Synthesis and biochemical activities of antiproliferative amino acid and phosphate derivatives of microtubule-disrupting β-lactam combretastatins

Article abstract of DOI:10.1016/j.ejmech.2013.01.016

The synthesis and biochemical activities of novel water-soluble β-lactam analogues of combretastatin A-4 are described. The first series of compounds investigated, β-lactam phosphate esters 7a, 8a and 9a, exhibited potent antiproliferative activity and caused microtubule disruption in human breast carcinoma-derived MCF-7 cells. They did not inhibit tubulin polymerisation in vitro, indicating that biotransformation was necessary for their antiproliferative and tubulin binding effects in MCF-7 cells. The second series of compounds, β-lactam amino acid amides (including 10k and 11l) displayed potent antiproliferative activity in MCF-7 cells, disrupted microtubules in MCF-7 cells and also inhibited the polymerisation of tubulin in vitro. This indicates that the β-lactam amides did not require metabolic activation to have antiproliferative effects, in contrast to the phosphate series. Both series of compounds caused mitotic catastrophe and apoptosis in MCF-7 cells. Molecular modelling studies indicated potential binding conformations for the β-lactam amino acid amides 10k and 11l in the colchicine-binding site of tubulin. Due to their aqueous solubility and potent biochemical effects, these compounds are promising candidates for further development as microtubule-disrupting agents.

Full text of DOI:10.1016/j.ejmech.2013.01.016

European Journal of Medicinal Chemistry 62 (2013) 705e721
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biochemical activities of antiproliferative amino
acid and phosphate derivatives of microtubule-disrupting
b-lactam combretastatins
,
b
a
a
a
Niamh M. O’Boyle a , Lisa M. Greene , Niall O. Keely , Shu Wang , Tadhg S. Cotter ,
*
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 Biomedical Sciences Institute, 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 synthesis and biochemical activities of novel water-soluble
b
-lactam analogues of combretastatin A-
Received 11 October 2012
Received in revised form
10 January 2013
Accepted 11 January 2013
Available online 18 January 2013
4 are described. The first series of compounds investigated,
b-lactam phosphate esters 7a, 8a and 9a,
exhibited potent antiproliferative activity and caused microtubule disruption in human breast
carcinoma-derived MCF-7 cells. They did not inhibit tubulin polymerisation in vitro, indicating that
biotransformation was necessary for their antiproliferative and tubulin binding effects in MCF-7 cells.
The second series of compounds,
antiproliferative activity in MCF-7 cells, disrupted microtubules in MCF-7 cells and also inhibited the
polymerisation of tubulin in vitro. This indicates that the -lactam amides did not require metabolic
b-lactam amino acid amides (including 10k and 11l) displayed potent
Keywords:
b
Antiproliferative
Beta-lactam
Combretastatin A-4
Prodrug
activation to have antiproliferative effects, in contrast to the phosphate series. Both series of compounds
caused mitotic catastrophe and apoptosis in MCF-7 cells. Molecular modelling studies indicated potential
binding conformations for the b-lactam amino acid amides 10k and 11l in the colchicine-binding site of
Solubility
Tubulin
tubulin. Due to their aqueous solubility and potent biochemical effects, these compounds are promising
candidates for further development as microtubule-disrupting agents.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
1.1. Phosphate ester prodrugs
Solubility limitations to oral drug delivery are frequently
encountered in drug development [1,2]. There are many for-
mulation and drug design strategies that can be used to overcome
solubility issues, such as use of surfactants and co-solvents. Design
of soluble prodrugs is a routine technique used to overcome solu-
Phosphate ester prodrugs are useful for phenolic and amino-
containing poorly water-soluble drugs to enhance their aqueous sol-
ubility. Phosphate prodrugs display excellent chemical stability and
rapid bioconversion invivo to the parent drugby phosphatases present
in the intestinal brush border or in the liver [1]. Examples of com-
mercially available phosphate prodrugs include the anti-epileptic
fosphenytoin [3], the antiviral fosamprenavir and the steroid pred-
nisolone phosphate [1]. Phosphate prodrugs of tubulin-binding agents
areknown,forexamplethoseofN-acetylcolchicine (1), combretastatin
A-4 (2), combretastatin A-1 (3) and phenstatin (4, Fig. 1). Phosphate
prodrug 1a (Fig. 1) demonstrated increased water-solubility and
delayed metabolism compared to 1. Analogue 1a showed comparative
effects to 1 in vivo but did not inhibit in vitro polymerisation of tubulin,
indicating that phosphate compound 1a itself is inactive and requires
enzymatic activation in vivo [6]. Prodrug 1a was in clinical trials in the
United States for the treatment of metastatic renal cell carcinoma, but
these were halted due to cardiotoxicity problems [7].
bility problems [3]. We have recently reported a series of b-lactams
with extremely potent antiproliferative effects [4,5]. These com-
pounds are amongst the most potent analogues of combretastatin
A-4 reported to date, but their further development is limited by
lack of aqueous solubility. Hence, we designed phosphate- and
amino acid-containing derivatives of the b-lactams to improve their
pharmacokinetic profile.
Abbreviations: CA-1, combretastatin A-1; CA-4, combretastatin A-4; DIPEA,
diisopropylethylamine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; NAC, N-acetylcolchicine; PBS, phosphate buffered saline.
* Corresponding author. Tel.: þ353 1 8962798; fax: þ353 1 8962793.
E-mail address: oboyleni@tcd.ie (N.M. O’Boyle).
Combretastatin prodrugs have been extensively investigated,
including phosphate prodrugs of combretastatin A-4 (CA-4, 2,
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.016

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Fig. 1. Phosphate prodrugs of tubulin-binding agents NAC, CA-4, CA-1 and phenstatin.
Fig. 1) [8], combretastatin A-1 (CA-1, 3, Fig. 1) [9], combretastatin
B-1 [9], stilstatins 1 and 2 [10] and iodocombretastatins [11]. Due
to the sparing aqueous solubility of 2, initial formulation attempts
were unsuccessful [8]. The phosphate ester prodrug CA-4P (2a,
Fig. 1) had excellent water solubility, good stability and good cell
growth inhibitory activity, with a mean panel GI50 of 6.89 nM
(compared to 6.61 nM for 2) in the NCI screen [8]. Prodrug 2a is
rapidly dephosphorylated to 2 in vivo with a half-life of a few
minutes [12]. It is under evaluation in phase 3 trials for treatment
of anaplastic thyroid cancer and in phase 2 trials for non-small cell
lung cancer and platinum-resistant ovarian cancer [13]. Phen-
statin (4, Fig. 1) is a benzophenone-type compound structurally
similar to 2 with potent antiproliferative and tubulin-binding
activity but poor aqueous solubility. A phosphate prodrug (4a,
Fig. 1) was prepared to improve water-solubility and had equiv-
alent antiproliferative activity compared to the parent compound
4 [14].
17 nM for tyrosine derivative, compared to 13 nM for 6, across
a panel of six human cell lines). The amide prodrugs of 6 displayed
little inhibition of tubulin polymerisation in vitro, indicating
a requirement for bioactivation [17].
b
-Lactams for further development were selected from our
previous studies on the basis of their potency in antiproliferative
assays and the presence of suitable functional groups (phenolic and
amino) for modification (7e11, Fig. 3) [4,5,18]. The aim of this work
was to synthesise water-soluble
b-lactams with potent anti-
proliferative effects for pharmaceutical use and to characterise
their biochemical effects.
2. Chemistry
Phosphate esters of three potent phenolic b-lactams (7, 8 and 9)
[4,5] were prepared in two steps (Scheme 1). Benzyl-protected
compound 9c was used to achieve selective esterification of the
phenol at the C-4 position of 9. Reaction with dibenzyl phosphite
was achieved using diisopropylethylamine (DIPEA) and dimethy-
laminopyridine (DMAP) to give 7b, 8b and 9b in yields up to 74%
(Scheme 1, step a). Introduction of the dibenzyl phosphite group to
1.2. Amino acid prodrugs
Like phosphate prodrugs, amino acid prodrugs also confer the
advantage of increased aqueous solubility to poorly soluble drug
molecules. Additionally, drugs with an amino acid residue may be
substrates for peptide transporters, allowing them to be taken up
by endogenous transporters in the intestinal epithelium [1]. Of
a series of amino acid prodrugs of CA-4 amino analogue 5 (Fig. 2),
serine derivative 5a retained potent antiproliferative activity (IC50
value of 27.2 nM in colon 26 cells compared to 2.8 nM for 5) while
improving the solubility in human plasma compared to 5 from
1.4 mg/mL to 3.3 mg/mL [15]. Prodrug 5a is in clinical trials for
advanced-stage soft tissue sarcoma, solid tumours and advanced
solid tumours [16]. Amino acid prodrugs of combretastatin A-2
amino derivative 6 (Fig. 2) have also been reported, with glycine
and tyrosine amides possessing the most potent antiproliferative
effects (mean IC50 value of 20 nM for glycine derivative 6a and
the phenol of the
signals integrating for 4 protons at approximately
b
-lactam was confirmed by the appearance of
5.15 ppm in the
d
1H NMR spectra of 7b, 8b and 9b, attributable to the benzylic CH2
protons. The final step was hydrogenolysis of the benzyl protecting
groups to prepare 7a, 8a and 9a in high yields (Scheme 1, step b).
The deprotection of 7b was initially achieved in 72% yield by
treatment with bromotrimethylsilane. Debenzylation (H2/PdC) of
the
b
-lactam intermediates in high yield was successful without
-lactam ring.
decomposition of the
b
Initial investigation into potential amino acid prodrugs of 7 were
directed at preparation of amino acid esters. The Cbz-protected
alanine ester 7e and Cbz-protected phenylalanine ester 7f, along
with N-acetyl glycine ester 7g, were obtained by DCC/DMAP cou-
pling of the phenolic moiety of 7 with the appropriate amino acids
(Scheme 2). Removal of the Cbz protecting group from 7e afforded
the alanine ester 7h. However, isolation of the corresponding
phenylalanine and glycine esters proved difficult due to very rapid
hydrolysis to the phenol 7. The preparation of a number of addi-
tional Cbz protected amino acid esters of 7 was investigated, but in
each case the deprotected ester was rapidly hydrolysed. These es-
ters are not suitable for further prodrug development. The acetate
and benzoate esters 7i and 7j were prepared by Staudinger reaction
of corresponding imines 12a and 12b with phenylacetyl chloride. A
basic ether derivative 7l was also synthesised (Scheme 2). The ether
linkage would not be susceptible to hydrolysis in vivo while the
basic side chain may improve the water solubility of the derivative.
Reaction of the imine 12c with 1,2-dibromoethane afforded the
bromide 12d, which was then treated with phenylacetyl chloride to
Fig. 2. Amino-acid prodrugs of amino analogues of CA-4 and CA-2.
produce the b-lactam product 7k. Reaction of the bromide 7k with

