Synthesis and biological activity of piperine derivatives as potential PPARγ agonists

Article abstract of DOI:10.2147/DDDT.S238245

Introduction: Peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in glucose, which is a ligand-mediated transcription factor. The lipid homeostasis often serves as a pharmacological target for new drug discovery and development. Materials and Methods: In the research, we synthesized a series of piperine derivatives and then used a fluorescence polarization-based PPARγ ligand screening assay to evaluate the agonistic activity of PPARγ. Then, we cultured human normal hepatocytes, which were treated with 100μM compounds 2a, 2t or 3d. Then, the levels of PPARγ gene were determined so as to show whether the compounds could activate or inhibit the expression of PPARγ. Results: A total of 30 piperine derivatives were synthesized and evaluated. Compound 2a was identified as a potential PPARγ agonist with IC₅₀ at 2.43 μM, which is 2 times more potent than the positive control rosiglitazone with IC₅₀ at 5.61μM. The human hepatocytes cells were cultured and treated with compounds 2a, 2t or 3d as described in the “Materials and Methods” section. We found that compounds 2a, 2t and 3d could activate PPARγ by 11.8, 1.9 and 7.0 times compared with the “blank”, with compound 2a activation being the most significant. Molecular docking studies indicated that the piperine derivative 2a stably interacts with the amino acid residues of the PPARγ complex active site, which is consistent with the results of the in vitro PPARγ ligand screening assay.

Full text of DOI:10.2147/DDDT.S238245

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Open Access Full Text Article
O R I G I N A L R E S E A R C H
Synthesis and Biological Activity of Piperine
Derivatives as Potential PPARγ Agonists
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
1–3
Introduction: Peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in
Yanli Wang
Yuan Yao4,5
glucose, which is a ligand-mediated transcription factor. The lipid homeostasis often serves
Jing Liu2,6
as a pharmacological target for new drug discovery and development.
Lili Wu7
Materials and Methods: In the research, we synthesized a series of piperine derivatives
and then used a fluorescence polarization-based PPARγ ligand screening assay to evaluate the
Tonghua Liu7
agonistic activity of PPARγ. Then, we cultured human normal hepatocytes, which were
Jian Cui1
treated with 100μM compounds 2a, 2t or 3d. Then, the levels of PPARγ gene were
David Yue-Wei Lee2
determined so as to show whether the compounds could activate or inhibit the expression
1School of Pharmacy, Minzu University of
of PPARγ.
China, Beijing 100081, People’s Republic
Results: A total of 30 piperine derivatives were synthesized and evaluated. Compound 2a
of China; 2Bio-Organic and Natural
was identified as a potential PPARγ agonist with IC50 at 2.43 μM, which is 2 times more
potent than the positive control rosiglitazone with IC50 at 5.61μM. The human hepatocytes
cells were cultured and treated with compounds 2a, 2t or 3d as described in the “Materials
and Methods” section. We found that compounds 2a, 2t and 3d could activate PPARγ by
11.8, 1.9 and 7.0 times compared with the “blank”, with compound 2a activation being the
most significant. Molecular docking studies indicated that the piperine derivative 2a stably
interacts with the amino acid residues of the PPARγ complex active site, which is consistent
with the results of the in vitro PPARγ ligand screening assay.
Products Laboratory, McLean Hospital,
Harvard Medical School, Boston, MA
02478, USA; 3Key Laboratory of Plant
Molecular Biology, Inner Mongolia
Autonomous Region Institute of
Biotechnology, Hohhot 010010, Inner
Mongolia, People’s Republic of China;
4State Key Laboratory of Reproductive
Regulation & Breeding of Grassland
Livestock, School of Life Sciences, Inner
Mongolia University, Hohhot 010070,
People’s Republic of China; 5Department
of Neurology, Inner Mongolia People’s
Hospital, Hohhot 010017, Inner
Keywords: PPARγ, piperine derivatives, ligand screening assay, molecular docking
Mongolia, People’s Republic of China;
6Natural Pharmacia International Inc.,
Burlington, MA 01803, USA; 7School of
Traditional Chinese Medicine, Beijing
University of Chinese Medicine, Beijing
100029, People’s Republic of China
Introduction
The peroxisome proliferator-activated receptors (PPARs) are ligand-activated
nuclear receptors (NRs) which play a pivotal role in the regulation of genes
associated with a wide range of physiological processes. In the chromatin, PPARs
are a heterodimer with retinoid X receptor (RXR) and play a role of specific PPAR
elements (PPRE) response to ligand binding. Ligand binding triggers the conforma-
tional change of LBD, which promotes the dissociation or recruitment of transcrip-
tion co-regulators, mainly through ligand-dependent activation function 2 (AF2).
