3-Vinylazetidin-2-Ones: Synthesis, antiproliferative and tubulin destabilizing activity in MCF-7 and MDA-MB-231 Breast Cancer Cells

Article abstract of DOI:10.3390/ph12020056

Microtubule-targeted drugs are essential chemotherapeutic agents for various types of cancer. A series of 3-vinyl-β-lactams (2-azetidinones) were designed, synthesized and evaluated as potential tubulin polymerization inhibitors, and for their antiproliferative effects in breast cancer cells. These compounds showed potent activity in MCF-7 breast cancer cells with an IC50 value of 8 nM for compound 7s 4-[3-Hydroxy-4-methoxyphenyl]-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin-2-one) which was comparable to the activity of Combretastatin A-4. Compound 7s had minimal cytotoxicity against both non-tumorigenic HEK-293T cells and murine mammary epithelial cells. The compounds inhibited the polymerisation of tubulin in vitro with an 8.7-fold reduction in tubulin polymerization at 10 μM for compound 7s and were shown to interact at the colchicine-binding site on tubulin, resulting in significant G2/M phase cell cycle arrest. Immunofluorescence staining of MCF-7 cells confirmed that β-lactam 7s is targeting tubulin and resulted in mitotic catastrophe. A docking simulation indicated potential binding conformations for the 3-vinyl-β-lactam 7s in the colchicine domain of tubulin. These compounds are promising candidates for development as antiproiferative microtubule-disrupting agents.

Full text of DOI:10.3390/ph12020056

Article
3
-Vinylazetidin-2-Ones: Synthesis, Antiproliferative
and Tubulin Destabilizing Activity in MCF-7 and
MDA-MB-231 Breast Cancer Cells
1
1,2,†
1,†
1
3
Shu Wang , Azizah M. Malebari , Thomas F. Greene , Niamh M. O’Boyle , Darren Fayne ,
3
4
4
1
Seema M. Nathwani , Brendan Twamley , Thomas McCabe , Niall O. Keely ,
Daniela M. Zisterer and Mary J. Meegan *
3
1,
1
School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Trinity Biomedical Sciences
Institute, 152-160 Pearse Street, 2 DO2R590 Dublin, Ireland; wangsh@tcd.ie (S.W.); melibaa@tcd.ie
(
A.M.M.); tgreene@tcd.ie (T.F.G.); Niamh.OBoyle@tcd.ie (N.M.O’B.); nkeely@tcd.ie (N.O.K.)
2
3
Department of Pharmaceutical Chemistry, College of Pharmacy, King Abdulaziz University,
Jeddah 21589, Saudi Arabia
School of Biochemistry and Immunology, Trinity College Dublin, Trinity Biomedical Sciences Institute,
1
52-160 Pearse Street, 2 DO2R590 Dublin, Ireland; FAYNED@tcd.ie (D.F.);
seema.nathwani@outlook.com (S.M.N.); dzistrer@tcd.ie (D.M.Z.)
School of Chemistry, Trinity College Dublin, 2 DO2R590 Dublin, Ireland; TWAMLEYB@tcd.ie (B.T.);
TMCCABE@tcd.ie (T.M.)
4
*
†
Correspondence: mmeegan@tcd.ie (M.J.M.); Tel.: +353-1-896-2798; Fax: +353-1-8962793
These authors contributed equally to this work.
Abstract: Microtubule-targeted drugs are essential chemotherapeutic agents for various types of
cancer. A series of 3-vinyl-β-lactams (2-azetidinones) were designed, synthesized and evaluated
as potential tubulin polymerization inhibitors, and for their antiproliferative effects in breast
cancer cells. These compounds showed potent activity in MCF-7 breast cancer cells with an IC50
value of 8 nM for compound 7s 4-[3-Hydroxy-4-methoxyphenyl]-1-(3,4,5-trimethoxyphenyl)-3-
vinylazetidin-2-one) which was comparable to the activity of Combretastatin A-4. Compound 7s
had minimal cytotoxicity against both non-tumorigenic HEK-293T cells and murine mammary
epithelial cells. The compounds inhibited the polymerisation of tubulin in vitro with an 8.7-fold
reduction in tubulin polymerization at 10 µM for compound 7s and were shown to interact at the
colchicine-binding site on tubulin, resulting in significant G2/M phase cell cycle arrest.
Immunofluorescence staining of MCF-7 cells confirmed that β-lactam 7s is targeting tubulin and
resulted in mitotic catastrophe. A docking simulation indicated potential binding conformations
for the 3-vinyl-β-lactam 7s in the colchicine domain of tubulin. These compounds are promising
candidates for development as antiproiferative microtubule-disrupting agents.
Keywords: Combretastatin A-4; β-lactam; 3-vinylazetidin-2-ones; antiproliferative activity;
tubulin; antimitotic.
1
. Introduction
Antimitotic agents such as taxol and the vinca alkaloids vinblastine and vincristine are a major
class of drugs used clinically in the treatment of many cancers [1–3]. Microtubule-destabilizing
Pharmaceuticals 2019, 12, 56; doi:10.3390/ph12020056
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agents (e.g., vinblastine) typically bind with tubulin at the vinca alkaloid site [4], while colchicine
exerts its biological effects at the intrasubunit interface within a tubulin dimer [5]. Stilbene-based
1
compounds have attracted the attention of chemists and pharmacologists due to their many
biological properties such as anticancer, antioxidant and anti-inflammatory activities, and are often
used in traditional medicine for a variety of therapeutic effects [6]. The combretastatins are a group
of stilbenes isolated from the South African bush willow tree Combretum caffrum[7], and are shown
to have outstanding potency in binding to the colchicine-binding site of tubulin and thus inhibiting
the formation of the mitotic spindle [8]. Combretastatin A-4 2a and Combretastatin A-1 2c
demonstrate exceptionally potent antiproliferative activity against a range of human cancer cell
lines (Figure 1) [7]. Additionally, antivascular effects are produced by these compounds in vivo
[
9,10]. Although some combretastatin compounds have progressed to clinical trials[11,12], there
are major problems associated with combretastatins including poor water solubility and cis/trans
isomerization during administration or storage, which results in an extensive loss of potency.
Water soluble prodrugs such as the combretastatin phosphate CA-4P, (fosbretabulin) 2b [13,14] are
currently in clinical trials for advanced anaplastic thyroid carcinoma [15], ovarian cancer [16], and
in combination with Bevacizumab for patients with advanced cancer [17]. Recently, the potential
combination therapy of CA-4P and vincristine in the treatment of hepatocellular carcinoma was
reported to show a beneficial effect in reducing doses of drugs with narrow therapeutic windows
[
18]. Ombrabulin is a serine prodrug whose derivatives display the same activity as CA-4 and has
completed a phase III clinical trial for the treatment of advanced stage soft tissue sarcoma [19,20].
There is ongoing interest in the clinical development of combretastatin A1 diphosphate (OXi 4503)
2
d [21]. The structurally related benzophenones phenstatin 3a, phenstatin phosphate 3b [22] and
the lignin podophyllotoxin 4 also destabilize microtubules [23].
Many heterocyclic scaffold structures have been introduced to replace the alkene of the
stilbene structure of CA-4 and to provide conformational restriction by locking the stilbene in the
cis configuration (Rings A and B) required for biological activity [24]. Small molecule tubulin
polymerization inhibitors have been reported in which the cis double bond of CA-4 has been
replaced by various heterocycles such as furan [25], indole[26,27], imidazole [28], isoxazole [29],
triazole [30], tetrazole [31], benzoxepine [32], pyrazole [33], pyridine [34], benzimidazole [35] and
related heterocycles [36]. While β-lactam antibiotics have occupied a central role in the treatment
of pathogenic bacteria, the antiproliferative activity of compounds containing the β-lactam
(
azetidin-2-one) ring has also been investigated [37–42]. The synthesis and antitumour activity of
a number of chiral β-lactam bridged CA-4 analogues have been reported [37,38]. Additional
impetus for research efforts on β-lactam chemistry has been provided by the use of β-lactams as
synthetic intermediates in organic synthesis [43].
We have previously investigated the antiproliferative and SERM (selective estrogen receptor
modulator) activity of the azetidin-2-one(β-lactam) scaffold [44] and also demonstrated the
effectiveness of 1,4-diarylazetidin-2-ones in breast cancer cell lines as tubulin targeting agents.
[
45,46]. These compounds also demonstrated both anti-angiogenic effects in MDA-MB-231 breast
adenocarcinoma cells. In addition, we established that these compounds inhibited the migration of
MDA-MB-231 cells indicating a potential anti-metastatic function for these compounds [47]. To
further our understanding of the antiproliferative activity of these compounds, we wished to
investigate the design, synthesis and evaluation of a series of azetidin-2-ones containing a vinyl
substituent at C3 of the azetidin-2-one ring, and to explore the effect of this hydrophobic
substituent on the biological activity of these compounds in which the cis configuration (Rings A
and B) is locked into the azetidin-2-one ring structure. The introduction of this vinyl substituent at
C-3 also allowed us to examine further chemical transformations of the alkene, and to determine
structure-activity relationships for the series. On this basis, we now aimed to investigate a new

