Piperlongumine (piplartine) and analogues: Antiproliferative microtubule-destabilising agents

Article abstract of DOI:10.1016/j.ejmech.2016.09.048

Piperlongumine (piplartine, 1) is a small molecule alkaloid that is receiving intense interest due to its antiproliferative and anticancer activities. We investigated the effects of 1 on tubulin and microtubules. Using both an isolated tubulin assay, and a combination of sedimentation and western blotting, we demonstrated that 1 is a tubulin-destabilising agent. This result was confirmed by immunofluorescence and confocal microscopy, which showed that microtubules in MCF-7 breast cancer cells were depolymerized when treated with 1. We synthesised a number of analogues of 1 to explore structure-activity relationships. Compound 13 had the best cytotoxic profile of this series, showing potent effects in human breast carcinoma MCF-7?cells whilst being relatively non-toxic to non-tumorigenic MCF-10a cells. These?compounds will be further developed as potential clinical candidates for the treatment of breast cancer.

Full text of DOI:10.1016/j.ejmech.2016.09.048

Accepted Manuscript
Piperlongumine (piplartine) and analogues: Antiproliferative microtubule-destabilising
agents
Mary J. Meegan, Seema Nathwani, Brendan Twamley, Daniela M. Zisterer, Niamh M.
O'Boyle
PII:
S0223-5234(16)30748-6
10.1016/j.ejmech.2016.09.048
EJMECH 8914
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 July 2016
Revised Date: 5 September 2016
Accepted Date: 6 September 2016
Please cite this article as: M.J. Meegan, S. Nathwani, B. Twamley, D.M. Zisterer, N.M. O'Boyle,
Piperlongumine (piplartine) and analogues: Antiproliferative microtubule-destabilising agents, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.048.
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Graphical Abstract

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Piperlongumine (Piplartine) and Analogues: Antiproliferative Microtubule-Destabilising
Agents
Mary J. Meegan,1 Seema Nathwani,2 Brendan Twamley,3 Daniela M. Zisterer,2 Niamh M.
O’Boyle1,2*
1School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland.
2School of Biochemistry & Immunology, Trinity Biomedical Sciences Institute, 152-160 Pearse
Street, Trinity College Dublin, Dublin 2, Ireland.
3School of Chemistry, Trinity College Dublin, Dublin 2, Ireland.
*Corresponding author; email oboyleni@tcd.ie
ABSTRACT
Piperlongumine (piplartine, 1) is a small molecule alkaloid that is receiving intense interest due
to its antiproliferative and anticancer activities. We investigated the effects of 1 on tubulin and
microtubules. Using both an isolated tubulin assay, and a combination of sedimentation and
Western blotting, we demonstrated that 1 is a tubulin-destabilising agent. This result was
confirmed by immunofluorescence and confocal microscopy, which showed that microtubules in
MCF-7 breast cancer cells were depolymerised when treated with 1. We synthesised a number of
analogues of 1 to explore structure-activity relationships. Compound 13 had the best cytotoxic
profile of this series, showing potent effects in human breast carcinoma MCF-7 cells whilst
being relatively non-toxic to non-tumorigenic MCF-10a cells. These compounds will be further
developed as potential clinical candidates for the treatment of breast cancer.
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KEYWORDS
Antiproliferative; anticancer; combretastatin A-4; microtubule-destabilizing; piperlongumine;
tubulin
1. INTRODUCTION
Cancer is the uncontrolled growth and spread of cells. Annually, 8.2 million deaths are
attributable to cancer, an estimated 13% of all deaths worldwide, and breast cancer accounts for
over half a million of these. The total number of new cases is expected to rise by 70% over the
next two decades [1]. The continuous development of new, cost-effective drug therapies is
crucial to increase the range of options available to patients, and to improve their long-term
prognosis.
Piperlongumine (also known as piplartine, 1, Figure 1) is an alkaloid isolated from a number of
Piper plant species, including the long pepper plant Piper longum L. It has a large number of
reported uses in traditional medicine, and has demonstrated cytotoxic and antitumour activity [2].
In particular, it was found to be selectively toxic to cancer cells by increasing the level of
reactive oxygen species (ROS) and inducing apoptotic cell death, although the exact mechanisms
of action remain unclear [3]. Compound 1 has been recently reported to be a direct STAT3
inhibitor [4], a proteasome inhibitor [5], to promote autophagy [6], and to modulate NF-κB and
NF-κB-regulated gene products [7].
Compound 1 is a chalcone-type molecule, consisting of two ring systems linked by a α-β-
unsaturated carbonyl chain. One of the rings is aromatic and substituted with three methoxy
groups, while the second is a piperidinone-type ring containing a conjugated alkene. Compound
1 is structurally similar to a number of known tubulin-targeting antiproliferative compounds.
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These include combretastatin A-4 (2) and chalcone 3 (Figure 1), both containing a
trimethoxyphenyl moiety [8]. An analogue of 2 with a substituted pyridone ring (4, Figure 1)
also displays potent antitubulin activity [9]. A hybrid of compounds 1 and 2 was recently
reported as a tubulin depolymerising agent [10]. Considering these results, we undertook to
investigate if 1 has inherent tubulin- and microtubule-targeting activity in breast cancer cells. We
also synthesised a number of analogues of 1 to investigate structure-activity relationships of the
trimethoxyphenyl group of 1 on the antiproliferative potency in breast cancer cells.
2. RESULTS and DISCUSSION
2.1. Chemistry
Previous research has examined the structure-activity relationships of the piperidinone ring of 1
[11]. Hence, we sought to examine the effects of modifying the trimethoxyphenyl ring. The
trimethoxyphenyl ring has repeatedly been shown to be crucial for the tubulin-depolymerising
ability of a number of compounds, including colchicine, podophyllotoxin and 2 [12-14]. These
trimethoxyphenyl-containing compounds bind at the colchicine-binding site on tubulin, mainly
located in the β-subunit. A number of analogues of 1 with different substituents were chosen for
synthesis, in order to better understand the contribution of the trimethoxyphenyl ring to the
antiproliferative activity of 1. Synthesis was achieved in two steps via the key intermediate 5,6-
dihydropyridin-2(1H)-one.
