Lead identification of conformationally restricted β-lactam type combretastatin analogues: Synthesis, antiproliferative activity and tubulin targeting effects

Article abstract of DOI:10.1016/j.ejmech.2010.09.033

The synthesis and study of the structure-activity relationships of a series of rigid analogues of combretastatin A-4 are described which contain the 1,4-diaryl-2-azetidinone (β-lactam) ring system in place of the usual ethylene bridge present in the natur

Full text of DOI:10.1016/j.ejmech.2010.09.033

European Journal of Medicinal Chemistry 45 (2010) 5752e5766
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Lead identification of conformationally restricted b-lactam type combretastatin
analogues: Synthesis, antiproliferative activity and tubulin targeting effects
Miriam Carr a, Lisa M. Greene b, Andrew J.S. Knox c, David G. Lloyd c, Daniela M. Zisterer b,
,
Mary J. Meegan a
*
a School of Pharmacy and Pharmaceutical Sciences, Centre for Synthesis and Chemical Biology, Trinity College Dublin, Dublin 2, Ireland
b School of Biochemistry & Immunology, Trinity College Dublin, Dublin 2, Ireland
c Molecular Design Group, School of Biochemistry & Immunology, Trinity College Dublin, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and study of the structureeactivity relationships of a series of rigid analogues of com-
bretastatin A-4 are described which contain the 1,4-diaryl-2-azetidinone (b-lactam) ring system in place of
Received 13 April 2010
Received in revised form
9 September 2010
Accepted 14 September 2010
Available online 22 September 2010
the usual ethylene bridge present in the natural combretastatin stilbene products. The 1,4-diaryl-2-aze-
tidinones are unsubstituted at C-3, or contain methyl substituent(s) at C-3. The most potent compounds
12d and 12e display antiproliferative activity at nanomolar concentrations when evaluated against the
MCF-7 and MDA-MB-231 human breast carcinoma cell lines. 12d exerts antimitotic effects through an
inhibition of tubulin polymerisation and subsequent G2/M arrest of the cell cycle in human MDA-MB-231
Keywords:
Combretastatin A-4 analogues
breast cancer cells, with similar activity to that of CA-4. These novel b-lactam compounds are identified as
potentially useful scaffolds for the further development of antitumour agents which target tubulin.
b-Lactam
Ó 2010 Elsevier Masson SAS. All rights reserved.
2-Azetidinone
Cytotoxicity
Tubulin
Structureeactivity
1. Introduction
microtubules dynamics at low concentrations; however it binds at
a separate site to the vinca alkaloids, the colchicine domain [5,6].
Microtubules are cytoskeletal structures that are formed by the
self-assembly of and tubulin heterodimers and are involved in
The combretastatins were obtained from the African willow tree
Combretum caffrum. Pettit et al. [7] showed that combretastatin A-4
(2, Fig. 1) (CA-4) potently inhibited microtubule activity by binding
to tubulin at the colchicine site, thereby interfering with cell
growth and proliferation. CA-4 acts as a vascular targeting agent
and induces blood flow reduction and subsequent tumour cell
death [4,8e10]. In contrast to colchicine, the anti-vascular effects of
CA-4 in vivo are apparent well below the maximum tolerated dose,
offering a wide therapeutic window. The disodium phosphate salt
of CA-4 3 has been developed with improved solubility [11,12] and
is currently in phase II clinical trials for the treatment of thyroid
cancer [13e15]. Hydrolysis in vivo by endogenous non-specific
phosphatases under physiological conditions affords CA-4 [11,12].
The cis-double bond in CA-4 is readily converted to the more stable
trans geometry during storage or use, resulting in a dramatic
decrease in antitumour activity [16e18].
a
b
many cellular functions [1]. Their most important role in the
formation of the mitotic spindle, which is intimately involved in cell
division. Antimitotic agents are one of the major classes of cytotoxic
drugs for cancer treatment and microtubules are a significant target
for many natural product anticancer agents [2]. The three charac-
terised binding sites of tubulin are the taxane domain, the vinca
domain and the colchicine domain and many compounds interact
with tubulin at these known sites.
Paclitaxel binds to tubulin at the taxane site and was the first
compound found to stabilise tubulin by promoting the assembly of
tubulin heterodimers into microtubules [3]. The vinca alkaloids,
vinblastine and vincristine bind at the vinca domain resulting in
depolymerisation of microtubules and destruction of mitotic
spindles at high concentrations [4]. Colchicine (1, Fig. 1) also
depolymerises microtubules at high concentrations and stabilizes
Many structural modifications to CA-4 have been reported
including variation of the A and B-rings substituents (Fig. 1)
[19e21]. Most modifications of the B-ring result in decreased
bioactivity; the exception is the removal of the 30-hydroxy group,
which results in a compound with almost the same potency as CA-4
* Corresponding author. Tel.: þ353 1 8962798; fax: þ353 1 8962793.
E-mail address: mmeegan@tcd.ie (M.J. Meegan).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.033

M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
5753
the appropriately substituted Schiff bases 11a and 11c which were
obtained by condensation of the appropriate amine and aldehyde.
Schiff bases 11a and 11c were then protected by treatment with
tert-butyldimethylchlorosilane to yield the respective silyl ether
Abbreviations
CA-4
DEPT
EGTA
EI
combretastatin A-4
products 11b and 11d. The racemic b-lactam products 12a and 12g
distortionless enhancement by polarization transfer
ethylene glycol tetraacetic acid
electron Impact
were obtained in moderate yields by reaction of imines 11b and 11d
with ethylbromoacetate in the presence of zinc and trimethyl-
chlorosilane using the conditions similar to those reported by
Palomo et al. [33].
ER
estrogen receptor
FACS
FBS
fluorescence-activated cell sorting
fetal bovine serum
The 1H NMR spectrum of 12a, the C-3 proton at
d
2.91(dd,
J ¼ 2.0 Hz, 15.0 Hz) is in a trans orientation to H-4 which appears as
a double doublet at
4.86 (J ¼ 2.0 Hz, 5.4 Hz) [34]. The second C-3
proton is observed at
3.49 (dd, J ¼ 5.4 Hz,15.4 Hz) and is cis to H-4.
The silyl protecting group was removed from both -lactams 12a
and 12g using TBAF to afford 12d (62%) and 12j (39%) respectively,
the -lactam analogues of CA-4. The related 3-methylazetidinones
12b and 12h were similarly obtained by Reformatsky reaction of
Schiff bases 11b and 11d using ethyl-2-bromopropionate as the
GTP
guanidine triphosphate
d
HMQC heteronuclear multiple quantum coherence
d
HRMS
IR
high resolution mass spectrometry
infra red
b
LRMS
MTT
low resolution mass spectra
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
nuclear magnetic resonance
nuclear overhauser effect
phosphate buffer saline
structureeactivity relationship
tetrahydrofuran
b
a
-
NMR
NOE
PBS
SAR
THF
TMS
bromoester (Scheme 1). These azetidinones were obtained as
mixtures of cis/trans isomers (3:2) which were not separated. The
3,3-dimethyl analogues 12c and 12i were prepared in a similar
manner by reaction of the Schiff bases 11b and 11d with ethyl-2-
bromoisobutyrate.
