Synthesis and evaluation of antiproliferative microtubule-destabilising combretastatin A-4 piperazine conjugates

Article abstract of DOI:10.1039/c9ob00558g

Microtubules are a validated clinical target for the treatment of many cancers. We describe the design, synthesis, biochemical evaluation, and molecular modelling studies of a series of analogues of the microtubule-destabilising agent, combretastatin A-4 (CA-4). Our series of 33 novel compounds contain the CA-4 core structure with modifications to the stilbene linking group, and are predominantly piperazine derivatives. Synthesis was achieved in a two-step process by firstly obtaining the acrylic acid via a Perkin reaction using microwave enhanced synthesis, followed by coupling using either DCC or Mukaiyama's reagent. All target compounds were screened for antiproliferative activity in MCF-7 breast cancer cells. Hydroxyl derivative (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl) propenone (4m) displayed potent antiproliferative activity (IC₅₀ = 190 nM). Two amino-containing derivatives, (E)-3-(3-amino-4-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4q) and (E)-3-(3-amino-4-methoxyphenyl)-1-(4-(p-tolyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4x), were the most potent with IC₅₀ values of 130 nM and 83 nM respectively. Representative compounds were shown to depolymerise tubulin, induce G₂/M arrest and apoptosis in MCF-7 cells but not peripheral blood mononuclear cells, and induce cleavage of the DNA repair enzyme poly ADP ribose polymerase (PARP) in MCF-7 cells. Modelling studies predict that the compounds bind to tubulin within the colchicine-binding site. These compounds are a valuable addition to the library of CA-4 analogues and 4m, 4q and 4x will be developed further as novel, water-soluble molecules targeting microtubules.

Full text of DOI:10.1039/c9ob00558g

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Org. Biomol. Chem., 2019, DOI: 10.1039/C9OB00558G.
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Synthesis and Evaluation of Antiproliferative Microtubule-Destabilising Combretastatin A-4
DOI: 10.1039/C9OB00558G
Piperazine Conjugates
1
,2
1
1
2
1
Niamh M. O’Boyle, ᶧ Gloria Ana, ᶧ Patrick M. Kelly, Seema M. Nathwani, Sara Noorani, Darren
2
2
3
2
1
Fayne, Sandra A. Bright, Brendan Twamley, Daniela M. Zisterer, and Mary J. Meegan
1School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, Trinity
College Dublin, 152−160 Pearse Street, Dublin 2 D02 R590, Ireland
2School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin,
1
52−160 Pearse Street, Dublin 2 D02 R590, Ireland
3School of Chemistry, Trinity College Dublin, Dublin 2, Ireland
ᶧAuthors contributed equally.
Abstract
Microtubules are a validated clinical target for the treatment of many cancers. We describe the design,
synthesis, biochemical evaluation, and molecular modelling studies of a series of analogues of the
microtubule-destabilising agent, combretastatin A-4 (CA-4). Our series of 33 novel compounds contain
the CA-4 core structure with modifications to the stilbene linking group, and are predominantly
piperazine derivatives. Synthesis was achieved in a two-step process by firstly obtaining the acrylic
acid via a Perkin reaction using microwave enhanced synthesis, followed by coupling using either DCC
or Mukaiyama’s reagent. All target compounds were screened for antiproliferative activity in MCF-7
breast cancer cells. Hydroxyl derivative (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-phenylpiperazin-1-
yl)-2-(3,4,5-trimethoxyphenyl) propenone (4m) displayed potent antiproliferative activity (IC = 190
5
0
nM). Two amino-containing derivatives, (E)-3-(3-amino-4-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-
-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4q) and (E)-3-(3-amino-4-methoxyphenyl)-1-(4-(p-
2
tolyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4x), were the most potent with IC50
values of 130 nM and 83 nM respectively. Representative compounds were shown to depolymerise
tubulin, induce G /M arrest and apoptosis in MCF-7 cells but not peripheral blood mononuclear cells,
2
and induce cleavage of the DNA repair enzyme poly ADP ribose polymerase (PARP) in MCF-7 cells.
Modelling studies predict that the compounds bind to tubulin within the colchicine-binding site. These
compounds are a valuable addition to the library of CA-4 analogues and 4m, 4q and 4x will be
developed further as novel, water-soluble molecules targeting microtubules.
1

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DOI: 10.1039/C9OB00558G
Keywords
Acrylic Acids; Cancer; Combretastatin A-4; Microtubules; Piperazine; Piperidine
Abbreviations
BCA: Bicinchoninic acid
CA-4: Combretastatin A-4
CA-4P: Combretastatin A-4 phosphate
DAMA-colchicine: N-Deacetyl-N-(2-mercaptoacetyl)colchicine
DCC: N,N'-Dicyclohexylcarbodiimide
PARP: Poly (ADP-ribose) polymerase
PBMC: Peripheral blood mononuclear cells
PBS: Phosphate-buffered saline
PI: Propidium iodide
Introduction
Combretastatin A-4 and a related drug, combretastatin A-1 (Figure 1), are natural products isolated
from the African bush willow tree Combretum caffrum. They are potent microtubule-disrupting agents
1
which bind at the colchicine-binding site of tubulin. Translation to clinical use has been hindered by
two main obstacles: limited water solubility and isomerisation to the less potent trans isomer. Many
strategies to improve their solubility have been investigated. The most successful formulation to date
has been the use of phosphate salts (CA-4P and CA-1 diphosphate, Figure 1).2-3 Both CA-4 and CA-4P
display anti-angiogenic activity.4-5 Phosphate prodrugs CA-1P and CA-4P (Figure 1) are in clinical trials,
with a particular interest for anaplastic thyroid cancer but also in advanced solid tumours, non-small
6
cell lung cancer and leukaemias. The cis isomer of CA-4 is required for antiproliferative activity, as the
trans isomer possesses greatly reduced potency.7-8 The reduced activity was recently reported to be
9
due to structural distortion of trans CA-4 in the colchicine-binding site of tubulin. Many synthetic
small molecule microtubule-disrupting agents are known. Microtubule-destabilising
agentsincorporating nitrogen-containing rings include piperazine-containing compound CB694,
pyrazoline analogues (e.g. pyrazoline 1a), and piperidinone-containing small molecule piperlongumine
(
piplartine) (Figure 1).10-12 A series of piperazine-containing microtubule-depolymerising colchicine
derivatives have been recently reported, with a chloro-containing piperazine showing the most potent
antiproliferative activity in three human cancer cell lines (gastric, lung and fibrosarcoma) (triazole 1b,
2