5,6-Dihydropyridin-2(1H)-one (6) was synthesised in two steps from but-3-en-1-amine via N-
(but-3-en-1-yl)acrylamide (5) (Scheme 1, steps i and ii). Compound 1 and seven analogues (7-
13) were obtained in moderate yields via a one-pot reaction between 6 and appropriately
substituted cinnamic acids, pre-activated with pivaloyl chloride (Scheme 1, steps iii and iv, 61-
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1
72% yield)[15]. H NMR coupling constants (15-16 Hz) indicated a trans configuration around
the linear α,β-unsaturated double bond. An X-ray crystallographic study of 12 confirmed the
structure, featuring a non-planar piperidinone ring and a 3,4-dimethoxyaryl ring (Figure 2). The
length of the double bond in the lactam ring agrees with the published structure for 1 (1.329 Å
compared to 1.322 Å) [16]. The C-N bonds in the lactam ring are unequal with lengths of 1.482
Å (C1-N6) compared to 1.390 Å (C5-N6). The 3-methoxy and 4-methoxy groups are
approximately co-planar with the aromatic ring (torsion angles 5.6 (2)° and 176.8 (13)°
respectively). The linear α,β-unsaturated double bond adopts the predicted trans configuration,
with a bond length of 1.335 Å (1.329 Å reported)[16].
2.2. Biochemistry
2.2.1. Cell Viability
Compound 1 was first tested for its effects on viability in two human cell lines, MCF-7 breast
cancer cells and Jurkat T-lymphocytes. The concentration of 1 required to inhibit the growth of
MCF-7 and Jurkat cells by 50% (IC50) was determined to be 1.2 ± 0.6 ꢀM and 1.4 ± 0.3 ꢀM
respectively at 48 hr (Figure 3A). These are similar to previously reported values for 1 (8 ꢀM in
MCF-7 cells and 5 ꢀM in Jurkat cells) [17, 18]. Cell viability results for 1 in a large number of
cancer cell lines have been determined and, in general, IC50 values range between 1-10 ꢀM [2].
IC50 values for compounds 7-13 were found to be between 3.4 and 9.6 ꢀM in MCF-7 cancer
cells, with 7, 12 and 13 showing the best activity (IC50 values of 3.5, 3.7 and 3.4 ꢀM
respectively)(Table 1). Mono-methoxylated compounds 8, 9 and 10 showed decreased potency
indicating that one methoxy group is detrimental to antiproliferative activity, regardless of its
position on the ring (2-, 3-, or 4-methoxy). Comparison of di-methoxylated compounds 11 and
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12 reveals that a 2,3-methoxy substitution pattern is preferable to 1,3-methoxy (IC50 values of
8.5 and 3.7 ꢀM respectively). These analogues were also assessed for toxicity using non-
tumorigenic MCF-10a mammary epithelial cell line (Figure 3B and Figure S1, Supporting
Information). None of the compounds had any effect on viability of MCF-10a cells at a
concentration of 1 ꢀM (24 and 48 hr) and minimal effects were noted at 5 ꢀM (>90% viability
for all compounds at 24 hr, and >80% viability at 48 hr). The best selectivity at 5 ꢀM was
observed for compounds 7, 12 and 13 (differences of 53, 46 and 48% in cell viability of MCF-
10a compared to MCF-7, respectively). Significant effects on MCF-10a cell viability were
observed at 10 ꢀM, equal to the effect on MCF-7 viability in most cases. Treatment with 1 (10
ꢀM) caused a 90% reduction in cell viability. This indicates poor selectivity for cancer cells at
this concentration, with the exception of compound 13. Compound 7 showed the highest toxicity
at 48 hr (Figure 3B). On the basis of combined results in MCF-7 and MCF-10a cells, compounds
12 and 13 were identified as the best candidates for future development.
2.2.2. Effect of Pre-treatment with Antioxidants on Cellular Viability
Compound 1 has been reported as an agent that increases the level of ROS in cells [3]. To
investigate the potential effects of our analogues 7-13 on ROS levels in MCF-7 breast cancer
cells, we employed a viability assay in which cells are pre-incubated with an antioxidant prior to
treatment with the compound of interest (Figure 4). This assay indicates potential involvement of
ROS in a compound’s mechanism of action. Two antioxidants were used: N-acetyl cysteine
(NAC) and the vitamin E derivative Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid). The antioxidant concentrations used were shown to have no effect on cell viability. Trolox
(100 ꢀM and 300 ꢀM) has been shown to be an effective antioxidant in MDA-MB-231 breast
cancer cells and MIA PaCa-2 pancreatic cancer cells [19]. We also evaluated a number of known
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tubulin-destabilising agents [colchicine, 2 and four chalcones (14-17, Figure 5)]. These
compounds were chosen on the basis of structural similarity to 1 (all with the exception of
colchicine contain a linear double bond linking two ring systems, one of which is substituted
with 2 or more methoxy groups), antiproliferative potency, and tubulin-targeting activity. These
chalcones were synthesised and characterised by published procedures (compound 14 [8], 15
[20], 16 [8], 17 [21]).
Firstly, the effects of 1 and representative compounds 7, 12 and 13, alone and in combination
with NAC and Trolox were assessed. Pre-treatment for 1 hr with NAC has a dramatic protective
effect on MCF-7 cells at all concentrations of all compounds tested (Figure 4). Viability
increases were generally greater than 50%, with the highest increase of 73% (compound 7, 10
ꢀM). No significant increases in cell viability were noted when cells were pre-treated with
Trolox. Similar results show that pre-treatment with NAC but not Trolox has an effect on activity
of 1 [19]. Compound 1 is a relatively reactive compound, due to the reactive α,β-unsaturated
carbonyl system contained in the piperidinone ring. It has been shown to directly bind to NAC
(200 equiv. in Hepes buffer; 24 hr reaction)[19] and to the small-molecule thiol methyl
thioglycolate (3 equiv. in DMSO; 72 hr reaction)[11]. It is possible that this reaction occurs in
the aqueous media of cell viability assay, thus inactivating the α,β-unsaturated carbonyl group of
1. This reaction is not possible with Trolox. The same considerations apply to analogues 7, 12
and 13. This is a potential reason for the different effects observed with the two different
antioxidants. The differing concentrations of NAC and Trolox may also play a part.
