Cytotoxic Amides from Fruits of Kawakawa, Macropiper excelsum

Article abstract of DOI:10.1055/s-0035-1546106

Cytotoxic amides have been isolated from the fruits of the endemic New Zealand medicinal plant kawakawa, Macropiper excelsum (Piperaceae). The main amide was piperchabamide A and this is the first report of this rare compound outside the genus Piper. Elev

Full text of DOI:10.1055/s-0035-1546106

Original Papers 1163
Cytotoxic Amides from Fruits of Kawakawa,
Macropiper excelsum*
1
2
1
1
3
2
Authors
Jeremy Lei , Elaine J. Burgess , Alistair T. B. Richardson , Bill C. Hawkins , Sarah K. Baird , Bruce M. Smallfield ,
2
1,2
John W. van Klink , Nigel B. Perry
1
Affiliations
Department of Chemistry, University of Otago, Dunedin, New Zealand
The New Zealand Institute for Plant & Food Research Limited, Department of Chemistry, University of Otago, Dunedin,
New Zealand
2
3
Department of Pharmacology & Toxicology, University of Otago, Dunedin, New Zealand
Key words
Abstract
change between two rotamers about the amide
bond, and they were chemically synthesized. In
view of the antitumor activity of the related pi-
perlongumine, all of these amides plus four syn-
thetic analogs were tested for cytotoxicity. The
most active was the piperine homolog piperdar-
dine, with an IC50 of 14 µM against HT 29 colon
cancer cells.
"
l amides
!
"
l Macropiper excelsum
Cytotoxic amides have been isolated from the
fruits of the endemic New Zealand medicinal
plant kawakawa, Macropiper excelsum (Pipera-
ceae). The main amide was piperchabamide A
and this is the first report of this rare compound
outside the genus Piper. Eleven other amides were
purified including two new compounds with the
unusual 3,4-dihydro-1(2H)-pyridinyl group. The
new compounds were fully characterized by
"
l Piperaceae
"
l cytotoxicity
"
l synthesis
Supporting information available online at
http://www.thieme-connect.de/products
2D NMR spectroscopy, which showed a slow ex-
Introduction
5]. Leaves were usually used for medicinal pur-
poses, but the fruits were also eaten “rejecting
the numerous seeds” [5]. Extracts of leaves
showed antimicrobial activity [6]. The roots were
also used medicinally [5], but they do not contain
the narcotic kavalactones (Plant & Food Research
unpublished results) found in kava roots [7–9].
The leaves of kawakawa contain myristicin as the
main volatile component [10] plus other volatile
phenyl propanoids, and mono- and sesquiter-
penes [11]. A rare bioactive lignan, diayangambin,
has been found in both wood [12] and leaves [13].
This compound exerts immunosuppressive and
anti-inflammatory effects in vivo [14]. We could
find no reports of kawakawa fruit chemistry, de-
spite fruits of other Piperaceae being rich sources
of bioactive amides such as pungent piperine 1
[15–17] and the antitumor piperlongumine/pi-
!
The islands of Aotearoa New Zealand were the last
major land mass on Earth to be settled by hu-
mans. Māori settlers arrived from Polynesia to
find a densely forested land, with almost no spe-
cies that they would have encountered previ-
ously, as the flora of New Zealand is about 80%
endemic [1]. However, the Māori recognized the
relationship of one plant to the Polynesian kava,
Piper methysticum, and named it kawakawa [2].
Kawakawa is classified as Macropiper excelsum
received
revised
accepted
March 12, 2015
April 14, 2015
April 19, 2015
(G.Forst.) Miq. (Piperaceae), a New Zealand en-
Bibliography
demic in a small Piper-related genus that also
contains eight other species across the Pacific [3].
There are two recognized subspecies of M. excel-
sum [3], ssp. psittacorum (Endl.) A.C. Sm. found
on Lord Howe Island, Norfolk Island, Kermadec Is-
land, and smaller islands off the north coast of the
North Island of New Zealand, and the main ssp.
excelsum, which grows as a shrub or small tree
on the North Island and warmer parts of the
South Island of New Zealand [4].
