Synthesis, biochemical and molecular modelling studies of antiproliferative azetidinones causing microtubule disruption and mitotic catastrophe

Article abstract of DOI:10.1016/j.ejmech.2011.07.039

The structure-activity relationships of antiproliferative β-lactams, focusing on modifications at the 4-position of the β-lactam ring, is described. Synthesis of this series of compounds was achieved utilizing the Staudinger and Reformatsky reactions. The

Full text of DOI:10.1016/j.ejmech.2011.07.039

European Journal of Medicinal Chemistry 46 (2011) 4595e4607
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biochemical and molecular modelling studies of antiproliferative
azetidinones causing microtubule disruption and mitotic catastrophe
,
a
b
a
c
Niamh M. O’Boyle a , Miriam Carr , Lisa M. Greene , Niall O. Keely , Andrew J.S. Knox ,
*
Thomas McCabe d, David G. Lloyd c, Daniela M. Zisterer b, Mary J. Meegan a
,
*
a School of Pharmacy and Pharmaceutical Sciences, Centre for Synthesis and Chemical Biology, Trinity College Dublin, Dublin 2, Ireland
b School of Biochemistry & Immunology, Trinity College Dublin, Dublin 2, Ireland
c Molecular Design Group, School of Biochemistry & Immunology, Trinity College Dublin, Dublin 2, Ireland
d School of Chemistry, Trinity College Dublin, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure-activity relationships of antiproliferative b-lactams, focusing on modifications at the 4-
Received 14 April 2011
Received in revised form
13 June 2011
Accepted 22 July 2011
Available online 28 July 2011
position of the
b-lactam ring, is described. Synthesis of this series of compounds was achieved
utilizing the Staudinger and Reformatsky reactions. The antiproliferative activity was assessed in MCF-7
cells, where the 4-(4-ethoxy)phenyl substituted compound 26 displayed the most potent activity with an
IC50 value of 0.22 mM. The mechanism of action was demonstrated to be by inhibition of tubulin poly-
merisation. Cell exposure to combretastatin A-4 and 26 led to arrest of MCF-7 cells in the G2/M phase of
the cell cycle and induction of apoptosis. Additionally, mitotic catastrophe for combretastatin A-4 and for
26 was demonstrated in breast cancer cells for the first time, as evidenced by the formation of giant,
multinucleated cells.
Keywords:
Antiproliferative
Azetidinone
Ó 2011 Elsevier Masson SAS. All rights reserved.
b
-lactam
Combretastatin
Mitotic catastrophe
Tubulin
1. Introduction
tubulin-binding agents that are not in clinical use due to problems of
toxicity. Colchicine was the first drug known to bind to tubulin and
Microtubules are a component of the mitotic spindle and are
essential to the mitotic division of cells. Tubulin is an hetero-
inhibit microtubule formation as early at the 1930s [1,4]. Colchicine
is not used clinically for the treatment of cancer due to gastroin-
testinal side-effects [4]. To date, there is no clinically approved drug
that binds to the colchicine domain of tubulin and much work
continues to be carried out in this area.
a-b
dimeric protein which is the main constituent of microtubules [1].
Tubulin is the target of numerous small molecule ligands that act by
interfering with microtubule dynamics. These ligands can be broadly
divided into two categories e those that inhibit the formation of the
mitotic spindle and those that inhibit the disassembly of the mitotic
spindle once it has formed [2]. Tubulin has three well-characterised
binding sites: the taxane domain, the vinca domain and the colchi-
cine domain and many compounds interact with tubulin at these
known sites. Paclitaxel (Taxol, 1, Fig. 1) binds to tubulin at the taxane
site, the vinca alkaloids, including vinblastine (2, Fig. 1), bind at the
vinca domain and colchicine (3, Fig. 1) binds at the colchicine
domain. Paclitaxel and vinblastine are in clinical use for many types
of cancer [3]. Colchicine and podophyllotoxin are colchicine domain
The combretastatins are a group of diaryl stilbenes isolated from
the stem wood of the South African tree Combretum Caffrum.
Traditionally, the root bark of Combretum Caffrum was powdered
and boiled and used by the Zulu tribe as a charm for harming an
enemy, but there is no written evidence of use of the plant for
treating cancer amongst the indigenous people of Africa [5]. A
number of constituent stilbenes were found to inhibit the growth of
cancer cells. Combretastatin A-4 (4, Fig. 1) demonstrated potent
antiproliferative activity against a number of human cancer cell
lines including multi-drug resistant cancer cell lines [4]. Stilbene 4
binds to the colchicine domain of tubulin and induces vascular
shutdown within tumours [6]. Clinically, a water-soluble prodrug,
combretastatin A-4-phosphate (CA4P, fosbretabulin, 5, Fig. 1) is
under evaluation in phase 3 trials for treatment of anaplastic
thyroid cancer and in phase 2 trials for non-small cell lung cancer
* Corresponding authors. Tel.: þ353 1 8962798; fax: þ353 1 8962793.
E-mail addresses: oboyleni@tcd.ie (N.M. O’Boyle), mmeegan@tcd.ie (M.
J. Meegan).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.039

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N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
a phenyl substituted ring at the 3-position of the azetidinone ring, it
Abbreviations
was of importance to extend the SAR by investigating a range of aryl
substituents at the 4-position. It was also valuable to further
CA-4
Combretastatin A-4
characterise the biochemical effects of both 4 and these b-lactam
CA-4P
Combretastatin A-4 phosphate
compounds. Herein we report novel findings for 4 and related b-
DAMA-colchicine N-Deacetyl-N-(2-mercaptoacetyl)-
colchicine
lactam analogues in MCF-7 breast cancer cells.
ER
Estrogen receptor
2. Results and discussion
GTP
LDH
mAb
MTT
Guanidine triphosphate
Lactate dehydrogenase
Monoclonal antibody
3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
Nuclear magnetic resonance
Phosphate buffered saline
Trimethylchlorosilane
2.1. Chemistry
The design of this series of compounds incorporated a 3,4,5-
trimethoxyphenyl ring at the N-1 position of the -lactam ring,
previously demonstrated to be the optimal substituent at this
position [15]. This mimics the A-ring of 4 (Fig. 1). The choice of
substituents at the 4-position was based on previously reported
potent derivatives of 4. The first step in the synthesis of the
b
NMR
PBS
TMCS
and platinum-resistant ovarian cancer [7]. In vivo, in isolated
tumour systems, vascular shutdown is seen within 20 minutes of
the start of infusion of 5. At 100 mg/kg, rapid and prolonged blood
flow shutdown is evident, and both human and murine tumour
models show extensive necrosis within 24 h. These effects were
also seen at doses between 25 and 1500 mg/kg, indicating the wide
therapeutic window [6]. In addition to 5, a second structurally
related prodrug (6, ombrabulin, Fig. 1, a water-soluble serine amino
acid prodrug) is in clinical trials [7, 8]. A related analogue from the
required
b-lactams was the formation of the imine precursors
14e25. This is achieved by condensation of the appropriately
substituted benzaldehydes and anilines (Schemes 1 and 2). The
desired imines were obtained in high yields. Synthesis of b-lactams
27e33 and 35e37 was carried out using the Staudinger reaction
with in situ generation of a ketene and subsequent reaction with
the appropriately substituted imine (method I, Schemes 1 and 2). A
modified Staudinger method (method II, Scheme 1) requiring
overnight reaction was used to obtain bromo-containing
b-lactam
combretastatin
A series, combretastatin A-1 diphosphate (7,
34 as method I was unsuccessful [17]. Where the appropriate
a
-
OXi4503, Fig. 1), is being evaluated in hepatic and solid tumours as
well as acute myeloid leukemia [7].
bromoacetate precursor was available, the Reformatsky reaction
was used for azetidinone synthesis (26, Scheme 1, method III). The
Many conformationally restricted analogues of 4 have been
reported, the majority of which replace the isomerisable cis-double
bond in 4 with a heterocycle. Reported heterocyclic CA-4 analogues
include imidazole 8 [9], tetrazole 9 [10] and benzoxepin 10 [11]
stereochemistry of the
b-lactam product obtained can vary
depending on numerous factors, including the reaction conditions,
the order of addition of the reagents and the substituents present
on both the imine and on the acid chloride [18e20]. The X-ray
(Fig. 2). The azetidin-2-one (b-lactam) ring [12] is an alternative
crystal structure of
protons H-3 and H-4 of the
(Fig. 3). This trans stereochemistry was observed for all
compounds synthesised with phenyl rings directly attached to
b
-lactam 26 shows the trans arrangement for
-lactam ring, with J values of 2 Hz
-lactam
scaffold for potent non-isomerisable combretastatin analogues
(11e13, Fig. 2) [13e16]. Having previously investigated compre-
hensive structure-activity relationships of these series with
b
b
Fig. 1. Tubulin binding agents.