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
707
Fig. 3. Antiproliferative b-lactams chosen for prodrug development. One enantiomer is illustrated.
methylamine afforded the required product 7l in low yield (30%) as
the basic reaction conditions resulted in extensive cleavage of the
(compounds 10d, 11f and 11g). The required lysine precursor was
available with both amino groups protected with the Fmoc group.
Products were obtained in yields of over 44% with the exception of
lysine-analogue 10c (6%).
b
-lactam ring.
In order to prepare further amino acid
b-lactams amides,
amino-containing azetidinones 10 and 11 [4] were coupled to
series of protected -amino acids. DCC, with DMAP and
Initially the Fmoc-group was cleaved using the base piperidine
[19,20]. When 5% piperidine in dichloromethane [21] was used to
a
L
HOBt$H2O, was used to couple either 10 or 11 to the carboxylate
moiety of amino acids to afford the products 10aef and 11aeh
(Scheme 3). Use of polymer-supported DCC gave higher yields
and led to more facile isolation of the product. This resin contains
1% cross-linked poly(styrene-co-divinylbenzene) with a typical
loading of 1.3 mmol/g of carbodiimide. The amino group of the
amino acid was protected during the coupling reaction with either
Fmoc or Cbz. Selected amino acid precursors (serine and tyrosine)
also required benzyl protection of their phenolic groups
remove the Fmoc-group from amino acid-b-lactam conjugates,
difficulties in purification of the product arose. A second method
using tetrabutylammonium fluoride (TBAF, 2 equiv.) and a thiol (1-
octanethiol, 10 equiv.) to scavenge the liberated dibenzofulvene
was investigated. This method removes the Fmoc group from
a variety of amino acid conjugates in up to 100% yield within 1 min
[22]. It was successfully applied to remove the Fmoc group from the
b-lactam derivatives 10aed and 11aeg (Scheme 3), giving easily
isolated products 10gej and 11ieo in high yield, in less than 10 min
Scheme 1. Preparation of
b-lactam phosphate esters 7a, 8a, 9a. Reagents and conditions: (a) dibenzyl phosphite, DIPEA, DMAP, CCl4, CH3CN (b) Pd/C, H2, EtOH:EtOAc (1:1)
(c) (i) BrSi(CH3)3, CH2Cl2 (ii) Na2S2O3. (Bn ¼ eCH2C6H5). *Only one enantiomer is illustrated.

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N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
Scheme 2. Synthesis of
b
-lactams 7ee7l. Reagents and conditions: (a) DCC, DMAP (b) H2, Pd, EtOh, EtOAc (c) Et3N, C6H5CH2COCl, CH2Cl2 (d) BrCH2CH2Br, (nBu)4NHSO4, NaOH; (e)
Et3N, C6H5CH2COCl, CH2Cl2; (f) MeNH2.
as monitored by TLC. Cbz- and Bn-groups were removed by
hydrogenolysis (H2/PdC) from intermediates 10e, 10f, 10j, 11h, 11n
and 11o to afford compounds 10k, 10l, 10m, 11p, 11q and 11r
respectively (Scheme 4).
aqueous solubility, while phosphate derivatives 7a, 8a and 9a were
soluble at 0.3 mg/mL, 0.14 mg/mL and 0.45 mg/mL respectively in
water, greater than the recommended minimum solubility of
0.05mg/mL whichisdesirableforpotentialdrugcandidates[23]. The
increase in solubility is reflective of the increase in hydrophilicity of
the molecules, as indicated by their lower cLogP values (Table 1). All
3. Solubility
amino acid
solubility compared to parent
b
-lactam amides assessed showed increased aqueous
-lactams 10 and 11 (Tables 2 and 3).
Selected compounds 9, 9a, 10, 10g, 11 and 11l were shown to be
stable in the pH range 4e9 and in human plasma, with half-lives
greater than 24 h (data not shown). The solubility of the novel b-
lactam derivatives was measured and compared to that of the parent
compounds. The phosphate derivatives showed increased aqueous
solubility compared to the insoluble parent compounds (Table 1).
b
The highest solubility was obtained with the alanine derivative 11j
(0.37 mg/mL), which again is favourable compared to the minimum
desired value of 0.05 mg/mL. The observed increase in solubility due
to the phosphate or amino acid group improves the pharmacokinetic
profile of the parent compound. Amide 7g and amine 7l were not
useful as water soluble compounds.
The phenolic b-lactams 7, 8 and 9 did not display any detectable

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
709
Scheme 3. Preparation of b-lactams 10aej and 11aeo. Reagents and conditions: (a) N-protected L-amino acid, anhydrous DMF, DCC (polymer supported), HOBt$H2O, stirring, rt; (b)
TBAF, 1-octanethiol, CH2Cl2, rt.
4. Biochemical results and discussion
approximately the same trend as their parent compounds, as 8 and
9 are more potent than 7, with 8a and 9a more potent than 7a.
4.1. Antiproliferative effects of phosphate ester and amino acid
The antiproliferative activity of b-lactam amino acid conjugates
amide
b
-lactams
10gei, 10kem, 11iem and 11per was assessed (Tables 2 and 3).
Derivatives of compound 10 were extremely potent (Table 2), with
alanine derivative 10h, phenylalanine derivative 10k and valine
derivative 10l showing activity greater than or equal to that of the
parent amino compound 10 (IC50 values of 27 nM, 35 nM and 51 nM
respectively compared to 51 nM for 10). Valine derivative 11l (IC50
value of 460 nM) was the most potent derivative of 11. Alanine (11j),
serine (11q) and tyrosine (11r) derivatives also had IC50 values in
the nanomolar range comparable to that of the parent compound
11 (760 nM, 780 nM and 740 nM respectively, 650 nM for 11).
Stilbene 2 was insoluble in water and the development of 2a
represented the most successful prodrug of 2; it retained the ac-
tivity of the parent compound, had improved solubility and good
stability [8]. Phosphate esters of three phenolic
b-lactam com-
pounds with potent IC50 values in antiproliferative assays (7, 8 and
9) were prepared to investigate if improvements in aqueous solu-
bility could be obtained without compromising antiproliferative
activity. The antiproliferative activities of parent compounds were
also previously demonstrated in a variety of cell types in the NCI
cell line screen [4,5]. The antiproliferative activity of all compounds
was assessed using human breast carcinoma MCF-7 cells.
4.2. Effects on tubulin polymerisation
The
b
-lactam phosphate esters 7a, 8a and 9a had IC50 values of
Inhibition of tubulin polymerisation for existing phosphate
prodrugs 1a, 2a and 4a differs, with intact 1a [6] and 2a [24] dis-
playing no intrinsic tubulin binding affinity while 4a displays 40%
of the activity of the parent compound 4 [14]. The effects of 5a on
71 nM, 20 nM and 22 nM respectively and had slightly decreased
potency compared to the corresponding parent compounds
(Table 1). The IC50 values for the phosphate esters follow

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N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
Scheme 4. Preparation of b-lactams 10ke10m and 11pe11r. Reagents and conditions: (a) H2, Pd/C, EtOAc:EtOH 1:1, rt.
in vitro tubulin polymerisation have not been reported, but it is
known that amino acid amide derivatives of 6, including 6a, do not
mAb demonstrated a well organised microtubular network in
control cells (Fig. 6, A þ E and Fig. 7, A þ D). Exposure to any of the
inhibit the polymerisation of tubulin at concentrations up to 40
[17]. It has previously been demonstrated that compounds 7, 8 and
9 inhibit the polymerisation of tubulin [4,5]. Representative -lac-
mM
four b-lactam conjugates 8a, 9a, 10k and 11l for 16 h led to a com-
plete loss of microtubule formation consistent with depolymerised
microtubules (Figs. 6 and 7). Additionally, cells treated with -lac-
b
b
tams 7a and 9a (phosphate esters) and 10k and 11l (amino acid
amides) were selected for in vitro tubulin polymerisation studies.
tams 8a, 9a, 10k and 11l contained multiple micronuclei e a phe-
nomenon described as mitotic catastrophe. Mitotic catastrophe is
a type of programmed cell death in response to DNA damage,
characterised by multinucleated cells [25]. We have previously
Phosphate
b
-lactam esters 7a and 9a did not inhibit the in vitro
M (Fig. 4). Given
polymerisation of tubulin at a concentration of 10
m
their potent antiproliferative effects in MCF-7 cells, this result in-
dicates that bioconversion is necessary for the biochemical effects
of the phosphate compounds. In contrast, the amino acid b-lactam
conjugates 10k and 11l inhibited in vitro polymerisation of tubulin
in a concentration dependent manner (shown for 11l in Fig. 5), with
IC50 values of 2.4 mM and 5.2 mM respectively. This indicates that
bioconversion is not necessary for these compounds, although
in vivo cleavage of the amide linkage is extremely likely to occur. For
example, it has been reported that aminopeptidases on the surface
of erythrocytes catalyse the cleavage of 5a to 5 [21].
noted mitotic catastrophe for related b-lactam derivatives [26]. The
findings are in agreement with previously published studies, where
2 induced mitotic catastrophe in non-small cell lung cancer cells
[27,28] and 2a in chronic lymphocytic leukaemia cells [29]. Mitotic
catastrophe has also been demonstrated for 2 and related de-
rivatives in human endothelial cells (HUVEC), human lung carci-
noma cells (H460) [30] and human breast cancer cells (MCF-7) [26].
The confocal imaging results confirm that both the phosphate and
amino acid
b-lactam conjugates are targeting tubulin.
4.4. Analysis of DNA content by flow cytometry
4.3. Immunofluorescence studies
We next examined the effects of 2, parent
b-lactams 7, 8 and 9
In addition to in vitro tubulin polymerisation studies, we
and -lactam derivatives 7a, 8a, 9a, 10k and 11l on the cell cycle of
b
investigated alterations in the microtubule network induced by
lactams 8a, 9a, 10k and 11l in MCF-7 cell culture by confocal mi-
croscopy. Confocal analysis of MCF-7 cells stained with -tubulin
b
-
MCF-7 cells by flow cytometric analysis of propidium iodide stained
cells (Fig. 8). In this whole-cell assay, both phosphate and amino
acid b-lactam prodrugs caused increases in the percentage of cells
a