DOI http://dx.doi.org/
0.1055/s-0035-1546106
1
Published online June 3, 2015
Planta Med 2015; 81:
1
163–1168 © Georg Thieme
"
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
plartine 2 (structures in l Fig. 1) [18,19].
The fruits of female kawakawa (M. excelsum is di-
oecious [20]) are compound spikes (drupes) car-
rying up to 150 seeds each [21]. They develop
from being green and rigid to orange and soft dur-
ing ripening [22]. We now report the identifica-
tion of 12 amides from these fruits, including the
rare piperchabamide 3 and two new variants, 4
and 5. The new compounds and four related com-
Correspondence
Dr. Nigel Perry
Plant & Food Research
University of Otago
Department of Chemistry
P.O. Box 56
Dunedin 9054
New Zealand
Phone: + 6434798354
nigel.perry@plantandfood.co.nz
Māori developed a wide range of medicinal and
food uses for kawakawa, especially pain relief [2,
* Dedicated to Professor Dr. Dr. h.c. mult. Adolf Nahrstedt
on the occasion of his 75th birthday.
Lei J et al. Cytotoxic Amides from… Planta Med 2015; 81: 1163–1168

1
164 Original Papers
sensitive 2D NOESY spectrum, two sorts of cross-peaks were
present: in phase cross-peaks between the paired signals and op-
posite phase cross peaks corresponding to the usual through
space NOE correlations. This effect was assigned to the separation
of chemical exchange and cross-relaxation effects described by
Davis and Bax [26], and the 2D NMR structure solution was com-
pleted on the two sets of signals.
"
HR‑ESI‑MS (l Table 1) supported a molecular formula of
"
C14H15NO for 4. The NMR data (l Table 2) and COSY and HMBC
spectra were appropriate for a cinnamoyl moiety, which was
"
confirmed by closely matching data (l Table 2) to those of cinna-
moyl piperidine 6 [27]. The remaining amide portion showed
connectivity and shifts of a 3,4-dihydro-1(2H)-pyridinyl group,
"
which was supported by our data (l Table 2) closely matching
the two sets of signals reported for similar compounds [28]. This
gave the proposed structure 4, with two slowly exchanging ro-
"
tamers (l Fig. 2). Increasing the temperature gave a broadening
1
"
of the H NMR signals, most obviously H3′ (l Table 2), but coales-
cence was not reached at the temperatures possible in CDCl or
3
(
CD ) CO.
3 2
The 3,4-dihydro-1(2H)-pyridinyl (piperideide) group is rare in
natural amides, with only one previous example (7) from Pipera-
ceae plants [29]. Surprisingly, only one set of NMR signals was
listed for 7, closely matching our data for the major rotamer of 4
"
(
l Table 2, the NMR spectra of 7 were in the same CDCl solvent,
3
but field strength and temperature were not stated) [29]. Patil et
al. also reported a crystal structure for 7, with the E conformation
"
about the amide bond (l Fig. 2). However, one cannot assume “…
that the molecular shape observed in a crystal is identical to that
to be found in fluid media.” [30]; see for example [31].
Fig. 1 Structures of kawakawa amides and related compounds.
Other reports of piperideide natural products have been from
plants in the family Asteraceae, e.g., [28,32]. Hofer analyzed the
solution conformations of piperideides and found that the E con-
formation about the amide bond was slightly (60:40) more sta-
ble than the Z form (l Fig. 2) [28]. Our NOESY results were not
clear-cut because of small chemical shift differences between
"
pounds have been synthesized, and the cytotoxic activities of the
natural and synthetic amides are reported.
key signals, but did support the E form of 4 as the major confor-
"
mation (l Fig. 2).
Results and Discussion
The minor amide 5 showed a molecular formula of C15H15NO by
3
!
HR‑ESI‑MS (l Table 1), i.e., with an additional CO2 compared
"
1
Ethanol extracts of kawakawa leaves and fruits were analyzed by
reversed-phase liquid chromatography (RPLC). The leaf extract
showed myristicin as the major component with diayangambin
also present, as expected from previous reports [10,13]. The fruit
extract showed a contrasting composition, with diayangambin
as the major component, a trace of myristicin, plus a series of oth-
er UV active peaks. A bulk extract of kawakawa fruits was frac-
tionated by RP and silica gel column chromatography, and then
preparative RPLC to give the main unknown component. HR-
with 4. The H NMR spectrum of 5 showed a dioxymethylene
"
substituted aromatic ring (l Table 2), which accounted for these
additional atoms, but was otherwise very similar to that of 4, in-
cluding the pairing of corresponding signals. However, this com-
pound 5 was somewhat unstable, so it was synthesized for full
characterization and for biological testing (see below).
