s), 130.35 (s), 125.42 (s), 115.74 (s), 110.36 (s), 78.38 (s), 73.41 (s),
68.17 (s), 55.18 (s), 45.59 (s), 41.75 (s), 36.53 (s), 26.95 (s), 25.97 (s), 24.03 (s).
HRMS: calcd for C17H21NO2 [M+H]+ 272.1645, found 272.1658.
4.4.2. 5-((5-(methyl(prop-2-yn-1-yl)amino)pentyl)oxy)-2,3-dihydro-1H-inden-1-one
(6b)
1
Yield 87%; pale yellow oil; H NMR (500 MHz, CDCl3) δ 7.68–7.62 (m, 1H), 6.89–
6.83 (m, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.34 (d, J = 2.3 Hz, 2H), 3.09 – 3.02 (m, 2H),
2.68–2.60 (m, 2H), 2.47–2.43 (m, 2H), 2.31 (s, 3H), 2.22 (t, J = 2.3 Hz, 1H), 1.88–
13
1.77 (m, 2H), 1.59–1.45 (m, 4H). C NMR (126 MHz, CDCl3) δ 205.45 (s), 164.87
(s), 158.28 (s), 130.30 (s), 125.41 (s), 115.73 (s), 110.31 (s), 78.40 (s), 73.38 (s),
68.33 (s), 55.53 (s), 45.58 (s), 41.82 (s), 36.52 (s), 29.04 (s), 27.29 (s), 25.96 (s),
23.92 (s). HRMS: calcd for C18H23NO2 [M+H]+ 286.1802, found 286.1813.
4.4.3.
5-((6-(methyl(prop-2-yn-1-yl)amino)hexyl)oxy)-2,3-dihydro-1H-inden-1-one
(6c)
Yield 78%; pale yellow solid; m.p. 52.7-53.2 oC; 1H NMR (500 MHz, CDCl3) δ 7.68–

7.64 (m, 1H), 6.87 (d, J = 4.9 Hz, 2H), 4.01 (t, J = 6.5 Hz, 2H), 3.34 (d, J = 2.2 Hz,
2H), 3.10–3.03 (m, 2H), 2.69–2.60 (m, 2H), 2.48–2.38 (m, 2H), 2.30 (s, 3H), 2.21 (t,
J = 2.2 Hz, 1H), 1.87–1.76 (m, 2H), 1.49 (dq, J = 14.5, 7.2 Hz, 4H), 1.39 (dd, J = 14.3,
7.5 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 205.43 (s), 164.92 (s), 158.27 (s), 130.30
(s), 125.41 (s), 115.74 (s), 110.34 (s), 78.48 (s), 73.31 (s), 68.41 (s), 55.64 (s), 45.57
(s), 41.84 (s), 36.52 (s), 29.12 (s), 27.53 (s), 27.19 (s), 26.02 (s), 25.96 (s). HRMS:
calcd for C19H25NO2 [M+H]+ 300.1958, found 300.1961.
4.4.4. 6-((6-(methyl(prop-2-yn-1-yl)amino)hexyl)oxy)benzofuran-3(2H)-one (6d)
1
Yield 86%; yellow oil; H NMR (500 MHz, CDCl3) δ 7.55 (d, J = 8.6 Hz, 1H), 6.63
(dd, J = 8.6, 1.7 Hz, 1H), 6.51 (d, J = 1.5 Hz, 1H), 4.61 (s, 2H), 4.01 (t, J = 6.4 Hz,
2H), 3.41 (s, 2H), 2.55 – 2.47 (m, 2H), 2.37 (s, 3H), 2.27 (s, 1H), 1.86 – 1.77 (m, 2H),
13
1.55 (dd, J = 15.0, 7.5 Hz, 2H), 1.51 – 1.46 (m, 2H), 1.43 – 1.36 (m, 2H). C NMR
(126 MHz, CDCl3) δ 196.72 (s), 175.69 (s), 166.88 (s), 124.18 (s), 113.28 (s), 111.20
(s), 95.88 (s), 74.67 (s), 73.06 (s), 67.77 (s), 54.53 (s), 44.44 (s), 40.66 (s), 27.96 (s),
26.20 (s), 26.12 (s), 24.97 (s). HRMS: calcd for C18H23NO3 [M+H]+ 302.1751, found
302.1764.
