Synthesis and biological evaluation of 1,4-diaryl-2-azetidinones as specific anticancer agents: Activation of adenosine monophosphate activated protein kinase and induction of apoptosis

Article abstract of DOI:10.1021/jm201344a

A series of novel 1,4-diaryl-2-azetidinones were synthesized and evaluated for antiproliferative activity, cell cycle effects, and apoptosis induction. Strong cytotoxicity was observed with the best compounds (±)-trans-20, (±)-trans-21, and enantiomers (+

Full text of DOI:10.1021/jm201344a

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of 1,4-Diaryl-2-azetidinones as
Specific Anticancer Agents: Activation of Adenosine Monophosphate
Activated Protein Kinase and Induction of Apoptosis†
Farida Tripodi,‡ Roberto Pagliarin,*,§ Gabriele Fumagalli,§ Alessandra Bigi,‡,⊥ Paola Fusi,‡ Fulvia Orsini,§
Milo Frattini,∥ and Paola Coccetti*,‡
‡Department of Biotechnology and Biosciences, University of Milan-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy
§Department of Organic and Industrial Chemistry, University of Milan, Via Venezian 21, 20133 Milan, Italy
∥Institute of Pathology, Via in Selva 24, 6600 Locarno, Switzerland
S
* Supporting Information
ABSTRACT: A series of novel 1,4-diaryl-2-azetidinones were
synthesized and evaluated for antiproliferative activity, cell cycle
effects, and apoptosis induction. Strong cytotoxicity was observed
with the best compounds ( )-trans-20, ( )-trans-21, and
enantiomers (+)-trans-20 and (+)-trans-21, which exhibited IC50
values of 3−13 nM against duodenal adenocarcinoma cells. They
induced inhibition of tubulin polymerization and subsequent G2/
M arrest. This effect was accompanied by activation of AMP-
activated protein kinase (AMPK), activation of caspase-3, and
induction of apoptosis. Additionally, the most potent compounds displayed antiproliferative activity against different colon cancer
cell lines, opening the route to a new class of potential therapeutic agents against colon cancer.
zoles) have been reported as replacements for the cis-double
bond and have been reviewed by different authors.4,7,8 Among
them, β-lactam derivatives, which are scaffolds with a similar
spatial arrangement between the two phenyl rings as observed
in the CA-4 molecule, have also been found to have less toxicity
but increased anticancer activity.9 In addition, a few groups
studied the mechanism of action of anticancer polyaromatic β-
lactams. In particular, Banik and colleagues showed that some
β-lactam derivatives exhibited cytotoxicity against the colon
cancer cell line HT-29 with an increase of G2 phase cells 24 h
after the treatment.10 Parallel to this group, other researchers
showed that some β-lactam derivatives induced DNA damage,
inhibited DNA replication, and activated the apoptotic death
program in human leukemic Jurkat T cells.11 Importantly, one
of these derivatives inhibited proliferation and induced
apoptosis in various human solid tumor cell lines (breast,
prostate, and head-and-neck).11 Several authors have recently
proposed products that maintain the structural elements of the
combretastatin, such as the presence of 3,4,5-trimethoxyphenyl
moiety. Sun et al.12 have prepared compounds 3−5 (Figure 1)
in the cis geometry only; compound 4 exhibits an IC50 of 29.19
nM against human renal adenocarcinoma. O’Boyle et al. have
proposed trans derivatives 613 and 9−139 in which in position 3
is noted the presence of a heterocyclic residue (6) or a phenyl
group (9) or a p-fluoro (10), a p-hydroxyl, or a p-aminophenyl
group (11−13). In particular, compound 12 shows an IC50 of
INTRODUCTION
■
Natural compounds are often used in traditional medicine for
their different and multiple therapeutic effects.1 Among them,
stilbene-based compounds are widely distributed in nature and
show a wide range of biological activities. Combretastatins
(Figure 1), a group of polyhydroxylated stilbenes isolated from
the South African tree Combretum caffrum, were shown to
inhibit the formation of the mitotic spindle by binding to the
colchicine-binding site of tubulin.2 In addition they have also
shown antivascular properties in vivo.3 In particular combre-
tastatin A-4 (CA-4) exhibits potent anticancer activity against a
panel of human cancer cells including multidrug resistant ones.4
Two problems, however, have limited their use as therapeutic
agents for a long time: their low water-solubility and cis/trans
isomerization which may occur during storage and admin-
istration, causing a dramatic loss of activity. The first problem
has been overcome by a water-soluble phosphate prodrug (CA-
4P, zybrestat, fosbretabulin) that is currently under inves-
tigation in human clinical trials (phase III) as an anticancer
drug.5,6 The second problem has been approached by designing
a variety of conformationally restricted cis-locked analogues.
Cis-restricted combretastatins may be obtained by introducing
a five-membered ring (carbocylic or heterocyclic) in place of
the olefinic bond. Besides the prevention of cis/trans
isomerization, these analogues may have the additional
advantage that a proper heterocyclic system might improve
the therapeutic potential of these drugs. A large number of
modified rings (combretazolones, furanones, cyclopentenones,
isoxazolines, isoxazoles, thiazoles, triazoles, tetrazoles, pyra-
Received: October 7, 2011
Published: February 13, 2012
© 2012 American Chemical Society
2112
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
Figure 1. Combretastatin A-4, resveratrol, and anticancer β-lactams.
0.8 nM on human MCF-7 breast cancer cells. Carr et al.14
synthesized β-lactams without substituents (7) or with the
presence of one (8) or two methyl groups in position 3 of the
four-membered ring. Compound 8, as a mixture of cis/trans
stereoisomers tested on MCF-7 cells, has an IC50 of 39 nM.
On this base, we focused our work on the preparation of a
small library of 1,4-diaryl-2-azetidinones bearing a hydroxyl or
an amino group in position 3 of the four-membered ring.15 We
justify the presence of these two substituents in position 3 with
the requirement to improve water solubility of the final
products.16 In addition, these groups could be further
derivatized, without affecting the aromatic rings, with molecules
that enhance water solubility (phosphates) or with specific
ligands useful to selectively target tumor cells. We designed four
series of compounds that mimic the natural compound CA-4.
The first series has a 3,4,5-trimethoxyphenyl residue in position
1 coupled with a 4-methoxy-3-hydroxyphenyl moiety in
position 4 of the azetidinone ring and a hydroxyl group (21)
or an amino group in position 3 (29) (Scheme 1). In the
second series a 3,5-dimethoxyphenyl substituent is coupled
with a 4-methoxyphenyl group in position 4 of the four-
membered ring (20, 28, Scheme 1), a substitution pattern also
present in resveratrol (Figure 1), the 3,4′,5-trihydroxystilbene
identified as a cancer chemopreventive agent.17,18 The other
two series have the inverted aryl moieties of the first (25, 33,
Scheme 1) and second series (24, 32, Scheme 1), respectively.
The natural compound CA-4 acts as an activator of either
adenosine monophosphate activated protein kinase (AMPK)19
or of the yeast homologous Snf1/AMPK.20 In the activated
state, AMPK down-regulates several anabolic enzymes and thus
shuts down the ATP-consuming metabolic pathways.21 AMPK
is activated upon phosphorylation on Thr172 by its upstream
kinase STK11 (LKB1) in response to an increase in cellular
AMP/ATP ratio.22 AMPK activation is cytotoxic to various
cancer cell types and inhibits tumor growth,23,24 supporting
AMPK as a tumor suppressor and a potential target for cancer
therapy and chemoprevention.25 Interestingly, many chemo-
therapic drugs can activate AMPK, mediating apoptotic effects;
for example, a recent paper suggests that AMPK activation is a
key player for doxorubicin-induced cancer cell apoptosis.26
In this study we have found that the new 1,4-diaryl-2-
azetidinones show specific antiproliferative activity against
duodenal and colon cancer cells, through activation of AMPK
and induction of the apoptotic pathway.
CHEMISTRY
■
The synthetic route to β-lactam analogues of combretastatins
and resveratrol, illustrated in Scheme 1, is based on the
Staudinger approach, which is a [2 + 2]-cycloaddition reaction,
between properly substituted imines and ketenes, the latter
easily obtained “in situ” from the corresponding acid chlorides
under basic conditions. The preparation of the imine precursors
14−17 was achieved in very good yields (94−98%) by
condensation of the properly substituted aldehydes and anilines
in ethanol, following two different workup procedures
depending on the imine solubility (Scheme 1).
The ketene precursor acetoxyacetyl chloride was used for the
preparation of compounds 20, 21, 24, 25, while phthalylglycyl
chloride was used for the preparation of compounds 28, 29, 32,
33. Among all the synthesized compounds, only compounds 26
and 27 have been obtained in traces, and after purification, the
poor yields did not allow the execution of activity tests.
In order to modulate the stereochemical outcome of the
reaction, two experimental protocols have been optimized to
prepare azetidinones 18−33 (Scheme 1). It is indeed well-
known that besides the type and number of substituents on
both the imine and the ketene, the stereochemistry of the
products of the Staudinger cycloaddition is significantly affected
by the experimental reaction conditions such as solvent choice,
temperature, and order of addition of the reagents.27 A mixture
of racemic cis and trans cycloadducts was obtained by dropwise
addition of a 5-fold excess of acid chloride to a methylene
chloride solution of imine and TEA. Stereochemistry was
2113
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of 3-Hydroxy- and 3-Amino-1,4-diaryl-2-azetidinones
a
Reagents and conditions: (a) Na2SO4, ethanol, 25 °C, 48 h; (b) CH3COOCH2COCl, Et3N, anhydrous CH2Cl2, 25 °C, 24 h; (c) NH2NH2·2HCl,
Et3N, MeOH, reflux, 4 h; (d) FtNCH2COCl, Et3N, anhydrous CH2Cl2, 25 °C, 24 h.
3
assigned by measuring the J coupling constants between H-3
and H-4 (cis = 4−5 Hz, trans = 1−2 Hz).
particular the imine and the ketene precursor, 2-acetoxy acetyl
chloride, were mixed at ice temperature, then heated at 100 °C
before adding TEA to afford, as racemic mixture, the trans
cycloadduct only, albeit in lower yields. The diastereoisomeric
cis−trans mixture was separated by flash chromatography.
With the aim of obtaining mainly the more active trans
stereoisomers, a different protocol was used, changing solvent,
temperature, and order of addition of the reagents.28 In
2114
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
a
Scheme 2. Resolution of ( )-3,4-trans-3-Hydroxy-1,4-diaryl-2-azetidinones
a
Reagents and conditions: (a) (L)-N-Boc- proline, DIPEA, HBTU, anhydrous acetonitrile, 24 h; (b) NH2NH2·2HCl, Et3N, MeOH, reflux, 4 h.
The resolution of the racemic trans mixture of 20 and 21 was
tested using different strategies. The best results were obtained
using, as chiral solving reagent, N-protected proline as
illustrated in Scheme 2. The resulting diastereoisomeric
mixtures were then separated by flash chromatography using
a properly selected elution system. After the cleavage of the
proline moiety, the enantiomeric purity was verified to be 98%
via chiral HPLC analysis by comparison with the corresponding
racemic mixtures.
