Design, synthesis and evaluation of novel tacrine-coumarin hybrids as multifunctional cholinesterase inhibitors against Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2013.03.051

A series of tacrine-coumarin hybrids (8a-t) were designed, synthesized and evaluated as multifunctional cholinesterase (ChE) inhibitors against Alzheimer's disease (AD). The screening results showed that most of them exhibited a significant ability to inhibit ChE and self-induced β-amyloid (Aβ) aggregation, and to act as metal chelators. Especially, 8f displayed the greatest ability to inhibit acetylcholinesterase (AChE, IC₅₀ = 0.092 μM) and Aβ aggregation (67.8%, 20 μM). It was also a good butyrylcholinesterase inhibitor (BuChE, IC₅₀ = 0.234 μM) and metal chelator. Besides, kinetic and molecular modeling studies indicated that 8f was a mixed-type inhibitor, binding simultaneously to active, peripheral and mid-gorge sites of AChE. These results suggested that 8f might be an excellent multifunctional agent for AD treatment.

Full text of DOI:10.1016/j.ejmech.2013.03.051

Accepted Manuscript
Design, synthesis and evaluation of novel tacrine-coumarin hybrids as multifunctional
cholinesterase inhibitors against Alzheimer's disease
Sai-Sai Xie, Xiao-Bing Wang, Jiang-Yan Li, Lei Yang, Ling-Yi Kong
PII:
S0223-5234(13)00211-0
10.1016/j.ejmech.2013.03.051
EJMECH 6089
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 January 2013
Revised Date: 21 March 2013
Accepted Date: 24 March 2013
Please cite this article as: S.-S. Xie, X.-B. Wang, J.-Y. Li, L. Yang, L.-Y. Kong, Design, synthesis
and evaluation of novel tacrine-coumarin hybrids as multifunctional cholinesterase inhibitors
against Alzheimer's disease, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.03.051.
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ACCEPTED MANUSCRIPT
Graphical abstract
A series of novel tacrine-coumarin hybrids were designed and synthesized as
multifunctional cholinesterase inhibitors. 8f was found to be a promising compound
for further study.

ACCEPTED MANUSCRIPT
Highlights
ꢀ
ꢀ
ꢀ
ꢀ
A series of tacrine-coumarin hybrids were designed and synthesized.
All compounds showed better ChE inhibitory activity than galanthamine.
All compounds could inhibit Aβ aggregation and chelate metal ions.
Compound 8f was identified as the most promising candidate for further study.

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Design, synthesis and evaluation of novel tacrine-coumarin hybrids as
multifunctional cholinesterase inhibitors against Alzheimer's disease
Sai-Sai Xie, Xiao-Bing Wang *, Jiang-Yan Li, Lei Yang, Ling-Yi Kong *
State Key Laboratory of Natural Medicines, Department of Natural Medicinal
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,
People’s Republic of China
*
Corresponding Author. Tel/Fax: +86-25-8327 1405; E-mail: cpu_lykong@126.com
(Ling-Yi Kong); xbwang@cpu.edu.cn (Xiao-Bing Wang)
1

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Abstract
A series of tacrine-coumarin hybrids (8a-t) were designed, synthesized and evaluated
as multifunctional cholinesterase (ChE) inhibitors against Alzheimer’s disease (AD).
The screening results showed that most of them exhibited a significant ability to
inhibit ChE and self-induced β-amyloid (Aβ) aggregation, and to act as metal
chelators. Especially, 8f displayed the greatest ability to inhibit acetylcholinesterase
(AChE, IC50 = 0.092 ꢀM) and Aβ aggregation (67.8 %, 20 ꢀM). It was also a good
butyrylcholinesterase inhibitor (BuChE, IC50 = 0.234 ꢀM) and metal chelator. Besides,
kinetic and molecular modeling studies indicated that 8f was a mixed-type inhibitor,
binding simultaneously to active, peripheral and mid-gorge sites of AChE. These
results suggested that 8f might be an excellent multifunctional agent for AD
treatment.
Key words: Alzheimer’s disease, coumarin, tacrine, metal chelator, cholinesterase,
β-amyloid aggregation.
2

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1
. Introduction
Alzheimer’s disease (AD), the most common form of dementia in elderly people, is a
progressive neurodegenerative disorder characterized by loss of cognitive ability,
severe behavioral abnormalities, and ultimately death [1, 2]. It is estimated that more
than 18 million people presently suffer from AD, and the number of patients is
expected to sharply increase to 70 million by 2050 [3]. The vast number of people
requiring constant care and related services will severely strain medical, monetary and
human resources. Consequently, the developing treatments that slow or halt the
disease progression have become imperative. Over the past decades, despite many
efforts devoted to research in AD, its etiology is still unclear. To date, several factors
including amyloid-β (Aβ) deposits, oxidative stress, dyshomeostasis of biometals and
low levels of acetylcholine (ACh) have been demonstrated to be associated with AD
pathogenesis, and several hypotheses based on these factors have been proposed to
explain the mechanism of AD development [4,5].
The classical hypothesis of AD is the cholinergic hypothesis [6], which suggests that
the cognitive and memory deterioration of AD are mainly caused by the low level of
choline, especially ACh in the brain, and sustaining or recovering the ACh can
alleviate these symptoms of AD [7, 8]. There are two types of cholinesterase (ChE)
responsible for the hydrolysis of ACh into acetic acid and choline, namely,
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) [9]. Since the AChE
accounts for approximately 80% of ACh hydrolysis in normal brains, it has received
much attention compared to the BuChE [10]. The AChE has a nearly 20 Å deep
narrow gorge which consists of two binding sites: a catalytic active site (CAS) at the
3