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1
3
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DOI: 10.1039/C9OB00558G
Figure 1). Combretastatin A-4-piperlongumine hybrids with potent antimicrotubule and mutant p53-
1
4
reactivating properties contain a piperidone-type ring. CA-4 amide derivatives are also described as
potent antiproliferative agents, including cis-restricted cyclopropyl-amides (e.g. 2a, Figure 1),15
1
6
17
imidazolone-amides (2b) and quinolones (2c). Recently, a series of CA-4 analogues with an indole
in place of the ‘B’ ring of CA-4 and an acylhydrazone-bridge in place of the stilbene double bond were
reported to possess antiproliferative activity in a panel of six cancer cell lines, inhibit tubulin
1
8
polymerisation and induce apoptosis (the latter in A549 human lung carcinoma cells). We previously
reported a number of endoxifen amide conjugates of CA-4 demonstrating potent antiproliferative
1
9
potency in MCF-7 cells. Many other combretastatin analogues are known and have been reviewed
comprehensively elsewhere.20-23
Piperidine and piperazine heterocycles are attractive pharmacological scaffolds and have been
exploited for their biological activity at various pharmacological targets.24-25 The piperazine ring system
2
4
is present in 7% of all FDA-approved small molecules between 1998 and 2009, whilst the piperidine
2
6
ring system is the most common nitrogen heterocycle in FDA-approved pharmaceuticals. It is used
both as a rigid 3D-diamine core structure and a modifying group to introduce an amine residue in
molecules. This approach is often taken to improve solubility or achieve changes in the ionization state
of novel drug substances. In our present work, a series of piperazine and piperidine amide derivatives
of CA-4 are described as potential anti-cancer agents. These compounds are designed to overcome
the inherent limitations of CA-4, namely poor solubility and cis/trans isomerism. Substitution of the
unsaturated alkene bridge of cis CA-4 with a heterocycle is strategic for overcoming isomerisation to
the inactive trans isomer. Introduction of a nitrogen heterocycle may also confer improved solubility
on the products. A two-step approach to synthesis of the target compounds was utilised. Acrylic acids
were first synthesised in a Perkin condensation using an optimised microwave enhanced method,
followed by coupling with piperazine or piperidine derivatives to yield the target compounds. The
novel CA-4 analogues were screened for their antiproliferative activity in MCF-7 breast cancer cells,
after which further biochemical investigations were performed for the most potent compounds.
Chemistry
Synthesis of Acrylic Acids
The acrylic acids required (3a – 3g, Scheme 1) were obtained using the Perkin condensation of an
aromatic aldehyde with a phenylacetic acid in the presence of acetic anhydride and triethylamine.27
Following decarboxylation, this reaction provided a stereoselective, high-yielding route to CA-4.28-29
The classical reaction consists of prolonged heating of the reagents at reflux, and so we investigated
the use of microwave enhanced chemistry to reduce the reaction time and increase the yield of acrylic
3

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acids. The effects of different reaction times and temperatures were initially investigated (Tables S1
DOI: 10.1039/C9OB00558G
and S2, Supporting information) and the conventional four hour reflux to synthesise 3a (40%) was
repeated using microwave enhanced technology (Scheme 1). The optimum reaction conditions were
found to be 30 min at 120 °C, giving a modest improvement in yield for 3a (45%). Longer reaction time
(>60 min) and higher temperatures (>120 °C) resulted in degradation and low yields.
Three acrylic acids (3a - 3c) were synthesised by both conventional reflux and microwave methods,
with the microwave method giving a higher yield than conventional reflux in a fraction of the time (5%
increase in yield for 3a, 22% for 3b, and 13% for 3c), and this method was subsequently used for all
acrylic acids prepared. Our results demonstrate that the use of microwave enhanced chemistry is an
efficient method for the preparation of acrylic acids via the Perkin reaction in improved yield, with an
eight-fold reduction in time, compared to the conventional reflux method.
The series of acrylic acids 3a – 3g synthesised contain the characteristic ring A (3,4,5-
trimethoxyphenyl) and ring B (4-methoxyphenyl, 3-amino-4-methoxyphenyl or 3-hydroxy-4-
methoxyphenyl) present in CA-4 and related colchicine derivatives (Scheme 1). The products were
1
characterised by H NMR spectroscopy, where the alkene bridge proton occurs as a singlet at approx.
δ 7.66 ppm and the proton of the carboxylic group is identified as a broad singlet at δ 12.65 ppm. In
1
3
the C NMR spectrum of 3a the alkene carbon signals are observed at δ 127.8 and 138.1 ppm, and
the carbonyl carbon at δ 168.7 ppm. Formation of the Z-isomer was not observed on examination of
NMR spectrum or TLC of crude product. An X-ray crystallographic study of 3a confirms the presence
of a double bond in the linkage between the two aromatic rings (bond length of 1.347 (3) Å between
C9 and C10 as labelled in Figure 2). The X-ray structure of 3a indicates E-geometry around the C9-C10
double bond. The A and B rings of 3a are not co-planar as indicated by a dihedral angle of 57.50 (7) °.
1H NMR nuclear Overhauser effect (NOE) experiments also indicated formation of the E isomer
(Supplementary information, Figure S5). Overall, our adapted Perkin reaction using microwave
enhanced chemistry was an efficient synthetic route to an array of acrylic acids necessary for the
second synthetic step, involving coupling of the acid to a variety of amine derivatives.
Synthesis of Piperazine Derivatives of the Acrylic Acid Scaffold
The required piperazine amide derivatives 4a – 4z were synthesised from acrylic acids 3a – 3d (Scheme
2
). The substituted piperazines chosen for coupling included N-phenyl, N-benzyl, N-acetyl and N-
cinnamyl. N,N'-Dicyclohexylcarbodiimide (DCC) was initially used for amide bond formation between
the piperazine amines and the carboxylic acid of the acrylic acids. Subsequently, DCC was replaced
with Mukaiyama’s reagent (2-chloro-1-methylpyridinium iodide) due to several advantages: a
reduction in reaction time (1 hr compared to > 20h), easier product purification, and, in some cases,
4

