Piperolein B, isopiperolein B and piperamide C9:1(8E): Total synthesis and cytotoxicities

Article abstract of DOI:10.1039/c3ra42060d

Total syntheses of the reported structures of piperolein B, isopiperolein B and piperamide C9:1(8E) have been achieved. The analytical data reported for piperolein B and piperamide C9:1(8E) match the synthetic values, however those for isopiperolein B do

Full text of DOI:10.1039/c3ra42060d

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Piperolein B, isopiperolein B and piperamide C9:1(8E):
total synthesis and cytotoxicities3
Cite this: RSC Advances, 2013, 3,
16681
Francis Kayamba,{ Christopher Dunnill,{ David J. Hamnett, Arantxa Rodr´ıguez,
Nikolaos T. Georgopoulos§* and Wesley J. Moran§*
Total syntheses of the reported structures of piperolein B, isopiperolein B and piperamide C9:1(8E) have
been achieved. The analytical data reported for piperolein B and piperamide C9:1(8E) match the synthetic
values, however those for isopiperolein B do not. The cytotoxicities of these three structurally similar
compounds against cancer cell lines of different tissue origins were evaluated and the results indicated
that these compounds show differential effects on cancer cell viability.
Received 26th April 2013,
Accepted 15th July 2013
DOI: 10.1039/c3ra42060d
www.rsc.org/advances
Introduction
Results and discussion
Studies of plants of the genus Piper have led to the isolation of
277 different amide alkaloids to date.1 Some species of Piper
plants are widely used in folk medicine and the isolated
amides have been shown to possess a wide range of biological
activities. Amongst these, piperolein B, 1 which can be isolated
from black pepper, Piper nigrum, has been reported to possess
larvicidal,2 hepatoprotective3 and enzymatic inhibition activity
(Fig. 1).4 It has also been shown to be a TRP agonist.5
Piperamide C9:1(8E), 2 was also tested for larvicidal activity
and was found to be six times more toxic than piperolein B, 1.
More recently, isopiperolein B, 3 was isolated.6 This was later
shown to have antibacterial activity7 and cytotoxic activity
towards the human cervical carcinoma cell line HeLa.8
To date there have been few total syntheses of members of
this family of amide alkaloids. However, Strunz and co-
workers have synthesised over a dozen of these compounds
using a multi-step aldol condensation–Grob-type fragmenta-
tion strategy,9 whilst several dimeric compounds have been
prepared through cycloaddition processes.10 We were inter-
ested in developing a short synthetic route into piperolein B, 1
and the closely related molecules 2 and 3 and investigating
their anti-cancer properties.
A simple synthetic strategy was conceived for the preparation
of these compounds (Scheme 1). Amidation of either 8-none-
noic acid or 9-decenoic acid with piperidine or pyrrolidine
provided the amides 6, 7 and 8 in good yields. Subsequent Ru-
catalyzed cross metathesis with styrene derivative 9 delivered
the three natural products 1, 2 and 3 in moderate yields. These
yields of isolated compounds were lower than expected due to
difficulties in separating the natural products from the dimer
of styrene 9.
The analytical data for piperolein B, 1 and piperamide
C9:1(8E), 2 matched the literature values. However, the data
for amide
3 did not match the literature values for
isopiperolein B (Table 1). Moreover, the literature values are
inconsistent with a pyrrolidine amide and more closely
resemble the data for a piperidine amide. This difference
can clearly be seen in the aliphatic region of the 1H NMR
Department of Chemical & Biological Sciences, School of Applied Sciences, University
of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK.
E-mail: w.j.moran@hud.ac.uk; n.georgopoulos@hud.ac.uk; Fax: +44 (0)1484 47
2182
Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
3
for compounds 1, 2, 3, 6, 7 and 8. See DOI: 10.1039/c3ra42060d
{ These authors contributed equally to this work.
§ Joint senior authors.
Fig. 1 Reported structures for three Piper alkaloids.