Table 1. IC50 of ( )-cis-20, ( )-trans-20, (+)-trans-20,
(−)-trans-20, ( )-cis-21, ( )-trans-21, (+)-trans-21,
(−)-trans-21, ( )-cis-25, ( )-trans-25, 4, 5, and CA-4 in
HuTu-80 Cells Measured by MTT Assay after 72 h of
a
Treatment
IC50, nM
(HuTu-80 cells, 72 h)
compd
( )-cis-20
( )-trans-20
(+)-trans-20
(−)-trans-20
( )-cis-21
( )-trans-21
(+)-trans-21
(−)-trans-21
( )-cis-25
( )-trans-25
4
124.6 27.01
9.05 1.57
8.02 0.71
RESULTS
83.55 14.33
28.79 3.36
13.36 1.47
3.05 1.42
■
Compounds ( )-trans-20, ( )-trans-21, (+)-trans-20,
and (+)-trans-21 Show in Vitro Antiproliferative Activ-
ities against Cancer Cell Lines. The new β-lactam
compounds were tested in HeLa cells (cervical adenocarcino-
ma) in order to evaluate their efficacy as antiproliferative
agents. The cells were treated with 10 μM of each compound,
and their viability was measured after 48 h by the MTT assay.
The analyses revealed that ( )-cis-20, ( )-trans-20, ( )-cis-21,
( )-trans-21, ( )-cis-25, and ( )-trans-25 were the most
promising, showing an effect on cell viability similar to that
observed with combretastatin A4 (Figure S1 of Supporting
Information). We thus decided to evaluate the antiproliferative
effect of these compounds on small bowel adenocarcinoma
(SBA, HuTu-80 cancer cells), using CA-4 and compounds 4
and 5, previously described in literature,12 as controls. All
compounds exhibited IC50 values ranging from a few
nanomolar to 2.5 μM (Table 1). Compounds ( )-trans-20
589.9 98.71
2549.00 383.25
405.5 48.65
528.55 27.93
101.17 6.75
1.37 0.13
5
CA-4
a
IC50 values are the compound concentrations required to inhibit cell
proliferation by 50%. Data are expressed as the mean standard
deviation from the dose−response curves of at least three independent
experiments performed in triplicate.
and ( )-trans-21 showed the most potent antitumor effect
toward HuTu-80 cells, with IC50 of about 9 and 13 nM,
respectively (Table 1). Enantiomerically pure (+)-trans-20,
2115
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
Table 2. IC50 of ( )-trans-20, (+)-trans-20, ( )-trans-21, (+)-trans-21, and CA-4 in SW48, SW480, SW620, T84, MCF-7, and
a
SKNBE Cell Lines Measured by MTT Assay after 72 h of Treatment
IC50, nM
compd
SW48
SW480
SW620
T84
MCF-7
SKNBE
( )-trans-20
(+)-trans-20
( )-trans-21
(+)-trans-21
CA-4
12.05 0.92
11.06 1.61
15.19 3.28
9.81 3.02
3.32 1.61
24.17 5.84
16.74 4.01
22.14 4.55
12.53 3.58
3.63 1.65
6.6 0.49
5.73 0.47
6.77 2.28
45.37 4.70
20.30 2.55
23.43 5.81
5.19 1.65
4.01 2.43
6.14 2.73
4.47 0.96
1.11 0.13
2.25 0.36
1.12 0.22
4.20 1.82
1.32 0.50
0.58 0.14
8.58 0.18
13.63 4.34
3.37 1.80
1.52 0.13
a
IC50 values are the compound concentrations required to inhibit cell proliferation by 50%. Data are expressed as the mean standard deviation
from the dose−response curves of at least three independent experiments performed in triplicate.
(−)-trans-20, (+)-trans-21, and (−)-trans-21 were obtained and
tested on the same cells. It is noteworthy that enantiomer
(+)-trans-20 retained the same cytotoxic activity as the racemic
solution (( )-trans-20). In contrast, the IC50 value of
(−)-trans-20 was about 10 times higher than that of
(+)-trans-20 (Table 1). Remarkably, the enantiomer
(+)-trans-21 showed an IC50 of 3 nM, whereas enantiomer
(−)-trans-21 was less active with an IC50 close to 600 nM
(Table 1).
Compounds ( )-trans-20, ( )-trans-21, (+)-trans-20, and
(+)-trans-21 were additionally tested against a panel of six
human cancer cell lines, including colon cancer cells (SW48,
SW480, SW620, T84), the breast cancer cell line MCF-7, and
the neuroblastoma cell line SKNBE (Table 2). The compounds
retained nanomolar cytotoxic activity against all cancer cells,
and enantiomerically pure compounds (+)-trans-20 and
(+)-trans-21 were more active than their respective racemic
mixtures in most cell lines (Table 2). In contrast, normal
FHs74 intestinal cells were not affected and only a marginal
inhibitory effect was observed on normal CCD841CoN colon
cells at 10 μM (data not shown), suggesting a cancer-specific
activity of these compounds.
Table 3. Inhibition of Tubulin Polymerization in Response
a
to Azetidinone Compounds
compd
tubulin polymerization IC50 (μM)
( )-trans-20
(+)-trans-20
( )-trans-21
(+)-trans-21
3.60 0.51
1.30 0.20
3.80 0.60
1.61 0.42
1.32 0.15
CA-4
a
IC50 values are the compound concentrations required to inhibit
tubulin polymerization by 50%. DMSO (0.2% v/v) was used as a
vehicle control. Data are expressed as the mean standard deviation
from the dose−response curves of at least two independent
experiments performed in triplicate.
of the caspase pathway. HuTu-80 cells were treated with 30 nM
( )-trans-20 and ( )-trans-21 for 24 h, and caspase-3 and
PARP (poly ADP-ribose polymerase) cleavage was examined
by Western blot analysis (Figure 2 and S2B of Supporting
Compounds ( )-trans-20, ( )-trans-21, (+)-trans-20,
and (+)-trans-21 Induce Apoptotic Cell Death. The effects
of ( )-trans-20 and ( )-trans-21 on cell cycle progression were
examined by analyzing the DNA content of HuTu-80 cells
treated with 30 nM of these compounds for 48 and 72 h (0.1%
DMSO and CA-4 were used as negative and positive controls,
respectively). Both ( )-trans-20 and ( )-trans-21 caused a
large increase of cells in the G2/M phase which is accompanied
by a corresponding reduction in the G1 phase (Figure S2A of
Supporting Information), as reported for the control CA-4.29,30
These results suggest that the compounds may act by
targeting the microtubules, like CA-4.1,2 The effects of the more
active compounds ( )-trans-20, ( )-trans-21, (+)-trans-20, and
(+)-trans-21 on the assembly of purified porcine tubulin were
then evaluated. The ability of CA-4 to effectively inhibit tubulin
polymerization was assessed as a positive control (Table 3). In
the assembly assay, all the azetidinone compounds showed an
IC50 value ranging from 1.3 to 3.8 μM, indicating that one of
the molecular targets of the active azetidinones is tubulin.
Strikingly, the enantiomerically pure compounds (+)-trans-20
and (+)-trans-21 were more potent than their racemic mixtures,
with an IC50 values close to that of CA-4 (Table 3 and as
previously reported8).
Figure 2. The new β-lactams induce apoptosis in cancer cells. HuTu-
80 cells were treated with 30 nM ( )-trans-20 and ( )-trans-21 for 24
h, and SW48 cells were treated with 30 nM (+)-trans-20 and (+)-trans-
21 for 16 h. Total protein extracts were subjected to Western blot
analysis to detect caspase-3. Vinculin was used as loading control.
Treatments with 0.1% DMSO and with 30 nM CA-4 were used as
negative and positive controls, respectively.
Information). Treatment with ( )-trans-20 and ( )-trans-21
strongly led to extensive formation of the typical fragments of
caspase-3 and PARP, indicative of apoptosis induction, similar
to that observed with CA-4 (Figure 2 and S2B of Supporting
Information).
Since SBA is a model of cancer superimposable on colon
cancer cells (CRC),31−33 we examined the induction of the
apoptotic pathway on the SW48 colon cancer cell line, which
displayed a strong growth inhibition induced by (+)-trans-20
and (+)-trans-21 (Table 2). On the basis of the evidence of the
similar molecular profiles of CRC and SBA,31−33 we expected
In addition, treatment of cells with ( )-trans-20 and
( )-trans-21 was able to induce the time-dependent appear-
ance of a hypodiploid peak (sub-G1) indicative of cell death
(Figure S2A of Supporting Information). Since sub-G1 cells do
not necessarily indicate apoptosis, we investigated the activation
2116
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
Figure 3. Compounds ( )-trans-20, ( )-trans-21, (+)-trans-20, and (+)-trans-21 induce AMPK activation. (A) HuTu-80 cells were treated with 30
nM ( )-trans-20 and ( )-trans-21 for 24 h, and SW48 cells were treated with 30 nM (+)-trans-20 and (+)-trans-21 for 16 h. Total protein extracts
were subjected to Western blot analysis to assay pT172-AMPK and total AMPK levels. (B) HuTu-80 cells were treated with 30 nM ( )-trans-20 and
( )-trans-21 for 24 h, and nuclear protein extracts were subjected to Western blot analysis to assay pS15-p53 and total p53 levels. Fibrillarin was
used as nuclear control. (C) HuTu-80 cells were treated with 30 nM ( )-trans-20 and ( )-trans-21 for 24 h, and total protein extracts were
subjected to Western blot analysis to assay pS792-Raptor and total Raptor levels. (D) HuTu-80 cells were treated with ( )-trans-20 and ( )-trans-
21 (30 nM) for 24 h and then mock treated or treated with 10 μg/mL insulin for 10 min. Total protein extracts were subjected to Western blot
analysis to assay pS473-Akt and total Akt levels. Vinculin was used as loading control. Treatments with 0.1% DMSO and with 30 nM CA-4 were
used as negative and positive controls, respectively.
to find similar results. Indeed, treatment with (+)-trans-20 and
(+)-trans-21 induced activation of caspase-3 (Figure 2). These
data clearly indicate that ( )-trans-20, ( )-trans-21, (+)-trans-
20, and (+)-trans-21 induce apoptotic cell death in cancer cells.
Compounds ( )-trans-20, ( )-trans-21, (+)-trans-20,
and (+)-trans-21 Induce AMPK Activation Pathway.
Previous studies showed that CA-4 activates AMPK in
HepG2 cells.19 We thus examined whether AMPK was also
activated by the new azetidinone derivatives, which are
structural analogues of CA-4.12 Compounds ( )-trans-20 and
( )-trans-21 induced phosphorylation of AMPK on Thr172 in
HuTu-80 cells, as well as (+)-trans-20 and (+)-trans-21 in
SW48 cells (Figure 3A). Since AMPK acts on the tumor-
suppressor p53 leading to apoptotic cell death,34,35 our analysis
was further deepened, investigating the level and the
phosphorylation state of the tumor suppressor protein p53. It
is known that AMPK activation induces phosphorylation of p53
on Ser15, which is required for AMPK-dependent cell cycle
arrest.34,36 Compounds ( )-trans-20 and ( )-trans-21, as well
as the control CA-4, induced phosphorylation of p53 on Ser15.
Moreover, p53 was found to be accumulated (Figure 3B).
Another downstream effector of AMPK is the mTOR binding
partner Raptor, whose phosphorylation by AMPK is required
for the inhibition of mTORC1 and cell cycle arrest induced by
energy stress.37 Raptor was found to be phosphorylated on
Ser792 upon treatment with ( )-trans-20 and ( )-trans-21
(Figure 3C).