Secondly, effects of antioxidant pre-treatment on cell viability were assessed for known tubulin-
targeting compounds. Cells pre-treated with NAC showed increased viability for all compounds
compared to cells that were not pre-treated (Figure 6). Highest increases were noted for 2 (100
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nM; 22%) and chalcone 14 (35%). Increases in viability were greater than 10% for all
concentrations of the six compounds evaluated. Similar results were obtained for a lower
concentration of 16 and 17 (1 ꢀM; data not shown). Cells pre-treated with Trolox showed
slightly increased viability but effects were generally minimal (Figure 6). The highest increase in
viability was 11.4% for 2 (10 ꢀM). Viability was increased by less than 10% for chalcones 14-
17.
The results from the viability assay of colchicine, 2 and chalcones 14-17 indicate potential ROS
involvement in cancer cell death. This has not been reported for 2 or chalcones 14-17 previously
and is worthy of further investigation. A number of known tubulin-targeting agents also have
effects on reactive oxygen species in cells. Paclitaxel, a microtubule-stabilising agent, has been
reported to generate ROS by activating plasma-membrane associated NADPH oxidase
(NOX)[22]. Vinca alkaloids, clinically used microtubule-destabilising agents, induce apoptosis
in lung adenocarcinoma cells via ROS accumulation [23]. Isothiocyanates known to induce
oxidative stress have subsequently been shown to induce degradation of cellular tubulin by
proteasomes [24]. An analogue of colchicine, Green-1, was reported as increasing ROS
production in pancreatic cancer cells and leukaemia cells but its structural modification led to
loss of its ability to depolymerise tubulin [25]. It appears that there is a link between ROS levels
and the behaviour of microtubules in cells. Given the advanced status of 2 in phase II clinical
trials, it is important to fully elucidate its cellular effects [21].
2.2.3. Effects of Piperlongumine on the Cell Cycle and Apoptosis
Further biochemical work was carried out to explore the type of cell death induced by 1. The
effects of 1 on the cell cycle of MCF-7 cells were evaluated. Flow cytometric analysis of
propidium iodide stained cells demonstrated simultaneous decreases in the G0G1 cell population
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[16 % and 15 % (24 hr), and 21 % and 19 % (48 hr) for 10 and 20 ꢀM respectively] and
increases in the G2/M cell population [13 %, 16 % and 12 % (24 hr), and 6 %, 15 % and 10 %
(48 hr) for 5, 10 and 20 ꢀM respectively] (Figure 7A). There was a small, statistically significant
increase in the percentage of apoptotic cells (sub-G0G1) after 48 hr (10 and 20 ꢀM of 1; 4.3 and
4.9% respectively).
Due to the increase in apoptotic cells, further effects of 1 on anti-apoptotic proteins of the Bcl-2
protein family were investigated, namely Bcl-2 and Mcl-1. These proteins contribute to an
increased apoptotic threshold in cancer cells and allow cells to survive in stressful environments.
Treatment with 1 (10 ꢀM) caused downregulation of the expression of Bcl-2 and Mcl-1 as
demonstrated by western blotting (Figure 7B). Expression of Bcl-2 was downregulated at 48 hr,
whereas expression of Mcl-1 was evidenced after 72 hr. These results are in agreement with
previous reports of 1-induced apoptosis in MCF-7 cells after 36 hr, evidenced by annexin V/PI
double-staining and PARP cleavage [26]. Compound 1 also reportedly induces both apoptosis
and necrosis in leukaemia cell lines [17], and apoptosis in Burkitt lymphoma cell lines [27],
prostate cancer cells [28], and triple-negative breast cancer cells [18].
Tubulin-destabilising agents often cause increased cell accumulation in the G2/M phase, due to
defective mitotic checkpoints, as found for 1 in MCF-7 cells above. A number of previous
studies have noted G2/M arrest upon treatment with 1, including in Chinese hamster lung
fibroblasts (V79),[29] OVCAR3 human ovarian cancer cells [30] and PC-3 prostate carcinoma
cells [28]. This suggests that 1 may potentially target tubulin. The structural similarity of 1 to a
number of known tubulin-destabilizing agents (Figure 1) strengthens this hypothesis. To
investigate further, specific tubulin assays were performed.
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2.2.4. Effects of Piperlongumine on Tubulin Polymerisation and Interaction with
Colchicine-Binding Site
The tubulin-targeting properties of 1 were firstly examined in vitro using isolated bovine tubulin
(Figure 8A). The effect of test compounds 1 and 7 on tubulin polymerization was determined by
reading absorbance at 340 nm over 60 min, as the extent of light-scattering by microtubules is
proportional to their degree of polymerisation [31]. Paclitaxel, a known microtubule-stabilising
agent, was used as a control and increased the final polymer mass (Figure 8A). Depolymerisation
of tubulin and reduction in the final polymer mass was noted for the higher concentration of 1
(25 ꢀM, Figure 8A). A similar but less pronounced effect was seen for compound 7 (25 ꢀM).
Compound 1 was selected for further investigation in MCF-7 breast cancer cells and Jurkat T-
lymphocytes.
A cellular assay based on sedimentation followed by western blotting were used to examine the
effects of 1 on tubulin polymerisation in both MCF-7 and Jurkat cells (Figure 8B). Polymerized
and depolymerized microtubules have different solubilities and localize preferentially in the
pellet or supernatant of lysed, centrifuged cells, respectively [32]. Paclitaxel (a microtubule-
stabilising agent) and nocodazole (a microtubule-destabilising agent) were used as controls. As
expected, tubulin from nocodazole-treated cells was depolymerized and detected almost wholly
in the supernatant, whereas tubulin from paclitaxel-treated cells was polymerized and detected
solely in the pellet (Figure 8B). Tubulin from cells treated with 1 (10 ꢀM) was found exclusively
in the supernatant, indicating complete depolymerization of tubulin (Figure 8B). Similar results
were obtained for 5 ꢀM and 20 ꢀM concentrations of 1. Equal amounts of tubulin were detected
in the supernatants and pellets of cells treated with 1 ꢀM of 1, indicating that this concentration
does not have an effect on tubulin polymerization in MCF-7 and Jurkat cells (data not shown).