During the re-isolation of compounds 4 and 5, nine known
amides were purified from kawakawa fruits, and identified by
1
HR‑ESI‑MS and by matching H NMR data with published spectra
"
"
"
ESI-MS supported a molecular formula of C14H15NO2 (l Table
(l Table 1). These compounds (l Fig. 1) contain other combina-
"
1
). 2D NMR spectra (data not shown) led us to structure 3, which
tions of amine and acid groups (l Fig. 1): piperidine with piperi-
has been reported as piperchabamide A with chemical shift data
matching ours [23]. Piperchabamide A 3 has only been reported
from Piper chaba [23] and Piper capense [24], and this is the first
report of any amides from Macropiper.
dic acid (piperine 1) or reduced (8) or homologous (9, 13, 14) ac-
ids and isobutylamine with some of the same acids (10, 11, 12).
Such combinatorial diversity of natural amides is common within
Piper species [25].
Two other amides (4 and 5) purified from this kawakawa fruits
extract showed unusual features in their NMR spectra and could
Because of the instability of the new amides 4 and 5, and their
low yield from kawakawa fruits, we decided to synthesize them.
Commercially available cinnamoyl chloride readily reacted with
three off-the-shelf piperidines to give 6, 15, and 16 (all known,
see l Table 1 and Table S1, Supporting Information) for struc-
ture-activity relationships (see below). 1,2,3,4-Tetrahydropyri-
dine is not commercially available, so this approach to the syn-
1
not be matched to any known Piper amides [17,25]. The H and
1
3
"
C NMR spectra of the main unknown 4 (l Table 2) showed pair-
"
ing of some signals, suggesting the presence of two closely re-
lated compounds in a ratio of about 3:1, even though the RPLC
analyses showed only one sharp peak. However, in the phase-
Lei J et al. Cytotoxic Amides from… Planta Med 2015; 81: 1163–1168

Original Papers 1165
Table 1 Amides isolated from fruits of kawakawa, M. excelsum, plus synthetic analogs.
Compound
Registry number
Sourcea
HR‑ESI‑MS (Da)b
NMR match
RPLC (min)c
Assay purity (%)d
Piperchabamide A 3
807618-20-8
N
252.0992
[23]
11.2f
> 99
(C14H15NNaO2 req
2
52.0995)
Piperideide 4
Piperideide 5
New
New
N, S
N, S
236.1033 (C14H15NNaO
req 236.1046)
–
–
11.2f
11.2f
> 99
> 99
280.0923
(
C15H15NNaO3 req
80.0944)
308.1231
C17H19NNaO3 req
08.1263)
310.1408
C17H21NNaO3 req
10.1414)
336.1527
C19H23NNaO3 req
36.1570)
298.1403
C16H21NNaO3 req
98.1414)
324.1531
C18H23NNaO3 req
24.1570)
2
Piperine 1
94–62–2
N, C
N
[42]
[42]
[43]
[44]
[45]
[46]
[47]
[24]
11.3
11.1
12.2
10.8
11.8
10.1
10.4
10.0
> 99
95
(
3
Piperanine 8
Piperdardine 9
23512–46–1
188426-70-2
23512–53–0
139906-29-9
(
3
N
90
(
3
1
1
0
1
N
95
(
2
N
99
(
3
Fagaramide 12
Z 92449–51–9
E 495–86–3
N
270.1078
> 99
99
2
:1 Z:E
(C14H17NNaO3 req
2
70.1101)
282.1077
C15H17NNaO3 req
82.1101)
282.1079
C15H17NNaO3 req
82.1101)
13
82857–82–7
N
(
2
14
111479-04-0
N
96
(
2
6
1
1
1
5422–81–1
55486–27–6
17077–45–1
188106-57-2
S
S
S
S
NRe
NRe
NRe
NRe
[27]
NFf
NFf
NFf
10.6
10.7
8.4
> 99
> 99
> 99
> 99
5
6
7
10.9
a
f
N = natural product; S = synthetic; C= commercial; b [M + Na]+; c Analytical RPLC retention time; d By RPLC; e not recorded; f not found, see Table S1, Supporting Information;
These compounds gave coincident peaks under the analytical RPLC conditions, but were separated under preparative RPLC conditions (5: 5.6 min; 3: 5.9 min; 4: 7.4 min)
thesis of the target compounds 4 and 5 was not feasible. There-
fore, access to 4 was attempted from the known imide 17 [33].