Besides, post-translational modifications can modulate the affinity of the receptor
for co-regulators to determine whether the target gene is induced or suppressed.1–3
There is no doubt that PPARs regulate genes involved in the metabolism of lipid
and glucose. In addition, the PPARs are crucial to regulating some biological effects
associated with vascular and inflammation feature.4–6
Correspondence: David Yue-Wei Lee
Bio-Organic and Natural Products
Laboratory, McLean Hospital, Harvard
Medical School, Boston, MA 02478, USA
Tel +1617 855 2038
Email dlee@mclean.harvard.edu
Jian Cui
The PPARs also responding to the endogenous ligands including fatty acid
metabolite and organic synthesis.7,8 PPARα, PPAR β/δ and PPARγ have been
identified, which were significantly expressed in a tissue form. The most widely
School of Pharmacy, Minzu University of
China, Beijing 100081, People’s Republic
of China
Email cuijian@muc.edu.cn
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work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For
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studied PPARγ isoforms are responsible for adipogenesis, methylenedioxy ring as the alternative surrogates to
couple with the piperine.
glucose homeostasis, and regulation of lipid metabolism
and gene transcription.9,10 PPARγ also has a critical role in
insulin sensitization. Therefore, PPARγ is generally recog-
nized as an ideal therapeutic measurement of diabetes and
dyslipidemia. Thiazolidinediones (TZDs) such as rosigli-
tazone (RSG, Figure 1) and pioglitazone, are high-affinity
ligands and full agonists of PPARγ, which have been
successfully used to treat type II diabetes mellitus.11
TZDs have side effects in clinical application, we get the
information of the PPARγ active mechanism and biological
functions with the assistant of these synthetic ligands.
TZDs as one type of PPARγ agonists have also been
used as a potential drug candidate to develop effective
and safe medicine.12–14
This study describes the synthesis and biological
evaluation of piperine derivatives with the FP-based
PPARγ ligand screening assay. Compound 2a was iden-
tified as a potential PPARγ agonist with IC50 at 2.43 μM,
which is 2 times more potent than the positive control
Rosiglitazone with IC50 at 5.61μM. Molecular docking
studies have shown that piperine derivative 2a fitted
nicely with the amino acid residues at the active site
of the PPARγ complex and is consistent with the result
of in vitro ligand assay.
Materials and Methods
Chemical Synthesis
Synthesis of Piperic Acid
Natural products have been served as rich sources
for the discovery and development of modern medi-
cine. Piperine (Figure 1) isolated from the traditional
Mongolian medicine of piper longum L., which is well
known for its extensive biological activities. It has
been reported that piperine attenuates the differentia-
tion of fat cell, which down-regulating the activity and
expression of PPARγ, prohibiting the process of
adipogenesis15,16 and increases the bioavailability of
many other drugs.17,18 However, piperine has an
acutely toxic effect,19 its biological applications are
limited due to the poor solubility in aqueous environ-
ments. According to related reports that piperoyl-amino
conjugates have better biological activity than
piperine,20 In addition, the results of piperine pharma-
cokinetic studies showed that after the piperine meta-
bolism by the human, it retains methylenedioxy ring
and conjugated double bonds while the piperidine ring
is modified to form a propionic acid group. Such
piperine derivatives may have the advantages of struc-
tural diversity, improved solubility, and reduced toxi-
city. Therefore, we wish to utilize amino acids and
Piperine (64g, 219.9 mmol), KOH (113.5g, 17.9mmol)
was refluxed with ethanol (1500mL) for 12h, the mixture
was allowed to stand for cooling, suction filtration under
reduced pressure, and the filter cake was washed with
ethanol (95%) to pH=7, and filtered under reduced pres-
sure to give crystals of brown piperic potassium salt. The
crystal was dried to obtain the product. The piperic potas-
sium salt was dissolved with water and then, gradually
acidified with dilute hydrochloric acid, formed yellow
precipitate, vacuum filtered, and washed with water
(1400mL) and recrystallized from acetone to afford yellow
crystalline compound.