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Finally, a second cellular assay to examine the effects of 1 on the microtubule network of MCF-7
cells was carried out using immunofluorescence and confocal microscopy. Cells were again
treated with either paclitaxel, known microtubule-destabilising agent 2, or 1. Vehicle-treated
cells displayed organised microtubule structures (Figure 9). Cells treated with the microtubule-
stabilising agent paclitaxel exhibited characteristic bundling, whereas cells treated with 2 showed
disruption of the microtubule network. Cells treated with a higher concentration of 1 (20 ꢀM)
showed a disorganised microtubule structure, consistent with depolymerised tubulin. A lower
concentration of 10 ꢀM caused less depolymerisation, with some polymerised microtubules still
present (Figure 9). These results provide further evidence that 1 is a direct microtubule-
destabilising agent.
Compound 1 is structurally similar to a number of tubulin-depolymerising agents, including 2,
which bind to the colchicine-binding site on tubulin. Potential binding of 1 at the colchicine-
binding site was therefore investigated using a whole-cell based assay. N,N’-Ethylene-
bis(iodoacetamide) (EBI) is an alkylating agent that cross-links cysteine residues at positions 239
and 354 in the colchicine-binding site of tubulin, forming a β-tubulin-EBI adduct. This adduct is
detectable by Western blotting as an immunoreactive band that migrates faster than β-tubulin.
Microtubule-destabilising agents that bind at the colchicine-site, such as 2, prevent the formation
of the β-tubulin-EBI adduct [33]. MCF-7 cells were treated with vehicle control, 2 (10 ꢀM) or 1
(40 ꢀM) for 2 h, followed by EBI for an additional 1.5 h (Figure 10). Control samples show the
presence of the β-tubulin-EBI adduct at a lower position, indicating that EBI has cross-linked
Cys239 and Cys354 on β-tubulin. Adduct formation was inhibited in cells treated with 2,
confirming that 2 binds to the colchicine-binding site, whereas 1 did not inhibit the formation of
the β-tubulin-EBI adduct. Similar results were found in HT-29 cells (Figure 10). This indicates
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that 1 does not bind at the colchicine-site of tubulin. It is also possible, due to the presence of
reactive α,β-unsaturated groups, that compound 1 itself could cross-link cysteine residues on
tubulin. It has a similar molecular weight to EBI (317 and 396 respectively) and so an adduct
between β-tubulin and 1 would migrate a similar distance to that of a β-tubulin−EBI adduct.
Further work is required to determine where, and how, 1 interacts with tubulin.
3. Conclusion
Piperlongumine (piplartine) 1, a small molecule which has been receiving huge interest of late,
has been shown for the first time to target tubulin, by destabilising microtubules in MCF-7 breast
cancer cells and Jurkat T-lymphocytes. This effect was confirmed by in vitro tubulin
polymerisation, sedimentation and western blotting, and combined immunofluorescence and
confocal microscopy. A series of analogues of 1 were synthesised and evaluated for
antiproliferative activity, from which compound 13 was identified as having the best toxicity
profile when comparing its effects on viability in MCF-7 breast cancer cells and non-tumorigenic
MCF-10a breast epithelial cells. It was also shown that combretastatin A-4 (2) and colchicine,
established tubulin-destabilising agents, may target the stress response to ROS in MCF-7 breast
cancer cells. The results of this work are an important contribution to further understanding the
intricate biochemical and biological mechanisms of action of 1.
4. EXPERIMENTAL METHODS
All reagents were commercially available and were used without further purification unless
otherwise indicated. Anhydrous dichloromethane was obtained by distillation from calcium
1
13
hydride immediately prior to use. H and C NMR spectra were obtained on a Bruker Avance
DPX 400 instrument at 20oC, 400.13 MHz for 1H spectra, 100.61 MHz for 13C spectra, in either
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CDCl3, CD3COCD3 or CD3OD (internal standard: TMS). HRMS for all final compounds were
obtained on a Micromass Time of Flight mass spectrometer (TOF) equipped with electrospray
ionization (ES) interface operated in positive ion mode at the High Resolution Mass
Spectrometry Laboratory by Mr. Brian Talbot in the School of Pharmacy, Trinity College
Dublin. TLC was performed using Merck Silica gel 60 TLC aluminium sheets with fluorescent
indicator visualizing with UV light at 254nm. Flash chromatography was carried out using
standard silica gel 60 (230-400 mesh) obtained from Merck. All products isolated were
homogenous on TLC. The purity of the tested compounds was determined by HPLC and, unless
otherwise stated, the purity level was >95%. Analytical high-performance liquid chromatography
(HPLC) was performed using a Waters 2487 Dual Wavelength Absorbance detector, a Waters
1525 binary HPLC pump and a Waters 717plus Autosampler. The column used was a Varian
Pursuit XRs C18 reverse phase 150 x 4.6mm chromatography column. Samples were detected
using a wavelength of 254 nm. All samples were analyzed using acetonitrile (70%): water (30%)
over 10 min and a flow rate of 1 mL/min.
4.1. N-(But-3-en-1-yl)acrylamide (5)(Ref. [15]). Triethylamine (2.95 mL, 21.12 mmol) was
added to a stirred solution of but-3-en-1-amine (1 g, 14.08 mmol) in DCM (20 mL) at 0 °C.
Acryloylchloride (1.2 equiv., 1.6 mL, 16.90 mmol) was added and the mixture was allowed to
stir at room temperature for 3 hr. The reaction mixture was diluted with water, and then extracted
into DCM (2 × 10 mL). The solvent was removed in vacuo and the crude product was purified
by flash column chromatography on silica gel using CH2Cl2: methanol (9:1) to give a yellow
1
liquid (56% yield). H NMR (CDCl3) δ 2.26 (2 H, q, J=6.7 Hz), 3.36 (2 H, q, J=6.7 Hz), 4.99 -
5.12 (2 H, m) 5.59 (1 H, dd, J=9.7, 1.0 Hz), 5.74 (1 H, ddt, J=17.1, 10.4, 6.7, 6.7 Hz), 6.11 (1 H,
dd, J=17.1, 10.4 Hz), 6.22 (1 H, dd, J=16.8, 1.5 Hz), 6.32 (1 H, s[br]); 13C NMR (CDCl3) δ 33.5,
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38.5, 117.0, 126.0, 130.9, 135.1, 165.7; HRMS (ESI): m/z calcd for C7H11NO + H+ (M + H)+:
126.0913; found: 126.0911.