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increased yields. Piperazine compounds 5a, 5c, 5d and 5e (Scheme 3, yields 22 – 66%), and piperidine
DOI: 10.1039/C9OB00558G
6
a (Scheme 4, yield 38%, 6b 81%) were similarly synthesised using Mukaiyama’s reagent. The stability
of selected compounds 4n and 4l at acidic, neutral and basic pH values was measured. For compound
n, there was 64%, 76%, and 50% remaining at 24 hr at pH values of 4, 7.4 and 9 respectively, whilst
4
for compound 4l, the corresponding values were 73%, 47%, and 63%.
Initial coupling between acrylic acid 3a and piperazine led to formation of a disubstituted piperazine,
where both amines reacted with the acrylic acid (compound 7, Scheme 5). Hence, Boc-piperazine was
used with protection in place for one of the amine groups. Preparation of compound 4d was achieved
by coupling of 3d with 1-Boc-piperazine to yield compound 4c, followed by deprotection using
trifluoroacetic acid. This reaction sequence was also used to successfully produce homopiperazine
derivatives 4z (Scheme 2) and 5f (Scheme 3), and piperazine derivative 5b (Scheme 3). Preparation of
amino-containing conjugates 4q, 4v and 4x from the nitro precursors 4p, 4u and 4w respectively was
successful by reduction with iron (Scheme 2).
1
The piperazine analogues have distinctive and characteristic signals observed in the H NMR spectrum.
1
The H NMR spectrum for the piperazine analogue 4a shows proton signals for the four CH groups as
2
two broad multiplets at δ 2.70 - 2.92 and 3.70 -3.80 ppm, and the alkene proton appears as a singlet
1
3
at δ 6.65 ppm. In the C NMR spectrum of 4a, the piperazine methylene carbon signals are not
1
observed possibly due to fast relaxation times, but from H NMR and HRMS (429.2041 required for
C H N O ), we concluded that 4a was obtained. The carbon signal for the N(CH ) carbons was not
2
3
29
2
6
2 2
1
3
observed in the C NMR spectra for some of the related piperazine analogues synthesised, e.g. 4m.
Biochemical Evaluations
Antiproliferative Activity: Effects on MCF-7 and MDA-MB-231 Cell Viability
The effects of selected analogues on the viability of MCF-7 breast cancer cells were determined by
Alamar Blue assay (Table 1). There are some notable trends in the structure-activity relationships. Of
our piperazine series, phenylpiperazine-substituted compounds display the best potency, for example
amino derivative 4q (IC = 0.13 μM). In all four examples in our series, they are more potent than the
5
0
corresponding benzyl compounds (compare 4l to 4r, 4m to 4s, 4n to 4t, and 4q to 4v, Table 1). They
are also better antiproliferative agents than the corresponding acetyl derivatives (4l has approx. 6-
fold greater activity than 4e; fluoro-substituted 4n is 4-fold more potent than 4g), and better than the
corresponding cinnamyl derivatives (4l has approx. 3-fold > activity than 4i; fluoro-substituted 4n is 7-
fold more potent than 4j). From these results, we can suggest the following general trend in potency:
Phenylpiperazine > acetylpiperazine ≅ cinnamylpiperazine > benzylpiperazine
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Amino-substituted p-tolylpiperazine 4x (IC50 = 0.083 μM) was the most potent of all piperazine
DOI: 10.1039/C9OB00558G
3
0
derivatives, see National Cancer Institute NCI60 cell line screen below . Phenyl-substituted
compound 4q had good potency (IC = 0.13 μM), along with 4x, suggesting that an amino group is
5
0
advantageous. The IC value obtained in-house for 4q compares favourably to that obtained in the
5
0
NCI60 cell line screen for MCF-7 cells (0.326 μM). This is also true for 4m (0.19 μM versus 0.31 μM).
Piperidine-substituted compound 6a also shows sub-micromolar antiproliferative potency in MCF-7
cells (IC = 0.39 μM), and is far more potent that our previously reported piperidine-containing CA-4
5
0
1
9
conjugates. The IC value obtained in-house for 6a compares favourably to that obtained in the
5
0
NCI60 cell line screen for MCF-7 cells (0.45 μM). Dimer 7 had no effects on the viability of MCF-7 cells
at concentrations up to 10 μM.
Direct comparison of structural isomers 4m and 4o (IC of 0.19 μM compared to no reduction in cell
5
0
viability at 10 μM, respectively) leads to the conclusion that a 3-methoxy-4-hydroxy substitution
pattern on ring B is much less potent than the corresponding 3-hydroxy-4-methoxy motif. The position
of the piperazine substituent on the alkene double bond, relative to the A and B rings, is crucial.
Compounds 5b – 5d (Scheme 3) do not cause any inhibition of MCF-7 cell viability at a concentration
of 10 μM. These compounds position the piperazine-substituted ring on the alkene carbon closest to
ring B. Potency is hugely improved when the substitution is made to the alkene carbon adjacent to
the 3,4,5-trimethoxyphenyl ring A. This can be clearly seen when comparing the antiproliferative
activity of corresponding compounds from both series, for example 5c with 4l (IC = 0.55 µM), and 5d
5
0
with 4m (IC = 0.19 µM). Compound 6a (IC = 0.39 µM) is far more potent than our previously
5
0
50
reported compound (E)-2-(4-methoxyphenyl)-1-(piperidin-1-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-
1
9
en-1-one, which is a structural isomer of 6a with interchanged A and B rings (IC > 50 μM). This trend
5
0
is also observed for antiproliferative activity of the acrylic acids used as starting materials: the
1
9
previously reported IC value for 3a in MCF-7 cells (7.8 μM) is lower than that for 3f (> 50 μM).
5
0
Triple-negative breast cancer is the most aggressive and lethal form of breast cancer, accounting for
around 10-15% of all breast cancers, and remains difficult to treat. Two selected compounds, 4q and
4
x (structural isomers), were evaluated for antiproliferative activity in the triple-negative breast
cancer cell line MDA-MB-231. They displayed good potency with IC values of 0.19 μM and 0.14 μM
5
0
respectively, indicating potential utility in this subtype of aggressive breast cancer.
NCI60 Cell Line Screen
Four of our novel compounds (piperazines 4m, 4q and 4x and piperidine 6a) were selected for
3
0
evaluation in the NCI60 5-dose cell line screen. This evaluated GI (50% growth inhibition), TGI (total
5
0
6

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DOI: 10.1039/C9OB00558G
growth inhibition) and LC (50% lethal concentration) in the NCI panel of 60 cell lines, using the
5
0
sulphorhodamine B (SRB) protein assay (Table 2 and Tables S3 - S6, supporting information). The GI50
measures the growth inhibitory potency of the test agent, while the LC value signifies a cytotoxic
5
0
effect.
Compound 4m showed good potency in all leukaemia cell lines, several CNS cell lines, and 7 of 9
melanoma cell lines (Table 2 and Table S3). Its best potency was in NCI-H522 non-small lung cell cancer
cells (GI = 0.13 µM) and MDA-MB-435 melanoma cells (GI = 0.15 µM). Compound 4q displayed
5
0
50
good antiproliferative activity in colon, breast, CNS, and leukaemia cell line panels. Like 4m and 6a,
MDA-MB-435 melanoma cells (GI50 = 0.14 µM) were particularly susceptible to the effects of
treatment with 4q. It also displayed good potency in A498 renal cells (GI = 0.14 µM) (Table S4). The
5
0
mean GI values demonstrate that 4x has the best overall potency (lowest GI mean value, 0.41 µM)
5
0
50
of the four compounds evaluated, with potent antiproliferative effects across many of the cell panels,
and particularly in NCI-H522 non-small lung cell cancer cells (GI50 = 0.096 µM), MDA-MB-435
melanoma cells (GI = 0.14 µM), and A498 renal cells (GI = 0.15 µM) (Supplementary Information,
5
0
50
Table S5). It displayed a similar GI value for MDA-MB-231 cells (0.378 μM) to our in-house value (0.14
5
0
μM). Compound 6a showed good potency in 6 of the 7 colon cell lines, half of the leukaemia cell lines,
and several CNS cell lines. Its best potency was in HCT-116 colon cancer cells (GI = 0.38 µM), OVCAR-3
5
0
ovarian cancer cells (GI50 = 0.32 µM), and MDA-MB-435 melanoma cells (GI50 = 0.26 µM)
Supplementary Information, Table S6).
(
Cytotoxicity (determined as the LC value) was determined to be greater than 100 µM in all cell lines
5
0
(4m, 6a), in all but one cell line (compound 4q), and in all but two cell lines (compound 4x), indicating
minimal cell death and toxicity of these compounds (Table 2). Further in-house toxicity studies are
described below.
Assessment of Toxicity to Non-Tumour Cells
An initial evaluation of the cytotoxicity of the first piperazine conjugate prepared, 4a, was completed
using peripheral blood mononuclear cells (PBMCs) prior to synthesis of the expanded series of
analogues. Further studies were carried out in breast-specific cells to assess toxicity of the most potent
amino-containing conjugates 4q and 4x on the MCF-10a non-tumorigenic breast epithelial cell line.
Compound 4a was investigated for its effects on the cell cycle in peripheral blood mononuclear cells
(PBMCs) after 72 hr of treatment (Figure 3). The chosen concentration was approximately twice the
IC50 value (4.4 μM) in MCF-7 cells. There were no significant differences in the percentages of
7