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series of novel 3-vinylazetidinones compounds with an improved biochemical profile particularly
in triple negative breast cancer for potential development in preclinical study of breast cancer as
tubulin destabilising agents. Therefore, we focused our efforts on the preparation of a library of
1
,4-diarylazetidin-2-ones which contain a vinyl substituent at C-3. The synthesis of phosphate
esters and amino acid amide type prodrugs of the most potent 1,4-diarylazetidin-2-ones were
examined, together with the antiproliferative and tubulin targeting effects.
Figure 1. Colchicine (1), Combretastatins (2a–2d), phenstatins (3a, 3b) and podophyllotoxin (4).
2
. Results and Discussion
2
.1. Chemistry: Synthesis of β-lactams
There are many synthetic routes available for the construction of the β-lactam ring [43,48]. The
choice of route depends on the structural features required in the final product. In the present work,
the Staudinger reaction between an imine and a ketene was chosen for the formation of the β-
lactam ring because of its ease of use, adaptability for use with structurally diverse imines and acid
chlorides, and readily available starting materials. A series of analogues with a variety of
substituents at C4 of the β-lactam ring B was synthesized from the appropriate imines. The
preparation of the Schiff bases 5a–5r was achieved by the condensation of the appropriately
substituted benzaldehyde with the 3,4,5-trimethoxyaniline in ethanol in the presence of a catalytic
amount of sulphuric acid, (Scheme 1). The 3,4,5-trimethoxy substituted A-Ring of CA-4 plays an
important role in inhibiting tubulin polymerisation, and is confirmed in the docking of CA-4 in
tubulin [49]. The substituents located at the para-position of C-4 aryl Ring B included halogens
(
compounds 5a–5c), nitro (5d), dimethylamino (5e), methyl (5g), alkoxyl (5h–5j), phenoxy (5k),
benzyloxy (5l), nitrile (5q) and thiomethyl (5r) together with naphthyl (compounds 5m and 5n). 5s
was similarly obtained by reaction of 4-methoxybenzaldehyde with 3,5-dimethoxyaniline. For the
synthesis of β-lactam derivatives with a phenolic hydroxy group to mimic Ring B of CA-4, it was
necessary to use the benzyl ether 5l and tert-butyldimethylsilyl ether 5o. A further series of Schiff
bases (6a–6k) was obtained from 3,4,5-trimethoxybenzaldehyde with appropriate anilines using
the same procedure as above, (Scheme 1). An example of the crystal structure of the imine 6k is
displayed in Figure 2, showing the E configuration of the imine N1-C2 bond (bond length 1.278(2)
Å) (Table 1).
A series of novel β-lactams (7a–7r) was obtained by reaction of imines 5a–5r with crotonyl
chloride using Staudinger reaction conditions requiring the slow addition of a solution of the
appropriate acid chloride to a refluxing solution of imine and TEA, (Scheme 2) [50,51]. One
enantiomer is illustrated in each case and products are obtained as a racemic mixture. β-Lactam
(
7s) containing the required Ring B phenolic group of CA-4 was successfully synthesised from the

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silyl ether imine 5o and crotonyl chloride to afford the silyl ether β-lactam 7o which was
deprotected in situ by treatment with tBAF to yield the phenol 7s (Scheme 2). This series of
compounds 7a–7r differ only in the substituent pattern of aryl ring at C-4 of the β-lactam ring B.
Many potentially useful CA-4 derivatives contain the amino substituent replacing the phenol
on ring B and have shown interesting biochemical activity[52]. We were interested in the
preparation of β-lactam CA-4 type compounds containing an amino substituent in Ring B, and the
subsequent conversion to a water-soluble prodrug by conjugation with an amino acid. The nitro
containing C-3-vinyl-β-lactam 7p was successfully reduced to the amino product 7t using zinc dust
in the presence of acetic acid (Scheme 2). To investigate the effect of replacement of the 3,4,5-
trimethoxy ring A of CA-4 with 3,5-dimethoxy substituted ring A, the β-lactam 7u was prepared
in a similar route from the imine 5s. Tripodi et al. reported that 3,5-dimethoxy substituted ring A
compounds demonstrated comparable activity to the β-lactam compounds containing the 3,4,5-
trimethoxy ring A of CA-4 [53]. A further series of β-lactam compounds (8a–8k), was also prepared
containing the 3,4,5-trimethoxyphenyl substituent (Ring A of the Combretastatin A-4) at C-4
position, (Scheme 2).
The products of the Staudinger reaction with imines and crotonyl chloride show IR
-1
absorptions at approximately ν 1750 cm characteristic of the carbonyl group of the β-lactam ring.
All of the β-lactams were obtained with exclusively trans stereochemistry, with coupling constants
of 1–3 Hz for the β-lactam ring protons (e.g., for compound 7s, H-4 is identified as a doublet δ 4.69,
J
3,4 = 2.52 Hz). Coupling constants of 5–6 Hz are usually observed for β-lactams with cis
stereochemistry [46].
Subsequent to our initial biochemical evaluation of the 3-vinyl-β-lactam CA-4 analogues, a
further series of 3-substituted β-lactams was prepared from 3-unsubstituted β-lactams by aldol
type reaction with a suitable electrophile [54,55]. We were particularly interested in the
introduction of modified alkene substituents at C-3, due to the exceptional biochemical activity
displayed by the 3-vinyl β-lactam 7s. Lithium enolates of 3-unsubstituted β-lactams 9a and 9b were
reacted with selected aldehydes and ketones to provide alcohol products 10a–10i, (Scheme 3). The
β-lactams 9a and 9b were obtained via the Reformatsky reaction of ethyl bromoacetate with imines
5
h and 5o using microwave conditions. Treatment of 9b with tBAF afforded the phenol 9c.
Similarly, for the preparation of compounds 10a, 10c, 10d, 10f, 10g, 10h the initially obtained
tBDMS ether intermediate was subsequently deprotected in situ using tBAF to yield the desired
phenolic product. The enolate chemistry is stereoselective, favouring trans stereochemistry for the
1
products. The presence of a diastereomeric mixture for products is confirmed from the H NMR
spectra (e.g., for 10h where H-3 and H-4 appear as two sets of doublets, δ 3.20 and δ 4.83
respectively, with J = 2.4 Hz, ratio H
3
/H 1.14:1.00). To investigate the role of the alcohol group at
4
C-5 in the biochemical activity of the products 10a-10h, the alcohol 10i was oxidised to the
corresponding ketone 11 using pyridinium chlorochromate. An alternative route to 11 was
identified where treatment of the 3-unsubstituted β-lactam 9b with LDA followed by addition of
acetyl chloride to gave the desired product 11 but only in low yields (11%) with the alcohol 10a
also isolated (22%), (Scheme 3).

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Scheme 1. Synthesis of imines 5a–5s, 6a–6k. Reagents and conditions: (a) EtOH, conc H
2
SO , reflux,
4
4
h, (67–100%).

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Scheme 2. Synthesis of β-lactams 7a–7u, 8a–8k; Reagents and conditions: (a) triethylamine, CH
2
Cl2,
reflux, 5 h, (17–61%); (b) TBAF, dry THF, 0 °C, 30 min, (20%); (c) Zn dust, acetic acid, 20 °C, 7 days,
43%).
(

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Scheme 3. Synthesis of azetidinones 10a–10i, 11–13. Scheme reagents and conditions: (a) Zn,
BrCH CO Et, microwave, 100 °C, 30 min, (37–39%) (b) LDA, THF, R COR , −78 °C, 30 min, (17–
8%); (c) TBAF, THF, 0 °C, 1.5 h (for compounds 10a, 10c, 10d, 10f, 10g, 10h, 11, 12, 13), (17–52%);
d) Ph P, DEAD, CH Cl2, 0 °C, 3 min, (52%); (e) PCC, CH Cl , 20 °C, 18 h, (7%) (f) LDA, THF,
COCl, −78 °C, 30 min, (11%); (g) OsO , pyridine, 0 °C 1 min, then 20 °C, 22 h, (39%).
2
2
1
2
3
(
3
2
2
2
CH
3
4
To further investigate the role of the 3-vinyl substitution pattern in the biochemical activity
of. β-lactams, a 3-ethylidene product 12 was investigated. The initial route attempted involved the
chlorination of the alcohol 10i using thionyl chloride followed by dehydrohalogentation with a
suitable base such as DBU. However, a more successful method to give the 3-ethylidene β-lactams
was the dehydration of the alcohol 10i under Mitsunobu conditions and subsequent deprotection
by treatment with tBAF to yield 12 in 63% yield overall, (Scheme 3). The Peterson olefination of 3-
unsubstituted β-lactams has also been reported by Kano et al. as an alternative route to 3-
ethylidene β-lactams [56], while the Mitsunobu reaction for the dehydration of alcohols has been
described by Plantan et al. in the synthesis of a trinem β-lactamase inhibitor [57]. The product 12
was obtained as a mixture of Z/E isomers in a 1:1 ratio. The configuration of the separated isomers
was determined by examining the chemical shifts associated with the C-6 methyl protons. The
further downfield doublet signal (δ 2.05, J = 4.16 Hz) is more deshielded, and so is assigned to the
Z isomer while the signal at δ 1.62, (J = 4.40 Hz) is assigned to the E isomer [51].
The introduction of a diol functionality at C-3 was now explored. The diol 13 was synthesised
1
in 39% yield by the oxidation of the alkene 7s with osmium tetroxide (Scheme 3). The H NMR
spectrum for 13 clearly illustrates the formation of a diastereomeric product. H-3 appears as a pair