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
711
Table 1
structurally related, conformationally constrained analogues of 2
[30e32]. Molecular modelling studies were performed to inves-
Antiproliferative activities and physiochemical properties of
esters.
b-lactam phosphate
tigate potential interactions for the intact
b-lactam amino acid
amides 10k and 11l, considering that they inhibited tubulin poly-
merisation without chemical or enzymatic modification. The
reported X-ray structure of tubulin cocrystallized with a colchicine
derivative, N-deacetyl-N-(2-mercaptoacetyl)colchicine (DAMA-
IC50 MCF-7
(nM)a
cLogPb
Solubility
(mg/mL)c
colchicine, PDB entry 1SA0) was used for the docking study [33]. b-
Lactams were isolated as racemic mixtures and the presence of two
enantiomers has been demonstrated [4]. Molecular modelling
studies were carried out with both enantiomers and results of the
R1¼
R2¼
7
7a
8
H
C6H5
C6H5
9.6 ꢀ 2.6 [4]
71 ꢀ 35
3.14
2.09
2.79
0d
best fitting 3S*,4R* enantiomer are illustrated. The b-lactam phos-
P(O)(OH)2
H
0.30
phate esters 7a, 8a and 9a had no effect in the in vitro tubulin
polymerisation assay, indicating that hydrolysis is required for their
effects, and therefore no molecular modelling was investigated for
those compounds.
7.0 ꢀ 2.0 [5]
0d
8a
P(O)(OH)2
20 ꢀ 8
1.74
0.45
Molecular docking studies of parent compounds 10 and 11
(Figs. 9A and 10A) predicted similar interactions to those previously
reported to be of importance for binding at the colchicine site,
including important interactions with Cys241 and Val318 [33,34].
Additionally, there is direct bond between compound 11 and
the Met259 residue. Both compounds are orientated in a similar
manner to DAMA-colchicine, with the 3,4,5-trimethoxyaryl A ring
and 4-methoxyaryl B ring occupying the same position in the
binding pocket (Figs. S1 and S3, Supplementary information).
Comparison of the docked structures of 10 and 10k indicated
9
9a
H
p-C6H4OH
p-C6H4OH
0.8 ꢀ 0.4 [4]
22 ꢀ 11
2.47
1.43
0d
0.14
P(O)(OH)2
a
IC50 values are half maximal inhibitory concentrations required to inhibit the
growth of MCF-7. Values represent the mean ꢀ S.E.M for at least three independent
experiments performed in triplicate. The IC50 value obtained for 2 in this assay was
5 nM for MCF-7 and is in good agreement with the reported values for 2 using the
MTT assay on human MCF-7 breast cancer cell line [31,39,40].
b
cLogP value calculated using ChemDraw Ultra and refers to cLogPoct/water
Solubility value taken as the maximum value from n ¼ 3.
Solubility is below the level of detection of the assay.
.
c
d
that the core b-lactam, A and B rings are orientated in the same way
in the binding pocket (Fig. 9A and B, and Figs. S1 and S2,
Supplementary information). All but one of the 24 binding con-
tacts for 10 are also predicted for 10k, the exception being an
interaction with Asn249. The phenylalanine substituent on ring C
extends further and makes numerous additional contacts, for
example with Lys254 and Lys352 (Fig. 9B). We have previously
shown that bulky substituents directly attached to ring C are det-
in the sub-G1 and G2/M phases, indicative of apoptosis and G2/M
arrest respectively. There were no significant differences in the
percentage of cells in sub-G1 and G2/M phases between the parent
b
-lactams and the derivatives.
rimental to activity [4]. In this series of b-lactams the potency of
5. Molecular modelling studies
10k in MCF-7 cells indicates that the linear chain linking the two
aromatic rings (ring C to the aromatic ring of phenylalanine) is
essential to maintain potent in vitro activity.
The docked structures of 11 and 11l are illustrated in Fig. 10.
Additional binding interactions associated with the valine moiety
are predicted for 11l, namely with Gln11, Gly143, Ser178, Glu183,
The tubulin binding and immunofluorescence studies clearly
demonstrate that tubulin is the target of the new
rivatives. Based on structural similarities between 2 and the
-lactams reported in this study, we propose that they interact with
b-lactam de-
b
the colchicine binding site of tubulin as demonstrated for 2 and
Table 2
Antiproliferative activity and solubility of
b-lactam amino acid amides derived from 10.
Structure
Amino
acid
IC50 (nM)a
cLogPb
Solubility (mg/mL)c
R¼
10g
10h
10i
10k
10l
H
Gly
Ala
Lys
Phe
Val
Ser
91 ꢀ 10
35 ꢀ 20
1.99
2.30
2.00
3.71
3.22
1.13
2.65
0.06
nd
CH3
(CH2)4NH2
CH2C6H5
CH(CH3)2
CH2OH
Parent
690 ꢀ 700
27 ꢀ 0.3
0d
0.14
0.004
nd
50 ꢀ 20
10m
10
860 ꢀ 300
50 ꢀ 20 [4]
0
compound
a
IC50 values are half maximal inhibitory concentrations required to inhibit the growth of MCF-7. Values represent the mean ꢀ S.E.M for at least three independent ex-
periments performed in triplicate.
b
cLogP value calculated using ChemDraw Ultra and refers to cLogPoct/water
.
c
Solubility value taken as the maximum value from n ¼ 3.
d
Solubility is below the level of detection of the assay; nd ¼ not determined.

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N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
b-lactam amino acid amides derived from 11.
Table 3
Antiproliferative activity and solubility of
Structure
Amino acid
IC50 MCF-7 (nM)a
cLogPb
Solubility (mg/mL)c
R¼
11i
11j
11k
11l
11m
11p
11q
11r
11
H
Gly
Ala
Phe
Val
Lys
Ile
1100 ꢀ 900
760 ꢀ 230
1130 ꢀ 500
460 ꢀ 200
9800 ꢀ 2000
1100 ꢀ 900
780 ꢀ 200
740 ꢀ 60
1.55
1.86
3.28
2.79
1.56
3.32
0.70
2.61
3.00
nd
CH3
0.37
CH2C6H5
CH(CH3)2
(CH2)4NH2
CH(CH3)C2H5
CH2OH
0d
0.11
nd
0.11
0.20
nd
Ser
Tyr
CH2C6H4OH
Parent compound (Fig. 3)
650 ꢀ 10 [4]
0d
5a
Ser
37e
0.87
0.11f
a
IC50 values are half maximal inhibitory concentrations required to inhibit the growth of MCF-7. Values represent the mean ꢀ S.E.M for at least three independent ex-
periments performed in triplicate.
b
cLogP value calculated using ChemDraw Ultra and refers to cLogPoct/water
.
c
d
e
f
Solubility value taken as the maximum value from n ¼ 3.
Solubility is below the level of detection of the assay; nd ¼ not determined.
In murine 26 colon cells [21].
Human plasma, pH 7.2e7.5 [21].
Tyr224 and Gln247. The predicted docking position for both the
core -lactam and A rings is similar for these two structures.
However, the positioning of the B and C rings of both compounds is
different (Figs. S3 and S4, Supplementary information). We have
previously noted this ‘flip’ in relative orientation of the B and C
useful information for the development of future antiproliferative
agents that interact with the colchicine binding site of tubulin,
possibly exploiting the additional binding interactions predicted for
10k and 11l.
b
rings for b-lactams when larger substituents are present on ring B
6. Discussion
[26]. The orientation predicted for 11l is consistent with our pre-
vious experience that bulky substitution to the B ring forces
a change in molecular orientation within the colchicine binding
site. This is not necessarily associated with a decrease in anti-
proliferative activity, as 11l is more potent in MCF-7 cells than 11.
However, we must once again emphasise that hydrolysis to yield
the parent amine compounds 10 and 11 is predicted to occur in vivo
prior to interaction with tubulin. These in silico studies provide
Our previously reported
b-lactams are amongst the most
potent analogues of 2 known, with selected compounds showing
sub-nanomolar antiproliferative activity (e.g. IC50 for 9 in MCF-7
cells is 0.8 nM). However, these compounds are not sufficiently
water soluble for further development and a prodrug strategy
was devised to improve this physiochemical characteristic.
Potentially useful prodrugs were synthesised including three
Fig. 4. Effects of compounds 7, 7a, 9 and 9a on the inhibition of tubulin polymer-
isation. Effects of compounds 7, 7a, 9 and 9a (10 mM) on in vitro tubulin polymerisation.
Fig. 5. Effects of compound 11l on the inhibition of tubulin polymerisation. Dosee
response effect of compound 11l on in vitro tubulin polymerisation. Purified bovine
tubulin and GTP were mixed in a 96-well plate. The reaction was started by warming
the solution from 4 ꢁC to 37 ꢁC. Ethanol (1%v/v) was used as a vehicle control. The
effect on tubulin assembly was monitored in a Spectramax 340PC spectrophotometer
at 340 nm at 30 s intervals for 60 min at 37 ꢁC. The graph shows one representative
experiment. Each experiment was performed in triplicate.
Purified bovine tubulin and GTP were mixed in a 96-well plate. The reaction was
started by warming the solution from 4 ꢁC to 37 ꢁC. Ethanol (1%v/v) was used as
a vehicle control. The effect on tubulin assembly was monitored in a Spectramax 340PC
spectrophotometer at 340 nm at 30 s intervals for 60 min at 37 ꢁC. The graph shows
one representative experiment. Each experiment was performed in triplicate.