4.4.5. 6-(4-(methyl(prop-2-yn-1-yl)amino)butoxy)-3,4-dihydronaphthalen-1(2H)-one
(6e)
1
Yield 91%; pale yellow oil; H NMR (500 MHz, CDCl3) δ 7.96 (d, J = 8.7 Hz, 1H),
6.78 (dd, J = 8.7, 1.9 Hz, 1H), 6.67 (s, 1H), 4.01 (t, J = 6.3 Hz, 2H), 3.34 (d, J = 1.8
Hz, 2H), 2.88 (t, J = 6.0 Hz, 2H), 2.60 – 2.54 (m, 2H), 2.47 (t, J = 7.3 Hz, 2H), 2.30 (s,
3H), 2.21 (s, 1H), 2.12 – 2.04 (m, 2H), 1.86 – 1.77 (m, 2H), 1.68 – 1.57 (m, 2H). 13C
NMR (126 MHz, CDCl3) δ 197.30 (s), 163.10 (s), 147.00 (s), 129.66 (s), 126.22 (s),
113.50 (s), 113.18 (s), 78.46 (s), 77.16 (s), 73.30 (s), 67.87 (s), 55.16 (s), 45.57 (s),
41.73 (s), 38.96 (s), 30.22 (s), 26.95 (s), 24.02 (s), 23.45 (s). HRMS: calcd for
C18H23NO2 [M+H]+ 286.1801, found 286.1815.
4.4.6.
6-((5-(methyl(prop-2-yn-1-yl)amino)pentyl)oxy)-3,4-dihydronaphthalen-1(2H)-one
(6f)
1
Yield 76%; pale yellow oil; H NMR (500 MHz, CDCl3) δ 7.96 (d, J = 8.7 Hz, 1H),

6.77 (dd, J = 8.7, 2.1 Hz, 1H), 6.66 (d, J = 1.4 Hz, 1H), 3.98 (t, J = 6.4 Hz, 2H), 3.32
(d, J = 2.1 Hz, 2H), 2.88 (t, J = 6.0 Hz, 2H), 2.59 – 2.54 (m, 2H), 2.42 (t, J = 7.1 Hz,
2H), 2.29 (s, 3H), 2.21 (t, J = 2.0 Hz, 1H), 2.11 – 2.03 (m, 2H), 1.85 – 1.75 (m, 2H),
1.56 – 1.42 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 197.31 (s), 163.14 (s), 147.00 (s),
129.65 (s), 126.18 (s), 113.48 (s), 113.14 (s), 78.47 (s), 73.27 (s), 68.02 (s), 55.50 (s),
45.55 (s), 41.79 (s), 38.94 (s), 30.20 (s), 29.03 (s), 27.28 (s), 23.88 (s), 23.43 (s).
HRMS: calcd for C19H25NO2 [M+H]+ 300.1958, found 300.1970.
4.4.7.
6-((6-(methyl(prop-2-yn-1-yl)amino)hexyl)oxy)-3,4-dihydronaphthalen-1(2H)-one
(6g)
1
Yield 74%; pale yellow oil; H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 8.7 Hz, 1H),
6.78 (dd, J = 8.7, 2.4 Hz, 1H), 6.67 (d, J = 2.2 Hz, 1H), 3.98 (t, J = 6.5 Hz, 2H), 3.34
(d, J = 2.3 Hz, 2H), 2.89 (t, J = 6.1 Hz, 2H), 2.58 (t, J = 6.5 Hz, 2H), 2.42 (t, J = 6.5
Hz, 2H), 2.30 (s, 3H), 2.21 (t, J = 2.3 Hz, 1H), 2.14–2.04 (m, 2H), 1.84–1.74 (m, 2H),
13
1.48 (m, 4H), 1.38 (m, 2H). C NMR (126 MHz, CDCl3) δ 197.35 (s), 163.21 (s),
147.02 (s), 129.68 (s), 126.19 (s), 113.50 (s), 113.19 (s), 78.45 (s), 73.31 (s), 68.11 (s),
55.63 (s), 45.54 (s), 41.81 (s), 38.98 (s), 30.24 (s), 29.13 (s), 27.50 (s), 27.18 (s),
26.00 (s), 23.47 (s). HRMS: calcd for C20H27NO2 [M+H]+ 314.2114, found 314.2127.
