Multifunctional thioxanthone derivatives with acetylcholinesterase, monoamine oxidases and β-amyloid aggregation inhibitory activities as potential agents against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2017.02.027

A series of 1-hydroxyl-3-aminoalkoxy-thioxanthone derivatives were designed, synthesized and evaluated as potential multifunctional agents against Alzheimer's disease (AD). The results indicated that most of these compounds exhibited good AChE and MAOs inhibitory activities, significant inhibition of self- and Cu²⁺-induced Aβ_1-42aggregation, and moderate to good antioxidant activities. Specifically, compound 9e displayed high inhibitory potency toward AChE (IC₅₀= 0.59 ± 0.02 μM), MAO-A and MAO-B (IC₅₀= 1.01 ± 0.02 μM and 0.90 ± 0.01 μM respectively), excellent efficiency to block both self- and Cu²⁺-induced Aβ_1-42aggregation (74.8 ± 1.2% and 87.7 ± 1.9% at 25 μM, respectively), good metal-chelating property and a low toxicity in SH-SY5Y cells. Furthermore, kinetic and molecular modeling studies revealed that compound 9e binds simultaneously to the catalytic active site and peripheral anionic site of AChE, and could penetrate the BBB. Collectively, these results suggested that 9e might be a potential multifunctional agent for further development in the treatment of AD.

Full text of DOI:10.1016/j.bmc.2017.02.027

Accepted Manuscript
Multifunctional thioxanthone derivatives with acetylcholinesterase, monoa-
mine oxidases and β-amyloid aggregation inhibitory activities as potential
agents against Alzheimer’s disease
Li Luo, Yan Li, Xiaoming Qiang, Zhongcheng Cao, Rui Xu, Xia Yang, Ganyuan
Xiao, Qing Song, Zhenghuai Tan, Yong Deng
PII:
S0968-0896(17)30050-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.027
BMC 13556
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 January 2017
10 February 2017
12 February 2017
Please cite this article as: Luo, L., Li, Y., Qiang, X., Cao, Z., Xu, R., Yang, X., Xiao, G., Song, Q., Tan, Z., Deng,
Y., Multifunctional thioxanthone derivatives with acetylcholinesterase, monoamine oxidases and β-amyloid
aggregation inhibitory activities as potential agents against Alzheimer’s disease, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.027
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Multifunctional thioxanthone derivatives with acetylcholinesterase, monoamine
oxidases and β-amyloid aggregation inhibitory activities as potential agents
against Alzheimer’s disease
Li Luoa,#, Yan Lia,#, Xiaoming Qianga, Zhongcheng Caoa, Rui Xua, Xia Yanga, Ganyuan Xiaoa, Qing
Songa, Zhenghuai Tanb,*, Yong Denga,*
aDepartment of Medicinal Chemistry, Key Laboratory of Drug Targeting and Drug Delivery System
of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, 610041,
China
bInstitute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan academy of
Chinese Medicine Sciences, Chengdu, 610041, China
# These authors contributed equally to this work.
*Corresponding Author.
Phone, +86-28-85258982; E-mail: tanzhh616@163.com (Zhenghuai Tan)
Phone, +86-28-85503790; E-mail: dengyong@scu.edu.cn (Yong Deng)

Abstract
A series of 1-hydroxyl-3-aminoalkoxy-thioxanthone derivatives were designed, synthesized and
evaluated as potential multifunctional agents against Alzheimer’s disease (AD). The results indicated
that most of these compounds exhibited good AChE and MAOs inhibitory activities, significant
inhibition of self- and Cu2+-induced Aβ1-42 aggregation, and moderate to good antioxidant activities.
Specifically, compound 9e displayed high inhibitory potency toward AChE (IC50 = 0.59 ± 0.02 μM),
MAO-A and MAO-B (IC50 = 1.01 ± 0.02 μM and 0.90 ± 0.01 μM respectively), excellent efficiency
to block both self- and Cu2+-induced Aβ1-42 aggregation (74.8 ± 1.2% and 87.7 ± 1.9% at 25 μM,
respectively), good metal-chelating property and a low toxicity in SH-SY5Y cells. Furthermore,
kinetic and molecular modeling studies revealed that compound 9e binds simultaneously to the
catalytic active site and peripheral anionic site of AChE, and could penetrate the BBB. Collectively,
these results suggested that 9e might be a potential multifunctional agent for further development in
the treatment of AD.
Keywords:
Alzheimer’s
disease;
thioxanthone
derivatives;
multifunctional
agents;
acetylcholinesterase inhibitors; anti-Aβ aggregation; monoamine oxidase inhibitors.

1. Introduction
Alzheimer’s disease (AD) is a multifaceted and progressive neurodegenerative disorder
characterized by memory loss, severe behavioral abnormalities, cognitive impairment and ultimately
death. And it is the most common manifestation of dementia in the elderly population.1 Although the
etiology of AD is not completely known, several factors have been considered to play significant
roles in the pathogenesis, including the development of deposits of β-amyloid (Aβ) and τ-protein,
dyshomeostasis of biometals, oxidative stress and deficit of acetylcholine (ACh). Several hypotheses
based on these factors have been proposed to explain the mechanism of AD development.2,3
Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are the two major forms of
cholinesterases in brain, and the AChE has attracted more attention since it could hydrolyze the
majority of ACh.4 According to the cholinergic hypothesis, the cognitive and memory deterioration
of AD is due to the deficit of ACh in specific regions of the brain.5 Currently, AChE inhibitors are
the most effective drugs for the treatment of AD.6 In addition, studies showed that AChE facilitates
amyloid fibril formation, obtaining stable AChE-Aβ complexes, which are more toxic than single Aβ
peptide.7 Dual-site inhibitors, which interact with both the catalytic active site (CAS) and peripheral
anionic site (PAS) of AChE can not only stimulate the cholinergic system, but also inhibit the
AChE-promoted Aβ production and aggregation.8 Thus, AChE is an important therapeutic target for
the treatment of AD.
Amyloid hypothesis, the most predominant hypothesis, asserts that an increased production and
accumulation of Aβ peptide in the brain was considered to initiate the pathogenic cascade, ultimately
lead to neuronal loss and dementia.9 Aβ1-40 and Aβ1-42 are the primary isoforms of Aβ peptides. Aβ1-42
displays a higher neuronal toxicity and aggregates more quickly than Aβ1-40, though the amount of
former is only 10% of the latter.10 Therefore, impeding the formation and deposition of Aβ1-42 is
thought to be a pivotal strategy for AD treatment.
Monoamine oxidase A and B (MAO-A and -B) are flavin adenine dinucleotide
(FAD)-containing enzymes, which show different substrate specificity and sensitivity to inhibitors.11
As known, most of the AD patients suffer from psychological symptoms such as depression and
psychosis. MAO-A inhibitors are used as antidepressants such as toloxatone and moclobemide, and
selective MAO-B inhibitors, such as rasagiline and selegiline, are beneficial for the treatment of
neurodegenerative disorders like AD.12 Besides, the biochemical activity of MAOs generates

hydrogen peroxide and gives rise to oxidative stress.12,13 Hence, MAOs are important factors that
involoved in AD pathogenesis.
Recently, studies has demonstrated that oxidative stress is a central event in initiating the Aβ
aggregation and τ-protein hyperphosphorylation, involved in the early stage of the pathologic
cascade.14,15 Consequently, drugs that specifically scavenge oxygen radicals might be useful for the
prevention or treatment of AD. In addition, there is increasing evidence that elevated concentrations
of the metal ions such as Cu2+, Zn2+, Fe2+ and Al3+ clearly exists in AD brains.16 The high levels of
these redox-active metals could promote the generation of reactive oxygen species (ROS) and
oxidative stress.17,18 Additionally, the abnormal accumulation of these metal ions is able to accelerate
the formation of Aβ plaques and neurofibrillary tangles (NFT), which could cause inflammation and
neurotoxicity.18,19 Accordingly, modulation of such biometals in brain has become a promising
therapeutic strategy for interdicting AD pathogenesis.
Due to the multifactorial pathogenesis of AD as mentioned above, the “multitarget-directed
ligands” (MTDLs) strategy seems more efficient. Compared to single-targeted drugs, it believes that
compounds with two or more distinct pharmacological properties closely related to the
neurodegenerative process are more effective in AD therapy.20-22
To date, it was reported that natural xanthones and their derivatives could inhibit both AChE
and BuChE, self-induced Aβ aggregation and serve as potential antioxidants and biometal
chelators.23-25 Thioxanthone (I, Figure 1, 9H-thioxanthen-9-one), a bioisostere of xanthone (II,
Figure 1), is the core structure of various natural occurring and synthetic compounds that exhibits
broad pharmacological activities such as anticancer26, antiparasitic27 and MAO-A inhibitory
activities.28 However, there are no reports about the research on thioxanthone derivates against AD.
Considering sulfur atom, as the isostere of oxygen in xanthone nucleus, is easier to lose electron and
can allows thioxanthones to display higher antioxidant potency. Furthermore, thioxanthones have
better lipid solubility and permeability. Therefore, thioxanthone could be used as a promising lead
compound in the design of multifunctional agents against AD. Recently, our group has reported a
7,4′-O-modified genistein derivative (III) as the potential multifunctional agent for AD treatment,
and described the benzylamine moieties as AChE inhibitory pharmacophores.29 However, this
compound showed low inhibitory effect on self-induced Aβ1-42 aggregation. Therefore, according to
the MTDLs strategy, we combined 1,3-dihydroxy-9H-thioxanthen-9-one (IV) with appropriate

secondary amines using different lengths of carbon spacers to design a series of novel
1-hydroxyl-3-aminoalkoxy thioxanthone derivatives (Figure 2). In this article, these new compounds
were synthesized and evaluated for their biological activities, including AChE, MAOs and Aβ
aggregation inhibitory activities, antioxidant and metal chelating effects.
Figure 1. Structures of thioxanthone (I) and xanthone (II)
Figure 2. Design strategy for 1-hydroxyl-3-aminoalkoxythioxanthone derivatives.
2. Results and discussion
2.1. Chemistry
The general route for the synthesis of 1-hydroxyl-3-aminoalkoxythioxanthone derivatives was
shown in Scheme 1. 2,2’-disulfanediyldibenzoic acid (1) was prepared according to the literature
using 2-aminobenzoic acid as a starting material.30 1,3-dihydroxy-9H-thioxanthen-9-one (2) was
synthesized in a yield of 46.4% via the cyclization of 1 and phloroglucinol in the presence of Eaton’s
reagent.31,32 Then, 3-hydroxyl group of compound 2 was alkylated in the presence of K2CO3 by the
addition of two equivalent amounts of 1,3-dibromopropane, 1,4-dibromobutane or

