Synthesis, biological evaluation, and molecular docking study of novel allyl-retrochalcones as a new class of protein tyrosine phosphatase 1B inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.01.034

We describe herein the design, synthesis, and biological evaluation of a series of novel protein tyrosine phosphatase 1B (PTP1B) inhibitor retrochalcones having an allyl chain at the C-5 position of their B ring. Biological screening results showed that the majority of these compounds exhibited an inhibitory activity against PTP1B. Thus, preliminary structure-activity relationship (SAR) and quantitative SAR analyses were conducted. Among the compounds, 23 was the most potent inhibitor, exhibiting the highest in vitro inhibitory activity against PTP1B with an IC₅₀ of 0.57 μM. Moreover, it displayed a significant hepatoprotective property via activation of the IR pathway in type 2 diabetic db/db mice. In addition, the results of our docking study showed that 23, as a specific inhibitor of PTP1B, effectively transformed the WPD loop from “close” to “open” in the active site. These results may reveal suitable compounds for the development of PTP1B inhibitors.

Full text of DOI:10.1016/j.bmc.2019.01.034

Accepted Manuscript
Synthesis, biological evaluation, and molecular docking study of novel allyl-
retrochalcones as a new class of protein tyrosine phosphatase 1B inhibitors
Yunjie Zhao, Yongkai Cao, Huizhen Chen, Fei Zhuang, Chao Wu, Goo Yoon,
Weiwei Zhu, Ying Su, Suqing Zheng, Zhiguo Liu, Seung Hoon Cheon
PII:
S0968-0896(18)32043-1
https://doi.org/10.1016/j.bmc.2019.01.034
BMC 14726
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 December 2018
26 January 2019
29 January 2019
Please cite this article as: Zhao, Y., Cao, Y., Chen, H., Zhuang, F., Wu, C., Yoon, G., Zhu, W., Su, Y., Zheng, S.,
Liu, Z., Cheon, S.H., Synthesis, biological evaluation, and molecular docking study of novel allyl-retrochalcones
as a new class of protein tyrosine phosphatase 1B inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.bmc.2019.01.034
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Synthesis, biological evaluation, and molecular docking study of novel
allyl-retrochalcones as a new class of protein tyrosine phosphatase 1B
inhibitors
Yunjie Zhao 1,2 #, Yongkai Cao 3,#, Huizhen Chen 4,#, Fei Zhuang 1, Chao Wu 1, Goo Yoon 5, Weiwei
Zhu 1, Ying Su 1, Suqing Zheng 1, Zhiguo Liu 1,*, Seung Hoon Cheon 2,*
1
Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical
University, 1210 University Town, Wenzhou, Zhejiang 325035, China.
2 College of Pharmacy and Research Institute of Drug Development, Chonnam National University,
77 Yongbong-Ro, Buk-Gu, Gwangju 61186, Korea.
3 Guangdong Key Laboratory for Genome Stability & Disease Prevention, School of Pharmaceutical
Science, Shenzhen University Health Science Center, Shenzhen, 518060, China.
4 College of Chemistry & Materials Engineering, Wenzhou University, Wenzhou, Zhejiang 325035,
China.
5
College of Pharmacy and Natural Medicine Research Institute, Mokpo National University,
Jeonnam 58554, Korea.
# These authors contribute equally to this work.
* Corresponding authors:
Zhiguo Liu, Professor
Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical
University, 1210 University Town, Wenzhou, Zhejiang 325035, China.
Tel. +8686699892
Fax. +8686699892
Email: lzgcnu@163.com
Seung Hoon Cheon, Professor
College of Pharmacy and Research Institute of Drug Development, Chonnam National University,
300 Yongbong-Dong, Buk-Gu, Gwangju 500-757, Korea.
Tel. +82625302929
Fax. +82625302949
Email: shcheon@jnu.ac.kr
1

Abstract
We describe herein the design, synthesis, and biological evaluation of a series of novel protein
tyrosine phosphatase 1B (PTP1B) inhibitor retrochalcones having an allyl chain at the C-5 position
of their B ring. Biological screening results showed that the majority of these compounds exhibited
an inhibitory activity against PTP1B. Thus, preliminary structure-activity relationship (SAR) and
quantitative SAR analyses were conducted. Among the compounds, 23 was the most potent inhibitor,
exhibiting the highest in vitro inhibitory activity against PTP1B with an IC50 of 0.57 µM. Moreover,
it displayed a significant hepatoprotective property via activation of the IR pathway in type 2
diabetic db/db mice. In addition, the results of our docking study showed that 23, as a specific
inhibitor of PTP1B, effectively transformed the WPD loop from “close” to “open” in the active site.
These results may reveal suitable compounds for the development of PTP1B inhibitors.
Keywords: Allyl-retrochalcone, Protein tyrosine phosphatase 1B, Drug design, Structure-
activity relationship.
1. Introduction
Phosphorylation and dephosphorylation are catalyzed by the coordinated actions of protein
kinases and phosphatases, respectively. They are among the most important modifications that
modulate protein function in nearly all biological systems to transfer information between distinct
cellular sites and to control a wide variety of cellular functions.1 Although the molecular mechanism
of insulin resistance and obesity is not fully understood, the binding of insulin to the insulin receptor
induces autophosphorylation of tyrosine residues in its intracellular domain, triggering a kinase
cascade.2 Protein tyrosine phosphatase 1B (PTP1B) has been shown to dephosphorylate the insulin
receptor and downregulate insulin signaling. It has been found that decreased phosphorylation of
protein tyrosine of the insulin receptor is caused by increased activity of protein tyrosine
phosphatase 1B instead of by a lack of tyrosine kinase activity.3 Consequently, a selective PTP1B
2

inhibitor will increase the half-life of phosphorylated insulin receptor and maintain the receptor in an
activated state.
PTP1B is an attractive target for the treatment of both type 2 diabetes and obesity. Animal studies
have shown that PTP1B-deficient mice show enhanced insulin sensitivity, improved glycemic
control, and resistance to high fat diet-induced obesity.4-6 It has also been shown that the
downregulation of PTP1B expression by a designed antisense oligonucleotide normalizes blood
glucose and improves insulin sensitivity without changing the diet of mice.7 These results attracted
considerable interest in the discovery of small-molecule PTP1B inhibitors to treat type 2 diabetes,
which is characterized by insulin resistance and beta-cell dysfunction. The discovery of clinically
useful PTP1B inhibitors, although heavily investigated worldwide, has proven to be very difficult
because of their limited selectivity and unfavorable pharmacokinetics.8,9 This difficulty suggests a
need to investigate compounds that can overcome these major hurdles in phosphatase inhibitor
research.10 Unlike kinase inhibitors, no phosphatase inhibitor has been approved by the FDA to treat
human diseases. Currently, two PTP1B inhibitors are being clinically developed, TTP814 and
trodusquemine (MSI-1436), which are currently in Phase II and Phase Ib trials for type 2 diabetes,
respectively.11,12
Figure 1. Structures of trodusquemine, licochalcone A, and licochalcone E.
Retrochalcone is an exceptional chemical template having multifarious biological activities.13-15 In
our previous study, which is a further development of our research program, licochalcones A and E
(Figure 1), each with an allyl group at position C-5 of the B ring, exhibit significant inhibitory
effects against PTP1B. Licochalcone A derivative with methylation at the 4'-hydroxy position exhibit
approximately two-fold higher activities than that of licochalcone A.16 From this finding, we can
3

conclude that an allyl group at position 5 of retrochalcones is essential in its protective effect against
PTP1B activities in vitro. Thus, we synthesized analogs of allyl-retrochalcones in order to evaluate
changes in bioactivity that result from the varied substituent groups attached to the phenol ring.
Interestingly, most of the synthetic allyl-retrochalcone derivatives displayed significant inhibitory
effects against PTP1B. Herein, we report the design, synthesis, and evaluation of new allyl-
retrochalcone derived from licochalcones A and E as PTP1B inhibitors.
2. Chemistry
The synthesis of the allyl-retrochalcone derivatives 1-39 is illustrated in Scheme 1 and 2.
Allylation of the commercially available 4-hydroxy-2-methoxybenzaldehyde (1a) with allyl bromide
to yield allyl phenyl ether 2a.17 Claisen rearrangement of 2a in boiling N,N-dimethylaniline solvent
afforded allyl-benzaldehyde 3a, which was further protected with MOMCl/NaH to obtain the
benzaldehyde 4a with an excellent yield. Coupling of 4a by aldol condensation with commercially
available or previously synthesized acetophenones in an ethanolic solution readily generated the
protective allyl-retrochalcone 5a. The final allyl-retrochalcone derivatives, 1, 3-8, and 10-30, were
obtained by deprotection of 5a under acidic conditions. Alternatively, protection of the 4-hydroxy
group of 3a with 3,4-dihydro-2H-pyran in THF generated Compound 6a, which was further coupled
with corresponding acetophenones to obtain Compounds 2 and 9 with catalyzation by KOH.
4