It was shown that other AMPK activators, such as metformin
and AICAR, induce diminished expression of pSer473-Akt,38
which plays a key role in multiple cellular processes such as cell
growth, cell survival, and cell cycle progression.39 We thus
tested the effect of ( )-trans-20 and ( )-trans-21 on Akt
phosphorylation, both in unstimulated cells and upon insulin
stimulation. No differences in the level of pSer473-Akt were
2117
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
observed, neither in the basal condition nor upon insulin
stimulation, suggesting that the inhibitory effect of the new
azetidinone compounds is not mediated by the Akt upstream
activation (Figure 3D).
Thus, we suggest that the new 1,4-diaryl-2-azetidinones are
promising novel anticancer drugs to be tested in preclinical
cancer models of CRC.
EXPERIMENTAL SECTION
■
DISCUSSION AND CONCLUSIONS
Chemistry: Experimental Methods. All commercially available
reagents were purchased by Sigma-Aldrich and used without further
purification. Preparative separations were usually performed by flash
column chromatography on silica gel (Merck grade 9385). Thin-layer
chromatography was conducted using silica gel (Merck, 10 cm × 5 cm,
■
In the present article we report the synthesis of new
azetidinone derivatives, with a strong antiproliferative activity
against cancer cells (Table 1 and 2). In our activity test, trans
azetidinone derivatives show better results than cis isomers, and
they can be prepared in good yields by thermodynamic reaction
conditions.27 Moreover, the presence of a hydroxyl group at
position 3 of the azetidinone ring gives a water solubility 5
times more elevated for ( )-trans-21 with respect to the natural
product combretastatin A4.16,40 The molecules ( )-trans-20
and ( )-trans-21 were identified as the most potent
compounds of the series having nanomolar activity against
several colon cancer cell lines (SW48, SW480, SW620, T84) as
well as on the breast cancer cell line MCF-7 and on the
neuroblastoma cell line SKNBE. These compounds, resolved in
the pure enantiomers, are more active than their respective
racemic mixtures in most cell lines, showing a positive
correlation between cytotoxic activity and inhibition of tubulin
polymerization (Tables 1−3).
1
silica gel 60 F254). H and 13C NMR spectra were recorded on a
Bruker AC 200 spectrometer at 200 and 50.3 MHz, respectively, in
CDCl3 solution unless stated otherwise, and chemical shifts were
represented as δ values relative to the internal standard TMS. ESI mass
spectra were recorded on Bruker Esquire 3000 Plus. The synthesized
compounds submitted to biological tests have a purity of ≥95%
determined by HPLC (Phenomenex, Nucleosil 250 mm × 3.2 mm
column, 5 μm, C18; mobile phase 0.05 M phosphate buffer, pH 7/
acetonitrile, 7:3; flow rate 1.5 mL/min), and they have been filtered in
sterile atmosphere with 0.20 μm filters. Chiral liquid chromatography
was carried out on selected compounds using a DAICEL Chirapak IB
4.0 mm column and Hewlett-Packard 1050 HPLC instrument. The
organic mobile phase was 2-propanol/Exane, 20:80. The flow rate was
0.8 mL/min, and detection was carried out at 225 nm. Water solubility
of ( )-trans-20 and ( )-trans-21 have been obtained after stirring a
suspension of each compound for 2 h at 30 °C. They have been
further centrifuged, and the liquid phase was analyzed by HPLC
(Supelcosil LC18, 250 mm × 4.6 mm, 5 μm; mobile phase 0.05 M
phosphate buffer, pH 7/acetonitrile, 5:5; flow rate 1 mL/min). The
concentrations have been calculated by intercepting the peak area
values with the calibration curves obtained by standard solutions of
compounds 20 and 21 in acetonitrile.
Pure enantiomer (+)-trans-21 exhibited an activity 200 times
higher than the (−)-trans-antipode (Table 1). In keeping with
literature data,12 our results confirm that compounds with the
3,4,5-trimethoxy or 3,5-dimethoxyphenyl ring on the N-1
position of azetidinone ring are significantly more active than
compounds with polymethoxylated moieties on the C-4
position.
Our data show that the mechanism of the antitumoral action
exerted by these new azetidinone compounds relies on the
inhibition of tubulin polymerization and the stimulation of the
apoptotic pathway, with the cleavage of the effector caspase-3
and the PARP protein (Figure 2 and Figure S2 of Supporting
Information). It is of high relevance that our compounds
induced AMPK activation (Figure 3), since AMPK is a novel
therapeutic target for cancer and metabolic diseases. Indeed,
when physiologically or pharmacologically activated, AMPK
acts in a tumor suppressor-like fashion.41 In addition, AMPK
inhibits the mTOR pathway through direct phosphorylation of
the mTOR-associated factor Raptor and induces cell cycle
arrest and/or apoptosis through phosphorylation of p53.22 The
mTOR signaling pathway is known to play important roles in
the protein synthesis machinery.42 Therefore, when AMPK is
activated, cell growth and proliferation might be inhibited as a
result of limitations in protein synthesis.
4-Methoxybenzylidene-(3,5-dimethoxyphenyl)amine 14.
Dry Na2SO4 (4 g) was added to a solution of 3,5-dimethoxyaniline
(3.0 g, 19.6 mmol) and 4-methoxybenzaldehyde (2.67 g, 19.6 mmol)
in ethanol (6 mL), and the mixture was stirred at room temperature
for 48 h. The mixture was filtered and the solvent removed under
reduced pressure to afford the imine 14 as a brown oil (5.0 g, 18.4
mmol, 94% yields). 1H NMR (CDCl3): δ = 8.41 (1H, s), 7.86 (2H, d,
J = 8.8 Hz), 7.00 (2H, d, J = 8.8 Hz), 6.40 (2H, d, J = 2 Hz), 6.36 (1H,
t, J = 2 Hz), 3.88 (3H, s), 3.83 (6H, s). EI-MS (m/z): 271 (M+).
3 - H y d r o x y - 4 - m e t h o x y b e n z y l i d e n e - ( 3 , 4 , 5 -
trimethoxyphenyl)amine 15. Dry Na2SO4 (4 g) was added to a
solution of 3,4,5-trimethoxyaniline (1.9 g, 10.1 mmol) and 3-hydroxy-
4-methoxybenzaldehyde (1.5 g, 10.1 mmol) in ethanol (6 mL), and
the mixture was stirred at room temperature for 48 h to afford a yellow
solid. The imine 15 was a solid mixed with Na2SO4. The mixture was
filtered, and the solid was washed with cold ethanol (10 mL). The
solid was suspended in methylene chloride and filtered to separate the
1
solubilized imine 2 from Na2SO4 (3.1 g, 9.9 mmol, 98% yield). H
NMR (CDCl3): δ 8.41 (1H, s), 7.63 (1H, s), 7.46 (1H, d, J = 7.2 Hz),
6.96 (1H, d, J = 7.2 Hz), 6.53 (2H, s), 5.77 (1H, s), 3.99 (3H, s), 3.92
(6H, s), 3.88 (3H, s). EI-MS (m/z): 318 (M+).
The ability of these compounds to inhibit cancer cell
proliferation can be of particular significance against small
bowel adenocarcinoma (SBA) and colorectal cancer diseases
(CRC), which display similar risk factors and share the same
carcinogenetic process,31−33 since compounds ( )-trans-20 and
( )-trans-21, as well as their enantiomers (+)-trans-20 and
(+)-trans-21, showed a strong antiproliferative activity against
these cancer cells (Tables 1 and 2). In addition, they showed
only a slight inhibitory effect on the two normal cell lines
analyzed (FHs74 intestinal cells and CCD841CoN colon cells),
even at very high concentrations (about 1000-fold higher than
the IC50 concentration estimated on most cancer cell lines
tested, data not shown). This specific anticancer activity is of
great interest considering that chemotherapies are limited by
their frequent and potentially severe dose-limiting toxicities.
3,5-Dimethoxybenzylidene-4-methoxyphenylamine 16. This
compound has been prepared following the same methodology
described for 14 (92% yield). δ = 8.41 (1H, s), 7.26 (2H, d, J = 8.9
Hz), 7.07 (2H, d, J = 2.0 Hz), 6.95 (2H, J = 8.9 Hz), 6.59 (1H, t, J =
2.0 Hz), 3.88 (6H, s), 3.85 (3H, s). EI-MS (m/z): 271 (M+).
3 , 4 , 5 - T r i m e t h o x y b e n z y l i d e n e - ( 3 - h y d r o x y - 4 -
methoxyphenyl)amine 17. This compound has been prepared
following the same methodology described for 15 (80% yield). δ =
8.36 (1H, s), 7.16 (2H, s), 6.92 (1H, d, J = 2.3 Hz), 6.89 (1H, d, J =
8.5 Hz), 6.81 (1H, dd, J = 2.3, 8.5 Hz), 5.86 (1H, s), 3.95 (3H, s), 3.92
(9H, s). EI-MS (m/z): 318 (M+).
General Methods for the Synthesis of Azetidin-2-ones
(Trans + Cis). Procedure a. ( )-3,4-trans-1-(3,5-Dimethoxy-
phenyl)-3-hydroxy-4-(4-methoxyphenyl)azetidin-2-one
(( )-trans-20) and ( )-3,4-cis-1-(3,5-Dimethoxyphenyl)-3-hy-
droxy-4-(4-methoxyphenyl)azetidin-2-one (( )-cis-20). A sol-
ution of 2-acetoxyacetyl chloride (7.44 g, 54.5 mmol) in anhydrous
2118
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
methylene chloride (30 mL) was added dropwise at 0 °C under
nitrogen to a stirred solution of imine 14 (2.97 g, 10.9 mmol) and
TEA (17.6 g, 24.3 mL, 174 mmol) in anhydrous methylene chloride
(90 mL). The solution was allowed to reach room temperature and
then stirred for 24 h. The solvent was concentrated under reduced
pressure and the residue was purified by flash chromatography (silica
gel; eluent n-hexane/ethyl acetate, 1/1) to afford a mixture of the two
diastereoisomers ( )-trans-18 and ( )-cis-18 as a white amorphous
solid (1.67 g, 4.5 mmol, 41% yield). A sample of this mixture was
further purified by gradient flash chromatography with n-hexane/ethyl
acetate, and the two diastereoisomers ( )-trans-18 and ( )-cis-18
were identified by analytical techniques.
The diastereoisomers ( )-trans-21 (7% yield) and ( )-cis-21 (40%
yield) were isolated as colorless amorphous compounds.
1
( )-trans-21. H NMR (CDCl3) δ: 6.92−6.85 (3H, m), 6.49 (2H,
s), 5.84 (1H, s), 4.72 (2H, bs), 3.87 (3H, s), 3.74 (3H, s), 3.62 (6H, s).
13C NMR (CDCl3) δ: 167.68, 154.05, 147.67, 146.94, 135.39, 133.80,
129.81, 118.80, 112.91, 111.68, 96.14, 84.14, 66.35, 61.57, 55.67. ESI-
MS (m/z): 398 (M + Na+).
1
( )-cis-21. H NMR (CDCl3): δ 7.10 (1H, d, J = 8.8 Hz), 7.00−
6.95 (2H, m), 6.81 (2H, s), 5.35 (1H, d, J = 5.0 Hz), 5.28 (1H, d, J =
5.0 Hz), 4.00 (3H, s), 3.86 (6H, s), 3.84 (3H, s). 13C NMR (CDCl3)
δ:167.25, 153.28, 145.02, 143.25, 133.51, 126.47, 119.17, 114.49,
111.14, 95.13, 76.50, 62.69, 59.65, 54.93. ESI-MS (m/z): 398 (M +
Na+).