4.4.8. 7-(3-(methyl(prop-2-yn-1-yl)amino)propoxy)chroman-4-one (6h)
Yield 81%; yellow solid; m.p. 55.1-56.7 oC; 1H NMR (500 MHz, CDCl3) δ 7.81 (d, J
= 8.8 Hz, 1H), 6.56 (dd, J = 8.8, 2.2 Hz, 1H), 6.39 (d, J = 2.2 Hz, 1H), 4.49 (t, J = 6.4
Hz, 2H), 4.03 (t, J = 6.3 Hz, 2H), 3.36 (d, J = 2.2 Hz, 2H), 2.73 (t, J = 6.4 Hz, 2H),
2.60 (t, J = 7.1 Hz, 2H), 2.33 (s, 3H), 2.23 (t, J = 2.3 Hz, 1H), 1.95 (p, J = 6.6 Hz, 2H).
13C NMR (126 MHz, CDCl3) δ 190.65 (s), 165.49 (s), 163.87 (s), 128.93 (s), 115.29
(s), 110.33 (s), 101.37 (s), 78.27 (s), 73.52 (s), 67.46 (s), 66.47 (s), 52.13 (s), 45.75 (s),
41.76 (s), 37.53 (s), 27.14 (s). HRMS: calcd for C16H19NO3 [M+H]+ 274.1438, found
274.1438.
4.4.9. 7-(4-(methyl(prop-2-yn-1-yl)amino)butoxy)chroman-4-one (6i)
1
Yield 71%; pale yellow oil; H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 8.8 Hz, 1H),
6.56 (dd, J = 8.8, 2.4 Hz, 1H), 6.38 (d, J = 2.3 Hz, 1H), 4.50 (t, J = 6.3 Hz, 2H), 4.00

(t, J = 6.3 Hz, 2H), 3.37 (d, J = 2.3 Hz, 2H), 2.74 (t, J = 5.0 Hz, 2H), 2.50 (t, J = 5.0
13
Hz, 2H), 2.33 (s, 3H), 2.23 (t, J = 2.4 Hz, 1H), 1.87–1.77 (m, 2H), 1.64 (m, 2H). C
NMR (126 MHz, CDCl3) δ 190.77 (s), 165.63 (s), 163.78 (s), 128.95 (s), 115.29 (s),
110.31 (s), 101.28 (s), 78.01 (s) 73.69 (s), 68.09 (s), 67.43 (s), 55.06 (s), 45.73 (s),
42.00 (s), 37.29 (s), 27.07 (s), 23.98 (s). HRMS: calcd for C17H21NO3 [M+H]+
288.1594, found 288.1568.
4.4.10. 7-((5-(methyl(prop-2-yn-1-yl)amino)pentyl)oxy)chroman-4-one (6j)
1
Yield 89%; yellow oil; H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 8.8 Hz, 1H), 6.55
(dd, J = 8.8, 2.4 Hz, 1H), 6.38 (d, J = 2.3 Hz, 1H), 4.50 (t, J = 6.4 Hz, 2H), 3.98 (t, J =
6.4 Hz, 2H), 3.37 (d, J = 2.3 Hz, 2H), 2.74 (t, J = 5.0 Hz 2H), 2.47 (t, J = 5.0 Hz, 2H),
2.33 (s, 3H), 2.24 (t, J = 2.4 Hz, 1H), 1.81 (q, J = 6.6 Hz, 2H), 1.60–1.52 (m, 2H),
1.48 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 190.76 (s), 165.61 (s), 163.90 (s),
128.97 (s), 115.30 (s), 110.32 (s), 101.29 (s), 78.27 (s), 73.69 (s), 68.35 (s), 67.49 (s),
55.53 (s), 45.59 (s), 41.82 (s), 37.57 (s), 28.98 (s), 27.38 (s), 23.92 (s). HRMS: calcd
for C18H23NO3 [M+H]+ 302.1751, found 302.1745.