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bottom of the gorge and a peripheral anionic site (PAS) near the entry of the gorge [11,
1
2]. Generally, inhibitors that bind to either one site can inhibit the AChE. However,
recent reports indicate that besides the ability of hydrolysis of ACh, the PAS has close
relation to the prevention of Aβ aggregation [13, 14]. Thus, AChE inhibitors binding
simultaneously to PAS and CAS have become more promising in AD treatment.
Moreover, although BuChE only play a secondary role in the healthy brains, in the
case of AD, it becomes a major modulator in regulating the ACh level [9, 15]. Several
lines of evidence indicate that, as the disease progresses, the activity of AChE
gradually decrease, while that of BuChE significantly increases in the hippocampus
and temporal cortex [14, 16]. Consequently, simultaneous inhibition of both AChE
and BuChE should provide additional benefits in the treatment of AD.
In contrast to cholinergic hypothesis, the recent amyloid hypothesis suggests that
accumulation of Aβ peptide aggregates in the brain is thought to play a central role in
the pathogenesis of AD, as its accumulation may result in a cascade of biochemical
events leading to neuronal dysfunction [17]. Aβ is a 39- to 43-residue peptides
generated by the sequential cleaving of the amyloid precursor protein (APP) by β- and
γ- secretases [18]. Aβ (1-40) and Aβ (1-42) are the main isoforms of Aβ peptides.
Though the amount of secreted Aβ (1-42) is only 10% of Aβ (1-40), Aβ (1-42) tends
to aggregate more rapidly and displays an enhanced neurotoxicity relative to Aβ (1-40)
[
19]. Therefore, the prevention of Aβ (1-42) aggregation attracts much current
attention.
In addition to these two hypotheses, there is another hypothesis, called metal
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hypothesis, which indicates that the metals (Fe, Cu and Zn) also play a role in
pathogenesis of AD [20]. It was observed that during disease progression from
moderate to severe AD, the metals progressively accumulate in AD patients [21]. The
abnormal accumulation of metals is closely associated with the formation of Aβ
plaques and neurofibrillary tangles (NFT), the hallmarks in brain of AD patients [22,
2
3]. Besides, the abnormally high levels of Cu and Fe in brain catalyze the production
of reactive oxygen species (ROS), which further elicit oxidative stress contributing to
the AD pathogenesis [23-25]. Thus, lowering the concentration of metals in brain by
chelating metals represents another rational therapeutic approach for halting AD
pathogenesis.
The current drugs for treatment of AD are mainly limited to three AChE inhibitors,
namely, donepezil, rivastigmine and galantamine. However, due to multifactorial
nature of AD, these drugs that modulate such a single target could only enable a
palliative treatment instead of curing or preventing the neurodegeneration [12, 26].
Thus, drugs discovery in AD is gradually turning to the design of multifunctional
molecules with two or more complementary biological activities beyond simple AChE
inhibition [27-30]. Considering the importance of BuChE, the common approach to
designing multifunctional molecules is to modify a known ChE inhibitor to provide it
with additional biological properties such as lowering the Aβ aggregation and
chelating metals.
Tacrine, the first ChE inhibitor approved by the FDA for AD treatment, was
withdrawn due to severe liver toxicity [31]. However, with increased research interest
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in design of multifunctional molecules, tacrine has become a widely used scaffold in
recent years [4]. Tacrine can inhibit both ChEs with potency in nanomolar range by
binding to their CAS, and it has a relatively low molecular weight suitable for
modification. It is convenient to connect tacrine and another fragment with additional
activities through a suitable linker to produce multifunctional ChE inhibitors. For
example, tacrine-melatonin hybrids and tacrine-ferulic acid hybrids have been
designed as potent ChE inhibitors with antioxidant property [32].
Tacrine-4-oxo-4H-chromene hybrids have been designed as multifunctional agents
capable of inhibiting ChE and β-secretase [14], and tacrine-8-hydroxyquinoline
hybrids have been designed to exhibit neuroprotective, cholinergic, antioxidant, and
copper-complexing properties [33] (Figure 1).
As naturally occurring phytochemicals, coumarins are widely present in many plant
species and have attracted much attention in recent years because they possess a wide
range of biological activities associated with neurological disorders, especially for AD
[
34-37]. The studies showed that naturally occurring as well as the chemically
synthesized coumarin analogs exhibited potent AChE inhibitory activity, and their
inhibitory activity was exerted by interacting with PAS of AChE [38, 39]. Many
compounds containing a coumarin scaffold, such as AP2238 [2] and ensaculin [40]
(Figure 1), were successfully used as AChE inhibitors. Besides its AChE inhibition, a
recent report indicated that coumarin analogs also exhibited Aβ anti-aggregation
potency, and functionalization of their aromatic center could manipulate the inhibition
mechanism [41].
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Despite these obvious advantages exhibited by tacrine and coumarin scaffolds in AD
drug design, few reports focus on designing multifunctional hybrids through
combining them into one molecule. Therefore, the aim of this study is to combine
them together to design a series of novel tacrine-coumarin hybrids (8a-t) as
multifunctional ChE inhibitors for AD treatment. The tacrine was selected to inhibit
the ChEs through CAS and the coumarin scaffold was used for its self-induced Aβ
aggregation inhibitory activity, as well as for its potential interaction with the PAS of
AChE due to its aromatic character. Since the mid-gorge of AChE that is lined with
aromatic residues could establish cation-π interaction with protonated nitrogen atoms
[
42], we considered connecting coumarin and tacrine fragments by piperazine
side-armed-alkane spacer, expecting to enhance the inhibition to AChE by binding to
mid-gorge site of AChE. Moreover, unlike the reported tacrine-based hybrids, whose
9
-amino group of tacrine was a secondary amine. In present work, the 9-amino group
was acylated by acetyl chloride because previous reports suggested that the acylation
of 9-mino group could promote the choline uptake in hippocampus synaptosome of
rat and decreases the toxicity of tacrine [43, 44]. Meanwhile, the produced amide
moiety also has the ability to chelate metal ions [45]. All designed compounds were
synthesized and evaluated for their ability to inhibit both ChEs and self-induced Aβ
aggregation, and to chelate metal ions. Moreover, to further investigate their
mechanism of interaction with AChE, kinetic analysis and molecular modeling were
also performed. Herein, we report the design, synthesis, evaluation and molecular
modeling of a series of novel tacrine-coumarin hybrids as multifunctional ChE
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inhibitors.
2
. Result and discussion
.1. Chemistry
2
Target compounds 8a–t were synthesized as shown in Scheme1.The synthesis of the
coumarin derivatives 2a-d was performed according to the well-known von
Pechmann reaction with slight modifications depending on the stability and reactivity
of the used reagents [46]. Compound 2a was obtained by condensing resorcinol and
2
-hydroxysuccinic acid with a large excess of concentrated sulphuric acid as catalyst
o
at 100 C, while compounds 2b and 2c were prepared under relatively mild condition
by condensing resorcinol with corresponding β-ketoesters (ethyl acetoacetate and
ethyl benzoylacetate) in the presence of a catalytic amount of concentrated sulfuric
o
acid in 1, 4-dioxane at 60 C. For compound 2d, it was obtained by condensing
o
resorcinol and malonic acid at 90 C with a Lewis acid, boron trifiuoride, as catalyst.
The methylation of 4-OH group of 2d with methanol and concentrated hydrochloric
acid afforded compound 2e. The obtained compounds 2a-c and 2e were then reacted
with α, ω-dibromoalkanes in the presence of potassium carbonate to provide the key
intermediates 3a-t.
On the other hand, reacting 4 with cyclohexanone through a Lewis acid mediated
cyclodehydration reaction gave compound 5 [47], which was then acylated with
chloroacetyl chloride to yield compound 6. Compound 6 was treated with excess
anhydrous piperazine in the presence of potassium iodide to give another key
intermediate 7, which was finally reacted with corresponding coumarin derivatives
8

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3
a-t to produce desired compounds 8a-t.
.2. Cholinesterase inhibitory activity
2
The inhibitory activities of hybrids 8a-t and related compounds 2a-c, 2e and 7 against
AChE (from electric eel) and BuChE (from equine serum) were measured according
to the spectrophotometric method of Ellman et al using tacrine and galanthamine as
reference compounds [48]. The IC50 values of all test compounds and their selectivity
index for AChE over BuChE are summarized in Table 1. It could be seen from the
table that all novel target compounds showed good inhibitory activity to both ChEs
with IC50 values ranging from micromolar to sub-micromolar. Among the target
compounds, 8f (IC50 = 0.092 ꢀM) showed the highest inhibitory activity against
AChE, and the potency was 3- and 29-times stronger than those of the reference
compounds tacrine (IC50 = 0.269 ꢀM) and galanthamine (IC50 = 2.67 ꢀM),
respectively. In contrast, compound 8j exhibited strongest inhibition against BuChE
with IC50 value of 0.099 ꢀM, which was 128-fold more potent than that of
galanthamine (IC50 = 12.7 ꢀM). The coumarin precursor compounds 2a-c and 2e
showed the AChE inhibitory activity with IC50 values ranging from 140 to 258 ꢀM,
while no inhibitory activity for BuChE was observed up to 500 ꢀM, suggesting that
coumarin moiety primarily bound to PAS of AChE, since there is no such domain
within BuChE according to previous reports [49]. The tacrine derivative 7 (IC = 145
5
0
ꢀM for AChE; IC50 = 22 ꢀM for BuChE; SI = 0.152) exhibited a trend that inhibit
both ChEs similar to that of tacrine (IC50 = 0.269 ꢀM for AChE; IC50 = 0.042 ꢀM for
BuChE; SI = 0.156 ꢀM), indicating that tacrine derivative 7 mainly bound to CAS of
9