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Fig. 2 1H NMR spectra showing the clear difference between piperidine and
pyrrolidine amides.
urothelial (urinary bladder) carcinoma cell line EJ. We have
previously reported that these cell lines are highly representa-
tive of the original tumors of origin and have characterized
their responses to anti-proliferative (pro-apoptotic) responses
in vitro.11 This allowed us not only to determine the effect of
the compounds on relevant representative cell lines but also
permitted cell type-specific distinctions to be made. In
addition to examining cytotoxic effects on cell cultures of
standard cell density, i.e. 5–6 6 103 cells per well,12 we also
tested for biological effects using cultures of lower cell density
(3 6 103 cells per well) in order to compare our findings to a
previous report in which cytotoxicity detection assays were
performed using relatively low density cultures of carcinoma
(HeLa) cells to study the effects of isopiperolein B (Fig. 3).8
Piperolein B, 1 and piperamide C9:1(8E), 2 showed little (if
any) detectable cytotoxicity at concentrations below 10 mM. In
comparison, amide 3 showed some cytotoxicity at 10 mM, more
significant effects at 30 mM and it caused complete cellular
death in all cultures at higher concentrations (100 mM). This
was independent of the initial seeding density of the cell
cultures. All compounds demonstrated cytotoxicity at concen-
trations over 30 mM but only compound 3 resulted in 100%
loss of viability in both cell lines and was hence the most
cytotoxic independent of cell culture density and cell type.
Interestingly, treatment with compound 1 at 100 mM caused
100% death only in HCT116 cells but not in EJ cells, indicating
a differential effect between the cell lines. This however was
only noticeable when cells were cultured at normal (but not
low) density. Such a cell type-dependent differential response
was even more pronounced in the case of compound 2; at high
doses, the compound showed almost 100% toxicity in HCT116
cells but the effect was much less pronounced in EJ cells which
showed 60% cell viability.
Scheme 1 Synthesis of alkaloids 1, 2 and 3.
spectra of amides 1, 2 and 3 (Fig. 2). The reported data for
isopiperolein B is more like 1 than 3. It remains unclear as to
what the actual structure of this alkaloid is.
As the potential anti-proliferative effects of these com-
pounds on carcinoma cells remain virtually unexplored, we
were interested in comparing and contrasting the biological
effects of these three structurally very similar amides on cancer
cells. Importantly, unlike the majority of previously published
studies that routinely involve testing compounds on single cell
lines, which in most cases are not closely representative of the
tumors of origin, we assessed the effect of compounds 1, 2,
and 3 on the colorectal carcinoma cell line HCT116 and the
Table 1 Comparison of NMR data reported for isopiperolein B and that
obtained for amide 3
1H lit. data
1H amide 3
13C lit. data
13C amide 3
1.35–1.50, 14H
1.29–1.50, 8H
1.56–1.71, 2H
1.85, 2H, pent
1.95, 2H, pent
2.12–2.21, 2H
2.26, 2H, t
3.41, 2H, t
3.47, 2H, t
5.93, 2H, s
6.04, 1H, dt
6.28, 1H, d
24.5
25.4
26.5
28.9
29.3
29.7
32.8
33.4
42.5
24.8
25.3
26.5
29.4
29.7
29.8
33.3
35.2
45.9
2.15, 2H, q
2.30, 2H, t
3.40, 2H, t
3.55, 2H, t
5.90, 2H, s
5.95–6.15, 1H
6.25, 1H, d
6.60–7.00, 3H
46.6
47.0
100.8
106.0
108.5
119.6
128.0
129.2
132.4
146.4
147.8
171.4
101.2
105.7
108.5
120.5
129.6
129.7
132.8
146.8
148.2
172.3
6.71–6.89, 3H
Morphological observation of the cultures by routine, phase
contrast microscopy indicated that compound 3 caused rapid
and extensive cell death within less than 24 h after treatment
and complete death in cultures of both cell lines by 48 h. The
effect of the other two compounds was slower, and particularly
in the case of compound 2 for EJ cells only a proportion of cells
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Fig. 3 The effect of the synthetic compounds 1, 2 and 3 on carcinoma cell viability. HCT116 and EJ carcinoma cells were grown at 3 6 103 cells per well (low cell
density) and 6 6 103 cells per well (normal cell density), before being treated with the indicated doses of compounds 1, 2 and 3. Cell viability (expressed as % in
relation to controls) was assessed 48 h later as described in the experimental section and results are expressed as mean % values (¡ standard error of the mean).
