Piperine Congeners as Inhibitors of Vascular Smooth Muscle Cell Proliferation

Article abstract of DOI:10.1055/s-0035-1546165

Successful vascular healing after percutaneous coronary interventions is related to the inhibition of abnormal vascular smooth muscle cell proliferation and efficient re-endothelialization. In the search for vascular smooth muscle cell anti-proliferative

Full text of DOI:10.1055/s-0035-1546165

Original Papers 1065
Piperine Congeners as Inhibitors of Vascular Smooth
Muscle Cell Proliferation*
Authors
Christina E. Mair1,2 #, Rongxia Liu1,4 #, Atanas G. Atanasov1, Laurin Wimmer3, Daniel Nemetz-Fiedler1, Nadine Sider1,
Elke H. Heiss1, Marko D. Mihovilovic3, Verena M. Dirsch1, Judith M. Rollinger1,2
1
Affiliations
Department of Pharmacognosy, University of Vienna, Vienna, Austria
Institute of Pharmacy/Pharmacognosy, University of Innsbruck, Innsbruck, Austria
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria
School of Pharmacy, Yantai University, Yantai, China
2
3
4
Key words
Abstract
amide (20). They showed IC50 values of 3.38, 6.00,
"
l Piper nigrum
!
and 7.85 µM, respectively. Furthermore, the syn-
thetic compound (2E,4E)-5-(4-fluorophenyl)-1-
(piperidin-1-yl)penta-2,4-dien-1-one (12) was
found to be cell type selective, by inhibiting vas-
cular smooth muscle cell proliferation with an
IC50 of 11.8 µM without influencing the growth
of human endothelial cells.
"
l Piperaceae
Successful vascular healing after percutaneous
coronary interventions is related to the inhibition
of abnormal vascular smooth muscle cell prolifer-
ation and efficient re-endothelialization. In the
search for vascular smooth muscle cell anti-pro-
liferative agents from natural sources we identi-
fied piperine (1), the main pungent constituent
of the fruits from Piper nigrum (black pepper). Pi-
perine inhibited vascular smooth muscle cell pro-
liferation with an IC50 of 21.6 µM, as quantified by
a resazurin conversion assay. Investigations of ten
piperamides isolated from black pepper fruits and
15 synthesized piperine derivatives resulted in
the identification of three potent vascular smooth
muscle cell proliferation inhibitors: the natural al-
kaloid pipertipine (4), and the two synthetic de-
rivatives (2E,4E)-N,N-dibutyl-5-(3,5-dimethoxy-
phenyl)penta-2,4-dienamide (14) and (E)-N,N-di-
butyl-3-(naphtho[2,3-d][1,3]dioxol-5-yl)acryl-
"
l Piperine
"
l vascular smooth muscle cells
"
l inhibition of VSMC prolifera-
tion
l SAR
"
"
l restenosis
Abbreviations
!
BrdU:
EBM:
GABAA:
HUVEC:
LDH:
5-bromo-2′-deoxyuridine
endothelial cell basal medium
γ-aminobutyric acid type A
human umbilical vein endothelial cell
lactate dehydrogenase
received
revised
accepted
March 12, 2015
May 1, 2015
May 5, 2015
PDGF:
RU:
platelet-derived growth factors
relative units
Bibliography
DOI http://dx.doi.org/
10.1055/s-0035-1546165
Published online July 1, 2015
Planta Med 2015; 81:
SAR:
VSMC:
structure activity relationship
vascular smooth muscle cell
1065–1074 © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Introduction
jury, mediators like cytokines and growth factors,
including PDGF, promote a change of the VSMC
phenotype, which does no longer regulate con-
traction but vascular reconstruction [2]. Abnor-
mal migration, proliferation and apoptosis of
VSMCs at this stage, are key elements in the devel-
opment of atherosclerosis and restenosis [3].
Drug-eluting stents are the state of the art treat-
ment of coronary artery diseases and aim to over-
come restenosis after percutaneous coronary in-
terventions by inhibiting VSMC proliferation [4].
Paclitaxel (taxol), a natural, microtubules stabiliz-
ing compound, and sirolimus, everolimus and zo-
tarolimus, i.e., rapamycin derivatives acting as
mTOR inhibitors, are the most prominent drugs
!
Correspondence
Atherosclerosis is the worldwide leading cause of
heart disease and stroke. In westernized societies
this disease condition is the underlying cause of
about 50% of all deaths [1]. VSMC proliferation
strongly contributes to initial atherosclerotic
plaque formation as well as to restenosis. Reste-
nosis means the pathological re-narrowing of the
blood vessel lumen and is a severe complication
occurring after angioplasty, stenting procedures
or bypass surgery. The major function of VSMCs
in their quiescent or contractile phenotype is to
regulate blood vessel diameter and blood flow in
normal mature blood vessels. After an initial in-
Judith M. Rollinger
Department of Pharmacognosy
Faculty of Life Sciences
University of Vienna
Althanstrasse 14
1090 Vienna
Austria
Phone: + 431427755255
Fax: +4314277855255
Judith.Rollinger@univie.ac.at
Correspondence
Atanas G. Atanasov
Department of Pharmacognosy
Faculty of Life Sciences
University of Vienna
Althanstrasse 14
1090 Vienna
Austria
Phone: + 431427755231
Fax: +4314277855231
Atanas.Atanasov@univie.ac.at
* Dedicated to Professor Dr. Dr. h.c. mult. Adolf Nahrstedt
on the occasion of his 75th birthday.
#These authors contributed equally to this work.
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074

1066 Original Papers
used therefore [5]. However, unresolved drug-related issues, like
(0.747 0.065 RU). All other compounds of this cluster, including
two piperamides with a pyrrolidinyl (2 and 7) instead of the pi-
peridinyl moiety, showed no significant enhancement of VSMC
inhibition.
impaired re-endothelialization or delayed thrombosis induction,
remain, since both drug classes not only inhibit VSMC prolifera-
tion but also endothelial cell growth [6–9]. Hence, identifying
new VSMC proliferation inhibitors is of high relevance.
Cluster B consists of only one compound, the piperidin-dimer
chabamide (8), which had no noteworthy inhibitory effect.
Cluster C (9–14) comprises compounds with variations of the ar-
omatic function. Removal or opening of the dioxole ring did not
significantly alter the VSMC inhibitory effect in comparison to 1
(11 and 13) or even decreased it (10). The same was observed
for compound 9, which in addition has a shortened linker region.
In contrast, when the dioxole was replaced by 3-fluoro (12), the
proliferation was inhibited significantly (0.426 0.079 RU). Intri-
guingly, supplementing the aromatic cycle with two methoxy
moieties in meta positions and additional replacement of the pi-
peridinyl ring by an n-dibutylamide (14) completely blocked the
VSMC proliferation (0.000 0.004 RU).