1
( )-trans-18. H NMR (CDCl3) δ: 7.23 (2H, d, J = 7.7 Hz), 6.85
General Methods for the Synthesis of Azetidin-2-ones
(Trans). Procedure b. ( )-3,4-trans-1-(3,5-Dimethoxyphenyl)-
3-hydroxy-4-(4-methoxyphenyl)azetidin-2-one (( )-trans-20).
The imine 14 (5.0 g, 18.4 mmol) and 2-acetoxyacetyl chloride (2.5
g, 1.97 mL, 18.4 mmol) were dissolved in dry toluene (50 mL), under
nitrogen and stirring, at 0 °C. The solution was allowed to reach room
temperature and then warmed to 100 °C. Dry TEA (2.0 g, 2.75 mL,
20.2 mmol) was added dropwise. The mixture was warmed at 100 °C
for 5 h. The solution was concentrated under reduced pressure and the
residue was purified by flash chromatography (silica gel; eluent n-
hexane/ethyl acetate, 1/1) to afford the diastereoisomer ( )-trans-18
(3.4 g, 9.2 mmol, 50% yield) as an amorphous white solid. Hydrazine
dichloride (2.85 g, 27.2 mmol) was added to a stirred solution of trans-
18 (2.5 g, 6.8 mmol) in methanol (30 mL) at 0 °C and under
nitrogen. TEA (5.5 g, 7.6 mL, 54 mmol) was added dropwise. The
mixture was allowed to reach room temperature and then refluxed at
for 4 h. The solvent was removed at reduced pressure, and the residue
was treated with a saturated solution of KHSO4 and extracted with
ethyl acetate (3 × 50 mL). The organic phase was dried over Na2SO4,
and the solvent, after filtration, was removed at reduced pressure. The
crude residue was purified by flash chromatography (silica gel; eluent
n-hexane/ethyl acetate, 1:1). ( )-trans-18 (2.2 g, 6.8 mmol, 37%
yield) was obtained as a white amorphous solid.
( )-3,4-trans-3-Hydroxy-4-(3-hydroxy-4-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)azetidin-2-one (( )-trans-21). The
compound ( )-trans-21 was prepared following the same method-
ology described for 20. The compound trans-19 was obtained with
35% yield. Compound 21 was purified by gradient flash chromatog-
raphy (silica gel; eluent n-hexane/tert-butyl methyl ether 1:1).
( )-trans-21 (94% yield) was obtained as a white amorphous solid.
Resolution of Racemic (trans)-Azetidin-2-ones. Resolution
of ( )-trans-20. HBTU (5.6 g, 15 mmol) was added, under nitrogen,
to a stirred solution of ( )-trans-20 (2.2 g, 6.8 mmol) and (L)-Boc-
prolina (3.0 g, 14 mmol) in dry acetonitrile (30 mL). DIPEA (72.3
mL, 420 mmol) was added dropwise, and the reaction mixture was
stirred for 24 h. The solvent was removed under reduced pressure, and
the residue was diluted with water and extracted with methylene
chloride (3 × 50 mL). The combined organic extracts were washed
with a saturated solution of KHSO4, with a saturated solution of
NaHCO3, with brine and then dried (Na2SO4). The solvent was
removed under reduced pressure and the residue purified by gradient
flash chromatography (silica gel; eluent n-hexane/ethyl acetate from
8/2 to 6/4) to afford a mixture of the two diastereoisomers 34a + 34b
as colorless solid (3.1 g, 5.9 mmol, 86% yield). The two
diastereoisomers were then separated by gradient flash chromatog-
raphy (silica gel) using n-hexane/tert-butyl methyl ether as eluent (n-
hexane/tert-butyl methyl ether from 8/2 to 2/8).
(2H, d, J = 7.7 Hz), 6.42 (2H, d, J = 2.3 Hz), 6.20 (1H, t, J =2.3 Hz),
5.30 (1H, d, J = 2 Hz), 4.82 (1H, d, J=2 Hz), 3.80 (3H, s), 3.70 (6H,
s), 2.17 (3H, s). 13C NMR (CDCl3) δ: 169.81, 162.20, 161.30, 160.30,
138.66, 127.80, 127.22, 114.75, 97.10, 96.43, 82.72, 63.74, 55.49,
55.45, 20.59. EI-MS (m/z): 371 (M+).
( )-cis-18. 1H NMR (CDCl3) δ: 7.23 (2H, d, J = 8.7 Hz), 6.87 (2H,
d, J = 8.7 Hz), 6.53 (2H, d, J = 2.2 Hz), 6.22 (1H, t, J = 2.2 Hz), 5.89
(1H, d, J = 4.9 Hz), 5.29 (1H, d, J = 4.9 Hz), 3.81 (3H, s), 3.73 (6H,
s), 1.74 (3H, s). 13C NMR (CDCl3) δ: 169.81, 162.43, 161.36, 160.15,
138.82, 129.32, 124.05, 114.14, 97.12, 96.31, 77.82, 61. 53, 55.52,
55.49, 20.02. EI-MS (m/z): 371(M+).
Hydrazine dihydrochloride (3.3 g, 31.5 mmol) was added, at 0 °C
and under nitrogen to a stirred solution of ( )-trans-18 and ( )-cis-18
(1.67 g, 4.5 mmol) in methanol (65 mL). TEA (9.11 g, 12.5 mL, 90
mmol) was added dropwise. The mixture was allowed to reach room
temperature and then refluxed for 4 h. The solvent was removed at
reduced pressure, and the residue was treated with a saturated solution
of KHSO4 and extracted with ethyl acetate (3 × 30 mL). The
combined organic phases were dried (Na2SO4), and the solvent was
removed at reduced pressure. The crude residue was purified by
gradient flash chromatography (silica gel; eluent n-hexane/tert-butyl
methyl ether from 7/3 to 3/7) to afford the diastereoisomers
( )-trans-20 (0.050 g, 0.15 mmol, 3.3% yield) and ( )-cis-20 (0.246 g,
0.75 mmol, 17% yield) as a colorless amorphous solid.
1
( )-trans-20. H NMR (CDCl3) δ: 7.25 (2H, d, J = 8.7 Hz), 6.88
(2H, d, J = 8.7 Hz), 6.42 (2H, d, J = 2.2 Hz), 6.14 (1H, t, J = 2.2 Hz),
4.81 (1H, d, J = 1.8 Hz), 4.69 (1H, d, J = 1.8 Hz), 3.80 (3H,s), 3.67
(6H, s). 13C NMR (CDCl3) δ: 167.77, 161.14, 160.09, 138.67, 128.13,
127.60, 114.45, 97.07, 96.52, 83.77, 65.82, 55.49. EI-MS (m/z): 329
(M+), 272.
1
( )-cis-20. H NMR (CDCl3) δ: 7.26 (2H, d, J = 8.6 Hz), 6.92
(2H, d, J = 8.6 Hz), 6.53 (2H, d, J =2.0 Hz), 6.19 (1H, t, J = 2.0 Hz),
5.21 (1H, d, J = 5.6 Hz), 5.14 (1H, d, J = 5.6 Hz), 3.79 (3H,s), 3.71
(6H, s). 13C NMR (CDCl3) δ: 167.67, 161.33, 160.27, 138,80, 128.90,
124.77, 114.80, 96.93, 96.41, 77.10, 65.69, 55.57. EI-MS (m/z) 329
(M+).
( )-3,4-trans-3-Hydroxy-4-(3-hydroxy-4-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)azetidin-2-one (( )-trans-21) and
(
)3,4-cis-3-Hydroxy-4-(3-hydroxy-4-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)azetidin-2-one (( )-cis-21). Com-
pounds ( )-trans-21 and ( )-cis-21 were prepared following the
same methodology described for 20. Compounds cis-19 and trans-19
were obtained with 60% yield.
1
( )-trans-19. H NMR (CDCl3) δ: 7.23 (1H, dd, J = 8.5, 2.2 Hz),
7.14 (1H, d, J = 2.2 Hz), 7.00 (1H, d, 8.5 Hz), 6.53 (2H, s), 5.38 (1H,
d, J = 1.7 Hz), 4.68 (2H,s), 4.58 (1H, d, J = 1.7 Hz), 3.85 (3H, s), 3.79
(3H, s), 3.74 (6H,s), 2.19 (3H, s), 1.76 (3H, s). ESI-MS (m/z): 517.
34a. Diastereoisomer with Rf = 0.36; on silica gel TLC plate, two
runs, eluent n-hexane/tert-butyl methyl ether, 4:6. [α]D +1.25 (c 30
mg/10 mL). 1H NMR (DMSO-d6, 80 °C, 500 MHz) δ: 7.38 (2H, d, J
= 8.5 Hz), 6.97 (2H, d, J = 8.5 Hz), 6.43 (2H, d, J = 2.2 Hz), 6.27 (1H,
t, J = 2.2 Hz), 5.48 (1H, bs), 5.12 (1H, d, J = 1.8 Hz), 4.32 (1H, dd, J =
8.6, 4.3 Hz), 3.79 (3H, s), 3.67 (6H, s), 3.41−3.37 (2H, m), 2.35−2.26
(1H, m), 2.06−1.98 (1H, m), 1.90−1.88 (2H, m), 1.41 (9H, s). 13C
NMR (CDCl3, 25 °C, 125 MHz) (two conformers): δ 171.68, 171.48,
161.03, 160.76, 160.45, 160.38, 159.51, 159.27, 153.81, 152.86, 137.80,
137.76, 127.2, 126.9, 126.96, 126.83, 126.41, 126.08, 113.96, 113.73,
1
( )-cis-19. H NMR (CDCl3) δ: 7,22 (1H, dd, J = 8.4, 2.0 Hz),
7.05 (1H, d, J = 2.0 Hz), 6.96 (1H, d, J = 8.4 Hz), 6.57 (2H, s), 5.90
(1H, d, J = 4.8 Hz), 5.28 (1H, d, J = 4.8 Hz), 4.89 (2H, s), 3.82 (3H,
s), 3.78 (3H, s), 3.73 (6H, s), 2.19 (3H, s), 1.76 (3H, s). 13C NMR
(CDCl3) δ: 170.37, 169.57, 166.00, 161.81, 153.81, 151.71, 139.27,
132.98, 127.17, 124.86, 122.54, 113.28, 95.45, 76.48, 61.20, 61.11,
60.48, 56.32, 20.62, 20.06. ESI-MS (m/z): 517 (M+).
Compounds 21 were purified by gradient flash chromatography
(silica gel; eluent n-hexane/tert-butyl methyl ether from 6/4 to 2/8).
2119
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
96.39, 96.28, 95.68, 95.58, 82.23, 82.11, 79.50, 79.38, 62.90, 57.87,
54.66, 45.94, 45.68, 30.30, 29.17, 27.74, 23.81, 23.0. ESI-MS (m/z):
549 (M + Na+).
and 79.37, 62.38 and 61.93, 60.54, 58.87 and 58.63, 56.46 and 56.23,
46.84 and 46.44, 38.79, 30.92 and 30.74, 29.97 and 29.77, 28.49 and
28.17, 24.48 and 24.37, 23.64, and 23.52. EI-MS (m/z): 769 (M+).