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of double doublets at δ 3.16 (0.7H) and δ 3.19 (0.3H) with coupling constants of 2.42 Hz and 5.55
Hz, while H-4 appears as two separate doublets at δ 4.90 (0.3H) and δ 5.00 (0.7H), J = 2.37 Hz.
The amino acid alanine was chosen for prodrug formation of the β-lactam 7t [58]. The
protected amino acid prodrug 14 was obtained from 7t using the coupling agent DCC with HOBt
in dry DMF (Scheme 4). The FMOC protecting group was easily removed from 14 by treatment
with 2N sodium hydroxide over 24 h to afford the amino acid prodrug conjugate 15 (57%).
Controlled esterification of the phenolic β-lactams 7s and 9c with dibenzyl phosphite using
diisopropylethylamine and dimethylaminopyridine afforded dibenzyl phosphate β-lactams 16a
and 16b respectively, (Scheme 4). The dimethyl and diethylethyl phosphate esters of compound
9
c, 16c and 16d respectively, were also prepared (Scheme 4). The phosphate 17a was obtained by
treatment of dibenzylphosphate ester 16a with bromotrimethylsilane. Hydrogenation of the
dibenzylphosphate ester 16b with palladium/carbon catalyst removed the dibenzyl protecting
groups and also reduced the double bond at C-3 position of the β-lactam ring to afford phosphate
1
7c. For the preparation of compound 17b, where removal of the benzyl protecting groups and
retention of the double bond was required, reaction of the dibenzyl phosphate ester 16b with
bromotrimethylsilane was effective.
Preliminary stability studies of the representative β−lactam 7s were carried out at acidic,
neutral and basic conditions (pH 4, 7.4 and 9) and in plasma using HPLC. The half-life (t ) was
½
determined to be greater than 24 h at pH 4, 7.4 and 9 and in plasma for compound 7s. The
phosphate esters 17b and 17c were also found to be stable over the range of pH and in plasma,
with half-life (t
7c was also investigated in whole blood. They were cleaved much more rapidly in whole blood
62% and 34% remaining after 6 h respectively) than in human plasma (94% and 92% remaining
½
) determined to be greater than 24 h. The cleavage of phosphate prodrugs 17b and
1
(
after 6 h respectively). Based on this stability study the β-lactam 7s would be suitable for further
development.

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Scheme 4. Synthesis of amino acid prodrugs 15,17a–c. Reagents and conditions: (a) Fmoc-L-
alanine, anhydrous DMF, DCC, HOBt.H O, 20 °C, 24 h (58%); (b) 2N NaOH aq, CH OH, CH Cl
0 °C, 24 h (57%); (c) for compounds 16a-16b, dibenzyl phosphate, DIPEA, DMAP, CCl
10 °C–20 °C, 3h (60–61%); for compound 16c, dimethyl phosphate, DIPEA, DMAP, CCl
10 °C–20 °C, 3h (52%); for compound 16d diethyl phosphate, DIPEA, DMAP, CCl , CH
2
3
2
2
,
2
–
–
4
, CH
3
CN,
CN,
4
, CH
3
4
3
CN, –10
°
6
C–20°C, 3h (79%); (d) for compounds 17a, 17b: bromotrimethylsilane, dry DCM, 45 min, 0 °C, (91–
3%); for compound 17c: H /Pd/C, ethanol-ethyl acetate, 1:1, 3 h, 20 °C, (98%).
2
2
.2. X-Ray Structural Study
The X-ray crystal structures of compounds 7h, 8i, 8k and 8h are displayed in Figure 3 and
confirm the structural assignment. The crystal data for the compounds are shown in Tables 1 and
. For each compound the two aryl rings at N-1 and C-4 position are in a pseudo cis arrangement
2
while the phenyl ring at C4 and the alkene group are on opposite sides of the β-lactam (trans
configuration). The structure of the compounds 7h, 8h, 8i and 8k clearly demonstrated a non-
coplanar configuration for rings A and B of the β-lactams, with the β-lactam ring providing a rigid
scaffold. For compound 7h even though both enantiomers are present, the compound crystallizes

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out in a chiral space group. The trans configuration of the aryl rings A and B at C-3 and C-4 is also
evident. The dihedral angle H3/H4 is observed for compounds 7h, 8h, 8i and 8k respectively, which
1
is consistent with the small trans coupling constant observed in the H NMR spectrum of 2.00 Hz,
2
.52 Hz, 2.48 Hz and 2.44 Hz respectively for these compounds. The β-lactam C=O bond lengths
are 1.209(3) Å, 1.214(3) Å, 1.2077(17) Å and 1.2077(17) Å for compounds 7h, 8h, 8i and 8k
respectively, which is consistent with data previously reported for the carbonyl bond length of
monocyclic β-lactams of 1.217(3) Å [59] and 1.207(2) Å [60]. The ring A/B torsional angles for
compounds 7h, 8h, 8i and 8k were observed as –59.5°, 59.7°, –73.5° and –77.0° respectively; these
values are significantly greater than those observed for the corresponding rings A/B in the DAMA-
colchicine 1b [5], Combretastatin A-4 2a [61] and related 4-arylcoumarin [62] as 53°, 55° and 48.3°
respectively (Table 2). The azetidinone N1-C4 bond length was observed at 1.372(3) Å, 1.376(3) Å,
1
1
.367(2) Å and 1.3767(18) Å for compounds 7h, 8h, 8i and 8k respectively, which compares with
.334(4)Å reported for the alkene C=C of combretastatin A-4 [61]. The C26-C27 alkene bond length
for 7h, 8h, 8i and 8k were observed at 1.303(3) Å, 1.3174 Å, 1.308(3) Å and 1.316(2) Å respectively,
while the alkene C=C bond length for iso-combretastatin CA-4 has been reported as 1.329(3) Å [63].
The C-N bonds lengths in the β-lactam ring are unequal with N1-C4 bond lengths of 1.487(3) Å,
1
.483(3) Å, 1.4774(19) Å and 1.4801(17) Å for compounds 7h, 8h, 8i and 8k respectively, compared
to 1.372(3) Å, 1.376(3) Å, 1.367(2) Å and 1.3767(18) Å for the N1/C2 bond in compounds 7h, 8h, 8i
and 8k respectively, indicating some degree of amide resonance [59].
Figure 2. ORTEP representation of the X-ray crystal structure of compound 6k with the thermal
ellipsoids set at 50% probability.

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(
A)
(
B)

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(
C)
(
D)
Figure 3. ORTEP representation of the X-ray crystal structure of (A) compound 7h, (B) compound
8
i, (C) compound 8j and (D) compound 8kwith the thermal ellipsoids set at 50% probability.
Table 1. Crystal data, details of data collections and refinement 6k, 7h, 8h, 8i, 8k.
Compound
6k
7h
8h
8i
8k
Empirical formula
C17
H
19NO
3
S
C21
H
23NO
5
C22
H
25NO
4
C21
H
23NO
5
C21
H
23NO S
4
M (g/mol)
Crystal System
SG
317.39
monoclinic
P2 (No. 4)
369.40
monoclinic
P2 (No. 4)
367.43
monoclinic
P2 /n (No. 14)
369.40
385.46
triclinic
triclinic
1
1
1
P1 (No. 2)
10.9022(5)
13.0315(6)
14.9787(8)
94.994(2)
105.024(2)°
108.284(2)°
1918.37(16)
100(2)
P1 (No. 2)
8.2131(4)
10.5047(5)
12.5704(6)
107.9218(16)°
96.7759(18)°
103.3909(18)°
982.64(8)
100(2)
a (Å)
7.8282(3)
20.1106(7)
7.2135(14)
26.440(5)
10.389(2)
-
b (Å)
7.7880(3)
9.1481(3)
c (Å)
13.2937(6)
22.6378(8)
α (°)
-
-
β (°)
106.4320(10)
110.6238(14)
101.41(3)
-
γ (°)
-
777.36(6)
100(2)
2
-
3897.9(2)
100(2)
8
3
V (Å )
1942.3(7)
150(2)
4
T (K)
Z
4
2

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Dcalc (g/cm3)
13 of 56
1.356
0.220 (Mo Kα)
27644
1.259
0.738 (Cu Kα)
59468
1.256
0.086 (Mo Kα)
14461
1.279
0.091 (Mo Kα)
29738
1.303
−
1
μ (mm )
Total reflns
Indep. reflns
R(int)
0.191 (Mo Kα)
14916
4394
14180
3492
9673
4903
0.0258
0.0602
0.0565
0.0532
0.0304
S
1.058
1.047
1.188
1.006
1.038
R
1
* [I > 2σ(I)]
0.0251
0.0364
0.0666
0.0509
0.0388
wR
2
* [all data]
Flack
0.0676
0.0945
0.1509
0.1191
0.0918
0.027(12)
1820354
CCDC number
1820355
1820358
1820356
1820357
*R
1
= ∑||Fo| − |F
c||/∑|F
o
|, wR
2
= [∑w(F
o
2
− F
c
2)2/∑w(F
) ] .
o
2 2 1/2
Table 2. X-Ray data and torsional angles for compounds 7h, 8h, 8i, 8k.
Ring Plane Normal
Ring A to Central
Torsion (°)a
177.0(2)
Ring B to Central
Torsion (°)b
137.7(2)
RingAB
Torsion (°)c
−59.5(3)
−53.8(5)
58.1(3)
Ring B Vinyl
Torsion (°)d
124.8(2)
Compound
AB Angle(°)
7
h*
82.79(8)
7
5.19(8)
00.51(8)
5.35(8)
179.4(2)
137.1(2)
123.8(2)
1
177.6(2)
−136.1(2)
−126.1(2)
−120.9(2)
−124.4(2)
127.75(18)
127.49(16)
122.95(15)
8
−167.5(2)
−138.9(2)
59.7(3)
8
h
93.14(7)
86.28(5)
−171.6(3)
−130.1(2)
59.7(3)
8
i§
170.67(17)
165.62(18)
162.50(17)
150.21(14)
155.26(16)
156.94(13)
−73.5(2)
−73.2(2)
−77.0(2)
8
5.68(6)
8
k
85.60(5)
a
b
c
d
C14-C13-N1-C2 C12-C5-C4-N1 C13-N1-C4-C5 C5-C4-C3-C26
C18-C17-N1-C2 C10-C5-C4-N1 C17-N1-C4-C5 C5-C4-C3-C26
C18-C17-N1-C2
C18-C17-N1-C2
C6-C5-C4-N1
C6-C5-C4-N1
C17-N1-C4-C5 C5-C4-C3-C26
C17-N1-C4-C5 C5-C4-C3-C26
C14-C13-N1-C2 C10-C5-C4-N1 C13-N1-C4-C5 C5-C4-C3-C26
*
Four independent molecules in the asymmetric unit. § = 2 independent molecules in the
asymmetric unit. Each angle given but only the first atom numbering scheme is outlined above.
2
.2. Biological Results and Discussion
2
.2.1. In vitro Antiproliferative Activities
The synthesized compounds were first evaluated for their antiproliferative activity against the
human breast cancer cell line MCF-7 and compared with CA-4 as a reference compound (IC50 = 3.9
nM).[64,65] The results are shown in Table 3 (7a–7n, 7p–7t, 8a–8k), and Table 4 (10a–h, 11–13, 15
and 17a–c). All β-lactams were evaluated as the trans isomer. The most potent compounds were
identified as 7s and 7t, with IC50 values of 8 νM and 17 nM respectively. Compound 7s is a direct
analogue of CA-4, while 7t is the corresponding amino compound and this type of substitution has
been demonstrated to confer potency in many CA-4 analogues[52]. Compounds having the
methoxy, ethoxy and thiomethyl substituents at C-4 of Ring B displayed potent antiproliferative
effects, with IC50 values of 20 nM, 37 nM and 51 nM respectively for compounds 7h, 7i and 7r