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DOI: 10.1039/C9OB00558G
apoptotic cells or G /M arrested cells in samples treated with vehicle control or 4a, indicating no
2
adverse effects on PBMCs (solid black bars, Figures 3A and 3B). This is in contrast to the effects on
cancerous MCF-7 cells, where compound 4a increased the percentages of apoptotic cells and G /M
2
arrested cells (15% and 41% increases respectively, white bars with spots, Figure 3A and 3B). This
indicated minimal toxicity and further analogues were subsequently synthesised.
Compounds 4q and 4x were assessed for their effects on MCF-10a cell viability at three concentrations
(
10, 1, and 0.1 μM) (Figure 3C). The percentage of viable cells was lowest for the 10 μM concentrations
71% and 63% for 4q and 4x respectively). At 0.1 μM, MCF-10a cell viability was 84% and 86% for 4q
(
and 4x respectively. This compares favourably when considering the IC values of the compounds in
5
0
MCF-7 breast cancer cells (0.126 and 0.083 μM), indicating a selective effect on cancerous cells and a
potential therapeutic window for treatment and supporting the results from the NCI60 cytotoxicity
screening. This selectivity is similar to clinically used tubulin-targeting drugs such as vincristine, which
target actively replicating cells. There is ongoing debate about the reasons behind the selective effect
of tubulin-targeting agents, and the long-held view that cancer cells are targeted as they are actively
replicating is under scrutiny as discussed elsewere.31-32
Effects of 4m and 4x on Cell Cycle Distribution in MCF-7 cells and Expression Levels of Apoptosis-
Associated Proteins
Based on the cell viability results, combined with the lack of toxicity to MCF-10a cells and PBMCs, the
effects of conjugates 4m and 4x on the MCF-7 cell cycle distribution was examined using flow
cytometry with propidium iodide staining. Tamoxifen, a selective oestrogen receptor modulator that
does not interact with microtubules and is used for the treatment of breast cancer, was used as a
control. The percentage of cells in each stage of the cell cycle was quantified, including the number of
apoptotic cells. Tamoxifen caused an increase in apoptotic cells, but did not cause an increase in the
percentage of MCF-7 cells in the G /M phase; this is expected for a compound that does not target
2
microtubules. After 24 hr treatment with compound 4m, there was a statistically significant decrease
in the percentage of cells in the G /G phase (19% decrease) with a corresponding increase in G /M
0
1
2
phase arrested cells (20% increase) (Figure 4A). There was also a small, non-statistically significant
increase in the percentage of apoptotic cells (sub-G ) at this time-point. At 48 hr and 72 hr, there were
1
statistically significant increases in the percentage of cells in the G /M phase (28% and 23%) and
2
apoptotic cells (10.5% and 20% increase respectively compared to control, Figure 3A), with statistically
significant decreases in the percentage of cells in G /G phase (Figure 4A). Similar effects on the cell
0
1
cycle were observed for compound 4x (Figure 4B). The percentage of cells in G /M arrest is
2
significantly higher in cells treated with 4x at all three time-points (54%, 56%, and 62%) than in control
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samples (29%, 20% and 25%). Increases in the percentage of apoptotic cells in cells treated with 4x
DOI: 10.1039/C9OB00558G
were also noted at all three time-points.
Due to the increase in apoptosis upon treatment with 4m noted in the cell cycle studies, the effects
of selected analogues 4m and 4n on the cleavage of PARP was examined by Western blotting. PARP is
a DNA repair protein that participates in a diverse range of physiological functions and is cleaved in
apoptosis, with increased levels of cleaved PARP indicating enhanced apoptosis. Both 4m and 4n
decreased the expression levels of full length PARP (116kDa) at 24 hr compared to control (Figure 4C)
with a concomitant increase in cleaved PARP (83kDa). Cleavage of PARP is a hallmark of apoptosis and
this result correlates with the cell cycle results confirming induction of apoptosis by these analogues.
Effects on Tubulin Polymerisation
CA-4 is a known tubulin depolymeriser, and G /M arrest (as noted for compounds 4a and 4m) is a
2
characteristic of microtubule-interacting agents. Additionally, the NCI COMPARE algorithm allowed
comparison of activities of 4m with compounds of known mechanism of antiproliferative action in the
NCI Standard Agents Database. Compound 4m showed high correlation to tubulin targeting agents
such as maytansine, rhizoxin and the clinically important anticancer drugs vincristine and vinblastine.
(Supplementary Information, Table S7). Given this, we investigated the effects of representative
analogues 4l, 4m and 4n on the polymerisation of isolated tubulin (Figure 5). This assay is a
turbidimetric assay where light (measured at 340 nm) is scattered by microtubules to an extent that
3
3
is proportional to the concentration of microtubule polymer. Paclitaxel (10 µM) was included as a
positive control and induced hyperpolymerisation of tubulin when compared to the vehicle control,
as seen by the increase in the Vmax between 0 and 10 mins, and by the increase in final mass of polymer
after 60 mins (Figure 5). Compounds 4l, 4m and 4n had the opposite effect, decreasing the Vmax in the
initial stages of polymerisation (0 – 20 mins) and resulting in a lower final mass of polymer compared
to vehicle control (as indicated by the lower OD value) after 60 mins. This indicates that the three
compounds act directly on tubulin as microtubule-destabilising agents.
Following the experiment above (Figure 5), the in vitro effects on MCF-7 breast cancer cell microtubule
structure of compound 4m were examined using fluorescent anti-tubulin antibodies and confocal
3
4
microscopy. Paclitaxel and phenstatin, a known depolymeriser of tubulin, were used as controls.
Untreated cells displayed a well-organised microtubule network (stained green) surrounding the MCF-
7
cell nuclei (stained blue) (Figure 6). Hyperpolymerisation of tubulin was noted in the paclitaxel-
treated sample, whereas complete depolymerisation was observed for the phenstatin-treated
sample. Cells treated with 4m also displayed disorganised microtubule networks, alongside multiple
9

Organic & Biomolecular Chemistry
Page 10 of 38
3
5
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DOI: 10.1039/C9OB00558G
micronuclei, a phenomenon known as mitotic catastrophe and previously observed by us and others
upon treatment with tubulin-targeting agents.36-38 Mitotic catastrophe was also noted for phenstatin-
treated cells.
Molecular Modelling of 4m, 4q, 4x and 6b in the Colchicine-Binding Site of Tubulin
Docking calculations using MOE 2016.0839 were undertaken on conjugates 4m, 4q, 4x and 6b
1
9
(
previously reported ) in the colchicine-binding site of tubulin (Figure 7). All compounds overlay their
B-rings on the C-ring of DAMA-colchicine (forming hydrogen-bond accepting interactions with Lys352),
collocate the trimethoxyphenyl substituted A-rings and position the heterocycle in an open region of
the tubulin binding site. 4m is the highest ranked compound and, in addition to the interactions
mentioned above, can possibly form π-π interactions between the phenylpiperazine group and Tyr224
(Figure 7A). The adjacent carbonyl group forms HBA interactions with Ala251 (Figure 7). The E isomers
of 4q and 4x can recapitulate the same interactions as 4m as the only difference between them is the
B-ring substitution of the hydroxyl group to an amine (Figures 7B and 7C). As amines are less capable
hydrogen bond acceptors, the compounds are predicted to have less potent binding affinity. The
docked pose of compound 6b (the piperidine analogue of 4q) maps the trimethoxyphenyl groups well
and the C ring methoxy groups also overlay nicely (Figure 7D). The amine of the C ring is not directed
towards Lys352 but rotation is possible around the phenyl-alkene bond. The piperidine side-chain is
located in the pocket bordered by Ser178 and Leu248.
Conclusions
A series of combretastatin analogues were prepared in a two-step reaction by use of an adapted
Perkin reaction using microwave technology followed by coupling with amines using Mukaiyama’s
reagent. Following characterisation, these conjugates were screened for antiproliferative activity in
MCF-7 breast cancer cells with a number of compounds, e.g. hydroxyl-containing compound 4m and
amino-containing derivatives, 4q and 4x, displaying submicromolar IC values. Two compounds, 4q
5
0
and 4x, also potently inhibited the viability of triple-negative MDA-MB-231 cells. Assessment of
toxicity using MCF10a cells and PBMCs indicated selectivity toxicity to cancerous cells, which is crucial
for potential drug candidates. Representative compounds from the series were shown to induce
apoptosis, G /M arrest and mitotic catastrophe in MCF-7 cells, and induce cleavage of PARP. A number
2
of compounds including 4m, 4q and 4x will be developed further for the treatment of breast cancer,
focusing in particular on the triple-negative subtype for which limited treatment options are available.
Experimental Note: All reagents were commercially available and were used without further
purification unless otherwise indicated. Anhydrous DCM was obtained by distillation from calcium
1
0