The diastereoisomers 35a and 35b were separately treated with
hydrazine dihydrochloride and purified by flash chromatography (silica
gel; eluent n-hexane/ethyl acetate, 1:1). The compound (+)-21
(83.5% yield) was obtained as a colorless amorphous solid. [α]D
+16.20 (CHCl3, c 75 mg/5 mL). The protocol reported above, applied
to 35b, afforded the enatiomer (−)-21 (58% yield). [α]D −15.43
(CHCl3, c 97 mg/10 mL). The enantiomeric purity of (+)-21 and
(−)-21 was verified via chiral HPLC by comparison with the racemic
mixture and was 98%.
34b. Diastereoisomer with Rf = 0.32; on silica gel TLC plate, two
runs, eluent n-hexane/tert-butyl methyl ether, 4:6. [α]D −5.94 (c 30
mg/10 mL). 1H NMR (DMSO-d6, 80 °C, 500 MHz) δ: 7.38 (2H, d, J
= 8.4 Hz), 6.98 (2H, d, J =8.4 Hz), 6.43 (2H, d, J = 2.2 Hz), 6.27 (1H,
t, J = 2.2 Hz), 5.48 (1H, bs), 5.12 (1H, d, J = 1.7 Hz), 4.35 (1H, dd, J =
8.5, 4.3 Hz), 3.79 (3H, s), 3.67 (6H, s), 3.43−3.36 (1H, m), 2.35−2.26
(1H, m), 2.06−1.99 (1H, m), 1.83−1.86 (2H, m), 1.39 (9H, s). 13C
NMR (CDCl3, 25 °C, 125 MHz) (two conformers): δ 171.35, 171.19,
161.10, 160.80, 160.42, 159.51, 159.29, 153.80, 152.86, 137.86, 137.73,
127.19, 126.89, 126.35, 126.13, 113.97, 113.82, 96.34, 95.52, 82.08,
79.63, 79.36, 62.71, 62.66, 58.39, 58.07, 54.66, 45.92, 45.65, 30.36,
29.32, 27.74, 23.92, 22.99. ESI-MS (m/z): 549 (M + Na+).
Hydrazine dichloride (0.53 g, 5 mmol) was added to a stirred
solution of 34b (0.33 g, 0.63 mmol) in methanol (30 mL) at 0 °C and
under nitrogen. TEA (1.0 g, 1.4 mL, 10 mmol) was added dropwise.
The mixture was allowed to reach room temperature and then refluxed
for 4 h. The solvent was removed at reduced pressure, and the residue
was treated with a saturated solution of KHSO4 and extracted with
ethyl acetate (3 × 30 mL). The combined organic phases were washed
with a saturated solution of NaHCO3, brine and then dried over
Na2SO4. The solvent was removed at reduced pressure, and the crude
residue was purified by flash chromatography (silica gel; eluent n-
hexane/ethyl acetate, 1:1). Compound (−)-20 (111 mg, 0.34 mmol,
54% yield) was obtained as a white amorphous solid. [α]D −29.64
(CHCl3, c 111 mg/10 mL). The protocol reported above, applied to
34a, afforded the enatiomer (+)-20 (81 mg, 52% yield); [α]D +29.10
(CHCl3, c 101 mg/5 mL). The enantiomeric purity of (+)-20 and
(−)-20 was verified via chiral HPLC by comparison with the racemic
mixture and was 97% .
Resolution of ( )-trans-21. Compounds (−)-trans-21 and
(+)-trans-21 were obtained following the same methodology described
for the isomers 20. The crude mixture of 35a and 35b was obtained
with 35% yield. The mixture was purified by gradient flash
chromatography (silica gel; eluent n-hexane/tert-butyl methyl ether,
1:1). Compounds 35a and 35b were obtained as a white amorphous
solid. The two diastereoisomers were then separated by gradient flash
chromatography (silica gel) using n-hexane/ethyl acetate as eluent (n-
hexane/ethyl acetate ratio from 8/2 to 1/1).
35a. Diastereoisomer with Rf = 0.66 on silica gel TLC plate; (two
runs, eluent, n-hexane/ethyl acetate, 1/1). [α]D −5.83 (MeOH, c 120
mg/10 mL). 1H NMR (DMSO-d6, 80 °C, 500 MHz) δ: 7.38 (1H, dd,
J = 8.4, 1.5 Hz), 7.23−7.14 (2H, m), 6.57 (2H, s), 5.52 (1H, bs), 5.18
(1H, d, J = 1.5), 4.45 (1H, dd, J = 8.76, 3.83 Hz), 4.33 (1H, dd, J =
8.61, 4.22 Hz), 3.80 (3H, s), 3.68 (6H, s), 3.64 (3H, s), 3.44−3.37
(4H, m), 2.36−2.29 (2H, m), 2.17−2.10 (1H, m), 2.00−1.87 (5H, m),
1.41 (18H, s). 13C NMR (CDCl3, 25 °C, 125 MHz) (two
conformers): δ 172.32 and 172.29, 171.08 and 170.84, 161.53 and
161.45, 154.08 and 153.97, 153.62, 153.30 and 153.17, 151.65 and
151.57, 139.63 and 139.50, 134.91 and 134.87, 132.71, 128.27 and
128.17, 126.30 and 126.22, 121.78, 113.92 and 113.85, 95.94, 82.36
and 82.31, 79.72 and 79.68, 62.21 and 62.07, 60.55, 58.84 and 58.76,
56.51 and 56.21, 46.85 and 46.67, 38.71, 30.91 and 30.87, 29.94, and
29.82, 28.53 and 28.38, 24.54 and 24.34, 23.69, and 23.55. EI-MS (m/
z): 769 (M+).
(
)-3,4-trans-4-(3,5-Dimethoxyphenyl)-3-hydroxy-1-(4-
methoxyphenyl)azetidin-2-one (( )-trans-24) and ( )3,4-cis-4-
(3,5-Dimethoxyphenyl)-3-hydroxy-1-(4-methoxyphenyl)-
azetidin-2-one (( )-cis-24). Compounds trans-24 and cis-24 were
prepared following the method described in procedure a. Starting from
imine 16 (0.87 g, 2.2 mmol), ( )-trans-24 (100 mg) and ( )-cis-24
(390 mg) were obtained with 14% and 55% yields, respectively. The
intermediate mixture of trans-22 and cis-22 was obtained with 18%
yield.
1
( )-trans-22. H NMR (CDCl3): δ 7.28 (2H, d, J = 8.9 Hz), 6.79
(2H, J = 8.9 Hz), 6.45−6.40 (3H, m), 5.40 (1H, d, J = 1.2 Hz), 4.82
(1H, d, J = 1.2 Hz), 3.73 (9H,s), 2.16 (3H, s). ESI-MS (m/z): 371
(M+).
( )-cis-22. 1H NMR (CDCl3): δ 7.28 (2H, J = 8.9 Hz), 6.79 (2H, J
= 8.9 Hz), 6.46−6.41 (3H, m), 5.94 (1H, d, J = 4.9 Hz), 5.23 (1H, d, J
= 4.9 Hz), 3.73 (9H, s), 1.76 (3H, s). 13C NMR (CDCl3): δ 169.88,
161.96, 161.51, 157.30, 135.60, 131.04, 119.54, 119.41, 115.05, 106.49,
101.30, 76.82, 62.13, 56.02, 20.63. ESI-MS: m/z 371 (M+).
1
( )-trans-24. H NMR (CDCl3): δ 7.23 (2H, d, J = 9 Hz), 6.77
(2H, d, J = 9 Hz), 6.48 (2H, d, J = 2.2 Hz), 6.42 (1H, tr, J = 2.2 Hz),
4.78 (1H, d, J = 1.5 Hz), 4.73 (1H, d, J = 1.5 Hz), 3.77 (6H, s), 3.76
(3H,s). 13C NMR (CDCl3): δ 165.86, 161.66, 156.37, 138.77, 129.69,
119.13, 114.54, 104.20, 100.60, 83.83, 65.87, 55.60. ESI-MS: m/z 329
(M+).
1
( )-cis-24. H NMR (CDCl3): δ 7.29 (2H, d, J = 8.8 Hz), 6.80
(2H, d, J = 8.8 Hz), 6.48 (3H, s), 5.16 (2H, s), 3.76 (9H, s). 13C NMR
(CDCl3): δ 165.94, 161.62, 156.67, 135.97, 130.69, 119.03, 114.58,
105.60, 100.73, 77.40, 62.70, 55.59. ESI-MS: m/z 329 (M+).
( )-3,4-trans-3-Hydroxy-1-(3- hydroxy-4-methoxyphenyl)-4-
(3,4,5-trimethoxyphenyl)azetidin-2-one (( )-trans-25) and
(
)-3,4-cis-3-Hydroxy-1-(3-hydroxy-4-methoxyphenyl)-4-
(3,4,5-trimethoxyphenyl)azetidin-2-one (( )-cis-25). Com-
pounds ( )-trans-25 and ( )-cis-25 were prepared following the
method described in procedure a. Starting from imine 17 (0.2 g, 0.38
mmol), compounds ( )-trans-25 (47 mg) and ( )-cis-25 (61 mg)
were obtained with 33% and 43% yields, respectively.
1
( )-trans-23. H NMR (CDCl3): δ 7.23 (1H, d, J = 2.4 Hz), 7.12
(1H, dd, J1 = 8.9 Hz, J2 = 2.4), 6.86 (1H, d, J = 8.9 Hz), 6.53 (2H, s),
5.40 (1H, d, J = 1.4 Hz), 4.86 (2H, s), 4.82 (1H, d, J = 1.4 Hz), 3.85
(3H, s), 3.82 (6H,s), 3.78 (3H, s), 2.20 (3H, s), 2.18 (3H, s). 13C
NMR (CDCl3): δ 170.06, 169.65, 165.69, 161.39, 153.89, 148.13,
139.16, 138.45, 130.32, 115.98, 112.97, 112.82, 103.22, 82.54, 64.12,
60.82, 60.25, 56.20, 20.49. ESI-MS: m/z 540 (M + Na+).
1
( )-cis-23. H NMR (CDCl3): δ 7.26 (1H, d, J = 2.5 Hz), 7.14
(1H, dd, J1 = 8.9 Hz, J2 = 2.5 Hz), 6.88 (1H, d, J = 8.9 Hz), 6.48 (2H,
s), 5.94 (1H, d, J = 4.9 Hz), 5.25 (1H, d, J = 4.9 Hz), 4.87 (2H, s),
3.88 (3H, s), 3.80 (6H, s), 3.79 (3H, s), 2.18 (3H), 1.76 (3H). 13C
NMR (CDCl3): δ 170.22, 169.35, 165.84, 161.74, 153.49, 148.38,
139.37, 138.45, 130.46, 127.68, 116.08, 113.02, 104.98, 76.68, 62.04,
61.43, 60.41, 56.40, 20.56, 20.15. ESI-MS: m/z 540 (M + Na+).