Scheme 1. General synthetic method of the allyl-retrochalcones 1-30. Reagents and conditions: a)
Allyl bromide, K2CO3, acetone, reflux, 6 h; b) N,N-diethylaniline, 200 °C, 10 h; c) NaH, MOMCl,
THF, 0 °C, 5 h; d) Various substituted acetophenones, KOH, EtOH-H2O (2:1), rt, 4-24 h; e) 6N-HCl,
MeOH-H2O (2:1), rt, 3-5 h; f) 3,4-dihydro-2H-pyran, PPTS, CH2Cl2, rt, 4 h.
The syntheses of the allyl-retrochalcones 31-39 are illustrated in Scheme 2. Aldol condensation
of 4a with 1-{4-{[2-(trimethylsilyl)ethoxy]methoxy}phenyl}ethanone in the presence of KOH
generated the retrochalcone 7a, which was subsequently treated with TBAF to give the key
intermediate 8a. Compounds 31-33 were prepared by benzoylation followed by MOM-deprotection.
Furthermore, Compound 8a was alkylated with alkyl bromide in acetone by using K2CO3 as a
catalyst, and then deprotection of the THP protective groups generated the esters 34, 36, and 38.
Finally, they were hydrolyzed with 10% NaOH and 6N-HCl to obtain the carboxylic acids 35, 37,
5

and 39. All of the new products were isolated by conventional work-up with satisfactory yields.
Analytical and spectral data of all synthesized compounds were in full agreement with the proposed
structures.
Scheme 2. General synthetic method of the allyl-retrochalcones 31-39. Reagents and conditions: a)
1-{4-{[2-(Trimethylsilyl)ethoxy]methoxy}phenyl}ethanone, KOH, EtOH-H2O (2:1), rt, overnight; b)
TBAF, THF, 65 °C, 20 min; c) 1) Ethyl 2-bromoacetate, ethyl 4-bromobutanoate, or ethyl 5-
bromopentanoate, as well as K2CO3 and acetone, 63 °C, 8 h; 2) 6N-HCl, MeOH-H2O (2:1), rt, 3-5 h.
d) NaOH (10%), MeOH, rt, 3 h, then 6N-HCl; e) 1) Various benzoyl chlorides, Et3N, THF, rt, 8 h; 2)
6N-HCl, MeOH-H2O (2:1), rt, 3-5 h.
3. Results and discussion
3.1 Inhibitory screening and preliminary structure activity relationship study
The inhibitory activities of the synthesized compounds against PTP1B were assayed using p-
6

nitrophenyl phosphate (pNPP) as a substrate, and the results are summarized in Table 1. Ursolic acid
(IC50 = 3.1 µM), a known PTP1B inhibitor, was used as a positive control.18 The all-synthetic
compounds, except compounds 4, 9, 27, and 37, inhibited PTP1B activity in a dose-dependent
manner, with IC50 values ranging from 0.5 to 24.8 µM. Most of these compounds displayed higher
activities than that of licochalcone A. The introduction of an allyl group, instead of a 1,1-
dimethylallyl group, at the C-5 position generated Compound 1, which displayed a two-fold higher
inhibitory activity than that of licochalcone A. In contrast, Compound 4, which bore a meta-hydroxyl
group on A ring, exhibited no inhibitory effect against PTP1B. Methylation of the obtained
Compounds 3 and 5 resulted in higher activities than those of Compounds 1 and 4, respectively; this
observation concurs with our previous results.16
Table 1. Inhibitory activities of the synthesized Compounds 1-39 against PTP1B enzyme.
7

Incorporation of various alkyl groups or allyl groups at the 4-hydroxy position of Compound 5a
yielded compounds with considerably higher activities than that of Compound 1. For example, the
introduction of the ethyl, allyl, and 3,3-dimethylallyl groups at the 4'-hydroxy position of Compound
5a yielded Compounds 7 (IC50 = 5.3 µM), 8 (IC50 = 5.4 µM), and 11 (IC50 = 5.8 µM), respectively,
which showed approximately a two-fold higher inhibitory activity than that of Compound 1 (IC50 =
9.4 µM). Insertion of an ortho hydroxyl group or methoxy group to the A ring of Compounds 3, 8,
and 11 generated Compounds 6, 10, 12, and 13. Interestingly, with an extended substitution at the 4'-
hydroxy group, the four retrochalcones, which exhibited a proportionate increase in potency
(especially Compound 12 that bore a prenyl group), showed exceptionally good inhibitory activity
against PTP1B (IC50 = 3.0 µM) compared to that of the positive control, ursolic acid. Consistent with
a previous study,16 protection of Compounds 1 and 10 at the 2'-hydroxy position of their B ring with
an THP protective group yielded Compounds 2 and 9, which showed equivalent or no inhibitory
activity against PTP1B. However, by inserting various allyl groups at the C-3' position, Compounds
14, 15, and 16 provided similar potency to that of Compound 3 (IC50 = 7.4 µM).
Among Compounds 17-19, which contained piperidine substitution, meta- and para-substituted
derivatives exhibited a more potent PTP1B inhibitory effect than the ortho-substituted allyl-
retrochalcone 18 (IC50 = 9.5 µM). Compound 20 (IC50 = 12.3 µM), which had morpholine at its C-4'
position, showed a two-fold lower activity than that of Compound 21 (IC50 = 6.3 µM), a pyrazole-
substituted retrochalcone. In Compounds 22-27, it was observed that the compounds with meta- or
para-dimethylamino substituents were more potent than those with ortho-dimethylamino
substituents. More importantly, among these compounds, the meta-substituted Compound 23 showed
the most significant inhibitory activity against PTP1B, with a low IC50 value of 0.57 µM, and
exhibited a six-fold higher activity than that of the positive control, ursolic acid.
Incorporation of a benzyl and isopropyl group at the 2'-hydroxy position of A ring generated
Compounds 25 (IC50 = 5.0 µM) and 26 (IC50 = 6.9 µM), but decreased their inhibitory effects. In
Compound 27, incorporation of a methanesulfonamide at its C-3' position resulted in a complete loss
of potency. However, Compounds 29 and 30, which were protected with diallyl groups, showed
8

strong inhibitory effects similar to that of p-allyl in Compound 8 (IC50 = 5.4 µM). Compound 1 was
benzoylated separately at the 4'-hydroxy group to obtain the retrochalcone derivatives 31-33, all of
which exhibited an approximately two-fold higher activity than the initial Compound 1.
Incorporation of a carboxylic acid residue, as in Compounds 35 and 37, did not appear to increase
the PTP1B inhibition. Compound 39, however, exhibited moderate potency. Moreover, the other
esters (Compounds 34, 36, and 38) displayed varying degrees of PTP1B inhibitory activity.
Especially, Compound 38 showed a potent inhibitory effect against PTP1B similar to that of the
positive control, ursolic acid. Figure 2 shows the structure-activity relationship of these allyl-
retrochalcones.
Figure 2. Structure-activity relationships of synthetic allyl-retrochalcones.
3.2 Quantitative evaluation of structure activity relationship (QSAR)
In an attempt to correlate their inhibitory activity against PTP1B with the structure
conformation of the synthesized allyl-retrochalcones, a quantitative evaluation of structure activity
relationship (QSAR) study was conducted. The materials and methods for this computer-assisted
QSAR study are described in our previous publications.19,20 Our data set consisted of 33 active allyl-
retrochalcones, which are shown in Table 1 along with their 50% inhibition concentration (IC50). We
used 37 compounds of a total set of 39 synthetic new inhibitors because six allyl-retrochalcones
9