Numerous attempts at forming the corresponding vinyl triflate
of 17 were unsuccessful with only trace amounts observed. In-
stead, access was provided by the treatment of cinnamoyl chlo-
ride with α-tripiperideine [34] in the presence of triethylamine,
affording 4 in a 39% yield. Under analogous reaction conditions,
treatment of the known 3,4-methylenedioxycinnamoyl chloride
ities were found using the HT29 colon cancer cell line, so IC50 val-
ues were determined for the three most active compounds.
Piperdardine 9 (IC50 13.7 µM) and piperchabamide A 3 (IC50
28.3 µM) both showed maximal toxicity at the highest concentra-
tion tested, but with piperine 1 (IC50 70.3 µM), 38% of cells still
retained viability at 400 µM. Piperdardine 9 has previously been
reported to be cytotoxic to a range of other cell lines (as piperar-
boricoline, assayed at 64 µM) [36]. Piperchabamide A 3 has been
evaluated for hepatoprotective [37] and antiparasitic [24] acti-
vites, but we could not find any previous reports of cytotoxic ac-
tivity. In terms of the mechanism of action, piperlongumine 2 and
analogs have been found to elevate cellular levels of reactive oxy-
gen species selectively in cancer cell lines with the electrophilic
functionalities O=C1–C2=C3 and O=C2′-C3′=C4′ (our numbering,
[35] with α-tripiperideine provided 5 in a 47% yield. Both syn-
thetic samples were spectroscopically identical to the natural
products from kawakawa fruits.
The cytotoxicities of the full panel of kawakawa-derived com-
pounds were tested in three cancer cell lines and in one “normal”,
non-transformed cell line. Various compounds showed some tox-
icity at 10 µM over 48 h, but the degree of toxicity varied between
cell lines (Figs. S5 and S6, Supporting Information). In the immor-
talized bone marrow-derived mesenchymal stem cell line
RCB2157, quite a different effect was found. There was a com-
plete lack of cytotoxicity with all compounds, although some did
slightly increase cell growth rates. Therefore kawakawa-derived
amides exhibit selectivity towards cancer cells. The highest toxic-
"
l Fig. 1), both important for activity [38]. Piperchabamide A 3
was the only amide tested with the O=C2′-C3′=C4′ functionality,
whereas synthetic 17 is a regioisomer lacking O=C2′-C3′=C4′ but
"
with O=C1–C2=C3 (l Fig. 1). Compound 17 was less active than 3
against colon cancer cells, but more active than 3 against breast
cancer cells. In prostate cancer cells, compound 17 caused a de-
crease in cell viability of 22.0 ± 4.9%, compared to the vehicle on-
ly-treated control, whereas 3 supported cancer cell growth, in-
Lei J et al. Cytotoxic Amides from… Planta Med 2015; 81: 1163–1168

1
166 Original Papers
4
5
Table 2 NMR data of kawakawa
amides 4 and 5a.