tetramethylsilane
The tert-butyldimethylsilyl protecting group is removed from
compounds 12b, c, h, and i using TBAF to yield the phenolic
products 12e, f, k, and l respectively. The diastereomeric compo-
sition of compound 12e was demonstrated by chiral HPLC, (rt
3.39 min and 4.11 min for trans isomers and 4.33 min and 4.66 min
for cis isomers, assigned from integration). The effect of product
in both tubulin polymerisation and colchicine binding assays
[18,22]. Substitution of the 30-OH with an amino group results in
a compound with equipotent bioactivity as CA-4 and good water
solubility [23]. The 3,4,5-trimethoxy substituted pattern in ring A is
optimal for bioactivity of CA-4 as it coincides with the trimethox-
yaryl ring of colchicine [20]. A large number of analogues with
a modified bridge have also been reported. These include saturation
of the olefinic bridge, and replacement with eNHCH2ꢀ, eCH2NHe,
eCONHe, eOCH2ꢀ, eCH2Oe, eOe and many others.
stereochemistry on the biochemical activity of the
b-lactams
reported in this study will be considered in the future development
of these compounds. (The stereoselective activity of the cholesterol
lowering b-lactam drug ezetimibe(1-(4-fluorophenyl)-(3R)-[-3-(4-
fluorophenyl)-(3S)-hydroxypropyl]-4S-(4-hydroxyphenyl)-2-azeti-
dinone) has been reported [35,36]).
Because of the observation that the 3,4,5-trimethoxy ring
substitution (Ring A) is optimal for bioactivity in all analogues of
CA-4 in literature, compounds 12def and 12jel were examined for
their antiproliferative effects in both MCF-7 and MDA-MB-231 cell
lines with the aim of discovering if the placement of this critical
3,4,5-trimethoxyphenyl ring on the N-1 or C-4 positions resulted in
significantly different antiproliferative activities, (see Results and
discussion section, Table 1). Results in both cell lines concurred,
showing the compounds with the 3,4,5-trimethoxyphenyl ring
(Ring A) on the N-1 position being significantly more potent than
the compound with the 3,4,5-trimethoxyphenyl substituent (Ring
A) on the C-4 position, as previously reported by Sun et al. with
Isomerisation of the cis-double bond in CA-4 has led to intensive
investigations of rigid ring modifications [10,19,20,24], including
imidazole 4 [25], 1,3-dioxolane 5 [26], furazan (1,2,5-oxadiazole) 6
[27], heteroarylcoumarin 7 [28] and 3-aroyl-6-methoxyindole 8
[29] analogues (Fig. 1). In addition to tumour cell growth inhibition,
most of these non-isomerisable compounds have been shown to be
capable of binding to and depolymerising tubulin. The anticancer
activity of some b-lactam-containing compounds has been repor-
ted [30], including the non-isomerisable combretastatin analogues
9 and 10 [31]. We have previously reported antiproliferative activity
of compounds containing the
antiestrogenic effects in MCF-7 cells [32]. It was decided to examine
a range of compounds containing the -lactam ring as potential
tubulin targeting agents. These novel compounds are unsubstituted
at C-3, or contain methyl substituent(s) at C-3. The -lactam ring
b-lactam scaffold which exhibited
other
established that positioning of the 3,4.5-trimethoxyphenyl ring on
the rigid -lactam ring system for these compounds, was an
important consideration and required that any further -lactams
b-lactam compounds [31]. From our initial investigations we
b
b
b
b
provides a useful scaffold structure with a similar spatial arrange-
ment between the two phenyl rings as observed in the cis confor-
mation of CA-4.
synthesised would contain the 3,4,5-trimethoxyphenyl ring at the
N-1 position.
Structural modification of the initially synthesised
products 12def was then investigated with a view to further opti-
mization of the CA-4 analogue -lactam template for antiproliferative
activity. The synthesis of products 12mex with modification of the C-
4 aryl ring substituents is shown in Scheme 2. The 3-unsubstituted
b-lactam
2. Results and discussion
b
2.1. Chemistry
b-
lactam 12d contains the 3-hydroxy-4-methoxyphenyl ring at the C-4
The general procedure for the synthesis of the required
b-lac-
position; which is also present in CA-4 (Ring B). A variety of different
tams is illustrated in Scheme 1. The compounds which were
initially chosen for synthesis contained the 3,4,5-trimethoxyphenyl
(Ring A) and 3-hydroxy-4-methoxyphenyl (Ring B) substituents as
b-lactams 12mev, were obtained by using different substitution
patterns on the C-4 phenyl ring (Ring B), e.g. 4-F, 4-OCH3, 2,3,4-tri-
methoxy, 3,4,5-trimethoxy and 3,4-dimethoxy. The aryl ring at C-4
was also replaced with the alternative 6-methoxy-2-naphthyl and 2-
thiophene ring systems (compounds 12wex). The required imines
the
b-lactam N-1 and C-4 substituents, which are present in
Combretastatin A-4. The Reformatsky
b-lactam synthesis requires

5754
M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
H3CO
H3CO
H3CO
O
N
H3CO
A
H
H3CO
H3CO
O
B
OCH3
H3CO
R
1 Colchicine
2 R = OH, Combretastatin A-4
3 R = OPO3Na2
OH
NH2
OCH3
H3CO
H3CO
H3CO
O
O
N
O
H3CO
H3CO
N
H3CO
H3CO
H3CO
N
N
H3CO
OCH3
OCH3
OH
5
6
4
OCH3
O
H3CO
H3CO
O
O
H3CO
H3CO
RO
NH2
NH
N
OCH3
H3CO
O
OCH3
OCH3
OCH3
OCH3
N
CH3
9
R = CH3
7
8
10 R = H
Fig. 1. Colchicine (1), combretastatin A-4 (2), combretastatin A-4 phosphate (3) and related antitubulin compounds 4e10.
11ee11l were prepared by condensation of 3,4,5-trimethoxyaniline
with the appropriate aldehyde. The -lactam products 12meo,12ret
2.2. Biochemical studies
b
and 12wex were obtained in moderate yields by reaction of imines
11ee11l with ethylbromoacetate in the presence of zinc and trime-
thylchlorosilane. Enantiomeric separation of the racemic product 12o
was achieved by chiral HPLC (rt 7.88 min and 8.92 min).
2.2.1. Antiproliferative activity in MCF-7 and MDA-MB-231 breast
cancer cells
The first aim of the biochemical evaluation of the compounds
was to determine if the position of the 3,4,5-trimethoxyphenyl ring
The 3-methyl and 3,3-dimethyl compounds 12p, 12q, 12u and
12v were obtained in a similar manner using ethyl-2-bromopropi-
on the
of colchicines and combretastatin type compounds, effected the
potency of these -lactam compounds. Compounds 12def and
b-lactam scaffold at N-1 or C-4, which is critical in the activity
onate and ethyl-2-bromoisobutyrate as the
a
-bromoesters respec-
b
tively. The nature of the Schiff base influences the yields obtained in
these Reformatsky reactions. While good yields were obtained (e.g.
upto 83% for compound 12p), we have found that afteroptimization,
lower yields of b-lactam products e.g. 12n (28%) and 12s (16%) are
consistently obtained from the Schiff bases 11f and 11i both having
ortho methoxy substituents, which may be causing some steric
12jel were screened for their antiproliferative activity using two
breast cancer cell lines, the ER expressing (ER dependent) MCF-7
human breast cancer and the ER lacking (ER independent) MDA-
MB-231 human breast cancer cell lines, using the MTT (tetrazolium)
based viability assay. The drug concentration required to inhibit the
cell growth by 50% (IC50) following incubation of the cells in the
culture medium for 72 h was determined and the results are dis-
played in Table 1. The IC50 values obtained for Combretastatin CA-4
in this assay are 31 nM for MCF-7 and 43 nM for MDA-MB-231 and
are in good agreement with the reported values for Combretastatin
CA-4 using the MTT assay on human MCF-7 and MDA-MB-231
breast cancer cell lines [7,37,38].
hindrance for the imine in forming the
b-lactam ring.