General Procedure for the Synthesis of Piperic
Conjugates (2a-2j)
Pre-dried dichloromethane (DCM) was added to Piperic
acid (2.18g, 10mmol), and then added thionyl chloride
(73.8mmol, 14.4mL), the contents were refluxed under
nitrogen atmosphere for 2 hours. Removed the excess
thionyl chloride under reduced pressure, and then got
acid chloride of piperine. Amino acid ester hydrochloride
(20mmol) in one portion to acid chloride of piperine in
DCM; and Et3N in DCM under ice-cooling, stirred 2h at
Figure 1 Chemical structures of piperine and RSG.
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Wang et al
room temperature. Then added water (50 mL), washed the
organic layer with water (2*25mL), dried over with anhy-
drous sodium sulfate, concentrated to give the crude pro-
duct. The crude product was dissolved in ethyl acetate as
few as possible, and the yellow solids were obtained by
slowly recrystallization with petroleum ether to get the
pure compounds (2a–2j).
Gene Expression Experiments to Activate
PPARγ Assay
Cell Culture
Human normal hepatocytes (HL-7702[L-02]) purchased
from the Biotechnology Research of Beijing BeNa
Chuanglian in China. Quickly put the cryopreservation
tube containing normal human hepatocytes into a 37 °C
water bath and shake it constantly, so that the liquid in the
tube will melt rapidly in 1–2 minutes. The cells suspension
was transferred into the culture bottle adding CM 2–1
medium (90% RPMI-1640 + 10% FBS. RPMI-1640:
1640 medium, containing glutamine). And the cells were
cultured in 5% CO2 incubator at 37 °C. The next day, the
culture medium was changed for routine culture and pas-
sage. The cells grew well and were monolayer adherent.
Depending on the number of cells, fresh culture medium
should be replaced once every 2–3 days, and the passage
should be 1:3 once every 4–5 days. When the cells grew to
about 70% of the dish area, starvation culture was carried
out. Then the cells were culture in RPMI-1640 medium
without serum, which made the cells in a low nutritional
state. It is for starvation culture. After 24 hours, the
100 μM compounds 2a, 2t or 3d was added in the cultures.
The “blank” indicated without drugs.
General Procedure for the Synthesis of Piperic
Conjugates (2k–2t)
Mixed neutral alumina (15g) with KF (10 g) in 200 mL of
water for the preparation of KF-Al2O3. Then removed water
at 50–60°C in a rotary evaporator, further dried this reagent
in a vacuum oven for 12h. Added Piperic amino acid ester
conjugates (1.65mmol, 0.5g) to KF-Al2O3(3.13mol, 40%
KF) and then stirred this dry mixture at room temperature
in a round bottom flask to ensure uniform mixing of solid
support KF-Al2O3 with the substrate. Transfer the uni-
formly dispersed powder to a 50mL eggplant bottle and
heat and stir at 50°C for 2h. Then the reaction mixture was
added water (5mL), stirred for 5min and then filtered.
Neutralized the filtrate by adding aqueous HCl and obtained
the precipitate filtered, dried it, obtained the pure piperic
amino acid conjugates compounds.
General Procedure for the Synthesis of
Derivatization of Bisphenol Piperic Amino Acid
(Ester) Conjugates (3a–3e;3k–3o)
Total RNA Extraction
Total RNAs of HL-7702[L-02] cells were isolated using
the Trizol reagent kit (TRIzol™ Plus, Invitrogen, USA),
following the manufacturer’s instructions and described
before. RNA integrity, purity, and concentration were
detected by electrophoresis and the NanoDrop 2000C
spectrophotometer (NanoDrop Technologies, Thermo
Scientific™, USA).23 The integrity and purity of RNA
samples met the requirements of RT-qPCR analysis.24
http://dx.doi.org/10.17504/protocols.io.qafdsbn.