4.4.11. 7-((6-(methyl(prop-2-yn-1-yl)amino)hexyl)oxy)chroman-4-one (6k)
1
Yield 73%; pale yellow oil; H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 8.8 Hz, 1H),
6.56 (dd, J = 8.8, 2.3 Hz, 1H), 6.38 (d, J = 2.3 Hz, 1H), 4.58–4.45 (m, 2H), 3.97 (t, J
= 6.4 Hz, 2H), 3.46 (s, 2H), 2.79–2.70 (m, 2H), 2.62–2.51 (m, 2H), 2.42 (s, 3H), 2.31
(s, 1H), 1.86–1.73 (m, 2H), 1.58 (dt, J = 15.0, 7.6 Hz, 2H), 1.52–1.45 (m, 2H), 1.40
(dd, J = 15.1, 8.1 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 190.69 (s), 165.63 (s),
163.92 (s), 128.98 (s), 115.26 (s), 110.39 (s), 101.31 (s), 68.37 (s), 67.50 (s), 55.47 (s),
45.34 (s), 41.52 (s), 37.57 (s), 29.01 (s), 27.07 (s), 25.94 (s), 26.93. HRMS: calcd for
C19H25NO3 [M+H]+ 316.1907, found 316.1913.
4.4.12. 7-((8-(methyl(prop-2-yn-1-yl)amino)octyl)oxy)chroman-4-one (6l)
1
Yield 89%; pale yellow oil; H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 8.8 Hz, 1H),
6.56 (dd, J = 8.8, 2.3 Hz, 1H), 6.38 (d, J = 2.3 Hz, 1H), 4.50 (t, J = 6.4 Hz, 2H), 3.97
(t, J = 6.5 Hz, 2H), 3.39 (d, J = 2.1 Hz, 2H), 2.74 (t, J = 5.0 Hz, 2H), 2.46 (t, J = 5.0
Hz, 2H), 2.35 (s, 3H), 2.25 (t, J = 2.3 Hz, 1H), 1.77 (m, 2H), 1.55–1.40 (m, 4H), 1.33
(br, 6H). 13C NMR (126 MHz, CDCl3) δ 190.68 (s), 165.69 (s), 163.91 (s), 128.95 (s),

115.21 (s), 110.41 (s), 101.29 (s), 77.90 (s), 73.84 (s), 68.52 (s), 67.48 (s), 55.73 (s),
45.47 (s), 41.71 (s), 37.57 (s), 29.50 (s), 29.34 (s), 29.07 (s), 27.37 (s), 26.01 (s).
HRMS: calcd for C21H29NO3 [M+H]+ 344.2220, found 344.2221.
4.4.13. 1-(4-(4-(methyl(prop-2-yn-1-yl)amino)butoxy)phenyl)ethan-1-one (6m)
1
Yield 81%; pale yellow oil; H NMR (500 MHz, DMSO-d6) δ 7.91 (d, J = 8.8 Hz,
2H), 7.02 (d, J = 8.8 Hz, 2H), 4.06 (t, J = 6.5 Hz, 2H), 3.29 (s, 3H), 3.10 (s, 1H), 2.39
(t, J = 7.1 Hz, 2H), 2.19 (s, 3H), 1.78–1.68 (m, 2H), 1.53 (dt, J = 14.6, 7.3 Hz, 2H),
1.34 (s, 2H). 13C NMR (126 MHz, DMSO) δ 196.32 (s), 162.55 (s), 130.51 (s),
129.75 (s), 114.28 (s), 75.77 (s), 67.70 (s), 54.45 (s), 44.89 (s), 41.18 (s), 29.80 (s),
26.42 (s), 26.31 (s), 23.24 (s). HRMS: calcd for C16H21NO2 [M+H]+ 260.1645, found
260.1651.