Pharmaceuticals 2019, 12, 56
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respectively. The halo substituted compounds, 7b and 7c and 4-methyl compound 7g were less
effective with IC50 values of 690 nM, 445 nM and 355 nM respectively. Selectivity in
antiproliferative effect was demonstrated by the 1 and 2-naphthyl compounds 7m (IC50=1.738 µM)
and 7n (IC50=68 nM). This result compares favourably with the naphthyl CA-4 analogues reported
by Medarde et al. in which the 2-naphthalene ring directly replaces the Ring B of CA-4 [66].
The IC50 of compound 7u containing the 3,5-dimethoxyphenyl Ring A was determined as 170
nM in MCF-7 cells, demonstrating retention of antiproliferative potency with slightly reduced
activity compared to the 3,4,5-trimethoxy ring A substituted compound 7h. This observation could
infer that the para-methoxy aryl group is less important for activity and the 3,5-dimethoxyaryl
substituted Ring A is favourable for interaction of the molecule with the colchicine binding site of
tubulin [53]. Compounds 8a–8k containing the 3,4,5-trimethoxyphenyl substituent (Ring A of CA-
4
) at the C-4 position were generally observed to have poorer antiproliferative activity than the
corresponding compounds 7a–7t, containing the 3,4,5-trimethoxyphenyl substituent (Ring A of the
Combretastatin A-4) at the N-1 position, (Table 3). The exceptions were compounds 8a (4-fluoro)
and 8j (4-NHCOCH
) with IC50 values of 1.066 µM and 4.024 µM respectively. The relative positions
3
of the 3,4,5-trimethoxyphenyl Ring A and Ring B on the β-lactam ring at positions N-1 and C-4
have a significant effect on the antiproliferative activity of the compounds as we previously
reported [45].
The effects of various structural modifications on the activity of the more potent 3-
vinylazetidinones were next explored, (Table 4). The most potent compound in this series is the 3-
styryl containing compound 10f, with IC50 = 46 nM. The alcohol 10a showed interesting activity (65
nM) while the introduction of an additional methyl group at C-5 to afford the alcohol 10d resulted
in reduced efficacy with IC50 = 544 nM. The diol 13 also proved noteworthy with IC50 = 69 nM. The
3
-acetyl compound 11 and 3-ethylidene compound 12 resulted in similar antiproliferative effects
IC50 = 414 nM and 502 nM respectively). Additional compounds containing the hydroxyalkene
substituent at C-3 (e.g., compounds 10b, 10c, 10e, 10g, 10h) were found to be moderately active
(
(
IC50 values 288–570 nM).
Table 3. Antiproliferative activities of β-lactams 7a–7n, 7p–7u, 8a–8k in human MCF-7 breast
cancer cells.
Compound Number Antiproliferative Activity IC50 (µM) a
7
a
8.314 ± 1.40
0.690 ± 0.11
0.445 ± 0.07
3.827 ± 0.53
4.047 ± 0.45
4.034 ± 0.42
0.355 ± 0.03
0.020 ± 0.0025
0.037 ± 0.0033
13.990 ± 1.81
57.041 ± 3.72
8.015 ± 0.63
1.738 ± 0.17
0.068 ± 0.01
0.618 ± 0.10
6.251 ± 5.05
7
b
7
c
7
d
7
7
e
f
7
g
7
h
7
7
i
j
7
k
7
l
7
7
7
7
m
n
p
q

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7
r
0.051 ± 0.001
0.008 ± 0.00071
0.017 ± 0.0018
0.170 ± 0.07.
1.066 ± 0.14
29.150 ± 1.14
10.400 ± 0.87
59.150 ± 4.16
68.840 ± 3.63
50.460 ± 4.25
43.130 ± 2.16
36.400 ± 2.13
65.120 ± 5.55
4.024 ± 0.64
>50
7
s
7
t
7
8
u
a
8
8
b
c
8
8
d
e
8
f
8
g
8
h
8
8
i
j
8
k
b
CA-4
0.0039 ± 0.00032
a
IC50 values are half maximal inhibitory concentrations required to block the growth stimulation
−
6
of MCF-7 cells. Values represent the mean ± SEM (error values × 10 ) for at least three experiments
performed in triplicate. bThe IC50 value obtained for CA-4 (0.0039 µM for MCF-7) is in good
agreement with the reported values [64,65].
The amino acid prodrug amide 15 was evaluated in MCF-7 breast cancer cells to determine if it
retained any antiproliferative activity when compared with the parent compound 7t which was
extremely potent with IC50 = 17 nM. The IC50 for 15 (3.251 µM) was lower than expected; however
metabolic activation in vivo may be required for the hydrolysis of the amide [67]. The phosphate
esters 17a–17c displayed impressive antiproliferative activity, with IC50 values of 22 nM, 27 nM and
2
1 nM respectively (Table 4). The IC50 values for the corresponding phenols 7s and 9c in MCF-7 cells
are 8 nM and 17 nM respectively. Comparison of the 3-vinyl 17b (IC50 = 27 nM) with the 3-ethyl 17c
IC50 = 21 nM) indicated that introduction of the 3-vinyl or 3-ethyl substituent, together with the 3-
(
unsubstituted 17a (22 nM) retains potency and optimum activity. The potent activity displayed for
the phosphate esters 17a–17c, together with the predicted improvement in water solubility, indicate
that these compounds are useful prodrugs for future development. Rapid in vivo dephosphorylation
would be expected to occur for the β-lactam phosphates 17a–c as observed for CA-4P [11].
Triple-negative breast cancers (TNBC) are characterised by the absence of estrogen receptors
(
ER-), progesterone receptors (PR-) and human epidermal growth factor receptor 2 (HER2-). TNBC
does not respond to hormonal therapy (such as tamoxifen or aromatase inhibitors) or therapies
that target HER2 receptors, such as Herceptin. Treatment options are limited leading to poor
prognosis, as indicated by low 5-year survival rates. A number of the more potent compounds
were evaluated in the triple negative MDA-MB-231 cell line (Table 5). Compound 7s was the most
effective of the series with an IC50 value of 10 nM. Compounds 7h, 7t, 17a, 17b and 17c were also
seen to be effective with IC50 values of 31 nM, 30 nM, 30 nM, 49 nM and 44 nM respectively, and
compared favourably with the positive CA-4 (control IC50 = 43 nM)[34,63,68].
Compound 7h was also evaluated in the triple-negative Hs578T breast cancer cell line and its
isogenic subclone Hs578Ts(i)8 cells to examine the activity of β-lactams as CA-4 analogues and as
anti-tubulin agents for metastasis. Hs578Ts(i)8 cells are 3-fold more invasive and 2.5-fold more
migratory than the parental cell line (Hs578T). In addition, Hs578Ts(i)8 cells had 30% more
CD44+/CD24-/low cells that could enhance the invasive properties but with a significantly
increased capacity to proliferate, migrate and produce tumours in vivo in nude mice [69].

