Design, synthesis and biological evaluation of multifunctional tacrine-curcumin hybrids as new cholinesterase inhibitors with metal ions-chelating and neuroprotective property

Article abstract of DOI:10.1016/j.bmc.2017.02.049

Total sixteen tacrine-curcumin hybrid compounds were designed and synthesized for the purpose of searching for multifunctional anti-Alzheimer agents. In vitro studies showed that these hybrid compounds showed good cholinesterase inhibitory activity. Particularly, the potency of K_3-2 is even beyond tacrine. Some of the compounds exhibited different selectivity on acetylcholinesterase or butyrylcholinesterase due to the structural difference. Thus, the structure and activity relationship is summarized and further discussed based on molecular modeling studies. The ORAC and MTT assays indicated that the hybrid compounds possessed pronounced antioxidant activity and could effectively protect PC12 cells from the H₂O₂/Aβ42-induced toxicity. Moreover, the hybrid compounds also showed positive metal ions-chelating ability in vitro, suggesting a potential to halt ion-induced Aβ aggregation. All the obtained results demonstrated that the tacrine-curcumin hybrid compounds, in particular compound K_3-2, can be considered as potential therapeutic agents for Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2017.02.049

Accepted Manuscript
Design, synthesis and biological evaluation of multifunctional tacrine-curcumin
hybrids as new cholinesterase inhibitors with metal ions-chelating and neuro-
protective property
Zhikun Liu, Lei fang, Huan Zhang, Shaohua Gou, Li Chen
PII:
S0968-0896(17)30015-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.049
BMC 13579
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 January 2017
22 February 2017
24 February 2017
Please cite this article as: Liu, Z., fang, L., Zhang, H., Gou, S., Chen, L., Design, synthesis and biological evaluation
of multifunctional tacrine-curcumin hybrids as new cholinesterase inhibitors with metal ions-chelating and
neuroprotective property, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.02.049
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Design,
synthesis
and
biological
evaluation
of
multifunctional
tacrine-curcumin hybrids as
new
cholinesterase inhibitors with metal ions-chelating and
neuroprotective property
Zhikun Liu†,‡, Lei fang‡,*, Huan Zhang†,‡, Shaohua Gou‡, Li Chen†,*
†Department of Natural Medicinal Chemistry, China Pharmaceutical University,
Nanjing 210009, PR China
‡Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of
Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR
China
ABSTRACT
Total sixteen tacrine-curcumin hybrid compounds were designed and synthesized
for the purpose of searching for multifunctional anti-Alzheimer agents. In vitro
studies showed that these hybrid compounds showed good cholinesterase inhibitory
activity. Particularly, the potency of K3-2 is even beyond tacrine. Some of the
compounds exhibited different
selectivity on acetylcholinesterase or
butyrylcholinesterase due to the structural difference. Thus, the structure and activity
relationship is summarized and further discussed based on molecular modeling studies.
The ORAC and MTT assays indicated that the hybrid compounds possessed
pronounced antioxidant activity and could effectively protect PC12 cells from the
H2O2/Aβ42-induced toxicity. Moreover, the hybrid compounds also showed positive
metal ions-chelating ability in vitro, suggesting a potential to halt ion-induced Aβ
aggregation. All the obtained results demonstrated that the tacrine-curcumin hybrid
compounds, in particular compound K3-2, can be considered as potential therapeutic
agents for Alzheimer’s disease.
KEY WORDS: Alzheimer's disease; tacrine-curcumin hybrids; cholinesterase
inhibitors; metal ions-chelating ability; neuroprotective property

1. Introduction:
Alzheimer’s disease (AD), a typical neurodegenerative brain disorder characteristic
of dementia, cognitive decline, severe behavioral disturbances and loss of basic
abilities in daily living, is established as the fourth leading cause of death in the
western countries following cardiovascular disease, cancer, and cerebrovascular
disease.[1,2] It is predicted that the number of the affected persons will reach 115.4
million worldwide by 2050.[3] Despite much attention paid to this disease, no curable
treatment was developed due to its multifactorial pathogenesis. Several hypotheses
based on its pathogenic factors have been put forward, including the decrease of
cholinergic neurotransmitter, amyloid beta (Aβ) accumulation, neurofibrillary tangles
(NFTs), oxidative stress, metal ions irregulation,[4,5] and so on. Among all of the
hypotheses, the cholinergic hypothesis has been successfully applied in the
development of cholinesterase inhibitors (ChEIs), since the cognitive and memory
disturbance is to some extent due to the decrease of acetylcholine (ACh). There is no
doubt that ChEIs can promote the level of synaptic ACh between synaptic nicotinic
and muscarinic receptors through inhibition of cholinesterases (ChEs),[6] but these
inhibitors cannot halt the progress of AD. Therefore only a palliative treatment effect
can be obtained.
Traditionally, the “one molecule, one target” paradigm, which is so-called
single-target molecule, can generally offer limited or transient benefits.[7] Facing the
multifactorial pathogenesis of AD, developing a molecule with multiple
pharmacological effects is a promising strategy in the research for new anti-AD drugs.
In our previous studies, we used tacrine (Figure 1), a potent acetylcholinesterase
inhibitor (AChEI) but suffering from serious hepatotoxicity and cholinergic side
effects,[8] as the lead compound to construct various hybrid molecules consisting of
two or more pharmacophores in one molecule. We found such hybrid compounds
could simultaneously target different pathogenic factors of AD and consequently
achieve improved therapeutic effects, suggesting that this may be a promising drug
design strategy.[9,10]

Curcumin (Figure 1), a well-known polyphenolic compound in Asian food
ingredient turmeric, was reported to have many important biological activities,
including anti-inflammatory, antimicrobial, and anti-carcinogenic activities.[11]
Encouragingly, substantial evidences also suggested its potential therapeutic effect on
AD through binding to Aβ oligomers/fibrils and altering Aβ aggregation, thus
reducing the Aβ-inducing neuron toxicity.[12,13] Besides, curcumin in monoanionic
form could be stabilized by chelating metal ions.[14] It was well literatured that the
imbalance of trace elements, such as copper (Cu2+) and iron (Fe2+) ions, made
amyloid precursor protein dysfunctional and increased the production of reactive
oxygen species (ROS) and oxidative stress which was to some extent related to the
progress of AD.[15-17] More importantly, some reported hybrids of curcumin or its
analogues conjugations showed multifunctional application in Alzheimer either in
detection or in therapy.[18-21] Taking these unique biochemical activities in
consideration, we tried to combine the pharmacophores of curcumin with tacrine to
form multi-target molecules, hoping such compounds could not only inhibit AChE,
but also eliminate excessive metal ions and ROS (Figure 1). To our interest, this
hypothesis was well proved in our studies.

Figure 1. Drug design rationale of the target compounds.
2. Results and discussion
2.1 Chemistry
The synthesis of F, L and K series was depicted in scheme 1. Tacrine and its
derivatives were prepared using reported method (Scheme 1).[22] To increase the yield
of compounds D, the silica gel chromatogram that was pretreated with concentrated
aqueous ammonia was applied for purification. The intermediate E was synthesized
through a condensation reaction of compounds D with the protected compound 1b,
which was further deprotected with p-toluenesulfonic acid monohydrate (PTSA) to
give the target compound F. The intermediate H was prepared with the compound E
through an aldehyde reaction in the presence of a catalyst amount of acitic acid, which
was further deprotected to give the series of L. The intermediate J was synthesized by
reducing compound H with hydrogen in the presence of Pd/C, which was further
treated with PTSA to obtain the final target compound K.

Scheme 1. The synthesis of the target compounds.
Reagents and conditions: (a) p-TosOH·H2O (cat), anhydrous toluene, reflux; (b) KOH,
dioxane/H2O, 35°C, 12h; (c) POCl3, reflux, 6 h; (d) pentanol, NH2(CH2)nNH2, reflux, 18 h; (e)
EDCI, HOBT, CH2Cl2, overnight, romm temp; (f) p-TosOH·H2O (cat), acetone-H2O, N2, 95°C,
overnight; (h) acitic acid (cat), morpholine (cat), CHCl3-toluene, reflux, overnight; (j) H2, Pd/C
(cat), ethanol, reflux, 8h; (k) p-TosOH·H2O (cat), acetone-H2O, N2, 95°C, overnight; (l)
p-TosOH·H2O (cat), acetone-H2O, N2, 95°C, overnight.
2.2 In vitro inhibition of AChE and BuChE
The decrease of synaptic ACh between central neurons in brain is one of the
distinguished characteristic of AD, thus inhibition of AChE to improve the level of