(Compounds 4, 6, 9, 27, 36, and 37) were excluded due to an inexact IC50 concentration, and
therefore could not be used in the multiple regression analysis. A statistically significant model of
inhibitory activity against PTP1B was obtained with three variables. A scatter plot of predicted
values versus experimental values is illustrated in Figure 3.
As shown in Figure 3, an excellent QSAR model was obtained with autocorrelation descriptors
(AATS5e, ATSC7c, and ATSC5e), which exhibited a relatively high regression coefficient (R2
=
adj
0.74) and was able to describe more than 76% variance in the experimental activity. Autocorrelation
descriptor is a molecular descriptor that encodes both the molecular structure and physicochemical
properties attributed to atoms as a vector.21 Autocorrelation of a Topological Structure (ATS) is also
known as the Moreau-Broto autocorrelation, and it describes how a property is distributed along a
topological structure. AATS5e is the average electronegativity-weighed Moreau-Broto graph spatial
autocorrelation coefficient of the fifth lag. ATSC7c is the centered Sanderson charges-weighed
Moreau-Broto graph spatial autocorrelation coefficient of the seventh lag, ATSC5e represents the
centered Sanderson electronegativity-weighed Moreau-Broto graph spatial autocorrelation
coefficient of the fifth lag.22,23 The QSAR results indicated that electronegativity and molecular
charges might play an important role in the inhibitory activities of the synthesized allyl-
retrochalcones against PTP1B.
10

Figure 3. Scatter plot of predicted activity against the corresponding experimental activity. N is the
number of compounds considered in the regression, R2 is the multiple correlation coefficient, R2adj is
the adjusted multiple correlation coefficient, s is residual standard error, and the F value is related to
the F-statistical analysis (Fischer test). Numbers in parentheses indicate the standard deviation of the
coefficients. p refers to the significance of the variables in the model.
On the basis of the SAR and QSAR results, we concluded that the inhibitory activities of the
allyl-retrochalcones against PTP1B may reveal valuable information regarding compound structure-
activity relationship for the development of novel PTP1B inhibitors.
3.3 Cellular screening of active Compound 23
Glucose influx into cells is regulated by the insulin receptor (IR) transduction pathway.
Dysregulation of its downstream molecules (e.g. insulin receptor substrate 1 and Akt) contribute to
insulin resistance in the liver.24,25 Considering that PTP1B is a negative regulator of the insulin
signaling pathway, active Compound 23 would activate insulin receptor substrate-1 (IRS-1) and
protein kinase B (Akt) to revert insulin resistance. Therefore, we assessed the effect of 23 in
11

palmitate acid (PA)-induced insulin resistant HepG2 cells. As shown in Figure 4A, incubation with
PA significantly downregulated phospho-IRS-1 (P-IRS-1) and phospho-Akt (P-Akt) in HepG2 cells,
compared with single insulin stimulation. However, treatment with Compound 23 at concentrations
of 5 and 10 µM markedly enhanced P-IRS1 and P-Akt, respectively. The results indicated that
Compound 23 can alleviate PA-induced insulin resistance in the liver.
A
64
32
16
8
p-IRS-1
p-AKT
PA
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p-AKT
AKT
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GAPDH
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C
Control
db/db
10 mg/kg
20 mg/kg
D
23
Control
10 mg/kg
db/db
20 mg/kg
p-IRS-1
IRS-1
Figure 4. Compound 23 alleviated palmitate acid (PA)-induced insulin resistance in vitro, and
relieved liver injury in vivo. (A) HepG2 cells were pretreated with or without 23 at the indicated
concentrations for 1 h, and then incubated with or without 0.15 mM PA for another 24 h. Finally, the
12

cells were stimulated with or without 100 nM insulin for 5 min before being harvested. The lysates
were subjected to an immunoblotting assay, as described in the Methods section. The blots were
quantified and plotted. **, P < 0.01; ***, P< 0.001. (B) Eight-week-old db/db mice were orally
administered 10 or 20 mg/kg of 23 or vehicle every other-day for 10 weeks. The mice were
sacrificed, and their serum and liver samples were collected. AKP, ALT, and TCH levels were
measured using standard assay kits, as mentioned in the Method section. (C) Liver samples were
subjected to an H&E staining assay. (D) Liver lysates were immunoblotted for p-IRS1. db/db vs.
Control. *, P < 0.05; ***, P < 0.001; 23 vs. db/db, ##, P < 0.01; ###, P < 0.001.
3.4 In vivo evaluation of Compound 23
Next, because insulin resistance and liver injury are closely associated, we investigated the
potential protective effect of Compound 23 against hyperglycemia-induced liver injury in type 2
diabetic db/db mice. As shown in Figure 4B, the levels of alkaline phosphatase (AKP) and alanine
aminotransferase (ALT), classical markers for liver function and integrity, respectively, were
remarkably increased in db/db mice. After treatment with Compound 23 at 10 and 20 mg/kg, AKP
and ALT contents markedly decreased. In addition, the administration of Compound 23 effectively
and significantly downregulated the level of total cholesterol (TCH) in the serum.
Histological examination results showed diffuse lipid accumulation and sporadic large lipid
droplets in the liver tissues of db/db mice. However, Compound 23 (10 and 20 mg/kg) treatment
significantly attenuated the extent of steatosis, as shown in Figure 4C. The size and number of these
lipid droplets in the liver were also notably reduced, suggesting that Compound 23 effectively
inhibited lipid accumulation in the liver and exerted a significant hepatoprotective effect. Meanwhile,
the levels of phosphorylated IRS-1 in liver extracts were closely related to the activation of IR
signaling observed in the livers, in spite of individual differences were observed (Figure 4D). These
data indicated that the activation of IR signaling pathway by Compound 23 protected mice from
liver injury.
13

3.5 Pharmacokinetic study for Compound 23
With these encouraging in vitro and in vivo results, Compound 23 was further subjected to
preliminary pharmacokinetic profiling in Sprague-Dawley (SD) rats. Compound 23 was
administered at 2 mg/kg intravenously or at 20 mg/kg orally to male SD rats. The pharmacokinetic
parameters are summarized in Figure 5A. Rats intravenously administered 23 exhibited prolonged
half-life (21.12 h) and low clearance rate (1.41 mL/min/kg). In contrast, rats orally administered 23
at 20 mg/kg showed moderate oral exposure (AUC0-∞= 1620.78 h.ng/mL), half-life (8.85 h), and
high Cmax (2148.83 ng/mL). Interestingly, there was a second peak at 4 h after intragastric
administration (Figure 5B-5C), probably because hepatoenteric circulation caused part of the drug to
be absorbed once again in the intestine.
Figure 5. (A) Pharmacokinetic parameters of Compound 23 in rats. (B) Mean plasma concentration-
time profile of Compound 23 at a single oral dose of 20 mg/kg. (C) Mean plasma concentration-time
profile of Compound 23 at a single intravenous dose of 2 mg/kg. Plots are mean ± SD (n = 3 in each
group).
3.6 Molecular docking and molecular dynamics simulation
14