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Compound 7h exhibited an excellent anti-proliferative activity in Hs578T cells (IC50 31 nM) and
interestingly retained potency in invasive Hs578Ts(i)8 cells (IC50 76 nM). The values for CA4 in
these cells were 8 nM and 20 nM respectively. These results could indicate the ability of β-lactams
as CA-4 analogues to inhibit tumour invasion and angiogenesis which are characteristic of tumour
growth and metastasis. These β-lactam compounds may provide potential development leads for
this subset of aggressive breast cancers. Compound 7s was also evaluated in the leukemia cell lines
HL-60 and K562 and was found to be extremely potent with IC50 values of 17 nM and 26 nM
respectively, comparing favourable with CA-4 [IC50 values of 4 nM (HL-60) and 4 nM (K562)].
The novel compounds 7h, 7s, 7t, 17b and 17c were selected for further investigation based on
analysis of their drug-like properties (Lipinski) from a Tier-1 profiling screen, together with
predictions of blood brain barrier partition, permeability, plasma protein binding, metabolic
stability and human intestinal absorption properties which confirmed that these compounds are
moderately lipophilic-hydrophilic drugs and are suitable candidates for further investigation
(
Tables S1 and S2, Supporting information).
Table 4. Antiproliferative activities of β-lactams 10a–h, 11–13, 15a and 17a–c in human MCF-7
a.
breast cancer cells
Compound Number Antiproliferative Activity IC50 (nM)a
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
65±15
292±50
5701±246
544±310
537±80
46±41
1
1
1
1
1
1
0g
0h
1
2
3
5
288±76
467±0.253
414±132
502±212
69±29
3251±270
22±1.5
27±2
21±1.5
39±3.2
1
1
1
7a
7b
7c
b
CA-4
a
IC50 values are half maximal inhibitory concentrations required to block the growth stimulation
−
6
of MCF-7 cells. Values represent the mean ± SEM (error values × 10 ) for at least three experiments
performed in triplicate. The IC50 value obtained for CA-4 (39 nM for MCF-7) is in good agreement
b
with the reported values [64,65].
Table 5. Antiproliferative activities of selected β-lactams in human MDA-MB-231 breast cancer
a
cells .
Compound Number Antiproliferative Activity IC50 (nM)a
7
b
191±16
31.7
b
7
h
i
61±7
77±9
10
b

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7
t
30±2
30±4
48.6
44±7
43
1
7a
b
1
7b
1
7c
c
CA-4
a
IC50 values are half maximal inhibitory concentrations required to block the growth stimulation
−
6
of MDA-MB-231 cells. Values represent the mean ± SEM (error values × 10 ) for at least three
experiments performed in triplicate. Antiproliferative activity against MDA-MB-231 from NCI
(
b
c
see Supplementary information). The IC50 value obtained for CA-4 in this assay is 43 nM for
MDA-MB-231 in good agreement with reported values for CA-4 in MTT assay on human MDA-
MB-231 breast cancer cell line [68],[34,63].
2
.2.2. Evaluation of β-Lactams in the NCI60 Cell Line Screen
A series of the more potent compounds 7h, 7s, 7t, 17b and 17c were evaluated in the National
Cancer Institute (NCI)/Division of Cancer Treatment and Diagnosis (DCTD)/Developmental
Therapeutics Program (DTP) [70], in which the activity of each compound was determined using
approximately 60 different cancer cell lines of diverse tumor origins. The results are summarized
in Tables S3, S4 and S5, Supplementary Information. The compounds were tested for inhibition of
growth (GI50) and cytotoxicity (LC50) in the NCI panel of cancer cell lines and showed excellent
broad-spectrum antiproliferative activity against tumor cell lines derived from leukemia, non-
small-cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, breast
cancer and prostate cancer [71] using the sulforhodamine B (SRB) protein assay [72], (Table S3, S4,
S5 Supplementary Information). The NCI results confirmed our in-house evaluations in MCF-7
cells with GI50 values for compounds 7h, 7s, 7t, 17b and 17c of 30.6, <10, <10, 39.4 and 25.1 nM
respectively.
Compound 7s, the most potent compound in our panel, demonstrated a mean GI50 value of 23
nM across all NCI cell lines tested. The GI50 values for 7s were in the sub-micromolar range for each
of the cell lines investigated, except for two cell lines (melanoma cell line UACC-257 and the breast
cancer cell line T-47D). For compound 7s the GI50 values obtained were below 10 nM for 28 of the
cell lines investigated and below 40 nM in all but eight of the panel cell lines tested. Activity was
demonstrated for compound 7s against all of the non-small cell lung (GI50 value 85.5 - <10 nM),
colon (GI50 value 429 - <10 nM), CNS (GI50 value 40.5 - <10 nM), ovarian (GI50 value 45.3 - <10 nM),
prostate (GI50 value <10 nM) and renal (GI50 value 40.2 - <10 nM) cancer cell lines tested. The mean
GI50 values over the full 60 cell line panel for compounds 7h, 7t and 17c of 52, 48 and 73 nM
respectively (see Supplementary Information, Tables S5) compares very favourably with the GI50
value for CA-4 of 99 nM.
LC50 values for compound 7s were greater than 100 µM in all but three cell lines tested
indicating minimal toxicity and the potential use of this compound for a wide range of therapeutic
applications (Tables S3, S4, S5 Supplementary Information). A similar result was obtained for
compound 7h with LC50 values > 100 µM in all cell lines tested.
The NCI COMPARE algorithm allows a comparison of the activities of β-lactams 7h and 7s
with compounds of a known mechanism of antiproliferative action in the NCI Standard Agents
Database. Compounds 7h and 7s showed high correlation to tubulin targeting agents such as
maytansine, rhizoxin and the clinically important anticancer drugs vincristine and vinblastine, (see
Supplementary Information, Tables S6 and S7).
2
.2.3. Evaluation of Toxicity of 7s in Normal Murine Mammary Epithelial Cells

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The cytotoxic effect of a selected number of 3-vinyl-β-lactams in MCF-7 cells at 10 µM
concentration was initially determined in the lactate dehydrogenase (LDH) assay[73]. The 3-vinyl-
β-lactams 7d, 7h, 7i, 7q, 7r and 7u resulted in low cytotoxicity with 7.2%, 2.4%, 8.5%, 4.5%, 4.5%
and 3.5% cell death respectively while compounds 7s and 7t displayed increased cytotoxicity of
1
6.1% and 25% cell death in this assay. The 3-(1-hydroxyl-1-methylethyl) and 3-(1-hydroxy-1-
phenylallyl) substituted compounds 10d and 10f resulted in 9.8% and 7.6% cell death respectively
while cell death of 8.4% was obtained for the 3-ethylidene compound 12. CA-4 was used as the
positive control in this assay and resulted in 11.8% cell death at 10 µM concentration.
The cytotoxicity of the most potent compound 7s on non-tumourigenic cell line HEK-293
normal human embryonic kidney) was also investigated. We demonstrated an IC50 value greater
(
than 5 µM in HEK-293T cells for 7s. Cell viability of HEK-293T cells was significantly higher than
MCF-7 cells at 10, 1 and 0.5 µM concentrations of compound 7s (Figure 4A), demonstrating the
lack of cellular toxicity of the compounds in these non-cancerous cells.
Further toxicity studies were carried out on the most potent compound β-lactam 7s in primary
cells (mouse mammary healthy epithelial cells) at two different cell concentrations (25,000 and
5
0,000 cells/mL), with CA4 as a positive control. The cells were harvested from mid- to late-
pregnant CD-1 mice and were cultured as previously reported [74,75]. Both CA-4 [76] and 7s were
not cytotoxic at concentrations up to 10 µM in the NCI cell line panel (See Tables S8 and S9
Supplementary Information). The IC50 values for both compounds 7s and CA-4 evaluated in
normal murine mammary epithelial cells was greater than 10 µM which indicated a minimal
toxicity for these compounds (Figure 4B). At both 25,000 cells/mL and 50,000 cells/mL and a
concentration of 10 µM, CA-4 was lethal to the highest percentage of cells. The percentage of viable
murine mammary epithelial cells at the IC50 value of each compound in MCF-7 cells (see Table 3)
was calculated in order to give an estimation of the toxicity at this value. At 50,000 cells/mL, over
9
0% of cells were viable after 72 h for compound 7s, (Figure 4B). At 25,000 cells/mL, the percentage
of cells remaining viable after treatment with compound 7s for 72 h was 93%, compared to 74% for
CA-4. (Supplementary Information Tables S8, S9). These results indicate a favourable toxicity
profile for 7s in comparison to CA4. This provides further evidence, in addition to the NCI60 LC50
values for 7s, that the β-lactam compound developed in this study is minimally toxic to cells that
are not proliferating.
(
A)

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(
B)
Figure 4. (A) Antiproliferative activity of β-lactam 7s in tumorigenic MCF-7 cells and non-
tumourigenic HEK-293T cells. Values represent the mean for two independent experiments.
Statistical analysis was performed using a non-paired two-tailed t-test (ns, not significant; **, p <
0
.01). (B) Cell viability for compound 7s and CA-4 in murine mammary epithelial cells. Mouse
mammary epithelial cells were harvested from mid- to late- pregnant CD-1 mice and cultured. The
isolated mammary epithelial cells were seeded at 50,000 cells/mL. After 24 h, they were treated
with 2 µL volumes of test compound which had been pre-prepared as stock solutions in ethanol to
furnish the concentration range of study, 1 nM–100 µM, and re-incubated for a further 72 h. Control
wells contained the equivalent volume of the vehicle ethanol (1%, v/v). The cytotoxicity was
assessed using alamar blue dye.
2
.2.4. Effect of β-Lactam 7s on Cell Cycle and Apoptosis
It is well recognised that tubulin destabilizing agents arrest the cell cycle in the G /M phase
2
due to cytoskeleton disruption and microtubule depolymeriztion. The effects of β-lactam 7s on cell
cycle events and induction of apoptosis in MCF-7 cells were next explored. Initial analysis by flow
cytometry of propidium iodide stained MCF-7 cells showed G
64% (10 nM) and 82% (100 nM)] (Figure 5C). A time dependent increase in the percentage of
apoptotic cells (sub-G ) after 72 h (14% and 26% respectively for 10 nM and 100 nM
concentration) was also evident compared to the vehicle control (6% at 72 h), (Figure 5A), with a
corresponding decrease of cells in the G0- phase of the cell cycle, (Figure 5C). The positive control
CA-4 (100 nM) showed 52% of cells in G M arrest at 48 h, and 9.4% in the sub-G population.
2
M arrest at 24 h by compound 7s
[
0
G1
G
2
1
0
G1
(
A)