Derivatives from cluster D (15–20) are characterized by a naph-
tho dioxole moiety. This compound class does not enhance the
VSMC inhibitory activity, unless the piperidinyl moiety was al-
tered to noncyclic substituents, like n-dipropyl- (19) or n-dibuty-
lamide (20), which clearly improved the anti-proliferative activ-
ity (0.573 0.064 and 0.005 0.010 RU, respectively). Similar to
compound 14, compound 20 was able to decrease VSMC prolifer-
ation at 30 µM to nearly zero. Both are characterized by the n-di-
butylamide feature.
Dried unripe fruits of Piper nigrum L. (Piperaceae), commonly
called black pepper, are referred to as “king of spices” and are
known in almost every cuisine worldwide. Responsible for its al-
luring odour are various monoterpenes, like linalool, limonene,
or sabinene [10] while the typical pungency of black pepper and
other Piper species is mainly attributed to the predominant pi-
peramide piperine (1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pen-
tadienyl]piperidine) [11,12]. Also other piperamides and diverse
unsaturated, long-chain fatty acid amides are reported to con-
tribute to the tingling sensation [13–15]. Further constituents of
P. nigrum and related species include lignans and flavonoids [16].
Besides its use as spice, black pepper is described to be endowed
with medicinal and disease preventing properties, e.g., anti-in-
flammatory, anti-oxidant, and bactericidal activity [11,17,18].
The main constituent piperine is reported to enhance drug bio-
availability and to modulate GABAA receptors, besides various
other pharmacological activities, including analgesic, anti-in-
flammatory, anti-pyretic, anti-convulsive, anti-oxidant, anti-de-
pressant, anti-apoptotic, anti-cancer, and anti-viral properties
[12,19–21]. In this study, we identified piperine (1) to be a prom-
ising inhibitor of VSMC proliferation, which was very recently al-
so described by others [22]. We used piperine as lead compound
to synthesize derivatives thereof and isolated structurally related
amides from the fruits of P. nigrum to deduce a qualitative SAR
with regard to the anti-proliferative effect in VSMCs.
Compounds from cluster E (21–25) were modified in the linker
region by replacing the carbon chain by an aryl spacer. Only 21
and 25, the first containing a naphthalene and the second a ben-
zene cycle as aryl spacer, revealed an increase in anti-prolifera-
tive activity with values of 0.542 0.119 and 0.682 0.188 RU, re-
spectively. In the case of 25, the piperidinyl moiety was addition-
ally altered in analogy to compound 19 (cluster D).
Results and Discussion
!
To sum up, at a concentration of 30 µM, the natural isolate 4 and
the two synthetic compounds 14 and 20, both comprising noncy-
clic n-dibutylamide substituents at the tertiary amide, were able
to stop VSMC proliferation nearly completely. Also 12 showed
promising activity below 0.49 RU, whereas 11, 15, 19, 21, 24,
and 25 inhibited proliferation in the range of 0.50 to 0.79 RU
and had therefore comparable efficacy to 1 (0.747 0.065 RU).
Subsequently, IC50s were determined for all derivatives with val-
In a biological assessment, we identified the piperamide piperine
"
(1) (l Table 1) from P. nigrum as a VSMC proliferation inhibitor
"
(l Fig. 1). In a concentration dependent manner, it revealed an
anti-proliferative effect in the resazurin conversion assay, with
an IC50 of 21.6 µM.
In order to identify compounds with enhanced activity and to de-
duce a SAR, nine additional piperamides were isolated from black
pepper (2–10). All of them have been described previously as
compounds of P. nigrum (2, 5, 10 [23]; 3 [24]; 4 [25]; 6 [26]; 7
[27]; 8 [28]; 9 [29]) and their structures were elucidated by com-
paring spectral data (NMR, MS, and IR) with those published be-
fore (1, 3 [30]; 2, 5, 7, [27]; 4 [25]; 6 [26]; 8 [31]; 9, 10 [32]). In
addition to the isolated compounds, 15 further congeners were
synthesized (11–25). All piperine analogues were subjected to
"
ues below 0.79 RU (l Table 1). Compounds 11, 15, 24, and 25
showed similar or higher IC50 values than piperine (1) and were
therefore not further investigated. Compounds 4, 14, and 20 were
highly active with IC50s of 3.38, 6.00, and 7.85 µM, respectively.
Also the synthetic derivatives 12 (11.8 µM), 19 (14.8 µM), and 21
(15.3 µM) decreased metabolic activity with lower IC50 values
than that of 1.
The used resazurin conversion assay is based on the ability of via-
ble cells to metabolize added resazurin into the highly fluores-
cent resorufin. Measured fluorescence correlates generally well
with cell number. However, redox-active chemicals or treat-
ments modulating the cellular metabolic capacity can potentially
interfere with this assay.
"
cell-based assays. Their chemical structures are shown in l Table
1 together with their anti-proliferative effect at 30 µM (expressed
in RU).
In general, the chemical structure of the lead compound piperine
(1) comprises three main parts: an aromatic moiety (1,3-benzo-
dioxole), a four carbon chain linker region with two conjugated
double bonds, and an amidic bound piperidinyl moiety. To derive
a SAR, the congeners were clustered by their type of structural
Therefore, to confirm the anti-proliferative effect, the two most
active compounds, the natural derivative 4 and the synthesized
compound 14, were additionally assayed with the 5-bromo-2′-
deoxyuridine (BrdU) incorporation assay, which detects integra-
tion of BrdU into newly synthesized DNA of dividing cells by im-
"
modification (l Table 1).
Cluster A (2–7) focuses on alterations of the linker region, like
carbon chain length and number of double bonds. Compound 4,
differing from the other derivatives in this group by having seven
C atoms with only one double bond in the linker chain, revealed a
significant increase in activity (0.014 0.018 RU) in contrast to 1
"
munoassay. As shown in l Fig. 2, the compounds blocked DNA
synthesis in treated cells with IC50 values of 4.39 (4) and
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074

Original Papers 1067
Table 1 Inhibition of VSMC proliferation at 30 µM of isolated (2–10) and synthesized (11–25) congeners of piperine (1), and IC50 values of the most potent
identified compound. Results were obtained with the resazurin conversion method and presented as means SD in RU (n = 3 for all data groups).
Compound,
cluster
Structure
Name
Inhibition of VSMC proliferation
at 30 µM
IC50 (µM)
(relative units)
0.747 0.065**
1, A
2, A
3, A
4, A
Piperine
21.6
Piperylin
0.938 0.114
0.942 0.043
0.014 0.018***
Piperoleine A
Pipertipine
3.38
5, A
6, A
7, A
8, B
Pipernonaline
0.804 0.059
0.803 0.094
1.024 0.089
0.819 0.089
Dehydropipernonaline
(2E,4E,8E)-9-(Benzo[d][1,3]dioxol-5-yl)-
1-(pyrrolidin-1-yl)nona-2,4,8-trien-1-one
Chabamide
9, C
N-trans Feruloylpiperidine
1.118 0.082
10, C
11, C
12, C
Feruperine
1.059 0.113
(2E,4E)-5-Phenyl-1-(piperidin-1-yl)penta-
2,4-dien-1-one
0.769 0.075***
0.426 0.079***
48.3
11.8
(2E,4E)-5-(4-Fluorophenyl)-1-(piperidin-
1-yl)penta-2,4-dien-1-one
cont.