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ChEs. Interestingly, the inhibitory activities of all precursors for AChE were much
lower than those of target compounds, suggesting that design of dual binding site
AChE inhibitor was more rational.
The linker length between coumarin moiety and piperazine played a significant role in
determining the inhibitory activity for AChE in present work. The results indicated
that all tacrine-coumarin hybrids 8a-t showed decreasing AChE inhibitory activity
with lengthening alkyl chain spacer from two to six carbon atoms except 8b. It (IC50
=
0
.133 ꢀM) possessed a three-carbon linker but showed a slightly higher potency than
a (IC50 = 0.150 ꢀM) having a two-carbon linker. This suggested that two-carbon
8
linker was suitable length for AChE inhibition. The effect of linker length on BuChE
inhibitory activity revealed a trend different from those observed for the AChE
inhibitory activity. Compounds 8a-e showed a trend for BuChE inhibition which was
opposite their trend for AChE inhibition. However, for the other compounds 8f-t, no
clear trend was observed with lengthening the linker length. Overall, the alkyl chain
with five or six carbon atoms seemed to be beneficial for BuChE inhibitory activity.
For example, 8e (IC50 = 0.210 ꢀM), 8j (IC50 = 0.099 ꢀM), 8n (IC50 = 0.730 ꢀM) and
8
s (IC50 = 1.27 ꢀM) possessing a six- or five-carbon linker gave the most potent
BuChE inhibitory activity in each subset. Moreover, the inhibitory activity of target
compounds was sensitive to the size of substituents at 4-position of coumarin ring. As
shown in Table 1, compounds 8f-j possessing methyl substituent at the 4-position of
coumarin moiety showed inhibitory activity for both ChEs better than compounds
8
a-e with small substituent H in most cases. For example, the inhibitory activities of
1
0

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8
j against AChE and BuChE (IC50 = 0.398 ꢀM for AChE; IC50 = 0.099 ꢀM for
BuChE) were 1.6-fold and 2.1-fold more potent, respectively, than those of 8e (IC50
=
0
.646 ꢀM for AChE; IC50 = 0.210 ꢀM for BuChE). Further increasing the size of
substituent from the methyl group to methoxy group afforded a series of hybrids 8k-o
that led to a slight decrease in AChE inhibitory activity, but a relatively large decrease
in BuChE inhibitory activity. Due to such results, compounds 8k-o gave the higher
selectivity for AChE over BuChE relative to other compounds, and compound 8k
showed the highest selectivity with a selectivity index of 34.3. Finally, the
introduction of a bulky group, phenyl group, to the 4-position of coumarin moiety
produced compounds 8p-t, which result in a large decrease in both ChEs inhibitory
activity. This might be attributed to the high hydrophobicity and/or the steric
hindrance of the phenyl ring. Taken together, the inhibitory activity of both ChEs was
in the order: Me > H > MeO > Ph, which suggested that the methyl group might be
the optimal size for ChE inhibition.
2
.3. Kinetic study of AChE inhibition
To investigate the AChE inhibitory mechanism for this class of tacrine-coumarin
hybrids, the most potent compound 8f was selected for kinetic study. The overlaid
reciprocal Lineweaver-Burk plots (Figure 2) showed that both increasing slopes and
increasing intercepts at increasing inhibitor concentration. This pattern indicated a
mixed-type inhibition and therefore revealed that compound 8f might be able to bind
to the catalytic active site (CAS) as well as peripheral anionic site (PAS) of AChE.
Replots of the slope versus concentration of 8f gave an estimate of competitive
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inhibition constant, Ki, of 0.081ꢀM.
.4. Molecular modeling studies
2
To further study the interaction mode of compound 8f for AChE, molecular docking
study was performed using software package MOE 2008.10. The X-ray crystal
structure of the TcAChE complex with bis(7)-tacrine (PDB code 2CKM) was applied
to build the starting model of AChE. As shown in Figure 3, the tacrine moiety of 8f
was bound to the CAS of AChE, and stacked against the phenyl ring of Phe 330 and
the indole ring of Trp 84 with the ring-to-ring distance of 3.87 and 4.57 Å,
respectively. The coumarin ring as in previous reports [2, 39] interacted with the
indole ring of Trp 279 of PAS via π-π stacking interactions with the distance of 4.18
Å. In the middle gorge, as we expected, the piperazine could bind to the mid-gorge
site of AChE through a cation-π interaction (4.41 Å) between its protonated nitrogen
atom and Tyr 334. Moreover, a hydrogen bond (O· · ·O distance: 2.36 Å) that was
formed between the oxygen atom of the side chain of 8f and Tyr 121(another residue
in middle gorge) further enhanced the binding to mid-gorge site. All these results
clearly indicated that 8f could simultaneously bind to CAS, PAS and mid-gorge site
of AChE, thereby demonstrating rationality of our molecular design.
2
.5. Inhibition of self-induced Aβ (1-42) aggregation
All compounds tested for both ChEs inhibition were also evaluated for their ability to
inhibit self-induced Aβ (1-42) aggregation through a thioflavin T-based fluorometric
assay [50]. Curcumin (Cur), a known active natural product for inhibition of Aβ (1-42)
self-aggregation, was used as reference compound. From the results summarized in
1
2

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Table 1 and Figure 4, it could be seen that these hybrids showed moderate to good
potencies (21.6 to 67.8 % at 20 ꢀM) relative to that of curcumin (42.4 % at 20 ꢀM).
Interestingly, compounds 8a-j having the high inhibitory activity for both ChEs also
exhibited the good potency on inhibition of self-induced Aβ (1-42) aggregation with
percentages of inhibition ranging from 52.7 to 67.8 %, which were better than that of
reference compound curcumin. Specially, 8f as the most potent AChE inhibitor
showed the highest inhibitory potency, with inhibitory percentage of 67.8 %. In
contrast, compounds 8k-t only showed percentages of inhibition ranging from 21.6 to
3
4.9 %, nearly 2-fold lower than that of the curcumin. These results indicated that,
similarly to ChE inhibition, the size of substituents was also an important factor for
inhibition of Aβ (1-42) self-aggregation. However, the linker length did not play a
role in determining the inhibition of Aβ (1-42) self-aggregation, since no significant
change of the percentages of inhibition was observed with increase in the spacer
length. Unexpectedly, most of the compounds 8k-t showed inhibitory potencies which
were even lower than those of their precursors 2c, 2e and 7, indicating that all
compounds independently inhibited the Aβ (1-42) self-aggregation.
2
.6. Metal chelating effect
2
+
2+
The chelating effect of all compounds for metals such as Cu and Fe in methanol
was studied by UV-vis spectrometry with wavelength ranging from 200 to 500 nm
2+
[
51, 52]. In Figure 5a, UV-vis spectra of 8f at increasing Cu concentrations were
shown as an example. The increase in absorbance, which could be better estimated by
an inspection of the differential spectra (Figure 5b), indicated that there was an
1
3