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(
B)
(
C)
Figure 5. Effect of compound 7s on the cell cycle and apoptosis in MCF-7 cells. Cells were treated
with either vehicle [0.1% ethanol (v/v)], 7s (10 nM and 100 nM) for 24 h, 48 h and 72 h. Cells were
then fixed, stained with PI, and analyzed by flow cytometry. Cell cycle analysis was performed on
histograms of gated counts per DNA area (FL2-A). The number of cells with (A) <2N (sub-G
1
), (B)
2
N(G ), and (C) 4N (G /M) DNA content was determined with CellQuest software. Values
0
G
1
2
represent the mean ± SEM for three independent experiments. Statistical analysis was performed
using two-way ANOVA (**, p < 0.01; ***, p < 0.001).
To characterize the mode of cell death induced by 7s in MCF-7 cells, analysis of apoptosis was
performed using propidium iodide (PI), which stains DNA and enters only dead cells, and
annexin-V, which binds selectively to phosphatidyl serine (Figure 6). Dual staining for annexin-V
and PI facilitates discrimination between live cells (annexin-V-/PI-), early apoptotic cells (annexin-
V+/PI-), late apoptotic cells (annexin-V+/PI+) and necrotic cells (annexin-V-/PI+). Each
concentration induced an accumulation of annexin-V positive cells when compared to the vehicle
control (5%), Figure 6. About 13.6% of cells were found to be apoptotic (annexin-V positive) when
treated with compound 7s at 10 nM for 72 h. With an increase in concentration of 7s, 31.9% of cells
were found to be apoptotic at 100 nM. The positive control CA-4 (50 nM) resulted in 34.6%
apoptotic cells. The observed effect of compound 7s on cell cycle resulting in G
M arrest followed
2
by apoptosis is typical of tubulin targeting compounds. However, we have previously reported
that prolonged exposure of colon cancer cells CT-26, CaCo-2 and HT-29 to our structurally related

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3
-aryl-β-lactams induced autophagy [77]; it is possible that autophagy may be the cell death
mechanism in the present case, because of the level of apoptosis observed.
(
A)
***
*
**
*
40
*
control
CA-4
3
0
7s (10 nM)
s (100nM)
7
20
10
0
s
s
i
i
s
s
s
si
o
t
o
to
p
p
o
o
opt
p
p
a
l a
y
l
e ap
lat
r
ta
a
to
E

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(
B)
Figure 6. Compound 7s potently induces apoptosis in MCF-7 cells (Annexin V/PI FACS). (A) Effect
of compound 7s and CA-4 on apoptosis in MCF-7 cells analysed by flow cytometry after double
staining of the cells with Annexin-V-FITC and PI. MCF-7 cells treated with 10 and 100 nM of
compound 7s or 50 nM of CA-4 for 72 h and collected and processed for analysis. (B) Quantitative
analysis of apoptosis. Values represent the mean ± SEM for three independent experiments.
Statistical analysis was performed using two-way ANOVA (**, p < 0.01; ***, p < 0.001).
2
.2.5. Tubulin Polymerization Studies
The effect of selected β-lactam CA-4 compounds (7h, 7i, 7s, 7t) which demonstrated the most
potent antiproliferative effects in vitro was assessed on the assembly of purified bovine tubulin.
CA-4 which effectively inhibits the assembly of tubulin was used as a positive control, while
paclitaxel was used to demonstrate effective tubulin polymerization. Tubulin polymerization was
determined for compounds 7h, 7i and 7t at 10 µM for 30 min and compound 7s at 1, 5 and 10 µM
for 60 min by measuring the increase in absorbance at 340 nm, (Figure 7A, Figure 7B) [78]. The
degree of light scattering by microtubules is proportional to their degree of polymerization. For
the paclitaxel control the vmax was found to be 89.4 mOD/min. The vmax value provides a sensitive
indication of the tubulin/ligand interactions for the tubulin polymerization. The most potent
antiproliferative compound 7s (10 µM) demonstrated a significant 8.7-fold reduction in vmax value
while exposure to CA-4 (10 µM) brings about a 5.28-fold reduction in the vmax value. Compound
7
s compares very favourably to CA-4 in this respect. These effects are in good agreement with the
antiproliferative data recorded for both CA-4 (IC50 = 4.2 nM) and 7s (IC50 = 8 nM) in the MCF-7 cell
line. The vmax value for compounds 7h, 7i and 7t was determined as 3.43, 3.84 and 0.92 mOD/min
respectively, together with the fold-reduction in the vmax values of 2.45, 2.19 and 9.15 respectively
for the tubulin polymerization with reference to ethanol control. These results confirm that the
molecular target of these antiproliferative 3-vinyl-β-lactams is tubulin and that they are
microtubule-destabilising agents.
The dose-dependent effect of 7s on tubulin polymerization is illustrated in Figure 7. Exposure
of the tubulin to 10 µM, 5 µM and 1 µM of 7s resulted in a dose-dependent fold reduction of vmax
of 8.70, 7.31 and 2.61 respectively while the IC50 value for 7s for the inhibition of polymerization
was calculated to be 1.37 µM, Figure 7. Taken together, these results demonstrate that for these
novel β-lactam containing CA4 analogues, antiproliferative activity against the MCF-7 cell line and
the inhibition of tubulin polymerization are closely related. It has also been shown that the most
potent antiproliferative compound synthesised (7s) inhibits tubulin polymerization to a greater
extent than CA-4.
2
.2.6. Immunofluorescence Microscopy
Alterations in the microtubule network induced by β-lactam 7s in MCF-7 cells were
investigated using immunofluorescence and confocal microscopy (Figure 8). A well organised
microtubular network was observed in MCF-7 control cells when stained with α-tubulin mAb
(
Figure 8) and in untreated cells (data not shown). Formation of microtubule bundles and pseudo
asters was demonstrated for cells when exposed to paclitaxel (a microtubule-stabilising agent),
Figure 8 [79]. A complete loss of microtubule formation was induced in cells exposed to CA-4 or
β-lactam 7s for 16 h. This effect is consistent with depolymerised microtubules. Following
treatment with CA-4 or β-lactam 7s, MCF-7 cells were observed to contain multiple micronuclei.
Mitotic catastrophe resulting from premature or inappropriate entry of cells into mitosis is a type

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of programmed cell death in response to DNA damage, and is characterised by multinucleated
cells [80]. CA-4 induced mitotic catastrophe has also been reported in non-small cell lung cancer
cells [81,82], human endothelial cells (HUVEC) [83], human lung carcinoma cells (H460) [83] and
human breast cancer cells (MCF-7) [84]. Taken together with the effects demonstrated above in
section 2.2.5 on the inhibition of polymerisation of isolated tubulin, the confocal imaging results
confirm that β-lactam 7s is targeting tubulin.
(
A)
(
B)
Figure 7. (A) Effect of compounds 7h, 7i, 7t on tubulin polymerization in vitro. (B) Tubulin
polymerization assay for compound 7s at 10 µM, 5µM and 1µM. Paclitaxel (10 µM) and CA4 (10
µM) were used as references while ethanol (1% v/v) was used as a vehicle control. Purified bovine
tubulin and GTP were mixed in a 96-well plate. The polymerization reaction was initated by
warming the solution from 4 °C to 37 °C. 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. DMSO. Fold

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inhibition of tubulin polymerization was calculated using the Vmax value for each reaction. The
results represent the mean for three separate experiments.
Figure 8. CA-4 and β-lactam (7s) depolymerise the microtubule network of MCF-7 cells. MCF-7
cells were treated with vehicle control [1% ethanol (v/v)], paclitaxel (1 µM), CA-4 (100 nM) or 7s
(trans isomer, 100 nM) for 16 h. Cells were fixed in 4% paraformaldehyde and stained with mouse
monoclonal anti-α-tubulin−FITC antibody (clone DM1A) (green), Alexa Fluor 488 dye and
counterstained with DAPI (blue). Images were captured by Leica SP8 confocal microscopy with
Leica application suite X software. Representative confocal micrographs of three separate
experiments are shown. Scale bar: 30 µM (top images); 10 µM (bottom images).
2
.2.7. Interaction of β-Lactam 7s with Colchicine Binding Site of Tubulin
The binding of the lead compound 7s to the colchicine binding site of tubulin was confirmed
in a whole cell-based assay. N,N’-ethylene-bis(iodoacetamide) (EBI) is an alkylating agent that
cross-links cysteine residues 239 and 354 in the colchicine-binding site of tubulin to form the β-
tubulin-EBI adduct that migrates faster than β-tubulin [85,86], and is detected by Western blotting.
However, when the MCF-7 cells are pre-treated with colchicine or a colchicine-site ligand such as
CA-4, the formation of the β-tubulin-EBI adduct is prevented. The MCF-7 cells were initially
treated with selected β-lactam 7s (10 µM) or CA- 4 for 2 h, then followed by addition of EBI for a
further 1.5 h (Figure 9). The presence of the β-tubulin-EBI adduct was demonstrated for the control
samples (no drug) at a lower position on the gel, indicating that EBI has cross-linked Cys239 and
Cys354 on β-tubulin. When the cells are treated with β-lactam 7s and CA-4, the EBI adduct
formation is inhibited, indicating that 7s is interacting with tubulin at the colchicine site of tubulin.
Figure 9. Colchicine binding assay: Effect of compound 7s on the inhibition of the bisthioalkylation
of Cys239 and Cys354 of β-tubulin by N,N’-ethylene-bis(iodoacetamide) (EBI) in MCF-7 cells. MCF-