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
4597
for a series of 3-unsubstituted
b
-lactam compounds [14].
b
-Lactam
NH2
H3CO
OH
H3CO
H2N
13 was chosen as the lead compound due to both its synthetic
accessibility and the potential to examine a number of related
analogues to examine the structure-activity relationships of
derivatives with variations at the 4-position of the
Ethoxy derivate 26 was the most potent compound in this series in
MCF-7 cells with an IC50 value of 0.22 M. This is a decrease in
activity of approximately 10-fold compared to 13, and is consistent
with previously reported ethoxy-substituted combretastatin
derivatives [26]. Surprisingly 27, the 4-fluoro compound displays
submicromolar activity which is not shown in a related 3-
unsubstituted compound previously reported [14]. However,
potent fluoro-containing analogues of 4 are known [27]. The 4-
OCH3
N
N
N
N
O
b-lactam ring. 4-
N
H3CO
OCH3
OCH3
N
H3CO
H3CO
m
H3CO
OCH3
OCH3
OCH3
8
9
10
HO
N
H2N
OCH3
OCH3
OCH3
O
O
dimethylamino
substituted
compound
28
and
3,4-
dimethoxyphenyl compound 33 also exhibit submicromolar
activity. The inclusion of the two trimethoxy ring systems (as in
compounds 29, 30, 31 and 32) gives less active compounds with
antiproliferative values in the high micromolar range regardless of
the substitution pattern of the methoxy groups at position 4. A
N
N
O
O
O
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
13
H3CO
bromine-containing analogue (34, IC50 ¼ 1.42
mM) was of interest
as a derivative of 4 which replaced the hydroxy group of Ring B with
a bromide substituent was reported to have moderate activity in
a range of cell lines [28]. In our series, this compound was not as
potent as either the lead compound 13 or 26. However, this
compound has the potential to be used as an intermediate in the
11
12
Fig. 2. Heterocyclic analogues of 4.
positions 3 and 4 of the ring, as evidenced by the coupling
constants, J3,4 x 2 Hz. No cis isomers were detected in this series,
probably due to steric hindrance between the 3- and 4-positions of
the azetidin-2-one ring.
synthesis of further boronic acid b-lactams, as has been reported for
potent boronic acid bioisosteres of 4 [29]. The 2-naphthalene
moiety has been demonstrated to be good surrogate for the B-
ring of the combretastatin series [30], and the equivalent
b-lactam
b
-Lactams 38, 39 and 40 are diphenyl substituted at the 4-
35 containing a 2-naphthalene ring at the 4-position shows sub-
position of the azetidinone ring. The imine precursors, obtained
from the appropriately substituted benzophenones, could not be
isolated under normal reflux conditions in ethanol. It has been
noted previously that it is more difficult to synthesise Schiff bases
from ketone precursors such as acetophenone and benzophenone
than from less sterically hindered aldehydes such as para-
methoxybenzaldehyde. Anhydrous conditions, lengthy reaction
times, high temperatures and the use of activated molecular sieves
are normally required [21]. A number of different reaction condi-
tions were attempted for these compounds. Reaction of benzo-
phenones with 3,4,5-trimethoxyaniline did not proceed when
using 4 Å molecular sieves, reflux conditions and anhydrous
toluene [22]. Refluxing with benzene, molecular sieves and sodium
bicarbonate also did not yield any of the desired imine [23]. An
micromolar activity with an IC50 value of 0.44
m
M.
R4
R1
R3
R5
N
R3
OCH3
R7
R2
R1
R4
R5
R6
a
R2
+
R7
CHO
NH2
OCH3
R6
R2
R=H unless otherwise indicated
14: R3=OCH2CH3, R6=R7=OCH3
15: R3=F, R6=R7=OCH3
R3
R1
R4
16: R3=N(CH3)2, R6=R7=OCH3
17: R1=R2=R3=R6=R7=OCH3
18: R1=R3=R5=R6=R7=OCH3
19: R1=R3=R4=R6=R7=OCH3
20: R3=R4=R6=R7=OCH3
b, c
R5
N
alternative, one-pot preparation of b-lactams using titanium (IV)
O
R7
chloride was explored [24]. This method uses half an equivalent of
TiCl4 in the condensation of the appropriate aniline and benzo-
phenone using tri-n-butylamine as the base. Subsequent reaction
with the appropriate acid chloride without isolation of the inter-
21: R2=Br, R3=R6=R7=OCH3
25: R2=R3=R4=R6=R7=OCH3
OCH3
R6
R=H unless otherwise indicated
Method I
mediate imine yielded the desired
yields (Scheme 3).
b-lactams 38, 39 and 40 in low
27: R3=F, R6=R7=OCH3
31: R2=R3=R4=R6=R7=OCH3
28: R3=N(CH3)2, R6=R7=OCH3 32: R1=R3=R4=R6=R7=OCH3
29: R1=R2=R3=R6=R7=OCH3
30: R1=R3=R5=R6=R7=OCH3
33: R3=R4=R6=R7=OCH3
Method II
34: R2=Br, R3=R6=R7=OCH3
2.2. Pharmacology and biochemical results
O
O
2.2.1. Antiproliferative activity in breast cancer cells
d
Compounds 26e40 were screened for their antiproliferative
activity in the ER expressing MCF-7 human breast cancer cell line
using the MTT assay [25]. The drug concentration required to
inhibit the cell growth by 50% (IC50) was determined and the results
are displayed in Table 1. Additional screening using the ER inde-
pendent MDA-MB-231 human breast cancer cell lines was carried
out for selected analogues.
The antiproliferative activity of the 3-phenyl substituted b-lac-
tams in MCF-7 and MDA-MB-231 breast cancer cells (Table 1) are in
general agreement with our previously reported results obtained
N
OCH3
OCH3
N
O
OCH3
OCH3
OCH3
H3CO
26
14
Scheme 1. Reagents and conditions: (a) ethanol, reflux, 3 h; (b) method I:
C6H5CH2COCl, (CH3CH2)3N, CH2Cl2, 3 h, reflux; (c) method II: C6H5CH2COCl,
(CH3CH2)3N, triphosgene, CH2Cl2, overnight; (d) method III: ethyl-
cetate, zinc, TMCS, benzene, microwave (only one enantiomer of
illustrated for clarity).
a
-bromophenyla-
b-lactam compounds

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N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
OCH3
H3CO
OCH3
a
b
+
N
N
OCH3
OCH3
O
NH2
CHO
OCH3
OCH3
OCH3
H3CO
35
22
H3CO
OCH3
O
OCH3
OCH3
OCH3
H3CO
a
b
+
N
OCH3
OCH3
N
O
NH2
OCH3
OCH3
OCH3
H3CO
23
36
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
a
b
+
N
O
N
CHO
NH2
H3CO
H3CO
H3CO
OCH3
OCH3
OCH3
37
24
Scheme 2. Preparation of
b-lactams 35, 36 and 37 via the Staudinger reaction. Reagents and conditions: (a) ethanol, reflux, 3 h; (b) C6H5CH2COCl, (CH3CH2)3N, CH2Cl2, reflux, 3 h
(only one enantiomer of
b-lactam compounds illustrated for clarity).
Other aryl substituent variations at the C-4 position were also
examined. Removal of the direct attachment of the phenyl ring to
breast cancer cells, with the exception of compounds 26 and 33.
Compound 26 had an IC50 value of 0.83 while 4-(3,4-
dimethoxyphenyl) substituted -lactam 33 had an IC50 value of
0.53 M which was nearly 7-fold less potent than 13 (Table 2).
mM
the
a factor of 100 (vinyl analogue 36 has an IC50 value of 1.99
compared to 0.034 M for 13). This is also true of the N-1 position,
where a methylene spacer introduced in compound 37 between the
core -lactam ring and the trimethoxyphenyl ring led to a decrease
in activity by over a thousand-fold (IC50 value of 38.6 M compared
to 0.034 M for 13). The introduction of the two phenyl rings at the
b-lactam core results in reduction of antiproliferative effect by
b
mM
m
m
b
m
m
C-4 position leads to a small decrease in antiproliferative activity
for 39 and 40, both of which contain methoxy groups on one or
both phenyl rings, showing antiproliferative effects at sub-
micromolar concentrations. Compound 38, without methoxy
groups at the 4-position, is less potent (IC50 ¼ 3.84
mM).
This series of analogues did not display submicromolar anti-
proliferative activity equipotent with either 4 or 13 in MDA-MB-231
Scheme 3. Preparation of 4,4-diphenyl substituted
b-lactams 38e40. Reagents and
conditions: (a) TiCl4, anhydrous toluene, tri-n-butylamine, 18 h (b) Tri-n-butylamine,
C6H5CH2COCl, reflux, 8 h.
Fig. 3. Ortep representation of the crystal structure of 26 with displacement ellipsoids
plotted at 50%.