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
713
Fig. 6. Compound 2 and
(1% ethanol (v/v), A þ E), or compound 2 (B þ F), 8a (C þ G) or 9a (D þ H) (all 100 nM) for 16 h. Cells were fixed in methanol and stained with
counterstained with DAPI (blue). Images were captured by confocal microscopy coupled with OLYMPUS FLUOVIEW software. Bar equal to 40 m. Representative confocal mi-
crographs 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.)
b
-lactams 8a and 9a depolymerise the microtubule network of MCF-7 cells resulting in mitotic catastrophe. MCF-7 cells were treated with vehicle control
a-tubulin mAbs (green) and
m
b
-lactam phosphate esters, two
b
-lactam esters and eighteen
The primary in vitro degradation pathway of concern for phos-
phates is hydrolysis, and the electronic environment and substi-
tution on the prodrug moiety may have profound effects on
stability [35]. Two previously reported prodrugs of 2 were found to
amino acid amides. A number of novel compounds exhibited
greatly improved aqueous solubility compared to the parent
compounds.
Fig. 7. Compound 2 and
(1% ethanol (v/v), A þ D), compound 10k (B þ E; 100 nM) or 11l (C þ F; 1
with DAPI (blue). Images were captured by confocal microscopy coupled with OLYMPUS FLUOVIEW software. Bar equal to 20
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.)
b
-lactams 10k and 11l depolymerise the microtubule network of MCF-7 cells resulting in mitotic catastrophe. MCF-7 cells were treated with vehicle control
M) for 16 h. Cells were fixed in methanol and stained with -tubulin mAbs (green) and counterstained
m. Representative confocal micrographs of three
m
a
m

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N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
of the b-lactam derivatives to the parent compounds is possible. Both
series of compounds had the same potent biochemical effects in
these assays. However, we noted a significant difference between the
phosphate and amino acid analogues in the tubulin polymerisation
assay, which uses isolated, purified tubulin and in which metabolic
enzymes are not present. In this assay, the phosphate prodrugs did
not inhibit tubulin polymerisation. The amino acid b-lactam conju-
gates, taking 10k and 11l as representative examples, inhibited
tubulin polymerisation in vitro in the absence of enzymatic action.
These results indicate that the phosphate derivatives are true pro-
drugs requiring enzymatic activation, while the intact amino acid
derivatives are microtubule poisons in their own right. However, due
to the widespread presence of aminopeptidases in the blood, it is
likely that they may be hydrolysed invivo before reaching their target
site of action. Regardless of this, we have demonstrated they can act
as microtubule depolymerisers in either form.
7. Conclusion
We describe the synthesis and biochemical effects of novel se-
ries of antiproliferative phosphate ester and amino acid amide b-
lactam derivatives, designed to overcome the pharmacokinetic
hurdle of poor aqueous solubility observed for previously reported
compounds [4]. The novel compounds displayed increased aqueous
solubility, which is an advantage for drug delivery. The strategic
modification of previously described b-lactam CA-4 derivatives by
inclusion of phosphate or amino acid moieties did not adversely
influence their antiproliferative and tubulin targeting properties, as
determined by antiproliferative assays, cell cycle assays and anal-
ysis of the microtubular network by confocal microscopy. Novel
derivatives had nanomolar antiproliferative IC50 values in MCF-7
cells and disrupted the microtubule network. The potent activity
Fig. 8. Effects of 2 and
centages of cells in (A) sub-G1 and (B) G2/M phase after treatment with
b
-lactams 7, 8, 9, 7a, 8a, 9a, 10k, 11l on the cell cycle. Per-
-lactams.
MCF-7 cells were untreated (U), treated with vehicle control (V, 1% ethanol (v/v)), or
compound 2, 7, 8, 9, 7a, 8a, 9a, 10k (100 nM) or 11l (1 M) for 48 h. Values represent
b
m
and microtubule disrupting effects of the
warrants further investigations.
b-lactam derivatives
the mean ꢀ SEM for at least three independent experiments. Using one-way anova
followed by Bonferroni’s multiple comparison test, there is no statistical difference
between all b-lactam compounds for percentage of cells in sub-G1. G2/M p < 0.001 for
all compounds compared to vehicle with no statistical difference between parent
compounds and derivatives.
8. Experimental methods
All reagents were commercially available and were used
without further purification unless otherwise indicated. IR spectra
were recorded as thin films on NaCl plates or as KBr discs on
a PerkineElmer Paragon 100 FT-IR spectrometer. 1H and 13C NMR
spectra were obtained on a Bruker Avance DPX 400 instrument at
20 ꢁC, 400.13 MHz for 1H spectra, 100.61 MHz for 13C spectra, in
CDCl3, CD3OD or DMSO-d6 (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 (HRMS) for all final target compounds were
obtained on a Micromass Time of Flight mass spectrometer
equipped with electrospray ionisation (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. Thin layer chromatography was performed using
Merck Silica gel 60 TLC aluminium sheets with fluorescent indi-
cator visualising with UV light at 254 nm. Flash chromatography
was carried out using standard silica gel 60 (230e400 mesh)
obtained from Merck. Analytical high-performance liquid chro-
matography (HPLC) to determine the purity of the final com-
pounds was performed using a Waters 2487 Dual Wavelength
Absorbance detector, a Waters 1525 binary HPLC pump, a Waters
In-Line Degasser AF and a Waters 717plus Autosampler. The col-
be stable in aqueous solution [36]. When the stability of phosphate
ester prodrugs 2a and 3a was evaluated in murine plasma, there
was little evidence of dephosphorylation even after 3 h. In contrast,
2a and 3a were rapidly dephosphorylated in tumour (MAC29) and
liver preparations [37]. It is expected that rapid in vivo dephos-
phorylation would occur for the
in a manner similar to that documented for 2a [12]. In vitro, a se-
lection of representative -lactam phosphate esters were demon-
strated to be stable in the pH range 4e9 and in plasma, with half-
lives greater than 24 h. The -lactam amino acid amides are also
b-lactam phosphates 7a, 8a and 9a
b
b
stable at three pH values (4, 7.4 and 9). These amides are expected
to be hydrolysed by peptidases in vivo in a similar fashion to 5a [21].
Further detailed stability studies on the
and amino acid amides are ongoing.
b-lactam phosphate esters
Biochemical profiling of the
b-lactam derivatives confirmed
their antiproliferative and tubulin-targeting effects. For both series
of compounds, antiproliferative activity was noted in the nano-
molar concentration range, with 8a, 9a, 7a and 10k having the
lowest IC50 values (20, 22, 71 and 27 nM respectively). Immuno-
fluorescence microscopy studies showed disruption of the micro-
tubule network and mitotic catastrophe for both the phosphate and
amino acid series of analogues. In cell-cycle analysis the percentage
of cells in sub-G1 (indicative of cell death) was significantly
increased compared to the control.
umn used was
a Varian Pursuit XRs C18 reverse phase
150 ꢂ 4.6 mm chromatography column. Samples were detected
using a wavelength of 254 nm. All samples were analysed using
acetonitrile (70%):water (30%) over 10 min and a flow rate of 1 mL/
min. The purity of the tested compounds was ꢃ95%. Polymer-
In the antiproliferative, immunofluorescence and cell cycle anal-
ysis assays, whole cells are used and it is expected that bioconversion

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
715
Fig. 9. 2D representation of the binding interactions of (A) 10 and (B) 10k with the colchicine binding site of tubulin.
supported DCC was obtained from (BiotageÒ). Syntheses of imine
12c [18] and -lactams 7, 8, 9, 10 and 11 [4,5] were achieved as
8.1. Chemical synthesis
b
previously reported. Experimental characterisation of compounds
8a, 8b, 9a, 9b, 10aed, 10fej, 10l, 10m, 11aec, 11eek, 11mer is
contained in the Supplementary information.
8.1.1. General procedure for synthesis of imines
The appropriate amine (10 mmol) was refluxed with the
appropriate aldehyde (10 mmol) in ethanol (50 mL) for 3 h. The

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N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
Fig. 10. 2D representation of the binding interactions of (A) 11 and (B) 11l with the colchicine binding site of tubulin.