Results are mean values for six technical replicates and are representative of three independent experiments.
cell type-dependent. Of the three compounds, 3, the reported
structure of isopiperolein B, was the most cytotoxic and
piperamide C9:1(8E), 2 was the least cytotoxic demonstrating
that the length of the connecting alkyl chain has an impact on
activity. Consideration of the structures and the observed
differences in their cytotoxic capacities could be used as a
basis for structure–activity studies as well as for detailed
biological investigations in order to understand the structural
basis for these activities. In this way, the design of compounds
with improved activities and pharmacological properties for
anticancer therapy should be possible.
appeared fully apoptotic (dead) and a fraction of these
remained alive.
Our findings indicate that the choice of initial cell density
might be important if subtle cytotoxicity capacities and cell
type-specific effects are to be revealed. The example of
compound 1 exemplifies this point: the use of low cell density
indicated complete cytotoxicity, yet the use of normal
(standard) density revealed a dose and cell type-specific
response. Thus the use of cultures that are of too low
confluence might mask cell type specific responses and also
suggest misleadingly high cytotoxic capacity.
It should also be noted that when treated at sub-lethal drug
concentrations, most cultures showed an increased level of
growth, i.e. over 100% cell viability in comparison to negative
controls. This is not an anomalous observation that was
specific to our experiments. Our recent in vitro findings
(unpublished) and observations by others13 both in vitro and in
vivo suggest that both normal and cancer epithelial cells
exhibit a biphasic response to cytotoxic chemicals, consisting
of increased growth at sub-cytotoxic drug concentrations, yet
dramatic cytotoxicity at higher doses. This appears to be an
inherent ability of epithelial cells to respond to such ‘insults’
as part of a repair response.13a
Conclusion
A simple synthetic strategy to three structurally related Piper
compounds has been developed and their cytotoxic properties
studied. Critically, the assigned structure of isopiperolein B
has been shown not to be that of amide 3, although compound
3 was the most cytotoxic compound of the three tested.
Experimental
1H NMR spectra were recorded at 400 MHz. Chemical shifts
are reported in ppm from tetramethylsilane with the solvent
resonance as the internal standard (CDCl3: 7.26 ppm). 13C
Collectively, our results demonstrate for the first time that
all of the three compounds exhibit differential effects on
carcinoma cells and these effects appear to be both dose- and
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and the residue purified by flash chromatography (2 : 1
petroleum ether–EtOAc).
NMR were recorded with complete proton decoupling.
Chemical shifts are reported in ppm from tetramethylsilane
with the solvent as the internal standard (CDCl3: 77.4 ppm).
Mass spectrometry (m/z) was performed in ESI mode, with only
molecular ions being reported. Infrared (IR) spectra nmax are
reported in cm21. All purchased reagents were used as
received without further purification. Petroleum ether refers
to the fraction boiling at 40–60 uC. All reactions were
performed under a N2 atmosphere.
Piperolein B, 1. Isolated as a yellow oil. IR (neat): 1037 (m),
1442 (s), 1489 (s), 1636 (s), 2927 (m) cm21. 1H NMR (400 MHz,
CDCl3): d 1.29–1.73 (14H, m), 2.17 (2H, q, J = 7.0 Hz), 2.31 (2H,
t, J = 7.5 Hz), 3.38 (2H, t, J = 5.4 Hz), 3.54 (2H, t, J = 5.4 Hz), 5.93
(2H, s), 6.03 (1H, dt, J = 16, 6.8 Hz), 6.28 (1H, d, J = 16 Hz),
6.69–6.78 (2H, m), 6.89 (1H, s). 13C NMR (100 MHz, CDCl3): d
25.0, 25.8, 26.0, 27.0, 29.4, 29.7, 29.8, 33.2, 33.9, 43.0, 47.1,
101.3, 105.8, 108.6, 120.6, 129.7 (2C), 132.8, 146.9, 148.3, 171.8.
MS: m/z (M + 23) 366.2 HRMS: m/z calc’d for C21H29NNaO3
265.1563, found 366.2025.