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
4599
Table 1
assessment of its drug-like properties via a Tier 1 profiling screen.
Compound 26 satisfies Lipinski’s ‘rule of five’ for drug-like prop-
erties e.g. molecular weight (433) is less than 500, the number of
oxygen/nitrogen atoms is less than 10, the number of hydrogen
bond donors is less than 5 and the cLog P value of 3.72 (<5), imply
that this is a moderately lipophilicehydrophobic drug and is
a suitable candidate for further investigation (in addition to
predictions of permeability, metabolic stability, Pgp substrate
status, blood-brain barrier partition, plasma protein binding and
human intestinal absorption properties which indicated the suit-
ability of these compounds for further development).
In this study, we investigated the tubulin-targeting properties of
26 by a turbidity assay and confocal microscopy. As expected, 26
(10 mM) completely inhibited the assembly of tubulin in a cell free
tubulin turbidity assay in a similar manner to that previously
reported for 4 (Fig. 4) thus confirming that the target of this series
of compounds is tubulin. In an attempt to identify the cellular
effects that may be relevant to the antiproliferative activity of 26,
we evaluated their activity on the alteration of the microtubule
network by tubulin immunostaining, comparing it to that of 4.
Antiproliferative effects of combretastatin
analogues 26e40 in MCF-7 cells.
b-lactam
Compound
IC50 value (m
M)a
26
27
28
29
30
31
32
33
34
35
36
37
38
39
0.22 ꢀ 0.1
0.35 ꢀ 0.1
0.85 ꢀ 0.9
1.12 ꢀ 1.2
18.7 ꢀ 12
69.8 ꢀ 82
46.9 ꢀ 18
0.46 ꢀ 0.4
1.42 ꢀ 0.9
0.44 ꢀ 0.08
1.99 ꢀ 1.04
38.6 ꢀ 16
3.84 ꢀ 2.2
0.43 ꢀ 0.17
0.64 ꢀ 0.10
40
4b
0.005 ꢀ 0.002
0.034 ꢀ 0.02
13 [15]
a
IC50 values are half maximal inhibitory concentrations
required to block the growth stimulation of MCF-7 cells.
Values represent the mean ꢀ S.E.M (error values ꢁ 10ꢂ6) for
Confocal analysis of MCF-7 cells stained with
a-tubulin mAb
at least three experiments performed in triplicate.
demonstrated a well organised microtubular network in control
cells (Fig. 5). Exposure to 4 (100 nM) or 26 (500 nM) for 16 h led to
a complete loss of microtubule formation consistent with depoly-
merised microtubules (Fig. 5). Additionally, cells treated with 4 and
26 increased in cell size and contained multiple micronuclei e
a phenomenon described as mitotic catastrophe. Mitotic catas-
trophe is a type of programmed cell death in response to DNA
damage, characterised by giant multinucleated cells [31]. These
findings are in agreement with previously published studies, where
5 induced mitotic catastrophe in chronic lymphocytic leukemia
cells [32]. Similarly, 4 and a combretastatin derivative induced
mitotic catastrophe dependent on spindle checkpoint and caspase-
3 activation in non-small cell lung cancer cells [33]. Mitotic catas-
trophe has also been demonstrated for 4 and related derivatives in
both human endothelial cells (HUVEC) and human lung carcinoma
cells (H460) [34,35]. Taken together, these results confirm tubulin
b
The IC50 value obtained for 4 in this assay is 0.005 mM
for MCF-7 which is in good agreement with the reported
values for 4 using the MTT assay on human MCF-7 breast
cancer cell line [35,44e46].
The cytotoxicity of selected analogues at 10 mM was determined
using an LDH assay. The range of cytotoxicity in MCF-7 cells was
between 3% for 33 and 10.4% cell death for dimethylamino-
containing analogue 28. These values are comparative to the
percentage of cell death of 5.5% at 10
cells. In MDA-MB-231 cells, cytotoxicity at 10
(analogues 27, 30, 31, 32 and 33) to 6.1% (compound 29), again
falling in the same low percentage range as 4 (4.3%). The cytotox-
icity for the most potent compound, 26, was 5.4%. This confirms
previous work in normal murine epithelial cells that both 4 and b-
lactam derivatives are minimally cytotoxic to non-proliferating
cells [15,16].
m
M observed for 4 in MCF-7
m
M ranged from 0%
as the molecular target of this series of b-lactam combretastatin
derivatives and demonstrate mitotic catastrophe in MCF-7 breast
cancer cells for 4 and 26 for the first time.
2.2.2. Tubulin polymerization and immunofluorescence
b-Lactam 26 was chosen for further study on the basis of its
potent antiproliferative activity in MCF-7 cells, together with an
Table 2
Antiproliferative effects of combretastatin
analogues in MDA-MB-231 cells.
b
-lactam
Compound
IC50 (m
M)a
26
27
28
29
30
31
32
0.83 ꢀ 0.002
1.14 ꢀ 0.2
5.66 ꢀ 2.4
1.96 ꢀ 0.09
5.74 ꢀ 4.9
65.6 ꢀ 2.6
38.9 ꢀ 12
0.53 ꢀ 0.02
0.043
33
4b
13 [15]
0.078 ꢀ 0.04
a
IC50 values are half maximal inhibitory concentra-
tions required to block the growth stimulation of MDA-
MB-231 cells. Values represent the mean ꢀ S.E.M (error
values ꢁ 10ꢂ6) for at least three experiments performed
Fig. 4. Tubulin polymerization for 26 (blue squares) and ethanol (vehicle control, red
squares). Effects of compound 26 (10 mM) 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 ꢃC to 37 ꢃC. Ethanol (1%v/v) was used as a vehicle control.
The effect on tubulin assembly was monitored in a Spectramax 340PC spectropho-
tometer at 340 nm at 30 s intervals for 60 min at 37 ꢃC. The graph shows one repre-
sentative experiment. Each experiment was performed in triplicate (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
in triplicate.
b
The IC50 value obtained for 4 in this assay is 0.043 mM
for MDA-MB-231 which is in good agreement with the
reported values for 4 using the MTT assay on the human
MDA-MB-231 breast cancer cell line [47,48].

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N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
Fig. 5. CA-4 (4) and
b-lactam 26 depolymerise the microtubule network of MCF-7 cells resulting in mitotic catastrophe. MCF-7 cells were treated with vehicle [1% ethanol (v/v)], CA-
4 (4) [100 nM] or 26 (500 nM) for 16 h. Cells were fixed in methanol and stained with
microscopy coupled with OLYMPUS FLUOVIEW software. Bar equal to 40 m. Representative confocal micrographs of three separate experiments are shown (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
a-tubulin mAbs (green) and counterstained with DAPI (red). Images were captured by confocal
m
2.2.3. Analysis of DNA content by flow cytometry
We next examined the effect of 4 and -lactam 26 on the cell
position of the
more optimally than in the case of colchicine. This binding orien-
tation differs from that previously observed for a related series of
lactams, where the aryl ring at the C-4 position of the -lactam
b-lactam is observed to fill the pocket of tubulin
b
cycle of MCF-7 cells by flow cytometric analysis of propidium
iodide stained cells. After 48 h, both 4 and 26 induced a significant
increase in the percentage of cells in the G2M phase of the cell cycle
(52% and 47.5% respectively compared to 19.7% in untreated cells,
P ¼ 0.02) together with a significant increase in apoptosis as
determined by quantification of the sub-G1 population of cells
(9.4% and 13.97% respectively compared to 1.65% in untreated cells)
(Table 3 and Fig. 6).
b-
b
filled the space occupied by the C-ring of colchicine [15,16].
Hydrogen bonding between sulfur and Ser178 is also depicted in
Fig. 7 in both the co-crystal of 41 and also the docked complex of 26
which uses oxygen as a H-bond acceptor in this case. It is likely that
a steric clash between the longer ethoxy side chain and the residues
in the binding site contributes to the changed binding conforma-
tion in comparison to 3, 4 and 13 where the 3-hydroxy-4-
methoxyphenyl ring (or C-4 4-methoxyphenyl ring in the case of
13) occupies the space now occupied by the C-3 phenyl ring of 26.
The difference in positioning of the ethoxy side chain of 26 and the
methoxy group of the C-ring of 41 may be significant for observed
antiproliferative activity of 26 in combretastatin-resistant cell lines
(data not shown).
2.3. Structural studies of
binding site of tubulin
b-lactam compound 26 in the colchicine-
The proposed mode of binding of
by virtual molecular docking using a published crystal structure of
DAMA-colchicine (41) bound to the colchicine domain of ab
b-lactam 26 was investigated
-
tubulin [36]. Fig. 7 illustrates the key interactions observed for 41 in
the active site of tubulin. It is clear that 41 appears to bind to the
interface of the ab heterodimer of tubulin. The key contacts
involved have been described by Knossow et al. with the trime-
thoxy A-ring of 41 interacting with Cys241 [36]. Crosslinking
studies with trimethoxy-substituted A-rings with more reactive
groups have previously shown the importance of Cys241 in the
binding process [36,37]. A similar binding orientation is observed
for the trimethoxyphenyl A-rings of 41 and 26 (Fig. 7). The posi-
tioning of the trimethoxy substituents of the A-rings differ slightly
due to the 3D conformation of the molecule but both can still
interact favorably with Cys241 to provide the anchoring interaction
in the binding site. These binding parallels may rationalize the
antiproliferative potency observed for 26. The C-ring at the 3-
Table 3
Flow cytometric analysis of both cell death (sub-G1) and the cell cycle in MCF-7 cells
exposed to 4 and
b
-lactam 26.a
Compound Sub-G1 (%) G1 (%)
S (%)
G2/M (%)
Polyploidy (%)
Untreated
Ethanol
(1% v/v)
4
1.65 ꢀ 0.38 60.22 ꢀ 3.1 8.77 ꢀ 0.9 19.72 ꢀ 1.2 8.83 ꢀ 2.5
1.26 ꢀ 0.2 63.76 ꢀ 4.1 9.05 ꢀ 1.6 18.31 ꢀ 1.7 7.01 ꢀ 3.8
9.43 ꢀ 2.1 23.57 ꢀ 5.9 10.66 ꢀ 2.4 52.02 ꢀ 5.9 8.36 ꢀ 1.9
13.97 ꢀ 3.2 21.18 ꢀ 3.0 9.49 ꢀ 0.27 47.53 ꢀ 4.7 7.40 ꢀ 2.2
26
a
Cell cycle analysis of MCF-7 cells untreated or treated with vehicle control (1%
(v/v) ethanol), 4 (100 nM) or 26 (500 nM) for 48 h. % MCF-7 cells in each cell cycle
phase are shown after exposure to compounds 4 or 26 for 48 h. Cells were analysed
with the FACScan flow cytometry. Cells in the sub-G1 peak are indicative of
apoptotic cells. Results show a typical experiment which has been repeated three
times. Values represent the mean ꢀ standard deviation for three experiments.