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Organic & Biomolecular Chemistry
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hydride, and anhydrous THF by distillation from benzophenone-sodium, both in inert atmospheres
DOI: 10.1039/C9OB00558G
immediately before use. IR spectra were recorded as thin films on NaCl plates or as KBr discs on a
1
13
Perkin-Elmer Paragon 100 FT-IR spectrometer. H and C NMR spectra were obtained on a Bruker
o
1
13
Avance DPX 400 instrument at 20 C, 400.13 MHz for H spectra, 100.61 MHz for C spectra. High
resolution molecular ion determinations (HRMS) (mass measurement accuracies of <  5 ppm) were
obtained at the Mass Spectrometry Laboratory, School of Pharmacy and Pharmaceutical Sciences,
Trinity College Dublin by Mr. Brian Talbot. Additional mass measurements were obtained by Dr. Martin
Feeney at the HRMS Laboratory in the Department of Chemistry, Trinity College Dublin. Microwave
experiments were performed in a Biotage Initiator microwave reactor. 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) from Merck. All
products isolated were homogenous on TLC. Analytical high-performance liquid chromatography
(HPLC) for purity testing was performed using a Waters 2487 Dual Wavelength Absorbance detector,
a Waters 1525 binary HPLC pump and a Waters 717 plus Autosampler. The column used was a Varian
Pursuit XRs C18 reverse phase 150  4.6 mm chromatography column. Samples were detected using
a wavelength of 254 nm. All samples were analyzed using acetonitrile (60%): water (40%) with a run-
time of 10 min and a flow rate of 1 mL/min. Final compounds were obtained with purity of ≥ 95%.
Compound 6b (Figure 7) was synthesised in 81% yield as previously reported.19 Experimental
characterisation for acrylic acids 3b – 3f, piperazine conjugates 4b – 4l, 4n, 4o, and 4r – 4v, 4y, 4z,
piperazine conjugates 5a – 5f, piperidine conjugate 6a, and piperazine dimer 7 is contained in the
online Electronic Supplementary Information for this article.
General method IA: Synthesis of acrylic acids. A mixture of the appropriate benzaldehyde (1 eq.),
phenylacetic acid (3 mmol, 1 eq.), acetic anhydride (2 mL) and triethylamine (1 mL) was refluxed for 4
h. Following acidification with concentrated hydrochloric acid (5 mL), the resulting solid was filtered
and recrystallised from methanol to yield the acrylic acid.
General method IB: Synthesis of acrylic acids. A mixture of the appropriate benzaldehyde (1 eq.),
phenylacetic acid (3 mmol, 1 eq.), acetic anhydride (2 mL) and triethylamine (1 mL) was reacted in the
microwave reactor at a 120 ⁰C and for 30 min. Following acidification with concentrated hydrochloric
acid (5 mL), the resulting solid was filtered off and recrystallised from methanol to yield the acrylic
acid.
General method II: Synthesis of amides. The appropriate acrylic acid (1.38 mmol) and HOBt (1 eq.,
1
.38 mmol) were dissolved in dry DCM (10 mL) under an inert atmosphere. DCC (1 eq., 1.38 mmol)
and DMF (1 mL) were added and the mixture was stirred for 30 min. Piperazine or required derivative
1
1

Organic & Biomolecular Chemistry
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dissolved in dry DCM (5 eq., 6.8 mmol, dissolved in 10 mL) was added and the mixture was stirred
DOI: 10.1039/C9OB00558G
overnight under an inert atmosphere. The reaction mixture was filtered. The organic layer was washed
with 2 M HCl (3 × 20 mL). The separated aqueous layer was basified with 2 M NaOH and extracted
with DCM. The combined organic layers were washed with water (20 mL), brine (20 mL), dried over
Na SO and evaporated to dryness in vacuo.
2
4
General method III: Synthesis of amides. To a suspension of acrylic acid (1.38 mmol, 1 eq) in
anhydrous dichloromethane (20 mL) was added 2-chloro-1-methylpyridinium iodide (Mukaiyama
reagent) (4.14 mmol, 3 eq) and the reaction mixture was stirred for 5 min at room temperature. To
this the appropriate amine (1.38 mmol, 1 eq) was added followed by the addition of triethylamine (6.9
mmol, 5 eq). The reaction mixture was stirred at room temperature for 1h and was then diluted with
1
0% hydrochloric acid (10 mL), washed with 10% sodium hydroxide (10 mL), water and brine and dried
over sodium sulphate.
General method IV: BOC-deprotection. The BOC-protected derivative (1 mmol) was dissolved in DCM
(25 mL), trifluoroacetic acid (5 mL) was added and the solution was stirred at RT for 1h. After reaction
completion (as monitored by TLC) the solution was washed with NaHCO (10 mL), water (10 mL) and
3
brine (10 mL) and dried over sodium sulphate. No further purification was required.
General Method V: Synthesis of amino-substituted derivatives. The appropriate nitro compound (1
eq, 0.37 mmol) was dissolved in ethanol (5 mL) with acetic acid (5 mL), water (2 mL) and 1 drop of
hydrochloric acid and iron powder (10 eq) was added. The mixture was vigorously stirred under reflux
for 12 h. After completion of the reaction, the mixture was filtered through Celite, diluted with NaHCO3
and extracted with dichloromethane; the organic layers were combined, washed with water and brine
and dried over sodium sulphate.
(
E)-2-(3,4,5-Trimethoxyphenyl)-3-(4-methoxyphenyl)acrylic acid (3a)40 was synthesised from
anisaldehyde and 3,4,5-trimethoxyphenylacetic acid by general methods IA and IB as fine yellow solid
-1
(
IA: 0.41 g, 40%; 0.46 g, IB: 45%). Mp 212 °C. IR: νmax (KBr) cm : 2994, 2942, 2630, 1664, 1604, 1582,
1
1
7
508, 1464, 1414, 1253, 1242, 1180, 1127, 1029, 1008, 830; H NMR (DMSO-d )  12.48 (br s, 1H),
6
.69 (s, 1H), 7.07 (d, 2H, J =9 Hz), 6.82 (d, 2H, J =9 Hz), 6.46 (s, 2H), 3.72 (s, 6H), 3.69 (s, 6H); 13C NMR
(
DMSO-d )  55.2, 55.9, 60.1, 106.5, 113.9, 126.7, 130.5, 132.1, 132., 136.9, 138.7, 153.9, 160.0, 168.5;
6
+
HRMS (EI): Found 345.1246 (M+H) , C H O requires 344.1260.
1
9
21
6
(
E)-3-(3-Hydroxy-4-methoxyphenyl)-1-piperazin-1-yl-2-(3,4,5-trimethoxyphenyl)propenone (4a)
was prepared from 3c and piperazine by general method II. The crude product was purified via flash
chromatography over silica gel (eluent ethyl acetate: DCM 1:2) to afford the product as a cream solid
1
2