ACh is a useful method to relieve its symptoms. Notably, it is now well demonstrated
that butyrylcholinesterase (BuChE), which is primarily expressed and secreted by
glial cells,[23] plays an important compensatory role in response to the decrease of
AChE activity in the brain as AD progresses.[24] Thereby, inhibition of BuChE is also
supposed beneficial for the treatment of AD. In order to determine whether the target
compounds retained the AChE and BuChE inhibitory activities of tacrine, we
screened all the target compounds for the cholinesterase inhibition in vitro using
Ellman’s assay. From the result shown in Table 1, we can see that all the target
compounds showed potent inhibition of AChE with the IC50 values ranging from 0.08
to 9.64 µM. Clearly, the inhibitory activity of compound F3 and K3-2 was very potent,
slightly higher than that of positive drug tacrine. Taking the different enzyme sources
in consideration, some of the compounds exhibited different selectivity on
acetylcholinesterase or butycholinesterase due to the structural difference. However,
no clear rule of the inhibition activity was found between the two isoenzymes.
Analyzing the structure–activity relationship, we found that, among all of the three
series, the series F generally showed higher inhibitory activity against both AChE and
BuChE than the other two series. Comparing their structures, it was found that the
series F is lack of aromatic ring at the end of the side chain. The smaller structures
make them suitable for the accommodation to the catalytic gorge of AChE since the
catalytic pocket of AChE is relative narrow. Most of the target compounds showed
higher anti-BuChE activity than that of AChE (except compounds F3, K3, and L3 with
six-carbon long linker), which probably is due to the structural difference of the active
site of the enzymes. According to the former reports, AChE and BuChE differ in their
amino acid composition at the midgorge level. Because AChE presents several
aromatic residues that are replaced by smaller aliphatic ones in BuChE, BuChE has a
larger pocket with respect to AChE and allows the ligand with large steric hindrance
to approach the active site.[8,25] Interestingly, the compounds with six-carbon long
linker between tacrine and the unsaturated ketone (F3, K3-2, L3-2) showed better
anti-AChE activity than those with shorter linker. Many previous studies revealed that
six-carbon long linker is an optimal length which could allow the tacrine part of the

hybrid compounds smoothly enter the catalytic active site (CAS) while let the end of
the side chain locate at the lip of the gorge, interacting with the peripheral anionic site
(PAS) of AChE. Indeed our docking studies also supported the findings.
Table 1 The inhibitory activity against Torpedo Californica AChE and BChE
Compounds
IC50 ( µM) S.E.M.a
AChE
Selectivity ratiob
BuChE
F1
F2
0.46±0.09
0.24±0.05
0.09±0.01
0.57±0.1
8.76±2.1
5.71±1.7
1.99±0.8
3.68±1.3
0.13±0.9
0.08±0.03
9.64±1.2
1.26±0.5
3.80±1.6
2.10±0.9
2.95±1.4
0.29±0.1
0.10±0.07
0.31±0.02
0.06±0.01
0.16±0.01
0.22±0.02
0.83±1.6
1.31±1.2
0.39±2.4
3.38±1.5
0.20±1.3
0.22±0.1
1.60±1.1
1.87±0.4
4.77±1.9
2.11±0.4
2.24±1.7
1.50±0.5
1.5
3.9
0.4
2.6
10.6
4.3
5.1
1.1
0.7
0.4
6.0
0.7
0.8
1.0
1.3
0.2
2.1
F3
F4
K1-1
K1-2
K2-1
K2-2
K3-1
K3-2
L1-1
L1-2
L2-1
L2-2
L3-1
L3-2
Tacrine
0.05±0.01
b
aData is the mean of at least three determinations. Selectivity ratio = (IC50 of
AChE)/(IC50 of BChE)
2.3 Molecular modeling study
In order to investigate the interaction mode of the synthesized compounds with the
target enzymes, we further performed molecular modeling study. It is well known that
AChE has a dumbbell-shape active site gorge composed of two active sites: the CAS
at the bottom and the PAS at the lip. The CAS pocket looks like a vessel: on its
bottom lies Trp84 which is crucial for binding of both substrates and inhibitors.

Moreover, halfway up the gorge is a narrow tunnel where some crucial amino acid
residues such as Phe330 and Tyr334 contribute often to stabilization of an inhibitor or
substrate. At the entrance of the gorge the PAS exists, which is built up mainly by the
residues Trp279 and Tyr70. Based on the mentioned knowledge we found that
compound F3 which has a relative small side chain on the tacrine template could
smoothly enter the CAS pocket and interact with Trp84 and Phe330 through a π-π
stacking interaction (Figure 2, A). In contrast, the large steric hindrance of the side
chain of K1-1 significantly hindered the ligand to reach the bottom of the CAS pocket
and consequently increased the docking energy (Figure 2, B). However, the large
steric hindrance seems not a problem for BuChE since this enzyme has a larger active
pocket than AChE. The docking results showed that K1-1 could easily reach the active
site and interacted with Trp82 residue (Figure 2, C). Interestingly, when the length of
the linker is extended to six-carbon long, resulting in compound K3-2, the ligand can
be perfectly located in the gorge of AChE with the benzene ring binding to CAS and
the tacrine part bind to PAS. Besides, the ligand was further stabilized by an H-bond
formed between the carbonyl group and Tyr121 residue (Figure 2, D). This action
mode is quite similar to that of bis(7)tacrine. Given the ability to inhibit both CAS and
PAS, it may be understandable why K3-2 showed the most potent activity in the
enzyme assay.

B
A
C
D
Figure 2. (A) The interaction of F3 and AChE (Protein Data Bank code 2CKM); (B)
the interaction of K1-1 and AChE (Protein Data Bank code 2CKM); (C) the interaction
of K1-1 and BuChE (Protein Data Bank code 4BDS); (D) the interaction of K3-2 and
AChE (Protein Data Bank code 2CKM). The pictures were generated from MOE.
2.4 Antioxidant activity
Currently more and more attention is paid to oxidative stress for its role in the
progression of AD. There are many evidences suggesting that oxidative stress is
involved in the early stage of AD and represents a key factor to initiate the
aggregation of β-amyloid and tau protein hyperphosphorylation. Accordingly,
antioxidants were observed to be able to attenuate the syndrome of AD and prevent
the progression of the disease. In order to prove the antioxidant capacities of the
hybrid molecules, the ability of target compounds (F3, L3-1, L3-2, K3-1, K3-2) to reduce
peroxyl radicals was determined by the ORAC (oxygen radical absorbance capacity)

assay.[26] Curcumin was selected as the positive control, and tacrine was also tested as
a negative standard. The results (Table 2) are expressed as Trolox-equivalent
antioxidant capacity (TEAC), that is, their relative ability (at concentrations between
0.1 and 10 µM) compared to the highly potent compound Trolox. The results
indicated that curcumin was a potent radical scavenger of peroxyl radicals, with a
TEAC value of 3.1. As for the hybrid compounds, L3-1, L3-2, K3-1, and K3-2 showed
potent ability to absorb the ROS with the TEAC values around 2, while F3 exhibited
only a weak activity. From the chemical point of view, the antioxidant property of
curcumin probably results from the enol and phenolic hydroxyl groups. Compounds
L3-1 and L3-2 showed weaker radical scavenging activity than K3-1 and K3-2,
respectively. The reason may be that L3-1 and L3-2 lack one enol hydroxyl group due to
the existence of the carbon-carbon double bond between the two carbonyl groups. The
phenolic hydroxyl group clearly plays an important role in the antioxidant activity.
Compound F3 without phenolic hydroxyl group showed a very weak activity with a
TEAC value of 0.4, only a little higher than that of tacrine.
Table 2. Oxygen radical absorbance capacity of tacrine and the hybrid compounds
expressed as TEAC.
Compound
F3
TEAC
0.4
SD
0.02
0.1
L3-1
1.8
L3-2
2.0
0.1
K3-1
2.2
0.05
0.08
0.02
0.2
K3-2
2.4
0.3
tacrine
Curcumin
3.1
2.5 Neuron protective effect
Curcumin is well known for its neuroprotective effect, which probably results from
the Aβ regulation and antioxidant property.[13,14] Given the newly synthesized

compounds also showed pronounced ROS absorbance capacity, it may be interesting
to investigate whether these hybrid compounds could protect neurons from the
exogenous toxins. Thus, using H2O2 or Aβ42 as the toxins, the neuroprotective effect
of compound K3-2 was determined in vitro by MTT assay. Curcumin was used as the
positive control. The results were shown in Figure 3. No toxicity was observed when
PC12 cells were cocultured with K3-2 only. In contrast, H2O2 (200 µM) or Aβ42 (5 µM)
caused a significant toxicity to PC12 cells (Figure 3, C1 and C2) as the viability of the
cells dropped from 1.0 to 0.40 and 0.58, respectively. Interestingly, when PC12 cells
were co-treated with compound K3-2 (0.1, 1, 5, 10 µM), the induced toxicity by the
exogenous toxins was significantly alleviated in a dose-dependent manner (Figure 3,
C3 and C4). As compared with curcumin, K3-2 showed comparable protective effect
against the H2O2-induced toxicity. Such activity probably can be attributed to the ROS
absorbance capacity of the substances. However, curcumin exhibited only a slight
protective effect in the assays employing Aβ42 as the exogenous toxin, much weaker
than that of K3-2. Different from the oxidative damage of H2O2, the toxicity caused by
Aβ42 is more complex, which includes the generation of abnormally high
concentration of ROS, activating the release of toxic cytokines, and causing
mitochondrial dysfunctions.[27] Clearly the hybrid compound showed a broader
protection spectrum than curcumin.