Because it is not known whether Compound 23 specifically targets the catalytic site or a second
phosphor-tyrosine-binding pocket near the catalytic site (which has been proposed to be a
considerable binding site to promote selectivity and affinity26), we adopted a complex of PTP1B and
a selective inhibitor as a reference structure to cross the two binding pockets (PDB ID 1Q1M). The
results of the docking study clearly showed that the active Compound 23 was a catalytic site-specific
inhibitor because the top 20 positions were all enriched in this pocket and in nearby areas.
For further insight into the intermolecular binding mode, we performed a 50-ns molecular
dynamics simulation. As shown in Figure 6A, the receptor and ligand converged after 20 ns and the
root mean square deviation of the backbone atoms was about 1.6 Å and 2.0 Å, respectively.
Unexpectedly, the ligand showed a sudden conformational transition from 10 to 13 ns, indicating
that the docking result was not the authentic final conformation because the flexibility of the protein
receptor is not considered in the half-rigid docking. By analyzing the trajectory, we found that
during the conformational transition of the ligand, the configuration of the catalytic pocket also
started to reform. Figures 6A and 6B clearly show that a conserved protein loop (the WPD-loop)
transformed from a closed (hydrolysis competent) to an open (hydrolysis incompetent) position,
whereas the protein tyrosine phosphatase (PTP) loop, which contains the catalytic cysteine CYS215,
showed no significant change. Previous research has shown that the WPD loop plays a central role in
the mechanism of PTP1B catalysis. In the apo form, WPD loop is usually in an “open” conformation,
whereas it closes over the active site upon ligand binding, and its motion is the rate-limiting step for
hydrolysis.27 The result of this simulation is closely consistent with the results that indicated that our
compound has favorable affinity to and reasonable binding mode targeting at the catalytic site of
PTP1B.
15

Figure 6. Conformational transition during simulation. (A) Backbone root mean square deviations
are shown as a function of time for ligand (blue) and receptor (yellow). (B) WPD loop transformed
from a closed (grey) to an open (red) position.
Although conformational changes that occur upon substrate binding have been well
characterized,28 the mechanisms of small-molecular inhibition are still ambiguous. Hence, we
performed molecular mechanics/generalized born surface area (MM/GBSA) and calculation of
binding free energy to determine the inherent driving force of conformational transition. Trajectories
of 2-10 and 40-50 ns were chosen as representatives to calculate energy before and after transition,
respectively. Total binding energies (which were -54.6740 ± 4.2343 kcal/mol before and -53.6279 ±
5.4268 kcal/mol after) did not change significantly, but conformation and per-residue energy
contribution exhibited considerable differences. Figures 7A-7B show the top 10 contributing
residues, whereas Figures 7C-7D show the corresponding values. The ligand obtained a further step
insert in the catalytic pocket, and the energy contributions of ARG221 and SER216 slightly
decreased. By examining energy components, we found that electrostatic contribution was the
dominant term, consistent with the formation of hydrogen bonds. Consequently, although docking
task can indicate binding site and provide binding structure, its inherent limitation is that it cannot
reveal the in-depth mechanism. During the molecular simulation, the electrostatic contribution of
ARG221 and SER216 was the driving force that facilitated the conformational transition of the
ligand, as well as the motion of the WPD loop. Thereafter, the hydrolysis ability of PTP1B in a
complex with Compound 23 to prevent the development of some diseases was no longer available.
16

Figure 7. Dynamic and energy changes before and after conformational transition. (A, C) Mapping
of the energetically important residues (yellow) before transition and the corresponding values. (B,
D) Mapping of the energetically important residues (grey) after transition and the corresponding
values.
4. Conclusion
In conclusion, in the present study, we synthesized and investigated the inhibitory activity of new
allyl-retrochalcones against PTP1B. Among the allyl-retrochalcone derivatives, compounds 7, 8, 11,
15, 25, 29, 30, 31, 32, and 39 exhibited moderate inhibitory effects, with IC50 values ranging from
4.6 to 5.9 µM. The other four compounds, 12, 13, 23, and 38, showed potent inhibition against
PTP1B compared with the positive control, ursolic acid (IC50 = 3.1 µM). Among all the synthesized
compounds, 23 was the most active PTP1B inhibitor, with an IC50 value of 0.57 µM, nearly six-fold
higher than that of the positive control. Additionally, the allyl-retrochalcones with benzoylation at
the 4-hydroxy group position showed promising inhibitory properties against PTP1B. The above
results suggested a starting point for further optimization of retrochalcones having an allyl group
substitution at position C-5 as PTP1B inhibitors. The results of quantitative SAR analyses suggested
17

that the electronegativity and molecular charges of the compounds were closely correlated with their
PTP1B inhibition.
Further studies on cellular activities revealed that Compound 23 could activate IRS-1 and Akt
to revert PA-stimulated insulin resistance in HepG2 cells. The results of our in vivo examination in
db/db mice identified Compound 23, the most potent compound, as a new active inhibitor with an
ability to prevent hyperglycemia-induced liver injury and insulin sensitization. The results of
docking analysis and molecular dynamics simulation indicated that Compound 23 had favorable
affinity to and reasonable binding mode targeting at the catalytic site of PTP1B. The novel
compounds reported in this study will provide new insights to the design and development of new
low-molecular-weight PTP1B inhibitors.
5. Experimental section
5.1 Chemistry
5.1.1 General
Solvents were distilled under positive pressure of dry argon before use and dried using standard
methods. Unless otherwise noted, chemicals were obtained from local suppliers and were used
without further purification. All reactions were monitored by thin-layer chromatography (250
silica gel 60 F254 glass plates). Column chromatography was conducted using a Merck silica gel 60
column (230-400 mesh ASTM; Merck KGaA, Darmstadt, Germany). Melting points were
determined using a Fisher-Johns melting apparatus and uncorrected. Proton nuclear magnetic
resonance (1H NMR and 13C NMR) spectra were recorded on a Bruker 500 MHz spectrometer.
Chemical shifts were presented as parts per million with tetramethylsilane (TMS) as an internal
reference. Electron-spray ionization mass spectra in positive mode (ESI-MS) data were recorded on
a Bruker Esquire 3000t spectrometer.
5.1.2 4-(Allyloxy)-2-methoxybenzaldehyde (2a). Potassium carbonate (68.1 g, 0.48 mol) was
18

added slowly to a solution of 4-hydroxy-2-methoxybenzaldehyde (1a; 25.0 g, 0.16 mol) and 3-
bromoprop-1-ene (38.1 g, 0.32 mol) in acetone (150 mL). The reaction mixture was refluxed for 6 h.
The resulting mixture was filtered and the filtrate was concentrated under reduced pressure. The
residue was then dissolved in EtOAc (50 mL) and washed with water (50 mL) and brine (50 mL).
The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by chromatography on silica gel to obtain Compound 2a (27.95 g, 91%) as a
1
light yellow liquid. H-NMR (400 MHz, acetone--d6) δ (ppm): 10.26 (s, 1H), 7.73 (s, J = 1.5 Hz,
1H), 6.74 (s, 1H), 6.72 (d, J = 1.5 Hz, 1H), 6.13-6.03 (m, 1H), 5.48 (dq, J = 17.3, 1.7 Hz, 1H), 5.33
(dq, J = 10.6, 1.4 Hz, 1H), 4.74 (d, J = 5.3 Hz, 2H), 3.98 (s, 3H).
5.1.3 5-Allyl-4-hydroxy-2-methoxybenzaldehyde (3a). A solution of 2a (25.0 g, 0.13 mol) in N,N-
diethylaniline (50 mL) was heated at 200 °C under nitrogen atmosphere for 10 h. The resulting
mixture was purified by chromatography on silica gel to obtain the desired compound, 3a (5.24 g,
1
21%), as brownish red solid. m.p: 99.6-101.3 °C. H-NMR (400 MHz, acetone--d6) δ (ppm): 10.26
(s, 1H), 7.57 (s, 1H), 6.65 (s, 1H), 6.20-5.79 (m, 1H), 5.11 (dd, J = 17.1, 2.0 Hz, 1H), 5.07-5.02 (m,
1H), 3.91 (s, 3H), 3.37 (d, J = 6.6 Hz, 2H).
5.1.4 5-Allyl-2-methoxy-4-(methoxymethoxy)benzaldehyde (4a). A solution of 60% sodium
hydride (2.2 g, 0.056 mol) in dry THF (100 mL) was added portionwise to a solution of 3a (5.24 g,
0.028 mol) in THF (10 mL), followed by chloromethyl methyl ether (4.3 mL, 0.056 mol) in an ice-
bath. After 5 h, the resulting mixture was quenched with saturated ammonium chloride solution and
extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with water (150 mL),
dried over MgSO4, and concentrated under reduced pressure. The residue was further purified by
chromatography
on
silica
gel
to
obtain
the
desired
5-allyl-2-methoxy-4-
19