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
717
reaction mixture was reduced in vacuo, and the resulting solution
was allowed to stand until solid product crystallised from solution.
The resulting imine was recrystallised from ethanol.
The product was obtained as a brown oil in 93% yield; IR (KBr disk)
nmax: 1747 cmꢄ1 -lactam eC]O); 1H NMR (400 MHz, DMSO-d6)
(b
d
3.59 (s, 3H, OCH3), 3.67 (s, 6H, 2ꢂ OCH3), 3.77 (s, 3H, OCH3), 4.43
(d, 1H, J ¼ 2.5 Hz, H3), 5.24 (d, 1H, J ¼ 2.5 Hz, H4), 6.63 (s, 2H, ArH),
7.09 (d, 1H, ArH, J ¼ 8.5 Hz), 7.35 (m, 1H, ArH), 7.36e7.42 (m, 5H,
8.1.1.1. 2-Methoxy-5-(((3,4,5-trimethoxyphenyl)imino)methyl)phenyl
acetate (12a). Compound (12a) was obtained from 3,4,5-
trimethoxyaniline and 5-formyl-2-methoxyphenyl acetate as
ArH), 7.51 (s, 1H, ArH); 13C NMR (100 MHz, DMSO-d6)
d 56.1 (OCH3),
56.2 (OCH3), 60.5 (OCH3), 62.2 (C3), 64.0 (C4), 95.5 (CH, Ar), 113.7
(CH, Ar), 119.6 (CH, Ar), 122.9 (CH, Ar), 127.9 (CH, Ar), 128.0 (CH, Ar),
128.2 (CH, Ar),128.7 (CH, Ar),129.4 (CH, Ar),129.7 (CH, Ar),133.5 (C,
Ar), 134.3 (C, Ar), 135.2 (C, Ar), 150.9 (C, Ar), 151.0 (C, Ar), 153.6 (C,
Ar), 165.8 (C]O); HRMS: calculated for C25H26NNaO9PS: 538.1243;
found 538.1252 [M þ Na]þ.
a white powder (64% yield); mp 120 ꢁC; IR (KBr disk) nmax
:
1608 cmꢄ1 (C]N); 1H NMR (400 MHz, CDCl3)
d 2.22 (s, 3H, CH3),
3.85 (m, 12H, OCH3), 6.51 (s, 2H, ArH), 7.08 (d, 1H, J ¼ 4.5 Hz, ArH),
7.28 (s, 1H, ArH), 7.79 (m, 1H, ArH), 8.39 (s, 1H, C]N); HRMS: cal-
culated for C19H22NO6: 360.1449; found 360.1430 [M þ H]þ.
8.1.1.2. 2-Methoxy-5-(((3,4,5-trimethoxyphenyl)imino)methyl)phe-
nyl benzoate (12b). Compound (12b) was obtained from 3,4,5-
trimethoxyaniline and 5-formyl-2-methoxyphenyl benzoate as
8.1.4. 2-Methoxy-5-(4-oxo-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-yl)phenyl 2-(((benzyloxy)carbonyl)amino)propanoate
(7e)
peach coloured crystals (75% yield); mp 120 ꢁC; IR (KBr disk) nmax
:
N-Benzyloxycarbonyl-
and DMAP (0.23 mmol) were stirred in anhydrous DCM (5 mL) at
ꢁC. A solution of the
-lactam 7 (0.23 mmol) in anhydrous DCM
L-alanine (0.23 mmol), DCC (0.23 mmol)
1611 cmꢄ1 (C]N); 1H NMR (400 MHz, CDCl3)
d 3.90 (m,12H, OCH3),
7.11 (s, 1H, ArH), 7.28 (s, 2H, ArH), 7.55 (m, 2H, ArH), 7.67
(m,1H, ArH), 7.83 (m, 2H, ArH), 8.24 (d, 2H, J ¼ 1 Hz, ArH), 8.43 (s,
1H, C]N); HRMS: calculated for C24H24NO6: 422.1604; found
422.1582[M þ H]þ.
0
b
(5 mL) was added dropwise over 5 min. The reaction was stirred for
16 h under a nitrogen atmosphere at ambient temperature. Reac-
tion was monitored via TLC. After 24 h, dichloromethane (50 mL)
was added and the mixture was filtered. The solvent was removed
by washing with water (5 ꢂ 50 mL). The organic solvent containing
the product was evaporated under reduced pressure. The product
was isolated by flash column chromatography (dichlor-
omethane:methanol gradient), to afford the product 7e (88%). IR
8.1.2. General procedure for synthesis of 7b, 8b and 9b
Carbon tetrachloride (85 mmol) was added to a solution of
appropriate phenol (17 mmol) in acetonitrile (100 mL) at 0 ꢁC. The
resulting solution was stirred for 10 min prior to addition of dii-
sopropylethylamine (35 mmol) and DMAP (1.7 mmol). Then,
dibenzyl phosphite (24.5 mmol) was added dropwise. When the
reaction was complete as indicated by TLC, KH2PO4 (aq., 0.5 M) was
added and the mixture was allowed to warm to room temperature.
An ethyl acetate extract (3 ꢂ 50 mL) was washed with saturated
NaCl (100 mL) followed by water (100 mL) and dried over anhy-
drous Na2SO4. The solvent was evaporated under reduced pressure
and the product isolated by flash column chromatography (hex-
ane:ethyl acetate gradient, 88:12 to 50:50).
(NaCl film) nmax: 1752.9 (
CDCl3)
4.30e4.32 (m, 1H, H3), 4.69 (1H, q, J ¼ 7 Hz, CH), 4.83e4.87 (m,
0.5H, CH, H4), 5.15e5.44 (m, 0.5H, CH, H4), 5.39e5.44 (2H, m,
OCH2), 6.61e6.64 (2H, m, ArH), 6.88e6.95 (m, 1H, ArH), 7.01e7.03
(m, 1H, ArH), 7.16e7.17 (m, 1H, ArH), 7.29e7.41 (m, 10H, ArH).13C
b
-lactam eC]O); 1H NMR (400 MHz,
1.62 (3H, d, J ¼ 7 Hz, CH3), 3.75e3.92 (m, 12H, 4ꢂ OCH3),
d
NMR (100 MHz, CDCl3)
d 18.7 (CH3), 18.8 (CH3), 49.7 (C), 56.1
(OCH3), 60.9 (OCH3), 63.4 (C3), 63.8 (C3), 64.9 (C4), 65.0 (CH2), 67.1
(CH2), 94.8 (CH, Ar), 94.9 (CH, Ar), 111.1 (CH, Ar), 112.0 (CH, Ar),113.1
(CH, Ar), 113.1 (CH, Ar), 117.8 (CH, Ar), 120.8 (CH, Ar), 124.6 (CH, Ar),
124.6 (CH, Ar), 127.5 (CH, Ar), 127.9 (CH, Ar), 128.0 (CH, Ar), 128.1
(CH, Ar), 128.2 (CH, Ar), 128.6 (CH, Ar), 129.0 (CH, Ar), 129.1 (CH, Ar),
129.9 (CH, Ar), 130.5 (CH, Ar), 133.6 (C, Ar), 133.7 (C, Ar), 134.5 (C,
Ar), 134.5 (C, Ar), 134.8 (C, Ar), 136.2 (C, Ar), 139.9 (C, Ar), 146.4 (C,
Ar), 146.9 (C, Ar), 151.2 (C, Ar), 153.5 (C, Ar), 153.6 (C, Ar), 155.6 (C,
Ar), 165.5 (C]O), 165.6 (C]O), 171.0 (C]O), 171.1 (C]O); HRMS:
calculated for C36H36N2O9Na: 663.2319; Found 663.2332
[M þ Na]þ.
8.1.2.1. Dibenzyl (2-methoxy-5-(4-oxo-3-phenyl-1-(3,4,5-trimethoxyp
henyl)azetidin-2-yl)phenyl) phosphate (7b). Compound (7b) was pre-
pared according to the general procedure above from 4-(3-hydroxy-
4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-
one (7) in 74.3% yield as a yellow oil; IR (NaCl film) nmax: 1750 cmꢄ1
(
b
-lactam eC]O); 1H NMR (400 MHz, CDCl3)
d
3.73 (s, 6H, 2ꢂ OCH3),
3.78 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.25 (d, 1H, J ¼ 2 Hz, H3), 4.78 (d,
1H, J ¼ 2 Hz, H4), 5.15e5.18 (m, 4H, CH2), 6.60 (s, 2H, ArH), 6.98 (d, 1H,
J ¼ 8 Hz, ArH), 7.20 (m, 2H, ArH), 7.33e7.41 (m, 15H, ArH); 13C NMR
(100 MHz, CDCl3)
d
55.6 (OCH3), 55.6 (OCH3), 60.5 (OCH3), 62.9 (C3),
8.1.5. 2-Methoxy-5-(4-oxo-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-yl)phenyl 2-(((benzyloxy)carbonyl)amino)-3-
phenylpropanoate (7f)
64.5 (C4), 69.5 (OCH2), 69.6 (OCH2), 94.3 (CH, Ar), 112.9 (CH, Ar), 119.3
(CH, Ar), 122.8 (CH, Ar), 127.0 (CH, Ar), 127.4 (CH, Ar), 127.5 (CH, Ar),
127.6 (CH, Ar), 128.2 (CH, Ar), 128.2 (CH, Ar), 128.6 (CH, Ar), 133.1 (C,
Ar), 134.0 (C, Ar), 153.1 (C, Ar), 165.0 (C]O); HRMS: calculated for
C39H38NO9PNa: 718.2182; found 718.2172 [M þ Na]þ.
N-Benzyloxycarbonyl-L-phenylalanine (0.23 mmol), DCC
(0.23 mmol) and DMAP (0.23 mmol) were stirred in anhydrous
DCM (5 mL) at 0 ꢁC. A solution of 7 (0.23 mmol) in anhydrous DCM
(5 mL) was added dropwise over 5 min. Following the method
described above for the preparation of 7e, the product 7f was
8.1.3. General method for synthesis of phosphates 7a, 8a and 9a
b-Lactams 7b, 8b or 9b (2 mmol) were dissolved in ethano-
isolated (79%). IR (NaCl film) nmax: 1754.7 (
NMR (400 MHz, CDCl3) 3.24e3.38 (m, 2H, CH2), 3.75e3.86 (m,
b
-lactam eC]O); 1H
l:ethyl acetate (50 mL; 1:1 mixture) and hydrogenated over 10% Pd/
C (1.2 g) until complete on TLC; typically less than 3 h. The catalyst
was removed by filtration through Celite, the solvent was evapo-
rated under reduced pressure and the product was isolated by flash
column chromatography (hexane:ethyl acetate gradient, 88:12 to
50:50).
d
12H, 4ꢂ OCH3), 4.32 (m, 1H, H3), 4.87 (m, 1H, H4), 4.95e4.99 (m,
1H, CH), 5.13 (s, 2H, CH2), 6.63 (s, 2H, ArH), 7.00e7.04 (m, 2H, ArH),
7.29e7.42 (m, 16H, ArH). 13C NMR (100 MHz, CDCl3)
d 37.6 (CH2),
37.7 (CH2), 54.3 (C), 55.5 (OCH3), 55.6 (OCH3), 60.5 (OCH3), 62.9
(C3), 64.5 (C4), 66.6 (OCH2), 94.3 (CH, Ar), 112.6 (CH, Ar), 112.7 (CH,
Ar), 120.4 (CH, Ar), 124.2 (CH, Ar), 126.8 (CH, Ar), 127.0 (CH, Ar),
127.6 (CH, Ar), 127.8 (CH, Ar), 128.1 (CH, Ar), 128.2 (CH, Ar), 128.3
(CH, Ar), 128.7 (CH, Ar), 128.8 (CH, Ar), 129.0 (CH, Ar), 129.1 (CH,
Ar), 129.4 (CH, Ar), 129.5 (CH, Ar), 133.1 (C, Ar), 134.0 (C, Ar), 134.1
8.1.3.1. 2-Methoxy-5-(4-oxo-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-yl)phenyl dihydrogen phosphate (7a). Compound (7a)
was prepared from 7b as described in the general method above.