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2+
interaction between Cu and 8f. A similar behavior was also observed between 8f
2+
and Fe . These observations indicated that the target compounds could effectively
2+
2+
chelate Cu and Fe , and therefore could serve as metal chelators to treat AD. The
ratio of ligand/metal ion in the complex was investigated by mixing a fixed amount of
compound 8f (25 ꢀM) with ascending amounts of metal ions (5-80 ꢀM); it was
possible to observe that the maximum intensity of differential spectra was reached at
1
:2 ratio, which was taken as an indication of the stoichiometry of the complex.
2
.7 SH-SY5Y neuroblastoma cell toxicity
On basis of the screening results above, the most potent compound 8f was selected to
further examine the potential toxicity effect on the human neuroblastoma cell line
SH-SY5Y. After exposing the cells to this compound for 24 h, the cell viability was
evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay.
The result indicated that 8f did not show significant effect on cell viability at 1-50 ꢀM
(
1 ꢀM: 96.6 ± 13.7 %; 5 ꢀM: 94.7 ± 9.2 %; 10 ꢀM: 104.3 ± 12.1 %; 25 ꢀM: 93.6 ±
0.6 %; 50 ꢀM: 91.5 ± 5.3 %). This suggested that 8f was nontoxic to SH-SY5Y cells
and might be a suitable multifunctional ChE inhibitor for treating AD.
. Conclusion
1
3
In conclusion, a series of tacrine-coumarin hybrids have been designed, synthesized
and evaluated as novel multifunctional ChE inhibitors. Most of these compounds
could effectively inhibit ChEs in the sub-micromolar range in vitro. Especially 8f
showed the most potent AChE inhibitory activity, which was 29-fold more active than
that of the reference compound galanthamine; it also showed a moderate BuChE
1
4

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inhibition with IC50 value of 0.234 ꢀM. Kinetic study implied that 8f could bind
simultaneously to PAS and CAS of AChE. The molecular modeling studies further
confirmed that 8f not only simultaneously bound to CAS and PAS but also to the
mid-gorge site of AChE. In inhibition of Aβ aggregation assay, compounds with no
substituent (8a-e) or methyl substituent (8f-j) at the 4-postiton of coumarin were more
potent inhibitors than curcumin. And the most AChE inhibitor, 8f, showed the greatest
inhibitory activity. In addition, 8f also showed metal-chelating property and low cell
toxicity. Altogether, the multifunctional effects of the new hybrids qualified them as
potential anti-AD drug candidates, and 8f might be considered as a promising lead
compound for further research.
4
. Experimental section
4
.1. Chemistry
All chemicals (reagent grade) used were purchased from Sinopharm Chemical
Reagent Co., Ltd.(China). Reaction progress was monitored using analytical thin layer
chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang Chemical
Plant, Qingdao, China) plates and the spots were detected under UV light (254 nm).
Melting point was measured on an XT-4 micromelting point instrument and
uncorrected. IR (KBr-disc) spectra were recorded by Bruker Tensor 27 spectrometer.
1
13
H NMR spectra (500 MHz) and C NMR spectra (125 MHz) were measured on a
Bruker ACF-500 spectrometer at 25°C and referenced to TMS. Chemical shifts are
reported in ppm (δ) using the residual solvent line as internal standard. Splitting
patterns are designed as s, singlet; d, doublet; t, triplet; m, multiplet. The purity of all
1
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compounds used for biological evaluation was confirmed to be higher than 95%
through analytical HPLC performed with Agilent 1200 HPLC System (Supporting
information, Table S1). Mass spectra were obtained on a MS Agilent 1100 Series
LC/MSD Trap mass spectrometer (ESI-MS) and a Mariner ESI-TOF spectrometer
(HRESIMS), respectively. Column chromatography was performed on silica gel
(90-150 ꢀm; Qingdao Marine Chemical Inc.)
4
.1.1. 7-Hydroxy-2H-chromen-2-one (2a)
A mixture of resorcinol (1.4 g, 12.7 mmol), 2-hydroxysuccinic acid (1.9 g, 14.0 mmol)
and conc. H SO (5 mL) was stirred at room temperature for 0.5 h. The above mixture
2
4
was heated to 100 °C and stirred for another 2.5 h. After cooling to room temperature,
ice water was added to the mixture and stirred for 30 min. The reaction mixture was
o
filtered and dried to afford 2a as white solid. Yield 71%, mp 234-236 C; IR (KBr) ν
-1
3
157, 2828, 1706, 1679, 1603, 1567, 1408, 1323, 1235, 1136, 989, 902, 850, 835 cm ;
1
3
H NMR (500 MHz, CD OD) δ 7.87 (d, 1H, J = 9.5 Hz), 7.48 (d, 1H, J = 8.5 Hz),
6
.82 (dd, 1H, J = 8.5 Hz, J = 2.0 Hz), 6.73 (d, 1H, J = 2.0 Hz), 6.21 (d, 1H, J = 9.5
+
Hz); ESI-MS m/z: 163.0 [M+H] .
.1.2. 7-Hydroxy-4-methyl-2H-chromen-2-one (2b)
To an ice-cold solution of resorcinol (2.0 g, 18.2 mol) in dioxane, conc. H
4
2
SO
4
(0.5
o
mL) was added dorpwise under 25 C. After the addition of conc. H SO , ethyl
2
4
o
acetoacetate (2.8 g, 21.8 mmol) was added, and the mixture was heated to 60 C for 4
h. Then, the mixture was poured into cold water, and the precipitate was filtered and
dried under reduced pressure. The resulting mixture was recrystallized from methanol
1
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o
to give 2b as white needle crystals. Yield 92 %, mp 186-187 C; IR (KBr): 3495, 3119,
-1 1
1
6
660, 1600, 1454, 1390, 1274, 938, 848 cm ; H NMR (500 MHz, acetone-d ): δ 7.61
(d, 1H, J = 9.0 Hz), 6.85 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.75 (d, 1H, J = 2.5 Hz),
-
6
.30 (s, 1H), 2.40 (s, 3H); ESI-MS m/z: 175.0 [M-H] .
.1.3. 7-Hydroxy-4-phenyl-2H-chromen-2-one (2c)
4
According to the procedure already described for 2b, 2c was obtained as yellow
crystals by reacting resorcinol (2.0 g, 18.2 mmol) with ethyl benzoylacetate (4.2 g,
o
2
1
1
1
4
1.8 mmol). Yield 90 %, mp 249-251 C; IR (KBr): 3102, 1692, 1598, 1377, 1110,
-1 1
000, 863, 860, 773 cm ; H NMR (500 MHz, CDCl ): δ 7.55-7.48 (m, 5H), 7.32 (d,
3
H, J = 8.5 Hz), 7.23(d, 1H, J = 2.5 Hz), 7.10 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz), 6.30 (s,
-
H); ESI-MS m/z: 236.9 [M-H] .
.1.4. 4, 7-Dihydroxy-2H-chromen-2-one (2d)
A mixture of resorcinol (8.8 g, 80 mmol), malonic acid (25.0 g, 240 mmol) and boron
trifluoride-diethyl etherate complex (40 mL) was stirred at 90 °C for 24 h. The crude
mixture was poured onto crushed ice, and the resulting precipitate was filtered,
washed with diethyl ether (50 mL), and then purified by silica gel chromatography
3 3
using CHCl /CH OH (15:1) as eluent to give compound 2d as red solid. Yield 23 %,
o
mp 265-267 C; IR (KBr): 3246, 1633, 1629, 1568, 1448, 1325, 1303, 1247, 1217,
-1 1
1
151, 1096, 1000, 851, 811, 799 cm ; H NMR (500 MHz, DMSO-d ): δ 12.20 (s,
6
1
6
4
H), 10.51 (s, 1H), 7.62 (d, 1H, J = 8.5 Hz), 6.75 (dd, 1H, J = 8.5 Hz, J =2.0 Hz),
-
.64 (d, 1H, J = 2.0 Hz), 5.36 (s, 1H); ESI-MS m/z: 176.7 [M-H] .
.1.5. 7-Hydroxy-4-methoxy-2H-chromen-2-one (2e)
1
7