Dissolved piperic conjugates (2a-2e;2k-2o) with anhydrous
DCM. Added BBr3 at −78 °C, stirred for 2h hours at 0°C, the
solvent was removed under reduced pressure. The product
was purified by column chromatography in DCM/MeOH
(10:1) to get the pure compounds (3a-3e;3k-3o).
FP-Based PPARγ Ligand Screening Assay
We selected the PPARγ Ligand Screening Assay Kit,
which provided a single-step assay to screen PPARγ
ligands based on fluorescence polarization (FP).21,22 In Relative Quantitative Real-Time PCR
this method, the ligand of PPARγ in conjunction with The cDNA synthesis was performed with 1 μg of total RNA
fluorescein and displacement probe. The fluorescent as template in a 20 μL reaction mixture including 4 μmol/L
probes were replaced by PPARγ ligands, agonists and of each primer using the TransScript® One-Step gDNA
antagonists, resulting in the reduction of FP. Firstly, we Removal and cDNA Synthesis SuperMix Kit (TransGens,
prepared the assay cocktail and measured every ligand China) as described in the manufacture’s protocol. The
concentration in duplicate, covered the plate and used for primers for RT-qPCR were designed, meeting the require-
60–90 min at room temperature. Secondly, we read the ments with Tm values of 60°C and products shorter than
data in the wavelengths of 470 nm and 530 nm, respec- 200bp. The pairs of primer PPARγ-RT-F (TATCGA
tively. The results were calculated by FP. According to the CCAGCTGAATCCAGAG) and PPARγ-RT-R (GGGGGTG
kit protocol, we calculated IC50 with the mP-concentra- ATGTGTTTGAACTTG); Human-actin-RT-F (CTCCATC
tion displacement curve (Cayman).
CTGGCCTCGCTGT) and Human-actin-RT-R (GCTGTC
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ACCTTCACCGTTCC) were used in RT-qPCR assay. The and the grid contained 14×8×44 autogrid points, which is
RT-qPCR assay was performed using the PerfectStart TM 0.375Å spacing. In the population, the number of individuals
was 150, the maximum number of energy evaluations was 25
million and the generations were 27,000, respectively.
Green qPCR SuperMix kit (TransGens, China). And the
Light Cycler 480 II Real-Time PCR System (Roche,
Switzerland) was used as a quantitative analysis detector.
The RT-qPCR procedure used default procedure (95 °C for
30 s, 45 cycles of 95°C for 10 s, 60 °C for 20 s) and the
melting curves were performed immediately as described
previously.23 The CT value of each reaction can be given
automatically. The relative standard curve was made by
template concentration (X-axis) and threshold cycles
(Y-axis). Amplification efficiency (E) was calculated from
equation 1, and gene dose was determined from equation 2,
as described previously. Then, the target gene relative
Results and Discussion
Chemistry
Following the synthetic scheme shown in Scheme 1, a
total of 30 piperine derivatives were synthesized (see
Supplementary materials). The basic skeleton of piper-
ine consists of methylenedioxyphenyl (MDP) moiety,
basic six-membered piperidine moiety attached to side-
chain via an amide and linkage side-chain which con-
sists of double bonds. This study aimed to establish
SAR focusing on the side chain of piperine. A series
of piperine derives shown in Table 1. Converted piper-
ine into acid by hydrolysis used ethanolic KOH.
Converted the acid into acid chloride used thionyl chlor-
ide, and then followed by condensation with the amine
moiety of amino acid in dichloromethane. Basically, the
part of piperine’s piperidine moiety was replaced by
different amino acid esters and substituted aniline to
obtain compounds 2a–2t. Boron tribromide can be
used for the demethylation of aryl methyl ethers in the
expressions were calculated by formula 2(-ΔΔCt) 24
The
.
β-actin gene was the reference gene. The expression level
of target genes was normalized to this reference gene. The
final data came from three test repetitions, each with three
technical repetitions.
Molecular Docking
We used the crystal structure (PDB: 4EMA) of the PPARγ
docking template combined with RSG. We used AutoDock
4.2.6 docking compounds 2a, 2t and 3d into the ligand-binding
domain.25 Implemented AutodockTools 1.5.6 to build the presence of many functional groups without affecting
autogrid box. Based on the known ligand, Set the grid center, these, it does not affect the cleavage of methylenedioxy
Scheme 1 Reagents and conditions.