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N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
(C, Ar), 135.0 (C, Ar), 135.1 (C, Ar), 139.3 (C, Ar), 150.9 (C, Ar), 153.1
(C, Ar), 155.2 (C, Ar), 164.9 (C]O), 165.0 (C]O), 169.1 (C]O), 169.2
(C]O); HRMS: calculated for C42H41N2O9: 717.2812; found
717.2813 [M þ H]þ.
8.1.9. [3-(2-Bromoethoxy)-4-methoxybenzylidene]-(3,4,5-
trimethoxyphenyl)amine (12d)
Imine 12c (5 mmol) and tertabutylammonium sulphate
(4.5 mmol) were dissolved in dibromoethane (50 mmol). Sodium
hydroxide solution (1 M, 40 mL) was added to the reaction mixture.
The reaction was vigorously stirred for 16 h. Upon completion, the
reaction mixture was diluted with dichloromethane (100 mL) and
saturated sodium hydrogen carbonate (100 mL). The organic phase
was separated, while the aqueous phase was extracted with
dichloromethane (2 ꢂ 100 mL). The organic phases were combined,
dried over Mg2SO4 and evaporated to dryness in vacuo. The
resulting crystals were recrystallised from ethanol to afford the
8.1.6. Acetylamino-acetic acid 2-methoxy-5-[4-oxo-3-phenyl-1-
(3,4,5-trimethoxy-phenyl)azetidin-2-yl]phenyl ester (7g)
N-Acetylglycine (0.23 mmol), DCC (0.23 mmol) and DMAP
(0.23 mmol) were stirred in anhydrous DCM (5 mL) at 0 ꢁC. A so-
lution of 7 (0.23 mmol) in anhydrous DCM (5 mL) was added
dropwise over 5 min. The reaction was stirred for 16 h under a ni-
trogen atmosphere at ambient temperature. Reaction was moni-
tored via TLC. After 24 h, dichloromethane (50 mL) was added and
the mixture was filtered. The solvent was removed by washing with
water (5 ꢂ 50 mL). The organic solvent containing the product was
evaporated under reduced pressure. The product was isolated by
flash column chromatography (dichloromethane:methanol gra-
product (83%); IR (KBr disk) nmax
(400 MHz, CDCl3)
:
1622.8 (C]N); 1H NMR
3.89e3.97 (m, 12H, 3ꢂ OCH3), 3.74 (t, 2H,
d
J ¼ 6.5 Hz, CH2Br), 4.46 (t, 2H, CH2O), 6.51 (s, 2H, ArH), 6.99 (d, 1H,
J ¼ 8.5 Hz, ArH), 7.39 (d, 1H, J ¼ 8 Hz, ArH), 7.62 (s, 1H, ArH), 8.40 (s,
1H, HC]N); 13C NMR (100 MHz, CDCl3)
d 29.1 (CH2Br), 55.6 (OCH3),
dient), to afford the product (72%). IR (NaCl film) nmax: 1753.3 (
b
-
60.6 (OCH3), 68.4 (CH2O), 97.6 (CH, Ar),110.8 (CH, Ar),111.2 (CH, Ar),
124.8 (CH, Ar), 128.8 (CH, Ar), 135.7 (C, Ar), 143.0 (C, Ar), 147.5 (C,
Ar), 153.1 (C, Ar), 158.4 (C]N); HRMS: calculated for C19H23NO5Br:
424.0760; found 424.075 [M þ H]þ.
lactam eC]O); 1H NMR (400 MHz, CDCl3)
d
2.07 (s, 3H, CH3), 3.73
(s, 6H, 2ꢂ OCH3), 3.79 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.29 (d, 1H,
J ¼ 2.5 Hz, H3), 4.32e4.33 (m, 2H, CH2), 4.86 (d, 1H, J ¼ 2 Hz, H4),
6.60 (s, 2H, ArH), 7.02 (d, 1H, J ¼ 8.5 Hz, ArH), 7.15 (m, 1H, ArH),
7.31e7.41 (m, 6H, ArH). 13C NMR (100 MHz, CDCl3)
d
22.5 (CH3), 40.7
8.1.10. 4-[3-(2-Bromoethoxy)-4-methoxyphenyl]-3-phenyl-1-
(3,4,5-trimethoxyphenyl)azetidin-2-one (7k)
(CH2), 55.6 (OCH3), 60.5 (OCH3), 62.9 (C3), 64.5 (C4), 94.3 (CH, Ar),
112.7 (CH, Ar), 120.2 (CH, Ar), 124.3 (CH, Ar), 126.9 (CH, Ar), 127.6
(CH, Ar), 128.6 (CH, Ar), 129.5 (CH, Ar), 133.1 (C, Ar), 133.9 (C, Ar),
134.0 (C, Ar), 139.3 (C, Ar), 150.8 (C, Ar), 153.1 (C, Ar), 165.0 (C]O),
167.7 (C]O), 169.9 (C]O); HRMS: calculated for C29H30NO8Na:
557.1900; found 557.1917 [M þ Na]þ.
Imine 12d (2.0 mmol) and triethylamine (2.6 mmol) were dis-
solved in anhydrous dichloromethane (10 mL). A solution of phe-
nylacetyl chloride (2.6 mmol) in anhydrous dichloromethane
(5 mL) was added slowly via syringe. The reaction was refluxed for
2 h, then allowed cool and evaporated to dryness. The residue was
purified by flash chromatography (hexane:ethyl acetate, 1:1)
8.1.7. 2-Methoxy-5-(4-oxo-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-yl)phenyl acetate (7i)
to afford 7k as a resin (29%); IR (NaCl film) nmax 1749.8 (
b
-lactam
C]O); 1H NMR (400 MHz, CDCl3)
d
3.64 (t, 2H, CH2Br, J ¼ 6.5 Hz),
The imine 12a (5 mmol) and triethylamine (15 mmol) were
added to anhydrous dichloromethane (50 mL) and the mixture
heated to reflux under nitrogen atmosphere. Acetyl chloride
(7.5 mmol) was injected dropwise through a septum, and the
mixture was allowed to reflux for 3 h. The reaction mixture was
then cooled, washed with water, (2 ꢂ 50 mL) and sodium bicar-
bonate solution (aq., 50 mL). The organic layer was dried with
anhydrous Na2SO4, and the solvent evaporated to dryness in vacuo.
The residue was purified by flash chromatography (hexane:ethyl
3.75 (s, 6H, OCH3), 3.80 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.29e4.34
(m, 3H, OCH2, H3), 4.86 (d, 1H, J ¼ 2.5 Hz), 6.63 (s, 2H), 6.93e6.96
(m, 2H), 7.05e7.08 (m, 1H), 7.34e7.40 (m, 5H, ArH); 13C NMR
(100 MHz, CDCl3)
d 28.4 (CH2Br), 55.6 (OCH3), 59.9 (OCH3), 60.5
(OCH3), 63.4 (C3), 64.5 (C4), 68.9 (OCH2), 94.4 (CH, Ar), 111.7 (CH,
Ar),112.1 (CH, Ar),119.7 (CH, Ar),126.9 (CH, Ar),127.5 (CH, Ar),128.6
(CH, Ar), 129.4 (CH, Ar), 130.5 (CH, Ar), 133.2 (C, Ar), 134.2 (C, Ar),
147.7 (C, Ar), 149.8 (C, Ar), 153.1 (C, Ar), 165.1 (C]O); HRMS: cal-
culated for C27H28NO6NaBr: 564.0998, found 564.1005 [M þ Na]þ.
acetate, 50:50) to afford the
from methanol (7%), Mp 70 ꢁC. IR (KBr disk) nmax: 1725.1 (
eC]O); 1H NMR (400 MHz, CDCl3)
2.12 (3H, s, CH3), 3.75 (6H, s,
b
-lactam product as colourless crystals
b
-lactam
8.1.11. 4-[4-Methoxy-3-(2-methylaminoethoxy)phenyl]-3-phenyl-
1-(3,4,5-trimethoxyphenyl)azetidin-2-one (7l)
d
2ꢂ OCH3), 3.80 (3H, s, OCH3), 3.87 (3H, s, 2ꢂ OCH3), 4.32 (1H, d,
J ¼ 2.5 Hz, H3), 4.86 (1H, d, J ¼ 2.5 Hz, H4), 6.64 (2H, s, ArH), 7.02
(1H, m, ArH), 7.13 (1H, d, J ¼ 2 Hz, ArH), 7.27e7.40 (6H, m, ArH);
HRMS: calculated for C27H27NO7Na: 500.1685; found: 500.1688
[M þ Na]þ.
b-Lactam 7k (0.2 mmol) and methylamine 2.0 M solution
(4.0 mmol) were dissolved in anhydrous tetrahydrofuran (10 mL) in
a high pressure tube. The sealed tube was heated to 60 ꢁC for 6 h.
The reaction mixture was allowed cool and sodium carbonate/so-
dium hydrogen carbonate pH 10 buffer solution was added (25 mL).
The reaction mixture was extracted with dichloromethane
(3 ꢂ 50 mL). The combined organic phases were dried over Mg2SO4
and evaporated to dryness in vacuo. The residue was purified using
flash chromatography (hexane:ethyl acetate, 1:1) to afford 7l as
8.1.8. 2-Methoxy-5-4-oxo-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-yl)phenyl benzoate (7j)
The imine 12b (5 mmol) and triethylamine (15 mmol) were
added to anhydrous dichloromethane (50 mL) and the mixture
heated to reflux under nitrogen atmosphere. Phenylacetyl chloride
(7.5 mmol) was injected dropwise through a septum, and the
mixture was allowed to reflux for 3 h, following the method
a resin (30%); IR (NaCl film) nmax 1746.6 (
(400 MHz, CDCl3) 2.70 (s, 3H, NCH3), 3.24 (m, 2H, CH2), 3.75 (s, 6H,
b
-lactam C]O); 1H NMR
d
OCH3), 3.79 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.22e4.30 (m, 3H, CH,
OCH2), 4,57 (broad s, 1H, NH), 4.86 (d, 1H, CH, J ¼ 2 Hz), 6.62 (s, 2H,
ArH), 6.93e6.96 (m, 2H), 7.05e7.08 (m, 1H), 7.33e7.41 (m, 5H, ArH);
described above for the preparation of 7i. The
was obtained as colourless crystals from methanol (8%), Mp 73 ꢁC.
IR (NaCl film) nmax: 1752.0 (
-lactam eC]O); 1H NMR (400 MHz,
CDCl3)
b-lactam product 7j
13C NMR (100 MHz, CDCl3)
d 34.0 (NCH3), 48.6 (CH2), 55.6 (OCH3),
b
55.9 (OCH3), 60.5 (OCH3), 63.3 (C3), 64.5 (C4), 66.0 (OCH2), 94.4 (CH,
Ar), 102.9 (CH, Ar), 111.5 (CH, Ar), 111.7 (CH, Ar), 119.7 (CH, Ar), 127.0
(CH, Ar), 127.5 (CH, Ar), 127.8 (CH, Ar), 127.9 (CH, Ar), 128.5 (CH, Ar),
128.6 (CH, Ar), 129.7 (CH, Ar), 130.5 (CH, Ar), 133.2 (C, Ar), 134.1 (C,
Ar), 147.7 (C, Ar), 149.5 (C, Ar), 153.1 (C, Ar), 165.2 (C]O); HRMS:
calculated for C28H33N2O6: 493.2334; found 493.2337 [M þ H]þ.
d
3.84 (s, 6H, 2ꢂ OCH3), 3.84 (s, 3H, OCH3), (s, 3H, OCH3),
4.36 (d, 1H, J ¼ 2.9 Hz, H3), 4.90 (d, 1H, J ¼ 2.9 Hz, H4), 6.63 (s, 2H,
ArH), 7.07e7.31 (m, 2H, ArH), 7.35e7.39 (m, 4H, ArH), 7.52e7.83 (m,
4H, ArH), 8.20e8.24 (m, 3H, ArH); HRMS: calculated for
C32H29NO7Na: 562.1842; found: 562.1841 [M þ Na]þ.