To further investigate the contribution of the
b
-lactam carbonyl
group to the activity of this compound class, the novel thione
analogues 13aec were prepared in moderate yield by reaction of
the compounds 12oeq with Lawesson’s reagent (2,4-bis-(4-
methoxyphenyl)-1,2,3,4-dithiaphosphetane-2,4-disulfide)
(Scheme 2). The characteristic C]S absorption band was observed
in the region 1590e1595 cmꢀ1 for these products.
From the results in Table 1 it is evident that the 3,4,5-trime-
thoxyphenyl ring located at the N-1 position of the b-lactam ring is

M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
5755
R1
OCH3
H3CO
R1
OCH3
OCH3
N
OCH3
OCH3
CHO
(i)
H3CO
(iii)
OCH3
NH2
11a R1 = OH
11b R1 = OSi(CH3)2C(CH3)3
(ii)
OCH3
OCH3
R2
R2
OH
(iv)
OSi(CH3)2C(CH3)3
OCH3
R1
R1
N
N
OCH3
O
O
OCH3
OCH3
OCH3
OCH3
12d R1 = R2 = H
12e R1 = CH3, R2 = H
12f R1 = R2 = CH3
12a R1 = R2 = H
12b R1 = CH3, R2 = H
12c R1 = R2 = CH3
OCH3
OCH3
OCH3
H3CO
H3CO
H3CO
OCH3
R1
N
R1
CHO
(i)
(iii)
OCH3
NH2
11c R1 = OH
11d R1 = OSi(CH3)2C(CH3)3
(ii)
OCH3
OCH3
OCH3
OCH3
R2
R2
(iv)
OCH3
OCH3
R1
R1
N
N
OH
OSi(CH3)2C(CH3)3
O
O
OCH3
OCH3
12j R1 = R2= H
12k R1 = CH3, R2 = H
12l R1 = R2= CH3
12g R1 = R2= H
12h R1 = CH3, R2 = H
12i R1 = R2= CH3
Scheme reagents and conditions: (i) EtOH, reflux, 2.5h.(ii) (CH3)3C(CH3)2SiCl, K2CO3, CH2Cl2, DBU,
20oC. (iii) BrCH2CO2Et, BrCH(CH3)CO2Et or Br(CH3)2CCO2Et, Zn, (CH3)3SiCl, C6H6, reflux.(iv)
(CH3CH2CH2CH2)4NF, THF, 0oC.
Scheme 1. Synthesis of compounds 12ael.
the structural arrangement for optimum activity as previously
observed by Sun et al. with other -lactam compounds [31]. All three
There is little difference in antiproliferative activity between the 3-
unsubstituted compound 12d and 3-methyl compound 12e, both
demonstrating low nanomolar activity in both MCF-7 cell line
(IC50 ¼ 17 nM and 10 nM respectively) and MDA-MB-231 cell line
(IC50 ¼ 54 and 47 nM respectively). These values are similar to that
obtained for CA-4 (IC50 ¼ 3 nM for MCF-7 and IC50 ¼ 43 nM for MDA-
MB-231 cell line). However, the introduction of the 3,3-dimethyl
substitution in compound 12f results in significant loss of activity
b
compounds 12def (in which the 3,4,5-trimethoxyphenyl ring is
located at the N-1 position) are much more potent in both cell lines
than the corresponding compounds 12k and 12l (where the 3,4,5-
trimethoxyphenyl ring is located at the C-4 position) and therefore
this template was chosen for the further compounds examined.
The 3-unsubstituted compound 12j was the most active of the
products with 3,4,5-trimethoxy ring located at the C-4 position
with IC50 ¼ 0.25
m
M for MCF-7 cells and IC50 ¼ 0.27
mM for the MDA-
(IC50value ¼ 0.130
m
M in MCF-7 cells). It is interesting tonote that the
MB-231 cell line.
most active compounds described here (12d, 12e) are considerably
The cytotoxic effect of these b-lactam compounds on prolifer-
more potent as antiproliferative agents than the previously reported
ating cells was determined in the lactose dehydrogenase (LDH)
3-hydroxy, 3-methoxy and 3-acetoxy substituted
b
-lactams [31].
assay [39]. Cytotoxicity values in the range 2e15% were obtained

5756
M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
Table 1
Antiproliferative effects of
b
-lactam compounds 12def, 12jel in MCF-7 cells and MDA-MB-231 cells.
OH
OCH3
OCH3
OCH3
R1
R2
R1
R2
OCH3
N
N
OCH3
OCH3
O
O
OCH3
OCH3
OH
12d-f
12j-l
Compound
Number
R1
R2
Antiproliferative
Cytotoxicity %
Antiproliferative
activitya, b MDA-MB-231
Cytotoxicity %
,
b
activitya MCF-7
deathc 10
mM
deathc 10
mM
cells IC50 value (
m
M)
cells IC50 value (mM)
12d
H
H
H
CH3
H
H
0.017 ꢁ 0.002
0.010 ꢁ 0.0032
0.25 ꢁ 0.08
0.130 ꢁ 0.054
2.96 ꢁ 0.61
4.04 ꢁ 2.35
0.0031
9
15
12
2.3
17
0.054 ꢁ 0.046
0.047 ꢁ 0.041
0.27 ꢁ 0.15
1.67 ꢁ 0.06
13.03 ꢁ 4.36
3.19 ꢁ 2.56
0.043
16
25
20
6.6
0
12ed
12f
CH3
CH3
H
CH3
CH3
e
12j
12kd
12l
CH3
e
11.5
5.5
12.1
4.3
2 (CA-4)
a
IC50 values are half maximal inhibitory concentrations required to block the growth stimulation of MCF-7 or MDA-MB-231 cells. Values represent the mean ꢁ S.E.M (error
values ꢂ 10ꢀ6) for three experiments performed in triplicate.
b
The IC50 values obtained for combretastatin CA-4 in this assay are 0.0031 mM for MCF-7 and 0.043 mM for MDA-MB-231 and are in good agreement with the reported
values for combretastatin CA-4 using the MTT assay on human MCF-7 and MDA-MB-231 breast cancer cell lines, (see Refs. [7,37,38]).
c
Lactate dehydrogenase assay: Following treatment of the cells, the amount of LDH was determined using LDH assay kit from Promega. Data is presented as % cell lysis at
compound concentration of 10
m
M, (see Ref. [39]).
d
3-Methyl substituted compounds 12e and 12k isolated and tested as cis/trans mixture.
for compounds 12def and j at a concentration of 10
cells and 6e25% for the same compounds in MDA-MB-231 cells at
a concentration of 10 M. CA-4 was determined to have cytotoxicity
m
M in MCF-7
heterocyclic 2-thiophene ring in 12w also resulted in much reduced
antiproliferative activity with an IC50 value of 21.6 M and notably
m
m
higher cytotoxicity of 20%. A similar trend was observed for anti-
proliferative activity of these compounds in the MDA cell line, with
compounds 12o and 12p again displaying the most potent anti-
proliferative activity (IC50 ¼ 210 and 127 nM respectively) with low
value of 5.6% in MCF-7 cells and 4.3% in MDA-MB-231 cells at 10
concentration.
mM
The antiproliferative results in MCF-7 and MDA-MB-231 breast
cancer cell lines for the second series of compounds synthesised
12mex, and 13aec are displayed in Table 2. All compounds contain
the 3,4,5-trimethoxyphenyl substituent located at N-1 with the C-3
position unsubstituted, or mono or dimethyl substituted. The effects
on antiproliferative activity of a number of aryl substitution patterns
are investigated for the aryl group at C-4, including 2,3,4-trimethoxy,
3,4,5-trimethoxy, 3,4-dimethoxy, 4-methoxy and 4-fluoro are dis-
played in Table 2. There is no significant loss in activity observed on
removal of the C-3 phenolic group from the most potent compounds
12d and 12e as shown in the results obtained for 12o and 12p with
IC50 values of 39 nM and 47 nM respectively. These compounds also
showed reduced cytotoxicity values in the LDH assay of 1.5% and 5.7%
cytotoxicity (1.1% and 4.5% respectively, at 10
mM concentration).