A
100
50
0
B
100
50
0

C2
C1
#
Figure 3. (A) The neuroprotective effect of K3-2 against H2O2-induced toxicity. p
<0.01 vs control, **p <0.01 vs H2O2 (200 µM), n = 3. (B) The neuroprotective effect

#
of K3-2 against Aβ42-induced toxicity. p <0.01 vs control, **p <0.01 vs Aβ (5 µM), n
= 3. (C1), picture of the cells after culture with H2O2 (200 µM) for 24h; (C2), picture
of the cells after culture with Aβ42 (5 µM) for 24h; (C3), picture of the cells after
culture with H2O2 (200 µM)+ K3-2 (10 µM) for 24h; (C4), picture of the cells after
culture with Aβ42 (5 µM) + K3-2 (10 µM) for 24h.
2.6 The ability of binding to ion
Metal ion dysfunction is a well-recognized cofactor in several neurodegenerative
disorders. The endogenous ion, such as Cu2+ and Fe2+, attracted great interest of
researchers investigating the etiology and the pathogenesis of AD for many years.[4,5]
Many evidence demonstrated that the misfolding progress associated with Aβ
accumulation was greatly influenced by ions (i.e. Cu2+, Fe2+, Zn2+ etc.).[17] Thus,
developing an agent that can eliminate excessive ions would be helpful for halting the
progress of AD. Considering the great ion-chelating ability of the β-diketone structure,
we further selected three compounds (i.e. F3, K3-2, L3-2) with the β-diketone structure
to test the ion-chelating ability using a UV spectroscopy method. From the results
showed in Figure 4, we could find that the absorbance of compound F3 evidently
increased due to the binding to Cu2+ or Fe2+ at the wavelength of 225, 250, 275, 335
and 355 nm with the increase of the ion concentration. The same performance was
also found for compound K3-1 at the same wavelengths. As for the compound L3-2 the
absorbance changed greatly at 275 nm and 375 nm when treated with Cu2+ or Fe2+.
According to former reports, the increase of the absorbance caused by the addition of
Cu2+/Fe2+ is probably due to the formation of a better coplanarity and larger rigid
π-conjugation system between curcumin derivatives (F3, K3-1, L3-2 ) and the ions.[28-30]
Thus, based on the obtained results we may assume that the hybrid compounds tested
in this study had great affinity to ions, which may be helpful for halting the
ions-induced Aβ accumulation and consequently beneficial for prevention of the
progress of AD.

Figure 4. The ions binding ability of F3, K3-2, and L3-2, expressed as the increase of
the absorbance with the concentration of Cu2+/Fe2+ rising from 2 to 50 µM (in detail:
2 µM，5 µM，10 µM，15 µM，20 µM，25 µM，30 µM，35 µM，40 µM，45 µM，
50µM).
3. Conclusion

In a conclusion, a series of tacrine and curcumin hybrids were designed and
synthesized with the hypothesis to obtain multifunctional compounds that could
simultaneously targets the ChE and Aβ pathways of AD. In Ellman’s assays, all of the
synthesized compounds showed positive inhibitory effects both on AChE and BuChE.
Particularly, the potency of K3-2 against AChE is even higher than that of tacrine,
which is probably because K3-2 is a dual inhibitor towards both CAS and PAS of
AChE. Furthermore, the hybrid compounds also exhibited pronounced oxygen radical
absorbance capacity duo to the introduction of curcumin part. In MTT assays, the
target compounds could effectively protect PC12 cells from H2O2- or Aβ42-induced
toxicity with a higher potency than curcumin. Moreover, the β-diketone moiety of the
target compounds also endowed them with remarkable ions-chelating ability,
suggesting a potential of the hybrid compound to halt ion-induced Aβ aggregation.
Altogether, the obtained results suggest that the tacrine-curcumin hybrid compounds,
in particular compound K3-2, can be considered as potential therapeutic agents for
AD.
4. Experimental section
4.1 Chemistry
4.1.1. General Chemical Methods.
All of the reagents and solvents used were purchased from Sinopharm Chemical
Reagent Co. The course of the reaction was monitored by thin layer chromatography
(TLC) on glass plates coated with silica gel 60 GF254 from Qingdao Haiyang
Chemical Co. Ltd. (China) with a UV lamp (254 nm). Melting points were determined
using a capillary apparatus (RDCSY-I). IR spectra were measured on KBr pellets with
a Nicolet IR200 FTIR spectrometer. All compounds synthesized were purified by
column chromatography using silica gel 60 (200-300 mesh), and confirmed by a
Bruker 300/500 MHz spectrometer and high resolution mass spectrum (Angilent
technologies LC/MSD TOF). The purity of individual compound used for the other
experiment was above 95%.
4.1.2. The procedure for the preparation of compound 1a.

To a mixture of ethyl acetoacetate (39.0 g, 0.3 mol) and ethylene glycol (55.8 g, 0.9
mol) in 200 ml toluene was added p-toluenesulfonic acid monohydrate (0.2 g, cat) and
the resulted solution was heated to reflux with water separator. The solution was
evaporated under reduced pressure after the completion of the reaction to gather the
fraction at 96-99°C and got 47.6 g colorless oil. Yield 91% .IR (KBr, cm-1): 2984,
2890, 1736, 1370, 1049
4.1.3. The procedure for the preparation of compound 1b.
To a mixture of potassium hydroxide (21.3g, 0.38mol) in 200ml distilled water,
was added 1a (17.4 g, 0.1 mol ) in 50 ml 1,4-dioxane and stirred at 35°C overnight.
The reaction solution was evaporated under reduced pressure to 100 ml after the
completion of the reaction and extracted with dichloromethane (100 ml ×2). The pH
value of the water layer was adjusted to 2 with 2M HCl and extracted with ether (200
ml ×3). The organic layer was dried with anhydrous sodium sulfate and evaporated
under reduced pressure to get the 6.9 g colorless oil target product. Yield 47%.
ESI-MS: m/z 145.0 [M-H]-; IR (KBr, cm-1): 3191, 2989, 2895, 1715, 1381.
4.1.4. General procedure A for the preparation of series E1-E4.
To a mixture of 1b in 20 ml anhydrous dichloromethane was added 1.2 equiv
amount of N-Ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI)
and 1.2 equiv amount of 1-Hydroxybenzotriazole(HOBT), then stirred for 1h. Then
equal equiv amount of compound D in 30 ml anhydrous dichloromethane was added
to the reaction solution and stirred overnight. After the completion of the reaction, 30
ml distilled water was added to the solution and pH value was adjusted to 8-9 with
saturated Na2CO3 solution. The organic layer was dried with anhydrous sodium
sulfate and evaporated under reduced pressure to get the crude product which was
further purified by column chromatography (CH2Cl2:MeOH=20:1).
4.1.4.1. The procedure for the preparation of compound E1.
White solid. Yield 47%. mp 123-125°C. ESI-MS: m/z 384.2 [M+H]+; IR (KBr,
1
cm-1): 3312, 3187, 2980, 1657, 1575, 1512, 1497, 1424, 1369, 1130, 1073; H NMR
(CDCl3, 300 MHz), δ (ppm): 8.01 (d, 1H, J = 7.14 Hz, arom), 7.93 (d, 1H, J = 7.93
Hz, arom), 7.54 (t, 1H, J = 7.60 Hz, arom), 7.35 (t, 1H, J = 7.60 Hz, arom), 3.99-3.89
(m, 4H, OCH2CH2O), 3.51 (m, 2H, NHCH2), 3.42 (m, 2H, CH2NHCO), 3.07 (br, 2H,
C4-H2), 2.77 (br, 2H, C1-H2), 2.62 (s, 2H, COCH2), 1.92 (br, 4H, C2-H2, C3-H2), 1.80
(m, 2H, NHCH2CH2), 1.40 (s, 3H, CH3).