1
(methoxymethoxy)benzaldehyde (4a, 5.24 g, 80%) as a yellow solid. m.p: 85.3-87.9 °C. H-NMR
(400 MHz, acetone--d6) δ (ppm): 10.15 (s, 1H), 7.40 (s, 1H), 6.76 (s, 1H), 6.01-5.63 (m, 1H), 5.24 (d,
J = 19.0 Hz, 2H), 4.93 (dd, J = 17.1, 1.5 Hz, 1H), 4.89 (d, J = 10.5 Hz, 1H), 3.81 (s, 3H), 3.35 (s,
3H), 3.21 (d, J = 6.6 Hz, 2H).
5.1.5 (E)-3-[5-Allyl-2-methoxy-4-(methoxymethoxy)phenyl]-1-phenylprop-2-en-1-one (5a).
KOH solution (44.0 mg, 0.8 mmol) in H2O (1 mL) was added dropwise to a stirred solution of 4a
(0.1 g, 0.42 mmol) in EtOH and H2O (9 mL, v/v 2:1) at room temperature. The reaction mixture was
stirred at room temperature for 4-24 h. The resulting mixture was diluted with H2O (15 mL) and
extracted with EtOAc (3× 20 mL). The combined organic layers were washed with brine (15 mL)
and dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue
was further purified by chromatography on silica gel to obtain the desired Compound 5a with a yield
of 35-89%.
5.1.6 General preparation procedure of the compounds 1, 3-8 and 10-30.
Ten drops of 6N-HCl was added to several solutions of 5a (0.1 mmol) in MeOH and H2O (10 mL,
v/v 2:1). The reaction mixture was stirred at room temperature for 3-5 h, quenched with saturated
aqueous NH4Cl solution (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic
layers were washed with water (20 mL) and brine (20 mL), dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by chromatography on silica gel to
obtain the target compounds below.
5.1.6.1 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (1).
Yellow solid, 42.64% yield, m.p: 101.1-102.4 °C. 1H-NMR (500 MHz, acetone--d6) δ (ppm): 8.98 (s,
20

1H), 8.73 (s, 1H), 7.92 (d, J = 15.7 Hz, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 15.6 Hz, 1H), 7.47
(s, 1H), 6.82 (d, J = 8.5 Hz, 2H), 6.47 (s, 1H), 5.90 (dd, J = 16.7, 10.1 Hz, 1H), 4.93 (dd, J = 17.1,
1.7 Hz, 1H,- -), 4.87 (d, J = 8.9 Hz, 1H), 3.74 (s, 3H, -OCH3), 3.22 (d, J = 6.5 Hz, 2H). 13C-NMR
(125 MHz, acetone-d6) δ (ppm): 188.4, 162.4, 159.9, 159.4, 139.4, 138.1, 131.9, 131.6 ×2, 131.4,
120.2, 119.6, 116.7, 116.1 ×2, 115.4, 99.9, 56.0, 34.2. ESI-MS m/z: 311.18 (M+H)+, calcd for
C19H18O4: 310.12.
5.1.6.2 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (3).
Yellow solid, 49.5% yield, m.p: 123.2-125.1 °C. 1H-NMR (500 MHz, acetone-d6) δ (ppm): 8.79 (s,
1H), 7.96 (s, 1H), 7.93 (d, J = 5.6 Hz, 1H), 7.91 (s, 1H), 7.57 (d, J = 15.6 Hz), 7.49 (s, 1H), 6.91 (d,
J = 8.8 Hz, 2H), 6.47 (s, 1H), 5.90 (dd, J = 16.7, 1.7 Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz), 4.87 (d, J
= 8.9 Hz, 1H), 3.76 (s, 3H), 3.74 (s, 3H), 3.22 (d, J = 6.5 Hz, 2H). 13C-NMR (125 MHz, acetone-d6)
δ (ppm): 188.5, 164.1, 159.9, 159.5, 139.6, 138.1, 132.7, 131.4, 131.3 ×2, 120.2, 119.5, 115.6, 115.4,
114.6 ×2, 99.9, 56.1, 55.8, 34.2. ESI-MS m/z: 325.14 (M+H)+, calcd for C20H20O4: 324.14.
5.1.6.3 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (4).
Yellow solid, 47.98% yield, m.p: 105.5-107.3 °C. 1H-NMR (500 MHz, acetone-d6) δ (ppm): 8.73 (s,
2H), 7.93 (d, J = 15.6 Hz, 1H), 7.51 (s, 1H), 7.48 (d, J = 2.3 Hz, 1H), 7.42 (d, J = 7.6 Hz, 1H), 7.38
(s, 1H), 7.23 (d, J = 7.9 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.49 (s, 1H), 5.90 (dd, J = 16.7, 6.5 Hz,
1H), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 3.74 (s, 3H), 3.22 (d, J = 6.5 Hz, 2H).
13C-NMR (125 MHz, acetone-d6) δ (ppm): 190.0, 160.0, 159.7, 158.6, 141.4, 140.3, 138.0, 131.5,
130.4, 120.4, 120.2, 119.6, 116.3, 115.5, 115.4, 99.9, 66.0, 56.0, 34.1. ESI-MS m/z: 311.24 (M+H)+,
calcd for C19H18O4: 310.12.
21

5.1.6.4 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-(3-methoxyphenyl)prop-2-en-1-one (5).
1
Yellow solid, 45.8% yield. m.p: 139.0-141.5 °C. H NMR (500 MHz, acetone-d6) δ (ppm): 8.91 (s,
1H), 7.93 (d, J = 15.6 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.51 (d, J = 2.1 Hz, 1H), 7.50 (s, 1H), 7.43
(d, J = 1.6 Hz, 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.04 (dd, J = 8.2, 2.1 Hz, 1H), 6.48 (s, 1H), 5.90 (dd, J
= 16.7, 6.5 Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 3.74 (s, 6H), 3.22 (d, J
= 6.5 Hz, 2H). 13C-NMR (125 MHz, acetone-d6) δ (ppm): 190.0, 160.9, 160.4, 159.8, 141.2, 140.5,
138.0, 131.5, 130.4, 121.4, 120.2, 119.5, 119.1, 116.3, 115.4, 113.8, 99.8, 56.0, 55.7, 34.1. ESI-MS
m/z: 325.32 (M+H)+, calcd for C20H20O4: 324.14.
5.1.6.5 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-
1-one (6). Yellow solid, 42.7% yield, m.p: 134.4-136.3 °C. 1H-NMR (500 MHz, acetone-d6) δ (ppm):
8.83 (s, 1H), 8.31 (s, 1H), 7.93 (d, J = 15.6 Hz, 1H), 7.59 (d, J = 1.8 Hz), 7.57 (d, J = 1.8 Hz, 1H),
7.52 (d, J = 1.6 Hz, 1H), 7.48 (s, 1H), 6.81 (d, J = 8.2 Hz, 1H), 6.48 (s, 1H), 5.90 (dd, J = 16.7, 6.5
Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.22 (d,
J = 6.5 Hz, 2H. 13C-NMR (125 MHz, acetone-d6) δ (ppm): 186.9, 158.4, 158.0, 150.5, 147.1, 138.9,
136.7, 130.7, 129.9, 122.5, 118.7, 118.0, 115.2, 113.9, 113.9, 110.6, 99.4, 54.9, 54.6, 32.7. ESI-MS
m/z: 341.07 (M+H)+, calcd for C20H20O5: 340.13.
5.1.6.6 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-(4-ethoxyphenyl)prop-2-en-1-one (7).
Yellow solid, 49.5% yield, m.p: 131.3-132.8 °C. 1H-NMR (500 MHz, acetone--d6) δ (ppm): 8.87 (s,
1H), 7.95 (s, 1H), 7.93 (d, J = 15.6 Hz, 1H), 7.57 (d, J = 15.6 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.49
(s, 1H), 6.89 (d, J = 8.7 Hz, 2H), 6.47 (s, 1H), 5.90 (dd, J = 16.7, 6.5 Hz, 1H), 4.93 (dd, J = 17.1, 1.7
Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 4.01 (dd, J = 13.9, 6.9 Hz, 2H), 3.76 (s, 3H), 3.22 (d, J = 6.5 Hz,
2H), 1.26 (t, J = 6.9 Hz, 3H). 13C-NMR (125 MHz, acetone-d6) δ (ppm): 188.5, 163.4, 159.8, 159.5,
22