Atomb
13C
1H
13C
1H
1
1
2
2
3
3
4
5
6
7
8
9
9
2
2
3
3
4
5
5
6
6
1
A
164.3
163.7
117.0
117.0
143.2
143.6
135.2
127.9
128.8
129.7
128.8
127.9
–
–
164.4
163.7
114.8
114.8
142.9
143.4
129.5
106.3
148.2c
149.1c
108.4
123.9
124.0
125.3
124.6
108.6
109.2
21.7
–
B
–
–
A
6.91 (d, 15.5)
6.90 (d, 15.5)
7.68 (d, 15.5)
7.75 (d, 15.5)
–
6.72 (d, 15.5)
6.70 (d. 15.5)
7.58 (d, 15.5)
7.64 (d, 15.5)
–
B
A
B
A + 4B
A + 5B
7.53 (br d, 8.0)
7.37 (m)
7.02 (d, 1.5)
–
A + 6B
A + 7B
7.37 (m)
–
A + 8B
A
7.37 (m)
6.79 (d, 8.0)
6.99 (br d, 8.0)
6.99 (br d, 8.0)
6.81 (br d, 8.5)
7.28 (br d, 8.5)
4.99 (br dt, 8.0, 3.5)
5.12 (br m)
2.09 (m)
7.53 (br d, 8.0)
–
B
′A
′B
′A
′B
′
125.3
124.7
108.8
109.5
21.7
6.83 (br d, 8.0)
7.30 (br d, 8.0)
5.02 (br dt, 8.0, 4.0)
5.16 (br m)
2.12 (m)
′A
′B
′A
′B
′′
22.0
1.90 (m)
22.0
1.86 (m)
22.2
22.2
1.86 (m)
40.8
3.79 (m)
3.79 (m)
–
40.8
3.75 (m)
44.0
43.9
3.75 (m)
–
101.4
5.96 (s)
a
c
In CDCl3; 1H at 500 MHz, shift in ppm (multiplicity, J in Hz); 13C at 125 MHz, shift in ppm; b A = major rotamer, B = minor, see l Fig. 2;
"
assignments uncertain
NMR system or at 500 MHz on a Varian 500 MHz AR premium
shielded spectrometer, from samples in CDCl3 at 25°C in 5 mm
Fig. 2 Proposed ro-
tamers of kawakawa
amides 4 and 5.
1
tubes. H chemical shifts are reported relative to the residual
1
3
CHCl3 singlet at δ 7.25 ppm and C chemical shifts relative to
the CDCl3 triplet at δ 77.00 ppm. CH Cl2 and tetrahydrofuran
2
(THF) were dried using a PureSolv MD-6 solvent purification sys-
tem. EtOH, H O, and EtOAc were distilled before use and all other
2
solvents and reagents were used as received. Analytical RPLC
"
analyses (l Table 1) were carried out using an Agilent HP1200,
controlled with Agilent OpenLab software, at 20°C on a C18 col-
umn [Phenomenex Luna ODS(3) 5 µm 100 Å 150 × 3 mm] with a
2
× 4 mm C18 guard column. Peaks were detected at 210, 254,
and 280 nm. The mobile phase components were A – MeCN and
B – H O, with 0.1% formic acid in both. A linear program was
2
used: A t0 min = 10%, t12.5 = 100%, t15 = 100%, t16 = 10%, t20 = 10%.
The flow rate was 0.5 mL/min, with an injection volume of 5 µL.
creasing viable cells by 9.3 ± 4.4%. (Fig. S5, Supporting Informa-
tion).
In conclusion, we have shown the presence of bioactive amides in
fruits of the endemic New Zealand medicinal plant kawakawa,
M. excelsum (Piperaceae). The main amide was the rare pipercha-
bamide A, which has selective cytotoxic activity.
Plant material
Kawakawa fruits were collected from Parapara River Access Road,
Golden Bay, Nelson, New Zealand (40°43′ 44′′ E 172°40′ 21′′ S)
from January 23rd to 25th, 2008. Identification was by B.M.S.,
and a foliage voucher specimen (code 080125–07) is lodged in
the Plant & Food Research Lincoln herbarium collection. Fruits
were stored at − 80°C until extracted.
Materials and Methods
!
Extraction and isolation
Frozen kawakawa fruits (43 g) were pulverized under liq N and
2
General
extracted with 96% EtOH (430 mL) overnight with shaking. The
filtered extract was evaporated to dryness (35°C) to give a green,
sweet-smelling gum (2.43 g). This was bound onto C18 (octadec-
yl-functionalized silica gel, Aldrich, 2.5 g) and subjected to RP
flash chromatography using a prepacked C18 column (Isolute,
10 g) preconditioned with EtOH, EtOH: H O (1:1), and H O. This
IR spectra were recorded on a Bruker Optics Alpha FTIR spec-
trometer with ATR, and UV spectra on a Jasco spectrometer.