The replacement of the
b
-lactam carbonyl group with the thione
in compounds 13aec resulted in a reduction in the observed anti-
proliferative activity; the most active thione compound 13c was
found to be IC50 ¼ 0.35
m
M for MCF-7 cells and IC50 ¼ 1.33
mM for
MDA-MB-231 breast cancer cells. This could be related to the
difference in lipophilicity (e.g. cLogP for 12q ¼ 3.36 compared with
cLogP for 13c ¼ 2.86 [40]).
The influence of the rigid b-lactam ring scaffold structure on the
activity of the above compounds was investigated. The anti-
proliferative effect of the two Schiff bases 11a and 11c (related in
structure to the most potent b-lactam 12d and the related 12j) was
respectively at 10
mM concentration. The related 3,3-dimethyl-2-
determined in MCF-7 breast cancer cells. Neither of the Schiff bases
azetidinones 12q and 12v showgood antiproliferative effects on MCF-
7 cells (IC50 ¼ 265 nM and 344 nM respectively). Introduction of the
3,4-dimethoxyphenyl substituent at C-4 results in intermediate
show any significant antiproliferative activity at concentrations up to
50 mM, which is in sharp contrast to the corresponding b-lactams 12d
and 12j. As already observed by Cushman et al. the lack of activity of
these Schiff bases probably derives from their trans geometry [41].
antiproliferative activity for compound 12t (IC50 value ¼ 1.64
with moderate activity also obtained for the 3-methyl substituted
analogue 12u (IC50 value ¼ 2.96 M) and also for compound 12x
(containing the 6-methoxy-2-naphthyl substituent at C-4), with IC50
value of 1.51 M. The introduction of the fluoro substituent in
mM),
m
2.2.2. Evaluation of G2/M arrest in MDA-MB-231 cells exposed to
compound 12d
m
The extent of G2/M arrest and apoptosis (sub-G1 peak) induced
by compound 12d in MDA-MB-231 cells was statistically quantified
by flow cytometric analysis. The fluorescent dye, propidium iodide
(PI) intercalates with the DNA and hence, the amount of fluores-
cence measured per cell is proportional to the DNA content. Cells
were harvested after 24, 48 or 72 h and analysed for DNA content
by flow cytometry. Table 3 shows the percentage of cells in each
phase of the cell cycle over the three different time scales.
12m resulted in loss of activity (IC50 value ¼ 25.9
m
M). The presenceof
two trimethoxyphenyl ring systems in the
b-lactam products
e.g. compound 12s resulted in a moderate reduction in activity
M). However for the related 1,4-bis(trimethoxyphenyl)
(IC50 ¼ 0.31
m
substituted compounds 12n and 12r a significant reduction in the
antiproliferative activitywasobservedwiththeIC50 values inthehigh
mM range. The replacement of aryl ring at C-4 position with the

M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
5757
R1
R1
OCH3
OCH3
H3CO
N
OCH3
OCH3
CHO
(i)
(ii)
NH2
OCH3
11e R1 = 4-F
11f R1 = 2,4,5-OCH3
11g R1 = 4- OCH3
11h R1 = 3,4,5-OCH3
11i R1 = 2,3,4-OCH3
11j R1 = 3,4-OCH3
R1
R3
R2
N
OCH3
O
OCH3
OCH3
OCH3
R1
R2
(iii)
12m R1 = 4-F, R2 = R3 = H
12o-q
N
OCH3
OCH3
12n R1 = 2,4,5-OCH3, R2 = R3 = H
12o R1 = 4- OCH3, R2 = R3 = H
S
12p
R
R
1 = 4- OCH3, R2 = CH3, R3 = H
1 = 4- OCH3, R2 = R3 = CH3
12q
OCH3
12r R1 = 3,4,5-OCH3, R2 = R3 = H
12s R1 = 2,3,4-OCH3, R2 = R3 = H
13a R1 = R2 = H
13b R1 = CH3, R2 = H
13c R1 = R2 =CH3
12t
R1 = 3,4-OCH3, R2 = R3 = H
12u R1 = 3,4-OCH3, R2 = CH3, R3 = H
12v R1 = 3,4-OCH3, R2 = R3 = CH3
CHO
OCH3
S
H3CO
OCH3
S
S
N
OCH3
OCH3
(ii)
N
OCH3
OCH3
(i)
O
NH2
OCH3
OCH3
11k
12w
H3CO
OCH3
OCH3
OCH3
(ii)
H3CO
OCH3
N
OCH3
N
OCH3
OCH3
O
CHO
(i)
OCH3
OCH3
NH2
OCH3
12x
11l
Scheme reagents and conditions: (i) EtOH, reflux, 2.5h. (ii) BrCH2CO2Et, BrCH(CH3)CO2Et or
Br(CH3)2CCO2Et, Zn, (CH3)3SiCl, C6H6, reflux.(iii) Lawesson's reagent, toluene, reflux, 3h
Scheme 2. Synthesis of compounds 12mex.
The results obtained for compound 12d show a large increase of
cells in the G2/M phase at concentrations 100 nM, 1 M and 10
indicate that this compound’s mechanism of action may indeed be by
targeting the microtubules. The ability of these -lactam compounds
m
mM
b
after 24 h of exposure (highlighted in Table 3). This increase is
accompanied by a corresponding reduction in the G1 phase. At
a concentration of 10 nM, there appears to be little effect on the cells
giving results similar to the vehicle value. After 48 h a significant
increaseinG2/M peak(highlighted) isagainseenat100 nMand above
but not to the same degree as seen after 24 h as highlighted inTable 3.
This is due to the parallel increase in sub-G1 phase indicating
induction of apoptosis. After 72 h a decrease in G2/M and in G1 phase
is accompanied by a large increase in sub-G1 peak. These results
to bind to and depolymerise tubulin was then investigated.
2.2.3. Tubulin polymerisation study
The effects of representative
b-lactam combretastatin A-4
analogues on the assembly of purified bovine tubulin were evalu-
ated. The assay was optimised using a polymerisation enhancer
(paclitaxel) and polymerisation suppressor (nocodazole) (data not
shown). Compound 12d which demonstrated potent anti-
proliferative effects (low nanomolar) in vitro and compound 12h

5758
M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
Table 2
Antiproliferative effects of b-lactam compounds 12mex, 13aec in MCF-7 cells and MDA-MB-231 cells.