4.4.14. 1-(4-((6-(methyl(prop-2-yn-1-yl)amino)hexyl)oxy)phenyl)ethan-1-one (6n)
Yield 74%; pale yellow oil;1H NMR (500 MHz, DMSO-d6) δ 8.00–7.80 (m, 2H),
7.09–6.93 (m, 2H), 4.04 (t, J = 6.5 Hz, 2H), 3.27 (s, 3H), 3.09 (s, 1H), 2.32 (t, J = 7.1
Hz, 2H), 2.17 (s, 3H), 1.77–1.68 (m, 2H), 1.43–1.36 (m, 4H), 1.35–1.27 (m, 4H). 13C
NMR (126 MHz, DMSO) δ 196.31 (s), 162.58 (s), 130.50 (s), 129.74 (s), 114.26 (s),
75.68 (s), 67.85 (s), 54.87 (s), 44.90 (s), 41.29 (s), 28.53 (s), 26.79 (s), 26.55 (s),
26.42 (s), 25.36 (s). HRMS: calcd for C18H25NO2 [M+H]+ 288.1958, found 288.1966.
4.4.15. 5-(3-methoxyphenoxy)-N-methyl-N-(prop-2-yn-1-yl)pentan-1-amine (6o)
Yield 84%; pale yellow oil; 1H NMR (500 MHz, DMSO-d6) δ 7.15 (t, J = 8.2 Hz, 1H),
6.50 (t, J = 2.5 Hz, 1H), 6.48 (t, J = 2.4 Hz, 1H), 6.46 (t, J = 2.3 Hz, 1H), 3.92 (t, J =
6.5 Hz, 2H), 3.72 (s, 3H), 3.10 (s, 1H), 2.35 (s, 2H), 2.18 (s, 3H), 1.69 (dd, J = 12.8,
5.9 Hz, 2H), 1.46–1.38 (m, 4H), 1.35 (d, J = 2.6 Hz, 2H). 13C NMR (126 MHz,
DMSO) δ 160.94 (s), 160.39 (s), 130.37 (s), 107.03 (s), 106.61 (s), 101.07 (s), 67.81
(s), 55.51 (s), 55.28 (s), 45.33 (s), 30.24 (s), 29.00 (s), 23.83 (s). HRMS: calcd for
C16H23NO2 [M+H]+ 262.1801, found 266.1809.
4.4.16. 1-(3-methoxy-4-(4-(methyl(prop-2-yn-1-yl)amino)butoxy)phenyl)ethan-1-one
(6p)
o
Yield 75%; pale yellow solid; m.p. 65.1-65.9 C; 1H NMR (500 MHz, CDCl3) δ 7.54
(dd, J = 8.3, 2.0 Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 4.10 (t, J

= 6.6 Hz, 2H), 3.90 (s, 3H), 3.35 (d, J = 2.4 Hz, 2H), 2.55 (s, 3H), 2.51 – 2.45 (m, 2H),
2.31 (s, 3H), 2.21 (t, J = 2.4 Hz, 1H), 1.89 (dd, J = 14.9, 6.8 Hz, 2H), 1.65 (dt, J =
13
15.0, 7.5 Hz, 2H). C NMR (126 MHz, CDCl3) δ 196.71 (s), 152.70 (s), 149.08 (s),
130.14 (s), 123.09 (s), 111.01 (s), 110.25 (s), 78.27 (s), 72.91 (s), 68.39 (s), 55.86 (s),
54.81 (s), 45.35 (s), 41.50 (s), 26.60 (s), 25.99 (s), 23.59 (s). HRMS: calcd for
C17H23NO3 [M+H]+ 290.1751, found 290.1749.
4.4.17.
1-(3-methoxy-4-((5-(methyl(prop-2-yn-1-yl)amino)pentyl)oxy)phenyl)ethan-1-one (6q)
1
Yield 79%; yellow oil; H NMR (500 MHz, CDCl3) δ 7.54 (dd, J = 8.3, 2.0 Hz, 1H),
7.50 (d, J = 2.0 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 4.10 (t, J = 6.6 Hz, 2H), 3.90 (s,
3H), 3.35 (d, J = 2.4 Hz, 2H), 2.55 (s, 3H), 2.51–2.45 (m, 2H), 2.31 (s, 3H), 2.21 (t, J
= 2.4 Hz, 1H), 1.89 (dd, J = 14.9, 6.8 Hz, 2H), 1.65 (dt, J = 15.0, 7.5 Hz, 2H). 13C
NMR (126 MHz, CDCl3) δ 196.98 (s), 152.98 (s), 149.29 (s), 130.28 (s), 123.49 (s),
111.09 (s), 110.55 (s), 78.51 (s), 73.31 (s), 68.97 (s), 56.13 (s), 55.54 (s), 45.61 (s),
41.84 (s), 28.96 (s), 27.30 (s), 26.34 (s), 23.88 (s). HRMS: calcd for C18H25NO3
[M+H]+ 304.1907, found 304.1908.