1,6-dibromohexane to give the key intermediates 3-5 respectively.32,33 Due to the hydrogen bond
between 1-hydroxyl and 9-carbonyl group, the reactivity of 1-hydroxyl group was much lower than
3-hydroxyl group and 3-alkylated products were obtained exclusively32. Finally, compound 3-5 were
reacted with corresponding secondary amines 6a-f to afford the target compounds 7a-f, 8a-f, and
9a-f.29 All the thioxanthone derivatives were characterized by 1H NMR, 13C NMR and ESI-MS.
Scheme 1. Synthesis of thioxanthone derivatives 7-9. Reagents and conditions: (i) NaNO2, conc.HCl,
H2O, 5 °C for 20 min; (ii) Na2S.9H2O, S, NaOH, 0-5 °C, then r.t. for 2 h; (iii) Phloroglucinol, P2O5,
CH3SO3H, 90 °C for 1.5 h; (iv) Br(CH2)nBr, K2CO3, acetone, reflux for 5-7 h; (v) NHR1R2 (6a-f),
K2CO3, CH3CN, reflux for 4-9 h.
2.2. Pharmacology
2.2.1. Inhibition studies on AChE and BuChE
The inhibitory activities of all the synthesized derivatives against AChE from Electrophorus
electricus (EeAChE) and BuChE from rat serum (RatBuChE) were determined by modified Ellman’s
method34, using donepezil as a reference compound. The target compounds showed inhibitory
activity against AChE with IC50 values ranging from submicromolar to micromolar. As shown in
Table 1, all target compounds showed much more AChE inhibitory potency than the lead compound
2 (IC50 = 38.90 ± 0.85 μM), which indicated that the introduction of the O-alkylbenzylamine group
was beneficial. Among them, compounds 7e, 9a, 9b and 9e exhibited excellent efficiency to inhibit
AChE (IC50 = 0.25 ± 0.01 μM, 0.32 ± 0.02 μM, 0.58 ± 0.03 μM, 0.59 ± 0.02 μM, respectively).

Moreover, the AChE inhibitory potency was related to the length of alkylene spacer chains. In
general, the derivatives with a 6-methylene linker showed better inhibition of AChE than those with
a 3- or 4-methylene chain. For example, compounds 9a-c (n = 6) were much more potent than
compounds 7a-c (n = 3) and 8a-c (n = 4). Compared with 9a (n = 6, IC50 = 0.32 ± 0.02 μM), the
AChE inhibitory activity of 8a (n = 4) was about 39-fold lower (IC50 = 12.41 ± 0.29 μM), and 7a (n
= 3) was almost 35-fold lower (IC50 = 11.20 ± 0.24 μM). Therefore, the alkyl chain with 6-methylene
may be optimal for the inhibition of AChE. It also revealed that the various benzylamine moieties
were closely related to the inhibitory activity. Compounds possessing an N-methyl group usually
displayed better AChE inhibitory activity than those with an N-ethyl group (AChE inhibitory activity:
7a > 7b, 8a > 8b, 9a > 9b). Furthermore, the adoption of 2-dimethylamino group on the benzylamine
moiety dramatically improved AChE inhibitory activity (7e, 8e and 9e; IC50 = 0.25 ± 0.01 μM, 2.88
± 0.07 μM and 0.59 ± 0.02 μM, respectively), while the introduction of 4-dimethylamino group
significantly decreased the potency (7f, 8f and 9f; IC50 = 17.70 ± 0.56 μM, 25.40 ± 0.94 μM and
33.80 ± 0.98 μM, respectively). In contrast, the adoption of 2-methoxy group showed no obvious
regularity. Overall, the results suggested that N-benzylmethylamine and N-(2-dimethylaminobenzyl)
ethylamine might be more appropriate for AChE inhibition. Almost all compounds exhibited weak
activity against BuChE. This result showed that these derivatives were potent high selective AChE
inhibitors. This selectivity profile was a limitation to some extent, but it might be beneficial to
diminish peripheral cholinergic side effects and provide lower toxicity. A typical case in point is
tacrine, which had severe side effects because of its poor selectivity.35 Based on the above results,
compound 9e was selected for kinetic analysis to investigate the type of inhibition.
Table 1. Inhibition of AChE, BuChE and MAOs activities by compound 2 and its derivatives,
reference compounds and 7,4′-O-modified genistein derivative (III).
IC50(μM) ± SDa
EeAChEb
Inhibition(%)c
RatBuChE
IC50(μM) ± SD
Compound
SId
MAO-A
MAO-B
11.20 ± 0.24
18.40 ± 0.49
28.70 ± 0.98
2.79 ± 0.13
n.a.e
16.00 ± 0.63
13.20 ± 0.59
6.31 ± 0.12
8.17 ± 0.23
1.73 ± 0.03
7.13 ± 0.32
6.09 ± 0.19
1.88 ± 0.07
9.25
1.85
1.04
4.35
7a
7b
7c
7d
31.8 ± 0.74
30.2 ± 0.56
17.3 ± 0.27

0.25 ± 0.01
17.70 ± 0.56
12.41 ± 0.29
22.00 ± 0.83
9.16 ± 0.36
15.50 ± 0.49
2.88 ± 0.07
25.40 ± 0.94
0.32 ± 0.02
0.58 ± 0.03
4.28 ± 0.19
11.50 ± 0.51
0.59 ± 0.02
33.80 ± 0.98
38.90 ± 0.85
0.14 ± 0.003
22.4 ± 0.54
n.a.e
5.94 ± 0.21
2.79 ± 0.14
0.57 ± 0.03
0.34 ± 0.02
0.62 ± 0.03
1.02 ± 0.06
1.54 ± 0.04
1.00 ± 0.02
0.57 ± 0.01
1.44 ± 0.06
1.03 ± 0.02
1.30 ± 0.03
1.01 ± 0.02
1.25 ± 0.09
0.28 ± 0.01
—
3.73 ± 0.15
3.55 ± 0.09
0.37 ± 0.02
0.27 ± 0.01
0.59 ± 0.02
0.88 ± 0.03
2.02 ± 0.11
2.20 ± 0.08
0.35 ± 0.02
0.33 ± 0.01
0.38 ± 0.01
0.46 ± 0.03
0.90 ± 0.01
0.39 ± 0.03
0.30 ± 0.02
—
1.59
0.79
1.54
1.26
1.05
1.16
0.76
0.45
1.63
4.36
2.71
2.83
1.12
3.21
0.93
—
7e
7f
n.a.e
8a
8b
8c
8d
8e
8f
n.a.e
5.62 ± 0.24
n.a.e
30.3 ± 0.89
7.15 ± 0.42
n.a.e
9a
9b
9c
9d
9e
9f
8.83 ± 0.31
14.9 ± 0.76
13.9 ± 0.42
28.7 ± 0.54
n.a.e
n.a.e
2
20.0
III
IC50 = 21.4 ±
Donepezil
0.0226 ± 0.0010
—
—
—
0.97 μM
Clorgyline
Rasagiline
Iproniazid
—
—
—
—
—
—
0.0027 ± 0.0005
2.61 ± 0.02
2.43 ± 0.11
0.00112
0.00997 ± 0.00072 261.79
0.985 ± 0.025 0.85
0.837 ± 0.012
a
IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50%
and are the mean of three independent experiments, each performed in triplicate. Data were
expressed as mean ± SD (SD = standard deviation).
b From Electrophorus electricus.
c BuChE from rat serum and tested compounds were used at 50 μM.
d SI = IC50 (MAO-A) / IC50 (MAO-B).
e n.a. = no active. Compounds defined “no active” means that percent inhibition is less than 5.0% at a
concentration of 50 μM in the assay conditions.
2.2.2. Kinetic study for the inhibition of AChE
In order to investigate the inhibitory mechanism for this family of compounds toward AChE, a

kinetic study was performed with compound 9e by using EeAChE. The Lineweaver-Burk reciprocal
plots (Figure 3) indicated that with increasing concentrations of 9e, both the slopes (decreased Vmax)
and intercepts (higher Km) were increased. This pattern revealed a mixed-type inhibition, which
implied that compound 9e was likely to bind both of the CAS and PAS of AChE.
Figure 3. Kinetic study on the mechanism of EeAChE inhibition by compound 9e. Merged
Lineweaver-Burk reciprocal plots of AChE initial velocity with increasing substrate concentration
(0.1-0.4 mM) in the absence or presence of 9e. Lines were derived from a weighted least-squares
analysis of data points.
2.2.3. Molecular modeling study of AChE
To clarify the binding modes between thioxanthone derivatives and AChE, a molecular
modeling study was carried out using the docking program, AutoDock 4.2 package with Discovery
Studio 2.5, based on the X-ray crystal structure of Torpedo californica (Tc) AChE (PDB: 1EVE).36-38
Considering the result of AChE inhibition assay and the kinetic study, compound 9e was chosen for
our molecular modeling investigation. Due to the flexible structure of 9e, there were many
conformational clusters, most of which showed the binding energy with -12 to -11 kcal/mol. Took
into account the binding energy and the conformational distribution, the most stable binding mode
was graphically inspected (Figure 4, binding energy: -12.34 kcal/mol, inhibit constant: 0.904 nM).
As shown in Figure 4, 9e occupied the entire enzymatic CAS, the mid-gorge sites and PAS. One
hydrogen bond was generated between the ligand 9e and the residue Arg289 (Arg289=O • • • HO-9e).