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074

1068 Original Papers
Table 1 Continued
Compound,
cluster
Structure
Name
Inhibition of VSMC proliferation
at 30 µM
IC50 (µM)
(relative units)
0.810 0.011***
13, C
(2E,4E)-5-(4-Methoxyphenyl)-1-(piperi-
din-1-yl)penta-2,4-dien-1-one
14, C
(2E,4E)-N,N-Dibutyl-5-(3,5-dimethoxy-
phenyl)penta-2,4-dienamide
0.000 0.004***
6.00
26.1
15, D
16, D
Naphtho[2,3-d][1,3]dioxol-6-yl(piperi-
din-1-yl)methanone
0.716 0.059***
0.836 0.176
Naphtho[2,3-d][1,3]dioxol-5-yl(piperi-
din-1-yl)methanone
17, D
18, D
(E)-3-(Naphtho[2,3-d][1,3]dioxol-6-yl)-1-
(piperidin-1-yl)prop-2-en-1-one
0.831 0.097***
0.828 0.057***
(E)-3-(Naphtho[2,3-d][1,3]dioxol-5-yl)-1-
(piperidin-1-yl)prop-2-en-1-one
19, D
20, D
(E)-3-(Naphtho[2,3-d][1,3]dioxol-5-yl)-N,
N-dipropylacrylamide
0.573 0.064***
14.8
7.85
(E)-N,N-Dibutyl-3-(naphtho[2,3-d][1, 3]
dioxol-5-yl)acrylamide
0.005 0.010***
21, E
22, E
(5-(Benzo[d][1,3]dioxol-5-yl)naphthalen-
1-yl)(piperidin-1-yl)methanone
0.542 0.119***
0.899 0.171
15.3
(5-(Benzo[d][1,3]dioxol-5-yl)thiophen-2-
yl)(piperidin-1-yl)methanone
cont.
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074

Original Papers 1069
Table 1 Continued
Compound,
cluster
Structure
Name
Inhibition of VSMC proliferation
at 30 µM
IC50 (µM)
(relative units)
0.974 0.055
23, E
(2-(Benzo[d][1,3]dioxol-5-yl)phenyl)(pi-
peridin-1-yl)methanone
24, E
25, E
(3-(Benzo[d][1,3]dioxol-5-yl)phenyl)(pi-
peridin-1-yl)methanone
0.790 0.041***
0.682 0.188***
46.0
22.3
3-(Benzo[d][1,3]dioxol-5-yl)-N,N-dipro-
pylbenzamide
** p < 0.01; *** p < 0.001
centration of 30 µM, compounds 14, 19, 20, and 21 influenced
HUVEC growth with values above 0.6 RU. The piperamide 4 in-
hibited HUVEC proliferation at 30 µM stronger than the other
compounds. However, the IC50 of 3.38 µM in VSMCs indicates a
"
much higher efficacy in this cell type (l Fig. 3). Interestingly,
compound 12 did not influence HUVEC viability at all up to
30 µM, which renders it a promising cell selective candidate.
All compounds with IC50 values below 16 µM (4, 12, 14, 19, 20,
and 21) were additionally tested for their cytotoxic effects by
measuring LDH inside cells and in cell supernatants. All com-
pounds were well tolerated up to 30 µM, except compounds 4
"
and 14, which showed some cytotoxic effects at 30 µM (l Fig. 4).
In this study, we identified six compounds (4, 12, 14, 19, 20, and
21) out of 24 investigated piperine derivatives with enhanced
anti-proliferative activity in VSMC compared to the natural scaf-
fold piperine (1) itself (IC50 of 21.6 µM). This major alkaloid from
black pepper was just recently also described by Lee et al. to in-
hibit VSMC migration at a concentration of 100 µM [22]. Piperine
and congeners are reported to modulate GABAA receptors and to
activate transient receptor potential vanilloid type 1 receptors
[33,34]. In pharmacokinetic studies performed in Wistar rats, 1
revealed an absolute oral bioavailability of 24% [35], and is re-
ported to be a potent bioavailability enhancer for drugs by inhib-
iting CYP3A4 and human P-glycoprotein [36–39]. Beside its mul-
titude of described activities, e.g. analgesic, anti-pyretic, anti-
convulsive, anti-oxidant, anti-depressant, anti-apoptotic, and
anti-viral [12,19,20], Kumar et al. demonstrated an anti-inflam-
matory activity for 1 by suppressing NF-kappa B and Ikappa B ki-
nase activation [40]. These reported activities together with our
findings render the piperine congeners a promising compound
class endowed with a beneficial pharmacological profile to com-
bat abnormal proliferation, migration and inflammation of
VSMCs as key elements in the development of atherosclerosis
and restenosis.
Fig. 1 Inhibition of VSMC proliferation in the presence of different con-
centrations of piperine (1). Values are normalized to the PDGF stimulated
vehicle control (0.1% DMSO). Results were obtained by the resazurin con-
version method and presented as means SD in relative units (n = 3 for all
data groups); *** p < 0.001, ** p < 0.01, n.s. not significant (ANOVA/Bon-
ferroni).
6.03 µM (14), which is in accordance to the IC50 values obtained
"
by the resazurin conversion assay (l Table 1).
An intact and functional endothelium is crucial for vascular
health. Optimal vasoprotection is featured by a compound that
potently inhibits VSMC proliferation and moreover does not in-
terfere with endothelial cell viability. Hence, we investigated our
six most active compounds (4, 12, 14, 19, 20, and 21) as to
whether they affect proliferation of HUVECs.
Among the investigated piperamides, compounds 4, 12, 14, and
20 were identified as potent VSMC proliferation blockers with
IC50 values of 3.38, 11.8, 6.00, and 7.85 µM, respectively. Whereas
"
As shown in l Fig. 3, none of the investigated derivatives inter-
acted with HUVEC proliferation at 10 µM or below. Only at a con-
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074

1070 Original Papers
Fig. 2 Proliferation of VSMCs, treated with differ-
ent concentrations of A pipertipine (4) and B
(2E,4E)-N,N-dibutyl-5-(3,5-dimethoxyphenyl)pen-
ta-2,4-dienamide (14) by quantification of BrdU in-
corporation. Values are normalized to the PDGF-
stimulated vehicle control (0.1% DMSO). Results are
presented as means SD in relative units (n = 3 for
all data groups); *** p < 0.001, n.s. not significant
(ANOVA/Bonferroni).