4.4.18. 2-methoxy-4-(4-(methyl(prop-2-yn-1-yl)amino)butoxy)benzaldehyde (6r)
Yield 79%; red solid; m.p. 83.6-84.2 oC; 1H NMR (500 MHz, CDCl3) δ 10.26 (s, 1H),
7.78 (d, J = 8.7 Hz, 1H), 6.52 (dd, J = 8.7, 1.9 Hz, 1H), 6.43 (d, J = 2.0 Hz, 1H), 4.04
(t, J = 6.3 Hz, 2H), 3.88 (s, 3H), 3.36 (s, 2H), 2.53–2.47 (m, 2H), 2.32 (s, 3H), 2.22 (t,
J = 2.3 Hz, 1H), 1.88–1.79 (m, 2H), 1.70–1.60 (m, 2H). 13C NMR (126 MHz, CDCl3)
δ 188.48 (s), 165.80 (s), 163.71 (s), 130.85 (s), 119.01 (s), 106.28 (s), 98.45 (s), 78.38
(s), 73.42 (s), 68.17 (s), 55.71 (s), 55.16 (s), 45.60 (s), 41.77 (s), 26.96 (s), 24.02 (s).
HRMS: calcd for C16H21NO3 [M+H]+ 276.1594, found 276.1601.
4.4.19. 2-methoxy-4-((5-(methyl(prop-2-yn-1-yl)amino)pentyl)oxy)benzaldehyde (6s)
o
1
Yield 82%; yellow solid; m.p. 51.2-51.6 C; H NMR (500 MHz, CDCl3) δ 10.24 (s,
1H), 7.76 (d, J = 8.7 Hz, 1H), 6.49 (dd, J = 8.7, 1.8 Hz, 1H), 6.41 (d, J = 1.9 Hz, 1H),
4.00 (t, J = 6.4 Hz, 2H), 3.87 (s, 3H), 3.33 (d, J = 2.2 Hz, 2H), 2.43 (t, J = 7.1 Hz, 2H),
2.37 (s, 1H), 2.29 (s, 3H), 2.21 (t, J = 2.2 Hz, 1H), 1.87–1.76 (m, 2H), 1.50 (t, J =
11.4 Hz, 4H). 13C NMR (126 MHz, CDCl3) δ 188.44 (s), 165.82 (s), 163.68 (s),

130.79 (s), 118.92 (s), 106.24 (s), 98.38 (s), 78.48 (s), 73.29 (s), 68.33 (s), 55.66 (s),
55.50 (s), 45.56 (s), 41.81 (s), 29.02 (s), 27.29 (s), 23.89 (s). HRMS: calcd for
C17H23NO3 [M+H]+ 290.1751, found 290.1764.
4.4.20. 2-methoxy-4-((6-(methyl(prop-2-yn-1-yl)amino)hexyl)oxy)benzaldehyde (6t)
Yield 73%; yellow power; m.p. 57.5-57.9 oC;1H NMR (500 MHz, CDCl3) δ 10.26 (s,
1H), 7.77 (d, J = 8.7 Hz, 1H), 6.51 (dd, J = 8.7, 1.5 Hz, 1H), 6.42 (d, J = 1.8 Hz, 1H),
4.00 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 3.34 (d, J = 2.1 Hz, 2H), 2.48–2.38 (m, 2H),
2.30 (s, 3H), 2.21 (t, J = 2.2 Hz, 1H), 1.85–1.74 (m, 2H), 1.55–1.44 (m, 4H), 1.39 (dd,
13
J = 14.5, 7.6 Hz, 2H). C NMR (126 MHz, CDCl3) δ 188.44 (s), 165.87 (s), 163.70
(s), 130.82 (s), 118.94 (s), 106.26 (s), 98.41 (s), 78.47 (s), 73.31 (s), 68.41 (s), 55.64
(s), 45.57 (s), 41.83 (s), 29.14 (s), 27.53 (s), 27.19 (s), 26.01 (s). HRMS: calcd for
C18H25NO3 [M+H]+ 304.1907, found 304.1921.