35b. Diastereoisomer with Rf = 0.57; on silica gel TLC plate (two
runs, eluent n-hexane/ethyl acetate, 1/1). [α]D −56.12 (MeOH, c 131
mg/10 mL). 1H NMR (DMSO-d6, 80 °C, 500 MHz) δ: 7.38 (1H, d, J
= 7.9 Hz), 7.20 (1H, d, J = 7.9 Hz), 7.16 (1H, bs), 6.56 (2H, s), 5.52
(1H, bs), 5.18 (1H, d, J = 1.5), 4.45 (1H, dd, J = 8.0, 3.91 Hz), 4.34
(1H, dd, J = 8.0, 4.27 Hz), 3.80 (3H, s), 3.66 (6H, s), 3.63 (3H, s),
3.44−3.37 (4H, m), 2.36−2.31 (2H, m), 2.17−2.15 (1H, m), 2.04−
2.00 (1H, m), 1.93−1.88 (4H, m), 1.37 (18H, s). 13C NMR (CDCl3,
25 °C, 125 MHz) (two conformers) δ: 172.38 and 171.99, 171.10 and
170.88, 161.46 and 161.37, 154.03 and 153.97, 153.71, 153.31 and
153.21, 151.65 and 151.59, 139.65 and 139.49, 134.89, 132.74 and
132.66, 128.28 and 128.17, 126.46 and 126.14, 122.42 and 122.26,
121.73 and 121.62, 113.79, 96.03 and 95.83, 82.29 and 82.05, 79.66
1
( )-trans-25. H NMR (CDCl3): δ 6.90 (1H, d, J = 2.4 Hz), 6.83
(1H, dd, J1 = 2.4, 8.6 Hz), 6.71 (1H, d, J = 8.6 Hz), 6.52 (2H, s), 5.31
(1H, s), 4.76 (1H, d, J = 1.6 Hz), 4.70 (1H, d, J = 1.6 Hz), 3.86 (3H,
s), 3.85 (3H, s), 3.82 (6H, s). 13C NMR (CDCl3): δ 166.28, 154.07,
146.05, 131.84, 131.22, 111.05, 109.69, 105.05, 102.98, 83.90, 66.18,
61.03, 56.40. ESI-MS: m/z 375 (M+).
1
( )-cis-25. H NMR (400 MHz, CDCl3): δ 6.98 (1H, d, J = 8.0
Hz), 6.95 (1H, s), 6.79 (1H, d, J = 8.0 Hz), 6.53 (2H, s), 5.69 (1H, s),
2120
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
5.19−5.15 (2H, m), 3.88 (6H, s), 3.85 (6H, s). 13C NMR (100 MHz,
CDCl3): δ 156.00, 147.00, 129.80, 111.68, 111.09, 105.28, 104.71,
63.29, 61.52, 56.89. ESI-MS: m/z 375 (M+).
3-Amino-4-(3,5-dimethoxyphenyl)-1-(4-methoxyphenyl)-
azetidin-2-one (( )-cis-32). Compounds ( )-trans-30 and ( )-cis-
30 were prepared following the procedures described for compound
26. Starting from imine 16 (1.74 g, 6.42 mmol), the mixture of isomers
trans-30 and cis-30 were isolated (0.55 g, 1.20 mmol 19% yield) after
purification by flash column chromatography (silica gel; eluent n-
hexane/ethyl acetate, 1:1).
(
)-3,4-trans-3-Amino-1-(3,5-dimethoxyphenyl)-4-(4-
methoxyphenyl)azetidin-2-one (( )-trans-28) and ( )-3,4-cis-
3-Amino-1-(3,5-dimethoxyphenyl)-4-(4-methoxyphenyl)-
azetidin-2-one (( )-cis-28) (Traces). A solution of phthalylglycyl
chloride (1.36 g, 6.08 mmol) in anhydrous methylene chloride (3 mL)
was added dropwise at 0 °C under nitrogen to a stirred solution of
imine 14 (0.375 g, 1.38 mmol) and TEA (1.23 g, 1.7 mL, 12.6 mmol)
in anhydrous methylene chloride (7 mL). The solution was
maintained at 0 °C for 1 h and then allowed to reach room
temperature and stirred for 24 h. HCl (1 N) was added (40 mL), and
the two phases were stirred for 40 min. The aqueuos phase was
separated and extracted twice with dichloromethane. Organic phases
were washed with a saturated solution of NaHCO3 dried with sodium
sulfate, filtered, and concentrated under reduced pressure, affording a
brown oil which was purified by flash column chromatography (silica
gel; eluent gradient of n-hexane/ethyl acetate from 7/3 to 4/6). The
isomer ( )-trans-26 only was isolated (0.289 g, 0.62 mmol, 46%
yield). Compound ( )-cis-26 was formed only in traces from the
1
( )-trans-30. H NMR (CDCl3): δ 7.91−7.89 (2H, m), 7.79−7.77
(2H, m), 7.32 (2H, d, J = 8.8 Hz), 6.84 (2H, d, J = 8.8 Hz), 6.51 (2H,
d, J = 2.4 Hz), 6.45 (1H, tr, J = 2.4 Hz), 5.30 (1H, d, J = 2.4 Hz), 5.26
(1H, d, J = 2.4 Hz), 3.78 (9H, s). 13C NMR (CDCl3): δ 166.99,
161.80, 156.74, 138.57, 134.73, 134.46, 131.93, 130.95, 123.99, 123.64,
118.84, 115.49, 104.32, 62.87, 61.66, 55.62. ESI-MS: m/z 481 (M +
Na+).
( )-cis-30. 1H NMR (CDCl3): δ 7.9−7.4 (4H, m), 7.41 (2H, d, J =
6.8 Hz), 7.00 (2H, d, J = 6.8 Hz), 6.45 (2H, d, J = 2.2 Hz), 6.20 (1H,
tr, J = 2.2 Hz), 5.63 (1H, d, J = 5.5 Hz), 5.35 (1H, d, J = 5.5 Hz), 3.78
(3H,s), 3.63 (6H, s). 13C NMR (CDCl3): δ 166.93, 160.90, 160.44,
156.71, 134.94, 134.73, 131.92, 131.49, 123.83, 123.63, 119.24, 114.57,
105.22, 62.18, 61.37, 55.55. ESI-MS: m/z 481 (M + Na+).
Compounds ( )-trans-30 and ( )-trans-30 were treated with
hydrazine dihydrochloride and afford after purification by flash
chromatography (silica gel; eluent n-hexane/ethyl acetate, 2/8) the
stereoisomer ( )-trans-32 (7% yield) as a yellow amorphous solid and
the stereoisomer ( )-cis-32 (8% yield).
1
reaction. ( )-trans-26: H NMR (CDCl3) δ 7.9−7.7 (4H, m), 7.32
(2H, J = 8.8 Hz), 6.82 (2H, J = 8.8 Hz), 6.50 (2H, d, J = 2.2 Hz), 6.44
(1H, tr, J = 2.2 Hz), 5.29 (1H, d, J = 2.7 Hz), 5.26 (1H, d, J = 2.7 Hz),
3.76 (9H, s). 13C NMR (CDCl3): δ 166.99, 161.80, 156.74, 138.57,
134.73, 131.93, 130.95, 123.98, 119.25, 114.57, 104.32, 100.94, 62.82,
61.65, 55.62. ESI-MS: m/z 481 (M + Na+).
1
( )-trans-32. H NMR (CDCl3): δ 7.30 (2H, d, J = 9.5 Hz), 6.80
(2H, d, J = 9.5 Hz), 6.48 (2H, d, J = 2.2 Hz), 6.42 (1H, t, J = 2.2 Hz),
4.55 (1H, d, J = 2.0 Hz), 4.06 (1H, d, J = 2.0 Hz), 3.76 (9H, s). 13C
NMR (CDCl3): δ 167.50, 161.56, 156.39, 139.64, 131.15, 118.85,
114.52, 103.95, 100.42, 69.90, 66.98, 55.59. ESI-MS: m/z 351(M +
Na+).
Hydrazine dihydrochloride (0.30 g, 2.8 mmol) was added, at 0 °C
and under nitrogen to a stirred suspension of ( )-trans-26 (0.6 mg,
1.3 mmol) in methanol (10 mL). TEA (1.6 g, 2.2 mL, 15.8 mmol) was
added dropwise. The mixture was allowed to reach room temperature
and then warmed at 50 °C for 5 h. The solvent was removed at
reduced pressure, and the residue was treated with 1 N HCl (40 mL)
and extracted with dichloromethane (3 × 10 mL). The aqueous phase
was made alkaline by 3 N NaOH and extracted with dichloromethane
(3 × 10 mL). The organic phase was dried (Na2SO4), and the solvent
was removed at reduced pressure. A solid was obtained, which was
purified by flash chromatography (silica gel; eluent n-hexane/ethyl
acetate, 2/8) to afford the stereoisomer ( )-trans-28 (0.36 g, 1.1
mmol, 85% yield) as a yellow solid. 1H NMR (CDCl3) δ 7.29 (2H, d, J
= 8.7 Hz), 6.93 (2H, d, J = 8.7 Hz), 6.49 (1H, d, J = 2.2 Hz), 6.41 (1H,
tr, J = 2.2 Hz), 4.64 (1H, d, J = 2.2 Hz), 4.10 (1H, d, J = 2.2 Hz), 3.75
(9H, s). 13C NMR (CDCl3): δ 161.65, 139.41, 118.92, 114.52, 104.02,
100.51, 69.27, 66.40, 55.59. ESI-MS: m/z 351 (M + Na+).
( )-cis-32. 1H NMR (CDCl3): δ 7.30 (2H, d, J = 9 Hz), 6.80 (2H,
d, J = 9 Hz), 6.42 (1H, t, J = 2.2 Hz), 6.39 (2H, d, J = 2.2 Hz), 5.14
(1H, d, J = 5.4 Hz), 4.59 (1H, d, J = 5.4 Hz), 3.76 (9H, s). 13C NMR
(CDCl3): δ 167.46, 161.67, 156.37, 137.22, 131.14, 118.82, 114.51,
105.22, 103.95, 100.25, 69.91, 66.96, 63.99, 62.59, 55.59. ESI-MS: m/z
351 (M + Na+).
(
)-3,4-trans-3-Amino-4-(3-hydroxy-4-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)azetidin-2-one (( )-trans-33) and
( )-3,4-cis-3-Amino-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (( )-cis-33). Compounds
( )-trans-31 and ( )-cis-31 were prepared following the procedures
described for compound 26. Starting from imine 17 (0.324 g, 1.02
mmol), the mixture of isomers ( )-trans-27 and ( )-cis-27 was
isolated (0.48 g, 0.7 mmol 68% yield) after purification by flash
column chromatography (silica gel; eluent n-hexane/ethyl acetate,
45:55).
(
)-3,4-trans-3-Amino-4-(3-hydroxy-4-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)azetidin-2-one (( )-trans-29). Com-
pounds ( )-trans-27 and ( )-cis-27 were prepared following the
procedures described for compound 26. Starting from imine 15 (0.25
g, 0.8 mmol), the isomer trans-27 only was isolated (0.34 g, 0.50 mmol
63% yield) after purification by flash column chromatography (silica
gel; eluent gradient of n-hexane/ethyl acetate from 85/15 to 40/60).
Compound cis-27 was formed only in traces from the reaction.
( )-trans-27: 1H NMR (CDCl3) δ 8−7.6 (8H, m), 7.29 (1H, dd, J1 =
8.4 Hz, J2 = 2.2 Hz), 7.19 (1H, d, J = 2.2 Hz), 7.00 (1H, d, J = 8.4 Hz),
6.57 (2H, s), 5.28 (2H, s), 4.71 (2H, s), 3.85 (3H, s), 3.76 (3H, s),
3.70 (6H, s). 13C NMR (CDCl3): δ 167.46, 166.96, 165.41, 162.03,
140.09, 134.79, 134.53, 133.40, 132.15, 131.86, 128.37, 126.40, 125.08,
124.27, 123.87, 121.47, 113.64, 95.50, 67.78, 61.07, 56.24, 38.89. ESI-
MS: m/z 691 (M+).