Fig. 3 HUVEC proliferation in the presence of dif-
ferent concentrations of compounds 4, 12, 14, 19,
20, and 21 (30, 10, and 3 µM). 1 µM of paclitaxel
(taxol) was used as positive control. Results were
obtained by the resazurin conversion method. Val-
ues are normalized to the vehicle control (0.1%
DMSO) and results are presented as means SD in
relative units (n = 3 for all data groups); *** p <
0.001, ** p < 0.01, n.s. not significant (ANOVA/
Bonferroni).
Fig. 4 Cytotoxicity of the compounds (2E,4E)-N,
N-dibutyl-5-(3,5-dimethoxyphenyl)penta-2,4-dien-
amide (14) and pipertipine (4) compared to digito-
nin (100 µg/mL) which is used as positive control.
Values are obtained by the LDH assay and present-
ed as means SD in relative units (n = 3 for all data
groups); *** p < 0.001, * p < 0.05, n.s. not signifi-
cant (ANOVA/Bonferroni).
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074

Original Papers 1071
4 and 14 showed some cytotoxicity at higher concentrations
(30 µM), compounds 12 and 20 were identified as non-cytotoxic
and selective inhibitors of VSMC proliferation. The obtained in-
sights into the piperamidesʼ SAR yields knowledge that enables
further development of more potent and cell type-specific VSMC
growth inhibitors, based on the natural alkaloid scaffold piperine.
volume 235–269 mL) were combined and subjected to silica gel
column chromatography (Merck silica gel 60 PF254, 87 cm ×
2 cm) using a gradient system of CH2Cl2/EtOAc/MeOH (CH2Cl2
1200 mL; CH2Cl2/EtOAc, v/v, 99:1; 98:2; 97:3; 96:4; 95:5;
90:10; 80:20, 500 mL each; 50:50, 350 mL; EtOAc 500 mL;
MeOH 500 mL) yielding in 10 fractions (S3F0-S3F9). Fraction
S3F7 (100.71 mg, elution volume 3483–3782 mL) was purified
via Sephadex LH-20 (mobile phase: CH2Cl2, 98 cm × 2 cm) to yield
11 fractions (S4F0-S4F10) and 2.0 mg of 9 (S4F8, elution volume
165–177 mL). Fraction S3F6 (49.98 mg, elution volume 3288–
3482 mL) was subjected to Sephadex LH-20 column chromatog-
raphy (mobile phase: CH2Cl2, 98 cm × 2 cm) to gain 10 fractions
(S5F0–S5F9) and 4.43 mg of 10 (S5F6, elution volume, 135–
156 mL). Fraction S3F5 (elution volume 3118–3287 mL) was
chromatographed over Sephadex LH-20 (mobile phase: CH2Cl2,
Materials and Methods
!
Isolation of amide alkaloids (1–10) from
Piper nigrum fruits
General experimental procedures: NMR experiments were per-
formed by using Bruker TXI600 NMR spectrometers, operating
at 600 MHz. The samples were measured in MeOD (calibrated to
the residual nondeuterated solvent signals). MS analysis was per-
formed on a Bruker Esquire 3000 plus ion-trap mass spectrome-
ter equipped with electrospray ionization (ESI) in the positive
and negative modes: spray voltage, 4.5 kV; sheath gas, N2, 30
psi; dry gas, N2, 6 L/min, 350°C; scanning range, m/z 50–1000.
Column chromatography was performed using Merck silica gel
60 (40–63 µm) and Sephadex® LH-20 (20–100 µm; Pharmacia).
Fast centrifugal partition chromatography was carried out on a
FCPC A200 instrument from Kromaton (column volume:
200 mL). Fractions obtained from all chromatographic steps were
analysed by TLC (mobile phase: toluene/diethyl ether/dioxane
(6.25:2.15:1.6); stationary phase: Merck silica gel 60 PF254, de-
98 cm × 2 cm) giving
8 fractions (S6F0–F7). Fraction S6F2
(12.17 mg, elution volume 88–103 mL) was further separated us-
ing semi-preparative HPLC resulting in 2.08 mg of 7 (elution vol-
ume: 64–69 mL; purified via Sephadex LH-20 with MeOH). Frac-
tions S4F3 (42.04 mg, elution volume 99–111 mL) and S4F4
(28.25 mg, elution volume 112–130 mL) were combined and sub-
jected to Sephadex LH-20 column chromatography (mobile
phase: MeOH, 83 cm × 2.5 cm) to give 7 fractions (S7F0–F6). From
fraction S7F4 (57.46 mg, elution volume 240–257 mL) 21.00 mg
of 2 were obtained by crystallization form methanol.
3.9 g of fraction S1F4 (elution volume 890–1170 mL) were frac-
tionated by fast centrifugal partition chromatography using a bi-
phasic equilibrated solvent mixture, consisting of n-hexane/ethyl
acetate/MeOH/H2O (2:6:3:2, v/v/v/v). The instrument was op-
erated in descending mode (lower phase as mobile phase) and
equilibrated at 800 rpm. The sample was dissolved in a mixture
of mobile and stationary phase (1:1, v/v) and separation was car-
ried out at a flow rate of 1 mL/min and a rotation speed of
1200 rpm in a counter clockwise direction to result in 14 frac-
tions (S8F1–F14). Fraction S8F5 (126.96 mg, elution volume
256–269 mL) was separated using semi-preparative HPLC gra-
dient (MeOH/H2O, v/v, 73:27 to 80:20 in 12 min; 80:20 to
85:15 in 28 min; 85:15 to 98:2 in 1 min) yielding in 11 fractions
(S9F1–F11). Fraction S9F4 (9.53 mg, elution volume 43.0–
45.2 mL) was identified as compound 3, fraction S9F5 (3.99 mg,
elution volume 47.5–49.5 mL) as compound 6, fraction S9F7
(3.51 mg, elution volume 51.7–54.7 mL) as compound 5, fraction
S9F8 (2.74 mg, elution volume 54.8–57.8 mL) as compound 8, and
S9F10 (3.46 mg, elution volume 61.0–64.0 mL) as compound 4.