Page 13 of 38
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-1
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DOI: 10.1039/C9OB00558G
(
0.095 g, 16%); MP: 83 °C. IR: νmax (KBr) cm : 3000, 2935, 2838, 1635, 1579, 1605, 1380, 1321, 1284,
1
1
1
177, 1284, 1151, 1031, 998, 905, 809, 830. H NMR (DMSO-d )  6.76 (s, 1H), 6.68 (s, 2H), 6.57 (s,
6
1
3
H), 6.55 (s, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.72 (s, 6H), 3.70 (br s, 4H), 2.88 (br s, 4H); C NMR (DMSO-
d6)  169.9, 152.9, 146.3, 144.8, 137.5, 134.3, 130.2, 129.9, 127.7, 121.5, 115.1, 115.0, 109.8, 105.4,
+
6
0.5, 55.7, 55.4. HRMS (EI): Found 429.2041 (M+H) , C H N O requires 429.2026.
2
3
29
2
6
(
E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)
propenone (4m) was prepared from 3c and 1-phenylpiperazine by general method II. The material
was purified via flash chromatography on silica gel (eluent, ethyl acetate: DCM, 1:1) to afford the
-1
product as a brown solid (0.194 g, 28%), MP 121-125 °C; IR: νmax (KBr) cm : 2994, 2884, 2779, 2742,
1
1
2
684, 1543, 1428, 1326, 1125, 1293, 1009, 982, 830, 839, 734, 645, 573; H NMR (DMSO-d )  7.30 (s,
6
H), 6.96 (s, 3H), 6.82 (s, 1H), 6.70 (s, 2H), 6.64 (s, 1H), 6.60 (s, 2H), 3.88 (s, 6H), 3.88-3.73 (m, 4H), 3.73
1
3
(
s, 6H), 3.20-3.07 (m, 4H); C NMR (DMSO-d )  169.9, 152.9, 146.0, 144.7, 137.5, 134.6, 130.3, 129.8,
6
1
27.8, 121.5, 116.5, 115.0, 109.7, 105.5, 60.5, 55.7, 55.4; HRMS (EI):. Found 527.2141 (M+Na)+,
C H N NaO requires 527.2158.
2
9
32
2
6
(
E)-3-(4-Methoxy-3-nitrophenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-
-one (4p) was prepared from 3d and phenylpiperazine by general method III. The crude product was
purified by flash column chromatography (eluent, dichloromethane: ethyl acetate, 1:1) and used
1
-1
without further purification (0.280 g, 38%); IR: νmax (KBr) cm : 3359, 2934, 2835, 1612, 1597, 1230,
1
1
121, 759, 693. H NMR (CDCl ) δ 7.70 (d, J = 2.2 Hz, 1H), 7.30 – 7.25 (m, 3H), 6.90 – 6.87 (m, 4H), 6.62
3
1
3
(
s, 1H), 6.54 (s, 2H), 3.91 (s, 3H), 3.85 (s, 3H), 3.71 (s, 6H. CH ), 3.09 (d, J = 61.5 Hz, 4H); C NMR (CDCl )
3
3
δ 169.3, 153.7, 152.2, 150.7, 139.2, 138.1, 134.9, 129.8, 129.2, 127.7, 127.2, 126.5, 120.7, 116.7, 113.0,
+
1
05.7, 61.0, 56.5, 56.2, 49.5; HRMS (EI): Found 556.2060 (M+Na) C H N O requires 556.2060.
2
9
31
3
7
(
E)-3-(3-Amino-4-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (4q) was prepared from 4p by general method V. The product was obtained as a yellow resin,
-1
1
(
0.025 g, 13%); IR: νmax (KBr) cm : 3361, 2934, 2835, 1597, 1579, 1411, 1230 1122, 1003, 693; H NMR
(
DMSO-d )  3.06 (br s, 4H), 3.60 (s, 6H), 3.64 (s, 3H), 3.69 (s, 3H), 5.72 (s, 2H), 6.36 (dd, J =8.3, 2.1 Hz,
6
1
H), 6.46 (s, 1H), 6.53 (d, J =2.1 Hz, 1H), 6.53 (s, 2H), 6.65 (d, J =8.3 Hz, 1H), 6.75 - 6.79 (m, 2H), 6.90
1
3
(
d, J =7.9 Hz, 2H), 7.18 (t, J =7.9 Hz, 2H). C NMR (DMSO-d )  55.3, 55.8, 55.9, 105.9, 110.1, 114.4,
6
1
15.9, 117.7, 119.3, 127.5, 128l1, 128.9, 129.8, 133.7, 137.2, 137.2, 146.3, 150.7, 152.8, 152.9, 169.2;
+
HRMS (EI): Found 504.2515 (M+H) C H N O requires 504.2498.
2
9
33
3
5
(
E)-3-(4-Methoxy-3-nitrophenyl)-1-(4-(p-tolyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (4w) was prepared from 3d and 1-(p-tolyl)piperazine by general method III. The crude
1
3

Organic & Biomolecular Chemistry
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product was purified by flash column chromatography over silica gel (eluent, ethyl acetate: DCM 1:1),
DOI: 10.1039/C9OB00558G
and was obtained as a yellow solid (0.158 g, 21%), MP: 89-92 °C. IR: νmax (ATR) cm-1: 2938, 2831, 1702,
1
1
7
6
614, 1578, 1504, 1528, 1439, 1411, 1279, 1235, 1123, 1003, 811, 774, 705, 667. H NMR (CDCl ) δ
3
.69 (d, J = 2.2 Hz, 1H), 7.27 (dd, J = 8.8, 2.2 Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.8 Hz, 1H),
.79 (d, J = 8.5 Hz, 2H), 6.61 (s, 1H), 6.53 (s, 2H), 3.91 (s, 3H), 3.85 (s, 3H), 3.70 (s, 6H), 3.64 (s, 2H), 3.02
1
3
(
d, J = 63.6 Hz, 4H), 2.25 (s, 3H); C NMR (CDCl ) δ 169.3, 153.7, 152.2, 148.6, 139.2, 138.5, 138.1,
3
1
34.9, 130.3, 129.7, 127.7, 127.2, 126.5, 117.0, 113.0, 105.7, 61.0, 56.5, 56.3, 50.2, 42.0, 21.0; HRMS
+
(
EI): Found 548.2389 (M+H) ; C H N O requires 548.2391.
3
0
34
3
7
(
E)-3-(3-Amino-4-methoxyphenyl)-1-(4-(p-tolyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (4x) was prepared from 4w by general method V and was obtained as a yellow oil (0.179 g,
-1
9
1%). IR: νmax (ATR) cm : 2995, 2894, 2829, 1695, 1578, 1508, 1410, 1231, 1121, 1003, 900, 812, 703.
1
H NMR (DMSO-d ) δ 2.16 (s, 3H), 2.98 (br s, 4H), 3.60 (s, 6H), 3.64 (s, 3H), 3.68 - 3.70 (m, 3H), 4.62 (br
6
s, 2H), 6.36 (dd, J =8.3, 2.1 Hz, 1H), 6.45 (s, 1H), 6.52 (s, 1H), 6.53 (s, 2H), 6.65 (d, J =8.3 Hz, 1H), 6.80
1
3
(
m, J =8.3 Hz, 2H), 7.00 (m, J =8.3 Hz, 2H); C NMR (DMSO-d ) δ 20.0, 54.9, 55.2, 55.8, 55.9, 105.9,
6
1
10.1, 114.4, 116.2, 117.7, 127.5, 128.3, 129.4, 129.7, 131.1, 133.7, 137.2, 146.3, 148.7, 152.8, 169.2;
+
HRMS (EI): Found 518.2648 (M+H) ; C H N O requires 518.2649.
3
0
36
3
5
X-Ray diffraction. X-ray structural analysis for 3a was performed on a Bruker APEX Kappa DUO at
00(2) K with an Oxford Cobra, with the sample mounted on a MiTeGen microloop using Cu Kα
1
4
1
radiation (λ = 1.54178 Å). Bruker APEX software was used to collect and reduce data and determine
4
2
the space group. Absorption corrections were applied using SADABS. Structures were solved with
the XT structure solution program43 using Intrinsic Phasing and refined with the XL refinement
package44 using Least Squares minimisation in Olex2.45 All non-hydrogen atoms were refined
anisotropically. Donor H atoms were refined with Uiso = 1.5 times Ueq of riding O atom and refined
with restraints (DFIX). All other hydrogen atoms were assigned to calculated positions using a riding
model with appropriately fixed isotropic thermal parameters. Crystallographic data for the structure
in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary
publication no. 1878160. Copies of the data can be obtained, free of charge, on application to CCDC,
1
2 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail:deposit@ccdc.cam.ac.uk).
Crystal Data for C H O (M =344.35 g/mol): triclinic, space group P-1 (no. 2), a = 7.7673(2) Å, b =
1
9
20
6
3
8
=
.9291(3) Å, c = 12.6819(4) Å, α = 99.235(2)°, β = 90.839(2)°, γ = 93.211(2)°, V = 866.54(5) Å , Z = 2, T
-1
3
100(2) K, μ(CuKα) = 0.818 mm , Dcalc = 1.320 g/cm , 9343 reflections measured (7.064° ≤ 2Θ ≤
1
4

