Coumarin-dithiocarbamate hybrids as novel multitarget AChE and MAO-B inhibitors against Alzheimer's disease: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bioorg.2018.09.010

A series of new coumarin-dithiocarbamate hybrids were designed and synthesized as multitarget agents for the treatment of Alzheimer's disease. Most of them showed potent and clearly selective inhibition towards AChE and MAO-B. Among these compounds, compound 8f demonstrated the most potent inhibition to AChE with IC₅₀ values of 0.0068 μM and 0.0089 μM for eeAChE and hAChE, respectively. Compound 8g was identified as the most potent inhibitor to hMAO-B, and it is also a good and balanced inhibitor to both hAChE and hMAO-B (0.114 μM for hAChE; 0.101 μM for hMAO-B). Kinetic and molecular modeling studies revealed that 8g was a dual binding site inhibitor for AChE and a competitive inhibitor for MAO-B. Further studies indicated that 8g could penetrate the BBB and exhibit no toxicity on SH-SY5Y neuroblastoma cells. More importantly, 8g did not display any acute toxicity in mice at doses up to 2500 mg/kg and could reverse the cognitive dysfunction of scopolamine-induced AD mice. Overall, these results highlighted 8g as a potential multitarget agent for AD treatment and offered a starting point for design of new multitarget AChE/MAO-B inhibitors based on dithiocarbamate scaffold.

Full text of DOI:10.1016/j.bioorg.2018.09.010

Accepted Manuscript
Coumarin- dithiocarbamate hybrids as novel multitarget AChE and MAO-B
inhibitors against Alzheimer’s Disease: Design, synthesis and biological eval-
uation
Qi He, Jing Liu, Jin-Shuai Lan, Jiaoli Ding, Yongbing Sun, Yuanying Fang,
Neng Jiang, Zunhua Yang, Liyuan Sun, Yi Jin, Sai-Sai Xie
PII:
S0045-2068(18)30840-X
https://doi.org/10.1016/j.bioorg.2018.09.010
YBIOO 2507
DOI:
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
7 August 2018
5 September 2018
7 September 2018
Please cite this article as: Q. He, J. Liu, J-S. Lan, J. Ding, Y. Sun, Y. Fang, N. Jiang, Z. Yang, L. Sun, Y. Jin, S-S.
Xie, Coumarin- dithiocarbamate hybrids as novel multitarget AChE and MAO-B inhibitors against Alzheimer’s
Disease: Design, synthesis and biological evaluation, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.09.010
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Coumarin- dithiocarbamate hybrids as novel multitarget AChE and MAO-B
inhibitors against Alzheimer’s Disease: Design, synthesis and biological
evaluation
‡
a
‡b
c
a
a
a
Qi He, Jing Liu , Jin-Shuai Lan , Jiaoli Ding , Yongbing Sun , Yuanying Fang ,
d
b
e
a
a
Neng Jiang , Zunhua Yang , Liyuan Sun , Yi Jin* , Sai-Sai Xie*
a
National Pharmaceutical Engineering Center for Solid Preparation in Chinese
Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang
3
30006, PR China
b
School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang
3
30006, PR China
c
Experiment Center of Teaching & Learning, Shanghai University of Traditional
Chinese Medicine, Shanghai 201203, PR China
d
Department of Pharmacy, Affiliated Tumor Hospital of Guangxi Medical University,
Nanning 530021, Guangxi, PR China
e
Guangxi Key Laboratory of Brain and Cognitive Neuroscience, Guilin Medical
University, Guilin 541004, PR China
*
Corresponding Author. E-mail: jinyixsnc@126.com (Y. Jin);
xiesaisainanchang@hotmail.com (S.-S. Xie);
‡
These authors contributed equally.

Abstract
A series of new coumarin-dithiocarbamate hybrids were designed and synthesized as
multitarget agents for the treatment of Alzheimer’s disease. Most of them showed
potent and clearly selective inhibition towards AChE and MAO-B. Among these
compounds, compound 8f demonstrated the most potent inhibition to AChE with IC50
values of 0.0068 μM and 0.0089 μM for eeAChE and hAChE, respectively.
Compound 8g was identified as the most potent inhibitor to hMAO-B, and it is also a
good and balanced inhibitor to both hAChE and hMAO-B (0.114 µM for hAChE;
0
.101 µM for hMAO-B). Kinetic and molecular modeling studies revealed that 8g
was a dual binding site inhibitor for AChE and a competitive inhibitor for MAO-B.
Further studies indicated that 8g could penetrate the BBB and exhibit no toxicity on
SH-SY5Y neuroblastoma cells. More importantly, 8g did not display any acute
toxicity in mice at doses up to 2500 mg/kg and could reverse the cognitive
dysfunction of scopolamine-induced AD mice. Overall, these results highlighted 8g as
a potential multitarget agent for AD treatment and offered a starting point for design
of new multitarget AChE/MAO-B inhibitors based on dithiocarbamate scaffold.
Keywords: Coumarin, dithiocarbamate, cholinesterase, monoamine oxidase,
Alzheimer’s disease.

1
. Introduction
’
Alzheimer s disease (AD) is one of the most disastrous neurodegenerative diseases
characterized by memory loss, degradation in language skills, behavioral
abnormalities and cognitive deficits [1]. Due to lacking daily life abilities, Alzheimer's
patients always lead to a heavy burden on both families and society. According to the
statistical report from Alzheimer’s Association, nearly 47 million people worldwide
are suffering from AD, and the number of patients will exceed 100 million by 2050
[
2]. Therefore, AD is regarded as a severe and urgent public health trouble that needs
to be tackled without delay.
For the sake of conquering AD, great efforts have been devoted to develop novel
drugs including chemical molecular and biological products by both academic
institutions and pharmaceutical companies over the past years [3]. However, most of
anti-AD drug candidates with promising therapy effects in the phase of preclinical
studies ended up with failure. To date, only four drugs (rivastigmine, galantamine,
donepezil, memantine) approved by the US Food and Drug Administration (FDA) are
used in clinic. These upset results of developing anti-AD drugs have been attributed to
the intricate and multifactorial etiopathogenesis of AD involving deficit of
acetylcholine (ACh)[4], β-amyloid protein (Aβ) deposits [5], aggregation of Tau
protein [6], oxidative stress [7] and metabolic homeostasis disruption of biometals [8].
Thus, the conventional paradigm of ‘one drug, one target’ may be not suitable enough
to treat this complicated disease. To address this issue, a multitarget-directed-ligand
(
MTDL) strategy means that one molecule that can simultaneously act on multitargets