4.5. MAO inhibition assay
The potential effects of all the synthesized compounds 6a-6t on MAOs activity was
investigated according to preciously reported fluorescence methods [57-58]. The
experiment was measuring the production of H2O2 from p-tyramine, using
recombinant human MAOs and Amplex Red assay kit. The enzyme of hMAO-A and
hMAO-B was obtained from Sigma-Aldrich (St. Louis, MO, USA), and p-tyramine
was purchased from Molecular Probes, Inc. (Eugene, Oregon, USA). Each synthetic
compound was adjusted with DMSO and PBS buffer solution to different final
concentrations (DMSO < 0.1%). The test compound was combined with hMAO-A (or
o
hMAO-B) and other substrate, and then incubated for 15 min at 37 C in a 96-well
black microtiter plate in the dark. The results were tested by a multidetection
microplate fluorescence reader at excitation/emission wavelengths of 545/590 nm.
Data were shown as mean ± SD of three independent experiments.
4.6 Molecular modeling studies
Molecular modeling studies were carried out using the Chemical Computing Group's
Molecular Operating Environment (MOE) software (Montreal, Canada, version
2015.10). The docking template structure of MAO-B was derived from the X-ray
crystal
structure
of
the
human
MAO-B
in
complex
with

7-(3-chlorobenzyloxy)-4-(methylamino)methyl-coumarin (PDB code 2V61) and all
water molecules were removed. The protein was energy minimized and 3D protonated
using the structure preparation module of MOE. Ligand file for the molecular docking
studies was prepared in MOE and energy minimized using Merck Molecular force
field (MMFF94x, RMSD gradient: 0.05 kcal mol-1 Å-1). Then, the optimized
geometry of ligand was saved in a molecular database file and docked into the active
site of the protein using the MOE-Dock program. The London dG was chosen as
initial scoring method and Rigid Receptor was selected as the final scoring method.
Finally, the geometry of docked complex was analyzed by the pose viewer utility in
MOE.
4.7. Reversibility and kinetic study of hMAO-B inhibition
The reversibility experiment of compound 6j for MAO-B was performed according
to a previously reported method [59]. Compound 6j at concentrations of 10×IC50 and
o
100×IC50 was treated with hMAO-B enzyme (0.75 mg/mL) for 30 min at 37 C. The
parallel control was without compound 6j under the same condition. Then, the
concentrations of compound 6j was diluted to 100-fold respectively. Pargyline at
concentration of 10×IC50 was diluted to 0.1 × IC50 by the same times.
Lineweaver-Burk reciprocal plots revealed hMAO-B inhibition mechanism of
compound 6j. Compound 6j at three different concentrations (2.5, 5 and 10 nM) were
for kinetic study. The initial catalytic rates of hMAO-B were tested in the presence or
absence of compound 6j at the corresponding concentrations with different
concentrations (0.05, 0.1, 0.5, 1.0, 1.5, and 3.0 mM) of p-tyramine. The results were
analyzed by Graph Pad Prism 4.03 software (San Diego, CA, USA).
4.8. Inhibition of self-induced Aβ1-42 aggregation
A thioflavin T (ThT)-binding assay [60] was used to measure the inhibition of
self-induced Aβ1-42 aggregation. Curcumin was the reference compounds. Aβ1-42 was
o
aliquoted and stored at -80 C. Before the experiment of compounds inhibiting
self-induced Aβ1-42 aggregation, Aβ1-42 stock solution was diluted to 50 mM with 50
mM phosphate buffer (ph = 7.4). Aβ1-42 solution (10 mL, 25 mM, final concentration)
o
in the presence or absence of compound (10 mL) was incubated at 37 C for 48 h.