General procedure for amidation
1-Propanephosphonic acid anhydride solution (T3P; 50% in
DMF, 1.39 mL, 4.8 mmol, 3 equiv.) was dissolved in CH2Cl2 (5
mL) and cooled to 0 uC. Triethylamine (1.3 mL, 9.6 mmol, 6
equiv.) and the carboxylic acid (1.6 mmol, 1 equiv.) were
added. The reaction mixture was left to stir at 0 uC for 0.5 h,
then piperidine or pyrrolidine (1.6 mmol, 1 equiv.) was added.
After stirring overnight at room temperature the reaction was
quenched with H2O (20 mL) and extracted with EtOAc (3 6 10
mL). The combined organics were dried (MgSO4), filtered and
concentrated under vacuum to provide the corresponding
amide. The crude products were deemed to be sufficiently
pure and no further purification was performed.
Piperamide C9:1(8E), 2. Isolated as a yellow oil. 1H NMR
(400 MHz, CDCl3): d 1.28–1.51 (6H, m), 1.57–1.70 (2H, m), 1.83
(2H, pent, J = 6.7 Hz), 1.93 (2H, pent, J = 6.5 Hz), 2.16 (2H, q, J =
7.0 Hz), 2.25 (2H, t, J = 7.0 Hz), 3.39 (2H, t, J = 6.7 Hz), 3.45 (2H,
t, J = 6.8 Hz), 5.92 (2H, s), 6.03 (1H, dt, J = 16, 6.5 Hz), 6.27 (1H,
d, J = 16 Hz), 6.69–6.76 (2H, m), 6.88 (1H, s). 13C NMR (100
MHz, CDCl3): d 24.8, 25.2, 26.5, 29.3, 29.6, 29.7, 33.2, 35.2,
46.0, 47.0, 101.2, 105.7, 108.5, 120.5, 129.6, 129.7, 132.8, 146.8,
148.2, 172.3. MS: m/z (M + 23) 352.2 HRMS: m/z calc’d for
C
20H27NaO3 352.1883, found 352.1871.
Proposed structure of isopiperolein B, 3. Isolated as a yellow
oil. IR (neat): 1036 (s), 1248 (s), 1444 (s), 1605 (m), 1728 (m),
1-(Piperidin-1-yl)non-8-en-1-one, 6. Isolated as a yellow oil.
1
2926 (m) cm21. H NMR (400 MHz, CDCl3): d 1.29–1.50 (8H,
1
IR (neat): 1434 (s), 1639 (s), 2854 (m), 2926 (s) cm21. H NMR
m), 1.56–1.71 (2H, m), 1.85 (2H, pent, J = 7.0 Hz), 1.95 (2H,
pent, J = 7.0 Hz), 2.12–2.21 (2H, m), 2.26 (2H, t, J = 7.9 Hz), 3.41
(2H, t, J = 6.5 Hz), 3.47 (2H, t, J = 7.0 Hz), 5.93 (2H, s), 6.04 (1H,
dt, J = 16, 7.0 Hz), 6.28 (1H, d, J = 16 Hz), 6.71–6.78 (2H, m),
6.89 (1H, s). 13C NMR (100 MHz, CDCl3): d 24.8, 25.3, 26.5,
29.4, 29.7, 29.8 (2C), 33.3, 35.2, 45.9, 47.0, 101.2, 105.7, 108.5,
120.5, 129.6, 129.8, 132.8, 146.8, 148.2, 172.2. MS: m/z (M + 23)
366.2 HRMS: m/z calc’d for C21H29NNaO3 366.2040, found
366.2027.
(400 MHz, CDCl3): d 1.21–1.39 (6H, m), 1.42–1.64 (8H, m), 1.98
(2H, q, J = 6.8 Hz), 2.26 (2H, t, J = 7.5 Hz), 3.34 (2H, t, J = 5.2
Hz), 3.49 (2H, t, J = 5.2 Hz), 4.86 (1H, d, J = 10 Hz), 4.93 (1H, d, J
= 17 Hz), 5.67–5.81 (1H, m). 13C NMR (100 MHz, CDCl3): d 24.8,
25.7, 25.8, 26.8, 29.0, 29.1, 29.6, 33.7, 34.0, 42.9, 47.0, 114.4,
139.3, 171.9. MS: m/z (M + 23) 246.2 HRMS: m/z calc’d for
C14H25NNaO 246.1828, found 246.1822.