4.2. 5,6-Dihydropyridin-2(1H)-one (6)(Ref. [15]). Grubbs-II catalyst (42 mg, 5 mol %) was
added to a solution of 5 (0.125 g, 1 mmol) in anhydrous DCM (160 mL) and refluxed for 6 hr
under inert conditions. The mixture was stirred for an additional 1 hr at room temperature in
open air to deactivate the catalyst. The reaction mixture was filtered through celite, concentrated,
and the residue was purified by column chromatography on silica gel using hexane/EtOAc (1:1)
to give a brown liquid (52% yield). 1H NMR (CDCl3) δ 2.35-2.36 (2H, m), 3.43 (2H, td, J = 7.0,
2.5 Hz), 5.91 (1H, dd, J=10.0, 2.1 Hz), 6.65 (1H, dt, J=10.0, 4.4 Hz); 13C NMR (CDCl3) δ 23.8,
39.7, 124.8, 141.4, 166.3; HRMS (ESI): m/z calcd for C5H7NO + Na (M + Na)+: 120.0425;
found: 120.0419.
4.3. Synthesis of compounds 7-13.
General Method I: To a solution of cinnamic acid analogue (0.875 mmol), in freshly distilled
THF (5 mL) was added triethylamine (0.10 mL). Pivaloyl chloride (0.69 mmol) was added at -
20°C and the reaction mixture was stirred for 45 min. To a solution of 6 (1.05 mmol) in freshly
distilled THF (5 mL) was added n-BuLi (1.2 equiv) at -78°C under inert atmosphere conditions
and the reaction was stirred for 45 min. Then, anhydride prepared from the above step was added
and the reaction mixture was stirred for 1h. The reaction mixture was quenched with saturated
NH4Cl (2 mL), extracted with ethyl acetate (2 × 10 mL), the organic layer was separated and
washed with sat. NaCl (2 x 6 mL) and dried over anhydrous Na2SO4. The residue was evaporated
in vacuo to give a crude product which was finally purified by column chromatography on silica
gel using hexane/EtOAc (6:4) as eluent to give yellow solid piperlongumine analogue.
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4.3.1. 1-Cinnamoyl-5,6-dihydropyridin-2(1H)-one (7) was obtained from 6 and cinnamic acid
as a colourless oil (72% yield). 1H NMR (CDCl3) δ 2.45 - 2.52 (2 H, m) 4.05 (2 H, t, J=6.43 Hz)
6.05 (1 H, dt, J=9.85, 1.71 Hz) 6.92 - 6.98 (1 H, m) 7.36 - 7.39 (3 H, m) 7.57 - 7.61 (2 H, m)
13
7.76 (1 H, d, J=15.34 Hz) ; C NMR (CDCl3) δ 24.79 (1 C, s) 41.60 (1 C, s) 121.84 (1 C, s)
125.83 (1 C, s) 128.32 (1 C, s) 128.74 (1 C, s) 130.02 (1 C, s) 135.07 (1 C, s) 143.59 (1 C, s)
145.44 (1 C, s) 165.78 (1 C, s) 168.96 (1 C, s); HRMS (ESI): m/z calcd for C14H13NO2 + Na (M
+ Na): 250.0838; found: 250.0845.
4.3.2. (E)-1-(3-(2-Methoxyphenyl)acryloyl)-5,6-dihydropyridin-2(1H)-one (8) was obtained
from 6 and 2-methoxycinnamic acid as a yellow oil (68% yield). 1H NMR (CDCl3) δ ppm 2.44 -
2.51 (2 H, m) 3.89 (3 H, s) 4.04 (2 H, t, J=6.53 Hz) 6.05 (1 H, d, J=10.04 Hz) 6.89 - 6.99 (3 H,
m) 7.34 (1 H, t, J=7.78 Hz) 7.53 - 7.64 (2 H, m) 8.10 (1 H, d, J=15.56 Hz) ; 13C NMR (CDCl3) δ
ppm 24.78 (1 C, s) 41.61 (1 C, s) 55.48 (1 C, s) 111.02 (1 C, s) 120.58 (1 C, s) 122.09 (1 C, s)
124.08 (1 C, s) 125.88 (1 C, s) 128.85 (1 C, s) 131.25 (1 C, s) 138.96 (1 C, s) 145.28 (1 C, s)
158.45 (1 C, s) 165.77 (1 C, s) 169.39 (1 C, s); HRMS (ESI): m/z calcd for C15H15NO3 + Na (M
+ Na): 280.0944; found: 280.0951.
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4.3.3. (E)-1-(3-(3-Methoxyphenyl)acryloyl)-5,6-dihydropyridin-2(1H)-one (9) was obtained
1
from 6 and 3-methoxycinnamic acid as a colourless oil (65% yield). H NMR (CDCl3) δ ppm
2.61 - 2.71 (2 H, m) 4.00 (3 H, d, J=2.51 Hz) 4.17 - 4.26 (2 H, m) 6.22 (1 H, d, J=9.54 Hz) 7.05 -
7.16 (2 H, m) 7.36 (1 H, d, J=6.53 Hz) 7.41 - 7.50 (1 H, m) 7.66 (1 H, dd, J=16.06, 2.51 Hz) 7.84
- 7.94 (1 H, m); 13C NMR (CDCl3) δ ppm 24.74 (1 C, s) 41.57 (1 C, s) 55.25 (1 C, s) 112.99 (1
C, s) 116.01 (1 C, s) 121.03 (1 C, s) 122.05 (1 C, s) 125.72 (1 C, s) 129.70 (1 C, s) 136.39 (1 C,
s) 143.48 (1 C, s) 145.53 (1 C, s) 159.75 (1 C, s) 165.75 (1 C, s) 168.90 (1 C, s); HRMS (ESI):
m/z calcd for C15H15NO3 + Na (M + Na): 280.0944; found: 280.0960.