( )-trans-31. 1H NMR (CDCl3): δ 8.0−7.8 (8H, m), 7.27 (1H, d, J
= 2.6 Hz), 7.16 (1H, dd, J1 = 2.6 Hz, J2 = 8.9 Hz), 6.88 (1H, d, J = 8.9
Hz), 6.56 (2H, s), 5.29 (1H, d, J = 2.6 Hz), 5.25 (1H, d, J = 2.6 Hz),
4.71 (2H, s) 3.86 (3H, s), 3.82 (6H,s), 3.80 (3H, s). 13C NMR
(CDCl3): δ 167.42, 166.97, 165.34, 161.98, 148.23, 139.77, 134.80,
134.45, 132.20, 131.89, 131.25, 130.85, 124.05, 123.86, 116.36, 113.12,
103.16, 63.12, 62.10, 61.03, 56.43, 38.87. ESI-MS: m/z 691 (M+).
( )-cis-31. 1H NMR (CDCl3): δ 7.91−7.75 (8H, m), 7.69 (1H, d, J
= 2.3 Hz), 7.38 (1H, dd, J1 = 2.3 Hz, J2 = 8.9 Hz), 6.94 (1H, d, J = 8.9
Hz), 6.49 (2H, s), 5.63 (1H, d, J = 5.4 Hz), 5.34 (1H, d, J = 5.4 Hz),
4.72 (2H, s), 3.84 (3H, s), 3.72 (6H, s), 3.65 (3H,s). ESI-MS: m/z 691
(M+).
Compound ( )-trans-27 was treated with hydrazine dihydro-
chloride and affords after purification by flash chromatography (silica
gel; eluent n-hexane/ethyl acetate, 2/8) the stereoisomer ( )-trans-29
Compounds ( )-trans-33 and ( )-cis-33 were obtained following
the procedures described for compound 26. Starting from a mixture of
compounds ( )-trans-31 and ( )-cis-31 (179 mg, 0.26 mmol),
isomers ( )-trans-33 (33% yield) and ( )-cis-33 (22% yield) were
isolated after purification by flash column chromatography (silica gel;
eluent hexane/ethyl acetate = 2:8).
1
(0.048 g, 0.13 mmol, 81% yield) as a yellow amorphous solid. H
NMR (CDCl3): δ 6.92 (1H, s), 6.89 (2H, s), 6.54 (2H, s), 4.54 (1H, d,
J = 2.2 Hz), 4.05 (1H, d, J = 2.2 Hz), 3.88 (3H, s), 3.76 (3H, s), 3.71
(6H,s). 13C NMR (CDCl3): δ 168.05, 153.65, 147.11, 146.51, 134.80,
133.80, 130.14, 118.14, 117.99, 112.33, 111.22, 95.38, 69.70, 66.70,
61.09, 56.22. ESI-MS: m/z 374 (M+).
1
( )-trans-33. H NMR (400 MHz, CDCl3): δ 6.94 (1H, d, J = 2.4
Hz), 6.79 (1H, dd, J1 = 8.7 Hz, J2 = 2.4 Hz), 6.72 (1H, d, J = 8.7 Hz),
6.53 (2H, s), 4.54 (1H, d, J = 2.0 Hz), 4.04 (1H, d, J = 2.0 Hz), 3.85
(3H, s), 3.82 (3H, s), 3.80 (6H, s). 13C NMR (100 MHz; CDCl3): δ
(
)-3,4-trans-3-Amino-4-(3,5-dimethoxyphenyl)-1-(4-
methoxyphenyl)azetidin-2-one (( )-trans-32) and ( )-3,4-cis-
2121
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
168.39, 154.53, 146.73, 144.33, 133.23, 132.16, 111.77, 109.75, 105.45,
10% glycerol) at 2.5 mg/mL and mixed with 1 mM GTP and varying
concentrations of the compounds. DMSO (0.2% v/v) was used as a
vehicle control. Tubulin assembly was monitored at 340 nm at 37 °C
in a Jasco V-530 spectrophotometer (Jasco Europe, Italy). The IC50
values were obtained using the software GraphPad Prism (San Diego,
CA) and were defined as the compound concentration that inhibited
the extent of assembly by 50% after a 30 min incubation. Assays were
performed in triplicate in two independent experiments.
103.29, 70.37, 67.53, 61.48, 56.84. ESI-MS: m/z 374 (M+).
1
( )-cis-33. H NMR (400 MHz; CDCl3): δ 6.98−6.94 (2H, m),
6.77 (1H, d, J = 8.4 Hz), 6.46 (2H, s), 5.13 (1H, d, J = 5.2 Hz), 4.58
(1H, d, J = 5.2 Hz), 3.87 (6H, s), 3.82 (6H, s). 13C NMR (100 MHz;
CDCl3): δ 168.29, 154.59, 146.64, 144.17, 138.69, 132.20, 130.60,
111.74, 109.87, 105.30, 104.48, 67.64, 64.36, 61.47, 56.85. ESI-MS: m/
z 374 (M+).
Biology: Materials and Methods. Reagents and Antibodies.
Anti-AMPK, anti-phospho-AMPK (Thr172), anti-caspase-3, anti-
PARP, anti-Raptor, anti-phospho-Raptor (Thr792), and anti-vinculin
for Western blotting analysis were purchased from Cell Signaling
Technologies (Beverly, MA). Anti-fibrillarin antibody, used as nuclear
extract control, was purchased from EnCor Biotechnology (Gaines-
ville, FL). Dulbecco’s modified Eagle’s medium (DMEM), Eagle's
minimal essential medium medium (EMEM), F12 medium, Leibovitz
medium, fetal bovine serum (FBS), L-glutamine, penicillin, and
streptomycin were obtained from Lonza (Basel, Switzerland). All
other reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Cell Cultures. HuTu-80 cells and HeLa cells were cultured using
DMEM supplemented with 10% (v/v) FBS, 4 mM L-glutamine, 100
U/mL penicillin, and 100 μg/mL streptomycin. FHs74 cells were
cultured using DMEM supplemented with 10% (v/v) FBS, 4 mM L-
glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 30 ng/mL
EGF, and 10 μg/mL insulin. CCD841CoN cells and MCF-7 cells were
cultured using EMEM/NEAA supplemented with 10% (v/v) FBS, 4
mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.
T84 cells were cultured using DMEM/F12 medium supplemented
with 5% (v/v) FBS, 4 mM L-glutamine, 100 U/mL penicillin, and 100
μg/mL streptomycin. SKNBE cells were cultured using EMEM/F12
medium supplemented with 10% (v/v) FBS, 4 mM L-glutamine, 100
U/mL penicillin, and 100 μg/mL streptomycin. All these cell lines
were maintained at 37 °C in a humidified 5% CO2 incubator. SW48,
SW480, and SW620 cells were cultured using Leibovitz’s medium
supplemented with 10% (v/v) FBS, 4 mM L-glutamine, 100 U/mL
penicillin, and 100 μg/mL streptomycin. These cell lines were
maintained at 37 °C in a humidified incubator.
Subcellular Fractionation and Total Protein Extraction. To
detect p53 levels and phosphorylation, nuclear extracts were prepared
using the NE-PER kit (Pierce Biotechnology, Rockford, IL) according
to the manufacturer’s protocol. To analyze all the other proteins, total
cell extracts were prepared using RIPA buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS)
plus 1 mM PMSF (phenylmethanesulfonyl fluoride), protease
inhibitor cocktail (Roche, Indianapolis, IN), and phosphatase inhibitor
cocktail (Sigma-Aldrich, St. Louis, MO).
Flow Cytofluorimetric Analysis. For flow cytometric analysis,
cells were trypsinized, washed with PBS, and fixed in 75% ethanol at 4
°C. Samples were stained with propidium iodide (Sigma-Aldrich, St.
Louis, MO) to analyze DNA content using a BD Biosciences FACScan
(Becton-Dickinson, San Jose, CA).
ASSOCIATED CONTENT
* Supporting Information
Figure S1 showing that the new azetidinone compounds exhibit
antiproliferative activity on HeLa cells and Figure S2 showing
that the new β-lactams induce apoptotic cell death in cancer
cells. This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*For R.P.: phone, +39-0250314118; e-mail, roberto.pagliarin@
unimi.it. For P.C.: phone, +39-0264483521; e-mail, paola.
coccetti@unimib.it.
■
Present Address
⊥IFOM-IEO Campus, Via Adamello, 16, 20139 Milan, Italy.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Lilia Alberghina for helpful discussions and
support and Neil Campbell for language editing. This work was
supported by a grant to P.C. (FAR 2008), to P.F. (FAR 2010),
to M.F. (Oncosuisse), and to F.O. (PRIN 2007, Grant
2007K29W5J and PRIN 2009, Grant 2009KFMP7Z). The
patent in ref 15 was supported by UNIMITT of University of
Milan, Italy, and by University of Milano-Bicocca, Italy.
DEDICATION
■
†We dedicate this work to the memory of our friend Alessandro
Gori, who believed and hoped in scientific research and whom
we thank for his encouragement.
ABBREVIATIONS USED
■
CA-4, combretastatin A-4; AMP, adenosine monophosphate;
AMPK, adenosine monophosphate activated protein kinase;
ATP, adenosine 5′-triphosphate; CRC, colorectal cancer;
DIPEA, N,N-diisopropylethylamine; DMSO, dimethyl sulf-
oxide; FBS, fetal bovine serum; Ft, ftalyl; EGF, epidermal
growth factor; EGTA, ethylene glycol tetraacetic acid; GTP,
guanidine triphosphate; HBTU, O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; IC50,
50% inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; NMR, nuclear magnetic
resonance; PARP, poly ADP-ribose polymerase; PIPES,
piperazine-N,N′-bis(2-ethanesulfonic acid); PMSF, phenylme-
thanesulfonyl fluoride; SBA, small bowel adenocarcinoma; SDS,
sodium dodecyl sulfate; TEA, triethylamine; TMS, tetrame-
thylsilane
Growth Inhibition Assay. The antiproliferative activity of the
azetidinone derivatives was measured by the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded
in 96-well plates. After 24 h of incubation in the appropriate medium,
cells were treated with different concentrations of each compound for
48−72 h, as indicated in the figure captions. Afterward, the MTT
solution was added, and then plates were incubated for 2 h at 37 °C.
The purple formazan crystals were solubilized. and the plates were
read on a model 550 microplate reader (BioRad Laboratories,
Hercules, CA) at 570 nm. The IC50 values were obtained using the
software GraphPad Prism (San Diego, CA) and were defined as the
concentration of drug causing a 50% inhibition in absorbance
compared to control cells. Assays were performed in triplicate in
three independent experiments.
REFERENCES
Tubulin Polymerization Assay. The effect of compounds on the
assembly of tubulin was determined spectrophotometrically by
monitoring the change in turbidity. Lyophilized purified porcine
brain tubulin (Cytoskeleton, Denver, CO) was resuspended in ice-cold
assembly buffer (80 mM PIPES, pH 6.9, 1 mM MgCl2, 2 mM EGTA,
■
(1) Yue, Q.-X.; Liu, X; Guo, D.-A. Microtubule-binding natural
products for cancer therapy. Planta Med. 2010, 76, 1037−1043.