High-resolution mass spectra (HR‑MS) were recorded on a
Bruker microTOFQ mass spectrometer using an electrospray ion-
ization (ESI) source in the positive mode. H, C, and 2D NMR
spectra were recorded at either 400 MHz on a Varian 400-MR
1
13
2
2
was developed using H O, 1:4 EtOH:H O, 1:1 EtOH:H O, 4:1
2
2
2
Lei J et al. Cytotoxic Amides from… Planta Med 2015; 81: 1163–1168

Original Papers 1167
EtOH:H O, EtOH, and EtOAc, two fractions per solvent mix.
m.p. > 260 = °C (lit. 80–81°C [40]), 16 as a white solid: m.p. 91°C
2
Fractions 7 and 8 eluted with 4:1 EtOH:H O were combined
(no lit. value found), and 17 as a white solid: m.p. 60°C (lit. 58–
2
(433 mg) and subjected to silica gel flash chromatography (10 g
59°C [33]).
Si gel 60, 200–400 mesh, 40–63 µm) on a column preconditioned
with toluene (AR grade). The column was developed using a 1–
Cell lines and cell culture
1
0% gradient with toluene:EtOAc (lab grade distilled), collecting
MDA‑MB‑231 breast cancer and PC3 prostate cancer cells were a
kind gift from Assoc. Prof. R. Rosengren, HT29 colon cancer cells
were from CellBank Australia, and the immortalized bone mar-
row-derived mesenchymal stem cell line RCB2157 (MSC) was
from the Riken BioResource Center through the National Bio-Re-
source Project of the MEXT, Japan. All cell lines were cultured in
DMEM with 5% or 10% (for the MSCs) heat-inactivated FBS, 100
units/mL penicillin, and 100 µg/mL streptomycin. Cells were in-
cubated at 37°C in humidified conditions with 5% CO2.
26 fractions. Fractions 16–17 (18 mg) and 18–19 (11 mg) gave red
spots at R 0.625 on Si TLC (Merck DC Kieselgel 60 F254, mobile
phase 7:3 toluene:EtOAc, visualized with vanillin H SO4 dip)
and were combined. The combined sample was subjected to pre-
parative RPLC (Phenomenex Luna 5 µ C18 100, 250 mm × 10 mm,
f
2
5
5
3
µm; guard column Merck 100 RP 18 LichoCart 25 mm × 4 mm,
µm) with a 65:35 MeCN:H O mobile phase at 5 mL/min,
2
0°C, 50 µL injections, and 210 and 254 nm detection. Three col-
lected fractions gave piperideide 5 (2 mg, preparative RPLC RT
5
.6 min), piperchabamide A 3 (6 mg, RT 5.9 min), and piperideide
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay
4
(4 mg, RT 7.4 min)
Piperchabamide A 3: colorless oil. H and 13C NMR data matching
1
The quantity of cells remaining 48 h after treatment of the cells
with the compounds or DMSO only (vehicle control) was com-
pared using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assay, which assesses mitochondrial and
endoplasmic reticulum dehydrogenase activity [41]. MTT was
made up to 5 mg/mL in PBS, added to cells at a final concentration
of 0.5 mg/mL, and incubated at 37°C for 3 h. DMSO was used to
dissolve the formazan crystals and the resulting absorbance was
read at 560 nm. The positive control chemotherapeutic drug cis-
platin (Sigma-Aldrich, purity ≥ 99.9%) was tested at the 48 h time
point for all cell lines. IC50 values were calculated as follows:
HT29 cells: 17.8 µM; MDA‑MB‑231 cells: 3.7 µM; PC3 cells:
4.4 µM and MSC cells 3.8 µM.
"
[23]; HR‑ESI‑MS, l Table 1.
Piperideide 4: colorless oil. UV (MeOH): λ
283 nm (ε 12700);
max
1
13
"
"
H and C NMR data, l Table 2; HR‑ESI‑MS, l Table 1.