4.1.4.2. The procedure for the preparation of compound E2.
White solid. Yield 50%. mp 104-106°C; ESI-MS: m/z 398.2 [M+H]+; IR (KBr,
cm-1): 3317, 3061, 2933, 1652, 1560, 1500, 1420, 1377, 1132, 1045; 1H NMR (CDCl3,
300 MHz), δ (ppm): 7.95-7.90 (m, 2H, arom), 7.55 (t, 1H, J = 7.61 Hz, arom), 7.33 (t,
1H, J = 7.61 Hz, arom), 3.96-3.90 (m, 4H, OCH2CH2O), 3.51 (t, 2H, J = 7.05 Hz,
NHCH2), 3.29 (m, 2H, CH2NHCO), 3.07 (br, 2H, C4-H2), 2.72 (br, 2H, C1-H2), 2.62
(s, 2H, COCH2), 1.93-1.91 (br, 4H, C2-H2, C3-H2), 1.69-1.60 (m, 4H, NHCH2CH2 ×
2), 1.44 (s, 3H, CH3).
4.1.4.3. The procedure for the preparation of compound E3 .
White solid. Yield 42%. mp134-137°C; ESI-MS: m/z 426.2 [M+H]+; IR (KBr,
1
cm-1): 3370, 3183, 2936, 1666, 1579, 1561, 1501, 1435, 1407, 1379, 1198, 1045; H
NMR (CDCl3, 300 MHz), δ (ppm): 7.95-7.90 (m, 2H, arom), 7.55 (t, 1H, J = 7.59 Hz,
arom), 7.35 (t, 1H, J = 7.59 Hz, arom), 3.96-3.88 (m, 4H, OCH2CH2O), 3.50 (t, 2H, J
= 7.06 Hz, NHCH2), 3.28 (m, 2H, CH2NHCO), 3.06 (br, 2H, C4-H2), 2.71 (br, 2H,
C1-H2), 2.56 (s, 2H, COCH2), 1.93-1.90 (br, 4H, C2-H2, C3-H2), 1.71-1.60 (m, 4H,
NHCH2CH2 × 2), 1.44 (br, 4H, NHCH2CH2CH2 × 2), 1.38 (s, 3H, CH3).
4.1.4.4 The procedure for the preparation of compound E4.
Whit solid. Yield 38%. m.p.63-66°C; ESI-MS: m/z 370.2 [M+H]+; IR (KBr,
cm-1): 3345, 3055, 2932, 1655, 1560, 1501, 1418, 1375, 1133, 1042; 1H NMR (CDCl3,
300 MHz), δ (ppm): 7.95(d, 1H, J = 7.13 Hz, arom), 7.84 (t, 1H, J = 7.60 Hz, arom),
7.50 (t, 1H, J = 7.60 Hz, arom), 7.27 (d, 1H, J = 7.92 Hz, arom), 3.94 (br, 4H,
OCH2CH2O), 3.62 (br, 2H, NHCH2), 3.28 (m, 2H, CH2NHCO), 3.04 (br, 2H, C4-H2),
2.71 (br, 2H, C1-H2), 1.85 (br, 4H, C2-H2, C3-H2), 1.35 (s, 3H, CH3).
4.1.5. General procedure B for the preparation of series F1 –F4.
To a solution of compound E in pure water and acetone (1:1,v/v) was added two
equiv amount of PTSA and stirred at 95°C overnight under the protection of nitrogen.
The solvent was removed in vacuum after the completion of the reaction. The pH of
the reaction was justified to 7-8 with saturated Na2CO3 solution and extracted with
dichloromethane. The organic layer was dried with anhydrous sodium sulfate and
evaporated under reduced pressure to get the crude product F which was further

purified by column chromatography (CH2Cl2:MeOH=20:1).
4.1.5. 1. The procedure for the preparation of compound F1.
White solid. Yield 91%. mp 157-160°C. IR (KBr, cm-1): 3264, 3060, 2926, 2857,
1720, 1651, 1562, 1502, 1412; 1H NMR (CDCl3, 300MHz), δ (ppm): 8,04 (d, 1H, J =
8.34 Hz, arom), 7.89 (d, 1H, J = 8.19 Hz, arom), 7.53 (t, 1H, J = 7.59 Hz, arom), 7.32
(t, 1H, J = 7.59 Hz, arom), 4.33 (s, 1H, CH=C), 3.49 (t, 2H, J = 6.06 Hz, NHCH2),
3.41 (m, 2H, CH2NHCO), 3.04 (br, 2H, C4-H2), 2.78 (br, 2H, C1-H2), 1.91-1.89 (br,
4H, C2-H2, C3-H2), 1.85 (s, 3H, CH3), 1.78 (m, 2H, NHCH2CH2); 13C NMR (CDCl3,
75 MHz), δ (ppm): 173.6, 160.9, 159.2, 153.4, 150.1, 131.0,126.3, 125.2, 122.9, 119.1,
118.9, 88.9, 48.2, 38.8, 36.4, 34.6, 33.9, 32.4, 27.6, 25.3. HRMS (ESI) m/z Calcd for
C19H24N3O2 [M+H]+ 326.18630 found 326.18644.
4.1.5. 2. The procedure for the preparation of compound F2.
White solid. Yield 93%. mp 79-82°C; IR (KBr, cm-1): 3523, 3394, 3198, 2920,
1
1719, 1629, 1556, 1505, 1435; H NMR (CDCl3, 300 MHz), δ (ppm): 7.95-7.89 (m,
2H, arom), 7.54 (t, 1H, J = 7.36 Hz, arom), 7.32 (t, 1H, J = 7.36 Hz, arom), 4.30 (s,
1H, CH=C), 3.49 (t, 2H, J = 6.59 Hz, NHCH2), 3.28 (m, 2H, CH2NHCO), 3.05 (br,
2H, C4-H2), 2.71 (br, 2H, C1-H2), 1.91 (br, 4H, C2-H2, C3-H2), 1.83 (s, 3H, CH3),
1.71-1.57 (m, 4H, NHCH2CH2 × 2); 13C NMR (CDCl3, 300 MHz), δ (ppm): 173.6,
161.1, 159.2, 153.6, 149.6, 130.9,126.4, 125.4, 122.9, 119.1, 118.9, 88.9, 48.0, 38.4,
36.5, 34.6, 33.9, 32.3, 27.7, 25.7, 25.3. HRMS (ESI) m/z Calcd for C20H26N3O2
[M+H]+ 340.20195 found 340.20203.
4.1.5.3. The procedure for the preparation of compound F3.
White solid. Yield 89%. mp 87-89°C; IR (KBr, cm-1): 3368, 2929, 2850, 1716,
1671, 1577, 1560, 1443; 1H NMR (CDCl3, 300 MHz), δ (ppm): 7.96-7.91 (m, 2H,
arom), 7.55 (t, 1H, J = 7.35 Hz, arom), 7.35 (t, 1H, J = 7.35 Hz, arom), 3.49(t, 2H, J =
7.18 Hz, NHCH2), 3.40 (s, 2H, CH2), 3.26 (m, 2H, CH2NHCO), 3.07 (br, 2H, C4-H2),
2.71 (br, 2H, C1-H2), 2.25 (s, 3H, CH3), 1.93-1.91 (m, 4H, C2-H2, C3-H2), 1.71-1.62
(m, 2H, NHCH2CH2), 1.57-1.47 (m, 2H, CH2CH2NHCO), 1.39-1.33 (m, 4H,
NHCH2CH2CH2 × 2); 13C NMR (CDCl3, 300 MHz), δ (ppm): 204.5, 164.8, 157.7,
150.3, 146.6, 127.9, 123.1, 122.3, 119.5, 115.2, 48.8, 38.8, 33.3, 31.1, 30.6, 28.8, 26.0,

24.2, 22.5, 22.2. HRMS (ESI) m/z Calcd for C21H28N3O2 [M+H]+ 354.21760 found
354.21771.
4.1.5.4. The procedure for the preparation of compound F4.
White solid. Yield 78%. mp 69-72°C; IR (KBr, cm-1): 3295, 2926, 2856, 1633,
1
1560, 1501, 1419; H NMR (CDCl3, 300 MHz), δ (ppm): 7.98 (d, 1H, J = 8.46 Hz),
7.88 (d, 1H, J = 8.66 Hz), 7.52 (t, 1H, J = 7.54 Hz), 7.29 (t, 1H, J = 7.54 Hz), 3.62 (br,
2H), 3.54 (s, 2H), 3.28 (br, 2H), 3.04 (br, 2H), 2.71 (br, 2H), 1.96 (s, 3H), 1.85 (br,
4H). HRMS (ESI) m/z Calcd for C23H32N3O2 [M+H]+ 382.24890 found 382.24877.
4.1.6. General procedure C for the preparation of series H.
To a solution of compound E in 5ml chloroform and 30 ml toluene was added two
eqiv p-hydroxybenzaldehyde or vanillin and acetic acid (cat.) and morphine (cat.).
Then, the solution was heated to reflux overnight with water separator and monitored
by TLC. After the completion of the reaction, the solution was filtered and the residue
was purified on silica gel column eluted with ethyl acetate and dichloromethane to
give the target product.
4.1.6.1. The procedure for the preparation of compound H1-1.
Orange-red solid. Yield 25%. mp 75-77°C; ESI-MS: m/z 488.2 [M+H]+; IR (KBr,
1
cm-1): 3355, 2932, 1732, 1649, 1605, 1573, 1509, 1438, 1376, 1244, 1169, 1044; H
NMR (CDCl3, 300 MHz), δ (ppm): 8.07-7.99 (m, 2H, arom), 7.94 (br, 1H, NH), 7.55
(t, 1H, J = 7.38 Hz, arom), 7.35 (t, 1H, J = 7.38 Hz, arom), 7.25 (d, 2H, J = 8.40 Hz,
arom), 6.82 (d, 2H, J = 8.40 Hz, arom), 6.68 (s, 1H, C=CH), 4.93 (br, 1H, NH), 3.94
(m, 4H, OCH2CH2O), 3.47-3.40 (m, 4H, NHCH2, CH2NHCO), 2.85-2.79 (m, 4H,
C1-H2, C4-H2), 2.63 (br, 2H, NHCH2CH2), 1.84-1.77 (m, 4H, C2-H2, C3-H2), 1.40 (s,
3H, CH3); 13C NMR (CDCl3, 75 MHz), δ (ppm): 170.0, 155.7, 154.3, 150.6, 146.9,
133.8, 131.2, 129.7, 129.5, 128.8, 128.6, 124.2, 122.1, 120.6, 117.3, 115.3, 107.8,
64.6, 46.3, 45.4, 36.6, 31.3, 27.9, 25.7, 23.8, 23.0.
4.1.6.2. The procedure for the preparation of compound H1-2
.
Yellow solid. Yield 15%. mp 134-136°C; ESI-MS: m/z 518.2 [M+H]+; IR (KBr,
cm-1): 3357, 2938, 1733, 1663, 1575, 1551, 1510, 1464, 1423, 1370, 1337, 1267, 1145,
1
1040; H NMR (DMSO-d6, 300 MHz), δ (ppm): 9.13 (br, 1H, NH), 8.18 (m, 1H, J =