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DOI: 10.1039/C9OB00558G
1
40.266°), 3226 unique (R = 0.0415, R
= 0.0450) which were used in all calculations. The final R
int
sigma
1
was 0.0518 (I > 2σ(I)) and wR was 0.1501 (all data).
2
Biochemistry Experimental.
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).
Cells were maintained at 37 °C in 95% air/5% CO2.
MCF-10a cells were obtained as a kind gift from Dr. Susan McDonnell, School of Chemical and
Bioprocess Engineering, University College Dublin and were cultured in Dulbecco's Modified Eagle
Medium: Nutrient Mixture F-12 (DMEM/F12; Gibco) supplemented with 5% horse serum (Invitrogen),
2
0 ng/mL epidermal growth factor (Merck Millipore), 0.5 μg/mL hydrocortisone (Sigma), 100 ng/mL
cholera toxin (Sigma), 10 μg/mL insulin (Sigma), and penicillin/streptomycin 5000 U/mL (1%)(Gibco).
Cell viability studies. MCF-7 and MCF-10a cells were seeded in 96-well plates at a density of 2.5 × 104
cells/mL (200 μL/well). After 24 h, cells were treated with a vehicle control [1% (v/v) ethanol] or a
range of drug concentrations. Plates were incubated for 72 h at 37 °C in 5% CO . AlamarBlue (20 μL)
2
(Invitrogen) was added 5 hr (MCF-7) or 7 hr (MCF-10a) 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 μL) and
AlamarBlue (20 μL), and were subtracted from the values obtained for the other wells. The percentage
cell viability was calculated for each drug concentration from FI /FIcontrol; FItest is the fluorescence
test
intensity of the drug and FIcontrol is the fluorescence intensity of the control. Experiments were
performed in triplicate on a minimum of two independent occasions for determination of the mean
values reported.
Cell cycle analysis by propidium iodide staining and flow cytometry. MCF-7 cells were seeded in T25
5
5
flasks at a density of 1 × 10 cells/mL (5 × 10 cells/flask). After 24 hr, cells were treated with a vehicle
control [0.1% (v/v) ethanol] or compounds 4a (5, 10 or 20 μM). 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 μL). 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 μL) 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 μL). Ribonuclease A (1 mg/mL; 25
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5

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μL) and propidium iodide (1 mg/mL; 75 μL) were added and the mixtures were incubated in the dark
DOI: 10.1039/C9OB00558G
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 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.
Generation of human peripheral blood mononuclear cells (PBMCs). Blood was obtained from healthy
donors (n=2) after informed consent, transferred into a 50 mL falcon tube and diluted 1:2 with PBS.
LymphoPrep was used to separate the blood into red blood cells, white blood cell ring and serum. The
blood was slowly added to 20 mL of ficoll pague plus. The tubes were centrifuged at 1700 g for 30 min.
The white blood cell ring was transferred into a new 50 mL tube. The volume was adjusted to 50 mL
and the samples were centrifuged again at 1700 g for 10 min. The supernatant was removed. This step
was repeated again, the pellet was then resuspended in 10 mL of complete Iscove's Modified
Dulbecco's (IMDM) media [10% FBS, 0.1% Ciprofloxacin (10 mg/mL)].
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.33
Evaluation of effects of treatment with novel conjugates on expression levels of PARP and cleaved
5
PARP. MCF-7 cells were seeded at a density of 5 × 10 cells/flask in T25 flasks. After 24 or 72 hr, whole
cell lysates were prepared from untreated cells or cells treated with vehicle control [ethanol (0.1%
v/v)] or compounds 4m or 4n (1 μM). Cells were harvested in RIPA buffer (880 µL) supplemented with
protease inhibitors (100 µL, Roche Diagnostics), phosphatase inhibitor cocktail 2 (10 µL, Sigma-Aldrich)
and phosphatase inhibitor cocktail 3 (10 µL, 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 PARP (1:1000) (Millipore) followed by incubation with a horseradish peroxidase-
conjugated anti-mouse antibody (1:1000) (Promega, Madison, WI, USA). All blots were probed with
1
6

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anti-GAPDH antibody (1:5000) (Millipore) to confirm equal loading. Proteins were detected using
DOI: 10.1039/C9OB00558G
chemiluminescent western blot detection (Clarity Western ECL substrate) (Bio Rad) on the ChemiDoc
MP System (Bio Rad). Experiments were performed on three independent occasions.
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 [0.1% ethanol (v/v)], paclitaxel (1 μM), phenstatin (1 μM) or 4m (10 μM) for 16 h. Following
treatment cells were gently washed in PBS, fixed for 20 min with ice-cold methanol 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 further 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.
Molecular modelling. The 1SA0 X-ray structure of bovine tubulin co-crystallised with N-deacetyl-N-(2-
mercaptoacetyl)-colchicine (DAMA-colchicine) was downloaded from the PDB.46 A UniProt Align
analysis confirmed a 100% sequence identity between human and bovine β tubulin. The crystal
structure was prepared using QuickPrep (minimised to a gradient of 0.001 kcal/mol/Å), Protonate 3D,
Residue pKa and Partial Charges protocols in MOE 2016 with the MMFF94x force field. Compounds
3
9
4
q, 4m and 6b were drawn in MOE, saved as mdb and processed in MOE. For each compound,
MMFF94x partial charges were calculated and each was minimised to a gradient of 0.001 kcal/mol/Å.
Default parameters were used for docking except that 300 poses were sampled for each compound
and the top 50 docked poses were retained for subsequent analysis.
Acknowledgements. The skilful technical assistance of Dr. John O’Brien (NMR), Dr. Gavin McManus
(confocal microscopy) and Barry Moran (flow cytometry) is gratefully acknowledged. Contributions
from Ms. Tanya Codd, Ms. Eadaoin Crowley and Ms. Sarah Fitzpatrick are also appreciated. The Trinity
Biomedical Sciences Institute (TBSI) 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).
This work was supported by the Irish Research Council (GOIPD/2013/188; NMO′B). This study was also
co-funded under the European Regional Development, and a Trinity College Dublin postgraduate
1
7

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research award (GA and PMK), and Agenzia Regionale per il Lavoro, Sardinia, Programme Master and
DOI: 10.1039/C9OB00558G
Back (G.A.). D.F. thanks the software vendors for their continuing support of academic research
efforts, in particular the contributions of Biovia, the Chemical Computing Group, and OpenEye
Scientific. The support and provisions of Dell Ireland, the Trinity Centre for High Performance
Computing (TCHPC) and the Irish Centre for High-End Computing (ICHEC) are also gratefully
acknowledged.
Electronic Supplementary Information. Experimental Characterisation for Acrylic Acids 3b – 3f;
Experimental Characterisation for Piperazine Conjugates 4b – 4l, 4n, 4o, 4r – 4v, 4y and 4z;
Experimental Characterisation for Piperazine Conjugates 5a – 5f; Experimental Characterisation for
1
Piperidine Conjugate 6a; Experimental Characterisation for Piperazine Dimer 7; H NMR spectra for
compounds 4m, 4q and 4x and NMR NOE experimental data for acrylic acid 3a; results of effects of
reaction time and temperature on the isolated yields of 3a; representative data from FACS assay for
cell-cycle analysis; full results from the NCI-60 cell line panel screen and COMPARE analysis. We have
also provided a pdb file for the overlay of compound 4q and DAMA-colchicine in 1SAO.
1
8