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
719
8.1.12. General method for synthesis of amides 10aef and 11aeh
To a stirred solution of the appropriate amino -lactam 10 or 11
(4.76 mmol) in anhydrous DMF (30 mL) were added DCC
(5.7 mmol), Cbz- or Fmoc-protected -amino acid (5.7 mmol) and
HOBt$H2O (7.3 mmol) at room temperature. After 24 h, ethyl ace-
tate (50 mL) was added and the mixture was filtered. DMF was
removed by washing with water (5 by 50 mL). The organic solvent
containing the product was evaporated under reduced pressure.
The product was isolated by flash column chromatography
(dichloromethane:methanol gradient).
(10 equiv.) and TBAF (2 equiv.). The mixture was stirred at room
temperature until complete on TLC, typically within 10 min. The sol-
vent was evaporated under reduced pressure and product was iso-
lated by flash column chromatography (dichloromethane:methanol
gradient, 100:0 to 90:10).
b
L
8.1.13.1. 2-Amino-N-(2-methoxy-5-(4-oxo-3-phenyl-1-(3,4,5-
trimethoxyphenyl)azetidin-2-yl)phenyl)-3-methylbutanamide (11l).
Compound (11l) was prepared from 11d by general method 2 and
was obtained as a yellow oil in 57% yield; IR (NaCl film) nmax
3380.25 cmꢄ1 (NH2), 1747.97 cmꢄ1 -lactam eC]O), 1676.35 cmꢄ1
(eC]O); 1H NMR (400 MHz, CDCl3)
0.88 (d, 3H, J ¼ 7 Hz), 1.05 (d,
:
(b
8.1.12.1. Benzyl (1-((4-(2-(4-methoxyphenyl)-4-oxo-1-(3,4,5-trimetho-
xyphenyl)azetidin-3-yl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)
carbamate (10e). Compound (10e) was prepared from 10 and Cbz-
protected phenylalanine following the general method above and
d
3H, J ¼ 7 Hz), 2.35 (m, 1H), 3.75e3.77 (m, 10H), 3.92 (s, 3H), 4.29e
4.37 (m, 1H), 4.78e4.92 (m, 1H), 6.65 (s, 2H), 6.79 (s, 1H), 6.93 (m,
1H), 7.13 (m, 1H), 7.29e7.36 (m, 5H), 8.66 (d, 1H, J ¼ 13 Hz), 10.00 (d,
was isolated as a yellow gel in 28% yield; IR (NaCl film) nmax
1743 cmꢄ1 -lactam eC]O), 1689 cmꢄ1 (eC]O); 1H NMR
(400 MHz, CDCl3)
:
1H, J ¼ 13 Hz); 13C NMR (100 MHz, CDCl3)
d 19.2 (CH3), 19.3 (CH3),
(b
30.4 (CH), 30.5 (CH), 55.1, 55.5 (OCH3), 55.6 (OCH3), 55.7 (OCH3),
60.4 (OCH3), 60.5 (OCH3), 63.4 (C3), 63.6 (C3), 64.2 (C4), 64.3 (C4),
94.4 (CH, Ar), 94.5 (CH, Ar), 110.0 (CH, Ar), 110.2 (CH, Ar), 110.3 (CH,
Ar), 111.0 (CH, Ar), 115.8 (CH, Ar), 117.5 (CH, Ar), 117.9 (CH, Ar), 120.0
(CH, Ar), 120.1 (CH, Ar), 126.9 (CH, Ar), 127.0 (CH, Ar), 127.3 (CH, Ar),
127.4 (CH, Ar), 127.5 (CH, Ar), 127.6 (CH, Ar), 128.3 (CH, Ar), 129.3
(CH, Ar), 129.4 (CH, Ar), 133.3 (C, Ar), 133.4 (C, Ar), 133.9 (C, Ar),
134.2 (C, Ar), 134.3 (C, Ar), 134.5 (C, Ar), 136.7 (C, Ar), 147.1 (C, Ar),
148.0 (C, Ar), 148.1 (C, Ar), 153.0 (C, Ar), 165.3 (C]O), 165.3 (C]O),
172.3 (C]O); HRMS: calculated for C30H36N3O6: 534.2604; found
534.2584 [M þ H]þ.
d
3.11e3.25 (m, 2H, CH2), 3.74 (s, 6H, 2ꢂ OCH3),
3.80 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.01 (m, 2H, CH2), 4.24 (d, 1H,
CH), 4.55 (d, 1H, CH), 4.82 (d, 1H, J ¼ 2.5 Hz, H4), 5.49 (s, 1H), 6.61 (s,
2H, ArH), 6.96 (d, 2H, J ¼ 9.0 Hz, ArH), 7.25e7.35 (m,18H, ArH), 7.67 (s,
1H, ArH); 13C NMR (100 MHz, CDCl3)
d 38.4 (CH2), 53.0 (CH), 54.9
(OCH3), 55.6 (OCH3), 60.5 (OCH3), 63.5 (C3), 64.1 (C4), 66.6 (OCH2),
66.9 (OCH2), 94.4 (CH, Ar), 114.2 (CH, Ar), 120.2 (CH, Ar), 125.4 (CH,
Ar), 126.8 (CH, Ar), 127.6 (CH, Ar), 127.7 (CH, Ar), 127.9 (CH, Ar), 128.1
(CH, Ar), 128.5 (CH, Ar), 128.7 (CH, Ar), 128.8 (CH, Ar), 133.2 (C, Ar),
134.0 (C, Ar), 135.4 (C, Ar), 135.7 (C, Ar), 153.0 (C, Ar), 159.5 (C, Ar),
165.2 (C]O), 168.6 (C]O); HRMS: calculated for C42H41N3O8Na:
738.2791; found 738.2777 [M þ Na]þ.
8.1.14. Synthesis of 7h, 10kem and 11per
The carboxybenzyl or benzyl protected compound 7e, 10e, 10f,
10j, 11h, 11n or 11o (2 mmol) was dissolved in ethanol:ethyl acetate
(50 mL; 1:1 mixture) and hydrogenated over 1.2 g of 10% palladium
on carbon until complete on TLC, typically less than 2 h. The catalyst
was filtered, the solvent was evaporated under reduced pressure
and the product was isolated by flash column chromatography
(dichloromethane:methanol gradient, 100:0 to 90:10).
8.1.12.2. (9H-Fluoren-9-yl)methyl (1-((2-methoxy-5-(4-oxo-3-
phenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-yl)phenyl)amino)-3-
methyl-1-oxobutan-2-yl)carbamate (11d). Compound (11d) was
prepared from 11 and Fmoc-protected valine following the general
method above and was isolated as a brown oil in 51% yield; IR (NaCl
film) nmax: 1748 cmꢄ1
(b
-lactam eC]O),1686 cmꢄ1 (C]O); 1H NMR
1.01e1.04 (m, 6H, 2ꢂ CH3), 2.20e2.28 (m, 1H,
(400 MHz, CDCl3)
d
CH), 3.75e3.88 (m, 12H, 4ꢂ OCH3), 4.18 (m, 2H), 4.24 (m, 1H, CH),
4.43e4.48 (m, 2H), 4.92 (s,1H, CH), 5.49 (d,1H, J ¼ 8.6 Hz), 6.66 (s, 2H,
ArH), 6.81 (s, 1H, ArH), 6.92e6.94 (m, 1H, ArH), 7.16e7.18 (m, 1H,
ArH), 7.31e7.45 (m, 9H, ArH), 7.62e7.63 (m, 2H, ArH), 7.77 (m, 2H,
ArH), 8.23 (s,1H, ArH), 8.54 (m,1H, ArH); 13C NMR (100 MHz, CDCl3)
8.1.14.1. 2-Methoxy-5-(4-oxo-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-yl)phenyl 2-aminopropanoate (7h). Compound (7h) was
prepared from compound 7e following the procedure above in
58% yield. IR (NaCl film) nmax: 1748.1 (
(400 MHz, CDCl3)
OCH3), 4.30e4.32 (m, 1H, H3), 4.83e4.88 (m, 1H, H4), 6.62e6.64
(m, 2H, ArH), 6.88e6.95 (m, 1H, ArH), 7.01e7.04 (m, 1H, ArH),
7.14e7.15 (m, 1H, ArH), 7.34e7.41 (m, 5H, ArH); 13C NMR
b
-lactam eC]O); 1H NMR
1.54e1.56 (m, 3H, CH3), 3.76e3.92 (m, 12H, 4ꢂ
d
d
17.5 (CH3), 18.8 (CH3), 30.7 (CH), 46.7 (CH), 55.1 (OCH3), 55.5
(OCH3), 55.6 (OCH3), 55.7 (OCH3), 60.5 (OCH3), 61.0 (OCH3), 63.3 (C3),
63.6 (C3), 64.3 (C4), 64.4 (C4), 66.8 (CH2), 94.4 (CH, Ar), 94.4 (CH, Ar),
94.5 (CH, Ar), 110.1 (CH, Ar), 110.3 (CH, Ar), 111.3 (CH, Ar), 111.4 (CH,
Ar), 115.8 (CH, Ar), 116.3 (CH, Ar), 117.9 (CH, Ar), 119.6 (CH, Ar), 120.8
(CH, Ar),120.9 (CH, Ar),124.6 (CH, Ar), 126.2 (CH, Ar),126.6 (CH, Ar),
126.8 (CH, Ar),127.0 (CH, Ar),127.1 (CH, Ar),127.2 (CH, Ar),127.3 (CH,
Ar),127.4 (CH, Ar),128.5 (CH, Ar),128.6 (CH, Ar),129.4 (CH, Ar),133.2
(C, Ar),133.4 (C, Ar),133.9 (C, Ar),134.1 (C, Ar),134.4 (C, Ar),136.1 (C,
Ar),140.8 (C, Ar),143.2 (C, Ar),143.3 (C, Ar),147.8 (C, Ar),153.0 (C, Ar),
156.0 (C, Ar), 165.3 (C]O), 172.1 (C]O); HRMS: calculated for
C45H45N3O8Na: 778.3104; found 778.3107 [M þ Na]þ.
(100 MHz, CDCl3)
d 20.6 (CH3), 56.0 (OCH3), 56.1 (OCH3), 60.9
(OCH3), 63.5 (C3), 63.8 (C3), 64.9 (C4), 65.0 (C4), 94.8 (CH, Ar), 94.9
(CH, Ar), 111.1 (CH, Ar), 112.1 (CH, Ar), 113.0 (CH, Ar), 117.9 (CH, Ar),
120.6 (CH, Ar), 124.5 (CH, Ar), 127.4 (CH, Ar), 127.9 (CH, Ar), 128.0
(CH, Ar), 129.0 (CH, Ar), 129.1 (CH, Ar), 129.9 (CH, Ar), 130.5 (CH,
Ar), 133.7 (C, Ar), 134.4 (C, Ar), 134.5 (C, Ar), 134.8 (C, Ar), 140.3 (C,
Ar), 146.4 (C, Ar), 146.9 (C, Ar), 153.5 (C, Ar), 153.6 (C, Ar), 165.6
(C]O); HRMS: calculated for C28H31N2O7: 507.2131; found
507.2152 [M þ H]þ.
8.1.13. General methods for removal of Fmoc protecting group for
preparation of 10gej and 11ieo
8.1.14.2. 2-Amino-N-(4-(2-(4-methoxyphenyl)-4-oxo-1-(3,4,5-
trimethoxyphenyl)azetidin-3-yl)phenyl)-3-phenylpropanamide (10k).
Compound (10k) was prepared from 10e following the procedure
General method 1: to the Fmoc-amino acid amide (2.1 mmol) at
roomtemperatureinCHCl3 (55mL)wasaddedpiperidine(2mL).The
mixture was stirred and monitored until complete on TLC. The sol-
vent was evaporated under reduced pressure. The product was iso-
lated by flash column chromatography (dichloromethane:methanol
gradient, 100:0 to 90:10).
above as a yellow oil in 51% yield; IR (NaCl film) nmax: 1742 cmꢄ1
(b-
lactam eC]O), 1684 cmꢄ1 (eC]O); 1H NMR (400 MHz, CDCl3)
2.82e2.88 (m, 1H, CH2), 3.35e3.39 (m, 1H, CH2), 3.79e3.85 (m,
d
13H, 4ꢂ OCH3, CH), 4.26 (s, 1H), 4.84 (d, 1H, J ¼ 2.5 Hz), 6.62 (s, 2H),
6.97 (d, 3H, J ¼ 8.5 Hz), 7.25e7.39 (m, 10H), 9.52 (s, 1H, NH); 13C
General method 2: to the Fmoc-amino acid amide dissolved in
CH2Cl2 (minimum volume, 1e2 mL) was added 1-octanethiol
NMR (100 MHz, CDCl3) d 40.0 (CH2), 54.4 (OCH3), 55.6 (OCH3), 56.2
(CH), 58.8 (CH), 60.5 (OCH3), 63.5 (C3), 64.2 (C4), 94.4 (CH, Ar), 114.2