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
4601
Fig. 6. Effect of 4 and 26 on the cell cycle. MCF-7 cells were left untreated (U) or treated with vehicle (E) [1% ethanol (v/v)], CA-4 (4) [100 nM], 26 [500 nM] for 48 h. Percentages of
(A) G2M arrested cells and (B) apoptotic (sub-G1) are indicated. Values represent the mean ꢀ SEM for at least three separate experiments. *P < 0.05. C, representative DNA profiles
are shown and mean percentage of cells in sub-G1 and G2M are indicated.
Structurally, similarity between the common features of 3, 4 and
antiproliferative -lactams has been demonstrated. Of particular
note is that the crystal structures of 4 [38], 5 [28], 13 [15] and 26
show non-coplanar ring systems. In the crystal structure of the
a microtubule-damaging mechanism and that additional mecha-
nisms involved in mitosis control should be considered. The
unusual effects of these compounds are significant and are under
further investigation.
b
b
-
lactam 26 (Fig. 3), the distance between the centroid of the C-3-
phenyl and N-1-phenyl rings is 7.338 Å, in comparison to 6.193 Å
for the distance between the centroids of C-3 and C-4 phenyl rings
and 5.146 Å between the N-1 and C-4 phenyl rings (Fig. 8). The
comparative distance between the centroids of the A- and C-rings
of the co-crystal structure of 41 is 4.516 Å. Surprisingly, the equiv-
alent distance between the centroids of A- and B-rings of 4
4. Experimental protocols
4.1. Chemistry
All reagents were commercially available and were used
without further purification unless otherwise indicated. Toluene
was dried by distillation from sodium and stored on activated
molecular sieves (4 Å) and dichloromethane was dried by distilla-
tion from calcium hydride prior to use. IR spectra were recorded as
thin films on NaCl plates or as KBr discs on a Perkin-Elmer Paragon
100 FT-IR spectrometer. 1H and 13C NMR spectra were obtained on
a Bruker Avance DPX 400 instrument at 20 ꢃC, 400.13 MHz for 1H
spectra, 100.61 MHz for 13C spectra, in CDCl3 (internal standard
tetramethylsilane) by Dr. John O’Brien and Dr. Manuel Ruether in
the School of Chemistry, Trinity College Dublin. High resolution
accurate mass determinations for all final target compounds were
obtained on a Micromass time of flight mass spectrometer (TOF)
equipped with electrospray ionization (ES) interface operated in
the positive ion mode at the High Resolution Mass Spectrometry
Laboratory by Dr. Martin Feeney in the School of Chemistry, Trinity
College Dublin. Elemental analysis was carried out in the micro-
analytical laboratory at University College Dublin, Belfield, Dublin
4. Thin layer chromatography was performed using Merck Silica gel
(5.191 Å) is closer in distance to that between N-1 and C-4 of b-
lactam compound 26, but despite this, the preferred orientation of
26 in the molecular docking with tubulin showed overlay of the N-1
and C-3 rings of 26 with the A- and B-rings of 41.
3. Conclusion
A novel series of antiproliferative agents are described. Struc-
tural and molecular modelling studies rationalize the observed
antiproliferative activities. Interesting effects on microtubule
dynamics were observed both for 4 and b-lactam derivative 26,
including mitotic catastrophe in MCF-7 breast cancer cells observed
for the first time. MCF-7 breast cancer cells treated with 4 and 26
undergo cell death both through microtubule disorganization and
mitotic catastrophe, as demonstrated by the presence of giant
multinucleated cells. These observations suggest that the activities
of 4 and 26 in MCF-7 cells might not be related solely to

4602
N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
Fig. 7. The crystal structure of tubulin with (A) DAMA-colchicine 41 and (B) docked X-ray of compound 26 in the same active site. Docked pose of 41 and
b-lactam 26 in the
colchicine-binding site of tubulin (PDB entry 1SA0). Hydrogens are not shown for clarity. Coloured by atom: Green (colchicine carbon); Pink ( -lactam carbon); red (oxygen); blue
b
(nitrogen). Residue numbers are those used by Ravelli et al. [36] (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
60 TLC aluminium sheets with fluorescent indicator visualizing
with UV light at 254 nm. Flash chromatography was carried out
using standard silica gel 60 (230e400 mesh) obtained from Merck.
All products isolated were homogenous on TLC. Analytical high-
performance liquid chromatography (HPLC) to determine the
purity of the final compounds was performed using a Waters 2487
Dual Wavelength Absorbance detector, a Waters 1525 binary HPLC
pump, a Waters In-Line Degasser AF, a Waters 717plus Autosampler
and a Varian Pursuit XRs C18 reverse phase 150 ꢁ 4.6 mm chro-
matography column. Samples were detected using a wavelength of
254 nm. All samples were analysed using acetonitrile (70%): water
(30%) over 10 min with a flow rate of 1 mL/min. Unless otherwise
indicated, the purity of the final products was ꢄ95%. Analyses
indicated by the symbols of the elements or functions were within
ꢀ0.4% of the theoretical values.
4.1.1. General preparation of Schiff bases (14e25)
A solution of the appropriately substituted aryl aldehyde
(0.1 mol) and the appropriately substituted aryl amine (0.1 mol) in
ethanol (50 mL) was heated at reflux for three hours. The reaction
mixture was reduced to 25 mL under vacuum. The mixture was left
to stand and the Schiff base product crystallized out of the solution.
The crude product was then recrystallized from ethanol and filtered
to yield the purified product. Schiff bases 15, 17, 19, 20 and 25 were
prepared and isolated as previously reported [14].
N-(4-Ethoxybenzylidene)-3,4,5-trimethoxyaniline 14. Preparation
Fig. 8. Distances between the centres of the three phenyl rings of b-lactam 26 (single
was as above from 3,4,5-trimethoxybenzenamine and 4-
crystal X-ray structure).