R4
R4
R3
R5
R3
R5
OCH3
R1
R2
R1
R2
R1
R2
R1
R2
S
R6
R6
N
N
N
N
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
S
O
O
O
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
12m-v
12w
12x
13a-c
.
Compound
Number
R1
R2
R3
R4
R5
R6
H
OCH3
H
H
H
OCH3
H
H
H
Antiproliferative
activity MCF-7
Cytotoxicity %
Antiproliferative
activity MDA-MB-231
Cytotoxicity % death 10 m
Mc
death 10 m
Mc
b
cells IC50 value
cells IC50 value (m
M)a,
b
(m
M)a,
12m
12n
12o
H
H
H
H
CH3
H
H
H
H
H
H
H
CH3
CH3
H
H
H
CH3
CH3
H
H
H
H
CH3
e
H
OCH3
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
e
F
25.95 ꢁ 11.95
63.74 ꢁ 19.8
0.039 ꢁ 0.013
0.047 ꢁ 0.024
0.265 ꢁ 0.010
167.9 ꢁ 121.1
0.31 ꢁ 0.036
1.64 ꢁ 0.02
2.96 ꢁ 0.61
0.344 ꢁ 0.116
21.6 ꢁ 14.5
1.51 ꢁ 0.56
1.12 ꢁ 0.10
0.89 ꢁ 0.21
0.35 ꢁ 0.012
0.0031
0
25.95 ꢁ 15.1
38.5 ꢁ 6.43
0.21 ꢁ 0.17
0.127 ꢁ 0.0096
1.15 ꢁ 0.16
94.39 ꢁ 18.55
2.83 ꢁ 1.98
0.97 ꢁ 0.008
8.84 ꢁ 0.50
1.46 ꢁ 0.83
34.47 ꢁ 1.50
13.18 ꢁ 1.61
4.50 ꢁ 2.08
9.26 ꢁ 2.07
1.33 ꢁ 0.11
0.043
6.3
0
1.1
4.5
16
0
9.0
15
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
e
2.2
1.5
5.7
12
1.9
8.0
5.0
7.5
14
20
13.5
16
3.2
5.5
5.6
12pd
12q
12r
12s
12t
12ud
12v
6.1
12.0
15
CH3
H
H
H
e
12w
12x
e
e
e
e
e
18
13a
H
H
H
H
e
H
H
H
e
OCH3
OCH3
OCH3
e
H
H
H
e
3.0
5.1
8.0
4.3
13bd
13c
CH3
CH3
e
2 (CA-4)
a
IC50 values are half maximal inhibitory concentrations required to block the growth stimulation of MCF-7 or MDA-MB-231 cells. Values represent the mean ꢁ S.E.M (error
values ꢂ 10ꢀ6) for three experiments performed in triplicate.
b
The IC50 values obtained for combretastatin CA-4 in this assay are 0.0031 mM for MCF-7 and 0.043 mM for MDA-MB-231 and are in good agreement with the reported
values for combretastatin CA-4 using the MTT assay on human MCF-7 and MDA-MB-231 breast cancer cell lines (see Refs. [7,37,38]).
c
Lactate dehydrogenase assay: Following treatment of the cells, the amount of LDH was determined using LDH assay kit from Promega. Data is presented as % cell lysis at
compound concentration of 10
m
M (see Ref. [39]).
d
3-Methyl substituted compounds 12p, 12u and 13b isolated and tested as cis/trans mixture.
which demonstrated poor antiproliferative effects (high micro-
molar) were assessed. The ability of combretastatin A-4 to effec-
tively inhibit the assembly of tubulin was assessed as a positive
control. Tubulin polymerisation was determined by measuring the
increase in absorbance over time at 340 nm. The nmax value offers
the most sensitive indicator of tubulin/ligand interactions, hence
nmax values were calculated for each test compound. Fold changes
in nmax values for polymerisation curves of each test compound
with reference to ethanol control were calculated and detailed in
Table 4. As anticipated the active -lactam combretastatin A-4
analogue 12d inhibited the polymerisation of tubulin whilst the
relatively inactive counterpart 12h did not significantly affect the
b
rate of tubulin polymerisation (Table 4). In more detail, the active
b-
lactam 12d when evaluated at 10 M concentration, reduced the
m
Table 3
Evaluation of G2/M arrest in MDA-MB-231 cells exposed to compound 12d.a
Concentration
Control
10 nM
Time (h)
24
24
24
24
24
48
48
48
48
72
72
72
72
72
Sub-G1 (%)
G1 (%)
S (%)
G2/M (%)
Polyploid (%)
5.71 ꢁ 3.47
3.72 ꢁ 1.97
4.88 ꢁ 0.85
7.08 ꢁ 4.20
3.18 ꢁ 0.25
9.80 ꢁ 1.55
11.57 ꢁ 3.06
37.50 ꢁ 0.71
51.27 ꢁ 6.31
12.92 ꢁ 2.61
15.25 ꢁ 2.03
63.10 ꢁ 20.06
50.53 ꢁ 14.78
46.49 ꢁ 17.49
51.20 ꢁ 2.37
48.33 ꢁ 4.18
18.95 ꢁ 6.60
19.22 ꢁ 6.19
29.30 ꢁ 5.95
50.34 ꢁ 11.09
45.25 ꢁ 7.37
10.76 ꢁ 0.34
14.44 ꢁ 2.28
44.13 ꢁ 2.31
48.24 ꢁ 2.79
13.90 ꢁ 3.62
14.29 ꢁ 2.20
17.14 ꢁ 3.31
10.46 ꢁ 4.19
12.55 ꢁ 2.54
6.05 ꢁ 0.38
6.01 ꢁ 2.90
3.72 ꢁ 0.71
10.50 ꢁ 0.77
13.36 ꢁ 2.58
8.90 ꢁ 0.85
4.47 ꢁ 0.06
9.10 ꢁ 2.75
9.08 ꢁ 2.32
5.34 ꢁ 4.32
7.76 ꢁ 0.58
9.66 ꢁ 1.84
20.18 ꢁ 3.18
25.61 ꢁ 0.98
59.43 ꢁ 3.13
63.16 ꢁ 4.86
55.36 ꢁ 1.49
18.81 ꢁ 1.04
23.71 ꢁ 7.81
36.42 ꢁ 3.42
27.25 ꢁ 4.45
21.51 ꢁ 4.86
17.68 ꢁ 4.58
12.62 ꢁ 9.22
21.24 ꢁ 12.05
21.81 ꢁ 12.40
4.53 ꢁ 1.66
2.84 ꢁ 0.59
4.87 ꢁ 4.39
4.31 ꢁ 3.17
3.85 ꢁ 3.83
5.62 ꢁ 3.06
6.10 ꢁ 0.62
4.61 ꢁ 0.86
2.28 ꢁ 0.06
5.48 ꢁ 3.26
3.55 ꢁ 1.57
2.99 ꢁ 1.89
5.97 ꢁ 4.71
3.38 ꢁ 2.84
100 nM
1
m
M
10
m
M
Control
10 nM
100 nM
10
m
M
Control
10 nM
100 nM
1
m
M
10
m
M
a
Cell cycle analysis of MDA-MB-231 cells treated with vehicle control (1% (v/v) ethanol), or 10 nM, 100 nM, 1
mM, and 10 mM(final concentrations) of compound 12d at 24
and 72 h, and at 10 nM, 100 nM and 10
m
M(final concentrations) of compound 12d at 48 h. % MDA-MB-231 cells in each cell cycle phase are shown after exposure to compound
12d. Cells were analysed with the FACScan flow cytometry. Cells in the sub-G1 peak are indicative of apoptotic cells. Results show a typical experiment which has been
repeated three times. Values represent the mean ꢁ standard deviation for three experiments.