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7
cells were treated with vehicle control [ethanol 0.1% (v/v)], CA-4 and 7s (10 µM) for 2 h; selected
samples were then treated with EBI (100 µM) for an additional 1.5 h. Cells were harvested, lysed
and analysed using sedimentation and Western blotting for β-tubulin and β-tubulin-EBI adduct.
2
.3. Molecular Modelling Studies
The 3-vinyl-β-lactam compound 7s represents the most potent compound synthesised in the
study with IC50 value of 8 nM in MCF-7 breast cancer cells. The tubulin binding and
immunofluorescence studies of 3-vinyl-β-lactam 7s have demonstrated that the colchicine binding
site of tubulin is the target for the compound. Flexible alignment of compound 7s with CA-4
resulted in a good degree of overlap between the trimethoxyphenyl rings (Ring A) and the phenolic
hydroxyl group of ring B (Figure 10A). The energy minimised structure of compound 7s
demonstrates the inter-atomic distances of the oxygens of the methoxy groups of ring A and ring
B as 9.17 Å, which is similar to that calculated for CA-4 (9.27 Å).
The X-ray structure of CA-4 co-crystallised with tubulin has been determined suggesting that
cis-CA-4 inhibits tubulin polymerization by preventing the transition from curved to straight
tubulin [49]. The X-ray structure of cis and trans stereoisomers of a 3-methyl-1,4-diarylazetidinone
[
87] co-crystallised with tubulin was reported by Zhou et al. [37,38]. In the present study the
potential interaction of our novel synthesised 3-vinyl-β-lactams with the colchicine binding site of
tubulin, a series of docking calculations using MOE 2018.0101 [88] was undertaken on both the
3
S/4R and 3R/4S enantiomers of the β–lactams 7s and 7t using the tubulin co-crystallised with
DAMA-colchicine X-ray crystal structure (PDB entry 1SA0) [5]. Only results for the 3S/4R studies
will be discussed as these stereoisomers were more highly ranked than the 3R/4S enantiomer and
this is also supported by the crystallographic evidence [37,38]. The 3S/4R enantiomers of the
hydroxyl 7s and amino 7t substituted analogues overlay their B-rings on the C-ring of DAMA-
colchicine, collocate the trimethoxyphenyl substituents, overlap the 3-hydroxyl/amino groups on
the DAMA-colchicine carbonyl oxygen atom and form HBA interactions with Lys β352 as shown
in Figure 10B and 10C. The 3,4,5-trimethoxyphenyl groups of all analogues are able to make
favourable van der Waals contacts within the lower subpocket delineated by Val β318 and Cys
β241. The β-lactam carbonyl oxygen atom can make an HBA interaction with the backbone amine
of Asp β251 for both analogues. For both compounds, the trans geometry at C3/C4 facilitates a more
favourable interaction of rings A and B with the residues of the β-tubulin colchicine binding site.
Protein-ligand interactions for 7s are illustrated in Figure 11. The enantioselective synthesis of 7s
and 7t are in progress which will provide the optimum configuration of these compounds to be
determined for biological activity.
(
A)

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(
B)
(C)
Figure 10. (A): Flexible alignment of 7s (yellow) and CA-4 (2a)(red). (B) and (C): Overlay of the X-
ray structure of tubulin cocrystallised with DAMA-colchicine (PDB entry 1SA0) on the best ranked
docked poses of the 3S/4R enantiomers of (B) 7s and (C) 7r. The B-ring substituted analogues both
overlap well on the C-ring of DAMA-colchicine. Ligands are rendered as tube and amino acids as
line. Tubulin amino acids and DAMA-colchicine are coloured by atom type, 7s orange and 7t green.
The atoms are coloured by element type, carbon = grey, hydrogen = white, oxygen = red, nitrogen
=
blue, sulphur = yellow. Key amino acid residues are labelled and multiple residues are hidden to
enable a clearer view.

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Figure 11. Protein-ligand interactions for the 3-vinyl-β-lactam compound (7s). 2D representation
of the ligand−protein interactions of 7s with the colchicine-binding site rendered using LigX
module of MOE.
3
. Conclusion
We have developed an interesting series of 3-vinylazetidinones which selectively modulate
the activity of the tubulin protein, resulting in significant cytotoxicity to cancer cells and minimum
cytotoxic effects to normal cells. Molecular modelling studies indicated that these compounds
could interact with the colchicine binding site of tubulin, and consequently disrupt tubulin
polymerization. X-Ray crystallographic studies confirmed that the torsional angle between Ring A
and Ring B of the β-lactam was similar to CA-4 and was important in maintaining antiproliferative
and tubulin disrupting activity. Biochemical evaluation of these compounds coupled with a
molecular modeling study contributes to our understanding of the attributes of the 3-
vinylazetidinones such as 7s and 7t that result in exceptional antiproliferative activity and dose-
dependent microtubule assembly inhibition. Analysis of DNA content by flow cytometry
demonstrated that the cells were arrested in the G
by an increase in the sub-G population, which was confirmed by Annexin-V staining.
Immunofluorescence staining with α-tubulin antibodies in MCF-7 cells demonstrated disorder and
2
/M phase; induction of apoptosis was confirmed
0
G1

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fragmentation of the microtubule network and disruption of mitotic spindle formation. The
phosphate prodrugs 17a–c were found to retain antitumour potency. The potent antiproliferative
activity of the 3-vinylazetidinones 7s and 7t in breast cancer cells MCF-7 and notably in the triple
negative MDA-MB-231 cell line reported in the present study compare very favourably with
examples from the related series of 3-arylazetidinone compounds previously reported by our
research group [45]. Vinyl substitution at C-3 of these azetidinones also results potent tubulin
destabilizing effects in these derivatives of combretastatin A-4.
In summary, these novel 3-vinyl-β-lactam analogues of CA-4 which we now report show
potent antiproliferative effects in preliminary in vitro investigations on MCF-7 and MDA-MB231
breast cancer cells. Further studies to establish the long-term effect of these compounds on cancer
cell growth, migration and the potential vascular disrupting effects of these molecules are ongoing.
4
. Experimental Section
4
.1. Chemistry
All reagents were commercially available and were used without further purification unless
otherwise indicated. Tetrahydrofuran (THF) was distilled immediately prior to use from
Na/Benzophenone under a slight positive pressure of nitrogen, toluene was dried by distillation
from sodium and stored on activated molecular sieves (4Å) and dichloromethane was dried by
distillation from calcium hydride prior to use. Uncorrected melting points were measured on a
Gallenkamp SMP 11 melting point apparatus. Infra-red (IR) spectra were recorded as thin film on
NaCl plates, or as potassium bromide discs on a Perkin Elmer FT-IR Spectum 100 spectrometer,
1
13
(
(
PerkinElmer Inc., 940 Winter Street, Waltham, MA, USA). H and C nuclear magnetic resonance
NMR) spectra were recorded at 27°C on a Brucker Avance DPX 400 spectrometer (Bruker, 40
1
13
Manning Road, Billerica, MA, USA), (400.13 MHz, H; 100.61 MHz, C) at 20 °C in either CDCl
3
(
internal standard tetramethylsilane TMS) or CD
3
OD by Dr. John O’Brien and Dr. Manuel Ruether
1
in the School of Chemistry, Trinity College Dublin. For CDCl
relative to the TMS peak at δ 0.00 ppm and C-NMR spectra were assigned relative to the middle
CDCl
3
, H-NMR spectra were assigned
1
3
1
13
3
triplet at δ 77.00 ppm. For CD
3
OD, H and C-NMR spectra were assigned relative to the
centre peaks of the CD
3
OD multiplets at δ 3.30 and 49.00 ppm respectively. Electrospray ionisation
mass spectrometry (ESI-MS) was performed in the positive ion mode on a liquid chromatography
time-of-flight (TOF) mass spectrometer (Micromass LCT, Waters Ltd., Manchester, UK) equipped
with electrospray ionization (ES) interface operated in the positive ion mode at the High Resolution
Mass Spectrometry Laboratory by Mr. Brian Talbot in the School of Pharmacy and Pharmaceutical
Sciences, Trinity College Dublin and Dr. Martin Feeney in the School of Chemistry, Trinity College
Dublin. Mass measurement accuracies of < ±5 ppm were obtained. Low resolution mass spectra
(
LRMS) were acquired on a Hewlett-Packard 5973 MSD GC-MS system in electron impact (EI)
mode, (Hewlett-Packard, 6280 America Center, San Jose, CA, USA). R values are quoted for thin
f
layer chromatography on silica gel Merck F-254 plates, unless otherwise stated, (Merck, 2000
Galloping Hill Road, Kenilworth, NJ, USA). Flash column chromatography was carried out on
Merck Kieselgel 60 (particle size 0.040–0.063 mm). Chromatographic separations were also carried
out on Biotage SP4 instrument, (Biotage AB, Box 8, Uppsala, Sweden). 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 and a
Waters 717plus Autosampler, (Waters, 34 Maple St, Milford, MA, USA). The column used was a
Varian Pursuit XRs C18 reverse phase 150 x 4.6 mm chromatography column (Agilent