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
4603
ethoxybenzaldehyde. Yield 64%, yellow crystalline solid, m.p. 98 ꢃC.
(5 mmol) and triethylamine (15 mmol) in anhydrous CH2Cl2
(50 mL) at reflux in an inert atmosphere. The solution was refluxed
for 10 hours and then washed with saturated sodium bicarbonate
solution (50 mL), dilute (10%) HCl (50 mL) and brine (50 mL). The
organic layer was dried and evaporated in vacuo.
IR nmax (KBr): 1604.72 cmꢂ1, 1586.85 cmꢂ1 (eC]Ne). 1H NMR (400
MHz, CDCl3)
(m, 2H), 6.47 (s, 2H), 6.97 (d, 2H, J ¼ 8.28 Hz), 7.83 (d, 2H, J ¼ 8.8 Hz),
8.40 (s, 1H). 13C NMR (100 MHz, CDCl3)
14.30, 55.65, 60.58, 63.22,
d 1.43e1.47 (t, 3H), 3.86 (s, 3H), 3.90 (s, 6H), 4.08e4.13
d
97.63, 114.23, 128.42, 130.03, 135.58, 147.90, 153.08, 158.74, 161.26.
Elemental analysis: C18H21NO4 (C, H, N).
N-(4-(Dimethylamino)benzylidene)-3,4,5-trimethoxyaniline 16.
Preparation was as above from 4-dimethylaminobenzaldehyde and
3,4,5-trimethoxyaniline. Yield 64%, yellow crystals, m.p. 83e84 ꢃC.
IR nmax (KBr): 1602.7 cmꢂ1 (C]N). 1H NMR (400 MHz, CDCl3):
4.1.2.2. Modified Staudinger reaction (method II). The appropriate
imine (10 mmol) and acetyl chloride (10 mmol) were added to
anhydrous CH2Cl2 (50 mL) under nitrogen and the mixture was left
stirring for 2 h. Triethylamine (10 mmol) was injected dropwise
and the mixture was stirred overnight. The mixture was washed
with distilled water (50 mL) (twice) and then with saturated
aqueous sodium bicarbonate solution (50 mL). The organic layer
was dried by filtration through anhydrous sodium sulfate. The
organic layer containing the product was collected and reduced in
vacuo.
d
3.08 (s, 6H, eNeCH3), 3.85 (s, 3H, OCH3), 3.89 (s, 6H, OCH3), 6.47
(s, 2H, ArH), 6.73 (d, 2H, J ¼ 9 Hz, ArH), 7.76 (d, 2H, J ¼ 9.04 Hz, ArH),
8.33 (s, 1H, eCH]N). 13C NMR (100 MHz, CDCl3):
39.42, 55.42,
d
55.61, 60.54, 97.64, 111.10, 123.54, 130.01, 135.14, 148.42, 152.08,
152.99, 159.43. HRMS: Calculated for C18H23N2O3: 314.3790; Found
315.1709 (Mþ þ H).
4-(4-Fluorophenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin-
2-one 27. Preparation was from 15 (5 mmol) as described in method
I. Evaporation of solvent yielded a brown solid residue which was
purified using column chromatography hexane:diethyl ether (1:1).
3,4,5-Trimethoxy-N-(2,4,6-trimethoxybenzylidene)aniline
18.
Preparation was as above from 2,4,6-trimethoxybenzaldehyde and
3,4,5-trimethoxyaniline. Yield 69%, Orange gel, IR nmax (NaCl film):
1659.4 cmꢂ1 (C]N). 1H NMR (400 MHz, CDCl3):
d
3.86 (s, 3H,
Yield 11%, Orange gel. IR nmax (KBr): 1706.1 cmꢂ1 (C]O,
b
-lactam).
OCH3), 3.90 (s, 9H, OCH3), 3.94 (s, 3H, OCH3), 3.96 (s, 3H, OCH3),
6.48 (s, 2H, ArH), 6.52 (s, 1H, ArH), 7.65 (s, 1H, ArH), 8.82 (s, 1H,
1H NMR (400 MHz, CDCl3):
d 3.72 (s, 6H, OCH3), 3.78 (s, 3H, OCH3),
4.26e4.27 (d, 1H, J ¼ 2.5 Hz, H3), 4.91e4.92 (d, 1H, J ¼ 2.5 Hz, H4),
6.61 (s, 2H, ArH), 7.14 (m, 2H, ArH), 7.31e7.45 (m, 7H, ArH). 13C NMR
eCH]N). 13C NMR (100 MHz, CDCl3):
d 56.01, 56.22, 56.38, d 60.95,
98.20, 108.84, 116.48, 136.78, 143.55, 148.87, 153.37, 155.04, 155.16.
N-(3-Bromo-4-methoxybenzylidene)-3,4,5-trimethoxyaniline 21.
Preparation was as above from 3,4,5-trimethoxyaniline and 3-
bromo-4-methoxybenzaldehyde. Yield 68%, Yellow crystalline
solid, m.p. 128 ꢃC. IR nmax (KBr): 1620.47 cmꢂ1, 1585.77 cmꢂ1 (eC]
(100 MHz, CDCl3): d 55.57, 60.51, 62.98, 64.66, 94.35, 115.86, 116.07,
126.97, 128.68, 132.80, 133.05, 133.99, 134.17, 153.15, 163.62, 164.88.
HRMS: C24H22FNO4Na requires 430.1430; found 430.1395
(Mþ þ Na).
4-(4-(Dimethylamino)phenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one 28. Preparation was from 16 (10 mmol) as described in
method I. Evaporation of solvent yielded a brown solid residue which
was purified using column chromatography hexane:diethyl ether
(1:1). Yield 6%, Orange powder, m.p. 118 ꢃC. IR nmax (NaCl film):
Ne). 1H NMR (400 MHz, CDCl3)
d 3.89 (s, 3H), 3.92 (s, 6H), 3.99 (s,
3H), 6.50 (s, 2H), 7.00 (d,1H, J ¼ 8.52 Hz), 7.79e7.81 (dd,1H), 8.18 (d,
1H, J ¼ 2 Hz), 8.38 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 55.68, 56.00,
60.59, 97.67, 111.13, 111.95, 129.22, 129.81, 132.82, 135.92, 147.24,
153.13, 157.00, 157.81. HRMS: C17H18NO4Br requires 380.0580;
found 380.0497; elemental analysis: C17H18BrNO4 (C, H, N, Br).
3,4,5-Trimethoxy-N-(naphthalen-2-ylmethylene)aniline 22. Prep-
aration was as above from 2-naphthaldehyde and 3,4,5-
trimethoxyaniline. Yield 77%, yellow crystals, m.p. 108e116 ꢃC. IR
1745.1 cmꢂ1 (C]O, -lactam).1HNMR(400 MHz, CDCl3):
b d2.99 (s, 6H,
N-CH3), 3.75 (s, 6H, OCH3), 3.80 (s, 3H, OCH3), 4.33 (d, 1H, J ¼ 2.5 Hz,
H3), 4.86,(d,1H, J ¼ 2.5 Hz,H4), 6.69(s, 2H, ArH), 6.76 (d,2H, J ¼ 8.5 Hz,
ArH), 7.31e7.33 (m, 2H, ArH), 7.35e7.39 (m, 5H, ArH). 13C NMR
(100 MHz, CDCl3):
d 9.96, 55.55, 55.61, 60.49, 63.81, 64.42, 94.45,
nmax (KBr): 1625.74, 1610.62 and 1583.40 cmꢂ1 (C]N).
d
3.88 (s, 6H,
112.26, 123.75, 126.74, 127.32, 127.52, 128.11, 128.32, 128.78, 129.19,
133.51, 134.21, 134.65, 150.34, 153.00, 153.21, 165.59. HRMS:
C26H30N2O4 requires 433.5116; found 433.2112 (Mþ þ H).
OCH3), 3.93 (s, 3H, OCH3), 6.57e7.25 (m, 9H, ArH), 8.67 (s, 1H, N]
CH). Elemental analysis: C20H19NO3 (C, H, N).
3,4,5-Trimethoxy-N-(3-(4-methoxyphenyl)allylidene)aniline 23.
Preparation was as above from 3-(4-methoxyphenyl)propenal and
3,4,5-trimethoxyaniline. Yield 57%, Yellow crystalline solid, m.p.
128 ꢃC. IR nmax (NaCl): 1626.36 cmꢂ1, 1582.90 cmꢂ1 (eC]Ne). 1H
3-Phenyl-4-(2,3,4-trimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one 29. Preparation was from 17 (3 mmol) as described
in method I. Evaporation of solvent yielded a brown solid residue,
which was purified using column chromatography hexane:diethyl
ether (1:1). Yield 10%, Yellow solid, m.p. 92 ꢃC. IR nmax (NaCl film):
NMR (400 MHz, CDCl3)
d 3.86e3.90 (m, 12H), 6.48 (s, 2H),
6.93e7.02 (m, 3H), 7.12e7.16 (m, 1H), 7.50 (d, 1H, J ¼ 9.04 Hz), 8.28
(d, 1H, J ¼ 9.04 Hz). HRMS: C19H22NO4 requires 328.1549; found
328.1545 (Mþ þ H).
1749.2 cmꢂ1 (C]O,
b
-lactam). 1H NMR (400 MHz, CDCl3):
d
3.74
(s, 6H, OCH3), 3.77 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.86 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3).
4.36 (d, 1H, J ¼ 2.5 Hz, H3), 5.22 (d, 1H,
J ¼ 2.5 Hz, H4), 6.63 (s, 2H, ArH), 7.05 (d, 1H, J ¼ 8.6 Hz, ArH), 7.15
(d, 1H, J ¼ 6.5 Hz, ArH),
7.25e7.35 (m, 5H, ArH). 13C NMR
(100 MHz, CDCl3): 55.56, 58.11, 60.40, 60.51, 61.12, 63.39, 94.30,
d
N-(4-Methoxybenzylidene)-1-(3,4,5-trimethoxyphenyl)methan-
amine
trimethoxybenzylamine and 4-methoxybenzaldehyde. Yield 54%,
Colourless crystals, m.p. 62 ꢃC. IR nmax (KBr): 1630.82,1603.44 cmꢂ1
1H NMR (400 MHz, CDCl3)
3.85e3.88 (m, 12H), 4.74 (s, 2H), 6.60
(s, 2H), 6.97 (d, 2H, J ¼ 9.04 Hz), 7.76 (d, 2H, J ¼ 8.52 Hz), 8.35 (s,1H).
13C NMR (100 MHz, CDCl3)
54.93, 55.63, 55.69, 60.39, 64.82, 76.31,
24.
Preparation
was
as
above
from
3,4,5-
d
d
.
107.34, 121.07, 126.59, 127.02, 127.30, 128.24, 128.76, 129.58,
130.18, 133.31, 134.55, 135.49, 153.07, 153.60, 160.03, 161.70,
165.46. HRMS: C27H29NO7Na requires 502.1842; found 502.1823
(Mþ þ Na).
d
d
76.63, 76.95, 103.46, 104.44, 113.56, 113.86, 128.55, 129.43, 131.56,
134.87, 136.41, 152.83, 160.87, 161.33. Elemental analysis:
C18H21NO4 (C, H, N).
3-Phenyl-4-(2,4,6-trimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one 30. Preparation was from 18 (3 mmol) as described
in method I. Evaporation of solvent yielded a brown solid residue,
which was purified using column chromatography, hexane:diethyl
ether (1:1). Yield 52%, Yellow solid, m.p. 112e114 ꢃC. IR nmax (KBr):
4.1.2. General methods for preparation of 3-substituted azetidin-2-
ones
1746.6 cmꢂ1 (C]O, -lactam). 1H NMR (400 MHz, CDCl3):
b d 3.73 (s,
4.1.2.1. Staudinger reaction (method I). A solution consisting of
acetyl chloride (7.5 mmol) in anhydrous CH2Cl2 (50 mL) was added
dropwise to a stirring solution containing the appropriate imine
3H, OCH3), 3.75 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.85 (s, 3H, OCH3),
3.86 (s, 6H, OCH3), 4.86 (d, 1H, J ¼ 2.5 Hz, H3), 5.52 (d, 1H, J ¼ 2.5 Hz,
H4), 6.07 (s, 2H, ArH), 6.64 (s, 2H, ArH), 7.34e7.37 (m, 5H, ArH). 13
C