related to disease, which has been put forward as a potential approach for the
treatment of AD [9-11].
Among the therapy targets against AD, acetylcholinesterase (AChE) has been
recognized as an important target based on cholinergic hypothesis [12]. The
hypothesis emphasizes the deficit of acetylcholine (ACh) in brain regions of
Alzheimer’s patients leads to memory and cognitive impairments, and reducing the
ACh hydrolysis by inhibiting AChE can alleviate these symptoms [13]. At present,
with exception of memantine, all FDA-approved drugs are AChE inhibitors (AChEIs).
The crystal structure of AChE shows that it consists of two binding sites: one is
catalytic anionic site (CAS) and the other is peripheral anionic site (PAS), which are
connected by a 20 Å deep gorge [14, 15]. Generally, inhibitors binding to either one
site can inhibit AChE. However, recent studies indicate that, in addition to
hydrolyzing ACh, AChE also plays a role in inducing the aggregation of Aβ through
the interaction of PAS with Aβ peptides [16]. Thus, AChE inhibitors, like donepezil,
that can simultaneously act on CAS and PAS appear to be more beneficial for AD
treatment [17]. Besides AChE, the other cholinesterase isoform, butyrylcholinesterase
(
BuChE), is also responsible for the ACh hydrolysis. Although several studies have
proved that the inhibition of BuChE is another available choice for the treatment of
AD, it is still a matter of controversial whether the inhibition on BuChE is safe and
effective enough as a treatment approach for AD, because BuChE prevalently
distributes in peripheral tissues, and its inhibition may cause peripheral cholinergic
side effect [18]. Therefore, the selective inhibition of AChE is more promising for AD

treatment.
In addition to AChE, monoamine oxidase (MAO) is also an efficient therapy target in
the treatment of AD. MAO is a flavin adenine dinucleotide (FAD)-containing enzyme
that is responsible for the oxidative deamination of various biogenic and xenobiotic
amines [19]. Based on substrate selectivity and inhibitor sensitivity, MAO has been
classified as two isoforms, namely MAO-A and MAO-B. Typically, MAO-A is
selectively and irreversibly inhibited by clorgyline and catalyzes the oxidation of
5
-HT, whereas MAO-B is irreversibly inhibited by L-deprenyl and deaminates the
benzylamine and 2-phenylethylamine [20]. Accumulated evidence shows that
MAO-B activity increases with age, especially in AD patients, a significant activity
rise is found in brain tissue, cerebral spinal fluid (CSF) as well as in platelets [21].
The elevated activity of MAO-B leads to an increased level of hydrogen peroxide and
oxidative free radicals, which give rise to neuronal damage [22]. Furthermore, the
activated MAO-B can also cause disorder of the cholinergic system, destroy
cholinergic neurons, and promote the formation of amyloid plaques [23]. Thus,
inhibition of MAO-B provides another potential approach for treating AD.
In view of the importance of AChE and MAO-B in the treatment of AD, designing
MTDLs with simultaneous inhibition of AChE and MAO-B receive much attention in
recent years. A lot of AChE/MAO-B inhibitors with good therapy effect have been
reported [24-26]. Among them, ladostigil designed by hybridization of rivastigmine
and rasagiline has been announced to enter phase III clinical trials[27], which
encourage us to further search for other new multitarget molecules with AChE and

MAO-B inhibitory activity.
Coumarins constitute a large family of natural products, which are widely present in
many plant species. In recent years, considerable attention has been paid to coumarins
due to their widely biological activities related to neurological disorders, especially
for AD [28; 29]. It has been demonstrated that coumarin can inhibit AChE through
binding to PAS of AChE [30]. Besides, it can occupy the substrate cavity of MAO-B,
and thus presenting potent MAO-B inhibitory activity [31]. Given such excellent
properties of coumarin on AChE and MAO-B inhibition, more and more MTDLs with
AChE and MAO-B inhibitory activity have been designed and synthesized based on
coumarin core [32]. In most cases for design of these MTDLs, the coumarin was
chosen to connect with a CAS binding moiety through a flexible linker lodged into the
mid-gorge of AChE [33]. Owing to the inhibitory activity of coumarin, the designed
compounds can not only exert AChE and MAO-B inhibitory activity but also
simultaneously interact with PAS and CAS of AChE. Over the past three years, our
group has reported a number of coumarin derivatives as multitarget AChE/MAO-B
inhibitors according to this method, and most of them showed promising inhibitory
activity [34, 35]. As an ongoing program to development of these multitarget
inhibitors, in this work we wanted to introduce new CAS binding moiety to connect
with coumarin fragment.
Dithiocarbamate is a versatile pharmacophore with a wide range of biological activity
profiles, such as anticancer, antibacterial and inhibition on carbonic anhydrase. Recent
years, although dithiocarbamate derivatives have been widely reported, few reports

focus on its activities related to neurodegenerative disease. Very recently, our group
found that dithiocarbamate moiety could interact with CAS of AChE [36]. Therefore,
we attempted to combine this moiety with coumarin to design a new series of
coumarin-dithiocarbamate hybrids as multitarget AChE/MAO-B inhibitors for the
treatment of AD. All designed compounds were synthesized and tested in vitro to
evaluate their inhibitory activities on ChEs and MAOs. The compound with good and
balanced inhibitory activity on both AChE and MAO-B was selected for further
evaluation including the ability to cross the blood-brain barrier (BBB), in vitro
toxicity on SH-SY5Y neuroblastoma cell, acute toxicity, and neuroprotective effects
in scopolamine-induced cognitive impairment in mice. In addition, kinetic and
molecular modeling studies were also conducted to investigate binding mechanism of
the selected compound with both AChE and MAO-B.
2
. Design of coumarin-dithiocarbamate hybrids
As shown in Figure 1, like our previous design, the coumarin moiety was chosen to
inhibit the MAO-B and interact with PAS of AChE due to its aromatic character. The
dithiocarbamate moiety was used for binding to CAS of AChE. Meanwhile, a flexible
linker was utilized to connect these two fragments, which could allow the designed
compounds to simultaneously act on CAS and PAS of AChE. In addition, a
3
,4-dimethylcoumarin and a piperidine dithiocarbamate moiety were exploited as
starting fragments in our initial step, because the previous studies suggested that these
two moieties could potently and selectively inhibit MAO-B and AChE, respectively
[
26, 36]. In order to get optimal linker length for balanced inhibition of AChE and

MAO-B, compounds with varied linker length were synthesized firstly. And once the
optimal length was determined, various substitutes and secondary amine groups were,
respectively, introduced to coumarin and dithiocarbamate moiety for SAR studies.
3
. Results and discussion.
3
.1 Chemistry
The synthetic rout for target compounds 7a-n and 8a-n is depicted in Scheme 1-2.
Following the procedures listed in Scheme 1, the coumarin derivatives 5a-e and 5h
were obtained by our previously reported methods [35]. Then, treating
2
,4-dihydroxybenzaldehyde with ethyl acetoacetate or diethyl malonate in the
presence of piperidine afforded compounds 5f and 5g. Before preparation of the
cycloalkyl-substituted 7-hydroxycoumarins 5i-k, the key intermediates 4a-c were
firstly prepared through reacting the corresponding cycloalkanone 3a-c with diethyl
carbonate in the presence of sodium hydride according to the reported procedures[37,
3
8]. Afterwards, 5i-k were obtained by the consideration of 4a-c with resorcinol by
o
using concentrated sulfuric acid as catalyst at 0 C to room temperature. All
synthesized coumarin derivatives 5a-k were subsequently reacted with the
corresponding α, ω-dibromoalkanes to give compounds 6a-n. Finally, compounds
6
a-n were treated with the appropriate secondary amines, carbon disulfide and
triethylamine in DMF to obtain the target compounds 7a-n and 8a-n [39].
.2 In vitro biological activity evaluation
3
The ChEs inhibitory potencies of target compounds 7a-n and 8a-n were firstly tested
on electric eel acetylcholinesterase (eeAChE) and equine serum butyrylcholinesterase