1-(Pyrrolidin-1-yl)non-8-en-1-one, 7. Isolated as a yellow oil.
IR (neat): 1425 (s), 1637 (s), 2925 (m) cm21. 1H NMR (400 MHz,
CDCl3): d 1.22–1.38 (6H, m), 1.59 (2H, pent, J = 7.1 Hz), 1.79
(2H, pent, J = 7.0 Hz), 1.90 (2H, pent, J = 6.4 Hz), 1.98 (2H, q, J =
7.0 Hz), 2.20 (2H, t, J = 7.5 Hz), 3.36 (2H, t, J = 6.8 Hz), 3.41 (2H,
t, J = 6.9 Hz), 4.82–4.98 (2H, m), 5.67–5.81 (1H, m). 13C NMR
(100 MHz, CDCl3): d 24.7, 25.1, 26.4, 29.0, 29.2, 29.6, 34.0, 35.0,
45.9, 46.9, 114.4, 139.3, 172.2. MS: m/z (M + 23) 232.2 HRMS:
m/z calc’d for C13H23NNaO 232.1672, found 232.1680.
Assessment of compound cytotoxicity
The colorectal carcinoma HCT116 and bladder carcinoma EJ
cell lines were used to assess the effect of the synthetic
compounds on cancer cell viability. Cells were seeded in 96-
well plates at low (3 6 103 cells per well) and normal (6 6 103
cells per well) density in culture medium, which was a 1 : 1 (v/
v) mixture of DMEM and RPMI (SigmaAldrich) supplemented
with 5% (v/v) fetal bovine serum. Cells were incubated at 37 uC
in a humidified atmosphere containing 5% (v/v) CO2 and
permitted to adhere overnight. Following synthesis, the
compounds were aseptically reconstituted in DMSO at a final
concentration of 20 mM. For testing, all compounds were
diluted to concentrations 0.1, 0.3, 1, 3, 10 and 30 and 100 mM
in culture medium. For each condition, six replicate samples
were prepared. Solvent-alone treatments were also included for
comparison (controls). Cells were then cultured for a period of
48 h before cell viability assays were performed. To assess cell
viability, the CellTiter 961 AQueous One Solution Cell
Proliferation Assay (Promega) was used, which is a colori-
metric MTS viability assay based on the principle that viable
cells have the ability to bioreduce the MTS tetrazolium
compound into a coloured formazan product which is soluble
in culture medium. Following drug treatment of cells for 48 h,
CellTiter reagent was added to each well (as recommended by
1-(Pyrrolidin-1-yl)dec-9-en-1-one, 8. Isolated as a yellow oil.
IR (neat): 1037 (m), 1442 (s), 1489 (s), 1636 (s), 2927 (m) cm21
.
1H NMR (400 MHz, CDCl3): d 1.22–1.41 (8H, m), 1.56–1.68 (2H,
m), 1.83 (2H, q, J = 6.8 Hz), 1.93 (2H, pent, J = 6.8 Hz), 1.98–
2.07 (2H, m), 2.23 (2H, t, J = 7.8 Hz), 3.39 (2H, t, J = 6.8 Hz),
3.44 (2H, t, J = 6.8 Hz), 4.91 (1H, d, J = 10 Hz), 4.97 (1H, d, J = 17
Hz), 5.72–6.86 (1H, m). 13C NMR (100 MHz, CDCl3): d 24.7,
25.2, 26.4, 29.2, 29.3, 29.6, 29.8, 34.1, 35.2, 45.9, 46.9, 114.5,
139.5, 172.2. MS: m/z (M + 23) 246.2 HRMS: m/z calc’d for
C14H25NNaO 246.1828, found 246.1831.
General procedure for cross metathesis
Amide (0.20 mmol, 1 equiv.) and 5-vinylbenzo[d][1,3]dioxole 9
(120 mg, 0.81 mmol, 4 equiv.) were dissolved in 1,2-DCE (5
mL). Grubbs’ second generation catalyst (18 mg, 0.020 mmol,
0.1 equiv.) was added and the mixture was heated at reflux for
2 days. The reaction mixture was concentrated under vacuum
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