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DOI: 10.1039/C9OB00558G
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2
1

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Tables.
Table 1. Antiproliferative Effects of CA-4-Piperazine and Piperidine Conjugates in MCF-7 Breast
Cancer Cells
R3
N
N
R1
R2
a
Compound
IC (μM)
5
0
O
H CO
3
OCH3
OCH3
R3
H
R1
OH
F
R2
4
4
4
a
b
e
OCH3 4.4 ± 0.8
OCH3 2.74 ± 1.1
OCH3 3.5 ± 0.9
OCH3 4.3 ± 0.6
OCH3 1.7 ± 0.5
OCH3 1.89 ± 0.6
H
C(O)CH3
C(O)CH3
C(O)CH3
H
4
f
OH
F
4
g
4
i
j
H
4
F
OCH3 3.23 ± 0.8
OCH3 11%c
4
k
NO2
4
l
C6H5
H
OCH3 0.55 ± 0.06
OCH3 0.19 ± 0.05
OCH3 0.44 ± 0.1
4
m
C6H5
OH
F
4
n
o
q
C6H5
4
C6H5
OCH3 OH
0%c
4
C6H5
NH2
H
OCH3 0.13 ± 0.03
OCH3 49%c
OCH3 49%c
OCH3 47%c
OCH3 0%c
4
r
CH C H
2
6
5
5
5
5
4
s
CH C H
OH
F
2
6
4
t
CH C H
2
6
4
v
CH C H
NH2
2
6
2
2

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DOI: 10.1039/C9OB00558G
4
x
CH3
NH2
OCH3 0.083 ± 0.01
39%c
4
z
NH
N
O
OCH3
H CO
3
OCH3
OCH3
R2
N
N
OCH3
IC50a
Compound
O
OCH3
OCH3
R1
OCH3
1
2
0%c
0%c
0%c
5
b
R = H, R = H
1
2
5
c
R = H, R = C H
6
5
1
2
5
d
R = OH, R = C H
6
5
NH
N
OCH3
OCH3
O
15% (at 1 µM)c
5
f
OCH3
OCH3
O
N
6
a
0.39 ± 0.04
OCH3
H CO
3
OCH3
OCH3
2
3

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DOI: 10.1039/C9OB00558G
O
NH2
N
6
b19
34%
OCH3
H CO
3
OCH3
OCH3
CA-4b
0.0039
a
IC values are half-maximal inhibitory concentrations required to block the growth stimulation of
5
0
b
MCF-7 cells. Values represent the mean for three experiments each performed in triplicate. The IC
value obtained for CA-4 is in good agreement with reported values.47-48 cIC values were not
5
0
5
0
calculated; the percentage shown is the inhibition of cell viability at a concentration of 10 μM unless
otherwise indicated.
Table 2. Summary of NCI60 screening results for compounds 4m, 4q, 4x, and 6a.30
Compound NSC code Mean GI (µM) Mean TGI (µM) a Mean LC (µM)
a
a
5
0
50
4
4
4
6
m
q
x
791047
791055
795434
795432
0.77
0.56
0.41
1.31
50
50
50
70
> 100
> 100
> 100
> 100
a
a
GI = 50% growth inhibition, TGI = total growth inhibition and LC = 50% lethal concentration.
5
0
50
2
4

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DOI: 10.1039/C9OB00558G
Figures.
Figure 1. Structures of microtubule-destabilising agent combretastatin A-4 and related compounds.
R2
O
R1
Cl
N
B
N
Cl
OCH3
OCH3
A
H CO
3
OCH3
OCH3
CB694
O
O
1
2
CA-4: R = OH, R = H
OCH3
1
2
CA-4P: R = OP(O)(ONa) , R = H
N
2
1
2
CA-1: R = R = OH
OCH3
1
2
CA-1P: R = R = OP(O)(ONa)
2
OCH3
H CO
3
Piperlongumine
O
N
Cl
N
O
O
N
NH
N
H CO
3
H CO
3
N
N
O
H CO
3
H CO
3
OCH3
Pyrazoline 1a
Triazole 1b
H
H
O
O
N
OCH3
O
N
OCH3
NH2
N
HN
H CO
3
O
H
HN
OCH3
H CO
3
H CO
3
OCH3
OCH3
H CO
3
OCH3
2b
OCH3
2a
2c
2
5

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Figure 2. X-ray crystallographic asymmetric unit of 3a showing conformation of the E isomer. Atomic
DOI: 10.1039/C9OB00558G
displacement shown at 50% probability and selected atoms labelled for clarity. Grey = carbon; white
hydrogen; red = oxygen. CCDC deposition number: 1878160.
=
H CO
3
OCH3
OCH3
H CO
3
A
B
HO
O
3a
2
6

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DOI: 10.1039/C9OB00558G
Figure 3. Effects of CA-4-piperazine conjugates on non-tumorigenic cells. 3A, B. Conjugate 4a induces
G /M arrest and apoptosis in MCF-7 cells, but not in blood cells from healthy donors. MCF-7 cells or
2
peripheral blood mononuclear cells (PBMC) from healthy donors (n=2) were treated for 72 h with
vehicle (ethanol), tamoxifen (10 μM, MCF-7 only) or 4a (10 μM). After the required incubation period,
cells were fixed in ethanol (1 mL) and PBS (100 µL) and stained with propidium iodide (PI). Cells were
subsequently analysed by flow cytometry to determine (A) the percentage of apoptotic cells, as
assessed by quantification of the sub-G peak, (B) and G /M-arrested cells. Values represent the mean
1
2
±
S.E.M. for three independent experiments for MCF-7 and two independent experiments ± range for
PBMC. Statistical analysis was performed using a one-way ANOVA with Tukey’s post-comparison test
**, p < 0.01; ***, p < 0.001). 3C. Effects of compounds 4q and 4x on the viability of MCF10a human
(
mammary epithelial cells. Cells were treated at the indicated concentrations for 72 hr. Cell viability
was expressed as a percentage of vehicle control [ethanol 1% (v/v)] and was measured by alamarBlue
assay (average of two independent experiments).
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7

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Figure 4A, B. Effects of 4m and 4x on the cell cycle distribution of MCF-7 cells. MCF-7 cells were
treated with either vehicle control (ethanol) (spotted bars) or compound 4m (1 µM) (Figure 4A, solid
black bars) or compound 4x (1 µM) (Figure 4B, solid black bars)for 24, 48 hr or 72 hr. Cells were then
fixed, stained with PI, and analysed by flow cytometry with CellQuest software. All values represent
the mean ± S.E.M. for three independent experiments. Statistical analysis was performed using a
paired t-test (*, p < 0.05; **, p < 0.01).
Figure 4C. Effects of 4m and 4n on expression of PARP. MCF-7 cells were treated with either vehicle
control (ethanol) or with piperazines 4m or 4n (1 µM) for 24 hrs. After the required time cells were
harvested and separated by SDS PAGE. The membrane was probed with anti-PARP or anti-cleaved
PARP antibodies. Results are representative of three separate experiments.
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8

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Figure 5. Effects of conjugates 4l, 4m and 4n on in vitro tubulin polymerization. Purified bovine
tubulin and GTP were mixed in a 96-well plate. The reaction was started by warming the solution from
4
to 37 °C. The effects of 4l (10 μM; blue), 4m (10 μM; red), 4n (10 μM; yellow) and paclitaxel (10 μM;
green circles) on tubulin assembly was monitored in a Spectramax 340PC spectrophotometer at 340
nm at 30 s intervals for 60 min at 37 °C. Ethanol (1% v/v; black squares) was used as a vehicle control.
Results are representative of three independent experiments.
2
9