8.19 Hz, arom), 8.04 (s, 1H, C=CH), 7.82 (m, 1H, J = 8.40 Hz, arom), 7.73 (br, 1H,
NH), 7.56 (t, 1H, J = 7.41 Hz, arom), 7.36 (t, 1H, J = 7.41 Hz, arom), 7.04 (s, 1H,
arom), 6.95 (d, 1H, J = 8.14 Hz, arom), 6.83 (d, 1H, J = 8.14 Hz, arom), 5.62 (br, 1H,
OH), 4.93 (br, 1H, NH), 3.83 (br, 4H, OCH2CH2O), 3.82 (s, 3H, OCH3), 3.36 (m, 2H,
NHCH2), 3.11 (m, 2H, CH2NHCO), 2.90 (br, 2H, C4-H2), 2.82 (br, 2H, C1-H2), 2.37
(br, 2H, NHCH2CH2), 1.77 (br, 2H, C3-H2), 1.65 (m, 2H, C2-H2), 1.35 (s, 3H, CH3);
13C NMR (DMSO-d6, 75 MHz), δ (ppm): 168.3, 153.2, 150.2, 147.2, 146.7, 145.9,
133.8, 129.0, 128.7, 128.1, 127.7, 123.6, 122.5, 122.3, 120.7, 117.1, 115.3, 113.9,
107.4, 63.8, 55.6, 45.0, 39.2, 35.9, 30.4, 27.8, 25.9, 24.1, 22.6.
4.1.6.3. The procedure for the preparation of compound H2-1
.
Orange-red solid. Yield 21%. mp 71-74°C; ESI-MS: m/z 502.2 [M+H]+; IR (KBr,
1
cm-1): 3369, 3177, 2986, 2888, 1668, 1626, 1438, 1407, 1378, 1269, 1046; H NMR
(CDCl3, 300 MHz), δ (ppm): 8.02 (br, 1H, arom), 7.94 (d, 1H, J = 8.31 Hz, arom),
7.60 (t, 1H, J = 7.41 Hz, arom), 7.39 (t, 1H, J = 7.41 Hz, arom), 7.30 (d, 2H, J = 7.77
Hz, arom), 6.89 (d, 2H, J = 7.77 Hz, arom), 6.60 (s, 1H, C=CH), 3.96 (m, 4H,
OCH2CH2O), 3.51 (br, 2H, NHCH2), 3.33 (m, 2H, CH2NHCO), 2.89-2.81 (m, 4H,
C4-H2, C1-H2), 2.09 (m, 2H, C3-H2), 1.89 (m, 2H, C2-H2), 1.68-1.67 (m, 4H,
NHCH2CH2 × 2), 1.42 (s, 3H, CH3).
4.1.6.4. The procedure for the preparation of compound H2-2
.
Orange-red solid. Yield 14%. mp 71-74°C; ESI-MS: m/z 532.2 [M+H]+; IR (KBr,
1
cm-1): 3334, 3068, 2926, 1668, 1577, 1508, 1464, 1394, 1323, 1126, 1029; H NMR
(CDCl3, 300 MHz), δ (ppm): 9.82 (br, 1H, NH), 8.09 (br, 1H, NH), 8.03 (d, 1H, J =
8.34Hz, arom), 7.90 (d, 1H, J = 8.52Hz, arom), 7.57 (t, 1H, J = 7.43 Hz, arom), 7.41
(d, 2H, J = 7.53 Hz, arom), 7.36 (t, 1H, J = 7.43 Hz, arom), 7.04 (s, 1H, arom), 7.01
(d, 2H, J = 7.53 Hz, arom), 6.94 (s, 1H, C=CH), 6.43 (br, 1H, OH), 3.95 (br, 4H,
OCH2CH2O), 3.90 (s, 3H, OCH3), 3.46 (t, 2H, J = 6.66 Hz, NHCH2), 3.29 (m, 2H,
CH2NHCO), 2.93 (br, 2H, C4-H2), 2.83 (m, 2H, C1-H2), 2.01 (m, 2H, C3-H2), 1.91
(m, 2H, C2-H2), 1.69-1.61 (m, 4H, NHCH2CH2 × 2), 1.37 (s, 3H, CH3); 13C NMR
(CDCl3, 300 MHz), δ (ppm): 169.0, 154.0, 151.8, 150.2, 146.1, 144.8, 134.7, 130.3,
129.6, 129.0, 128.4, 127.4, 124.2, 123.0, 121.8, 117.5, 114.1, 112.7, 108.8, 64.5, 55.9,

48.8, 46.3, 38.9, 28.8, 27.9, 27.2, 25.7, 23.7, 23.0.
4.1.6.5. The procedure for the preparation of compound H3-1
.
Orange-red solid. Yield 24%. mp 63-65°C; ESI-MS: m/z 530.3 [M+H]+; IR (KBr,
1
cm-1): 3362, 2929, 2857, 1732, 1649, 1605, 1573, 1508, 1435, 1375, 1242, 1043; H
NMR (DMSO-d6, 300 MHz), δ (ppm): 9.58 (br, 1H, NH), 8.15 (d, 1H, J = 8.43 Hz,
arom), 8.02 (s, 1H, C=CH), 7.81 (d, 1H, J = 8.10 Hz, arom), 7.57-7.55 (m, 2H, arom),
7.33 (d, 2H, J = 8.58 Hz, arom), 6.82 (d, 2H, J = 8.58 Hz, arom), 5.55 (br, 1H, OH),
3.83 (br, 4H, OCH2CH2O), 3.33 (m, 2H, NHCH2), 2.98 (m, 2H, CH2NHCO),
2.86-2.81 (m, 4H, C1-H2, C4-H2), 1.76 (br, 2H, C3-H2), 1.52 (br, 2H, C2-H2), 1.33 (s,
3H, CH3), 1.25 (br, 8H, HNCH2CH2CH2 × 2); 13C NMR (DMSO, 300 MHz), δ (ppm):
167.7, 156.5, 153.2, 150.4, 146.7, 133.6, 130.9, 129.0, 128.2, 128.1, 127.3, 123.5,
122.5, 120.6, 116.9, 115.1, 107.5, 63.8, 47.8, 45.0, 40.0, 38.9, 38.2, 30.4, 28.9, 27.8,
26.0, 24.1, 22.6.
4.1.6.6. The procedure for the preparation of compound H3-2
.
Orange-red solid. Yield 27%. mp 115-117°C; ESI-MS: m/z 560.3 [M+H]+; IR
(KBr, cm-1): 3310, 2931, 2857,1670, 1647, 1578, 1512, 1465, 1424, 1306, 1262, 1124,
1031; 1H NMR (CDCl3, 300 MHz), δ (ppm): 9.82 (br, 1H, NH), 8.05 (d, 1H, J = 7.77
Hz, arom), 7.92 (d, 1H, J = 8.28 Hz, arom), 7.57 (t, 1H, J = 7.54 Hz, arom), 7.41 (t,
1H, J = 7.54 Hz, arom), 7.26 (s, 1H, arom), 7.02-7.00 (m, 2H, arom), 6.94 (s, 1H,
C=CH), 6.40 (br, 1H, OH), 3.95 (br, 4H, OCH2CH2O), 3.89 (s, 3H, OCH3), 3.45 (t,
2H, J = 7.13 Hz, NHCH2), 3.24 (m, 2H, CH2NHCO), 2.92 (br, 2H, C4-H2), 2.82 (m,
2H, C1-H2), 2.16 (s, 3H, CH3), 1.92 (m, 2H, C3-H2), 1.66 (m, 2H, C2-H2), 1.53-1.39
(br, 8H, HNCH2CH2CH2 × 2); 13C NMR (CDCl3, 300 MHz), δ (ppm): 168.9, 151.8,
147.2, 146.1, 144.8, 130.3, 129.1, 128.5, 127.4, 124.1, 123.0, 122.0, 114.4, 114.0,
112.8, 108.8, 107.9, 64.6, 56.0, 55.9, 49.2, 46.3, 39.1, 31.4, 29.5, 27.9, 26.6, 25.6,
23.7, 23.0.
4.1.7. General procedure D for the preparation of series J.
To a solution of the compound H in 30 ml anhydrous ethanol was added Pd/C (5%)
and heated to reflux. Then the solution was bubbled with hydrogen for 8h and
monitored by TLC. The solution was filtered to remove the residue after the