All pure compounds obtained from semi-preparative HPLC were
purified over Sephadex LH-20. The physical and spectroscopic
data of compounds 1 to 10 agreed with those published previ-
ously for piperine (1) [30], piperylin (2) [27], piperoleine A (3)
[30], pipertipine (4) [25], pipernonaline (5) [27], dehydropiper-
nonaline (6) [26], (2E,4E,8E)-9-(benzo[d][1,3]dioxol-5-yl)-1-
(pyrrolidin-1-yl)nona-2,4,8-trien-1-one (7) [27], chabamide (8)
[31], N-trans feruloylpiperidine (9) [32], and feruperine (10)
[32]. Copies of the original NMR spectra are obtainable from the
corresponding author. The purity of all compounds was checked
using TLC and LC‑MS and revealed to be > 95% in all cases.
tected with staining reagents vanillin/H2SO4 at vis, UV254
,
UV366). HPLC was performed on a Shimadzu UFLC‑XR instrument
with a photodiode array (PDA) detector. LC-parameters: station-
ary phase: Phenomenex HyperClone, 150 × 4.60 mm, 5 µm; tem-
perature: 35°C; mobile phase: water (A); methanol (B); flow rate
1.0 mL/min; UV detection wavelength: 205, 254, 270, 360 nm; in-
jection volume: 10 µL; gradient: 85% A, 15% B; 5 min; 45% A, 55%
B; 30 min; 32% A, 68% B; 41 min; 2% A, 98% B; 50 min; 2% A, 98%
B. Semi-preparative HPLC was performed on a Dionex (Thermo
Scientific) UltiMate 3000 instrument with a photodiode array de-
tector (DAD). LC-parameters: stationary phase: Phenomenex
Gemini-NX 250 × 10.00 mm, 5 µm; temperature: 35°C; mobile
phase: water (A); methanol (B); flow rate 2.0 mL/min; UV detec-
tion wavelength: 270 nm. All chemicals and solvents used were
analytical grade.
Plant material: Black pepper (dried unripe fruits of P. nigrum) was
obtained from Dr. Kottas – Heilkräuter (Charge KLA80342). A
voucher specimen (JR-20121114-A11) is deposited in the Her-
barium of the Department of Pharmacognosy, University of Vien-
na, Austria.
General Procedure: 500 g dried ground material were defatted
with 500 mL of n-hexane (ultrasonic bath, 1 h) and subsequently
macerated with dichloromethane (at RT, for 72 h) followed by
maceration with MeOH (at RT, three times for 72 h each). After
removal of the solvent under vacuum, a combined CH2Cl2/MeOH
(48.21 g) extract was obtained and subjected to silica gel column
chromatography (Merck silica gel 60 PF254, 37 cm × 3.5 cm) using
a gradient system of CH2Cl2 and MeOH (CH2Cl2 1000 mL; CH2Cl2/
MeOH, v/v, 98:2; 96:4; 90:10; 50:50; MeOH; 500 mL each) to
give 10 fractions (S1F1-S1F10). From fraction S1F4 (elution vol-
ume 890–1170 mL) 1.6 g 1 could be gained by crystallization
from methanol. Fraction S1F5 (2.2 g, elution volume 1171–
1561 mL) was further separated by means of Sephadex LH-20
column chromatography (mobile phase: MeOH, 81 cm × 2.5 cm)
to give 8 fractions (S2F1-S2F8). Fractions S2F4 and S2F5 (elution
Synthesis of piperine analogs
We have previously reported the synthesis of compounds 14 [41]
and 15–25 [33] including procedures in full experimental detail
"
(l Fig. 5). Purity of all synthesized compounds was > 95% as de-
termined by HPLC or NMR.
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074

1072 Original Papers
Fig. 5 A Synthesis of piperine analogues 11–13;
B Synthesis of compounds (14–25) previously re-
ported by our group.
General experimental procedures for the synthesis of compounds
“long method” (2 min at 100°C, 18°C/min until 280°C, 10 min at
280°C).
11–13: Unless otherwise noted, chemicals were purchased from
commercial suppliers and used without further purification.
Flash column chromatography was performed on silica gel 60
from Merck (40–63 mm), whereas most separations were per-
formed by using a Büchi SepacoreTM MPLC system with a 9 g col-
umn. Evaporation of solvents was carried out either on standard
rotary evaporators or on a Christ RVC 2–25 plus centrifugal evap-
orator with attached cooling system (Christ 2–4 LOplus). For TLC
aluminum-backed silica gel was used. Melting points were deter-
mined by using a Kofler-type Leica Galen III micro hot stage mi-
croscope (Aigner-Unilab Laborfachhandel GmbH) and are uncor-
rected. For compounds unknown in the literature high-resolution
MS was performed by E. Rosenberg at Vienna University of Tech-
nology, Institute for Chemical Technologies and Analytics; all
samples were analyzed by LC‑IT‑TOF‑MS in positive or negative
ion detection mode with the recording of MS and MS/MS spectra.
NMR-spectra were recorded on a Bruker AC 200 (200 MHz) or a
Bruker Avance 400 (400 MHz) spectrometer and chemical shifts
are reported in ppm. For assignment of 13C multiplicities standard
13C, DEPT or APT spectra were recorded. GC–MS runs were per-
formed on a Thermo Finnigan Focus GC/DSQ II with a standard
capillary column BGB 5 (ID = 30 m, 0.32 mm; Fisher Scientific
GmbH) using standardized temperature programs: “short meth-
od” (2 min at 100°C, 18°C/min until 280°C, 3 min at 280°C) and
General procedure: Palladium(II)acetate (4 mol%, 0.2 mmol,
2.2 mg), tri-o-tolylphosphine (8 mol%, 0.4 mmol, 6 mg), arylbro-
mide (0.5 mmol) and (piperidin-1-yl)pentadienone (0.5 mmol)
were placed in a screw-cap vial and set under argon. Acetonitrile
(1 ml) was added followed by triethylamine (1.5 equiv.,
0.75 mmol, 0.1 ml). The reaction mixture was heated to 70°C for
48 h and monitored with TLC and GC/MS. After letting the reac-
tion cool to room temperature, the crude reaction mixture was
adsorbed on silica and directly subjected to flash column chro-
matography (eluent LP/EA 20%).
(2E,4E)-5-Phenyl-1-(piperidin-1-yl)penta-2,4-dien-1-one (11): Re-
action stopped after 16 hours; 39% yield (47 mg, 0.19 mmol), pale
yellow solid, m.p. 85–87°C, Rf 0.4 (LP/EA 2:1), 1H NMR (CDCl3,
200 MHz): δ = 1.55–1.70 (m, 6H, CH2), 3.52–3.62 (m, 4H, CH2),
6.77–6.98 (m, 2H), 7.22–7.38 (m, 3H), 7.42–7.49 (m, 3H); 13C
NMR (CDCl3, 50 MHz): δ 24.6 (t), 25.6 (t), 26.7 (t), 43.2 (t), 46.9
(t), 120.9 (d), 126.9 (d), 127.0 (d), 128.5 (d), 128.7 (d), 136.4 (s),
138.5 (d), 142.3 (d), 165.3 (s), HR ESIMS m/z 242.1544 [M+H]+
(calculated for C16H19NO 242.1539, diff 2.06 ppm).