(
eqBuchE) by the Ellman’s spectrophotometric mothed (Table 1-2) [40], then six
compounds 7c, 7g, 8f-g and 8j-k were selected to determine their inhibitory activities
towards human acetylcholinesterase (hAChE) owing to their good and selective
inhibition on both eeAChE and MAO-B (Table 3). The inhibitory capabilities against
MAOs of the tested compounds were assessed directly on human MAOs following a
fluorescence-based Amplex Red method [41, 42]. Donepezil and two MAO inhibitors,
rasagiline and iproniazid, were used as positive control to reflect the inhibitory
potencies of all resulting compounds.
As shown in Table 1 and Table 2, most compounds exhibited potent inhibitory
activity on AChE with IC50 values ranging from micromolar to nanomolar. Among
these compounds, compound 8f showed the most potent inhibition on eeAChE with
IC50 value of 0.0068 μM, which was 6-fold more potent than that of the reference
compound donepezil (IC50 = 0.041 μM). For MAO-B, compounds also presented
moderate to good inhibitory activities. Compound 8g was the most potent inhibitor for
MAO-B in this series, showing the IC50 value of 0.101 μM, which was higher than
those of rasagiline (IC50 = 0.138 μM) and iproniazid (IC50 = 7.48 μM). In addition, all
compounds displayed excellent selectivity towards AChE and MAO-B over BuChE
and MAO-A. Considering inhibition of BuChE and MAO-A may lead to unexpected
side effects in the peripheral tissues, these compounds may be more beneficial for AD
treatment. Meanwhile, due to the poor inhibition of compounds on both BuChE and
MAO-A, it was difficult to draw a clear structure-activity relationships (SARs) for
them. Thus, the discussion of the SARs was mainly made on AChE and MAO-B.

Our previous studies indicated that the linker length tethering coumarin to CAS
binding moiety played a crucial role in determining AChE and MAO-B inhibitory
activity [34,35]. Therefore, the effect of linker length was firstly investigated by
preparing compounds 7a-d with different linker lengths (m = 2-5). It can be seen from
the Table 1 that a pronounced enhancement of inhibitory activity for AChE is
observed with progressive elongation of the methylene linker from two to four carbon
atoms. Compound 7b (IC50 = 1.39 µM) with a three-carbon atom linker provided
much better inhibitory activity for AChE than compound 7a with a two-carbon atom
linker (IC50 = 20.85 µM). When the linker length was extended to four carbon atoms,
the obtained compound 7c (IC50 = 0.082 µM) presented the most potent inhibitory
activity in this subset. However, further lengthening the linker length to five carbon
atoms did not lead to an increase in AChE inhibition. Compound 7d (IC50 = 0.088 µM)
showed a little decreased inhibition compared to compound 7c. Similar tendency was
also found in MAO-B inhibition, in general, compounds with the longer linker
displayed stronger inhibitory activity. However, different from AChE inhibition, the
linker length with odd number of carbon atoms seemed more beneficial for MAO-B
inhibition, as compounds 7d (m = 5) and 7b (m =3) showed more potent inhibitory
activity than compound 7c (m = 4). Notably, compound 7d with a five-carbon atom
linker also exhibited a strong inhibitory activity for MAO-A (IC50 = 0.654 µM), which
indicated that a more flexible linker that was longer than four carbon atoms might
give rise to a lower selectivity for MAO-B. In fact, the inhibitory activity on MAO-A
can give rise to an unexpected side effect of “cheese reaction” [43]. Therefore, taking

the inhibitory activity and selectivity on both AChE and MAO-B into consideration,
the linker containing four carbon atoms (m = 4) was chosen as optimal length to
connect coumarin and dithiocarbamate moiety.
When the optimal linker length was determined, different substituents were
introduced to the position(s) 3 and/or 4 of coumarin ring to explore the possible
effects on both enzyme inhibition. As shown in Table 1, with exception of compound
7
g, which have a chloro group at position 3 and a methyl group at position 4 of
coumarin ring, exhibited improved inhibitory activity on both AChE and MAO-B
IC50 = 0.061 μM for AChE; IC50 = 0.363 μM for MAO-B), all compounds showed
(
decreased inhibitory activity compared to compound 7c. The introduction of various
substituents to coumarin ring did not have a significant influence on AChE inhibition,
as the IC50 values of most compounds were varied very closely. Only compound 7k
(
IC50 = 15.48 μM) with a phenyl substituent at 4-posiotn of coumarin led to a large
decrease in inhibiting AChE. This suggested that the high steric hindrance on
coumarin region could not be well accommodated in active site of AChE. In contrast,
a wider activity range could be observed for MAO-B, which indicated that more
pronounced effect of the substituents on the MAO-B inhibition. In fact, this result
might be consistent with our design that coumarin was the main fragment responsible
for MAO-B inhibition.
In compassion to compound 7c, removing the two methyl groups (7e) or 4-substiuted
methyl group alone (7f) on coumarin ring resulted in a decreased activity for MAO-B,
which suggested that the methyl group at position 4 of coumarin seemed beneficial

for maintaining the inhibitory activity. Keeping the methyl group at position 4
unchanged and replacing the other methyl group with an electron-withdrawing
substituent, chloro group, increased the inhibition for MAO-B. The obtained
compound 7g was found to be the strongest inhibitor in this series. However,
introduction of a strong electron-withdrawing substituent, trifluoromethyl group, to
coumarin ring afforded compound 7h, which gave the poorest activity in this series
(
IC50 = 42.12 μM). Unlike previous reports that introducing methyl ketone or ethyl
ester group to position 3 of coumarin moiety could improve the inhibitory activity on
MAO-B[44], compounds 7i (IC50 = 12.29 μM) and 7j (IC50 = 39.0 μM) displayed
weak inhibition in our present study. Moreover, like AChE inhibition, the presence of
a phenyl group at 4-posiotn of coumarin was also not favorable for MAO-B inhibition.
Compound 7k (IC50 = 17.57 μM), which possess a bulky phenyl group on coumarin
ring, exerted a less inhibitory activity for MAO-B. In addition, given the above results
that compounds bearing di-substitution pattern on 3 and 4- positions of coumarin
showed more potent inhibition for MAO-B in comparison to mono-substituted
compounds, three compounds 7l-n with different size of 3, 4-fused cyclic substituents
were also synthesized to further extend the SARs. Among these compounds,
compound 7m (IC50 = 1.24 μM) having a six-membered ring on coumarin was more
potent than its homologues 7l (IC50 = 4.0 μM) and 7n (IC50 = 21.40 μM), which
suggested that the spatial arrangement of cyclohexyl allows for a better positioning in
the active site of the MAO-B. After completing these investigations, compound 7g
with good and balanced inhibitory activity on AChE and MAO-B was fished out for