The long chain of methylene folded in a conformation in the gorge that allowed it to interact with
Tyr334, Phe330 and Phe331via the hydrophobic interaction. Moreover, the benzylamine moiety of 9e
was observed to adopt a parallel π-π interaction with Trp84. The docking study indicated that 9e
could stably bind to the CAS and PAS of AChE, consistent with our kinetic analysis result.
Figure 4. Docking pose of compound 9e (colored by atom type) into the binding site of TcAChE
(PDB: 1EVE), highlighting the protein residues that participate in the main interactions with the
inhibitor. Picture was generated with Discovery Studio 2.5. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
2.2.4. Inhibition studies on recombinant human MAO-A and -B
The inhibitory activity against human MAO-A and -B was measured using clorgyline,
iproniazid and rasagiline as reference compounds. As shown in Table 1, most of these compounds
were effective in inhibiting MAO in submicromolar range, retaining the activities of the lead
compound 2 (IC50 = 0.28 ± 0.01 μM to MAO-A, 0.30 ± 0.02 μM to MAO-B). Among them,
compound 8b was the most potent inhibitor for both MAO-A and -B with IC50 values of 0.34 ± 0.02
μM and 0.27 ± 0.01 μM, respectively. The results exhibited that the derivatives with a 3-methylene

linker were less active than those with a 4- or 6-methylene chain. Besides, the structure-activity
relationship analysis indicated that the inhibitory potency of MAO-B rose as the length of chain
increased (such as 7e, 8e and 9e; IC50 = 3.73 ± 0.15 μM, 2.02 ± 0.11 μM and 0.90 ± 0.01 μM,
respectively), but this conclusion for MAO-A was different. It also revealed that compounds bearing
various benzylamine moieties showed no significant difference on MAO inhibitory potency.
2.2.5. Molecular modeling study of MAOs
The interaction mode of these target compounds to MAOs were performed based on the X-ray
crystal structures of human MAO-A (PDB: 2Z5X) and MAO-B (PDB: 2V60). From the results of
inhibition studies on AChE and MAOs, compound 9e was chosen as a representative for the
molecular modeling investigation. The most stable binding modes of 9e in MAO-A and -B active
sites were graphically inspected (Figure 5). As shown in Figure 5A, the thioxanthone ring was close
to the enzymatic cofactor FAD in MAO-A. One hydrogen bond was generated between the ligand 9e
and FAD (9e-OH • • • O=C-FAD). Furthermore, the thioxanthone ring was observed to adopt parallel
π-π interactions with Tyr407 and Tyr444. In Figure 5B, the thioxanthone ring was located far from
the FAD in MAO-B. Two hydrogen bonds were formed between the 1-OH of 9e and the active site of
MAO-B (Ile199=O • • • HO-9e; Tyr326-OH • • • O-9e). In addition, the benzylamine moiety of 9e
interacted with Tyr398 and Tyr435 via parallel π-π interactions. Therefore, the docking studies
explained the high MAO inhibitory activity of these derivatives, which consistent with the
conclusion resulting from the human MAO inhibition assay.
Figure 5. Representation of compound 9e docked into the binding sites of MAO-A (A) and -B (B),
highlighting the protein residues that participate in the main interactions with the inhibitor. Ligand 9e

and FAD are displayed in grey and red respectively. Hydrogen-bonds are shown with the green
dotted lines. Picture was generated with Discovery Studio 2.5. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
2.2.6. Inhibition of self- and Cu2+-induced Aβ1-42 aggregation
All synthesized compounds were tested for their ability to inhibit self- and Cu2+-induced Aβ1-42
aggregation by using thioflavin T fluorescence method.39,40 Curcumin was used as the reference
compound. The results summarized in Table 2 indicated that these derivatives exhibited excellent
inhibitory activities of both self- and Cu2+-induced Aβ1-42 aggregation. Furthermore, the
representative compound 9e exhibited good inhibitory potency of self- and Cu2+-induced Aβ1-42
aggregation with the percentage of 74.8 ± 1.2% and 87.7 ± 1.9%, respectively. Compared with lead
compound 2 (9.5 ± 0.3% and 31.4 ± 1.2% respectively), the introduction of the O-alkylbenzylamine
group greatly increased Aβ1-42 aggregation inhibitory efficiency. For the inhibition of self-induced
Aβ1-42 aggregation, compound 8d (n = 4) exhibited the highest inhibitory activity with inhibitory
percentage of 89.4 ± 2.1%. Interestingly, compound 7d (68.2 ± 4.3%) and 9d (78.7 ± 3.5%)
exhibited the highest inhibitory activity among the derivatives with a 3-methylene linker and
6-methylene linker, respectively. It revealed that compounds 7d, 8d and 9d featuring a
N-(2-methoxybenzyl)ethylamine group on the terminal side of carbon chain may be optimal for the
inhibition of self-induced Aβ1-42 aggregation. Regarding the inhibition of Cu2+-induced Aβ1-42
aggregation, compounds that featuring a N-(4-dimethylaminobenzyl)ethylamine group on the
terminal side of carbon chain may be beneficial for the inhibition of Cu2+-induced Aβ1-42
aggregation. For example, 7f (85.4 ± 4.3%) and 9f (91.3 ± 4.7%) exhibited the highest inhibitory
activity among the derivatives with a 3-methylene and 6-methylene linker, respectively. Generally,
almost all the compounds exhibited better inhibitory activities of Cu2+-induced Aβ1-42 aggregation
than that of self-induced Aβ1-42 aggregation. This result might be reasonably attributed to the
chelating effect, which greatly reduced the speed and degree of Cu2+-induced Aβ1-42 aggregation.
Besides, the results also showed that the derivatives with a 4-methylene linker possessed better
inhibitory activities than those with a 3- or 6-methylene chain, which were different from the
conclusion of AChE inhibition assays. Therefore, the length of the alkyl chain also played an
important role in inhibiting the aggregation of Aβ1-42
.

Table 2. Inhibition of self- and Cu2+-induced Aβ1-42 aggregation activities, oxygen radical absorbance
capacity (ORAC, Trolox equivalents) by compound 2 and its derivatives, reference compounds and
7,4′-O-modified genistein derivative (III).
% Inhibition of Aβ1-42 aggregationa
Compound
ORACd
Self-inducedb
Cu2+-inducedc
40.5 ± 1.6
46.9 ± 2.1
45.2 ± 2.6
68.2 ± 4.3
46.9 ± 1.1
64.8 ± 2.1
79.5 ± 4.5
75.8 ± 1.2
79.9 ± 2.0
89.4 ± 2.1
72.1 ± 1.8
77.1 ± 1.3
47.5 ± 2.6
50.6 ± 2.7
68.0 ± 3.2
78.7 ± 3.5
74.8 ± 1.2
76.3 ± 3.1
9.5 ± 0.3
81.1 ± 2.9
70.9 ± 1.5
77.8 ± 3.9
80.0 ± 2.1
75.7 ± 3.1
85.4 ± 4.3
88.6 ± 2.0
82.1 ± 3.9
88.0 ± 2.3
82.0 ± 2.7
82.1 ± 4.2
81.9 ± 6.5
76.8 ± 1.8
76.4 ± 2.8
86.5 ± 2.3
86.3 ± 3.0
87.7 ± 1.9
91.3 ± 4.7
31.4 ± 1.2
77.8 ± 4.0
70.0 ± 2.7
0.48 ± 0.01
0.59 ± 0.03
0.22 ± 0.01
0.68 ± 0.02
0.35 ± 0.01
1.38 ± 0.10
1.18 ± 0.09
1.00 ± 0.08
1.04 ± 0.05
0.57 ± 0.04
0.95 ± 0.06
1.50 ± 0.11
0.37 ± 0.03
0.17 ± 0.01
0.66 ± 0.02
0.83 ± 0.07
0.74 ± 0.02
1.37 ± 0.05
0.42 ± 0.02
0.30 ± 0.01
2.43 ± 0.04
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
8f
9a
9b
9c
9d
9e
9f
2
35.0 ± 1.0
36.2 ± 0.9
III
Curcumin
a For inhibition of Aβ aggregation, the thioflavin-T fluorescence method was used.
b Inhibition of self-induced Aβ1-42 aggregation by tested inhibitors at 25 μM.
c
Inhibition of Cu2+-induced Aβ1-42 aggregation. The concentration of tested compounds and Cu2+
were 25 μM.
d
The mean ± SD of the three independent experiments. Data are expressed as μM of Trolox
equivalent/μM of tested compound.
2.2.7. Evaluation for antioxidant activity