720
N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
(CH, Ar), 119.7 (CH, Ar), 124.1 (CH, Ar), 126.6 (CH, Ar), 126.9 (CH, Ar),
127.6 (CH, Ar), 127.8 (CH, Ar), 128.1 (CH, Ar), 128.2 (CH, Ar), 128.3
(CH, Ar),128.4 (CH, Ar), 128.7 (CH, Ar),128.9 (CH, Ar),129.3 (CH, Ar),
129.9 (CH, Ar), 133.2 (C, Ar), 133.9 (C, Ar), 136.2 (C, Ar), 136.9 (C, Ar),
153.0 (C, Ar), 159.4 (C, Ar), 159.5 (C, Ar), 164.7 (C]O), 165.3 (C]O),
171.7 (C]O), 174.0 (C]O); HRMS: calculated for C34H36N3O6:
582.2604; found 582.2613 [M þ H]þ.
8.3.3. Immunofluorescence and confocal microscopy
For immunofluorescence, MCF-7 cells were seeded at 1 ꢂ105 per
well on BD falcon four well chamber glass slides (BD Biosciences, San
Jose, USA). Cells were treated with vehicle [1% ethanol (v/v)]; 2, 8a,
9a, 10k, [100 nM] or 11l [1 mM] for 16 h. Following treatment, cells
were washed gently in PBS, fixed for 30 min in methanol at ꢄ20 ꢁC.
Following washes in PBST (PBS and 0.1% Triton-X-100), 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 isothiocyanate (FITC) conjugated
rabbit anti-mouse (Dakocytomation, UK); 1:100 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 micro-
scope coupled with OLYMPUS FLUOVIEW Ver 1.5 software. All im-
ages in each experiment were collected on the same day using
identical parameters.
8.2. Solubility measurement
Solubility testing was carried out on selected samples 7, 7a, 8,
8a, 9, 9a, 10, 10g, 10i, 10k, 10l, 11, 11j, 11k, 11l, 11p and 11q as fol-
lows: an amount of compound approximately equal to 5 mg was
accurately weighed and suspended in a corresponding amount of
distilled water in a vial (Wheaton sample vials with rubber lined
caps) to give a concentration of 1 mg/mL. This suspension was
placed on a shaking plate (IKAÒ MTS 2/4 digital) and agitated at
300 shakes/minute for 24 h. After 24 h, the vials were removed
from the shaking plate. The solution/suspensions were filtered and
the filtrate was assayed by HPLC using the conditions described
above for purity testing to determine the amount of compound in
solution.
8.3.4. Determination of DNA content by flow cytometry
MCF-7 cells were seeded at 1 ꢂ 105 cells/cm3. After 24 h, cells
were treated with vehicle [1% ethanol (v/v)]; 2, 7a, 8a, 9a, 10k,
8.3. Biochemical methods
[100 nM] or 11l [1 mM] for 48 h. Cells were harvested by cen-
trifugation at 800ꢂ g for 10 min. Cell pellets were re-suspended in
PBS and fixed in 70% ethanol: PBS overnight at ꢄ20 ꢁC. Following
centrifugation cell pellets were re-suspended in PBS supplemented
with 0.5 mg/mL RNase and 0.15 mg/mL propidium iodide (PI). Fol-
lowing a 30 min incubation at 37 ꢁC 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).
8.3.1. 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. Cells were trypsinised
and seeded at a density of 2.5 ꢂ 104 cells/mL in a 96-well plate and
incubated at 37 ꢁC, 95% O2/5% CO2 atmosphere for 24 h. After this
time they were treated with 2
had been pre-prepared as stock solutions in ethanol to furnish the
concentration range of study, 1 nMe100 M, and re-incubated for
mL volumes of test compound which
m
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 100
and 50 L MTT (1 mg/mL solution in PBS) added. Cells were incu-
bated for 2 h in darkness at 37 ꢁC. At this point solubilisation was
begun through the addition of 200 L DMSO and the cells main-
tained 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
compound added were drawn.
mL phosphate buffered saline (PBS)
m
8.4. Computational procedures
m
For ligand preparation, all compounds were built in Molecular
Operating Environment 2011.10 (MOE 2011.10) [38] and energy
minimised. Conformers of these compounds were generated in MOE
2011.10 using the conformation search option. The stochastic method
was chosen with an iteration limit of 100. All other parameters were
remained unchanged. For the receptor preparation, the PDB entry
1SA0 was downloaded from the Protein Data Bank (PDB) [33]. All
waters were retained in both isoforms. Addition and optimisation of
hydrogen positions for these waters was carried out using MOE
2011.10 ensuring all other atom positions remained fixed. Using the
reported X-ray structure of tubulin co-crystallised with a colchicine
derivative, DAMA-colchicine (PDB entry-1SA0), possible binding ori-
8.3.2. Tubulin polymerisation assay
The effect of selected compounds on the polymerisation of pu-
rified bovine brain tubulin was determined spectrophotometrically
by monitoring the change in turbidity. Lyophilised tubulin (Cytos-
keleton, 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 plate
containing the designated concentration of drug or vehicle. Sam-
ples were mixed well and tubulin assembly was monitored at
A340nm at 30 s intervals for 60 min at 37 ꢁC in a Spectramax 340PC
spectrophotometer (Molecular Devices). IC50 values were calcu-
lated at 30 min using GraphPad Prism software (GraphPad Soft-
ware, Inc.).
entations of the b-lactam ligands were probed with the docking pro-
gram MOE 2011.10. Docking was carried out using MOE 2011.10, using
the DAMA-colchicine to identity the binding site. Each conformation
was subsequently docked using the Triangle Matcher placement
methodologysetting. Allthedefaultparameters remained unchanged.
Acknowledgements
Funding from the Health Research Board and postgraduate
research awards from Trinity College Dublin are gratefully

N.M. O’Boyle et al. / European Journal of Medicinal Chemistry 62 (2013) 705e721
721
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acknowledged. Sincere thanks to Dr. Gavin Mc Manus (School of
Biochemistry and Immunology, Trinity College) for his technical
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.016.
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