further modification.
Compounds 8a-n were designed to explore the influences of different terminal amine
groups on inhibitory activity towards the target enzymes. Initially, we thought that
changing the dithiocarbamate moiety of compound 7g would mainly impact the AChE
inhibition, because this moiety was designed as a CAS binding moiety to interact with
AChE. However, to our surprise, the replacement of terminal piperidyl group in
compound 7g with other secondary amine groups also led to great impact on the
inhibition for MAO-B. Substituted piperidine groups were firstly introduced to
investigate their effects on inhibitory activity. Compounds 8a-e were obtained by
modification at position 4 of piperidine with different substituents. Compared to
unsubstituted piperidine compound 7g, introduction of corresponding 4-substituents
resulted in a dramatic reduced inhibitory activity against AChE, which made the IC50
values range from 1.54 µM (8c) to 18.46 µM (8b). The similar negative effect on
inhibitory activity towards MAO-B was also observed, as four out of five compounds
(
8a-e) displayed much lower activities than compound 7g. Among them, compound
b bearing 4-phenylpiperidine presented the worst inhibitory activity on both AChE
8
and MAO-B (IC50 = 18.46 µM for AChE; IC50 = 31.12 µM for MAO-B). However,
introduction of 4-methyl group to piperidine (8e) brought a significant potency
improvement on MAO-B (IC50 = 0.347 µM), which proved that methyl group might
be a potential substituent to keep the good inhibitory activity on MAO-B. Shifting the
methyl group from position 4 to position 2 of piperidine led to compound 8f (IC50
=
0
.0068 μM), giving a highly surprise that this minor change resulted in a 650-fold

improvement in AChE inhibition. Conversely, the MAO-B inhibitory activity of
compound 8f was 2.5-fold decreased compared to 4-methylpiperidyl hybrid 8e (8f:
IC50 = 0.876 µM vs 8e: IC50 = 0.347 µM), which indicated the ortho-methyl
substitution of the nitrogen atom in piperidine played a pivotal role in the inhibitory
activity for AChE and MAO-B. Further installation of another methyl group to
position 6 of piperidine ring obtained 2, 6-dimethylpiperidinyl derivative 8g.
Although this compound exhibited less AChE inhibitory activity than 8f, its activity
on MAO-B was remarkably improved. Such change rendered compound 8g (IC50
.044 µM for AChE; IC50 = 0.101 µM for MAO-B) a more balanced inhibitor than
compound 7g.
=
0
The effects of replacement of the piperidine group with other alkyl amines or cyclic
amines were also explored by preparing compounds 8h-n. The alkyl amine
compounds 8h-j showed continuous enhancement to both AChE and MAO-B
inhibition with gradually increasing the alkyl groups. Compound 8j (IC50 = 0.167 µM
for AChE; IC50 = 0.788 µM for MAO-B) bearing a diethylamine group exhibited
more potent activity than its congeners 8h (IC50 = 11.67 µM for AChE; IC50 = 2.07
µM for MAO-B) and 8i (IC50 = 0.217 µM for AChE; IC50 = 7.49 µM for MAO-B).
Contraction of the piperidine ring to pyrrole ring produced compound 8k (IC50
=
0
.386 µM for AChE; IC50 = 0.542 µM for MAO-B), which resulted in a little decrease
in both AChE and MAO-B inhibition compared to compound 7g. However, inserting
oxygen or nitrogen atoms into piperidine ring, affording compounds 8l-n, led to a
dramatic drop in both enzyme inhibitory activity. Especially for MAO-B, the IC50

values of compounds 8l-n were found to be higher than 25 μM. This suggested that
the presence of heteroatoms at terminal of piperidine ring was not tolerated for
compound locating into active sites of both AChE and MAO-B.
3
.3 In vitro inhibition on human ChEs
In order to evaluate the inhibitory activities of designed compounds on ChEs more
reasonably, six compounds 7c, 7g, 8f-g, and 8j-k having relatively good inhibitory
activity and selectivity on eeAChE and MAO-B were selected for further assay on
human AChE. From the Table 3, it can be seen that all of them still maintain the high
inhibitory activity and excellent selectivity on hAChE with IC50 values ranging from
sub-micromolar to low nanomolar. In particular, compound 8f with best inhibition on
eeAChE also presented the most potent inhibitory activity on hAChE. It showed the
IC50 value of 0.0089 µM, which was nearly 2.4-fold more potent than that of
donepezil (IC50 = 0.021 µM) under the same conditions.
3
.4. In vitro blood-brain barrier permeation assay
The good permeability for blood-brain barrier (BBB) is a significant factor for drugs
that can successfully act on the central nervous system (CNS). Thus, it is essential to
investigate whether the present compounds could penetrate the BBB. Compounds 7c,
7
g, 8f-g and 8j-k with strong inhibitory activity on both hAChE and hMAO-B were
selected to determine their permeabilities for BBB by the parallel artificial membrane
permeation assay of blood-brain barrier (PAMPA-BBB)[45]. The permeability of 9
commercial drugs with their reported values were used to validate the assay (Table 4).
A plot of experimental data versus the reported values gave a good linear correlation,

2
P
e
(exp.) = 0.9050 P (bibl.) - 0.2568 (R = 0.9759) (Figure 2). From this equation
e
and considering the limit established by Di et al. for BBB permeation, we determined
6
the following ranges of permeability: compound with P
e
(×10 cm/s) > 3.36
6
represented high BBB permeation (CNS+), compound with P
e
(×10 cm/s) < 1.55
6
represented low BBB permeation (CNS-), compound with 1.55 < P
e
(×10 cm/s) <
3
.36 represented uncertain BBB permeation (CNS±). It can be seen from the Table 5
that, with exception of compound 8f that shows an uncertain BBB permeation, all
6
compounds exhibit P
e
(×10 cm/s) values higher than 3.36, suggesting that they can
cross the BBB and may reach the therapeutic target in CNS. After all above biological
evaluation, compound 8g possessing good BBB permeability as well as potent and
well-balanced inhibition on hAChE and hMAO-B (IC50 = 0.114 and 0.101 µM,
respectively) was selected as the optimal candidate for further study.
3
.5 Kinetic study of inhibition on AChE.
In order to investigate the inhibition mechanism of compound 8g, an enzyme kinetic
study was carried out on hAChE. After plotting the reciprocal of enzyme velocity (l/v)
versus the reciprocal of substrate concentration (l/S), the established
Lineweavere-Burk reciprocal plots showed that both increasing slopes and intercepts
at increasing inhibitor concentration (37, 74 and 148 nM) (Figure 3). The pattern
suggested that there was a mixed-type inhibition mechanism between hAChE and
compound 8g, which implied that 8g might be able to interact with both catalytic
active site (CAS) and peripheral anionic site (PAS) of AChE.
3
.6 Reversibility and kinetic study of hMAO-B inhibition