4.3.4. (E)-1-(3-(4-Methoxyphenyl)acryloyl)-5,6-dihydropyridin-2(1H)-one (10) was obtained
from 6 and 4-methoxycinnamic acid as a white powder (67% yield). 1H NMR (CDCl3) δ 2.47 (2
H, d, J=5.52 Hz) 3.84 (3 H, s) 4.04 (2 H, t, J=6.53 Hz) 6.05 (1 H, d, J=10.04 Hz) 6.87 - 6.97 (3
13
H, m) 7.42 (1 H, d, J=15.56 Hz) 7.55 (2 H, d, J=8.53 Hz) 7.74 (1 H, d, J=15.56 Hz); C NMR
(CDCl3) δ 24.80 (1 C, s) 41.61 (1 C, s) 55.34 (1 C, s) 114.19 (1 C, s) 119.37 (1 C, s) 125.92 (1 C,
s) 127.83 (1 C, s) 130.03 (1 C, s) 143.63 (1 C, s) 144.41 (1 C, s) 145.31 (1 C, s); HRMS (ESI):
m/z calcd for C15H15NO3 + Na (M + Na): 280.0944; found: 280.0951.
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4.3.5. (E)-1-(3-(2,4-Dimethoxyphenyl)acryloyl)-5,6-dihydropyridin-2(1H)-one (11) was
obtained from 6 and 2,4-dimethoxycinnamic acid as a yellow oil (63% yield). 1H NMR (CDCl3)
δ 2.42 - 2.49 (2 H, m) 3.87 (4 H, s) 3.84 (3 H, s) 4.03 (2 H, t, J=6.43 Hz) 6.01 - 6.06 (1 H, m)
6.44 (1 H, d, J=2.07 Hz) 6.49 (1 H, dd, J=8.50, 2.28 Hz) 6.88 - 6.95 (1 H, m) 7.46 - 7.57 (2 H,
13
m) 8.05 (1 H, d, J=15.76 Hz); C NMR (CDCl3) δ 24.82 (1 C, s) 41.60 (1 C, s) 55.41 (1 C, s)
55.49 (1 C, s) 98.31 (1 C, s) 105.16 (1 C, s) 117.33 (1 C, s) 119.55 (1 C, s) 126.02 (1 C, s)
130.33 (1 C, s) 139.23 (1 C, s) 145.02 (1 C, s) 159.91 (1 C, s) 162.62 (1 C, s) 165.78 (1 C, s)
169.60 (1 C, s); HRMS (ESI): m/z calcd for C16H17NO4 + Na (M + Na): 310.1050; found:
310.1044.
4.3.6. (E)-1-(3-(3,4-Dimethoxyphenyl)acryloyl)-5,6-dihydropyridin-2(1H)-one (12) was
obtained from 6 and 3,4-dimethoxycinnamic acid as a pale yellow powder (64% yield). 1H NMR
(CDCl3) δ 2.48 (2 H, tdd, J=6.48, 6.48, 4.25, 1.87 Hz) 3.92 (6 H, d, J=2.07 Hz) 4.05 (2 H, t,
J=6.63 Hz) 6.05 (1 H, dt, J=9.74, 1.97 Hz) 6.86 (1 H, d, J=8.29 Hz) 6.94 (1 H, dt, J=9.85, 4.20
Hz) 7.11 (1 H, d, J=2.07 Hz) 7.17 (1 H, dd, J=8.50, 1.87 Hz) 7.42 (1 H, d, J=15.34 Hz) 7.73 (1
H, d, J=15.34 Hz); 13C NMR (CDCl3) δ 24.80 (1 C, s) 41.61 (1 C, s) 55.88 (1 C, s) 55.93 (1 C, s)
109.89 (1 C, s) 110.93 (1 C, s) 119.54 (1 C, s) 122.93 (1 C, s) 125.90 (1 C, s) 128.10 (1 C, s)
143.93 (1 C, s) 145.34 (1 C, s) 149.10 (1 C, s) 150.99 (1 C, s) 165.85 (1 C, s) 169.05 (1 C, s);
HRMS (ESI): m/z calcd for C16H17NO4 + Na (M + Na): 310.1050; found: 310.1043.
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4.3.7. (E)-1-(3-(2,4,5-Trimethoxyphenyl)acryloyl)-5,6-dihydropyridin-2(1H)-one (13) was
1
obtained from 6 and 2,4,5-trimethoxycinnamic acid as a bright yellow powder (61% yield). H
NMR (CDCl3) δ 2.47 (2 H, tdd, J=6.43, 6.43, 4.35, 1.87 Hz) 3.88 (6 H, d, J=1.66 Hz) 3.93 (3 H,
s) 4.04 (2 H, t, J=6.43 Hz) 6.04 (1 H, dt, J=9.74, 1.76 Hz) 6.50 (1 H, s) 6.92 (1 H, dt, J=9.74,
4.25 Hz) 7.10 (1 H, s) 7.46 (1 H, d, J=15.76 Hz) 8.11 (1 H, d, J=15.76 Hz); 13C NMR (CDCl3) δ
24.83 (1 C, s) 41.63 (1 C, s) 56.01 (1 C, s) 56.45 (1 C, s) 56.50 (1 C, s) 96.96 (1 C, s) 110.91 (1
C, s) 115.85 (1 C, s) 119.30 (1 C, s) 126.01 (1 C, s) 138.89 (1 C, s) 143.24 (1 C, s) 145.10 (1 C,
s) 152.04 (1 C, s) 154.08 (1 C, s) 165.84 (1 C, s) 169.45 (1 C, s); HRMS (ESI): m/z calcd for
C17H19NO5 + Na (M + Na): 340.1155; found: 340.1160.
4.4. Crystal Structure Report for Compound 12. A specimen of C16H17NO4, approximate
dimensions 0.050 mm x 0.140 mm x 0.300 mm, was used for the X-ray crystallographic analysis.
The X-ray intensity data were measured at 100(2)K using an Oxford Cryosystems low
temperature device using a MiTeGen micromount. Bruker APEX software was used to correct
for Lorentz and polarization effects. A total of 360 frames were collected. The total exposure
time was 7.00 hours. The integration of the data using a monoclinic unit cell yielded a total of
20430 reflections to a maximum θ angle of 26.80° (0.79 Å resolution), of which 2947 were
independent (average redundancy 6.932, completeness = 99.5%, Rint = 6.24%, Rsig = 3.74%) and
2189 (74.28%) were greater than 2σ(F2).The final cell constants of a = 10.5660(8) Å, b =
15.5439(12) Å, c = 8.4713(6) Å, β = 96.3752(17)°, volume = 1382.70(18) Å3, are based upon the
refinement of the XYZ-centroids of reflections above 20 σ(I).Data were corrected for absorption
effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent
transmission was 0.919. The calculated minimum and maximum transmission coefficients (based
on crystal size) are 0.6850 and 0.7454. CCDC deposition number: 1485303.