The antioxidant activities of these derivatives were evaluated by the ORAC-FL method41 and the
results were summarized in Table 2. Their ability to scavenge radicals was provided as a Trolox (a
vitamin E analog) equivalent, with their relative potency at 5 μM compared with Trolox. The
antioxidant capacity of these compounds was determined by their competition with fluorescein in the
radical capture, using a fluorescence microplate reader. Most of the target compounds demonstrated
moderate to good antioxidant activities compared to Trolox. Curcumin, as another reference,
possessed better antioxidant activity because that contained two phenolic hydroxyl groups and could
tautomerize to enol form. Among these tested compounds, 7f, 8f and 9f showed the most potent
antioxidant activity of this family with ORAC-FL values of 1.38 ± 0.10, 1.50 ± 0.11 and 1.37 ± 0.05
Trolox equivalents, respectively. It revealed that the N-(4-dimethylaminobenzyl) ethylamine moiety
seemed to be a potent substitution pattern to improve the antioxidant activity. However, the length of
the linker did not show distinct influence on the potency.
2.2.8. Studies of metal-chelating properties
Based on the above studies, metal-chelating properties of these derivatives toward biometals
such as Cu2+, Zn2+, Fe2+ and Al3+ were studied by ultraviolet-visible (UV-Vis) spectrometry, with 9e
as the representative compound. The spectrum of 9e was shown in Figure 6A. When CuCl2 was
added, red shifts occurred in the absorption wavelength from 302 nm and 324 nm to 330 nm and 342
nm, respectively. Interestingly, the maximum absorption at 264 nm exhibited a blue shift to 255 nm,
and the absorbance dropped significantly. These results indicated the formation of a 9e-Cu2+
complex. When FeSO4 was added, the spectrum changed significantly, and the absorbance increased
obviously, suggesting a possible interaction between 9e and Fe2+. However, no significant change
was observed with the addition of AlCl3 or ZnCl2. The chelating effect could be reasonably attributed
to the 9-carbonyl and 1-hydroxyl group at the thioxanthone25 as well as the benzylamine moiety
side-chain. And it also explained the high potency of the derivatives to block the Cu2+-induced Aβ1-42
aggregation.
The stoichiometry of 9e-Cu2+ complex was determined using the molar ratio method, by
preparing the methanol solutions of compound 9e with increasing amounts of CuCl2. The UV spectra
was used to obtain the absorbance of the 9e complex at different concentrations of CuCl2 at 342 nm.
The results showed that absorbance linearly increased initially and then plateaued (Figure 6B). The

two straight lines intersected at a mole fraction of 1.50, revealing a 1:1.5 stoichiometry for complex
9e-Cu2+.
Figure 6. (A) UV spectrum of compound 9e (37.5 μM in methanol) alone or in the presence of
CuCl2, FeSO4, ZnCl2 or AlCl3 (37.5 μM for all metals in methanol). (B) Determination of the
stoichiometry of complex-Cu2+ by using the molar ratio method of titrating the methanol solution of
compound 9e with ascending amounts of CuCl2. The final concentration of tested compound was
37.5 μM, and the final concentration of Cu2+ ranged from 3.75 to 150 μM.
2.2.9. Cytotoxic effect on SH-SY5Y cells
To examine the cytotoxicity of the representative compound 9e, the human neuronal cell line
SH-SY5Y cells were treated with 9e at three different concentrations (1, 10 and 100μM) for 24 h.
The cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT)
assays42,43. As shown in Figure 7, cell viability reached 99.7% and 96.8% when 9e was tested at 1
μM and 10 μM, respectively. With increased concentration to 100 μM, 9e exhibited a decrease of cell
viability (68.1%). These results showed that compound 9e had a wide therapeutic safety range.

Figure 7. Effect of compound 9e on cell viability in human SH-SY5Y cells. Data are mean values ±
SEM of three independent experiments.
2.2.10. In vitro blood-brain barrier permeation assay
The ability to cross the blood-brain barrier (BBB) and penetrate into the brain is an important
property for compounds in the treatment of AD. The parallel artificial membrane permeation assay of
the blood-brain barrier (PAMPA-BBB) was conducted to predict the in vivo BBB permeability of our
derivatives44,45. Compounds 9b and 9e were chosen as representatives for BBB permeation assay. To
validate the assay, the experimental permeabilities of 11 commercial drugs were compared with
reported values (Table 3). And a plot of experimental data versus the bibliographic values displayed
a good linear correlation: Pe (exp.) = 0.8792 × Pe (bibl.) − 0.0616 (R2 = 0.9555) (Figure 8). With the
consideration of the limit established by Di et al.44, we determined that compounds with Pe values
above 3.46 × 10-6 cm/s could cross the BBB (Table 4). The results in Table 5 indicated that
compound 9b and 9e could penetrate the BBB and reach the therapeutic targets in central nervous
system (CNS).
Table 3. Permeability Pe (× 10-6 cm/s) in the PAMPA-BBB assay for 11 commercial drugs used
in the experiment validation.
Commercial drugs
Verapamil
Oxazepam
Bibla
16
PBS/EtOH (70:30)b
16.20 ± 0.36
9.20 ± 0.21
10
Diazepam
Clonidine
Imipramine
Testosterone
16
5.3
13
10.90 ± 0.23
5.50 ± 0.16
11.20 ± 0.22
15.50 ± 0.25
17

Caffeine
1.3
0.9
2.5
0.1
0.12
1.22 ± 0.05
0.55 ± 0.01
0.84 ± 0.02
0.33 ± 0.01
0.17 ± 0.03
Enoxacine
Piroxicam
Norfloxacin
Theophylline
a Taken from Ref.44.
b Data are the mean ± SD of three independent experiments.
Figure 8. Lineal correlation between experimental and reported permeability of commercial drugs
using the PAMPA-BBB assay. Pe (exp.) = 0.8792 × Pe (bibl.) − 0.0616 (R2 = 0.9555)
Table 4. Ranges of permeability of PAMPA-BBB assays (Pe × 10-6 cm/s).
High BBB permeation predicted (CNS +)
Uncertain BBB permeation (CNS +/-)
Low BBB permeation predicted (CNS -)
Pe > 3.46
3.46 > Pe > 1.70
Pe < 1.70
Table 5. Permeability results Pe (× 10-6 cm/s) from the PAMPA-BBB assay for selected thioxanthone
derivatives with their predicted penetration into the CNS.
Compd.a
Pe (× 10-6 cm/s)b
Prediction
CNS +
4.12 ± 0.18
9b
3.49 ± 0.34
CNS +
9e
a Compounds were dissolved in tartaric acid aqueous solution and diluted with PBS/EtOH (70:30).
The final concentration of each compound was 100 μg/mL.
b Data are the mean ± SD of three independent experiments.

3. Conclusion
In summary, most of these 1-hydroxyl-3-aminoalkoxythioxanthone derivatives exhibited good
AChE and MAOs inhibitory activities, significant efficiency to block both self- and Cu2+-induced
Aβ1-42 aggregation, and moderate to good antioxidant activities. Overall, compound 9e displayed
excellent and balanced activities: AChE (IC50 = 0.59 ± 0.02 μM), MAO-A and MAO-B (IC50 = 1.01
± 0.02 μM and 0.90 ± 0.01 μM respectively), self- and Cu2+-induced Aβ1-42 aggregation (74.8 ± 1.2%
and 87.7 ± 1.9% at 25 μM, respectively), good metal-chelating properties and low toxicity in
SH-SY5Y cells. Furthermore, kinetic and molecular modeling studies indicated that compound 9e
showed a mixed-type inhibition, binding to both CAS and PAS of AChE, and could penetrate the
BBB. Therefore, according to the MTDLs strategy, the multifunctional properties made 9e a
promising candidate for further studies directed to the development of novel drugs in the treatment of
AD.
4. Experimental section
4.1. Chemistry
All chemical reagents and solvents were obtained from commercial sources and used without
further purification. Melting points were determined on YRT-3 melting-point apparatus (China) and
are uncorrected. Reactions were monitored by thin-layer chromatography (TLC) on silica gel GF254
plates from Qingdao Haiyang Chemical Co. Ltd. (China) and spots were visualized with UV lamp
1
13
(254 nm) or in iodine chamber. H NMR and C NMR spectra were recorded on a Varian INOVA
400 NMR spectrometer or Varian INOVA 600 NMR spectrometer at room temperature using CDCl3
or DMSO-d6 as the sovent. Chemical shifts were expressed in parts per millions (ppm) using
tetramethylsilane (TMS) as the internal standard. Coupling constants were reported in hertz. Column
chromatography was accomplished on silica gel (230-400 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd. (China). The purity of these compounds was determined by analytical reverse
phase high-performance liquid chromatography (HPLC) on a Shimadzu LC-10Avp plus system with
a Kromasil C18 column (4.6 mm×250 mm, 5μm). The mobile phase was a mixture of methanol and
water (both containing 0.1% formic acid) at a flow rate of 0.7 mL/min. All target compounds
possessed purity higher than 95%. MS spectra were performed on Agilent-627 TOF LC-MS