As we know, reversible inhibitors of MAO-B have superior advantages over the
irreversible inhibitors in the view of AD treatment. Thus, investigating whether
compound 8g is a/an reversible or irreversible inhibitor of MAO-B is very necessary.
The investigation was carried out by recovering the enzymatic activity after dilution
of the enzyme-inhibitor complexes, and an irreversible inhibitor, pargyline, was used
as reference compound[46]. Before starting experiment, MAO-B was pre-incubated
with compound 8g at concentrations of 0, 10 and 100 × IC50 for 30 min. Then, these
incubations were diluted 100-fold to obtain concentrations of 0, 0.1 and 1 × IC50. If
compound is a reversible inhibitor, enzymatic activity is expected to recover to
approximately 90 % after dilution to 0.1 × IC50, and 50 % after dilution to 1 × IC50. If
compound is an irreversible inhibitor, enzymatic activity is expected not to recover
after diluting the enzyme-inhibitor complex. As shown in Figure 4, after diluting
compound 8g to 0.1× IC50 and 1 × IC50, the MAO-B catalytic activities are recovered
to levels of 87 % and 46% of control value, respectively. This behavior suggested that
compound 8g was a reversible inhibitor for MAO-B. In contrast, after incubation of
MAO-B with the irreversible inhibitor pargyline at 10 × IC50, and dilution of it to 0.1
×
IC50, the enzyme activity was not recovered (less than 10% of control).
To further explore the interaction mechanism of compound 8g with hMAO-B, an
enzyme kinetic study similar as AChE was carried out. The Lineweavere-Burk
reciprocal plots were established according to initial rates of the MAO-B-catalyzed
oxidation for different concentrations of p-tyramine in the presence of three different
concentrations (63, 126, and 252 nM) of compound 8g (Figure 5). The plots were

linear and intersected at the y-axis, which indicated compound 8g was a competitive
inhibitor for hMAO-B.
3
.7. Molecular modeling studies
To further study the binding modes of compound 8g with hAChE and hMAO-B,
docking studies were performed using the Molecular Operating Environment (MOE
2
008.10) software package.
3
.7.1 Docking study of compounds 8g with hAChE
The docking mode of 8g on hAChE was investigated based on the X-ray crystal
structure of the recombinant human acetylcholinesterase (hAChE) complexed with
donepezil (PDB code 4EY7). As shown in Figure 6, similar to our previous study, the
coumarin moiety can occupy the PAS of hAChE and establish a π-π stacking
interaction between its phenyl ring and the indole ring of Trp 286 (3.75 Å). In the
middle gorge, the side chain connecting coumarin with dithiocarbamate moiety folded
in a conformation in gorge that allowed it to interact with Phe 338, Tyr 341 and Phe
2
97 via hydrophobic interactions. Besides, the oxygen atom in the side chain also
formed a hydrogen bond with the residue Tyr 124 in middle gorge (3.20 Å), which
further enhanced the binding ability to mid-gorge site. At last, the
piperidinyldithiocarbamate moiety of compound 8g was bound to the CAS, exhibiting
a hydrophobic interaction with residues Gly 448, Trp 86, Tyr 337, His 447 and Gly
1
21. Taken together, all these results suggested that compound 8g could occupy the
entire enzyme active sites and is a dual binding site inhibitor to hAChE.
.7.2 Docking study of compounds 8g with hMAO-B
3

The binding mode of compound 8g with respect to MAO-B was investigated based on
the X-ray crystal structure of the human monoamine oxidase B (hMAO-B) in
complex with 7-(3-chlorobenzyloxy)-4-(methylamino)methyl-coumarin (PDB code
2
V61). It can be seen from the Figure 7 that compound 8g crosses both the substrate
cavity and the entrance cavity of MAO-B. The coumarin moiety was located into the
substrate cavity, which left the lactone ring close to the FAD cofactor, and it was
stabilized by hydrophobic interactions with Tyr 398, Phe 343, Tyr 60 and Tyr 435.
Besides, the carbonyl oxygen of coumarin moiety also formed a hydrogen bond with
Tyr 188 (2.84 Å). The piperidinyldithiocarbamate moiety occupied the entrance cavity
and interacted with Phe 168, Leu 167, Leu 164, Pro 102, Pro 104, Ile 316, Trp199 and
Phe 103 through van der waals and hydrophobic interactions.
Overall, the above docking studies of compound 8g with both hAChE and hMAO-B
provided an explanation for kinetic assays and demonstrated the rationality of our
molecular design.
3
.8 Cytotoxicity of human neuroblastoma SH-SY5Y cells.
In order to evaluate the safety of compound 8g, cell toxicity experiment was
performed on human neuroblastoma cells SH-SY5Y, and donepezil was taken as the
reference compound. After treatment of SH-SY5Y cells with different concentrations
of compound 8g or donepezil for 24 h, the cell viability was determined by
3
-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay[47]. As shown in
Figure 8, compound 8g, like donepezil, shows negligible toxicity to SH-SY5Y cells
at test concentrations (6.25-100 µM), which indicate that it is a safe agent for the

treatment of AD.
.9. Acute toxicity study
3
Acute toxicity of compound 8g was carried out on KM mice (n = 10 per group, half
male and half female) according to the reported method[48]. Compound 8g was
delivered to mice by oral administration at three doses of 625, 1250 and 2500 mg/kg.
After drug administration, mice were observed continuously for the first 4 h for any
abnormal behavior and mortality changes, intermittently for the next 24 h, and
occasionally thereafter for 14 days for the onset of any delayed effects. During the
treatment period, no toxicity effects such as death, body weight reduction (Figure 9),
obvious decline in water or food consumption, or significant abnormal behaviors were
observed. In addition, all animals were sacrificed on the 14th day, and possible toxic
damage on heart, liver and kidneys was examined macroscopically. The results
indicated that compound 8g did not cause any toxic effect on mice at dose up to 2500
mg/kg.
3
.10. In vivo efficacy evaluation
The satisfactory therapeutic results in vitro of compound 8g encouraged us to further
determine whether it could improve memory impairment in vivo. The therapeutic
effect of compound 8g was tested using the scopolamine-induced cognitive deficit
mouse model [49]. A step-down passive avoidance test was performed to provide an
evaluation on the effect of cognitive improvement by compound 8g [50], and the
marketed drug, donepezil, was used as positive reference. As shown in Figure 10, the
model group present much shorter latency and more number of errors than control