M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
5759
Table 4
Inhibition of tubulin polymerisation in response to
analogues.
b-lactam combretastastin
Compound
Concentration
Fold inhibition of tubulin
polymerisationa
(nmax ꢁ SEM)
Ethanol (Control)
[1% v/v]
1
CA-4
12d
12n
10
10
10
m
m
m
M
M
M
6.0 ꢁ 1.4
10.2 ꢁ 2.3
1.2 ꢁ 0.2
a
Effects of 12d and 12n on in vitro tubulin polymerisation. Purified bovine tubulin
and GTP were mixed in a 96-well plate. The reaction was started by warming the
solution from 4 C to 37 C. CA-4 (10 mM) was used as a reference, while ethanol (1%v/v)
was used as a vehicle control. The effect on tubulin assembly was monitored in
a Spectramax 340PC spectrophotometer at 340 nm at 30 s intervals for 60 min at 37 C.
Fold inhibition of tubulin polymerisation was calculated using the nmax value for each
reaction. The results represent the mean ꢁ standard error of the mean for three
separate experiments.
nmax value for the rate of tubulin polymerisation from 6- to 10-fold.
This value is comparable if not superior to the rate of inhibition of
tubulin assembly (6-fold) observed with combretastatin A-4. These
results suggest that the molecular target of the active
combretastatin A-4 analogues is indeed tubulin.
b-lactam
2.3. Molecular modeling studies
Fig. 2. Docked pose of
b-lactam 12d overlayed with colchicine in the tubulin binding
site (PDB entry 1SA0). Hydrogen-bonds were created with an svl script through MOE.
To investigate potential binding modes of these
b-lactam
compounds, a docking study was carried out using the most potent
compound in the series, the 3-unsubstituted -lactam 12d. Using the
b
unsubstituted compound 12d and 3-methyl substituted compound
12e (each with 3-hydroxy-4-methoxyaryl substitution pattern at C-
4) were identified as the most potent compounds of the series
having low nanomolar activity in both MCF-7 and MDA-MB-231
breast cancer cells. The most effective compound 12d was shown to
arrest cells in the G2/M phase of the cell cycle. 12d was also shown
to inhibit the polymerisation of tubulin with improved efficacy
when compared with combretastatin CA-4.
However, as these compounds were evaluated in this study as
racemates, the effect of product stereochemistry on the biochem-
ical activity will be relevant in the future development of these
compounds. The b-lactam ring provides a scaffold structure with
a similar spatial arrangement between the two phenyl rings
reported X-ray structure of tubulin co-crystallised with the colchicine
derivative, N-deacetyl-N-(2-mercaptoacetyl)colchicine (DAMA-
colchicine, PDB entry 1SA0) [42], possible binding orientations for
12d were probed with the docking program FREDv2.2.3 (Openeye
Scientific Software) [43]. 3-D conformations were enumerated using
CORINAv3.4 (Molecular Networks GMBH) [44] for 12d and com-
bretastatin A-4 followed by multiple conformations using OME-
GAv2.2.1 (Openeye Scientific Software) [45]. Each conformation was
subsequently docked and scored with Chemgauss3 as outlined
previously by our laboratory [46] The top binding poses were refined
using the LigX procedure (MOE e Chemical Computing Group) [47].
Postdock analysis (SVL script; MOE) of the docked poses for 12d
revealed that approximately 10% of the population appeared to dock
in a similar orientation to colchicine as illustrated in Fig. 2.
as observed in cis conformation of combretastatin CA-4. These
For comparison, the docked pose of CA-4 overlayed with N-
deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-colchicine) in
the tubulin binding site (PDB entry 1SA0) is illustrated in Fig. 3. The
strong hydrogen-bonding to Thr179 observed for the 3-hydroxy
substituent of 12d is also observed with combretastatin A-4 as
depicted in Fig. 3. (The X-ray crystal structure of the disodium
phosphate salt of combretastatin A-4 (3) suggests that the confor-
mationof this stilbeneis not planar. Thecrystal structurereveals that
the planes of the two phenyl rings are inclined to each other, sug-
gesting a low-energy conformation that may be the one involved in
binding at the tubulin receptor site [48]). Importantly, an additional
H-bond is seen to occur with residue Lys352 of the active site for 12d,
colchicine and CA-4. Superimposition of 12d, CA-4 and colchicine as
they docked within the active site of 1SA0, illustrates the similar
nature ofpositioningof the trimethoxysubstituents(ring A) andalso
3-hydroxy-4-methoxy substituents of the B-ring. These binding
parallels may rationalise the potency observed for 12d in its tubulin
effects which is seen to be close to that reported for CA-4.
3. Conclusion
We have synthesised a series of
b-lactam compounds which
Fig. 3. Docked pose of CA-4 overlayed with colchicine in the tubulin binding site (PDB
show potent antiproliferative activity in breast cancer cells. The 3-
entry 1SA0).

5760
M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
conformationally restricted
b
-lactam structures, which unlike the
113.57, 122.26, 129.27, 135.50, 146.78, 147.59, 150.79, 153.21
(Aromatic C), 159.42 (CH]N). HRMS: Found: 318.1345; C17H20NO5
requires 318.1341(Mþ þ H).
cis-stilbene CA-4 are not easily isomerised, are promising lead
compounds in the development of new anticancer agents. Further
studies will determine the possible antiangiogenic effects of these
compounds.
4.1.2. (4-Fluorobenzylidene)-(3,4,5-trimethoxyphenyl)amine (11e)
Preparation was as above from 4-fluorobenzaldehyde (10 mmol,
1.24 g) and 3,4,5-trimethoxyaniline (10 mmol, 1.37 g). Yield 72%,
4. Experimental
pale yellow crystals, m.p. 87e90 ꢃC. IR nmax (KBr) cmꢀ1
:
4.1. Chemistry
1626.1 cmꢀ1 (C]N). 1H NMR (400 MHz, CDCl3):
d 3.87 (s, 3H,
OeCH3), 3.90 (s, 6H, 3ꢂ OeCH3), 6.48 (s, 2H, Ar-H), 7.14e7.29 (m,
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
underaslight positive pressureof nitrogen, and toluenewas driedby
distillation from sodium and stored on activated molecular sieves (4
A). IR spectra were recorded as thin films on NaCl plates or as KBr
2H, Ar-H), 7.88 (m, 2H, Ar-H), 8.44 (s, 1H, eCH]N). 13C NMR
(100 MHz, CDCl3):
d 55.98 (OeCH3), 60.88 (OeCH3), 98.00, 106.60,
115.73, 115.98, 130.56, 130.65, 131.51, 136.30, 147.58, 153.46,
(Aromatic C), 158.04 (CH]N). Elemental Analysis: Found: C, 66.28;
H, 5.54; N, 4.73; F, 16.87. C16H16FNO3 requires C, 66.43; H, 5.57; N,
4.84; F, 16.59%.