Spectrometer.
4.1.1. Synthesis of 2,2’-disulfanediyldibenzoic acid (1)
2,2’-disulfanediyldibenzoic acid (1) was prepared according to the literature,30 which was used
without further purification. 95.8% yield.
4.1.2. Synthesis of 1,3-dihydroxy-9H-thioxanthen-9-one (2)
A mixture of phosphorous pentoxide (4.13 g, 29.1 mmol) and methanesulfonic acid (30 mL)
was heated to 90 °C and stirred for 0.5 h under an argon atmosphere until a clear solution (Eaton’s
reagent) was obtained. Anhydrous phloroglucinol (1.11 g, 8.79 mmol) and
2,2’-disulfanediyldibenzoic acid (1, 1.14 g, 3.63 mmol) were then added in one portion. The reaction
mixture was stirred at 90 °C for 1.5 h. After cooling to room temperature, the reaction mixture was
poured into ice-water and stirred for 1 h at room temperature. The resulting solid was collected by
filtration, washed with water to adjust the pH to approximately 6, and dried at 70 °C to give 2 as a
reddish brown solid. The crude product was purified by silica gel column chromatography using
petroleum ether/ethyl acetate (12:1, v/v) as eluent to obtain compound 2 (1.03 g, 46.4%) as a yellow
1
solid. H NMR (400 MHz, DMSO-d6) δ 14.37 (s, 1H), 11.10 (s, 1H), 8.43 (d, J = 7.6 Hz, 1H),
7.80-7.72 (m, 2H), 7.60-7.54 (m, 1H), 6.65 (s, 1H), 6.32 (s, 1H).
4.1.3. General procedure for the synthesis of (3-5)
To a mixture of 1,3-dihydroxy-9H-thioxanthen-9-one (2, 300 mg, 1.23 mmol) and anhydrous
potassium carbonate (170 mg, 1.23 mmol) in acetone (5 mL), appropriate dibromoalkane derivative
(2.46 mmol) was added. The reaction mixture was refluxed for 5-7 h under an argon atmosphere
until the starting material disappeared, the solvent was evaporated under reduced pressure. Water (20
mL) was added to the oil residue and the mixture was extracted with dichloromethane (15 mL × 4).
The combined organic phases were washed with saturated aqueous sodium chloride (30 mL), dried
over sodium sulfate and filtered, concentrated under reduced pressure to obtain crude compound. The
residue was purified by silica gel chromatography using petroleum ether/ethyl acetate (30:1) as
eluent to afford the desired products 3-5.
4.1.3.1. 3-(3-bromopropoxy)-1-hydroxy-9H-thioxanthen-9-one (3)
It was synthesized from 1,3-dihydroxy-9H-thioxanthen-9-one (2) and 1,3-dibromopropane
1
according to the general procedure. 77.3% yield; yellow solid; mp 131.1-132.8 °C. H NMR (400
MHz, CDCl3) δ 14.44 (s, 1H), 8.55 (dd, J = 1.2, 8.4 Hz, 1H), 7.60 (t, J = 8.4 Hz, 1H), 7.48 (t, J = 8.4

Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 4.20 (t, J = 6.0
Hz, 2H), 3.61 (t, J = 6.4 Hz, 2H), 2.38−2.32 (m, 2H).
4.1.3.2. 3-(4-bromobutoxy)-1-hydroxy-9H-thioxanthen-9-one (4)
It was synthesized from 1,3-dihydroxy-9H-thioxanthen-9-one (2) and 1,4-dibromobutane
1
according to the general procedure. 80.4% yield; yellow solid; mp 118.9-120.4 °C. H NMR (400
MHz, CDCl3) δ 14.44 (s, 1H), 8.55 (dd, J = 1.2, 8.0 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.48 (t, J = 8.0
Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.42 (d, J = 2.4 Hz, 1H), 4.08 (t, J = 6.0
Hz, 2H), 3.50 (t, J = 6.4 Hz, 2H), 2.11−2.04 (m, 2H), 2.01−1.95 (m, 2H).
4.1.3.3. 3-((6-bromohexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (5)
It was synthesized from 1,3-dihydroxy-9H-thioxanthen-9-one (2) and 1,6-dibromohexane
1
according to the general procedure. 83.1% yield; yellow solid; mp 101.6-103.2 °C. H NMR (400
MHz, CDCl3) δ 14.45 (s, 1H), 8.55 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz,
1H), 7.45 (t, J = 8.0 Hz, 1H), 6.56 (d, J = 2.4 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H), 4.05 (t, J = 6.4 Hz,
2H), 3.44 (t, J = 6.8 Hz, 2H), 1.93-1.87 (m, 2H), 1.86-1.81 (m, 2H), 1.58-1.51 (m, 4H).
4.1.4. General procedure for the synthesis of secondary amines (6a-f)
Compounds 6a-f were prepared as previously described.46
4.1.5. General procedure for the synthesis of 1-hydroxyl-3-aminoalkoxythioxanthen derivatives
(7a-f, 8a-f and 9a-f)
Anhydrous potassium carbonate (44.2 mg, 0.32 mmol) and appropriate intermediates 3-5 (0.21
mmol) were added to a solution of the corresponding secondary amines 6a-f (0.32 mmol) in
acetonitrile (2 mL). The reaction mixture was refluxed for 4-9 h. Upon completion of the reaction,
the solvent was evaporated under reduced pressure. Water (5 mL) was added to the residue and the
mixture was extracted with dichloromethane (5 mL × 4). The combined organic phases were washed
with saturated aqueous sodium chloride (20 mL), dried over anhydrous sodium sulfate and then
concentrated at reduced pressure. The resultant oil was purified via silica gel chromatography to give
compounds 7a-f, 8a-f and 9a-f, respectively.
4.1.5.1. 3-(3-(benzyl(methyl)amino)propoxy)-1-hydroxy-9H-thioxanthen-9-one (7a)
It was prepared from 3-(3-bromopropoxy)-1-hydroxy-9H-thioxanthen-9-one (3) and
N-benzylmethylamine (6a) according to the general procedure, then purified on a silica gel
chromatography eluted with petroleum ether/acetone (5:1) to obtain the pure product 7a. 70.5% yield;

1
a yellow oil. Purity: 99.1%. H NMR (400 MHz, CDCl3) δ 14.44 (s, 1H), 8.54 (d, J = 8.0 Hz, 1H),
7.58 (t, J = 8.0 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.31−7.24 (m, 5H), 6.50 (s,
1H), 6.40 (s, 1H), 4.02 (t, J = 6.4 Hz, 2H), 3.54 (s, 2H), 2.56 (t, J = 6.8 Hz, 2H), 2.26 (s, 3H ),
13
2.04−1.98 (m, 2H); C NMR (100 MHz, CDCl3) δ 184.0, 167.2, 164.1, 140.1, 138.4, 137.1, 132.5,
129.1, 129.0 (2C), 128.4, 128.2 (2C), 127.1, 126.1, 125.3, 109.4, 102.1, 99.9, 66.4, 62.3, 53.2, 42.1,
26.8. HR-ESI-MS: Calcd. for C24H24NO3S [M + H]+: 406.1477, found: 406.1459.
4.1.5.2. 3-(3-(benzyl(ethyl)amino)propoxy)-1-hydroxy-9H-thioxanthen-9-one (7b)
It was prepared from 3-(3-bromopropoxy)-1-hydroxy-9H-thioxanthen-9-one (3) and
N-benzylethylamine (6b) according to the general procedure, then purified on a silica gel
chromatography eluted with petroleum ether/acetone (5:1) to obtain the pure product 7b. 76.1% yield;
1
a yellow oil. Purity: 98.8%. H NMR (400 MHz, CDCl3) δ 14.45 (s, 1H), 8.55 (dd, J = 1.2, 8.0 Hz,
1H), 7.60 (t, J = 8.0 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.33−7.28 (m, 5H),
6.49 (d, J = 2.4 Hz, 1H), 6.40 (d, J = 2.4 Hz, 1H), 4.07 (t, J = 6.4 Hz, 2H), 3.59 (s, 2H), 2.62−2.53
13
(m, 4H), 1.97−1.91 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl3) δ 183.9, 167.2,
164.1, 140.0, 139.6, 137.1, 132.4, 129.1, 128.7 (2C), 128.2, 128.1 (2C), 127.1, 126.1, 125.2, 109.4,
102.1, 99.9, 66.5, 58.1, 51.0, 47.4, 26.7, 11.8. HR-ESI-MS: Calcd. for C25H26NO3S [M + H]+:
420.1633, found: 420.1658.
4.1.5.3. 1-hydroxy-3-(3-((2-methoxybenzyl)(methyl)amino)propoxy)-9H-thioxanthen-9-one (7c)
It was prepared from 3-(3-bromopropoxy)-1-hydroxy-9H-thioxanthen-9-one (3) and
N-(2-methoxybenzyl)methylamine (6c) according to the general procedure, then purified on a silica
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product 7c.
1
68.1% yield; a yellow oil. Purity: 98.5%. H NMR (400 MHz, CDCl3) δ 14.43 (s, 1H), 8.55 (d, J =
8.0 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.36 (d, J =
7.2 Hz, 1H), 7.26 (t, J = 7.2 Hz, 1H), 6.93 (t, J = 7.2 Hz, 1H), 6.87 (d, J = 7.2 Hz, 1H), 6.54 (d, J =
2.4 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 4.13 (t, J = 6.0 Hz, 2H), 3.89 (s, 3H), 3.71 (s, 2H), 2.76-2.70
(m, 2H), 2.37 (s, 3H), 2.15-2.12 (m, 2H); 13C NMR (150 MHz, CDCl3) δ 184.0, 167.2, 164.0, 157.8,
140.2, 137.1, 132.5, 131.2, 129.1, 129.0, 128.2, 126.1, 125.3, 120.4, 110.5, 110.4, 109.2, 101.9,
100.1, 66.4, 55.4, 55.0, 53.5, 42.0, 26.3. HR-ESI-MS: Calcd. for C25H26NO4S [M + H]+: 436.1583,
found: 436.1595.
4.1.5.4. 3-(3-(ethyl(2-methoxybenzyl)amino)propoxy)-1-hydroxy-9H-thioxanthen-9-one (7d)