###
group ( P < 0.001). With exception of the low dose group of compounds 8g, the
latency and number of errors of all groups exhibited a significant difference compared
**
***
to model group ( P < 0.01,
P < 0.001, respectively). After treatment with
compound 8g, the latency and number of errors were reversed in a dose-dependent
manner. High dose group (20 mg/kg) and medium dose group (10 mg/kg) exhibited
longer latencies (134.9 s, 127.1 s vs 122.7s) and less number of errors (1.25, 1.4, vs
1
.6) than donepezil group (10 mg/kg). Although the low dose group (5mg/kg) did not
present significant difference with the model group, the latency and number of errors
were obviously improved (94.8 s vs 63.2s, 2.75 vs 3.83). This in vivo study further
proved that compound 8g might be a promising compound for the treatment of AD.
4
. Conclusion
In this study, a series of new hybrids based on coumarin and dithiocarbamate scaffolds
were designed, synthesized and evaluated as multitarget AChE/MAO-B inhibitors.
The results indicated that most of designed compounds exhibited potent and selective
inhibitory activity on AChE and MAO-B. The SAR analysis suggested that the linker
length between coumarin and dithiocarbamate moieties played an important role in
both AChE and MAO-B inhibition, and the four-carbon atom linker was the optimal
length for compound that potently and selectively inhibited the both enzymes. The
substituents on coumarin moiety showed more significantly effects on MAO-B
inhibition than those on AChE, and the di-substitution pattern on coumarin seemed
more beneficial for improving the inhibitory activity of MAO-B. In contrast, the
terminal amine groups could largely influence the inhibitory activity for both AChE

and MAO-B. Compounds with piperidine or methyl-substituted piperidines were
more favorable to the inhibitory activity. Interestingly, the position of methyl
substitution on piperidine group has an essential effect on inhibiting AChE. When the
methyl group was shifted from 4-position to 2-positon of the piperidine ring, a
6
50-fold improvement in AChE inhibition was observed, and the obtained compound
8
f presented the most potent AChE inhibitory activity in this series (IC50 = 0.0068 μM
for eeAChE; IC50 = 0.0089 μM for hAChE).
Of these compounds, compound 8g with potent and balanced inhibitory activity for
AChE and MAO-B as well as good ability to penetrate the BBB was selected as a
promising compound for further studies. Kinetic and molecular modeling study
suggested compound 8g was a mixed-type inhibitor, binding simultaneously to CAS,
PAS and mid-gorge site of AChE, and it was also a competitive inhibitor, which could
occupy the substrate and entrance cavities of MAO-B. In vitro cytotoxicity assay
indicated that compound 8g showed no toxicity to SH-SY5Y cells at 6.25–100 μM.
More importantly, our in vivo study proved that 8g did not display any acute toxicity
in mice at doses up to 2500 mg/kg, and mice treated with 8g (20 and 10 mg/kg, p.o.)
could significantly prolong the latency and reduce number of errors in the step-down
passive avoidance test. Taken together, these results highlighted compound 8g as a
potential multitarget agent for the treatment of AD. As far as we know,
dithiocarbamate moiety have never been used for design of multitarget AChE/MAO-B
inhibitors. Therefore, these results may provide a starting point for design of new
multitarget AChE/MAO-B inhibitors based on dithiocarbamate scaffold.

5
. Experimental section
5
.1 Chemistry
Starting chemical reagents and solvents used in synthesis were purchased from
Sinopharm Chemical Reagent Co., Ltd. (China). The reaction progress was routinely
checked by thin layer chromatography (TLC) on glass-packed precoated silica gel
GF254 (Qingdao Haiyang Chemical Plant, Qingdao, China) plates. Column
chromatography was performed using silica gel (90-150 µm; Qingdao Marine
Chemical Inc.). IR spectra were obtained with a PerkinElmer Spectrum Two
1
13
spectrophotometer (KBr disks). H NMR spectra (600 MHz) and C NMR spectra
151MHz) were recorded on a Bruker ACF-600 spectrometer at room temperature
using CDCl or DMSO-d as the solvent. Chemical shifts (δ) are reported in parts per
(
3
6
million (ppm) using the tetramethylsilane (TMS) as internal standard. The coupling
constants J are presented in hertz (Hz). Proton coupling patterns were expressed with
following abbreviations: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), m
(
multiplet), br s (broad signal). Melting points were measured on an XT-4
micromelting point apparatus and are uncorrected. The purity of all compounds for
biological evaluation was confirmed to be higher than 95% by analytical HPLC
performed on a Waters ACQUITY Arc HPLC system equipped with a 2998 PDA
detector. (Column: Hypersil ODS2, 5 µm particle size, 4.6 mm × 150 mm; mobile
phase: A = CH
54 nm; 10 µL injection). High resolution mass spectra were conducted on an AB
Sciex Triple TOF 5600 spectrometer (HR-ESI-MS).
3
OH, B = H O, isocratic elution, A = 70%; flow rate = 1 mL/min; λ =
2
2

Synthetic procedures and spectroscopic data of all the intermediates 4a-c, 5a-k and
6
a-n are available as Supporting Information.
5
.2. General procedure for synthesis of final compounds 7a-n.
To a mixture of piperidine (1.69 mmol) and triethylamine (1.54 mmol) dissolved in N,
N-dimethylformamide (DMF), carbon disulfide (1.85 mmol) was added dropwise.
The mixture was stirred for 5 min and a solution of 6a-n (1.54 mmol) in N,
N-dimethylformamide was added. The reaction was allowed to stir for 12 h at room
temperature. When the reaction was completed, 30 mL of water was added and the
mixture was extracted with ethyl acetate (4 × 18 mL). The ethyl acetate layer was
collected, dried over anhydrous sodium sulfate, filtered and then evaporated to
dryness to give crude product, which was purified by silica gel column
chromatography using petroleum ether/ ethyl acetate (8 : 1 to 15:1) as eluent.
5
.2.1.
thioate (7a)
Yield 84%; white solid; m.p. 139-140 C; IR (KBr): ν = 2944, 2853, 1714, 1612, 1508,
2-((3,4-dimethyl-2-oxo-2H-chromen-7-yl)oxy)ethyl
piperidine-1-carbodi-
o
-
1
1
1
478, 1450, 1432, 1283, 1231, 1182, 1091, 857, 755 cm . H NMR (600 MHz,
CDCl ) δ: 7.49 (d, J = 8.9 Hz, 1H), 6.90 (dd, J = 8.9, 2.5 Hz, 1H), 6.84 (d, J = 2.5 Hz,
H), 4.30 (t, J = 6.4 Hz, 4H), 3.91 (br s, 2H), 3.78 (t, J = 6.4 Hz, 2H), 2.37 (s, 3H),
3
1
2
1
3
3
13
.18 (s, 3H), 1.77 – 1.61 (m, 6H). C NMR (151 MHz, CDCl ) δ: 194.43, 162.41,
3
60.46, 153.48, 146.16, 125.29, 119.11, 114.46, 112.12, 101.77, 67.02, 53.30, 51.49,
+
5.57, 29.71, 25.44, 24.26, 15.09, 13.19. HRMS: calcd for C19
78.1119, found 378.1150. HPLC purity, 99.44%.
H
24NO
3
2
S [M + H]