ACCEPTED MANUSCRIPT
A mixture of 2d (1.0 g, 5.6 mmol) and conc. HCl (5 mL) in methanol (40 mL) was
refluxed for 1.5 hours. The solvent was removed under vacuum, and the residue was
purified by silica gel chromatography using CH
2
Cl
2
/MeOH (25:1) as eluent to afford
o
compound 2e as yellow solid. Yield 55%, mp 275-276 C; IR (KBr): 2419, 3225,
-1 1
1
693, 1602, 1546, 1439, 1398, 1241, 1151, 973, 864, 837, 806 cm ; H NMR (500
MHz, DMSO-d ): δ 10.54 (s, 1H), 7.62 (d, 1H, J = 8.5 Hz), 6.79 (dd, 1H, J = 8.5 Hz,
6
J = 2.0 Hz), 6.69 (d, 1H, J = 2.0 Hz), 5.67 (s, 1H), 3.96 (s, 3H); ESI-MS m/z:
+
1
93.1[M+H] .
4
.1.6. General procedures for the preparation of compounds 3a-t
A mixture of 2a-c/2e (5.0 mmol) with suitable α, ω-dibromoalkanes (50 mmol) and
anhydrous K CO (1.4 g, 10 mmol) in acetone (15 mL) was refluxed under stirring for
h. After cooling, the reaction mixture was filtered, and the filtrate was evaporated
2
3
4
under reduced pressure. The obtained residue was purified by silica gel
chromatography with hexane/acetone (20:1) as eluent to give compounds 3a-t as
white solid.
4
.1.6.1. 7-(2-Bromoethoxy)-2H-chromen-2-one (3a)
o
Yield 65%, mp 134-135 C; IR (KBr) ν 3083, 3039, 2975, 2929, 1732, 1610, 1509,
-1 1
1
394, 1288, 1229, 1124, 1070, 1011, 991, 891,833 cm ; H NMR (500 MHz, CDCl
3
)
δ
7.65 (d, 1H, J = 9.5 Hz), 7.41 (d, 1H, J = 8.5 Hz), 6.88 (dd, 1H, J = 8.5 Hz, J = 2.5
Hz), 6.82 (d, 1H, J = 2.5 Hz), 6.29 (d, 1H, J = 9.5 Hz), 4.36 (t, 2H, J = 6.0 Hz), 3.67 (t,
+
2
H, J = 6.5 Hz); ESI-MS m/z: 269.1 [M+H] .
4
.1.6.2. 7-(3-Bromopropoxy)-2H-chromen-2-one (3b)
1
8

ACCEPTED MANUSCRIPT
o
Yield 71%, mp 106-107 C; IR (KBr) ν 3088, 3041, 2950, 2882, 1723, 1620, 1508,
-
1
1
1
7
401, 1292, 1239, 1072, 1019, 981, 889, 836 cm ; H NMR (500 MHz, CDCl ) δ
3
.64 (d, 1H, J = 9.0 Hz), 7.39 (d, 1H, J = 8.0 Hz), 6.86 (dd, 1H, J = 8.0 Hz, J = 2.5
Hz), 6.84 (d, 1H, J = 2.5 Hz), 6.27 (d, 1H, J = 9.5 Hz), 4.18 (t, 2H, J = 6.0 Hz), 3.61 (t,
+
2
H, J = 6.5 Hz), 2.34-2.38 (m, 2H); ESI-MS m/z: 283.0 [M+H] .
4
. 1.6.3. 7-(4-Bromobutoxy)-2H-chromen-2-one (3c)
o
Yield 65%, mp 64-65 C; IR (KBr) ν 3082, 3035, 2964, 2924, 2878, 1724, 1622, 1511,
-1 1
1
400, 1293, 1237, 1133, 1045, 1011, 891, 851, 829 cm ; H NMR (500 MHz, CDCl
3
)
δ
7.64 (d, 1H, J = 9.5 Hz), 7.37 (d, 1H, J = 8.5 Hz), 6.84 (dd, 1H, J = 8.5 Hz, J = 2.5
Hz), 6.80 (d, 1H, J = 2.5 Hz), 6.26 (d, 1H, J = 9.5 Hz), 4.20 (t, 2H, J = 6.0 Hz), 3.49 (t,
+
2
H, J = 6.5 Hz), 2.06-2.11 (m, 2H), 1.98-2.02 (m, 2H); ESI-MS m/z: 297.1 [M+H] .
4
.1.6.4. 7-((5-Bromopentyl)oxy)-2H-chromen-2-one (3d)
o
Yield 66%, mp 61-63 C; IR (KBr) ν 3072, 3050, 2947, 2872, 1729, 1611, 1506, 1470,
-
1 1
1
392, 1349, 1280, 1230, 1119, 1036, 1021, 981, 889, 847, 825 cm ; H NMR (500
MHz, CDCl 7.64 (d, 1H, J = 9.5 Hz), 7.37 (d, 1H, J = 8.5 Hz), 6.84 (dd, 1H, J =
.5 Hz, J = 2.5 Hz), 6.80 (d, 1H, J = 2.5 Hz), 6.26 (d, 1H, J = 9.5 Hz), 4.03 (t, 2H, J =
3
) δ
8
6
2
4
.0 Hz), 3.45 (t, 2H, J = 6.5 Hz), 1.92-1.98 (m, 2H), 1.83-1.89 (m, 2H), 1.62-1.68 (m,
+
H); ESI-MS m/z: 311.2 [M+H] .
.1.6.5. 7-((6-Bromohexyl)oxy)-2H-chromen-2-one (3e)
o
Yield 60%, mp 58-60 C; IR (KBr) ν 3080, 3041, 2942, 2899, 2862, 1724, 1621, 1509,
-1 1
1
398, 1292, 1235, 1140, 1099, 1031, 1002, 891, 842, 828 cm ; H NMR (500 MHz,
CDCl3) 7.64 (d, 1H, J = 9.5 Hz), 7.37 (d, 1H, J = 8.5 Hz), 6.84 (dd, 1H, J = 8.5 Hz,
δ
1
9