139.5, 138.1, 132.5, 131.4, 131.3, 120.1, 119.4, 116.5, 115.3, 115.0 ×2, 99.8, 64.4, 56.0, 34.1, 14.9.
ESI-MS m/z: 339.23 (M+H)+, calcd forC21H22O4: 338.15.
5.1.6.7 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[4-(allyloxy)phenyl]prop-2-en-1-one (8).
Yellow solid, 56.87% yield, m.p: 103.4-105.2 °C. 1H NMR (500 MHz, acetone-d6) δ (ppm): 8.85 (s,
1H), 7.94 (dd, J = 11.3, 2.6 Hz, 3H), 7.56 (d, J = 15.6 Hz, 1H), 7.48 (s, 1H), 6.92 (d, J = 8.2 Hz, 2H),
6.47 (s, 1H), 6.00-5.83 (m, 2H), 5.30 (d, J = 17.3 Hz, 1H), 5.14 (d, J = 10.4 Hz, 1H), 4.93 (dd, J =
17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 4.53 (d, J = 4.4 Hz, 2H), 3.74 (s, 3H), 3.22 (d, J = 6.5 Hz,
2H). 13C-NMR (125 MHz, acetone-d6) δ (ppm): 188.5, 163.0, 159.5, 139.7, 138.0, 134.2, 133.7,
131.9, 131.4, 131.3, 129.6, 120.2, 119.4, 117.8, 116.6, 115.5, 115.3, 99.9, 69.3, 66.9, 56.06, 34.1.
ESI-MS m/z: 351.22 (M+H)+, calcd for C22H22O4 350.15.
5.1.6.8 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[4-(allyloxy)-2-hydroxyphenyl]prop-2-en-
1-one (10). Yellow solid, 47.98% yield, m.p: 129.1-132.2 °C. 1H-NMR (500 MHz, acetone-d6) δ
(ppm): 8.07 (d, J = 15.6 Hz, 1H), 7.93 (d, J = 9.4 Hz, 1H), 7.65 (dd, J = 9.7, 5.8 Hz, 1H), 7.53 (s, 1H,
Ar-H), 6.50 (s, 1H), 6.43 (d, J = 15.6 Hz, 1H), 6.33 (s, 1H), 6.01-5.80 (m, 2H), 5.30 (d, J = 17.2 Hz,
1H), 5.15 (d, J = 9.1 Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 4.53 (d, J =
4.4 Hz, 2H), 3.74 (s, 3H), 3.22 (d, J = 6.5 Hz, 2H), 13C-NMR (125 MHz, acetone-d6) δ (ppm): 193.3,
187.4, 160.6, 160.1, 140.8, 138.0, 134.9, 132.9, 132.5, 131.9, 120.3, 118.0, 117.7, 116.2, 115.4,
109.3, 102.5, 99.7, 99.3, 69.6, 56.1, 34.1. ESI-MS m/z: 367.23 (M+H)+, calcd for C22H22O5: 366.15.
5.1.6.9
(E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-{4-[(3-methylbut-2-en-1-
1
yl)oxy]phenyl}prop-2-en-1-one. Yellow solid, 45.2% yield, m.p: 108.2-111.1 °C. H NMR (500
MHz, acetone-d6) δ (ppm): 8.92 (s, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.98-7.95 (m, 2H), 7.94 (s, 1H),
23

7.63-7.58 (m, 1H), 7.51 (d, J = 4.9 Hz, 3H), 7.25 (d, J = 8.5 Hz, 2H), 6.44 (s, 1H), 5.90 (dd, J = 16.7
Hz, 6.5 Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz), 4.87 (d, J = 8.9 Hz, 1H), 3.73 (s, 3H), 3.22 (d, J = 5.8
Hz, 2H), 1.80 (s, 3H, -CH3), 1.68 (s, 3H, -CH3). 13C-NMR (125 MHz, acetone-d6) δ (ppm): 188.8,
165.2, 160.1, 159.7, 158.4, 155.3, 140.4, 137.9, 131.8, 131.6, 130.4, 130.1, 129.6, 127.5, 122.94,
119.6, 119.3, 116.2, 115.4, 99.8, 56.0, 41.5 ×2, 34.1. ESI-MS m/z: 379.55 (M+H)+, calcd for
C24H26O4: 378.18.
5.1.6.10
(E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-{2-methoxy-4-[(3-methylbut-2-en-1-
1
yl)oxy]phenyl}prop-2-en-1-one (12). Yellow solid, 47.4% yield, m.p: 116.3-118.2 °C. H-NMR
(500 MHz, acetone-d6) δ (ppm): 8.75 (s, 1H), 8.41 (s, 1H), 8.05 (d, J = 15.6 Hz, 1H), 7.77-7.73 (m,
2H), 7.68 -7.57 (m, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.58 (s, 1H), 5.89 (dd, J = 16.7, 6.5 Hz, 1H), 5.23
(m, 1H), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 4.65 (d, J = 6.4 Hz, 2H), 3.83 (s,
3H), 3.22 (d, J = 6.5 Hz, 2H), 1.80 (s, 3H), 1.68 (s, 3H). 13C-NMR (125 MHz, acetone-d6) δ (ppm):
188.4, 159.8, 159.4, 152.0, 148.5, 143.2, 139.3, 138.1, 132.1, 131.4, 123.9, 120.1, 120.3, 119.3,
115.6, 115.4, 115.3, 112.1, 99.8, 66.9, 56.5, 41.5 ×2, 34.1. ESI-MS m/z: 395.09 (M+H)+, calcd for
C24H26O5: 394.18.
5.1.6.11 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[4-(allyloxy)-2-methoxyphenyl]prop-2-
1
en-1-one (13). Yellow solid, 56.3% yield, m.p: 120.7-122.8 °C. H-NMR (500 MHz, acetone--d6) δ
ppm: 8.92 (s, 1H), 8.46 (s, 1H), 8.05 (d, J = 15.6 Hz, 1H), 7.76 -7.75 (m, 2H), 7.65-7.57 (m, 2H),
6.92 (d, J = 8.2 Hz, 1H), 6.58 (s, 1H), 5.89 (dd, J = 16.7, 6.5 Hz, 1H), 5.23 (m, 1H), 4.93 (dd, J =
17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 4.65 (d, J = 6.4 Hz, 2H), 3.91 (s, 3H), 3.83 (s, 3H), 3.22
(d, J = 6.5 Hz, 2H), 1.80 (s, 3H), 1.68 (s, 3H). 13C-NMR (125 MHz, acetone-d6) δ (ppm): 188.4,
159.8, 159.4, 152.0, 148.5, 139.3, 138.1, 132.1, 131.4, 123.9, 120.1, 119.4, 116.6, 115.3, 115.3,
24