5
.2.2.
3-((3,4-dimethyl-2-oxo-2H-chromen-7-yl)oxy)propyl
piperidine-1-carbodithioate (7b)
o
Yield 87%; white solid; m.p. 138-139 C; IR (KBr): ν = 2940, 2859, 1695, 1607, 1510,
-
1 1
1
7
475, 1427, 1286, 1241, 1177, 1086, 859, 761 cm . H NMR (600 MHz, DMSO) δ:
.70 (d, J = 9.6 Hz, 1H), 6.96 (dd, J = 9.6, 2.5 Hz, 1H), 6.94 (d, J = 2.5 Hz, 1H), 4.22
(
br s, 2H), 4.15 (t, J = 6.2 Hz, 2H), 3.90 (br s, 2H), 3.39 (t, J = 7.2 Hz, 2H), 2.37 (s,
13
3
H), 2.15 – 2.09 (m, 2H), 2.08 (s, 3H), 1.69 – 1.61 (m, 2H), 1.60 – 1.54 (m, 4H). C
NMR (151 MHz, DMSO) δ: 193.93, 161.72, 160.93, 153.49, 147.32, 126.65, 118.36,
14.14, 112.82, 101.42, 67.32, 52.76, 51.36, 33.25, 28.46, 24.03, 24.03,22.87, 15.39,
1
+
1
3.38. HRMS: calcd for C20
purity, 98.65%.
.2.3. 4-((3,4-dimethyl-2-oxo-2H-chromen-7-yl)oxy)butyl piperidine-1-carbodithioate
7c)
Yield 89%; white solid; m.p. 89-91 C; IR (KBr): ν = 2937, 2853, 1702, 1617, 1564,
H26NO
3
S
2
[M + H] 392.1276, found 392.1312. HPLC
5
(
o
-
1 1
1
7
502, 1426, 1296, 1241, 1156, 1090, 845, 759 cm . H NMR (600 MHz, CDCl
3
) δ:
.47 (d, J = 8.8 Hz, 1H), 6.83 (dd, J = 8.8, 2.5 Hz, 1H), 6.78 (d, J = 2.5 Hz, 1H), 4.30
(
t, J = 6.7 Hz, 2H), 4.04 (t, J = 5.9 Hz, 2H), 3.89 (br s, 2H), 3.39 (t, J = 7.1 Hz, 2H),
1
3
2
.36 (s, 3H), 2.17 (s, 3H), 2.02 – 1.84 (m, 4H), 1.76 – 1.60 (m, 6H). C NMR (151
MHz, CDCl
3
) δ:195.59, 162.53, 160.94, 153.56, 146.30, 125.22, 118.86, 114.14,
112.44, 101.14, 67.89, 52.93, 51.30, 36.67, 29.71, 28.28, 25.99, 25.56, 24.33, 15.09,
+
1
3.16. HRMS: calcd for C21
H28NO
3
S [M + H] 406.1432, found 406.1472. HPLC
2
purity, 99.71%.

5
.2.4.
5-((3,4-dimethyl-2-oxo-2H-chromen-7-yl)oxy)pentyl
piperidine-1-carbodithioate (7d)
o
Yield 86%; white solid; m.p. 91-92 C; IR (KBr): ν = 2938, 2853, 1719, 1606, 1504,
-
1 1
1
7
470, 1431, 1290, 1241, 1182, 1093, 859, 761 cm . H NMR (600 MHz, DMSO) δ:
.67 (d, J = 9.5 Hz, 1H), 6.92 (dd, J = 9.5, 2.6 Hz, 1H), 6.91 (d, J = 2.6 Hz, 1H), 4.20
(
br s, 2H), 4.05 (t, J = 6.4 Hz, 2H), 3.87 (br s, 2H), 3.25 (t, J = 7.3 Hz, 2H), 2.35 (s,
13
3
H), 2.06 (s, 3H), 1.80 – 1.71 (m, 2H), 1.71 – 1.59 (m, 4H), 1.60 – 1.46 (m, 6H). C
NMR (151 MHz, DMSO) δ: 194.39, 161.73, 161.17, 153.51, 147.33, 126.59, 118.23,
13.98, 112.76, 101.34, 68.44, 52.71, 51.27, 36.56, 28.65, 28.51, 25.30, 24.05, 24.05,
1
+
2
4
5
2.87, 15.37, 13.36. HRMS: calcd for C22
20.1608. HPLC purity, 97.71%.
H30NO
3
2
S [M + H] 420.1589, found
.2.5. 4-((2-oxo-2H-chromen-7-yl)oxy)butyl piperidine-1-carbodithioate (7e)
o
Yield 87%; white solid; m.p. 83-85 C; IR (KBr): ν = 2937, 2851, 1743, 1602, 1578,
-
1 1
1
7
500, 1480, 1283, 1234, 1148, 1079, 864, 752 cm . H NMR (600 MHz, CDCl
3
) δ:
.64 (d, J = 9.4 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 6.84 (dd, J = 8.6, 2.3 Hz, 1H), 6.80
(
d, J = 2.3 Hz, 1H), 6.24 (d, J = 9.4 Hz, 1H), 4.30 (br s, 2H), 4.07 (t, J = 6.0 Hz, 2H),
.90 (br s, J = 6.0 Hz, 2H), 3.40 (t, J = 7.1 Hz, 2H), 2.01 – 1.88 (m, 4H), 1.78 – 1.59
3
1
3
(
m, 6H). C NMR (151 MHz, CDCl
3
) δ: 195.50, 162.24, 161.27, 155.88, 143.48,
1
2
3
5
28.76, 112.98, 112.93, 112.48, 101.41, 68.04, 52.92, 51.30, 36.60, 29.70, 28.21,
+
6.00, 25.55, 24.32. HRMS: calcd for C19
78.1166. HPLC purity, 98.63%.
H24NO
3
S [M + H] 378.1119, found
2
.2.6. 4-((3-methyl-2-oxo-2H-chromen-7-yl)oxy)butyl piperidine-1-carbodithioate (7f)

o
Yield 80%; white solid; m.p. 105-107 C; IR (KBr): ν = 2934, 2862, 1716, 1620, 1570,
-
1 1
1
7
507, 1476, 1293, 1224, 1148, 1074, 849, 750 cm . H NMR (600 MHz, DMSO) δ:
.79 (s, 1H), 7.51 (d, J = 8.6 Hz, 1H), 6.94 (d, J = 2.2 Hz, 1H), 6.90 (dd, J = 8.6, 2.2
Hz, 1H), 4.21 (t, J = 6.4 Hz, 2H), 4.07 (t, J = 6.1 Hz, 2H), 3.87 (br s, 2H), 3.30 (t, J =
7
4
1
2
.0 Hz, 2H), 2.04 (s, 3H), 1.87 – 1.73 (m, 4H), 1.67 – 1.59 (m, 2H), 1.58 – 1.49 (m,
13
H). C NMR (151 MHz, DMSO) δ: 194.23, 162.06, 161.21, 154.81, 140.30, 128.94,
21.47, 113.27, 113.09, 101.33, 68.19, 52.75, 51.30, 36.30, 30.46, 28.13, 26.23, 25.65,
+
4.04, 16.94. HRMS: calcd for C20
H26NO
3
S
2
[M + H] 392.1276, found 392.1348.
HPLC purity, 99.32%.
.2.7.
piperidine-1-carbodithioate (7g)
Yield 89%; yellow solid; m.p. 119-121 C; IR (KBr): ν = 2941, 2854, 1730, 1600,
5
4-((3-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy)butyl
o
-
1
1
1
508, 1471, 1433, 1288, 1245, 1143, 1076, 830, 753 cm . H NMR (600 MHz,
CDCl ) δ: 7.51 (d, J = 8.9 Hz, 1H), 6.89 (dd, J = 8.9, 2.5 Hz, 1H), 6.81 (d, J = 2.5 Hz,
H), 4.30 (br s, 2H), 4.06 (t, J = 6.0 Hz, 2H), 3.89 (br s, 2H), 3.39 (t, J = 7.1 Hz, 2H),
3
1
2
1
5
13
.54 (s, 3H), 2.02 – 1.84 (m, 4H), 1.78 – 1.61 (m, 6H). C NMR (151 MHz, CDCl ) δ:
3
95.50, 161.98, 157.50, 153.13, 148.01, 125.85, 117.71, 113.33, 113.23, 101.33, 68.11,
2.91, 51.28, 36.58, 29.71, 28.19, 25.98, 25.56, 24.33, 16.19. HRMS: calcd for
+
C
20
H
25ClNO
3
S
2
[M + H] 426.0886, found 426.0934. HPLC purity, 99.02%.
5
.2.8.
4-((2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)oxy)butyl
piperidine-1-carbodithioate (7h)
Yield 84%; white solid; m.p. 111-113 C; IR (KBr): ν = 2937, 2860, 1736, 1610, 1514,
o