(2E,4E)-5-(4-Fluorophenyl)-1-(piperidin-1-yl)penta-2,4-dien-1-
one (12): 58% yield (75 mg, 0.29 mmol), pale yellow solid,
m.p. 77°C, Rf 0.1 (LP/EA 20%), 1H NMR (CDCl3, 200 MHz): δ =
1.48–1.67 (m, 6H), 3.49–3.59 (m, 4H), 6.44 (d, J = 14.7 Hz, 1H),
6.75 (s, 1H), 6.78 (d, J = 3.0 Hz, 1H), 6.98 (t, J = 8.6 Hz, 2H), 7.31–
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074

Original Papers 1073
7.44 (m, 3H); 13C NMR (CDCl3, 50 MHz): δ 24.6 (t), 25.6 (t), 26.7
(t), 43.3 (t), 46.9 (t), 115.7 (d, JC–F = 21.8 Hz), 120.9 (d), 126.76
Assessment of cytotoxicity: Quiescent VSMCs were pre-treated for
2
30 min with compound, or vehicle (0.1% DMSO) as indicated, and
subsequently stimulated for 24 h with PDGF‑BB (20 ng/mL).
Afterwards the supernatant of the treated cells was assessed for
soluble cytosolic protein LDH activity, as an indicator for loss of
cell membrane integrity and cell death [43,50]. For estimation of
the total LDH, identically treated samples were incubated for
45 min in the presence of 1% Triton X-100. The released and the
total LDH enzyme activity was measured for 30 min in the dark in
the presence of 4.5 mg/mL lactate, 0.56 mg/mL NAD+, 1.69 U/mL
diaphorase, 0.004% (w/v) bovine serum albumin, 0.15% (w/v) su-
crose, and 0.5 mM 2-p-iodophenyl-3-nitrophenyl tetrazolium
chloride. The enzyme reaction was stopped by adding 1.78 mg/
mL oxymate and the absorbance was measured at 490 nm. Poten-
tial effects on cell viability were estimated as percentage of extra-
cellular LDH enzyme activity. The cytotoxic natural product digi-
tonin (100 µg/mL) was used as a positive control.
5
3
4
(d, JC–F = 2.5 Hz), 128.5 (d, JC–F = 8.1 Hz), 132.7 (d, JC–F
=
1
2.9 Hz), 137.1 (d), 142.1 (d), 162.8 (d, JC–F = 249.3 Hz), 165.3 (s),
HR ESIMS m/z 260.1450 [M + H]+ (calculated for C16H18FNO
260.1445, diff 1.92 ppm).
(2E,4E)-5-(4-Methoxyphenyl)-1-(piperidin-1-yl)penta-2,4-dien-1-
one (13): 59% yield (80 mg, 0.29 mmol), pale yellow solid,
m.p. 93°C, Rf 0.06 (LP/EA 20%), 1H NMR (CDCl3, 200 MHz): δ
1.48–1.70 (m, 6H), 3.52–3.62 (m, 4H), 3.80 (s, 3H), 6.43 (d,
J = 14.6 Hz, 1H), 6.77–6.80 (m, 2H), 6.84–6.95 (m, 4H), 7.34–7.42
(m, 2H), 7.46 (dd, J = 6.6, 3.7 Hz, 1H); 13C NMR (CDCl3, 50 MHz):
δ = 24.7 (t), 25.6 (t), 26.7 (t), 43.2 (t), 46.9 (t), 55.3 (q), 114.0 (d),
119.6 (d), 125.0 (d), 128.3 (d), 129.3 (s), 138.2 (d), 142.8 (d),
160.0 (s), 165.5 (s), HR ESIMS m/z 272.1655 [M + H]+ (calculated
for C17H21NO2 272.1645, diff 3.76 ppm).
Bioactivity evaluation
Statistical analysis: Statistics were performed by ANOVA/Bonfer-
roni test, using GraphPad PRISM software, version 4.03. The
number of experiments is given in the figure legends, and a prob-
ability value < 0.05 was considered significant.
Chemicals and reagents: Rat aortic VSMCs were purchased from
Lonza. HUVECs (HUVECtert) were provided by Dr. Hannes Stock-
inger (Medical University of Vienna) [42,43]. Paclitaxel (taxol)
and digitonin were obtained from Sigma-Aldrich with a purity
of ≥ 95% and ~ 50%, respectively.
Cell culture: VSMCs were grown in Dulbeccoʼs modified essential
medium – F12 (1:1) supplemented with 20% fetal calf serum and
gentamycin. For all tests, VSMCs were seeded in 96-well plates at
5 × 103 cells/well and incubated for 24 h (37°C, 5% CO2). Then the
cells were serum-starved for another 24 h to render them quies-
cent. HUVECtert were cultivated in EBM supplemented with 10%
fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 1%
amphotericin B and EBM™ SingleQuots®, containing recombi-
nant human epidermal growth factor, hydrocortisone, gentami-
cin sulphate, amphotericin B and 0.4% bovine brain extract.
Resazurin conversion assay: Quiescent VSMCs were pre-treated
for 30 min with compound or vehicle (0.1% DMSO) as indicated,
and subsequently stimulated with PDGF‑BB (20 ng/mL) for 48 h.
To measure the number of metabolically active VSMCs by resa-
zurin conversion [44–46], cells were washed with phosphate-
buffered saline and incubated for 2 h in serum-free medium con-
taining 10 µg/mL resazurin. Total metabolic activity was mea-
sured by monitoring the increase in fluorescence at a wavelength
of 590 nm using an excitation wavelength of 535 nm in a 96-well
plate reader (Tecan GENios Pro). HUVECtert were seeded in 96-
well plates at 5 × 103 cells/well and incubated for 24 h (37°C, 5%
CO2). Then, HUVECtert [42,43] were treated with compound or
vehicle (0.1% DMSO) as indicated and incubated for another
48 h. Afterwards, cells were washed with phosphate-buffered sa-
line and incubated in culture medium containing 10 µg/mL resa-
zurin for 2 h. The detection step was performed as described
above. Results were expressed in RU compared to the fluores-
cence of PDGF/vehicle-treated control cells, which was set to 1.
Paclitaxel (taxol) was used as a reference compound, and inhib-
ited the proliferation of VSMC and HUVECtert with IC50 = 108 nM
and 4 nM, respectively [46].
Acknowledgements
!
This work was supported the European Union Seventh Frame-
work Program (EU‑FP7) Marie Curie Fellowship (R.L.) and the
University of Vienna “Back to Research Grant” (R.L.), as well as
by the Austrian Science Fund (FWF): P24587 (C.E.M.), S10704
(NFN-project “Drugs from Nature Targeting Inflammation”),
P23317-B11 and P25971-B23.
Conflict of Interest
!
The authors declare no conflict of interest.