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Technologies, 5301 Stevens Creek Blvd, Santa Clara, CA, USA). Samples were detected using a
wavelength of 254 nm. Imines 5a [87], 5b [89], 5c [46], 5d [46], 5e [84], 5f [90], 5g [91], 5h [45], 5i
[
84], 5m [46], 5n [84], 5o [45], 5p [45], 5q [46], 5r [46], 5s [53], 6a [92], 6b [93], 6c [94], 6f [45], 6g [95],
h [96], 6i [45], 6j [97], 6k [96] were prepared as previously reported (See Supplementary
Information).
6
4
.1.1. 3-(tert-Butyldimethylsilyloxy)-4-methoxybenzaldehyde.
To solution of 3-hydroxy-4-methoxybenzaldehyde (20 mmol) and tert-butyl-
dimethylsilylchloride (24 mmol) in dry CH Cl (60 mL) under a nitrogen atmosphere, 1,8-
a
2
2
diazabicyclo[5.4.0]undec-7-ene (DBU) (32 mmol) was added dropwise via syringe. The resulting
mixture was stirred at room temperature under a nitrogen atmosphere until reaction was complete
on thin layer chromatography. The solution was then diluted with CH
successively with water (60 mL), 0.1M HCl (60 mL) and saturated aqueous NaHCO
retaining the organic layer each time, before drying over anhydrous Na SO4. The solvent was
removed under reduced pressure to yield the protected benzaldehyde, yield 82%.[45] IR (NaCl,
2
Cl
2
(80 mL) and washed
(60 mL),
3
2
-
1 1
film) νmax: 1692 (C=O) cm . H NMR (400 MHz, CDCl ): δ 0.19 (s, 6H), 1.02 (s, 9H), 3.91 (s, 3H), 6.97
(
3
1
3
d, J = 8.56 Hz, 1H), 7.38 (d, J = 2.00 Hz, 1H), 7.48–7.51 (m, 1H), 9.84 (s, 1H). C NMR (100 MHz,
CDCl
3
): δ -5.07, 17.97, 25.19 (OTBDMS), 55.13, 110.71, 119.63, 125.82, 129.75, 145.12, 156.16, 190.48.
+
HRMS: found 266.1349 (M ); C14
H
22
O3Si requires 266.1338.
4
.1.2. General method I: Preparation of imines 5a–5s, 6a–6k.
The appropriately substituted benzaldehyde (10 mmol) and corresponding substituted aniline
10 mmol) were heated reflux in ethanol (40 mL) for 4 h with a catalytic amount of concentrated
(
sulphuric acid. The volume of reaction was then reduced to approximately 10 mL in vacuo. The
Schiff base precipitated from solution upon standing at room temperature overnight. The solid
product obtained was filtered and purified by recrystallisation from ethanol.
4
.1.2.1. (E)-1-(4-Butoxyphenyl)-N-(3,4,5-trimethoxyphenyl)methanimine (5j).
Preparation as described above from 4-butoxybenzaldehyde and 3,4,5-trimethoxyaniline. The
product was obtained as pale yellow solid, yield 67%, Mp: 107–108 °C. IR (KBr) νmax: 1607 (C=N)
-1 1
cm . H NMR (400 MHz, CDCl
3
): δ 1.01 (t, J = 7.26Hz, 3H), 1.51–1.56 (m, 2H), 1.79–1.84 (m, 2H),
3
2
1
.88 (s, 3H), 3.92 (s, 6H), 4.06 (t, J = 6.48 Hz, 2H), 6.42 (br s, 2H), 7.00 (d, J = 8.52 Hz, 2H), 7.87 (br s,
1
3
H), 8.41 (s, 1H). C NMR (100 MHz, CDCl
3
): δ 13.87, 19.24, 31.22, 56.13, 61.04, 67.93, 98.11, 114.76,
+
28.57, 130.65, 136.12, 148.17, 153.56, 159.24, 162.09. HRMS: found 344.1859 (M +H); C20
H
26NO
4
requires 344.1862.
4
.1.2.2. (E)-1-(4-Phenoxyphenyl)-N-(3,4,5-trimethoxyphenyl)methanimine (5k).
Preparation as described above from 4-phenoxybenzaldehyde and 3,4,5-trimethoxyaniline.
The product was obtained as pale yellow solid, yield 74%, Mp 86–88 °C. IR (KBr) νmax: 1631 (C=N)
-
1 1
cm . H NMR (400 MHz, CDCl ): δ 3.88 (s, 3H), 3.92 (s, 6H), 6.51 (s, 2H), 7.08–7.10 (m, 5H), 7.39–
3
1
3
7
.41 (m, 2H), 7.89 (d, J = 7.52 Hz, 2H), 8.45 (s, 1H). C NMR (100 MHz, CDCl ): δ 55.68, 60.58, 97.68,
3
1
17.80, 119.34, 123.78, 124.51, 129.53, 130.10, 135.83, 147.48, 153.12, 155.57, 158.32, 160.06. HRMS:
+
found 364.1534 (M +H); C22
H
22NO requires 364.1549.
4
4
.1.2.3. (E)-1-(4-(Benzyloxy)phenyl)-N-(3,4,5-trimethoxyphenyl)methanimine (5l).

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Preparation as described above from 4-(benzyloxy)benzaldehyde and 3,4,5-trimethoxyaniline.
The product was obtained as colourless solid, yield 79%, Mp 113–115 °C. IR (KBr) νmax: 1623 (C=N)
-
1 1
cm . H NMR (400 MHz, CDCl
3
): δ 3.88 (s, 3H), 3.92 (s, 6H), 5.17 (s, 2H), 6.51 (s, 2H), 7.09 (d, J = 7.84
1
3
Hz, 2H), 7.41–7.46 (m, 5H), 7.89 (d, J = 7.84 Hz, 2H), 8.42 (s, 1H). C NMR (100 MHz, CDCl
3
): δ
5
1
5.67, 60.58, 69.66, 97.65, 114.69, 127.90, 127.06, 127.75, 128.23, 128.30, 128.66, 130.16, 135.97, 147.19,
+
53.10, 158.63, 161.07. HRMS: found 378.1713 (M +H); C23
H
24NO requires 378.1705.
4
4
.1.2.4. (E)-N-(4-Nitrophenyl)-1-(3,4,5-trimethoxyphenyl)methanimine (6d).
Preparation as described above from 3,4,5-trimethoxybenzaldehyde and 4-nitroaniline. The
-1
product was obtained as yellow solid, yield 72%, Mp 161–162 °C. IR (KBr) νmax: 1627 (C=N) cm .
1
H NMR (400 MHz, CDCl ): δ 3.96 (s, 3H), 3.97 (s, 6H), 7.20 (s, 2H), 7.27 (d, J = 8.52 Hz, 2H), 8.28 (d,
3
1
3
J = 9.04 Hz, 2H), 8.34 (s, 1H). C NMR (100 MHz, CDCl
1
3
3
): δ 55.82, 55.85, 60.62, 105.93, 120.86, 124.62,
+
30.12, 141.48, 144.97, 153.12, 155.56, 161.70. HRMS: found 317.1135 (M +H); C16
H
17
N
2
O5 requires
17.1137.
4
.1.2.5. (E)-N-(4-(tert-Butyl)phenyl)-1-(3,4,5-trimethoxyphenyl)methanimine (6e).
Preparation as described above from 3,4,5-trimethoxybenzaldehyde and 4-(tert-butyl)aniline.
-1 1
The product was obtained as red solid, yield 100%, Mp 81–82 °C. IR (KBr) νmax: 1623 (C=N) cm . H
NMR (400 MHz, CDCl ): δ 1.37 (s, 9H), 3.94 (s, 3H), 3.97 (s, 6H), 7.15–7.21 (m, 4H), 7.44 (d, J = 8.52
Hz, 2H), 8.40 (s, 1H). C NMR (100 MHz, CDCl
1
3
3
1
3
3
): δ 30.98, 34.07, 55.82, 60.55, 105.29, 120.10, 125.61,
+
31.31, 140.41, 148.59, 148.70, 153.05, 158.78. HRMS: found 328.1899 (M +H); C20
H
26NO requires
3
28.1913.
4
.1.3. General method II: Preparation of 2-azetidinones 7a–7u, 8a–8k
To a stirring, refluxing solution of the imine (5 mmol) and triethylamine (6 mmol) in
anhydrous dichloromethane (40 mL), a solution of crotonyl chloride (6 mmol) in anhydrous
dichloromethane (10 mL) was injected dropwise through a rubber septum over 45 min under
nitrogen. The reaction was heated at reflux for 5 h and stirred at room temperature overnight,
continuously under nitrogen. The reaction mixture coled and washed with water (2 × 100 mL), with
the organic layer being retained each time. The reaction was dried over anhydrous sodium sulfate
and the solvent was then removed under reduced pressure. The crude product was purified by
flash chromatography over silica gel (eluent: n-hexane : ethyl acetate, 4:1).
4
.1.3.1. 4-(4-Fluorophenyl)-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin-2-one (7a)
Preparation as described above from crotonyl chloride and (4-fluorobenzylidene)-3,4,5-
trimethoxyphenylamine (5a). The product was obtained as yellow solid, yield 36%, Mp 147–149
-1 1
°
C. IR (KBr) νmax: 1749 (C=O) cm . H NMR (400 MHz, CDCl
3
): δ 3.73 (s, 6H), 3.74–3.75 (m, 1H), 3.78
(
7
1
s, 3H), 4.78 (d, J = 2.52 Hz, 1H), 5.35–5.43 (m, 2H), 5.99–6.08 (m, 1H), 6.53 (s, 2H), 7.09–7.13 (m, 2H),
1
3
.36–7.38 (m, 2H). C NMR (100 MHz, CDCl ): δ 55.57, 60.51, 60.51, 63.51, 94.20, 115.76, 115.97,
3
19.74, 127.18, 127.25, 129.81, 132.63, 133.16, 134.09, 153.10, 161.10, 164.56. HRMS: found 356.1303
+
(
M -H); C20
H
19FNO requires 356.1298.
4
4
.1.3.2. 4-(4-Chlorophenyl)-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin-2-one (7b)
Preparation as described above from crotonyl chloride and (4-chlorobenzylidene)-(3,4,5-
trimethoxyphenyl)amine (5b) to afford the product as a yellow solid, yield 30%, Mp 104 °C. IR
-1 1
(
KBr) νmax: 1754 (C=O) cm . H NMR (400 MHz, CDCl ): δ 3.70–3.71 (m, 1H), 3.72 (s, 6H), 3.77 (s,
3