discs on a PerkineElmer Paragon 100 FT-IR spectrometer.1H and 13
C
NMR spectra were obtained on a Bruker Avance DPX 400 instrument
at 20 ꢃC, 400.13 MHz for 1H spectra, 100.61 MHz for 13C spectra, in
either CDCl3, CD3COCD3 or CD3OD (internal standard tetrame-
thylsilane). Low resolution mass spectra were run on a Hew-
lettePackard 5973 MSD GCeMS system in an electron impact mode,
while high resolution accurate mass determinations for all final
target compounds were obtained on a Micromass Time of Flight
mass spectrometer(TOF) equipped with electrospray ionization (ES)
interface operated in the positive ion mode at the High Resolution
Mass Spectrometry Laboratory by Dr. Martin Feeney in the Depart-
ment of Chemistry, Trinity College Dublin. Thin layer chromatog-
raphy was performed using Merck Silica gel 60 TLC aluminium
sheets with fluorescent indicator visualizing with UV light at
254 nm. Flash chromatography was carried out using standard silica
gel 60 (230e400 mesh) obtained from Merck. All products isolated
were homogenous onTLC. Chiral liquid chromatography was carried
out on selected compounds using a Chiral-AGPÔ 150 ꢂ 4.0 mm
column supplied by ChromTech Ltd. (now supplied by Chiral Tech-
nologies Europe) with a Chiral e AGPÔ guard column. The HPLC
system consisted of the following components: a Waters 1525
binary HPLC pump, a Waters 2487 Dual Wavelength Absorbance
Detector, a Waters In-Line Degasser AF and a Waters 717 plus
Autosampler. Gradient elution was used beginning with 10% of
organic phase and finishing with 90% of organic phase over a period
of 20 min. The organic mobile phase was 2-propanol and the
aqueous phase was a sodium phosphate buffer. The sodium phos-
phate buffer, consisting of 10 mM sodium dihydrogen orthophos-
phate dihydrate (NaH2PO4) in HPLC-grade water, was made up to pH
7.0 using sodium hydroxide. The flow rate was 0.5 ml/min and
detection was carried out at 225 nm. Compounds 11c [48], 11g [31]
and 11h [49] were prepared as previously reported.
4.1.3. (2,4,5-Trimethoxybenzylidene)-(3,4,5-trimethoxyphenyl)
amine (11f)
Preparation as described above from 2,4,5-trimethox-
ybenzaldehyde (10 mmol, 1.48 g) and 3,4,5-trimethoxyaniline
(10 mmol, 1.37 g). Yield 78%, yellow crystals, m.p. 123 ꢃC. IR nmax
(KBr) cmꢀ1: 1619.5 cmꢀ1 (C]N). 1H NMR (400 MHz, CDCl3):
d 3.84
(s, 3H, OeCH3), 3.89 (s, 6H, 2ꢂ OeCH3), 3.90(s, 3H, OeCH3), 3.94 (s,
3H, OeCH3), 3.95 (s, 3H, OeCH3), 6.50 (s, 2H, Ar-H), 6.53 (s, 1H, Ar-
H), 7.26 (s, 1H, Ar-H), 8.80 (s, 1H, eCH]N). 13C NMR (100 MHz,
CDCl3):
d 55.42 (OeCH3), 55.81(OeCH3), 56.06(OeCH3), 57.93
(OeCH3), 60.62 (OeCH3), 95.71, 97.87, 108.47,122.48,116.78, 125.43,
143.06, 143.39, 153.08, 154.52, (Aromatic C), 155.35 (CH]N).
Elemental Analysis: Found: C, 62.68; H, 6.31; N, 3.05. C19H23NO6,
requires C, 63.16; H, 6.37; N, 3.88%.
4.1.4. (2,3,4-Trimethoxybenzylidene)-(3,4,5-trimethoxyphenyl)
amine (11i)
Preparation as described above from 2,3,4-trimethox-
ybenzaldehyde (10 mmol, 1.48 g) and 3,4,5-trimethoxyaniline
(10 mmol, 1.37 g). Yield 74%, yellow crystals, m.p. 178 ꢃC. IR nmax
(KBr) cmꢀ1: 1619.6 cmꢀ1 (C]N).1H NMR (400 MHz, CDCl3):
d 3.87 (s,
3H, OeCH3), 3.91 (s, 9H, 3ꢂ OeCH3), 3.93 (s, 3H, OeCH3), 3.98 (s, 3H,
OeCH3), 6.48 (s, 2H, Ar-H), 6.80 (d, 1H, J ¼ 8.8 Hz, Ar-H), 7.85 (d, 1H,
J ¼ 9.0 Hz, Ar-H), 8.74 (s, 1H, CH]N). 13C NMR (100 MHz, CDCl3):
d
55.96 (OeCH3), 60.82 (OeCH3), 60.87 (OeCH3), 61.96 (OeCH3),
98.15, 107.71, 122.45, 123.57, 135.96, 141.64, 148.61, 153.38, 154.47,
155.49 (Aromatic C), 155.25 (CH]N),. Elemental Analysis: Found: C,
63.06; H, 6.44; N, 3.82. C19H23NO6 requiresC, 63.10; H, 6.40; N, 3.90%.
4.1.5. (3,4-Dimethoxybenzylidene)-(3,4,5-trimethoxyphenyl)amine
(11j)
Preparation as described above from 3,4-dimethox-
ybenzaldehyde (10 mmol, 1.34 g) and 3,4,5-trimethoxyaniline
(10 mmol, 1.37 g). Yield 73%, yellow crystals, m.p. 102e104 ꢃC. IR
nmax (KBr) cmꢀ1: 1606.9 cmꢀ1 (C]N). 1H NMR (400 MHz, CDCl3):
4.1.1. 2-Methoxy-5-[(3,4,5-trimethoxyphenylamino)methyl]phenol
(11a)
A solution of 3-hydroxy-4-methoxybenzaldehyde (10 mmol,
1.36 g) and 3,4,5-trimethoxyaniline (10 mmol, 1.37 g) in ethanol
(50 mL) was heated to reflux for 3 h. The reaction mixture was
reduced to 25 mL under vacuum, and the solution transferred to
a beaker. The mixture was left to stand for 3 h and the Schiff base
product crystallised out of the solution. The crude product was then
re-crystallised from ethanol and filtered to yield the purified
product. Yield 84%, pale yellow crystals, m.p. 176e178ꢃ C. IR nmax
(KBr) cmꢀ1: 1602.6 cmꢀ1 (C]N), 3069.2 cmꢀ1 (OH). 1H NMR
d
3.87 (s, 3H, OeCH3), 3.90 (s, 6H, 2ꢂ OeCH3), 3.95 (s, 3H, OeCH3),
3.99 (s, 3H, OeCH3), 6.48 (s, 2H, Ar-H), 6.93 (d, 1H, J ¼ 8.4 Hz, Ar-H),
7.31e7.33 (m, 1H, Ar-H), 7.60 (s(br), 1H, Ar-H), 8.39 (s, 1H, eCH]N).
13C NMR (100 MHz, CDCl3):
d 55.89 (OeCH3), 55.97 (OeCH3), 60.90
(OeCH3), 97.97, 108.35, 110.35, 124.31, 129.25, 135.97, 148.05, 151.92,
153.42 (Aromatic C) 159.13 (CH]N). HRMS: Calculated for
C21H22NO5: 332.1498; Found: 332.1485, (Mþ þ H).