4604
N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
NMR (100 MHz, CDCl3):
58.88, 60.42, 89.76, 90.38, 93.72, 105.11, 126.83, 127.09, 128.29,
133.76, 136.13, 152.83, 159.86, 161.36, 163.83, 165.83.
d
54.44, 54.89, 55.05, 55.27, 55.48, 55.51,
4.31 (d, 1H, J ¼ 1.5 Hz, H3), 4.58e4.60 (m, 1H, H4), 6.26e6.32 (m, 1H,
ArH), 6.81e6.85 (t, 3H), 6.90 (d, 2H, J ¼ 8.5 Hz), 7.37e7.39 (m, 7H).
13C NMR (400 MHz, CDCl3)
d 54.91, 55.65, 60.57, 61.76, 63.28, 94.22,
3-Phenyl-1,4-bis(3,4,5-trimethoxyphenyl)azetidin-2-one 31. Prep-
aration was from 25 (3 mmol) as described in method I. Evaporation
of solvent yielded a brown solid residue, which was purified using
column chromatography, DCM:EtOAc (19:1). Yield 7%, Yellow
113.76, 123.98, 127.06, 127.46, 127.80, 128.57, 133.86, 153.12, 164.73.
HRMS: C27H27NO5Na requires 468.1787; found 468.1785 (Mþ þ Na).
4-(4-Methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxybenzyl)azeti-
din-2-one 37. Preparation was from 24 as described in method I.
powder, m.p. 231 ꢃC. IR nmax (KBr): 1747.2 cmꢂ1 (C]O,
b
-lactam). 1H
Yield 7.3%, Clear oil. IR nmax (NaCl film): 1751.30 cmꢂ1 (C]O,
b-
NMR (400 MHz, CDCl3):
d
3.74 (s, 6H, OCH3), 3.79 (s, 3H, OCH3), 3.83
lactam). 1H NMR (400 MHz, CDCl3)
d 3.79 (m, 6H, OCH3), 3.85 (s, 6H,
(s, 6H, OCH3), 3.87 (s, 3H, OCH3), 4.31 (d, 1H, J ¼ 2.5 Hz, H3), 4.81
OCH3), 4.22 (d, 1H, J ¼ 2 Hz), 4.36 (d, 1H, J ¼ 2 Hz), 4.88 (d, 1H,
(d, 1H, J ¼ 2.5 Hz, H4), 6.62 (d, 4H, J ¼ 5 Hz, ArH), 7.34e7.39 (m, 5H,
J ¼ 15.0 Hz), 6.41 (s, 2H), 6.96 (d, 2H, J ¼ 8.4 Hz), 7.17e7.31 (m, 11H,
ArH). 13C NMR (100 MHz, CDCl3):
d
55.60, 55.87, 60.47, 60.53, 64.10,
ArH). 13C NMR (400 MHz, CDCl3)
d 21.49, 44.75, 55.39, 56.03, 60.88,
64.48, 94.42, 102.28, 127.03, 127.58, 128.25, 128.66, 129.05, 133.26,
134.17, 134.19, 153.09, 153.59, 165.20. HRMS: C27H29NO7Na requires
502.1842; Found 502.1849 (Mþ þ Na).
62.80, 65.03, 105.31, 114.50, 125.32, 127.33, 127.69, 127.93, 128.25,
128.92, 129.06, 131.34, 135.18, 137.32, 137.87, 153.43, 159.98, 168.45.
HRMS: C26H27NO5Na requires 456.1787; found 456.1798 (Mþ þ Na).
3-Phenyl-4-(2,4,5-trimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one 32. Preparation was from 19 (4 mmol) as described
in method I. Evaporation of solvent yielded a brown solid residue,
which was purified using column chromatography, DCM. Yield 6%,
Yellow powder, m.p. 126e128 ꢃC. IR nmax (NaCl film): 1740.0 cmꢂ1
4.1.2.3. Reformatsky microwave reaction (method III). For prepara-
tion of 4-(4-ethoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)aze-
tidin-2-one 26. Zinc dust (6.9 mmol) was placed in 10 mL
microwave vial and 5 mL of anhydrous benzene was added and the
vial capped. TMCS (0.325 mL) was added. The reaction was stirred
at 25 ꢃC, 50 W, for 15 min and then heated at 100 ꢃC, 200 W for
3 min. The vessel was allowed to cool before addition of N-(4-
ethoxybenzylidene)-3,4,5-trimethoxyaniline 14 (5 mmol) and
(C]O, b d 3.74 (s, 6H, OCH3),
-lactam). 1H NMR (400 MHz, CDCl3):
3.77 (d, 6H, J ¼ 4.52 Hz, OCH3), 3.82 (s, 3H, OCH3), 3.91 (s, 3H,
OCH3), 4.40 (d,1H, J ¼ 2 Hz, H3), 5.31 (d,1H, J ¼ 2 Hz, H4), 6.58 (s,1H,
ArH), 6.66 (s, 2H, ArH), 6.85 (s, 1H, ArH), 7.27e7.35 (m, 5H, ArH). 13C
NMR (100 MHz, CDCl3):
d
55.54, 55.60, 56.02, 56.31, 57.59, 60.52,
ethyl-a-bromophenylacetate (6 mmol). Reaction was carried out at
61.11, 62.84, 94.25, 96.93, 110.05, 115.65, 126.65, 127.73, 128.23,
129.83, 133.39, 133.92, 134.01, 143.15, 149.56, 151.50, 153.03, 165.64.
HRMS: C27H29NO7Na requires 502.1842; Found 502.1835
(Mþ þ Na).
100 ꢃC, 200 W, 30 min. It was then poured over 20 mL of saturated
NH4Cl and 20 mL of 25% NH4OH. CH2Cl2 (20 mL) is used to extract
the organic layer which is further washed with 20 mL 0.1 N HCl and
20 mL of water. The organic layer is separated and dried using
anhydrous sodium sulfate. 4-(4-Ethoxyphenyl)-3-phenyl-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one 26 was obtained in 7% yield as
4-(3,4-Dimethoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one 33. Preparation was from 20 (4 mmol) as described
in method I. Evaporation of solvent yielded a brown solid residue,
which was purified using column chromatography, DCM:EtOAc
(19:1). Yield 5%, Orange solid, m.p. 106e108 ꢃC. IR nmax (NaCl film):
a
white crystalline material, m.p. 109 ꢃC. IR nmax (KBr):
1754.92 cmꢂ1 (C]O, -lactam). 1H NMR (400 MHz, CDCl3)
1.41e1.44 (t, 3H, CH2), 3.72 (s, 6H, OCH3), 3.77 (s, 3H, OCH3), 4.28
b
d
1738.9 cmꢂ1 (C]O,
b
-lactam). 1H NMR (400 MHz, CDCl3):
d
3.72 (s,
(d, 1H, J ¼ 2 Hz, H3), 4.86 (d, 1H, J ¼ 2 Hz, H4), 6.60 (s, 2H, ArH), 6.93
6H, OCH3), 3.78 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.89 (s, 3H, OCH3),
4.31 (d, 1H, J ¼ 2.5 Hz, H3), 4.86 (d, 1H, J ¼ 2.5 Hz, H4), 6.63 (s, 2H,
ArH), 6.89e6.91 (m, 2H, ArH), 6.99e7.01 (q, 1H, J ¼ 2 Hz, 6 Hz, ArH),
(d, 2H, J ¼ 8.52 Hz), 7.32e7.40 (m, 7H). 13C NMR (100 MHz, CDCl3)
d
14.35, 55.56, 60.51, 63.13, 63.43, 64.58, 94.39, 114.74, 126.88,
126.98, 127.43, 128.58, 128.68, 133.31, 134.00, 134.36, 146.43, 153.05,
158.90, 165.22. HRMS: C26H27NO5Na requires 456.1787; found
456.1800 (Mþ þ Na).
7.31e7.40 (m, 5H, ArH). 13C NMR (100 MHz, CDCl3):
d 55.58, 60.44,
63.68, 64.47, 94.34, 107.90, 111.01, 118.31, 126.94, 127.43, 128.56,
129.31, 133.27, 134.23, 148.94, 149.32, 153.01, 165.21. HRMS:
C26H27NO6Na requires 472.1736; found 472.1752 (Mþ þ Na).
4-(3-Bromo-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one 34. Preparation was from 21 as described in method
II. Yield 3.7%, Light off-white powder, m.p. 82 ꢃC. IR nmax (KBr):
4.1.2.4. General method for synthesis of 4-diphenyl substituted
lactams 38, 39 and 40. TiCl4 (2.0 mmol,1 M in CH2Cl2) was added to
solution of the appropriately substituted benzophenone
b-
a
(4.0 mmol) and trimethoxyaniline (4.0 mmol) in anhydrous toluene
(40 mL). Tri-n-butylamine (12.2 mmol) was added. The resulting
mixture was stirred overnight under nitrogen. Tri-n-butylamine
(8.4 mmol) and phenylacetyl chloride (4.4 mmol) were added
sequentially. The mixture was brought to reflux and refluxed for
8 h. The mixture was cooled to room temperature, the reaction was
quenched with water, and the mixture was transferred to a sepa-
rating funnel, diluted with ethyl acetate, washed with 1 M HCl,
saturated NaHCO3, water, and brine, dried over anhydrous sodium
sulfate and purified using flash column chromatography over silica
gel (eluent: hexane/ethyl gradient).
1751.57 cmꢂ1 (C]O, -lactam). 1H NMR (400 MHz, CDCl3)
b d 3.77 (s,
6H, OCH3), 3.81 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 4.29 (d, 1H,
J ¼ 2 Hz), 4.85 (d, 1H, J ¼ 2 Hz), 6.61 (s, 2H, ArH), 6.97 (s, 1H, ArH),
7.34e7.41 (m, 6H, ArH), 7.64 (s, 1H, ArH). 13C NMR (400 MHz, CDCl3)
d
55.65, 55.92, 60.53, 62.64, 64.65, 94.39, 111.98, 112.09, 125.68,
126.97, 127.63, 128.68, 130.47, 130.62, 133.03, 133.95, 153.16, 155.79,
164.89. HRMS: C25H24BrNO5 requires 498.0916; found 498.0926
(Mþ þ H).
4-(Naphthalen-2-yl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azeti-
din-2-one 35. Preparation was from 22 as described in method I.
Yield 5.4%, IR nmax (KBr): 1751.19 cmꢂ1 (C]O,
b
-lactam). 1H NMR
3,4,4-Triphenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one
Preparation was as above from benzophenone. Yield 0.4%, Yellow
powder, m.p. 165 ꢃC. IR nmax (KBr): 1749.57 cmꢂ1 (C]O,
-lactam).
1H NMR (400 MHz, CDCl3)
3.60 (s, 6H, OCH3), 3.79 (s, 3H, OCH3),
38.
(400 MHz, CDCl3):
1H, H4), 6.61 (s, 2H, ArH), 7.29e7.96 (m, 12H, ArH). 13C NMR
(400 MHz, CDCl3): 56.06, 60.96, 64.38, 65.06, 94.89, 122.96,
d 3.70e3.78 (9H, OCH3), 4.41 (m,1H, H3), 5.11 (m,
b
d
d
134.97, 153.59, 165.58. HRMS: C28H25NO4Na requires 462.1681;
5.18 (s, 1H, H3), 6.65 (s, 2H, ArH), 6.90e6.92 (m, 2H, ArH), 7.03e7.13
Found 462.1673 (Mþ þ Na).
(m, 8H, ArH), 7.38e7.41 (m, 3H, ArH), 7.60e7.62 (m, 2H, ArH). 13C
4-(4-Methoxystyryl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azeti-
din-2-one 36. Preparation was from 23 as described in method I.
NMR (100 MHz, CDCl3) d 55.88, 60.93, 72.44, 73.31, 95.97, 127.27,
127.46, 127.56, 127.94, 128.09, 128.50, 128.87, 129.21, 129.57, 132.79,
133.96, 134.21, 135.43, 140.56, 153.10, 166.61. HRMS: C30H27NO4Na
requires 488.1838; found 488.1856 (Mþ þ Na).
Yield 6%, Yellow oil. IR nmax (NaCl film): 1747.40 cmꢂ1 (C]O,
b-
lactam). 1H NMR (400 MHz, CDCl3)
d 3.82e3.85 (m, 12H, OCH3),