112.1, 108.7, 106.3, 102.3, 99.8, 66.9, 56.5, 56.0, 34.1, 31.2. ESI-MS m/z: 409.24 (M+H)+, calcd for
C25H28O5: 408.19.
5.1.6.12 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-(3-allyl-4-methoxyphenyl)prop-2-en-1-
one (14). Yellow-green solid, 60.43% yield, m.p: 119.7-121.9 °C. 1H-NMR (125 MHz, acetone-d6) δ
(ppm): 8.83 (s, 1H), 7.92 (d, J = 15.6 Hz, 1H), 7.87 (dd, J = 8.5, 2.1 Hz, 1H), 7.77 (s, 1H), 7.56 (d, J
= 15.6 Hz, 1H), 7.44 (s, 1H), 6.92 (d, J = 8.6 Hz, 1H), 6.46 (s, 1H), 5.97-5.78 (m, 2H), 4.98-4.91 (m,
2H), 4.88 (dd, J = 16.6, 10.7 Hz, 2H), 3.77 (s, 3H), 3.72 (s, 3H), 3.23 (d, J = 5.8 Hz, 4H). 13C-NMR
(125 MHz, acetone-d6) δ (ppm): 189.8, 161.7, 159.8, 159.5, 139.6, 138.0, 137.4, 132.4, 131.4, 130.8,
129.6, 129.4, 120.1, 119.6, 116.5, 116.1, 115.4, 110.8, 99.9, 56.1, 56.0, 34.8, 34.1. ESI-MS m/z:
366.06 (M+H)+, calcd for C23H24O4: 364.17.
5.1.6.13
(E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[4-methoxy-3-(3-methylbut-3-en-2-
1
yl)phenyl]prop-2-en-1-one (15). Yellow solid, 45.33% yield, m.p: 123.5-125.1 °C. H-NMR (500
MHz, acetone-d6) δ (ppm): 8.90 (s, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.87 (dd, J = 8.5, 2.1 Hz, 1H), 7.77
(s, 1H), 7.56 (d, J = 15.6 Hz, 1H), 7.44 (s, 1H), 6.92 (d, J = 8.6 Hz, 1H), 6.46 (s, 1H), 5.90 (dd, J =
16.7, 6.5 Hz, 1H), 4.98-4.91 (m, 2H), 4.88 (dd, J = 16.6, 10.7 Hz, 2H), 3.77 (s, 3H), 3.72 (s, 3H),
3.23 (d, J = 5.8 Hz, 2H), 1.82 (s, 3H), 1.75 (s, 3H). 13C-NMR (125 MHz, acetone-d6) δ (ppm): 189.7,
161.7, 159.8, 159.3, 149.00, 139.4, 138.0, 137.2, 132.4, 131.4, 130.8, 129.7, 120.1, 119.6, 116.4,
116.1, 115.4, 111.2, 99.9, 56.2, 56.0, 34.3, 34.1, 21.2, 20.1. ESI-MS m/z: 393.34 (M+H)+, calcd for
C25H28O4: 392.20.
5.1.6.14
(E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[4-methoxy-3-(3-methylbut-2-en-1-
yl)phenyl]prop-2-en-1-one (16). Yellow solid, 34.33% yield, m.p: 139.2-141.5 °C. 1H-NMR (500
25

MHz, acetone-d6) δ (ppm): 8.79 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.85 (dd, J = 8.4, 2.6 Hz, 1H), 7.74
(s, 1H), 7.56 (d, J = 15.6 Hz, 1H), 7.41 (s, 1H), 6.91 (d, J = 8.5 Hz, 1H), 6.48 (s, 1H), 5.90 (dd, J =
16.7, 6.5 Hz, 1H), 5.78 (s, 1H) 4.98-4.91 (m, 2H), 4.88 (dd, J = 16.6, 10.7 Hz, 2H), 3.77 (s, 3H),
3.72 (s, 3H), 3.23 (d, J = 5.8 Hz, 2H), 1.85 (s, 3H), 1.72 (s, 3H). 13C-NMR (125 MHz, acetone-d6) δ
(ppm): 189.6, 162.5, 159.4, 140.7, 138.4, 138.0, 132.4, 131.4, 130.8, 129.7, 121.6, 120.1, 119.6,
116.3, 116.1, 114.4, 111.1, 99.9, 56.2, 56.0, 34.3, 34.1, 25.3, 20.1, 14.6. ESI-MS m/z: 393.13
(M+H)+, calcd for C25H28O4: 392.20.
5.1.6.15 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[4-(piperidin-1-yl)phenyl]prop-2-en-1-
one (17). Yellow solid, 36.97% yield, m.p: 179.1-181.2 °C. 1H NMR (500 MHz, DMSO-d6) δ (ppm):
10.12 (s, 1H), 7.97 (d, J = 8.8 Hz, 2H), 7.91 (d, J = 15.6 Hz, 1H), 7.64 (d, J = 6.0 Hz, 1H), 7.62 (s,
1H), 6.99 (d, J = 7.8 Hz, 2H), 6.54 (s, 1H), 5.90 (dd, J = 16.7, 6.5 Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz,
1H), 4.87 (d, J = 8.9 Hz, 1H), 3.82 (s, 3H), 3.40 (d, J = 19.4 Hz, 4H), 3.22 (d, J = 6.5 Hz, 2H), 1.62-
1.33 (m, 6H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 186.4, 159.4, 158.2, 153.5, 137.2 ×2, 137.2,
130.2, 129.9 ×2, 119.9, 118.0, 114.9 ×2, 114.4, 113.1, 99.8, 65.0, 55.5 ×2, 47.9, 33.1, 24.9, 23.8.
ESI-MS m/z: 378.31 (M+H)+, calcd for C24H27NO3: 377.20.
5.1.6.16 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[2-(piperidin-1-yl)phenyl]prop-2-en-1-
one (18). Yellow solid, 29.34% yield, m.p: 175.3-177.1 °C. 1H-NMR (500 MHz, acetone--d6) δ
(ppm): 9.00 (s, 1H), 7.83 (d, J = 16.0 Hz, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.50 (d, J = 3.2 Hz, 1H),
7.41 (d, J = 17.0 Hz, 1 H), 7.28 (d, J = 28.6 Hz, 1H), 7.01 (s, 1H), 6.89 (s, 1H), 6.49 (s, 1H), 5.90
(dd, J = 16.7, 6.5 Hz, 1H), 4.92 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 3.74 (s, 3H),
3.22 (d, J = 6.5 Hz, 2H), 2.88 (d, J = 2.1 Hz, 4H), 1.64-1.48 (m, 6H), 13C-NMR (125 MHz, DMSO-
d6) δ (ppm): 193.4, 167.9, 159.6, 136.7, 133.4, 132.4, 131.9, 130.3, 129.5 ×2, 120.2, 116.3, 115.5,
26

99.9, 66.8, 56.0 ×2, 55.2, 34.0, 32.6, 31.3, 26.9, 24.7, 23.3. ESI-MS m/z: 378.41 (M+H)+, calcd for
C24H27NO3: 377.20.
5.1.6.17 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[3-(piperidin-1-yl)phenyl]prop-2-en-1-
1
one (19). Yellow solid, 45.89% yield, m.p: 178.3 -179.8 °C. H NMR (500 MHz, acetone-d6) δ
(ppm): 9.03 (s, 1H), 7.88 (d, J = 16.0 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.51 (d, J = 3.2 Hz, 1H),
7.41 (d, J = 17.0 Hz, 1 H), 7.33 (d, J = 18.6 Hz, 1H), 7.01 (s, 1H), 6.91 (s, 1H), 6.51 (s, 1H), 5.90
(dd, J = 16.7, 6.5 Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 3.71 (s, 3H),
3.22 (d, J = 6.5 Hz, 2H), 2.88 (d, J = 2.1 Hz, 4H), 1.64-1.48 (m, 6H). 13C-NMR (125 MHz, DMSO-
d6) δ (ppm): 194.6, 167.9, 159.6, 133.4, 132.5, 131.9, 130.3, 129.6, 128.5, 120.2, 116.4, 115.5, 114.4,
99.9, 66.9, 56.0, 55.1 ×2, 34.0, 32.6, 31.3, 26.9, 24.7, 23.3. ESI-MS m/z: 378.18 (M+H)+, calcd for
C24H27NO3: 377.20.
5.1.6.18 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-(4-morpholinophenyl)prop-2-en-1-one
1
(20). Yellow solid, 45.84% yield, m.p: 163.7-165.0 °C. H-NMR (500 MHz, DMSO--d6) δ (ppm):
10.13 (s, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 15.6 Hz, 1H), 7.72 (s, 1H), 7.66 (d, J = 4.6 Hz,
1H), 7.02 (d, J = 8.8 Hz, 2H), 6.53 (s, 1H), 5.90 (dd, J = 16.7, 6.5 Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz,
1H), 4.87 (d, J = 8.9 Hz, 1H), 3.82 (s, 3H), 3.77-3.70 (m, 4H), 3.32-3.29 (m, 4H), 3.22 (d, J = 6.5 Hz,
2H), 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 186.5, 159.8, 158.2, 153.7, 137.4, 131.4, 130.1,
129.9, 128.2, 118.9, 117.9, 114.9, 113.3, 112.9, 98.8, 66.8, 64.9, 56.5, 46.8, 33.1, 29.9, 19.6, 13.4.
ESI-MS m/z: 380.20 (M+H)+, calcd for C23H25NO4: 379.18.
5.1.6.19 (E)-1-[4-(1H-pyrazol-1-yl)phenyl]-3-(5-allyl-4-hydroxy-2-methoxyphenyl)prop-2-en-1-
one (21). Brownish yellow solid, 42.1% yield, m.p: 135.4-137.6 °C. 1H NMR (500 MHz, acetone-d6)
27