Notes: (i) KOH, ethanol, reflux, 12h; HCl (ii) SOCl2, DCM, reflux, 12h (iii) DCM, (2a-2j), Et3N, rt, 2h (iv) KF- Al2O3, 50°C, 2h (v) BBr3, DCM, -78°C, 2h.
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Table 1 Synthesis of Piperine Derivatives
Compounds
2a
R
Compounds
2k
R’
2b
2c
2L
2m
2d
2e
2n
2o
2f
2p
2g
2h
2q
2r
2i
2s
2j
2t
3a
3k
(Continued)
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Table 1 (Continued).
Compounds
3b
R
Compounds
3L
R’
3c
3m
3d
3e
3n
3o
groups. Halogenation was done with boron tribromide to ligand would result in a larger reduction in the MP value
give piperine catechol 3a-3e, 3k-3o.
of the well. So, plotting mP vs ligand concentration
allowed the construction of an IC50 curve with a broad
dynamic range. As shown in Figure 2, thirteen compounds
(2a, 2e, 2i, 2m, 2n, 2p, 2q, 2r, 2s, 2t, 3a, 3b, 3c, 3d) were
found to exhibit potent agonistic activity. As shown in
Figure 3, the MP values of three compounds (2a, 2t, 3d)
FP-Based PPARγ Ligand Screening Assay
We used a convenient FP-based PPARγ ligand screening
assay to assess the agonistic potency of 30 piperine
derivatives.26,27 We chose RSG as a PPARγ agonist as a
positive control. The addition of large amounts of a PPARγ decreased significantly with the increasing of the
80
70
60
50
40
30
20
10
0
compounds
Figure 2 FP-based PPARγ ligand screening assay of piperine derivatives.
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Figure 3 Compounds 2a, 2t, 3d showed potent effects of FP comparable to RSG.
logarithm of the concentration, and this trend was consis- relative expression of the PPARγ activity increased by 11.8,
tent with the positive control. However, the MP values of 1.9 and 7.0 times in compounds 2a, 2t and 3d, respectively,
compared with the “blank” (Figure 4). These results indi-
cated that compounds 2a, 2t, and 3d could activate PPARγ,
in which three compounds, compound 2a activation was the
most significant.
negative control (DMSO) were consistent with the change
of concentration. As shown in Table 2, the compound 2t
(IC50=1.03 μM) showed the most potent effects of FP, the
compound 2a (IC50=2.43 μM) showed the remarkable
agonistic activity on PPARγ, they were both more potent
than RSG (IC50=5.61 μM) and piperine(IC50=18.35 μM).
Besides, the compound 3d (IC50=79.32μM) showed mod-
Molecular Docking
Firstly, according to the PPARγ complex’s X-ray structure,
erate agonistic activity on PPARγ. Among the 30 small- the binding mode of RSG has been analyzed. The TZD of
RSG and the three amino acid residues (His323, Ser289,
and Tyr473) forms an H-bonding interaction. The RSG’s
hydrophobic tail interacts with the residues of Ile341,
Val339, Phe363, Leu353, Phe368, Met364 and Leu330.
So, we have chosen low binding energy and a similar
binding mode for the study to dock poses. In Table 3,
the in vitro agonistic activity showed good correlations
with the molecular properties. The potent compounds
proved useful for examining the piperine derivatives
because they displayed high values of tPSA and LogP,
we did the experiment to identify the complex for the
docking study. The binding energy of RSG was at −5.3
kcal/mol, while that of 2a was at −1.2 kcal/mol. This
discrepancy showed that the chemical structure of piperine
derivative and RSG play a significant role in the binding
pocket and it is conceivable that the receptor was differ-
molecule piperine derivatives (2a-2t, 3a-3e and 3k-3o),
piperine derivatives exhibited biological activity better
than piperine. Especially, we founded the group of the
amino acid-modified piperine contribute the most to the
agonist potency (2t > 2a> 3d). Compounds 2t and 2a are
being investigated in vivo and results will be reported
separately.