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
4605
4-(4-Methoxyphenyl)-3,4-diphenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one 39. Preparation was as above from (4-
methoxyphenyl)phenylmethanone. Yield 20%, White powder,
MCF-7 and MDA-MB-231 cells were seeded in 96-well plates,
incubated for 24 h and then treated with compounds as described
for the antiproliferative assay above. After 72 h, 20
solution (10ꢁ) was added to the ‘blank’ wells and left for 1 h to
ensure 100% death. A total of 50 L was removed from each well
and transferred to a new 96-well plate. A total of 50 L of substrate
mix from the LDH assay kit was added and the plate was incubated
in the dark at room temperature for 30 min. After this period, 50
mL of lysis
m.p. 171 ꢃC. IR nmax (KBr): 1742.06 cmꢂ1 (C]O, -lactam). 1H NMR
b
(400 MHz, CDCl3)
d
3.63 (s, 6H, OCH3), 3.80 (s, 3H, OCH3), 3.84 (s,
m
3H, OCH3), 5.16 (s,1H, H3), 6.69 (s, 2H, ArH), 6.88e6.96 (m, 4H, ArH),
7.03e7.15 (m, 7H, ArH), 7.38e7.44 (m, 1H, ArH), 7.60 (d, 2H,
m
J ¼ 8.8 Hz, ArH). 13C NMR (100 MHz, CDCl3)
d
54.94, 55.45, 60.48,
mL
72.02, 72.72, 95.50, 112.36, 113.62, 126.74, 126.97, 127.04, 127.40,
127.62, 127.70, 127.94, 128.38, 128.68, 128.87, 129.09, 129.94, 131.83,
132.44, 133.54, 133.72, 133.32, 152.64, 159.09, 166.26.
of stop solution was added to each well before reading the absor-
bance at a wavelength of 490 nm using a Dynatech MR5000 plate
reader. Percentage cell death was calculated at 10 mM.
4,4-Bis-(4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one 40. Preparation was as above from bis-(4-
methoxyphenyl)methanone. Yield 17%, White powder, m.p.
4.2.3. Tubulin polymerization assay
The effect of a selected analogue 26 on the polymerization of
purified bovine brain tubulin was determined spectrophotometri-
cally by monitoring the change in turbidity. Lyophilised tubulin
(Cytoskeleton, Denver, CO) was re-suspended in ice cold G-PEM
buffer (80 mM PIPES pH 6.9, 0.5 mM MgCl2, 1 mM EGTA, 1 mM GTP,
10.2% (v/v) glycerol) and added to wells on a half volume 96-well
165 ꢃC. IR nmax (KBr): 1742.60 cmꢂ1 (C]O,
b
-lactam). 1H NMR
(400 MHz, CDCl3)
d 3.63 (s, 6H, OCH3), 3.73 (s, 3H, OCH3), 3.80 (s,
3H, OCH3), 3.84 (s, 3H, OCH3), 5.12 (s, 1H, H3), 6.63 (d, 2H,
J ¼ 8.8 Hz), 6.68 (s, 2H, ArH), 6.90e6.95 (m, 6H, ArH), 7.09e7.11 (s,
3H, ArH), 7.51 (d, 2H, J ¼ 9.28 Hz, ArH). 13C NMR (100 MHz, CDCl3)
d
54.72, 54.94, 55.45, 60.48, 71.86, 72.31, 95.48, 112.31, 113.57,
plate containing the designated concentration of drug (10 mM) or
127.04, 127.35, 127.67, 128.79, 129.08, 129.85, 132.22, 132.50, 133.55,
152.62, 158.05, 159.00, 166.34. HRMS: C32H31NO6Na requires
548.2049; found 548.2062 (Mþ þ Na).
vehicle. Samples were mixed well and tubulin assembly was
monitored at A340nm at 30 s intervals for 60 min at 37 ꢃC in a Spec-
tramax 340PC spectrophotometer (Molecular Devices).
4.2. Biochemical evaluation of activity
4.2.4. Immunofluorescence and confocal microscopy
For immunofluorescence, MCF-7 cells were seeded at 1 ꢁ105
per well on BD falcon four well chamber glass slides (BD Biosci-
ences, San Jose, USA). Cells were treated with vehicle [1% ethanol
(v/v)], 4 [100 nM], 26 [500 nM] for 16 h. Following treatment cells
were washed gently in PBS, permabilised with PBS and 0.1% Triton-
X-100, fixed for 30 min in methanol at ꢂ20 ꢃC. Following washes in
PBS cells were blocked in 5% BSA diluted in PBST (blocking buffer).
Cells were then incubated with mouse anti-tubulin (DM1A (Merck
Chemicals Ltd)); 1:20 for 1 h at room temperature. Following
washes in PBST cells were incubated with fluorescein iso-
thiocyanate (FITC) conjugated rabbit anti-mouse (Dakocytomation,
UK); 1:200 for 1 h at room temperature. 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). Confocal images were
captured using the OLYMPUS 1X81 microscope coupled with
OLYMPUS FLUOVIEW Ver 1.5 software. All images in each experi-
ment were collected on the same day using identical parameters.
4.2.1. Antiproliferative studies
All assays were performed in triplicate for the determination of
mean values reported. Compounds were assayed as the free bases
isolated from reaction. The human breast tumour cell line MCF-7
was cultured in Eagles minimum essential medium in a 95%O2/5%
CO2 atmosphere with 10% fetal bovine serum, 2 mM
L-glutamine
and 100 g/mL penicillin/streptomycin. The medium was supple-
m
mented with 1% non-essential amino acids. MDA-MB-231 cells
were maintained in Dulbecco’s Modified Eagle’s medium (DMEM),
supplemented with 10% (v/v) Fetal bovine serum, 2 mM L-gluta-
mine and 100 mg/mL penicillin/streptomycin (complete medium).
Cells were trypsinised and seeded at a density of 2.5 ꢁ104 cells/mL
in a 96-well plate and incubated at 37 ꢃC, 95%O2/5% CO2 atmo-
sphere for 24 h. After this time they were treated with 2 mL volumes
of test compound which had been pre-prepared as stock solutions
in ethanol to furnish the concentration range of study,
1 nMe100 mM, and re-incubated for a further 72 h. Control wells
contained the equivalent volume of the vehicle ethanol (1% v/v).
The culture medium was then removed and the cells washed with
4.2.5. Determination of DNA content by flow cytometry
100
mL phosphate buffered saline (PBS) and 50
mL MTT added, to
MCF-7 cells were seeded at 5 ꢁ105 cells/mL in T25 flasks. After
24 h, cells were treated with vehicle [1% ethanol (v/v)], 4 [100 nM]
or 26 [500 nM] for 48 h. Cells were harvested by centrifugation at
800 ꢁ g for 10 min. Cell pellets were re-suspended in PBS and fixed
in 70% ethanol: PBS overnight at ꢂ20 ꢃC. Following centrifugation
cell pellets were re-suspended in PBS supplemented with 0.5 mg/
mL RNase and 0.15 mg/mL propidium iodide (PI). Following a 30-
min incubation at 37 ꢃC in the dark the PI fluorescence was
measured on a linear scale using a FACSCalibur flow cytometer
(Becton Dickinson, San Jose, CA). The amount of PI fluorescence is
directly proportional to the amount of DNA present in each cell. The
relative content of DNA indicates the distribution of a population of
cells throughout the cell cycle. For example, cells in G0G1 are
diploid and have a DNA content of 2N. Cells with the G2M phases
have a DNA content of 4N, while cells in S-phase have a DNA
content between 2N and 4N. Apoptotic cells are sub-diploid (<2N).
Data collection was gated to exclude cell debris and cell aggregates.
At least 10,000 cells were analysed per sample. All data were
recorded and analysed using the CellQuest software (Becton
Dickinson).
reach a final concentration of 1 mg/mL MTT added. Cells were
incubated for 2 h in darkness at 37 ꢃC. At this point solubilization
was begun through the addition of 200 mL DMSO and the cells
maintained at room temperature in darkness for 20 min to ensure
thorough colour diffusion before reading the absorbance. The
absorbance value of control cells (no added compound) was set to
100% cell viability and from this graphs of absorbance versus cell
density per well were prepared to assess cell viability and from
these, graphs of percentage cell viability versus concentration of
subject compound added were drawn.
4.2.2. Cytotoxicity
Cytotoxicity was determined using the CytoTox 96 non-
radioactive cytotoxicity assay by Promega following the manufac-
turer’s protocol [39]. The assay quantitatively measures lactate
dehydrogenase (LDH) a stable cytosolic enzyme that is released
upon cell lysis. Released LDH in culture supernatant is measured in
a 30 min coupled enzymatic assay, which results in the conversion
of a tetrazolium salt (INT) into a red formazan product [40].