ACCEPTED MANUSCRIPT
J = 2.0 Hz), 6.80 (d, 1H, J = 2.0 Hz), 6.25 (d, 1H, J = 9.5 Hz,), 4.02 (t, 2H, J = 6.5
Hz), 3.43 (t, 2H, J = 7.0 Hz), 1.88-1.92 (m, 2H), 1.82-1.85 (m, 2H), 1.51-1.53 (m, 4H);
+
ESI-MS m/z: 325.1 [M+H] .
4
.1.6.6. 7-(2-Bromoethoxy)-4-methyl-2H-chromen-2-one (3f)
o
Yield 67%, mp 104-105 C; IR (KBr) ν 3072, 2955, 1722, 1617, 1389, 1368, 1264,
-
1 1
1
1
156, 1137, 1075, 1010, 988, 845, 836 cm ; H NMR (500 MHz, CDCl
3
) δ 7.53 (d,
H, J = 9.0 Hz), 6.90 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.82 (d, 1H, J = 2.5 Hz,), 6.16
(s, 1H), 4.36 (t, 2H, J = 6.5 Hz), 3.67 (t, 2H, J = 6.0 Hz), 2.40 (s, 3H); ESI-MS m/z:
+
2
83.1 [M+H] .
4
.1.6.7. 7-(3-Bromopropoxy)-4-methyl-2H-chromen-2-one (3g)
o
Yield 63%, mp 82-83 C; IR (KBr) ν 3070, 2964, 2878, 1712, 1611, 1391, 1372, 1261,
-1 1
1
153, 1137, 1071, 1023, 982, 849, 826, 804 cm ; H NMR (500 MHz, CDCl )
δ
7.51
3
(d, 1H, J = 9.5 Hz), 6.87 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.84 (d, 1H, J = 2.5 Hz),
6
.15 (s, 1H), 4.18 (t, 2H, J = 5.5 Hz), 3.61 (t, 2H, J = 6.0 Hz), 2.40 (s, 3H), 2.33-2.36
+
(
m, 2H); ESI-MS m/z: 297.0 [M+H] .
4
.1.6.8. 7-(4-Bromobutoxy)-4-methyl-2H-chromen-2-one (3h)
o
Yield 63%, mp 58-60 C; IR (KBr) ν 3073, 2957, 2851, 1748, 1610, 1391, 1370, 1282,
-1 1
1
150, 1137, 1068, 1040, 993, 856, 817, 750 cm ; H NMR (500 MHz, CDCl
3
) δ 7.50
(d, 1H, J = 8.5 Hz), 6.86 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.80 (d, 1H, J = 2.5 Hz),
6
.13 (s, 1H), 4.06 (t, 2H, J = 6.0 Hz), 3.49 (t, 2H, J = 6.5 Hz), 2.41 (s, 3H), 2.06-2.10
+
(
m, 2H), 1.98-2.00 (m, 2H); ESI-MS m/z: 311.1[M+H] .
4
.1.6.9. 7-((5-Bromopentyl)oxy)-4-methyl-2H-chromen-2-one (3i)
2
0

ACCEPTED MANUSCRIPT
o
Yield 60%, mp 59-60 C; IR (KBr) ν 3065, 2942, 2910, 2873, 1718, 1608, 1389, 1365,
-1 1
1
264, 1199, 1138, 1069, 1014, 994, 879, 842, 818 cm ; H NMR (500 MHz, CDCl
3
)
δ
7.49 (d, 1H, J = 9.5 Hz), 6.86 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.81 (d, 1H, J = 2.5
Hz), 6.13 (s, 1H), 4.03 (t, 2H, J = 6.5 Hz), 3.45 (t, 2H, J = 6.5 Hz), 2.39 (s, 3H),
1
.92-1.95 (m, 2H), 1.84-1.87 (m, 2H), 1.63-1.66 (m, 2H); ESI-MS m/z: 325.2
+
[
M+H] .
4
.1.6.10. 7-((6-Bromohexyl)oxy)-4-methyl-2H-chromen-2-one (3j)
o
Yield 67%, mp 59-61 C; IR (KBr) ν 3068, 2946, 2915, 2868, 2843, 1719, 1614, 1386,
-
1
1
1
7
367, 1293, 1148, 1073, 1013, 937, 876, 799 cm ; H NMR (500 MHz, CDCl
3
) δ
.49 (d, 1H, J = 9.5 Hz), 6.86 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.81 (d, 1H, J = 2.5
Hz), 6.13 (s, 1H), 4.02 (t, 2H, J = 6.5 Hz), 3.43 (t, 2H, J = 7.0 Hz), 2.40 (s, 3H),
1
.88-1.94 (m, 2H), 1.81-1.87 (m, 2H), 1.52-1.53 (m, 4H); ESI-MS m/z: 339.1
+
[
M+H] .
4
.1. 6.11. 7-(2-Bromoethoxy)-4-methoxy-2H-chromen-2-one (3k)
o
Yield 62%, mp 121-123 C; IR (KBr) ν 3075, 3019, 2945, 2885, 1724, 1625, 1396,
-
1
1
7
294, 1250, 1151, 1121, 1027, 960, 844, 836, 804 cm ; H NMR (500 MHz, CDCl
3
) δ
.70 (d, 1H, J = 9.0 Hz), 6.83 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.73 (d, 1H, J = 2.5
Hz), 5.57 (s, 1H), 4.36 (t, 2H, J = 6.0 Hz,), 3.96 (s, 3H), 3.67 (t, J = 6.5 Hz, 2H);
+
ESI-MS m/z: 299.1 [M+H] .
4
.1.6.12. 7-(3-Bromopropoxy)-4-methoxy-2H-chromen-2-one (3l)
o
Yield 60%, mp 110-112 C; IR (KBr) ν 3075, 3022, 2943, 2880, 1730, 1627, 1394,
-1
1
1
295, 1248, 1145, 1120, 1023, 966, 845, 814 cm ; H NMR (500 MHz, CDCl ) δ
3
2
1

ACCEPTED MANUSCRIPT
7
.71 (d, 1H, J = 8.5 Hz), 6.84 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz), 6.81 (d, 1H, J = 2.5
Hz), 5.57 (s, 1H), 4.17 (t, 2H, J = 6.0 Hz), 3.97 (s, 3H), 3.61 (t, 2H, J = 6.5 Hz),
+
2
.34-2.37 (m, 2H); ESI-MS m/z: 313.2 [M+H] .
4
.1.6.13. 7-(4-Bromobutoxy)-4-methoxy-2H-chromen-2-one (3m)
o
Yield 64%, mp 93-95 C; IR (KBr) ν 3080, 3012, 2944, 2870, 1728, 1620, 1397, 1296,
-1 1
1
248, 1156, 1113, 1033, 981, 845, 836, 816 cm ; H NMR (500 MHz, CDCl
3
) δ 7.71
(d, 1H, J = 8.5 Hz), 6.85 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.81 (d, 1H, J = 2.5 Hz),
5
.57 (s, 1H), 4.06 (t, 2H, J = 6.0 Hz), 3.97 (s, 3H), 3.49 (t, 2H, J = 6.5 Hz), 2.06-2.11
+
(
m, 2H), 1.98-2.02 (m, 2H); ESI-MS m/z: 326.0 [M+H] .
4
.1.6.14. 7-((5-Bromopentyl)oxy)-4-methoxy-2H-chromen-2-one (3n)
o
Yield 68%, mp 77-78 C. IR (KBr) ν 3080, 3010, 2944, 2868, 1727, 1619, 1396, 1296,
-
1 1
1
7
248, 1157, 1103, 1043, 998, 981, 850, 836, 806 cm ; H NMR (500 MHz, CDCl ) δ
3
.70 (d, 1H, J = 9.0 Hz), 6.85 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.81 (d, 1H, J = 2.5
Hz), 5.56 (s, 1H), 4.04 (t, 2H, J = 6.5 Hz), 3.97 (s, 3H), 3.45 (t, 2H, J = 6.5 Hz),
1
.92-1.97 (m, 2H), 1.84-1.88 (m, 2H), 1.62-1.68 (m, 2H); ESI-MS m/z: 341.0
+
[
M+H] .
4
.1.6.15. 7-((6-Bromohexyl)oxy)-4-methoxy-2H-chromen-2-one (3o)
o
Yield 65%, mp 78-79 C; IR (KBr) ν 3078, 3033, 2939, 2855, 1717, 1615, 1393, 1281,
-
1
1
1
7
246, 1156, 1106, 1027, 1008, 971, 852, 835 cm ; H NMR (500 MHz, CDCl
3
) δ
.69 (d, 1H, J = 8.5 Hz), 6.83 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.78 (d, 1H, J = 2.5
Hz), 5.56 (s, 1H), 4.03 (t, 2H, J = 6.5 Hz), 3.97 (s, 3H), 3.43 (t, 2H, J = 7.0 Hz),
.89-1.92 (m, 2H), 1.82-1.85 (m, 2H), 1.51-1.53 (m, 4H); ESI-MS m/z: 355.1
1
2
2