-
1 1
1
7
472, 1430, 1346, 1284, 1240, 1123, 1033, 852 cm . H NMR (600 MHz, CDCl
3
) δ:
.61 (d, J = 9.0 Hz, 1H), 6.92 (dd, J = 9.0, 2.5 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H), 6.61
(
s, 1H), 4.30 (br s, 2H), 4.09 (t, J = 6.1 Hz, 2H), 3.90 (br s, 2H), 3.40 (t, J = 7.2 Hz,
1
3
2
H), 2.05 – 1.86 (m, 4H), 1.79 – 1.61 (m, 6H). C NMR (151 MHz, CDCl
95.44, 162.96, 159.48, 156.35, 141.62 (q, J = 32 Hz), 126.32, 121.63 (q, J = 273Hz ),
13.73, 112.11, 106.97, 101.92, 68.25, 52.96, 51.29, 36.52, 29.71, 28.11, 25.94, 25.55,
3
) δ:
1
1
+
2
4.26. HRMS: calcd for C20
H
23
F
3
NO
3
S [M + H] 446.0993, found 446.1039. HPLC
2
purity, 99.21%.
5
.2.9. 4-((3-acetyl-2-oxo-2H-chromen-7-yl)oxy)butyl piperidine-1-carbodithioate (7i)
o
Yield 84%; yellow solid; m.p. 128-130 C; IR (KBr): ν = 2920, 2853, 1725, 1677,
-
1
1
1
613, 1503, 1471, 1279, 1214, 1142, 1036, 868, 770 cm . H NMR (600 MHz,
CDCl ) δ: 8.49 (s, 1H), 7.53 (d, J = 8.7 Hz, 1H), 6.89 (dd, J = 8.7, 2.3 Hz, 1H), 6.81
d, J = 2.3 Hz, 1H), 4.30 (br s, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.90 (br s, 2H), 3.40 (t, J
3
(
1
3
=
7.2 Hz, 2H), 2.71 (s, 3H), 2.04 – 1.86 (m, 4H), 1.80 – 1.53 (m, 6H). C NMR (151
MHz, CDCl
3
) δ: 195.58, 195.44, 164.71, 159.82, 157.79, 147.85, 131.50, 120.55,
114.21, 111.98, 100.80, 68.40, 52.96, 51.33, 36.49, 31.94, 30.61, 29.71, 28.10, 25.56,
+
2
4.32. HRMS: calcd for C21
H25NO
4
S [M + Na] 442.1025, found 442.1081. HPLC
2
purity, 98.97%.
5
3
.2.10. Ethyl 2-oxo-7-(4-((piperidine-1-carbonothioyl)thio)butoxy)-2H-chromene-
-carboxylate (7j)
o
Yield 87%; white solid; m.p. 113-114 C; IR (KBr): ν = 2927, 2856, 1748, 1692, 1615,
-1
1
1
550, 1500, 1485, 1286, 1223, 1143, 1043, 861, 795 cm . H NMR (600 MHz,

CDCl
3
) δ: 8.50 (s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 6.88 (dd, J = 8.7, 2.3 Hz, 1H), 6.80
d, J = 2.3 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 4.30 (br s, 2H), 4.10 (t, J = 6.1 Hz, 2H),
.90 (br s, 2H), 3.40 (t, J = 7.2 Hz, 2H), 2.02 – 1.86 (m, 4H), 1.78 – 1.62 (m, 6H),
(
3
1
1
3
13
.40 (t, J = 7.2 Hz, 3H). C NMR (151 MHz, CDCl
3
) δ: 195.43, 164.57, 163.50,
57.57, 157.21, 149.02, 130.70, 113.98, 111.57, 100.88, 68.35, 61.70, 52.96, 51.29,
6.51, 31.93, 29.70, 28.09, 25.97, 25.54, 24.32, 14.30. HRMS: calcd for C22
5 2
H28NO S
+
[
M + H] 450.1331, found 450.1407. HPLC purity, 98.64%.
5
.2.11. 4-((2-oxo-4-phenyl-2H-chromen-7-yl)oxy)butyl piperidine-1-carbodithioate
(7k)
o
Yield 81%; white solid; m.p. 115-116 C; IR (KBr): ν = 3075, 2934, 2849, 1715, 1602,
-
1 1
1
7
1
2
471, 1429, 1281, 1231, 1157, 1043, 860, 754 cm . H NMR (600 MHz, CDCl ) δ:
3
.53 – 7.49 (m, 3H), 7.46 – 7.42 (m, 2H), 7.37 (d, J = 8.9 Hz, 1H), 6.88 (d, J = 2.5 Hz,
H), 6.79 (dd, J = 8.9, 2.5 Hz, 1H), 6.21 (s, 1H), 4.30 (br s, 2H), 4.08 (t, J = 6.0 Hz,
H), 3.90 (br s, 2H), 3.40 (t, J = 7.1 Hz, 2H), 2.01 – 1.86 (m, 4H), 1.79 – 1.64 (m, 6H).
1
3
C NMR (151 MHz, CDCl
3
) δ: 195.52, 162.21, 161.30, 156.01, 155.86, 135.63,
1
5
29.58, 128.83,128.83, 128.40, 128.40,127.97, 112.68, 112.46, 111.80, 101.67, 68.06,
2.93, 51.29, 36.61, 29.71, 28.22, 26.00, 25.56, 24.33. HRMS: calcd for C25
3 2
H28NO S
+
[
M + H] 454.1432, found 454.1470. HPLC purity, 99.22%.
5
.2.12.
4-((4-oxo-1,2,3,4-tetrahydrocyclopenta[c]chromen-7-yl)oxy)butyl
piperidine-1-carbodithioate (7l)
Yield 81%; yellow solid; m.p. 85-87 C; IR (KBr): ν = 2930, 2864, 1707, 1609, 1470,
o
-1 1
1
432, 1283, 1233, 1159, 1035, 859, 762 cm . H NMR (600 MHz, DMSO) δ: 7.52 (d,