completion of the reaction. The solvent was removed under reduced pressure to obtain
the target product J.
4.1.7. 1. The procedure for the preparation of compound J1-1.
Brown viscous product. Yield 98%. ESI-MS: m/z 490.2 [M+H]+; IR (KBr, cm-1):
3353, 2933, 1649, 1613, 1562, 1513, 1446, 1377, 1260, 1044; 1H NMR (DMSO, 300
MHz), δ (ppm): 8.15 (d, 1H, J = 7.07 Hz, arom), 7.80 (d, 1H, J = 8.27 Hz, arom), 7.73
(br, 1H, NH), 7.55 (t, 1H, J = 7.48 Hz, arom), 7.36 (t, 1H, J = 7.48 Hz, arom), 7.01 (d,
2H, J = 7.98 Hz, arom), 6.67 (d, 2H, J = 7.98 Hz, arom), 5.53 (br, 1H, NH), 4.33 (br,
1H, OH), 3.83 (br, 4H, OCH2CH2O), 3.44 (m, 2H, NHCH2), 3.41 (m, 2H,
CH2NHCO), 3.31 (t, 1H, J = 7.26 Hz, CH), 3.11 (m, 2H, C4-H2), 2.69 (br, 2H, C1-H2),
2.37 (d, 2H, J = 7.26 Hz, CH-CH2), 1.70-1.51 (m, 6H, C2-H2, C3-H2, NHCH2CH2),
1.35 (s, 3H, CH3).
4.1.7. 2. The procedure for the preparation of compound J1-2.
Brown viscous product. Yield 98%. ESI-MS: m/z 520.2 [M+H]+; IR (KBr, cm-1):
1
3360, 2934, 1650, 1562, 1515, 1449, 1377, 1272, 1041; H NMR (DMSO-d6, 300
MHz), δ (ppm): 8.63 (br, 1H, NH), 8.16 (d, 1H, J = 7.95 Hz, arom), 7.74 (br, 1H, NH),
7.82 (d, 1H, J = 8.01 Hz, arom), 7.56 (t, 1H, J = 7.25 Hz, arom), 7.38 (t, 1H, J = 7.25
Hz, arom), 6.72 (s, 1H, arom), 6.67 (d, 1H, J = 7.71 Hz, arom), 6.60 (d, 1H, J = 7.71
Hz, arom), 5.54 (br, 1H, OH), 3.83 (m, 4H, OCH2CH2O), 3.66 (br, 2H, NHCH2), 3.45
(t, 1H, J = 7.23 Hz, CH), 3.39 (br, 2H, CH2NHCO), 3.30 (s, 3H, OCH3), 3.10 (m, 2H,
C4-H2), 2.76 (br, 2H, C1-H2), 2.37 (d, 2H, J = 7.23 Hz, CH-CH2), 1.73-1.54 (m, 6H,
C2-H2, C3-H2, NHCH2CH2), 1.35 (s, 3H, CH3).
4.1.7.3. The procedure for the preparation of compound J2-1.
Brown viscous product. Yield 97%. ESI-MS: m/z 504.2 [M+H]+; IR (KBr, cm-1):
3383, 2933, 2853, 1658, 1613, 1563, 1513, 1443, 1377, 1239, 1044; 1H NMR (CDCl3,
300 MHz), δ (ppm): 8.04 (d, 1H, J = 8.04 Hz, arom), 7.93 (d, 1H, J = 8.23 Hz, arom),
7.56 (t, 1H, J = 7.50 Hz, arom), 7.36 (t, 1H, J = 7.50 Hz, arom), 6.90 (d, 2H, J = 7.83
Hz, arom), 6.72 (d, 2H, J = 7.83 Hz, arom), 3.98 (br, 4H, OCH2CH2O), 3.48 (m, 2H,
NHCH2), 3.41 (m, 2H, CH2NHCO), 3.24 (m, 2H, C4-H2), 2.86 (t, 1H, J = 6.85 Hz,
CH), 2.57 (br, 2H, C1-H2), 2.51 (d, 2H, J = 6.85 Hz, CH-CH2), 1.76-1.56 (m, 8H,

C2-H2, C3-H2, NHCH2CH2 × 2), 1.37 (s, 3H, CH3).
4.1.7.4. The procedure for the preparation of compound J2-2.
Brown viscous product. Yield 96%. ESI-MS: m/z 534.2 [M+H]+; IR (KBr, cm-1):
1
3359, 2931, 1654, 1577, 1515, 1452, 1263, 1032; H NMR (DMSO-d6, 300 MHz), δ
(ppm): 9.76 (br, 1H, NH), 8.62 (br, 1H, NH), 8.12 (d, 1H, J = 8.01 Hz, arom), 7.80 (d,
1H, J = 8.34 Hz, arom), 7.54 (t, 1H, J = 7.53 Hz, arom), 7.41 (t, 1H, J = 7.53 Hz,
arom), 7.38 (s,1H, arom), 6.95 (d, 1H, J = 7.38 Hz, arom), 6.59 (d, 1H, J = 7.38 Hz,
arom), 3.82 (br, 4H, OCH2CH2O), 3.66 (s, 3H, OCH3), 3.43 (t, 1H, J = 7.25 Hz, CH),
3.36-3.30 (m, 4H, NHCH2, CH2NHCO), 3.00 (br, 2H, C4-H2), 2.68 (br, 2H, C1-H2),
2.47 (d, 2H, J = 7.25 Hz, CH-CH2), 1.75 (br, 4H, C3-H2, C2-H2), 1.55-1.39 (m, 4H,
NHCH2CH2 × 2), 1.33 (s, 3H, CH3).
4.1.7.5. The procedure for the preparation of compound J3-1.
Brown viscous product. Yield 97%. ESI-MS: m/z 532.3 [M+H]+; IR (KBr, cm-1):
3353, 3167, 2921, 2855, 1662, 1573, 1508, 1456, 1393, 1268, 1044; 1H NMR (DMSO,
300 MHz), δ (ppm): 9.11 (br, 1H, NH), 8.13 (d, 1H, J = 8.46 Hz, arom), 7.80 (d, 1H, J
= 8.34 Hz, arom), 7.55 (t, 1H, J = 7.54 Hz, arom), 7.36 (t, 1H, J = 7.54 Hz, arom),
7.00 (d, 2H, J = 8.28 Hz, arom), 6.67 (d, 2H, J = 8.28 Hz, arom), 5.37 (br, 1H, NH),
3.83 (br, 4H, OCH2CH2O), 3.38 (m, 2H, NHCH2), 3.08 (t, 1H, J = 7.64 Hz, CH), 3.01
(m, 2H, CH2NHCO), 2.67 (m, 2H, C4-H2), 2.53 (d, 2H, J = 7.64 Hz, CH-CH2), 2.34
(br, 2H, C1-H2), 1.87-1.66 (m, 4H, C2-H2, C3-H2), 1.53-1.47 (m, 4H, NHCH2CH2 ×
2), 1.33 (s, 3H, CH3), 1.29-1.26 (m, 4H, NHCH2CH2CH2 × 2).
4.1.7. 6. The procedure for the preparation of compound J3-2.
Brown viscous product. Yield 95%. ESI-MS: m/z 561.3 [M+H]+; IR (KBr, cm-1):
3369, 2933, 2858, 1651, 1571, 1517, 1456, 1272, 1034; 1H NMR (CDCl3, 300 MHz),
δ (ppm): 8.10 (d, 1H, J = 7.96 Hz, arom), 7.89 (d, 1H, J = 8.34 Hz, arom), 7.55 (t, 1H,
J = 7.60 Hz, arom), 7.43 (t, 1H, J = 7.60 Hz, arom), 7.01 (s, 1H, arom), 6.75 (d, 1H, J
= 7.24 Hz, arom), 6.57 (d, 1H, J = 7.24 Hz, arom), 3.93 (br, 4H, OCH2CH2O), 3.59 (s,
3H, OCH3), 3.44 (m, 2H, NHCH2), 3.12 (t, 1H, J = 7.56 Hz, CH), 3.05 (m, 2H,
CH2NHCO), 2.71 (m, 2H, C4-H2), 2.59 (d, 2H, J = 7.56 Hz, CH-CH2), 2.46 (br, 2H,
C1-H2), 1.79 (br, 4H, C2-H2, C3-H2), 1.51-1.43 (m, 4H, NHCH2CH2 × 2), 1.35 (s, 3H,