It was prepared from 3-(3-bromopropoxy)-1-hydroxy-9H-thioxanthen-9-one (3) and
N-(2-methoxybenzyl)ethylamine (6d) according to the general procedure, then purified on a silica gel
chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product 7d. 80.6%
1
yield; a yellow oil. Purity: 98.3%. H NMR (400 MHz, CDCl3) δ 14.43 (s, 1H), 8.55 (d, J = 8.0 Hz,
1H), 7.60 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 7.6 Hz,
1H), 7.22 (t, J = 7.6 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.51 (d, J = 1.6 Hz,
1H), 6.40 (d, J = 1.6 Hz, 1H), 4.09 (t, J = 6.0 Hz, 2H), 3.82 (s, 3H), 3.66 (s, 2H), 2.68-2.61 (m, 4H),
13
2.04-1.98 (m, 2H), 1.10 (t, J = 6.8 Hz, 3H); C NMR (150 MHz, CDCl3) δ 184.0, 167.2, 164.2,
157.7, 140.1, 137.1, 132.4, 130.3, 129.1, 128.2, 128.0, 126.1, 125.3, 120.3, 110.2, 110.0, 109.1,
102.1, 100.0, 66.6, 55.3, 51.4, 49.4, 47.8, 26.7, 11.7. HR-ESI-MS: Calcd. for C26H28NO4S [M + H]+:
450.1739, found: 450.1723.
4.1.5.5.
3-(3-((2-(dimethylamino)benzyl)(ethyl)amino)propoxy)-1-hydroxy-9H-thioxanthen-
9-one (7e)
It was prepared from 3-(3-bromopropoxy)-1-hydroxy-9H-thioxanthen-9-one (3) and
2-((ethylamino)methyl)-N,N-dimethylaniline (6e) according to the general procedure, then purified
on a silica gel chromatography eluted with petroleum ether/ethyl acetate (1:2) to obtain the pure
product 7e. 63.6% yield; a yellow oil. Purity: 96.9%. 1H NMR (600 MHz, CDCl3) δ 14.44 (s, 1H),
8.55 (d, J = 7.8 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.58-7.54 (m, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.46 (t,
J = 7.8 Hz, 1H), 7.18 (t, J = 7.2 Hz, 1H), 7.08 (d, J = 7.2 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H), 6.50 (d, J
= 2.4 Hz, 1H), 6.40 (d, J = 2.4 Hz, 1H), 4.07 (t, J = 6.6 Hz, 2H), 3.68 (s, 2H), 2.67 (s, 6H), 2.57-2.55
(m, 2H), 1.98-1.94 (m, 2H), 1.68-1.62 (m, 2H), 1.08 (t, J = 6.8 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ 184.0, 167.3, 164.2, 153.0, 140.1, 137.1, 133.7, 132.5, 130.2, 129.1, 128.2, 127.5, 126.2, 125.3,
123.2, 118.9, 109.2, 102.1, 100.0, 66.6, 52.7, 49.5, 47.8, 45.2 (2C), 26.4, 10.1. HR-ESI-MS: Calcd.
for C27H31N2O3S [M + H]+: 463.2055, found: 463.2097.
4.1.5.6.
3-(3-((4-(dimethylamino)benzyl)(ethyl)amino)propoxy)-1-hydroxy-9H-thioxanthen-
9-one (7f)
It was prepared from 3-(3-bromopropoxy)-1-hydroxy-9H-thioxanthen-9-one (3) and
4-((ethylamino)methyl)-N,N-dimethylaniline (6f) according to the general procedure, then purified
on a silica gel chromatography eluted with petroleum ether/ethyl acetate (1:2)to obtain the pure
1
product 7f. 58.1% yield; a yellow oil. Purity: 98.9%. H NMR (400 MHz, CDCl3) δ 14.43 (s, 1H),

8.55 (d, J = 7.6 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H),
7.17 (d, J = 8.4 Hz, 2H), 6.55 (d, J = 8.4 Hz, 2H), 6.50 (d, J = 2.4 Hz, 1H), 6.39 (d, J = 2.4 Hz, 1H),
4.05 (t, J = 6.4 Hz, 2H), 3.53 (s, 2H), 2.89 (s, 6H), 2.61-2.57 (m, 4H), 1.98-1.94 (m, 2H), 1.09 (t, J =
13
6.4 Hz, 3H); C NMR (150 MHz, CDCl3) δ 184.0, 167.2, 164.2, 149.7, 140.0, 137.1, 133.2, 132.4,
129.9, 129.1, 128.2, 126.1, 125.3, 122.0, 120.0, 112.3, 109.1, 102.1, 100.0, 66.5, 57.3, 48.7, 47.3,
40.6 (2C), 26.6, 11.6. HR-ESI-MS: Calcd. for C27H31N2O3S [M + H]+: 463.2055, found: 463.2105.
4.1.5.7. 3-(4-(benzyl(methyl)amino)butoxy)-1-hydroxy-9H-thioxanthen-9-one (8a)
It was prepared from 3-(4-bromobutoxy)-1-hydroxy-9H-thioxanthen-9-one (4) and
N-benzylmethylamine (6a) according to the general procedure, then purified on a silica gel
chromatography eluted with petroleum ether/acetone (5:1) to obtain the pure product 8a. 73.9% yield;
1
a yellow oil. Purity: 97.5%. H NMR (400 MHz, CDCl3) δ 14.44 (s, 1H), 8.55 (dd, J = 1.2, 8.0 Hz,
1H), 7.59 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.33−7.25 (m, 5H),
6.54 (d, J = 2.0 Hz, 1H), 6.41 (d, J = 2.0 Hz, 1H), 4.02 (t, J = 6.8 Hz, 2H), 3.53 (s, 2H), 2.45 (t, J =
13
6.8 Hz, 2H), 2.24 (s, 3H ), 1.87−1.82 (m, 2H), 1.74−1.69 (m, 2H); C NMR (150 MHz, CDCl3) δ
183.9, 167.2, 164.1, 140.1, 138.9, 137.0, 132.4, 129.1, 129.0 (2C), 128.2 (2C), 128.1, 126.9, 126.1,
125.2, 109.1, 102.1, 99.9, 68.2, 62.3, 56.5, 42.1, 26.6, 23.6. HR-ESI-MS: Calcd. for C25H26NO3S [M
+ H]+: 420.1633, found: 420.1685.
4.1.5.8. 3-(4-(benzyl(ethyl)amino)butoxy)-1-hydroxy-9H-thioxanthen-9-one (8b)
It was prepared from 3-(4-bromobutoxy)-1-hydroxy-9H-thioxanthen-9-one (4) and
N-benzylethanamine (6b) according to the general procedure, then purified on a silica gel
chromatography eluted with petroleum ether/acetone (5:1) to obtain the pure product 8b. 76.0% yield;
1
a yellow oil. Purity: 97.7%. H NMR (400 MHz, CDCl3) δ 14.44 (s, 1H), 8.55 (d, J = 7.2 Hz, 1H),
7.59 (t, J = 7.2 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.36−7.25 (m, 5H), 6.52 (d,
J = 2.0 Hz, 1H), 6.41 (d, J = 2.0 Hz, 1H), 3.98 (t, J = 6.4 Hz, 2H), 3.61 (s, 2H), 2.56−2.52 (m, 4H),
1.85−1.79 (m, 2H), 1.68-1.65 (m, 2H), 1.08 (t, J = 6.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 183.8,
167.2, 164.0, 140.1, 138.9, 137.0, 132.4, 129.0, 128.9 (2C), 128.1 (2C), 128.0, 126.9, 126.0, 125.2,
109.0, 102.0, 99.9, 68.1, 57.9, 52.3, 47.2, 26.6, 23.2, 11.6. HR-ESI-MS: Calcd. for C26H28NO3S [M +
H]+: 434.1790, found: 434.1763.
4.1.5.9. 1-hydroxy-3-(4-((2-methoxybenzyl)(methyl)amino)butoxy)-9H-thioxanthen-9-one (8c)