(2) Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Antimitotic natural
products combretastatin A-4 and combretastatin A-2: studies on the
2122
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
mechanism of their inhibition of the binding of colchicine to tubulin.
Biochemistry 1989, 28, 6984−6991.
(22) Shackelford, D. B.; Shaw, R. J. The LKB1-AMPK pathway:
metabolism and growth control in tumour suppression. Nat. Rev.
Cancer 2009, 9, 563−575.
(3) Lippert, J. W. Vascular disrupting agents. Bioorg. Med. Chem.
2007, 15, 605−615.
(23) Buzzai, M.; Jones, R. G.; Amaravadi, R. K.; Lum, J. J.;
DeBerardinis, R. J.; Zhao, F.; Viollet, B.; Thompson, C. B. Systemic
treatment with the antidiabetic drug metformin selectively impairs
p53-deficient tumor cell growth. Cancer Res. 2007, 67, 6745−6752.
(24) Zakikhani, M.; Dowling, R. J. O.; Sonenberg, N.; Pollak, M. N.
The effects of adiponectin and metformin on prostate and colon
neoplasia involve activation of AMP-activated protein kinase. Cancer
Prev. Res. 2008, 1, 369−375.
(25) Fay, J. R.; Steele, V.; Crowell, J. A. Energy homeostasis and
cancer prevention: the AMP-activated protein kinase. Cancer Prev. Res.
2009, 2, 301−309.
(26) Ji, C.; Yang, B.; Yang, Y.-L.; He, S.-H.; Miao, D.-S.; He, L.; Bi,
Z.-G. Exogenous cell-permeable C6 ceramide sensitizes multiple
cancer cell lines to doxorubicin-induced apoptosis by promoting
AMPK activation and mTORC1 inhibition. Oncogene 2010, 29, 6557−
6568.
(27) Jiao, L.; Liang, Y.; Xu, J. Origin of the relative stereoselectivity of
the beta-lactam formation in the Staudinger reaction. J. Am. Chem. Soc.
2006, 128, 6060−6069.
(28) Endo, M.; Droghini, R. Synthesis of trans-1-p-methoxyphenyl-3-
acetoxy-4-phenylazetidin-2-one. A key starting β-lactam for 2′-epi-
taxol. Bioorg. Med. Chem. Lett. 1993, 3, 2483−2486.
(29) Shen, C.-H.; Shee, J.-J.; Wu, J.-Y.; Lin, Y.-W.; Wu, J.-D.; Liu, Y.-
W. Combretastatin A-4 inhibits cell growth and metastasis in bladder
cancer cells and retards tumour growth in a murine orthotopic bladder
tumour model. Br. J. Pharmacol. 2010, 160, 2008−2027.
(30) Wu, R.; Ding, W.; Liu, T.; Zhu, H.; Hu, Y.; Yang, B.; He, Q.
XN05, a novel synthesized microtubule inhibitor, exhibits potent
activity against human carcinoma cells in vitro. Cancer Lett. 2009, 285,
13−22.
(31) Overman, M. J.; Kopetz, S.; Wen, S.; Hoff, P. M.; Fogelman, D.;
Morris, J.; Abbruzzese, J. L.; Ajani, J. A.; Wolff, R. A. Chemotherapy
with 5-fluorouracil and a platinum compound improves outcomes in
metastatic small bowel adenocarcinoma. Cancer 2008, 113, 2038−
2045.
(4) Nam, N.-H. Combretastatin A-4 analogues as antimitotic
antitumor agents. Curr. Med. Chem. 2003, 10, 1697−1722.
(5) West, C. M. L.; Price, P. Combretastatin A4 phosphate. Anti-
Cancer Drugs 2004, 15, 179−87.
(6) www.clinicaltrials.gov.
(7) Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.;
Genazzani, A. A. Medicinal chemistry of combretastatin A4: present
and future directions. J. Med. Chem. 2006, 49, 3033−3044.
(8) Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.;
Hannick, S. M.; Gherke, L.; Credo, R. B.; Hui, Y.-H.; Marsh, K.;
Warner, R.; Lee, J. Y.; Zielinski-Mozng, N.; Frost, D.; Rosenberg, S.
H.; Sham, H. L. Potent, orally active heterocycle-based combretastatin
A-4 analogues: synthesis, structure−activity relationship, pharmacoki-
netics, and in vivo antitumor activity evaluation. J. Med. Chem. 2002,
45, 1697−1711.
(9) O’Boyle, N. M.; Carr, M.; Greene, L. M.; Bergin, O.; Nathwani, S.
M.; McCabe, T.; Lloyd, D. G.; Zisterer, D. M.; Meegan, M. J. Synthesis
and evaluation of azetidinone analogues of combretastatin A-4 as
tubulin targeting agents. J. Med. Chem. 2010, 53, 8569−8584.
(10) Banik, B. K.; Banik, I.; Becker, F. F. Novel anticancer β-lactams.
Top Heterocycl. Chem 2010, 22, 349−373.
(11) Smith, D. M.; Kazi, A.; Smith, L.; Long, T. E.; Heldreth, B.;
Turos, E.; Dou, Q. P. A novel beta-lactam antibiotic activates tumor
cell apoptotic program by inducing DNA damage. Mol. Pharmacol.
2002, 61, 1348−1358.
(12) Sun, L.; Vasilevich, N. I.; Fuselier, J. A.; Hocart, S. J.; Coy, D. H.
Examination of the 1,4-disubstituted azetidinone ring system as a
template for combretastatin A-4 conformationally restricted analogue
design. Bioorg. Med. Chem. Lett. 2004, 14, 2041−2046.
(13) O’Boyle, N. M.; Greene, L. M.; Bergin, O.; Fichet, J. B.;
McCabe, T.; Lloyd, D. G.; Zisterer, D. M.; Meegan, M. J. Synthesis,
evaluation and structural studies of antiproliferative tubulin-targeting
azetidin-2-ones. Bioorg. Med. Chem. 2011, 19, 2306−2325.
(14) Carr, M.; Greene, L. M.; Knox, A. J.; Lloyd, D. G.; Zisterer, D.
M.; Meegan, M. J. Lead identification of conformationally restricted β-
lactam type combretastatin analogues: synthesis, antiproliferative
activity and tubulin targeting effects. Eur. J. Med. Chem. 2010, 45
(12), 5752−5766.
(32) Delaunoit, T.; Neczyporenko, F.; Limburg, P. J.; Erlichman, C.
Small bowel adenocarcinoma: a rare but aggressive disease. Clin.
Colorectal Cancer 2004, 4, 241−248 (discussion, 249−251).
(33) Blaker, H.; Aulmann, S.; Helmchen, B.; Otto, H. F.; Rieker, R. J.;
̈
Penzel, R. Loss of SMAD4 function in small intestinal adenocarcino-
mas: comparison of genetic and immunohistochemical findings.
Pathol. Res. Pract. 2004, 200, 1−7.
(15) Pagliarin, R; Orsini, F.; Montano, G.; Tripodi, F.; Coccetti, P.;
Fusi, P. Patent MI2011A001463, 2011.
(16) Water solubility for ( )-trans-20 is 95 μM, and water solubility
for ( )-trans-21 is 1700 μM (see Experimental Section).
(17) Petrovski, G.; Gurusamy, N.; Das, D. K. Resveratrol in
cardiovascular health and disease. Ann. N.Y. Acad. Sci. 2011, 1215, 22−
33.
(18) Kondratyuk, T. P.; Park, E.-J.; Marler, L. E.; Ahn, S.; Yuan, Y.;
Choi, Y.; Yu, R.; van Breemen, R. B.; Sun, B.; Hoshino, J.; Cushman,
M.; Jermihov, K. C.; Mesecar, A. D.; Grubbs, C. J.; Pezzuto, J. M.
Resveratrol derivatives as promising chemopreventive agents with
improved potency and selectivity. Mol. Nutr. Food Res. 2011, 55,
1249−1265.
(19) Zhang, F.; Sun, C.; Wu, J.; He, C.; Ge, X.; Huang, W.; Zou, Y.;
Chen, X.; Qi, W.; Zhai, Q. Combretastatin A-4 activates AMP-
activated protein kinase and improves glucose metabolism in db/db
mice. Pharmacol. Res. 2008, 57, 318−323.
(20) Coccetti, P.; Montano, G.; Lombardo, A.; Tripodi, F.; Orsini, F.;
Pagliarin, R. Synthesis and biological evaluation of combretastatin
analogs as cell cycle inhibitors of the G1 to S transition in
Saccharomyces cerevisiae. Bioorg. Med. Chem. Lett. 2010, 20, 2780−
2784.
(34) Jones, R. G.; Plas, D. R.; Kubek, S.; Buzzai, M.; Mu, J.; Xu, Y.;
Birnbaum, M. J.; Thompson, C. B. AMP-activated protein kinase
induces a p53-dependent metabolic checkpoint. Mol. Cell 2005, 18,
283−293.
(35) Khanal, P.; Oh, W.-K.; Yun, H. J.; Namgoong, G. M.; Ahn, S.-
G.; Kwon, S.-M.; Choi, H.-K.; Choi, H. S. p-HPEA-EDA, a phenolic
compound of virgin olive oil, activates AMP-activated protein kinase to
inhibit carcinogenesis. Carcinogenesis 2011, 32, 545−553.
(36) Imamura, K.; Ogura, T.; Kishimoto, A.; Kaminishi, M.; Esumi,
H. Cell cycle regulation via p53 phosphorylation by a 5′-AMP activated
protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-
ribofuranoside, in a human hepatocellular carcinoma cell line. Biochem.
Biophys. Res. Commun. 2001, 287, 562−567.
(37) Gwinn, D. M.; Shackelford, D. B.; Egan, D. F.; Mihaylova, M.
M.; Mery, A.; Vasquez, D. S.; Turk, B. E.; Shaw, R. J. AMPK
phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell
2008, 30, 214−226.
(38) Priebe, A.; Tan, L.; Wahl, H.; Kueck, A.; He, G.; Kwok, R.;
Opipari, A.; Liu, J. R. Glucose deprivation activates AMPK and induces
cell death through modulation of Akt in ovarian cancer cells. Gynecol.
Oncol. 2011, 122, 389−395.
(39) Luo, J.; Manning, B. D.; Cantley, L. C. Targeting the PI3K-Akt
pathway in human cancer. Cancer Cell 2003, 4, 257−262.
(21) Hardie, D. G.; Carling, D.; Carlson, M. The AMP-activated/
SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic
cell? Annu. Rev. Biochem. 1998, 67, 821−855.
2123
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124

Journal of Medicinal Chemistry
Article
(40) Water solubility of combretastatin A4 is 350 μM. Lee, M.;
Brockway, O.; Dandavati, A.; Tzou, S.; Sjoholm, R.; Satam, V.;
Westbrook, C.; Mooberry, S. L.; Zeller, M.; Babu, B.; Lee, M. A novel
class of trans-methylpyrazoline analogs of combretastatins: synthesis
and in-vitro biological testing. Eur. J. Med. Chem. 2011, 46, 3099−
3104.
(41) Wang, W.; Guan, K.-L. AMP-activated protein kinase and
cancer. Acta Physiol. 2009, 196, 55−63.
(42) Wullschleger, S.; Loewith, R.; Hall, M. N. TOR signaling in
growth and metabolism. Cell 2006, 124, 471−484.
2124
dx.doi.org/10.1021/jm201344a | J. Med. Chem. 2012, 55, 2112−2124