CH3), 1.27-1.23 (m, 4H, NHCH2CH2CH2 × 2).
4.1.8. General procedure D for the preparation of series K.
To a solution of compound J in pure water and acetone (1:1,v/v) was added 2
equivalents PTSA and stirred at 95°C overnight under the protection of nitrogen. The
solvent was removed in vacuum after the completion of the reaction. The pH of the
reaction was justified to 7-8 with saturated Na2CO3 solution and extracted with DCM.
The organic layer was dried with anhydrous sodium sulfate and evaporated under
reduced pressure to get the target product K.
4.1.8. 1. The procedure for the preparation of compound K1-1
.
Yellow solid. Yield 90%. mp 118-121°C; IR (KBr, cm-1): 3283, 2925, 2857, 1717,
1
1650, 1613, 1560, 1513, 1446, 1360, 1261, 1099, 1026; H NMR (DMSO-d6, 300
MHz), δ (ppm): 9.28 (br, 1H), 8.39 (d, 1H, J = 8.73 Hz,), 8.14 (d, 1H, J = 8.52 Hz),
7.87 (t, 1H, J = 7.63 Hz), 7.57 (t, 1H, J = 7.63 Hz), 7.19 (d, 2H, J = 8.08 Hz), 6.73 (d,
2H, J = 8.08 Hz), 3.87 (t, 1H, J = 7.57 Hz), 3.31 (br, 4H), 3.18 (d, 2H, J = 7.57 Hz),
3.10 (m, 2H), 2.71 (br), 2.11 (s, 3H), 1.89-1.80 (m, 4H), 1.59 (br, 2H); 13C NMR
(DMSO-d6, 75 MHz), δ (ppm): 202.9, 179.1, 166.4, 155.8, 152.9, 139.1, 132.2, 130.0,
128.8, 127.9, 125.4, 124.9, 115.2, 115.0, 111.9, 53.8, 51.7, 44.7, 40.3, 35.7, 29.9, 29.8,
23.9, 22.7, 21.5. HRMS (ESI) m/z Calcd for C27H32N3O3 [M+H]+ 446.24382 found
446.24399.
4.1.8.2. The procedure for the preparation of compound K1-2.
Pale yellow solid.Yield 89 %. mp 106-109°C; IR (KBr, cm-1): 3279, 2962, 1717,
1
1651, 1560, 1515, 1428, 1361, 1261, 1098, 1032; H NMR (DMSO-d6, 300 MHz), δ
(ppm): 8.79 (br, 1H), 8.40 (d, 1H, J = 8.58 Hz), 8.21 (d, 1H, J = 8.25 Hz), 7.86 (t, 1H,
J = 7.57 Hz), 7.56 (m, 1H), 7.14 (s, 1H), 6.72 (br, 2H), 3.87 (t, 1H, J = 7.63 Hz), 3.75
(s, 3H), 3.40 (br, 2H), 3.32 (br, 4H), 3.18 (d, 2H, J = 7.63 Hz), 2.68 (m, 2H), 2.11 (s,
3H), 1.89-1.80 (m, 4H), 1.60 (br, 2H); 13C NMR (DMSO-d6, 75 MHz), δ (ppm): 202.9,
166.3, 156.7, 147,4, 145.0, 132.3, 132.2, 129.6, 127.9, 125.4, 124.9, 121.6, 115.1,
113.4, 111.6, 55.6, 51.1, 44.7, 35.7, 35.5, 29.9, 29.8, 24.1, 23.9, 22.8, 20.6. HRMS
(ESI) m/z Calcd for C28H34N3O4 [M+H]+ 476.25438 found 476.25449.
4.1.8.3. The procedure for the preparation of compound K2-1.

Pale yellow solid. Yield 98 %. mp 131-134°C; IR (KBr, cm-1): 3279, 2927,
1
1718, 1638, 1584, 1516, 1449, 1360, 1261, 1174, 1033; H NMR (DMSO-d6, 300
MHz), δ (ppm): 9.27 (br, 1H), 8.38 (d, 1H, J = 8.67 Hz), 8.12 (br, 1H), 7.85 (t, 1H, J =
7.65 Hz), 7.56 (t, 1H, J = 7.65 Hz), 7.49 (d, 1H, J = 7.98 Hz), 7.19 (d, 2H, J = 8.19
Hz), 6.72 (d, 2H, J = 8.19 Hz), 3.83 (t, 1H, J = 7.63 Hz), 3.33 (br, 4H), 3.08 (m, 2H),
2.71 (d, 2H, J = 7.63 HZ), 2.28 (m, 2H), 2.11 (s, 3H), 1.89-1.70 (m, 4H), 1.58-1.45 (br,
4H); 13C NMR (DMSO-d6, 75 MHz), δ (ppm): 202.9, 165.8, 155.8, 146.9, 145.1,
132.1, 130.0, 128.9, 127.9, 125.4, 124.8, 119.8, 115.0, 113.5, 111.6, 51.2, 47.0, 40.3,
38.6, 38.1, 29.8, 27.2, 26.1, 24.0, 22.9, 20.7. HRMS (ESI) m/z Calcd for C28H34N3O3
[M+H]+ 460.25947 found 460.25933.
4.1.8.4. The procedure for the preparation of compound K2-2
.
Pale yellow solid.Yield 85 %. mp 109-112°C; IR (KBr, cm-1): 3330, 2926, 2855,
1
1717, 1650, 1576, 1515, 1455, 1261, 1109, 1029; H NMR (DMSO-d6, 300 MHz), δ
(ppm): 8.77 (br, 1H), 8.38 (d, 1H, J = 8.25 Hz), 8.20 (d, 1H, J = 8.49 Hz), 8.12 (br, 1H,
NH), 7.85 (t, 1H, J = 7.54 Hz), 7.56 (t, 1H, J = 7.54 Hz), 7.12 (s, 1H), 6.71 (br),
3.82 (t, 1H, J = 7.38 Hz), 3.74 (s, 3H), 3.28 (m, 4H), 3.08 (m, 2H), 2.70 (d, 2H, J =
7.38 Hz), 2.62 (m, 2H), 2.11 (s, 3H), 1.91-1.72 (m, 4H), 1.61-1.45 (br, 4H); 13C NMR
(DMSO-d6, 75 MHz), δ (ppm): 202.9, 165.8, 156.9, 147.4, 144.9, 132.2, 129.8, 129.6,
128.7, 125.5, 124.8, 121.5, 115.0, 113.4, 111.5, 55.6, 51.2, 47.0, 40.3, 38.7, 38.1, 29.8,
27.3, 26.1, 24.1, 22.9, 20.7. HRMS (ESI) m/z Calcd for C29H36N3O4 [M+H]+
490.27003 found 490.27015.
4.1.8.5. The procedure for preparation of the compound K3-1
.
Pale yellow solid. Yield 92 %. mp 69-72°C; IR (KBr, cm-1): 3275, 2927, 2856,
1
1716, 1651, 1571, 1510, 1456, 1361, 1261, 1100, 1033; H NMR (DMSO-d6, 300
MHz), δ (ppm): 9.25 (br, 1H), 8.34 (d, 1H, J = 8.46 Hz), 8.06 (d, 1H, J = 8.25 Hz),
7.80 (t, 1H, J = 7.17 Hz), 7.54 (t, 1H, J = 7.17 Hz), 7.14 (d, 2H, J = 8.16 Hz), 6.71 (d,
2H, J = 8.16 Hz), 3.74 (t, 1H, J = 7.48 Hz), 3.29 (m, 4H), 3.16 (d, 2H, J = 7.48 Hz2),
3.02 (m, 2H), 2.72 (m, 2H), 2.11 (s, 3H), 1.90-1.79 (m, 4H), 1.68-1.23 (m, 8H); 13C
NMR (DMSO-d6, 75 MHz), δ (ppm): 202.9, 165.7, 155.7, 154.3, 147.6, 131.4, 130.0,
129.2, 127.9, 126.2, 125.4, 121.7, 115.0, 112.3, 51.2, 47.4, 40.3, 38.6, 38.3, 29.8, 28.7,

25.8, 25.7, 24.2, 23.2, 20.8, 20.7. HRMS (ESI) m/z Calcd for C30H38N3O3 [M+H]+
488.29077 found 488.29090.
4.1.8.6. The procedure for the preparation of compound K3-2.
Pale yellow solid. Yield 93 %. mp 94-96°C; IR (KBr, cm-1): 3279, 2961, 2854,
1
1717, 1648, 1575, 1521, 1456, 1361, 1261, 1097, 1029; H NMR (DMSO-d6, 300
MHz), δ (ppm): 8.78 (br, 1H), 8.38 (d, 1H, J = 8.64 Hz), 8.20 (d, 1H, J = 8.28 Hz),
7.85 (t, 1H, J = 7.62 Hz), 7.56 (t, 1H, J = 7.62 Hz), 7.12 (s, 1H), 6.71 (br, 2H), 3.81 (t,
1H, J = 7.46 Hz), 3.74 (s, 3H), 3.33 (m, 4H), 3.02 (m, 2H), 2.70 (d, 2H, J = 7.46 Hz),
2.60 (m, 2H), 2.11 (s, 3H), 1.91-1.71 (m, 4H), 1.60-1.23 (m, 8H); 13C NMR (DMSO-
d6, 300 MHz), δ (ppm): 202.9, 165.7, 154.6, 147.4, 144.9, 132.0, 129.7, 127.9, 125.4,
124.7, 121.5, 119.9, 115.3, 113.4, 111.5, 55.6, 51.2, 47.3, 40.3, 38.6, 38.3, 30.5, 29.8,
28.7, 25.6, 24.0, 22.8, 21.0, 20.8. HRMS (ESI) m/z Calcd for C31H40N3O4 [M+H]+
518.30133 found 518.30150.
4.1.9. General procedure C for preparation of the compound L
To a solution of compound H in pure water and acetone (1:1,v/v) was added 2
equivalents PSTA and stirred at 95°C overnight under the protection of nitrogen. The
solvent was removed in vacuum after the completion of the reaction. The pH of the
reaction was justified to 7-8 with saturated Na2CO3 solution and extracted with
dichloromethane. The organic layer was dried with anhydrous sodium sulfate and
evaporated under reduced pressure to get the target product L.
4.1.9.1. The procedure for the preparation of compound L1-1
Yellow solid. Yield 89%. mp 93-96°C. IR (KBr, cm-1): 2926, 1716, 1651, 1603,
1575, 1508, 1417, 1361, 1261, 1097, 1022; 1H NMR (DMSO-d6, 300 MHz), δ (ppm):
9.98 (br, 1H), 8.41 (d, 1H, J = 8.55 Hz), 8.22 (d, 1H, J = 8.67 Hz), 7.88 (t, 1H, J =
7.38 Hz), 7.60 (t, 2H, J = 7.38 Hz), 7.46 (d, 2H, J = 8.34 Hz), 7.10 (s, 1H), 6.88 (d,
2H, J = 8.34 Hz), 3.85 (m, 2H), 3.31 (m, 2H), 3.17 (m, 2H), 2.86 (br, 2H), 2.79 (br,
2H), 2.09 (s, 3H), 1.88-1.84 (m, 4H); 13C NMR (DMSO-d6, 75 MHz), δ (ppm): 202.9,
166.3, 158.1, 155.4, 147.7, 133.7, 132.5, 131.8, 127.9, 127.1, 126.4, 125.4, 125.2,
124.2, 119.9, 115.5, 112.3, 51.1, 44.8, 40.3, 35.7, 29.9, 26.4, 24.5, 21.9. HRMS (ESI)
m/z Calcd for C27H30N3O3 [M+H]+ 444.22817 found 444.22825.