It was prepared from 3-(4-bromobutoxy)-1-hydroxy-9H-thioxanthen-9-one (4) and
N-(2-methoxybenzyl)methylamine (6c) according to the general procedure, then purified on a silica
gel chromatography eluted with dichloromethane/methanol (15:1) to obtain the pure product 8c.
80.1% yield; a yellow oil. Purity: 98.3%. 1H NMR (400 MHz, CDCl3) δ 14.44 (s, 1H), 8.55 (dd, J =
0.8, 7.6 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.41 (d, J
= 7.2 Hz, 1H), 7.31−7.29 (m, 1H), 6.96 (t, J = 7.2 Hz, 1H), 6.89 (d, J = 7.2 Hz, 1H), 6.54 (d, J = 2.4
Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 4.04 (t, J = 6.4 Hz, 2H), 3.83 (s, 3H), 3.74 (s, 2H), 2.66-2.62 (m,
13
2H), 2.38 (s, 3H), 1.88-1.84 (m, 4H); C NMR (150 MHz, CDCl3) δ 183.9, 167.2, 164.0, 157.9,
140.2, 137.0, 132.5, 131.4, 129.3, 129.0, 128.1, 126.1, 125.3, 120.5, 110.5, 110.3, 109.2, 102.0, 99.9,
68.0, 56.3, 55.4, 54.7, 41.5, 26.6, 23.0. HR-ESI-MS: Calcd. for C26H28NO4S [M + H]+: 450.1739,
found: 450.1699.
4.1.5.10. 3-(4-(ethyl(2-methoxybenzyl)amino)butoxy)-1-hydroxy-9H-thioxanthen-9-one (8d)
It was prepared from 3-(4-bromobutoxy)-1-hydroxy-9H-thioxanthen-9-one (4) and
N-(2-methoxybenzyl)ethanamine (6d) according to the general procedure, then purified on a silica
gel chromatography eluted with dichloromethane/methanol (15:1) to obtain the pure product 8d.
1
73.9% yield; a yellow oil. Purity: 99.0%. H NMR (400 MHz, CDCl3) δ 14.43 (s, 1H), 8.55 (d, J =
8.0 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.50-7.44 (m, 3H), 7.28-7.24 (m, 1H), 6.96 (t, J = 7.2 Hz, 1H),
6.87 (d, J = 7.2 Hz, 1H), 6.53 (s, 1H), 6.40 (s, 1H), 4.03-3.98 (m, 2H), 3.84 (s, 5H), 2.69-2.66 (m,
13
4H), 1.84-1.80 (m, 4H), 1.25-1.18 (m, 3H); C NMR (150 MHz, CDCl3) δ 183.9, 167.3, 164.0,
157.8, 140.2, 137.1, 132.5, 131.0, 129.1, 128.7, 128.2, 126.2, 125.3, 120.6, 110.4, 110.3, 109.2,
102.0, 99.9, 68.1, 55.4, 52.3, 50.9, 47.5, 26.7, 22.7, 11.0. HR-ESI-MS: Calcd. for C27H30NO4S [M +
H]+: 464.1896, found: 464.1937.
4.1.5.11.
3-(4-((2-(dimethylamino)benzyl)(ethyl)amino)butoxy)-1-hydroxy-9H-thioxanthen-
9-one (8e)
It was prepared from
3-(4-bromobutoxy)-1-hydroxy-9H-thioxanthen-9-one (4) and
2-((ethylamino)methyl)-N,N-dimethylaniline (6e) according to the general procedure, then purified
on a silica gel chromatography eluted with petroleum ether/ethyl acetate (1:2) to obtain the pure
1
product 8e. 55.3% yield; a yellow oil. Purity: 95.8%. H NMR (600 MHz, CDCl3) δ 14.44 (s, 1H),
8.55 (d, J = 7.8 Hz, 1H), 7.81-7.78 (m, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.47 (t,
J = 7.8 Hz, 1H), 7.29-7.20 (m, 1H), 7.16-7.08 (m, 2H), 6.51(d, J = 1.8 Hz, 1H), 6.39 (d, J = 1.8 Hz,

1H), 4.09 (t, J = 6.6 Hz, 2H), 2.78 (s, 2H), 2.67 (s, 6H), 2.58-2.54 (m, 4H), 2.06-1.64 (m, 4H),
13
1.27-1.23 (m, 3H); C NMR (150 MHz, CDCl3) δ 183.9, 167.2, 163.9, 153.5, 140.3, 137.1, 132.5,
131.2, 129.1, 129.0, 128.1, 126.2, 125.3, 124.0, 119.6, 115.2, 109.3, 102.0, 99.9, 67.8, 59.5, 51.8,
47.2, 45.0 (2C), 44.9, 26.5, 22.0. HR-ESI-MS: Calcd. for C28H33N2O3S [M + H]+: 477.2212, found:
477.2183.
4.1.5.12.
3-(4-((4-(dimethylamino)benzyl)(ethyl)amino)butoxy)-1-hydroxy-9H-thioxanthen-
9-one (8f)
It was prepared from
3-(4-bromobutoxy)-1-hydroxy-9H-thioxanthen-9-one (4) and
4-((ethylamino)methyl)-N,N-dimethylaniline (6f) according to the general procedure, then purified
on a silica gel chromatography eluted with petroleum ether/ethyl acetate (1:2) to obtain the pure
1
product 8f. 58.9% yield; a yellow oil. Purity: 98.4%. H NMR (600 MHz, CDCl3) δ 14.44 (s, 1H),
8.55 (dd, J = 1.2, 7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 7.8 Hz,
1H), 7.28-7.24 (m, 2H), 6.66 (d, J = 8.4 Hz, 2H), 6.54 (d, J = 2.4 Hz, 1H), 6.40 (d, J = 2.4 Hz, 1H),
4.02 (t, J = 5.4 Hz, 2H), 3.79 (s, 2H), 2.91 (s, 6H), 2.80-2.71 (m, 4H), 1.85-1.84 (m, 4H), 1.25-1.20
13
(m, 3H); C NMR (150 MHz, CDCl3) δ 183.9, 167.2, 163.8, 150.3, 140.2, 137.0, 132.5, 130.9,
129.0, 128.3, 128.1, 126.1, 125.8, 125.3, 112.4, 112.2, 109.2, 101.9, 99.9, 67.7, 56.4, 51.1, 46.7, 40.3
(2C), 26.4, 21.8, 10.4. HR-ESI-MS: Calcd. for C28H33N2O3S [M + H]+: 477.2212, found: 477.2158.
4.1.5.13. 3-((6-(benzyl(methyl)amino)hexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (9a)
It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (5) and
N-benzylmethylamine (6a) according to the general procedure, then purified on a silica gel
chromatography eluted with petroleum ether/acetone (4:1) to obtain the pure product 9a. 68.9% yield;
1
a yellow oil. Purity: 98.1%. H NMR (400 MHz, CDCl3) δ 14.44 (s, 1H), 8.55 (d, J = 8.0 Hz, 1H),
7.59 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.34−7.28 (m, 5H), 6.55 (d,
J = 2.0 Hz, 1H), 6.42 (d, J = 2.0 Hz, 1H), 4.02 (t, J = 6.4 Hz, 2H), 3.50 (s, 2H), 2.45 (t, J = 6.8 Hz,
2H), 2.26 (s, 3H ), 1.84−1.77 (m, 2H), 1.65−1.59 (m, 2H), 1.51−1.44 (m, 2H), 1.43−1.39 (m, 2H);
13C NMR (150 MHz, CDCl3) δ 184.0, 167.3, 164.2, 140.1, 137.9, 137.1, 132.4, 129.2 (2C), 129.1,
128.3 (2C), 128.2, 127.2, 126.1, 125.3, 109.1, 102.2, 99.9, 68.4, 62.0, 57.0, 41.9, 28.9, 27.0, 26.8,
25.8. HR-ESI-MS: Calcd. for C27H30NO3S [M + H]+: 448.1946, found: 448.1905.
4.1.5.14. 3-((6-(benzyl(ethyl)amino)hexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (9b)

It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (5) and
N-benzylethanamine (6b) according to the general procedure, then purified on a silica gel
chromatography eluted with petroleum ether/acetone (5:1) to obtain the pure product 9b. 62.5% yield;
1
a yellow oil. Purity: 98.4%. H NMR (400 MHz, CDCl3) δ 14.45 (s, 1H), 8.55 (d, J = 8.0 Hz, 1H),
7.60 (t, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.39−7.25 (m, 5H), 6.57 (d,
J = 2.0 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 4.03 (t, J = 6.4 Hz, 2H), 3.65 (s, 2H), 2.59 (q, J = 6.8 Hz,
2H), 2.51 (t, J = 6.4 Hz, 2H ), 1.83−1.77 (m, 2H), 1.57−1.50 (m, 2H), 1.48−1.42 (m, 2H), 1.41−1.37
13
(m, 2H), 1.10 (t, J = 6.8 Hz, 3H); C NMR (100MHz, CDCl3) δ 183.9, 167.3, 164.2, 140.1, 138.5,
137.1, 132.4, 129.2, 129.0 (2C), 128.2 (2C), 128.1, 127.0, 126.1, 125.3, 109.1, 102.1, 99.9, 68.4,
57.8, 52.7, 47.1, 28.9, 27.0, 26.5, 25.7, 11.3. HR-ESI-MS: Calcd. for C28H32NO3S [M + H]+:
462.2103, found: 462.2163.
4.1.5.15. 1-hydroxy-3-((6-((2-methoxybenzyl)(methyl)amino)hexyl)oxy)-9H-thioxanthen-9-one
(9c)
It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (5) and
N-(2-methoxybenzyl)methylamine (6c) according to the general procedure, then purified on a silica
gel chromatography eluted with dichloromethane/methanol (15:1) to obtain the pure product 9c.
1
71.1% yield; a yellow oil. Purity: 98.1%. H NMR (400 MHz, CDCl3) δ 14.44 (s, 1H), 8.55 (d, J =
8.0 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.50−7.44 (m, 3H), 7.32 (t, J = 7.6 Hz, 1H), 6.98 (t, J = 7.6 Hz,
1H), 6.89 (d, J = 7.6 Hz, 1H), 6.56 (d, J = 2.0 Hz, 1H), 6.42 (d, J = 2.0 Hz, 1H), 4.02 (t, J = 6.4 Hz,
2H), 3.88 (s, 2H), 3.85 (s, 3H), 2.72-2.68 (m, 2H ), 2.45 (s, 3H), 1.83−1.78 (m, 4H), 1.51−1.47 (m,
13
2H), 1.45−1.41 (m, 2H); C NMR (150 MHz, CDCl3) δ 183.9, 167.2, 164.1, 157.9, 140.2, 137.1,
132.5, 132.0, 129.8, 129.0, 128.1, 126.1, 125.3, 120.7, 110.6, 110.4, 109.1, 102.1, 99.9, 68.2, 56.4,
55.4, 55.3, 54.2, 40.8, 28.7, 26.8, 25.6. HR-ESI-MS: Calcd. for C28H32NO4S [M + H]+: 478.2052,
found: 478.2013.
4.1.5.16. 3-((6-(ethyl(2-methoxybenzyl)amino)hexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (9d)
It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (5) and
N-(2-methoxybenzyl)ethanamine (6d) according to the general procedure, then purified on a silica
gel chromatography eluted with dichloromethane/methanol (15:1) to obtain the pure product 9d.
1
73.8% yield; a yellow oil. Purity: 98.2%. H NMR (600 MHz, CDCl3) δ 14.45 (s, 1H), 8.54 (d, J =
7.8 Hz, 1H), 7.59 (t, J = 7.8 Hz, 2H), 7.49 (d, J = 7.8 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.32 (t, J =