δ (ppm): 8.78 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.85 (dd, J = 8.7, 2.6 Hz, 1H), 7.78 (s, 1H), 7.56 (d, J
= 15.6 Hz, 1H), 7.41 (s, 1H), 7.29 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.2 Hz, 1H), 6.91 (d, J = 8.5 Hz,
1H), 6.48 (s, 1H), 5.90 (dd, J = 16.7, 6.5 Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz,
1H), 3.72 (s, 3H), 3.22 (d, J = 6.5 Hz, 2H), 13C-NMR (125 MHz, acetone-d6) δ (ppm): 188.79,
167.25, 160.21, 159.57, 158.42, 155.35, 140.34, 138.99, 134.57, 131.89, 130.43, 130.16, 129.63,
122.94, 119.68, 119.27, 116.22, 115.42, 109.35, 99.89, 56.03, 34.13. ESI-MS m/z: 362.30 (M+H)+,
calcd for C22H20N2O3: 360.15.
5.1.6.20 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[4-(dimethylamino)phenyl]prop-2-en-1-
one (22). Yellow solid, 48.3% yield, m.p: 151.6-153.1 °C. 1H-NMR (500 MHz, acetone-d6) δ (ppm):
8.91 (s, 1H), 8.03 (dd, J = 12.1, 9.5 Hz, 3H), 7.71 (d, J = 15.6 Hz, 1H), 7.61 (s, 1H), 6.80 (d, J = 8.9
Hz), 6.61 (s, 1H), 6.04 (dt, J = 16.7, 8.3 Hz), 5.09 (d, J = 17.1 Hz, 1H), 5.02 (d, J = 10.0 Hz, 1H),
3.88 (s, 3H), 3.38 (d, J = 6.3 Hz, 2H), 3.10 (s, 6H). 13C-NMR (125 MHz, acetone-d6) δ (ppm): 189.1,
168.7, 168.3, 142.5, 141.2, 139.4, 135.4, 129.7, 129.4, 127.2, 122.1, 118.4, 118.2, 115.9, 113.3,
113.1, 97.0, 64.0, 53.6, 44.2, 31.7. ESI-MS m/z: 338.34 (M+H)+, calcd for C21H23NO3: 337.17.
5.1.6.21 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[3-(dimethylamino)phenyl]prop-2-en-1-
one (23). Yellow solid, 59.36% yield, m.p: 159.8-162.8 °C. 1H NMR (500 MHz, CDCl3) δ (ppm):
8.91 (s, 1H), 8.05 (s, 1H), 8.03 (d, J = 2.6 Hz, 1H), 8.01 (d, J = 6.4 Hz, 1H), 7.71 (d, J = 15.6 Hz,
1H), 7.61 (s, 1H), 6.80 (d, J = 8.9 Hz, 2H), 6.61 (s, 1H), 5.90 (dd, J = 16.7, 6.5 Hz, 1H), 4.93 (dd, J
= 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 3.88 (s, 3H), 3.22 (d, J = 6.5 Hz, 2H), 3.10 (s, 6H).
13C-NMR (125 MHz, acetone-d6) δ (ppm): 188.2, 168.7, 168.3, 142.5, 141.0, 139.4, 135.6, 129.7,
129.4, 127.1, 122.1, 118.4, 118.2, 115.9, 113.4, 113.9, 97.0, 64.0, 53.7, 44.2, 31.7. ESI-MS m/z:
339.01 (M+H)+, calcd for C21H23NO3: 337.17.
28

5.1.6.22 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[2-(dimethylamino)phenyl]prop-2-en-1-
1
one (24). Yellow solid, 60.15% yield, m.p: 148.6-150.2 °C. H-NMR (500 MHz, CDCl3) δ (ppm):
8.87 (s, 1H), 8.05 (d, J = 5.4 Hz, 1H), 8.03 (d, J = 2.6 Hz, 2H), 7.99 (d, J = 6.4 Hz, 1H), 7.77 (d, J =
15.6 Hz, 1H), 7.61 (s, 1H), 6.80 (d, J = 8.9 Hz, 1H), 6.58 (s, 1H), 5.90 (dd, J = 16.7 Hz, 10.1, 6.5 Hz,
1H), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 3.88 (s, 3H), 3.22 (d, J = 6.5 Hz, 2H),
3.10 (s, 6H). 13C-NMR (125 MHz, acetone-d6) δ (ppm): 188.4, 169.4, 167.3, 142.3, 141.0, 138.6,
134.6, 129.6, 128.3, 126.2, 122.1, 118.5, 118.2, 115.9, 113.4, 113.9, 97.0, 63.2, 53.8, 44.2, 31.7.
ESI-MS m/z: 338.22 (M+H)+, calcd for C21H23NO3: 337.17.
5.1.6.23 (E)-3-(5-Allyl-4-hydroxy-2-methoxyphenyl)-1-[3-(dimethylamino)phenyl]prop-2-en-1-
one (25). Yellow solid, 59.36% yield, m.p: 167.3-169.5 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm):
8.89 (s, 1H), 8.04 (s, 1H), 8.03 (d, J = 2.6 Hz, 1H), 8.01 (d, J = 6.4 Hz, 1H), 7.71 (d, J = 15.6 Hz,
1H), 7.66 (d, J = 4.6 Hz, 2H), 7.61 (s, 1H), 7.02 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.2 Hz, 1H), 6.80 (d,
J = 8.9 Hz, 2H), 6.61 (s, 1H), 5.90 (dd, J = 16.7, 6.5 Hz), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J
= 8.9 Hz, 1H), 3.82 (s, 3H), 2.88 (s, 6H). 13C-NMR (125 MHz, acetone-d6) δ (ppm): 189.3, 160.6,
159.5, 157.7, 140.1, 134.3, 130.1 ×2, 129.7, 128.2, 121.6, 121.3, 120.3, 115.9, 112.4, 107.4, 101.4,
99.9, 72.5, 45.9, 34.1. ESI-MS m/z: 414.02 (M+H)+, calcd for C27H27NO3: 413.20.
5.1.6.24 (E)-3-(5-Allyl-4-hydroxy-2-isopropoxyphenyl)-1-[3-(dimethylamino)phenyl]prop-2-en-
1
1-one (26). Yellow solid, 48.98% yield, m.p: 137.4-139.6 °C. H-NMR (500 MHz, acetone--d6) δ
(ppm): 8.79 (s, 1H), 8.05 (d, J = 15.7 Hz, 1H), 7.92 (d, J = 15.6 Hz, 1H), 7.83 (d, J = 16.0 Hz, 2H),
7.76 (s, 1H), 7.53 (d, J = 4.9 Hz, 1H), 7.48 (s, 1H), 7.41 (d, J = 17.0 Hz, 1H), 6.45 (s, 1H), 5.90 (dd,
J = 16.7, 6.5 Hz, 1H), 4.93 (dd, J = 17.1, 1.7 Hz, 1H), 4.87 (d, J = 8.9 Hz, 1H), 3.82 (s, 3H), 2.88 (s,
29