ACCEPTED MANUSCRIPT
+
[
M+H] .
4
.1.6.16. 7-(2-Bromoethoxy)-4-phenyl-2H-chromen-2-one (3p)
o
Yield 71%, mp 108-109 C; IR (KBr) ν 3024, 2967, 1726, 1616, 1375, 1272, 1242,
-1
1
1
154, 1118, 1074, 1001, 960, 851, 830, 819, 767, 700 cm ; H NMR (500 MHz,
CDCl 7.50-7.53 (m, 3H), 7.43-7.45 (m, 2H), 7.38 (d, 1H, J = 9.0 Hz), 6.87 (d, 1H,
3
) δ
J = 2.5 Hz), 6.79 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.23 (s, 1H), 4.38 (t, 2H, J = 6.0
+
Hz), 3.69 (t, 2H, J = 6.5 Hz); ESI-MS m/z: 345.1 [M+H] .
4
.1.6.17. 7-(3-Bromopropoxy)-4-phenyl-2H-chromen-2-one (3q)
o
Yield 68%, mp 79-80 C; IR (KBr) ν 3067, 2931, 1710, 1609, 1378, 1264, 1212, 1155,
-1 1
1
3
124, 1076, 1003, 862, 839, 770, 706 cm ; H NMR (500 MHz, CDCl ) δ 7.50-7.54
(m, 3H), 7.43-7.45 (m, 2H), 7.39 (d, 1H, J = 9.0 Hz), 6.91 (d, 1H, J = 2.5 Hz), 6.80
(dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.23 (s, 1H), 4.20 (t, 2H, J = 6.5 Hz), 3.61 (t, 2H, J =
+
6
.5 Hz), 2.34-2.39 (m, 2H); ESI-MS m/z: 359.0 [M+H] .
4
.1.6.18. 7-(4-Bromobutoxy)-4-phenyl-2H-chromen-2-one (3r)
o
Yield 72%, mp 89-90 C; IR (KBr) ν 3071, 2958, 1714, 1603, 1374, 1282, 1207, 1156,
-
1
1
1
7
124, 1045, 1010, 991, 864, 823, 769, 738 cm ; H NMR (500 MHz, CDCl
3
) δ
.50-7.52 (m, 3H), 7.43-7.45 (m, 2H), 7.38 (d, 1H, J = 9.0 Hz), 6.88 (d, 1H, J = 2.5
Hz), 6.79 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.22 (s, 1H), 4.08 (t, 2H, J = 6.5 Hz), 3.50
(t, 2H, J = 6.5 Hz), 2.05-2.12 (m, 2H), 1.97-2.02 (m, 2H); ESI-MS m/z: 373.1
+
[
M+H] .
4
.1.6.19. 7-((5-Bromopentyl)oxy)-4-phenyl-2H-chromen-2-one (3s)
o
Yield 75%, mp 65-67 C; IR (KBr) ν 3073, 2954, 2869, 1719, 1604, 1376, 1270, 1206,
2
3

ACCEPTED MANUSCRIPT
-
1
1
1
7
152, 1033, 1003, 982, 856, 830, 775, 708 cm ; H NMR (500 MHz, CDCl
3
) δ
.50-7.52 (m, 3H), 7.42-7.44 (m, 2H), 7.38 (d, 1H, J = 9.0 Hz), 6.88 (d, 1H, J = 2.5
Hz), 6.78 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.21 (s, 1H), 4.05 (t, 2H, J = 6.5 Hz), 3.45
(t, 2H, J = 6.5 Hz), 1.92-1.98 (m, 2H), 1.83-1.89 (m, 2H), 1.62-1.68 (m, 2H); ESI-MS
+
m/z: 387.0 [M+H] .
4
.1.6.20. 7-((6-Bromohexyl)oxy)-4-phenyl-2H-chromen-2-one (3t)
o
Yield 69%, mp 70-72 C; IR (KBr) ν 3056, 2946, 2866, 1716, 1604, 1375, 1276, 1205,
-
1
1
1
7
153, 1020, 1006, 943, 858, 840, 775, 704 cm ; H NMR (500 MHz, CDCl
3
) δ
.50-7.52 (m, 3H), 7.42-7.45 (m, 2H), 7.38 (d, 1H, J = 9.0 Hz), 6.88 (d, 1H, J = 2.5
Hz), 6.79 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 6.21 (s, 1H), 4.04 (t, 2H, J = 6.5 Hz), 3.43
(t, 2H, J = 7.0 Hz), 1.89-1.94 (m, 2H), 1.82-1.87 (m, 2H), 1.52-1.57 (m, 4H); ESI-MS
+
m/z: 401.1 [M+H] .
4
.1.7. 9-Amino-1, 2, 3, 4-tetrahydroacridine (5)
To a solution of anthranilonitrile (5.0 g, 43.3 mmol) and cyclohexanone (5.0 mL) in
sodium-dried toluene (110.0 mL), boron trifluoride diethyl etherate (6.4 mL) was
added slowly via syringe. The mixture was then stirred at reflux for 24 h. On cooling,
the toluene was decanted and, to liberate the product, the remaining solids were
treated with NaOH (2 M, 120.0 mL) and heated at reflux for 24 h. After cooling, the
3
organic components were extracted with CHCl , the organic layers were combined
and dried, and the solvent was evaporated under reduced pressure to give compound 5
o
as yellow solid. Yield 92%, mp 182-184 C; IR (KBr) ν 3483, 3373, 3310, 3121, 2938,
-1 1
2
875, 1647, 1566, 1500, 1439, 1423, 1373, 1302, 978, 768 cm ; H NMR (500 MHz,
CDCl ): δ 7.95 (d, 1 H, J = 8.5 Hz), 7.71 (d, 1H, J = 8.5 Hz), 7.62 (td, 1H, J = 8.5 Hz,
3
2
4