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The structure was solved and refined using the Bruker SHELXTL Software Package, using the
space group P21/c, with Z = 4 for the formula unit, C16H17NO4.The final anisotropic full-matrix
least-squares refinement on F2 with 192 variables converged at R1 = 4.30%, for the observed
data and wR2 = 10.20% for all data. The goodness-of-fit was 1.045. The largest peak in the final
difference electron density synthesis was 0.184 e-/Å3 and the largest hole was -0.290 e-/Å3 with
an RMS deviation of 0.058 e-/Å3. On the basis of the final model, the calculated density was
1.380 g/cm3 and F (000), 608 e-.
4.5. Cell culture. MCF-7 cells were obtained from the ECACC and were cultured in Minimum
Essential Media with GlutaMAX™-I (Gibco) supplemented with heat-inactivated foetal bovine
serum (10%) (Gibco), penicillin/streptomycin 5000 U/mL (1%) (Gibco) and non-essential amino
acids (1%) (Sigma). Jurkat cells were cultured in RPMI 1640 with GlutaMAX™-I (Gibco)
supplemented with heat-inactivated foetal bovine serum (10%) (Gibco) and
penicillin/streptomycin 5000 U/mL (1%) (Gibco). Cells were maintained at 37 °C in 95% air/5%
CO2.
MCF-10a cells were obtained as a kind gift from Dr. Susan McDonnell, UCD School of
Chemical and Bioprocess Engineering and were cultured in Dulbecco's Modified Eagle Medium:
Nutrient Mixture F-12 (DMEM/F12; Gibco) supplemented with 5% horse serum (Invitrogen), 20
ng/mL epidermal growth factor (Merck Millipore), 0.5 ꢀg/mL hydrocortisone (Sigma), 100
ng/mL cholera toxin (Sigma), 10 ꢀg/mL insulin (Sigma), and penicillin/streptomycin 5000 U/mL
(1%)(Gibco).
4.6. Cell viability studies. Cells were seeded in 96-well plates at a density of 2.5 × 104 cells/mL
(200 ꢀL/well). After 24 h, cells were treated with a vehicle control [0.1% (v/v) ethanol] or a
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range of drug concentrations. Plates were incubated for 48 h at 37 °C in 5% CO2. AlamarBlue
(20 ꢀL) (Invitrogen) was added 5 h prior to the endpoint of the assay. Fluorescence was read
using the Spectramax Gemini plate reader with excitation at 544 nm and emission at 590 nm.
Blank fluorescence values were obtained from wells containing only media (200 ꢀL) and
alamarBlue (20 ꢀL), and were subtracted from the values obtained for the other wells. The
percentage cell viability was calculated for each drug concentration from FItest/FIcontrol; FItest is
the fluorescence intensity of the drug and FIcontrol is the fluorescence intensity of the control.
Experiments were performed in sextuplicate on three independent occasions for determination of
the mean values reported.
4.7. Cell cycle analysis by propidium iodide staining and flow cytometry. Cells were seeded
in T25 flasks at a density of 1 × 105 cells/mL (5 × 105 cells/flask). After 24 hr, cells were treated
with a vehicle control [0.1% (v/v) ethanol] or compound 1 (5, 10 or 20 ꢀM). After treatment for
the appropriate time, cell media was removed and cells were trypsinised. Cell media, trypsinised
cells and PBS washings were combined and centrifuged at 800g for 10 min. The supernatant was
discarded and cells were resuspended in ice-cold PBS (200 ꢀL). Fixing agent [ice-cold 70%
ethanol in PBS (2 mL)] was added slowly while gently vortexing and cell suspensions were
stored at – 20 °C for 24 hr. PBS (2 mL) and FBS (5 ꢀL) were added and cell suspensions were
centrifuged at 800g for 10 min. The supernatant was carefully removed and cells were
resuspended in BD FACSflow (400 ꢀL). Ribonuclease A (1 mg/mL; 25 ꢀL) and propidium
iodide (1 mg/mL; 75 ꢀL) were added and the mixtures were incubated in the dark at 37 °C for 30
min. Samples were analysed using filter FL-2 on the BD Accuri C6 flow cytometer (BD
Biosciences). Data collection was gated to exclude cellular debris and cell aggregates. At least
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10,000 cells per sample were analysed. Data was analysed using BD CellQuest™. Experiments
were performed on three independent occasions for determination of the mean values reported.
4.8. Tubulin Polymerization. Tubulin polymerisation was carried out using an assay kit
supplied by Cytoskeleton [Tubulin polymerization HTS assay using >97% pure porcine tubulin,
OD-based (BK004P)], based on the principal that light is scattered by microtubules to an extent
that is proportional to the concentration of the microtubule polymer. Compounds that interact
with tubulin alter its polymerisation, and this can be detected using a spectrophotometer. The
absorbance at 340 nm at 37°C is monitored. The assay was performed as described in version 2.2
of the tubulin polymerisation assay kit manual [31].
4.9. Sedimentation assay and western blotting (tubulin polymerisation). Tubulin
depolymerization was quantified by using a modified version of a previously documented
method.[32] MCF-7 cells were seeded in 6-well plates (1 × 106 cells/well) and then treated with
vehicle [0.1% ethanol (v/v)] or indicated concentrations of compound 1 (1, 10, 20 µM),
paclitaxel (1 µM) or nocodazole (1 µM) for 4 hours. Cells were harvested into MT-preserving
buffer (0.1 M PIPES (pH 6.9), 2 M glycerol, 5 mM MgCl2, 2 mM EGTA, 0.5% Triton X-100,
and protease inhibitors (Roche Diagnostics Ltd, UK) supplemented with 4 ꢀM paclitaxel to
maintain stability of assembled microtubules during isolation. The supernatant containing
unpolymerised tubulin was clarified by centrifugation (20,000g for 45 min) and separated from
the pellet containing polymerized tubulin. The pellet was washed once in MT-preserving buffer
before being denatured in PARP buffer (300 ꢀL)(62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2%
SDS, 0.01% bromophenol blue, 6M urea). Samples were stored at -80 °C. Before use 1M DTT
(20 µL) was added to both the supernatant and pellet samples. 2X Laemmli buffer (180 µL)(62.5
mM Tris-HCl, pH 6.8, 6 M urea, 2% SDS, 10% glycerol, and 0.01% bromphenol blue) was
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added to the supernatants. All samples were boiled at 100 °C for 3 min and loaded equally (30
µg protein). Proteins were separated on 12% SDS-PAGE gels and transferred onto PVDF
membranes (Millipore). Membranes were blocked in 5% non-dry fat milk/TBST for 1 h, anti-α-
tubulin primary antibody (1:2500) (Millipore) for 2 h and anti-mouse HRP-conjugated secondary
antibody (1:2500) (Promega) for 1 h at RT. All blots were probed with anti-GAPDH antibody
(1:5000) (Millipore) to confirm equal loading. Proteins were detected using chemiluminescent
western blot detection (Clarity Western ECL substrate) (Bio Rad) on the ChemiDoc MP System
(Bio Rad). Experiments were performed on three independent occasions.