The Compounds 2a, 2t, and 3d Can
Activate the Gene Expression of PPARγ
We cultured human normal hepatocytes, which were treated
with 100 μM compounds 2a, 2t or 3d. And then the levels
of PPARγ gene expression were determined. So as to show
whether the compounds could activate or inhibit the expres-
sion of PPARγ. The human hepatocytes HL-7702[L-02]
cells were cultured and treated with compounds 2a, 2t or entially sensitized by the binding moieties of RSG and
3d as described in the method section. We found that the piperine. There was a good correlation between piperine
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Table 2 IC50s of Piperine Derivatives in FP-Based PPARγ Ligand
Table 3 The Molecular Properties and Binding Energy of
Screening Assay
Selected Piperine Derivatives
Compounds
2a
Chemical Structure
IC50(µM)
Compounds Molecular
Properties
Binding Energy kcal/mol
2.43
tPSA
LogP
2a
73.86
96.89
95.6
1.83
2.79
1.61
2.78
3.21
−1.2
−2.4
−2.2
−6.5
−5.3
2t
2t
1.03
3d
Piperine
RSG
38.77
71.00
hydrophobic tail was encompassed from Val339, Leu340,
Ile341, Try327, Leu330 by a surface formed. The 2t inter-
acted with residues of Tyr473, His449, Ser342 to form the
PPARγ active sites’ hydrogen bonds. The 3d interacted
with residues of Ser289 to form hydrogen bonds to the
PPARγ active sites His449. (Figure 5A–E).
3d
79.32
Piperine
RSG
18.35
5.61
Conclusion
PPARγ is considered as a therapeutic target for the
treatment of diabetes and dyslipidemia, which modu-
lates the transcription of genes responsible for glucose
homeostasis, adipose differentiation, and lipid metabo-
lism. In this study, we synthesized 30 piperine deriva-
tives and evaluated with quick FP-based PPARγ ligand
screening assay to agonistic activity with an easy. A
preliminary structure-activity relationship was estab-
lished. Among the derivatives tested, ligand 2a and 2t
derivatives with similar chemical structures. But, among
the three agonists (2a, 2t, and 3d), compound, 2a exhib-
ited the highest PPARγ activity and the highest binding
energy. The 2a interacted with Ser289’s residues to form
the PPARγ active sites’ hydrogen bonds and its exhibited potent PPARγ agonistic activity than RSG
18.0
**
11.8
16.0
14.0
12.0
10.0
**
7.0
8.0
6.0
4.0
*
1.9
1.0
2.0
0.0
Blank
2a
2t
3d
Figure 4 The relative expression level of PPARγ in compound of 2a, 2t and 3d.
Notes: The values are the average of three experiments. The standard error bars are as indicated. The significance of discrepancy between data by single factor analysis of
variance. The “*” showed significant differences between blank and compounds, “*”p-value<0.05; “**”p-value<0.001.
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Figure 5 The binding modes of agonists with PPARγ.
Notes: H-binding interactions of RSG and other agonists were shown in yellow dotted line, respectively. (A) 2a, (B) 2t, (C) 3d, (D) piperine, (E) RSG.
5. Varga T, Czimmerer Z, Nagy L. PPARs are
a
unique set
and piperine. By test the gene expression of PPARγ in
human normal hepatocytes, we found that compound
2a, 2t, and 3d could activate PPARγ, compound 2a
activation was the most significant. Molecular docking
studies indicated that the piperine derivative 2a stably
interacts with the amino acid residues of the PPARγ
complex active site, which is consistent with the results
of the in vitro PPARγ ligand screening assay. The
compound of 2a’s further chemical modifications and
in vivo evaluation is in progress.
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Acknowledgments
This research was supported by the Bio-Organic and
Natural Products Laboratory, McLean Hospital, Harvard
Medical School and the China Scholarship Council.
12. Ohashi M, Gamo K, Oyama T, Miyachi H. Peroxisome proliferator-
activated receptor gamma (PPARγ) has multiple binding points that
accommodate ligands in various conformations: structurally similar
PPARγ partial agonists bind to PPARγ LBD in different conforma-
tions. Bioorg Med Chem Lett. 2015;25:2758–2762. doi:10.1016/j.
bmcl.2015.05.025
Disclosure
The authors report no conflicts of interest in this work.
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