ACCEPTED MANUSCRIPT
J = 1.0 Hz), 7.57 (td, 1H, J = 7.0 Hz, J = 1.0 Hz), 3.18 (t, 2H, J = 6.5 Hz,), 2.90 (t, 2H,
-
J = 6.0 Hz), 1.95-2.00 (m, 2H), 1.83-1.88 (m, 2H); ESI-MS m/z: 199.1 [M+H] .
4
.1.8. 2-Chloro-N-(1, 2, 3, 4-tetrahydroacridin-9-yl)acetamide (6)
A solution of chloroacetyl chloride (0.5 mL) in CH Cl (10 mL) was added dropwise
2
2
to an ice-cold solution of triethylamine (2.5 mmol, 1.8 mL) and 5 (1.0 g, 5.0 mmol) in
CH Cl (30 mL). After completed addition the solution was stirred for 2 h at room
temperature. When the reaction was completed, it was diluted with CH Cl , washed
2
2
2
2
with water, followed by brine solution. The organic layer was dried over anhydrous
Na SO and concentrated under reduced pressure. The obtained residue was purified
2
4
by silica gel chromatography using CH
2
Cl
2
/MeOH (100: 1) as eluent to give
o
compound 6 as white solid. Yield 65%, mp > 250 C; IR (KBr) ν 3420, 3236, 3015,
-
1 1
2
1
942, 2868, 1665, 1527, 1407, 1216, 762 cm ; H NMR (500 MHz, CDCl
3
): 8.06 (d,
H, J = 8.5 Hz), 7.80 (d, 1H, J = 8.0 Hz), 7.66 (td, 1H, J = 7.5 Hz, J = 1.5 Hz), 7.52
(td, 1H, J = 7.5 Hz, J = 1.0 Hz), 4.37 (s, 2H), 3.18 (t, 2H, J = 6.5 Hz), 2.85 (t, 2H, J =
-
6
.5 Hz), 1.96-2.00 (m, 2H), 1.88-1.92 (m, 2H); ESI-MS m/z: 275.0 [M-H] .
4
.1.9. 2-(Piperazin-1-yl)-N-(1, 2, 3, 4-tetrahydroacridin-9-yl) acetamide (7)
A mixture of 6 (0.2 g, 0.7 mmol), anhydrous piperazine (0.9 g, 10.5 mmol) and KI
0.06 g, 0.4 mmol) in ethanol (30 mL) was refluxed for 1 h. After cooling, the solvent
was removed under vacuum. The obtained residue was dissolvent in CH Cl and then
(
2
2
washed with water. The organic phase was dried over anhydrous Na
concentrated under reduced pressure. The crude product was purified by silica gel
chromatography with CH Cl /MeOH (20: 1) as eluent to afford compound 7 as white
2 4
SO and
2
2
2
5

ACCEPTED MANUSCRIPT
o
solid. Yield 72%, mp 73-75 C; IR (KBr) ν 3420, 3013, 2944, 2871, 2828, 1664, 1528,
-
1 1
1
427, 1356, 1231, 770 cm ; H NMR (500 MHz, CDCl
8.0 Hz), 7.79 (d, 1H, J = 8.0 Hz), 7.65 (td, 1H, J = 7.0 Hz, J = 1.5 Hz), 7.49 (td, 1H,
J = 8.5 Hz, J = 1.0 Hz), 3.23 (s, 2H), 3.04 (t, 2H, J = 6.5 Hz), 2.81 (t, 4H, J = 5.0 Hz),
3
): 9.79 (s, 1H), 7.98 (d, 1H, J
=
2
.77 (t, 2H, J = 6.5 Hz), 2.56 (br s, 4H), 1.86-1.91 (m, 2H), 1.79-1.82 (m, 2H);
-
ESI-MS m/z: 325.1 [M+H]
4
.1.10. General procedures for the preparation of compounds 8a-t
A mixture of 7 (1.5 mmol), anhydrous K CO (1.7 mmol) and the corresponding
2
3
coumarin derivatives 3a-t (1.8 mmol) in dry acetonitrile (15 mL) was refluxed for 8 h.
After cooling to the room temperature, the mixture was filtered, and the filtrate was
evaporated under vacuum. The obtained residue was purified by silica gel
3
chromatography with CHCl /MeOH (50:1) as eluent to yield target compounds 8a-t.
4
2
.1.10.1.
-(4-(2-((2-Oxo-2H-chromen-7-yl)oxy)ethyl)piperazin-1-yl)-N-(1,2,3,4-tetrahydroacri
din-9-yl)acetamide (8a)
Yield 44%, pale yellow oil; IR (KBr) ν 3424, 3299, 2933, 2868, 2820, 1728, 1612,
1
485, 1428, 1401, 1351, 1281, 1230, 1201, 1158, 1122, 1018, 996, 835, 760, 729, 615
-1 1
cm ; H NMR (500 MHz, CDCl
3
): δ 8.05 (d, 1H, J = 8.5 Hz), 7.74 (d, 1H, J = 8.5
Hz), 7.61-7.66 (m, 2H), 7.49 (t, 1H, J = 8.0 Hz), 7.38 (d, 1H, J = 9.0 Hz), 6.87 (dd,
1
2
H, J = 9.0 Hz, J = 2.5 Hz), 6.83 (d, 1H, J = 2.5 Hz), 6.26 (d, 1H, J = 9.5 Hz), 4.22 (t,
H, J = 5.5 Hz), 3.35 (s, 2H), 3.17 (t, 2H, J = 6.5 Hz), 2.93 (t, 2H, J = 5.5 Hz), 2.88
(br s, 4H), 2.82 (t, 2H, J = 6.5 Hz), 2.80 (br s, 4H), 1.95-2.00 (m, 2H), 1.86-1.91 (m,
2
6

ACCEPTED MANUSCRIPT
1
3
2
1
1
3
H); C NMR (125 MHz, CDCl ): δ 168.47, 161.84, 161.04, 159.89, 155.91, 146.71,
43.26, 138.34, 128.95, 128.80, 127.08, 126.02, 123.69, 121.88, 113.36, 112.95,
12.79, 101.60, 66.61, 61.79, 56.73, 53.86, 53.72, 33.89, 25.80, 22.65, 22.53; ESI-MS
+
+
m/z: 513.3 [M+H] ; HRMS: calcd for C H N O [M+H] , 513.2502, found
30
33
4
4
5
4
2
13.2496.
.1.10.2.
-(4-(3-((2-Oxo-2H-chromen-7-yl)oxy)propyl)piperazin-1-yl)-N-(1,2,3,4-tetrahydroac
ridin-9-yl)acetamide (8b)
Yield 56%, colorless oil; IR (KBr) ν 3444, 3299, 2932, 2874, 2818, 1730, 1612, 1489,
1
430, 1401, 1350, 1280, 1230, 1200, 1158, 1123, 1096, 1015, 996, 834, 763, 729, 615
-1
1
cm ; H NMR (500 MHz, CDCl
3
): δ 8.08 (d, 1H, J = 7.5 Hz), 7.75 (br s, 1H),
7
8
6
2
1
1
5
.61-7.67 (m, 2H), 7.54 (t, 1H, J = 6.0 Hz), 7.38 (d, 1H, J = 9.5 Hz), 6.84 (dd, 1H, J =
.5 Hz, J = 2.0 Hz), 6.81 (d, 1H, J = 2.0 Hz), 6.26 (d, 1H, J = 9.5 Hz), 4.14 (t, 2H, J =
.0 Hz), 3.44 (s, 2H), 3.18 (t, 2H, J = 6.5 Hz), 2.88-2.82 (m, 6H), 2.60-2.66 (m, 6H),
1
3
3
.09 (br s, 2H), 1.91-2.00 (m, 2H), 1.87-1.91 (m, 2H); C NMR (125 MHz, CDCl ): δ
68.34, 162.12, 161.08, 159.79, 155.93, 146.69, 143.29, 138.37, 129.13, 128.79,
27.16, 126.14, 123.68, 121.95, 113.26, 112.78, 112.68, 101.57, 66.51, 61.64, 54.75,
3.66, 53.16, 33.67, 26.47, 25.79, 22.56, 22.47; ESI-MS m/z: 527.3 [M+H]+; HRMS:
+
calcd for C H N O [M+H] , 527.2659, found 527.2653.
31
35
4
4
4
2
.1.10.3.
-(4-(4-((2-Oxo-2H-chromen-7-yl)oxy)butyl)piperazin-1-yl)-N-(1,2,3,4-tetrahydroacri
din-9-yl)acetamide (8c)
2
7