7.8 Hz, 1H), 7.00 (t, J = 7.8 Hz, 1H), 6.89 (d, J = 7.8 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.42 (d, J =
2.4 Hz, 1H), 4.05 (t, J = 6.6 Hz, 2H), 3.97 (s, 2H), 3.86 (s, 3H), 2.87-2.83 (m, 2H ), 2.77-2.73 (m,
2H), 1.82−1.78 (m, 4H), 1.51−1.46 (m, 2H), 1.41−1.36 (m, 2H), 1.28−1.25 (m, 3H); 13C NMR (100
MHz, CDCl3) δ 183.9, 167.2, 164.0, 157.8, 140.2, 137.0, 132.4, 131.9, 129.8, 129.0, 128.1, 126.1,
125.2, 120.8, 110.5, 110.4, 109.1, 102.0, 99.8, 68.1, 55.4, 52.1, 50.4, 47.1, 28.7, 26.8, 25.5, 24.8,
10.1. HR-ESI-MS: Calcd. for C29H34NO4S [M + H]+: 492.2209, found: 492.2256.
4.1.5.17. 3-((6-((2-(dimethylamino)benzyl)(ethyl)amino)hexyl)oxy)-1-hydroxy-9H-thioxanthen-
9-one (9e)
It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (5) and
2-((ethylamino)methyl)-N,N-dimethylaniline (6e) according to the general procedure, then purified
on a silica gel chromatography eluted with petroleum ether/ethyl acetate (1:2) to obtain the pure
1
product 9e. 49.8% yield; a yellow oil. Purity: 95.6%. H NMR (400 MHz, CDCl3) δ 14.44 (s, 1H),
8.55 (dd, J = 0.8, 7.6 Hz, 1H), 7.82-7.80 (m, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H),
7.45 (t, J = 7.6 Hz, 1H), 7.30-7.27 (m, 1H), 7.16-7.11 (m, 2H), 6.55(d, J = 2.0 Hz, 1H), 6.41 (d, J =
2.0 Hz, 1H), 4.03 (t, J = 6.6 Hz, 2H), 4.00 (s, 2H), 2.83-2.72 (m, 4H), 2.67 (s, 6H), 1.82-1.75 (m, 2H),
1.72-1.69 (m, 2H), 1.50−1.42 (m, 2H), 1.39−1.32 (m, 2H), 1.25−1.22 (m, 3H); 13C NMR (100 MHz,
CDCl3) δ 183.9, 167.3, 164.1, 153.3, 140.2, 137.1, 133.8, 132.5, 130.9, 129.1, 128.5, 126.1, 125.3,
123.8, 123.1, 119.4, 109.1, 102.1, 99.9, 68.3, 52.5, 51.6, 47.2, 45.4 (2C), 29.6, 28.8, 26.9, 25.7, 10.5.
HR-ESI-MS: Calcd. for C30H37N2O3S [M + H]+: 505.2525, found: 505.2517.
4.1.5.18. 3-((6-((4-(dimethylamino)benzyl)(ethyl)amino)hexyl)oxy)-1-hydroxy-9H-thioxanthen-
9-one (9f)
It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-thioxanthen-9-one (5) and
4-((ethylamino)methyl)-N,N-dimethylaniline (6f) according to the general procedure, then purified
on a silica gel chromatography eluted with petroleum ether/ethyl acetate (1:2) to obtain the pure
1
product 9f. 51.0% yield; a yellow oil. Purity: 98.7%. H NMR (400 MHz, CDCl3) δ 14.44 (s, 1H),
8.53 (dd, J = 0.8, 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz,
1H), 7.27 (d, J = 8.8 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H), 6.54 (d, J = 2.4 Hz, 1H), 6.41 (d, J = 2.4 Hz,
1H), 4.01 (t, J = 6.4 Hz, 2H), 3.75 (s, 2H), 2.94 (s, 6H), 2.74 (q, J = 6.8 Hz, 2H), 2.62 (t, J = 7.2 Hz,
2H ), 1.83-1.76 (m, 2H), 1.69-1.67 (m, 2H), 1.51-1.43 (m, 2H), 1.41-1.35 (m, 2H), 1.23 (t, J = 6.8 Hz,
13
3H); C NMR (100 MHz, CDCl3) δ 183.9, 167.3, 164.1, 150.2, 140.2, 137.1, 133.8, 132.5, 130.8,

129.1, 128.1, 126.1, 125.3, 123.1, 122.0, 112.3, 109.1, 102.1, 99.9, 68.2, 56.5, 51.7, 46.5, 40.4 (2C),
28.8, 26.8, 25.6, 25.2, 10.4. HR-ESI-MS: Calcd. for C30H37N2O3S [M + H]+: 505.2525, found:
505.2503.
4.2. Biological activity
4.2.1. Inhibitory activities of AChE and BuChE
AChE and BuChE inhibitory activities were determined according to the modified Ellman’s
method34 using AChE from Electrophorus electricus (EeAChE, Sigma-Aldrich Co.) and BuChE
from rat serum (RatBuChE). Donepezil was used as a reference compound. For EeAChE inhibition
assay, the reaction mixture (100 µL) consisted of acetylthiocholine iodide (ATCI, 1 mM, 30 µL)
(J&K Scientific), phosphate buffer solution (0.1 mM, pH = 8.0, 40 µL), different concentrations of
test compounds (20 µL) and EeAChE (0.05 U/mL, final concentration, 10 µL). Then
5,5’-dithiobis-2-nitrobenzoic acid (DTNB, 0.2%, 30 µL) (J&K Scientific) was added and the mixture
was incubated at 37 °C for 15 min. The absorbance was measured at 412 nm in a Varioskan Flash
Multimode Reader (Thermo Scientific). For BuChE inhibition assays, 25% rat serum (10 µL) and
S-butylthiocholine iodide (BTCI, 1 mM, 30 µL) (TCI Shanghai Development) were used and the
assay was carried out in a phosphate buffer (0.1 mM, pH = 7.4). Changes in absorbance were
detected at 405 nm.34,47 The other procedure was similar with the method described above. IC50
values were calculated as the concentration of compound that produces 50% AChE or BuChE
activity inhibition. Results are expressed as the mean ± SD of at least three different experiments
performed in triplicate.
4.2.1.1. Kinetic study of AChE Inhibition
Kinetic study of AChE inhibition was performed using purified EeAChE based on a reported
method.47 A mixture of phosphate buffer (0.1 M, pH = 8.0), EeAChE (0.5 U/mL), DTNB (0.2%) and
different concentrations of tested compounds solution was added and incubated at 37 °C for 15 min.
Then substrate in different concentrations was added (0.1-0.4 mM) quickly. Kinetic characterization
of the hydrolysis of ATCh catalyzed by EeAChE was done at 412 nm. The parallel control
experiments were performed without inhibitor in the assay. The plots were assessed by a weighted
least-squares analysis that assumed the variance of velocity (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 9e

in a weighted analysis and Ki was determined as the intercept on the negative x-axis.
4.2.1.2. Molecular Docking of AChE.
Docking studies were performed using AUTODOCK 4.2 program. The crystal structure of
AChE (PDB: 1EVE) complexed with donepezil was obtained from the Protein Data Bank after the
water molecules and original inhibitors were removed. The 3D Structure of 9e was built and
optimized by molecular mechanics. The inhibitor was prepared furtherly by removing the hydrogen
atoms, adding Gasteiger charges and their atomic charges to skeleton atoms, assigning the proper
atomic types. Autotors was used to define which bonds were rotatable in ligands. 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 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. All maps were calculated with 0.375 Å spacing between grid
points. The center of the grid box was placed at the center of donepezil with coordinates x = 2.023, y
= 63.295, z = 67.062. The dimensions of the active site box were set at 60 × 60 × 60 Å. Flexible
ligand docking was performed for the compounds. Other than the referred parameters above, the
other parameters were used as default. Each docking system was derived from 100 runs of the
AUTODOCK using the Lamarckian genetic algorithm (LGA). Finally, a cluster analysis was
performed on the docking results using a root mean square (RMS) tolerance of 1.0. Graphic
manipulations and visualizations were done by Autodock Tools or Discovery Studio 2.5 software.
Took into account the binding energy and the conformational distribution, the most stable binding
mode of 9e to AChE was graphically inspected (binding energy: -12.34 kcal/mol, inhibit constant:
0.904 nM).
4.2.2. Inhibition studies of MAO-A and MAO-B
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.48,49 Recombinant
human MAO-A and –B were purchased from Sigma-Aldrich and stored at -80 °C. All the enzymatic
reactions were performed in potassium phosphate buffer (100 mM, pH = 7.4, containing 20.2 mM
KCl) to a final volume of 500 μL containing kynuramine (45 μM for MAO-A and 30 μM for MAO-B)
and various concentrations of test compounds (0-100 μM) with the concentration of DMSO lower