Synthetic piperideide 4 (see below): yellow crystals, m.p. 50°C. UV
(MeCN): λmax 280 nm (ε 15400); IR (film) 2956, 1641, 1602,
−
1
1
13
"
1
416, 979, 761 cm ; H and C NMR data, l Table 2; HR‑ESI‑MS,
"
l Table 1.
1
"
Piperideide 5: colorless oil. H NMR data, l Table 2; HR‑ESI‑MS,
"
l Table 1.
Synthetic piperideide 5 (seebelow): pale yellowcrystals, m.p. 82°C.
UV (MeCN): λmax 329, 293 nm (ε 18600, 11900); IR (film) 2902,
−1
1
13
1637, 1595, 1488, 1446, 1250, 1031, 796 cm ; H and C NMR
"
"
data, l Table 2; HR‑ESI‑MS, l Table 1.
Another batch of frozen kawakawa fruits (376 g) were pulverized
under liq N2 and extracted with CHCl3 (1.75 L) overnight with
shaking. The separated extract was evaporated to dryness to give
a green gum (2.9 g). This was fractionated by various stages of sil-
ica gel flash chromatography and then preparative RPLC, as
above, to give further samples of piperchabamide A 3 and piper-
ideide 4, plus the known amides piperine 1 and 8–14 identified in
Statistical analysis
The results are presented as mean ± standard deviation (SD) of
three independent experiments. Absorbance values were nor-
malized to vehicle only control (DMSO) for each experiment and
a one-way ANOVA followed by a Dunnettʼs post hoc test was used
to analyze the data. Statistical significance was determined as
p < 0.05. To calculate IC50 values, concentrations were log trans-
formed and the resulting graph analyzed by nonlinear regres-
sion.
"
l Table 1.
Syntheses of 4–6 and 15–17
Synthesis of piperideide 4: Triethylamine (1.7 mL, 12.0 mmol) and
dodecahydro-4a,8a,12a-triazatriphenylene (0.5 g, 2.0 mmol)
were added to a solution of cinnamoyl chloride (1.0 g, 6.0 mmol)
in THF (3 mL) [34]. The reaction mixture was stirred at 60°C for
Supporting information
H and C NMR spectra of compounds 4 and 5, cytotoxicity re-
sults against four cell lines, and H NMR data for compounds 6,
15, 16, and 17 are available as Supporting Information.
1
13
1
1
6 h before being diluted with H O (20 mL) and extracted with
2
EtOAc (3 × 15 mL). The combined organic phase was washed with
aqueous NaHCO3 (sat.) (3 × 15 mL) and brine (15 mL), and then
Acknowledgements
!
dried over anhydrous MgSO . The solvent was removed under re-
4
duced pressure to provide the crude product as an orange oil
We thank Manawhenua Ki Mohua for permission to collect and J.
Ward-Holmes, C. Griffiths, M. Reiher and M. Little for assistance
with collecting kawakawa fruits, H. Smallfield for preparing her-
barium voucher samples, and I. Stewart and J. Lyburn for as-
sistance with the NMR work and for high-resolution MS.
A.T.B.R. thanks the Department of Chemistry, University of Ota-
go, for a summer studentship. This work was partly supported by
the New Zealand Foundation for Research, Science and Technol-
ogy (now the Ministry of Business Innovation and Employment;
contract C02X0601).
(1.0 g). The crude product was purified using a silica gel column
(20% EtOAc/40–60 pet ether) to give 4 (0.5 g, 39%, see properties
above).
Synthesis of piperideide 5: Triethylamine (0.5 mL, 3.2 mmol) and
dodecahydro-4a,8a,12a-triazatriphenylene (150 mg, 3.2 mmol)
were added to a solution of 3, 4-methylenedioxycinnamoyl chlo-
ride (335 mg, 1.6 mmol) in THF (1 mL). The reaction mixture was
worked up and purified, as above, to give 5 (190 mg, 47%, see
properties above).
Compounds 6, 15, 16, and 17 were prepared by the reaction of
cinnamoyl chloride with the appropriate amine to give 6 as a
white solid: m.p. 121°C (lit. 114–116°C [39]), 15 as a white solid:
Lei J et al. Cytotoxic Amides from… Planta Med 2015; 81: 1163–1168

1
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533–535
Conflict of Interest
!
The authors declare no conflict of interest.
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