4606
N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 46 (2011) 4595e4607
pharmacokinetics, and in vivo antitumor activity evaluation, J. Med. Chem. 45
4.3. X-ray crystallography
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The X-ray crystallography data for compound 26 was collected
on a Rigaku Saturn 724 CCD Diffractometer. A suitable crystal was
selected and mounted on a glass fiber tip and placed on the goni-
ometer head in a 123K N2 gas stream. The data set was collected
using Crystalclear-SM 1.4.0 software and 1680 diffraction images, of
0.5ꢃ per image, were recorded. Data integration, reduction and
correction for absorption and polarization effects were all per-
formed using Crystalclear-SM 1.4.0 software. Space group deter-
mination, structure solution and refinement were obtained using
Crystal structure ver. 3.8 and Bruker Shelxtl Ver. 6.14 software [41].
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Crystal Data for 26: C208H216N8O40, MW3469.89 (unit cell),
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Eur. J. Med. Chem. 45 (2010) 5752e5766.
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antiproliferative tubulin-targeting azetidin-2-ones, Bioorg. Med. Chem. 19
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Monoclinic, Space group C2/c; a ¼ 20.90(3), b ¼ 17.62(2),
c ¼ 14.02(2)Aꢀ,
b
¼ 119.95(5);
U ¼ 4474(12)(Å)3;
Z ¼ 1;
Dc ¼ 1.287 mg mꢂ3; m ¼ 0.089 mmꢂ1; Range for data collection ¼
2.12e25.00; Reflections collected 16136 , Unique Reflections 3934
[Rint ¼ 0.165]; Data/restraints/parameters 3934/0/294; Goodness-
of-fit on F2 1.194; R indices (all data) ¼ R1 ¼0.0746, wR2 ¼ 0.1678;
Final R indices [I > 2s(I)] ¼ R1 ¼0.0646, wR2 ¼ 0.1596. Cambridge
Crystallographic Data Centre (CCDC ID: 815023).
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4.4. Molecular modelling
PDB entry 1SA0 [36] was downloaded and only chains A and B
with co-crystallized N-deacetyl-N-(2-mercaptoacetyl)-colchicine
(DAMA-colchicine, 41) were retained. The X-ray co-ordinates of
compound 26 were used as input for a docking simulation carried
out using Molegro Virtual Docker [42]. A search space of 15 Å
around colchicine was created and the binding site defined by
cavity detection within Molegro. No docking template was used to
prevent search bias. A grid resolution of 0.30 Å and searching and
scoring using MolDock score was enabled for 10 runs. All other
parameters were kept as default and 10 poses were generated for
each run. The top scoring docked solution was retained for analysis
in MOE [43].
Acknowledgements
Sincere thanks to Dr. Gavin Mc Manus (School of Biochemistry
and Immunology, TCD) for his technical assistance on the confocal
microscope.
A Trinity College Dublin postgraduate research
studentship (Code 1252) and a one-year postgraduate research
award for continuing students (Code 7017) are gratefully
acknowledged. Support from the Health Research Board is also
gratefully acknowledged.
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