4.10. Evaluation of expression levels of anti-apoptotic proteins Bcl-2 and Mcl-1. MCF-7
cells were seeded at a density of 5 × 105 cells/flask in T25 flasks. After 48 or 72 hr, whole cell
lysates were prepared from untreated cells or cells treated with vehicle control [ethanol (0.1%
v/v)] or compound 1 (10 ꢀM). Cells were harvested in RIPA buffer supplemented with protease
inhibitors (Roche Diagnostics), phosphatase inhibitor cocktail 2 (Sigma-Aldrich) and
phosphatase inhibitor cocktail 3 (Sigma-Aldrich). Equal quantities of protein (as determined by a
BCA assay) were resolved by SDS-PAGE (12%) followed by transfer to PVDF membranes.
Membranes were blocked in 5% non-dry fat milk/TBST for 1 hr. Membranes were incubated in
the relevant primary antibodies at 4 °C overnight, washed, incubated in horseradish peroxidase
conjugated secondary antibody for 1 hr at rt, and washed again. Enhanced chemiluminescence
was used for detection of protein expression. Western blot analysis was performed using
antibodies directed against Mcl-1 (1:1000) (Millipore) or Bcl-2 (1:500) (Millipore) followed by
incubation with a horseradish peroxidase-conjugated anti-mouse antibody (1:1000) (Promega,
Madison, WI, USA). All blots were probed with anti-GAPDH antibody (1:5000) (Millipore) to
confirm equal loading. Proteins were detected using chemiluminescent western blot detection
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(Clarity Western ECL substrate) (Bio Rad) on the ChemiDoc MP System (Bio Rad).
Experiments were performed on three independent occasions.
4.11. Immunofluorescence and confocal microscopy. Confocal microscopy was used to study
the effects of drug treatment on MCF-7 cytoskeleton. For immunofluorescence, MCF-7 cells
were seeded at 1 × 105 cells/mL on eight chamber glass slides (BD Biosciences). Cells were
either untreated or treated with vehicle [1 % ethanol (v/v)], paclitaxel (1 ꢀM), compound 2 (100
nM) or compound 1 (10 or 20 ꢀM) for 16 h. Following treatment cells were gently washed in
PBS, fixed for 20 min with 4 % paraformaldehyde in PBS and permeabilised in 0.5 % Triton X-
100. Following washes in PBS containing 0.1 % Tween (PBST), cells were blocked in 5 %
bovine serum albumin diluted in PBST. Cells were then incubated with mouse monoclonal anti-
α-tubulin−FITC antibody (clone DM1A) (Sigma) (1:100) for 2 hr at rt. Following washes in
PBST, cells were incubated with Alexa Fluor 488 dye (1:450) for 1 hr at rt. Following washes in
PBST, the cells were mounted in Ultra Cruz Mounting Media (Santa Cruz Biotechnology, Santa
Cruz, CA) containing 4,6-diamino-2-phenolindol dihydrochloride (DAPI). Images were
captured by Leica SP8 confocal microscopy with Leica application suite X software. All images
in each experiment were collected on the same day using identical parameters. Experiments were
performed on three independent occasions.
4.12. Colchicine-binding site assay. MCF-7 cells were seeded at a density of 1 × 106 cells/well
in 6-well plates and incubated overnight. Cells were treated with vehicle control [ethanol (0.1 %
v/v)], colchicine, compound 2 or compound 1 (all 10 ꢀM) for 2 h. After this time, selected wells
were treated with N,N’-ethylene-bis(iodoacetamide) (EBI) (Santa Cruz Biotechnology) for 1.5 h.
Following treatment, cells were twice washed with ice-cold PBS and lysed by addition of
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Laemmli buffer. Samples were separated by SDS-PAGE, transferred to polyvinylidene difluoride
membranes and probed with β-tubulin antibodies (Sigma-Aldrich).
4.13. Cell viability assay including pre-treatment with N-acetyl cysteine or Trolox. N-Acetyl
cysteine (Sigma) was dissolved in sterile water (100 mM). Trolox (Sigma) was dissolved in
ethanol (1 mM). Fresh solutions were prepared for each experiment. MCF-7 cells were seeded in
96-well plates at a density of 2.5 × 104 cells/mL. After 23 h, cells were pre-treated with NAC or
Trolox (2 ꢀL) for 1 hr. The remainder of the assay was carried out as described for the ‘Cell
viability assay’ (above).
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Supplementary material. Effects of compounds 7-13 on the viability of MCF10a human
mammary epithelial cells.
Acknowledgements. This work was supported by the Irish Research Council (GOIPD/2013/188;
NMO′B). The Trinity Biomedical Sciences Institute is supported by a capital infrastructure
investment from Cycle 5 of the Irish Higher Education Authority’s Programme for Research in
Third Level Institutions (PRTLI). The skilful technical assistance of Dr. Gavin McManus
(confocal microscopy) and Barry Moran (flow cytometry) are gratefully acknowledged.
Abbreviations
EBI: N,N’-Ethylene-bis(iodoacetamide)
NAC: N-Acetyl Cysteine
PI: Propidium Iodide
ROS: Reactive Oxygen Species
STAT3: Signal transducer and activator of transcription 3
Trolox: 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
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