References
1 Lusis AJ. Atherosclerosis. Nature 2000; 407: 233–241
2 Rudijanto A. The role of vascular smooth muscle cells on the patho-
genesis of atherosclerosis. Acta Med Indones 2007; 39: 86–93
3 Ross R. The pathogenesis of atherosclerosis: a perspective for the
1990s. Nature 1993; 362: 801–809
4 Katz G, Harchandani B, Shah B. Drug-eluting stents: the past, present,
and future. Curr Atheroscler Rep 2015; 17: 11
5 Windecker S, Remondino A, Eberli FR, Juni P, Raber L, Wenaweser P, Togni
M, Billinger M, Tuller D, Seiler C, Roffi M, Corti R, Sutsch G, Maier W,
Luscher T, Hess OM, Egger M, Meier B. Sirolimus-eluting and paclitaxel-
eluting stents for coronary revascularization. N Engl J Med 2005; 353:
653–662
6 Raja SG. Drug-eluting stents and the future of coronary artery bypass
surgery: facts and fiction. Ann Thorac Surg 2006; 81: 1162–1171
7 Degertekin M, Serruys PW, Foley DP, Tanabe K, Regar E, Vos J, Smits PC,
van der Giessen WJ, van den Brand M, de Feyter P, Popma JJ. Persistent
inhibition of neointimal hyperplasia after sirolimus-eluting stent im-
plantation: long-term (up to 2 years) clinical, angiographic, and intra-
vascular ultrasound follow-up. Circulation 2002; 106: 1610–1613
8 Iakovou I, Schmidt T, Bonizzoni E, Ge L, Sangiorgi GM, Stankovic G, Airoldi
F, Chieffo A, Montorfano M, Carlino M, Michev I, Corvaja N, Briguori C,
Gerckens U, Grube E, Colombo A. Incidence, predictors, and outcome of
thrombosis after successful implantation of drug-eluting stents. Jama
2005; 293: 2126–2130
5-bromo-2′-deoxyuridine (BrdU) incorporation assay: Quiescent
VSMCs were pre-treated for 30 min with compound, or vehicle
(0.1% DMSO) as indicated and stimulated with PDGF (20 ng/mL)
for 2 h. Then BrdU was added to estimate de novo DNA synthesis
in VSMCs [47–49]. 22 h later, the BrdU incorporation was quanti-
fied according to the manufacturerʼs instructions (Roche Diag-
nostics).
9 Mehilli J, Byrne RA, Tiroch K, Pinieck S, Schulz S, Kufner S, Massberg S,
Laugwitz KL, Schömig A, Kastrati A. Randomized trial of paclitaxel- ver-
sus sirolimus-eluting stents for treatment of coronary restenosis in si-
rolimus-eluting stents: the ISAR-DESIRE 2 (intracoronary stenting and
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074

1074 Original Papers
angiographic results: drug eluting stents for in-stent restenosis 2)
study. J Am Coll Cardiol 2010; 55: 2710–2716
Erker T, Ecker GF, Mihovilovic MD, Hering S. Efficient modulation of
gamma-aminobutyric acid type A receptors by piperine derivatives.
J Med Chem 2014; 57: 5602–5619
10 Jagella T, Grosch W. Flavour and off-flavour compounds of black and
white pepper (Piper nigrum L.) I. Evaluation of potent odorants of black
pepper by dilution and concentration techniques. Eur Food Res Tech-
nol 1999; 209: 16–21
11 Meghwal M, Goswami TK. Piper nigrum and Piperine: an update. Phyt-
other Res 2013; 27: 1121–1130
12 Vasavirama K, Upender M. Piperine: a valuable alkaloid from Piper spe-
cies. Int J Pharm Pharm Sci 2014; 6: 34–38
13 Labruyère B. Determination of pungent constituents of Piper nigrum.
J Agr Food Chem 1966; 14: 469–472
14 Srinivasan K. Black pepper and its pungent principle-piperine: A re-
view of diverse physiological effects. Crit Rev Food Sci Nutr 2007; 47:
735–748
15 Dawid C, Henze A, Frank O, Glabasnia A, Rupp M, Büning K, Orlikowski D,
Bader M, Hofmann T. Structural and sensory characterization of key
pungent and tingling compounds from black pepper (Piper nigrum L.).
J Agric Food Chem 2012; 60: 2884–2895
16 Meghwal M, Goshwami T. Chemical composition, nutritional, medicinal
and functional properties of black pepper: A review. Open Access Sci
Rep 2012; 1: 1–5
34 Khom S, Strommer B, Schoffmann A, Hintersteiner J, Baburin I, Erker T,
Schwarz T, Schwarzer C, Zaugg J, Hamburger M, Hering S. GABAA recep-
tor modulation by piperine and a non-TRPV1 activating derivative. Bi-
ochem Pharmacol 2013; 85: 1827–1836
35 Sahu PK, Sharma A, Rayees S, Kour G, Singhi A, Khullar M, Magotra A,
Paswan SK, Gupta M, Ahmed I, Roy S, Tikoo MK, Sharma SC, Singh S, Singh
G. Pharmacokinetic study of Piperine in wistar rats after oral and intra-
venous administration. Int J Drug Delivery 2014; 6: 82–87
36 Bhardwaj RK, Glaeser H, Becquemont L, Klotz U, Gupta SK, Fromm MF.
Piperine, a major constituent of black pepper, inhibits human P-glyco-
protein and CYP3A4. J Pharmacol Exp Ther 2002; 302: 645–650
37 Khatri S, Ahmed FJ, Rai P. Formulation and evaluation of floating gastro-
retentive capsules of acyclovir with piperine as a bioenhancer. Pharma
Innovation 2015; 3: 78–81
38 Motiwala MN, Rangari VD. The effect of piperine on oral bioavailability
and pharmacokinetics of paclitaxel in rats. Int J Pharm Phytopharm Res
2014; 3: 399–403
39 Wadhwa S, Singhal S, Rawat S. Bioavailability enhancement by piper-
ine: a review. Asian J Biomed Pharm Sci 2014; 4: 1–8
17 OʼMahony R, Al-Khtheeri H, Weerasekera D, Fernando N, Vaira D, Holton
J, Basset C. Bactericidal and anti-adhesive properties of culinary and
medicinal plants against Helicobacter pylori. World J Gastroenterol
2005; 11: 7499–7507
40 Kumar S, Singhal V, Roshan R, Sharma A, Rembhotkar GW, Ghosh B. Pi-
perine inhibits TNF-alpha induced adhesion of neutrophils to endothe-
lial monolayer through suppression of NF-kappaB and IkappaB kinase
activation. Eur J Pharmacol 2007; 575: 177–186
18 Agbor GA, Vinson JA, Oben JE, Ngogang JY. Comparative analysis of the in
vitro antioxidant activity of white and black pepper. Nutr Res 2006; 26:
659–663
19 Singh VK, Singh P, Patel AMA, Yadav KKM. Piperine: delightful surprise
to the biological world, made by plant “pepper” and a great bioavaila-
bility enhancer for our drugs and supplements. World J Pharm Res
2014; 3: 2084–2098
20 Jiang ZY, Liu WF, Zhang XM, Luo J, Ma YB, Chen JJ. Anti-HBV active con-
stituents from Piper longum. Bioorg Med Chem Lett 2013; 23: 2123–
2127
21 Zaugg J, Baburin I, Strommer B, Kim HJ, Hering S, Hamburger M. HPLC-
based activity profiling: discovery of piperine as a positive GABA(A) re-
ceptor modulator targeting a benzodiazepine-independent binding
site. J Nat Prod 2010; 73: 185–191
22 Lee KP, Lee K, Park WH, Kim H, Hong H. Piperine inhibits platelet-de-
rived growth factor-BB-induced proliferation and migration in vascu-
lar smooth muscle cells. J Med Food 2015; 18: 208–215
23 Subehan, Usia T, Kadota S, Tezuka Y. Alkamides from Piper nigrum L.
and their inhibitory activity against human liver microsomal cyto-
chrome P450 2D6 (CYP2D6). Nat Prod Commun 2006; 1: 1–7
24 Okumura Y, Narukawa M, Iwasaki Y, Ishikawa A, Matsuda H, Yoshikawa
M, Watanabe T. Activation of TRPV1 and TRPA1 by black pepper com-
ponents. Biosci Biotechnol Biochem 2010; 74: 1068–1072
25 Siddiqui BS, Gulzar T, Begum S. Amides from the seeds of Piper nigrum
Linn. and their insecticidal activity. Heterocycles 2002; 57: 1653–1658
26 Lee SW, Rho MC, Park HR, Choi JH, Kang JY, Lee JW, Kim K, Lee HS, Kim YK.
Inhibition of diacylglycerol acyltransferase by alkamides isolated from
the fruits of Piper longum and Piper nigrum. J Agric Food Chem 2006;
54: 9759–9763
27 Wei K, Li W, Koike K, Pei Y, Chen Y, Nikaido T. New amide alkaloids from
the roots of Piper nigrum. J Nat Prod 2004; 67: 1005–1009
28 Wei K, Li W, Koike K, Chen Y, Nikaido T. Nigramides AS, dimeric amide
alkaloids from the roots of Piper nigrum. J Org Chem 2005; 70: 1164–
1176
29 Inatani R, Nakatani N, Fuwa H. Structure and synthesis of new phenolic
amides from Piper nigrum L. Agric Biol Chem 1981; 45: 667–673
30 Strunz GM, Finlay H. Concise, efficient new synthesis of pipercide an in-
secticidal unsaturated amide from Piper nigrum and related com-
pounds. Tetrahedron 1994; 50: 11113–11122
41 Wimmer L, Schonbauer D, Pakfeifer P, Schoffmann A, Khom S, Hering S,
Mihovilovic MD. Developing piperine towards TRPV1 and GABAA re-
ceptor ligands – synthesis of piperine analogs via Heck-coupling of
conjugated dienes. Org Biomol Chem 2015; 13: 990–994
42 Schiller HB, Szekeres A, Binder BR, Stockinger H, Leksa V. Mannose 6-
phosphate/insulin-like growth factor 2 receptor limits cell invasion by
controlling alphaVbeta3 integrin expression and proteolytic pro-
cessing of urokinase-type plasminogen activator receptor. Mol Biol Cell
2009; 20: 745–756
43 Fakhrudin N, Waltenberger B, Cabaravdic M, Atanasov AG, Malainer C,
Schachner D, Heiss EH, Liu R, Noha SM, Grzywacz AM, Mihaly-Bison J,
Awad EM, Schuster D, Breuss JM, Rollinger JM, Bochkov V, Stuppner H,
Dirsch VM. Identification of plumericin as a potent new inhibitor of
the NF-kappaB pathway with anti-inflammatory activity in vitro and
in vivo. Br J Pharmacol 2014; 171: 1676–1686
44 Kurin E, Atanasov AG, Donath O, Heiss EH, Dirsch VM, Nagy M. Synergy
study of the inhibitory potential of red wine polyphenols on vascular
smooth muscle cell proliferation. Planta Med 2012; 78: 772–778
45 Joa H, Vogl S, Atanasov AG, Zehl M, Nakel T, Fakhrudin N, Heiss EH, Picker
P, Urban E, Wawrosch C, Saukel J, Reznicek G, Kopp B, Dirsch VM. Identi-
fication of ostruthin from Peucedanum ostruthium rhizomes as an in-
hibitor of vascular smooth muscle cell proliferation. J Nat Prod 2011;
74: 1513–1516
46 Liu R, Heiss EH, Sider N, Schinkovitz A, Groblacher B, Guo D, Bucar F, Ba-
uer R, Dirsch VM, Atanasov AG. Identification and characterization of
[6]-shogaol from ginger as inhibitor of vascular smooth muscle cell
proliferation. Mol Nutr Food Res 2015; 59: 843–852
47 Schwaiberger AV, Heiss EH, Cabaravdic M, Oberan T, Zaujec J, Schachner
D, Uhrin P, Atanasov AG, Breuss JM, Binder BR, Dirsch VM. Indirubin-3′-
monoxime blocks vascular smooth muscle cell proliferation by inhibi-
tion of signal transducer and activator of transcription 3 signaling and
reduces neointima formation in vivo. Arterioscler Thromb Vasc Biol
2010; 30: 2475–2481
48 Blažević T, Schwaiberger AV, Schreiner CE, Schachner D, Schaible AM,
Grojer CS, Atanasov AG, Werz O, Dirsch VM, Heiss EH. 12/15-lipoxyge-
nase contributes to platelet-derived growth factor-induced activation
of signal transducer and activator of transcription 3. J Biol Chem
2013; 288: 35592–35603
49 Liu R, Heiss EH, Guo D, Dirsch VM, Atanasov AG. Capsaicin from chili
(Capsicum spp.) inhibits vascular smooth muscle cell proliferation.
F1000Res 2015; 4: 26
31 Rukachaisirikul T, Prabpai S, Champung P, Suksamram A. Chabamide, a
novel piperine dimer from stems of Piper chaba. Planta Med 2002; 68:
853–855
32 Correa EA, Hogestatt ED, Sterner O, Echeverri F, Zygmunt PM. In vitro
TRPV1 activity of piperine derived amides. Bioorg Med Chem Lett
2010; 18: 3299–3306
50 Sepp A, Binns RM, Lechler RI. Improved protocol for colorimetric detec-
tion of complement-mediated cytotoxicity based on the measurement
of cytoplasmic lactate dehydrogenase activity. J Immunol Methods
1996; 196: 175–180
33 Schoffmann A, Wimmer L, Goldmann D, Khom S, Hintersteiner J, Baburin
I, Schwarz T, Hintersteininger M, Pakfeifer P, Oufir M, Hamburger M,
Mair CE et al. Piperine Congeners as… Planta Med 2015; 81: 1065–1074