4.1.9.2. The procedure for the preparation of compound L1-2.
Orange-red solid. Yield.92%. mp 99-101°C. IR (KBr, cm-1): 3340, 2932, 1729,
1650, 1575, 1512, 1424, 1372, 1261, 1121, 1032; 1H NMR (DMSO-d6, 300 MHz), δ
(ppm): 9.54 (br, 1H), 8.40 (d, 1H, J = 8.46 Hz), 8.29 (br, 1H), 8.22 (d, 1H, J = 8.34
Hz), 7.83 (t, 1H, J = 7.53 Hz), 7.72 (s, 1H), 7.56 (t, 1H, J = 7.53 Hz), 7.14 (s, 1H),
7.06 (d, 1H, J = 8.28 Hz), 6.91 (d, 1H, J = 8.28 Hz), 3.82 (s, 3H), 3.31 (m, 4H), 3.16
(m, 2H), 2.90 (br, 2H), 2.80 (br, 2H), 2.11 (s, 3H), 1.85-1.79 (m, 4H); 13C NMR
(DMSO-d6, 75 MHz), δ (ppm): 202.9, 166.3, 154.8, 147.4, 144.3, 133.3, 131.9, 128.0,
127.1, 125.0, 124.0, 123.5, 121.0, 116.4, 115.5, 114.3, 113.0, 55.6, 51.1, 44.8, 40.3,
35.7, 29.9, 26.6, 24.8, 22.0. HRMS (ESI) m/z Calcd for C28H32N3O4 [M+H]+
474.23873 found 474.23866.
4.1.9.3. The procedure for the preparation of compound L2-1.
Orange-red solid. Yield 95%. mp 89-92°C. IR (KBr, cm-1): 3311, 2924, 2856, 1716,
1633, 1573, 1508, 1435, 1373, 1262, 1105, 1025; 1H NMR (DMSO-d6, 300 MHz), δ
(ppm): 9.83 (br, 1H), 8.31 (d, 1H, J = 8.64 Hz), 8.05 (d, 1H, J = 8.97 Hz), 7.81 (s, 1H),
7.72 (t, 1H, J = 7.66 Hz), 7.49 (t, 1H, J = 7.66 Hz), 7.40 (d, 2H, J = 8.31 Hz), 6.87 (d,
2H, J = 8.31 Hz), 3.62 (m, 2H), 3.25 (m, 2H), 3.04 (m, 2H), 2.86 (br, 2H), 2.80 (br,
2H), 2.09 (s, 3H), 1.79 (br, 2H), 1.65 (m, 2H), 1.44 (m, 2H); 13C NMR (DMSO-d6, 75
MHz), δ (ppm): 202.9, 165.8, 157.4, 153.1, 131.4, 131.0, 130.7, 130.5, 129.9, 127.9,
127.2, 125.4, 124.9, 124.4, 123.5, 115.3, 114.2, 51.2, 47.3, 40.3, 38.1, 29.8, 27.5, 26.2,
25.2, 22.2. HRMS (ESI) m/z Calcd for C28H32N3O3 [M+H]+ 458.24382 found
458.24377.
4.1.9.4. The procedure for the preparation of the compound L2-2.
Orange-red solid. Yield 91%. mp 82-85°C. IR (KBr, cm-1): 2961, 1716, 1653, 1577,
1514, 1463, 1429, 1261, 1101, 1030; 1H NMR (DMSO-d6, 300 MHz), δ (ppm): 9.50
(br, 1H), 8.38 (d, 1H, J = 8.58 Hz), 8.16 (d, 1H, J = 8.28 Hz), 8.08 (br, 1H), 7.82 (t,
1H, J = 7.65 Hz), 7.70 (s, 1H), 7.55 (t, 1H, J = 7.65 Hz), 7.13 (s, 1H), 7.06 (d, 1H, J =
7.98 Hz), 6.90 (d, 1H, J = 7.98 Hz), 3.82 (s, 3H), 3.75 (m, 2H), 3.32 (br, 2H), 3.07 (m,
2H), 2.90 (br, 2H), 2.80 (br, 2H), 2.10 (s, 3H), 1.81 (br, 2H), 1.70 (m, 2H), 1.46 (m,
2H); 13C NMR (DMSO-d6, 75 MHz), δ (ppm): 202.9, 165.8, 154.5, 147.4, 139.6,

132.9, 131.8, 128.4, 127.9, 127.2, 125.4, 124.9, 124.0, 123.4, 115.5, 114.2, 113.1,
55.6, 51.2, 47.2, 40.3, 38.1, 29.8, 27.3, 26.1, 24.8, 22.0. HRMS (ESI) m/z Calcd for
C29H34N3O4 [M+H]+ 488.25438 found 488.25455.
4.1.9.5. The procedure for the preparation of compound L3-1.
Orange-red solid. Yield 88%.mp 76-79°C. IR (KBr, cm-1): 3065, 2920, 2849, 1716,
1
1647, 1603, 1575, 1509, 1436, 1358, 1261, 1170, 1102; H NMR (DMSO-d6, 300
MHz), δ (ppm): 13.22 (br, 1H), 9.96 (br, 1H), 8.38 (d, 1H, J = 8.58 Hz), 8.23 (d, 1H, J
= 8.37 Hz), 8.09 (br, 1H), 7.80 (t, 1H, J = 7.82 Hz), 7.53 (t, 1H, J = 7.82 Hz), 7.43 (d,
2H, J = 8.40 Hz), 7.13 (s, 1H), 6.89 (d, 2H, J = 8.40 Hz), 3.71 (m, 2H), 3.02 (br, 2H),
2.86 (m, 2H), 2.79 (br, 2H), 2.11 (s, 3H), 1.79 (br, 2H), 1.69 (br, 2H), 1.38-1.22 (br,
8H); 13C NMR (DMSO-d6, 75 MHz), δ (ppm): 203.2, 165.7, 157.8, 148.9, 132.5,
131.6, 131.5, 128.3, 127.9, 126.8, 125.4, 124.8, 123.9, 121.9, 116.8, 115.4, 113.2,
112.7, 51.2, 47.5, 40.3, 38.3, 29.9, 28.7, 26.7, 25.8, 25.7, 24.9, 22.1. HRMS (ESI) m/z
Calcd for C30H36N3O3 [M+H]+ 486.27512 found 486.27525.
4.1.9.6. The procedure for the preparation of compound L3-2.
Orange-red solid.Yield 92%. mp 82-85°C. IR (KBr, cm-1): 3345, 2961, 2854, 1716,
1651, 1576, 1513, 1456, 1419, 1261, 1098, 1027; 1H NMR (DMSO-d6, 300 MHz), δ
(ppm): 13.19 (br, 1H), 9.45 (s, 1H), 8.35 (d, 1H, J = 8.52 Hz), 8.17 (d, 2H, J = 8.40
Hz), 8.04 (br, 1H), 7.80 (t, 1H, J = 8.14 Hz), 7.52 (t, 1H, J =7.47 Hz), 7.26 (br, 1H),
7.12 (s, 1H), 7.04 (d, 1H, J = 8.16 Hz), 6.89 (d, 1H, J = 8.16 Hz), 3.81 (s, 3H), 3.67
(m, 2H), 3.02 (br, 2H), 2.89 (m, 2H), 2.79 (br, 2H), 2.11 (s, 3H), 1.81 (br, 2H), 1.68
(br, 2H), 1.38-1.23 (br, 8H); 13C NMR (DMSO-d6, 75 MHz), δ (ppm): 203.0, 165.7,
153.9, 149.2, 147.3, 147.1, 132.3, 131.2, 129.0, 127.9, 127.4, 124.6 123.7, 123.3,
122.4, 117.1, 115.4, 114.2, 113.5, 55.6, 51.3, 47.5, 40.3, 38.3, 29.9, 28.7, 26.8, 25.8,
25.7, 25.0, 22.1. HRMS (ESI) m/z Calcd for C31H38N3O4 [M+H]+ 516.28568 found
516.28560.
4.2. Biological studies.
4.2.1 Cholinesterase inhibition assay in vitro
The cholinesterase inhibitory activity of the target compounds were measured using
Ellman’s assay.[31] AChE (E.C.3.1.1.7, Type VI-S, from Electric Eel) ,BChE