(400 MHz, DMSO-d6):
d
3.70 (s, 3H, OeCH3), 3.81 (s, 3H, OeCH3),
4.1.6. Thiophen-2-ylmethylene-(3,4,5-trimethoxyphenyl)amine
(11k)
3.85 (s, 6H, 2ꢂ OCH3), 6.57 (s, 2H, Ar-H), 7.03 (d, 1H, J ¼ 8.2 Hz, Ar-
H), 7.29 (dd, 1H, J ¼ 2.0, 8.2 Hz, Ar-H), 7.42 (d, 1H, J ¼ 2.0 Hz, Ar-H),
8.48 (s, 1H, CH]N), 9.33 (s, 1H, OH). 13C NMR (100 MHz, DMSO):
Preparation as described above from thiophene-2-carbaldehyde
(10 mmol, 1.12 g) and 3,4,5-trimethoxyaniline (10 mmol, 1.37 g).
d
55.83 (OeCH3), 56.09 (OeCH3), 60.09 (OeCH3), 98.50, 111.57,
Yield 81%, pale yellow crystals, m.p. 169 ꢃC. IR nmax (KBr) cmꢀ1
:

M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
5761
1617.8 (C]N), 1584.53 cmꢀ1. 1H NMR (400 MHz, CDCl3):
d
3.88 (s,
5 mmol) and the resulting mixture was stirred at room temperature
for 15 min and then under reflux for a further 2 min. The suspen-
sion was cooled and (3-(tert-butyldimethylsilanyloxy)-4-methox-
ybenzylidene)-(3,4,5-trimethoxyphenyl)amine (11b) (7 mmol,
3.017 g) and ethylbromoacetate (1.33 mL, 12 mmol) were succes-
sively added. The reaction mixture was refluxed under nitrogen for
8 h and then cooled in an ice-water bath. It was then poured over
20 mL of saturated NH4Cl and 20 mL of 25% NH4OH. The mixture
was extracted with dichloromethane (20 mL) and the organic layer
was further washed with HCl (20 mL, 0.1 N) and water (20 mL). The
organic layer was separated and dried over anhydrous sodium
sulphate. The solvent was evaporated under reduced pressure, and
3H, OCH3), 3.92 (s, 6H, 2ꢂ OCH3), 6.52 (s, 2H, Ar-H), 7.16e7.18 (m,
1H, Ar-H), 7.52e7.55 (m, 2H, Ar-H), 8.61 (s, 1H, HC]N). 13C NMR
(100 MHz, CDCl3):
d 55.65 (OeCH3), 60.56 (OeCH3), 97.82, 127.38,
129.90, 131.83, 135.96, 142.17, 146.81, 153.30 (Aromatic C), 151.95
(CH]N). HRMS Calculated for C14H16NO3S: 278.0851; Found:
278.0852, (Mþ þ H). Elemental analysis: Found: C, 60.62; H, 5.44; N,
5.01; C14H15NO3S requires C, 60.63; H, 5.45; N, 5.05%.
4.1.7. (6-Methoxynaphthalen-2-ylmethylene)-(3,4,5-
trimethoxyphenyl)amine (11l)
Preparation as described above from 6-methoxynaphthal-
dehyde (10 mmol, 1.86 g) and 3,4,5-trimethoxyaniline (10 mmol,
the b-lactam was purified by flash chromatography over silica gel
1.37 g). Yield 80%, yellow crystals, m.p. 40 ꢃC. IR nmax (KBr) cmꢀ1
:
(eluent: dichloromethaneeethyl acetate; 19:1) Yield 21%, yellow
1616.4 cmꢀ1 (C]N). 1H NMR (400 MHz, CDCl3):
d 3.88 (s, 3H,
crystals, m.p. 90e91 ꢃC. IR nmax (KBr) cmꢀ1: 1748.1 cmꢀ1 (C]O). 1H
OeCH3), 3.91 (s, 6H, 2ꢂ OeCH3), 3.93 (s, 3H, OeCH3), 6.53 (s, 2H,
NMR (400 MHz, CDCl3): d 0.15 (s, 3H, CH3eSieCH3), 0.16(s, 3H,
Ar-H), 7.17 (m, 2H, Ar-H), 7.82 (m, 2H, Ar-H), 8.08e8.12 (m, 2H, Ar-
CH3eSieCH3), 0.88 (s, 9H, Ce(CH3)3), 2.91 (dd, 1H, J ¼ 2.0 Hz,
15.0 Hz, H-3), 3.49 (dd, 1H, J ¼ 5.4 Hz, 15.4 Hz, H-3), 3.70 (s, 6H, 2ꢂ
OeCH3), 3.75 (s, 3H, OeCH3), 3.79 (s, 3H, OeCH3), 4.86, (dd, 1H,
J ¼ 2.0 Hz, J ¼ 5.4 Hz, H-4), 6.54 (s, 2H, Ar-H), 6.81e6.83 (m, 2H, Ar-
H), 8.58 (s, 1H, N]CH). 13C NMR (100 MHz, CDCl3):
d 54.95
(OeCH3), 55.67 (OeCH3), 60.60 (OeCH3), 97.72, 105.61, 119.02,
124.08, 127.09, 128.00, 129.89, 130.57, 131.32, 135.82, 136.09, 147.72,
153.14, 158.63 (Aromatic C), 159.36 (N]CH). HRMS: Calculated for
C21H22NO4: 352.1549; Found: 352.1549 (Mþ þ H).
H), 6.92e6.95 (m, 1H, Ar-H). 13C NMR (100 MHz, CDCl3):
d
ꢀ5.13
(CH3eSieCH3), 13.65 (CH3eCeCH3), 25.20 (Ce(CH3)3), 46.39 (CH2),
53.57 (CH), 54.91 (OeCH3), 55.41 (OeCH3), 55.69 (OeCH3), 94.04,
111.20, 111.81, 115.26, 119.05, 129.99, 133.57, 143.98, 150.72, 152.72
(Aromatic C), 164.08 (C]O). HRMS: Calculated for C25H36NO6Si:
474.2311; Found: 474.2312 (Mþ þ H).
4.1.8. [3-(tert-Butyldimethylsilanyloxy)(4-methoxybenzylidene)-
(3,4,5-trimethoxyphenyl)amine (11b)
To a suspension of 2-methoxy-5-[(3,4,5-trimethoxyphenylamino)
methyl]phenol (11a) (0.02 mol, 6.34 g) and dimethyl-tert-butyl-
chlorosilane (0.024 mol) in dry DCM (60 mL) was added 1,8-diazo-
bicyclo[5.4.0] undec-7-ene (DBU) (0.032 mol). The resulting mixture
was stirred at room temperature until complete on thin layer chro-
matography. The solution was then diluted with DCM (80 mL) and
washed with water (60 mL), 0.1 M HCl (60 mL) and finally with
saturated aqueous NaHCO3 (60 mL). The organic layer was removed
and dried using anhydrous sodium sulphate. Evaporation of the
solvent afforded the product which was recrystallised from ethanol.
4.1.11. 4-([3-tert-Butyldimethylsilanyloxy]-4-methoxyphenyl)-3-
methyle1-(3,4,5-trimethoxy phenyl)azetidin-2-one (12b)
Preparation as described above from 3-(tert-butyldimethylsila-
nyloxy)-(4-methoxyphenyl)-(3,4,5-trimethoxybenzylidene)amine
(11a) (10 mmol, 1.8312 g) and ethyl-2-bromopropionate (12 mmol,
1.55 mL) and isolated as mixture of diastereomers. Yield 61%, brown
solid which was used without further purification. IR nmax
(film) cmꢀ1: 1745.6 cmꢀ1 (C]O,
CDCl3):
b
-lactam). 1H NMR (400 MHz,
d 0.04 (s, 3H, SieCH3), 0.05 (s, 3H, SieCH3), 0.91 (s, 9H, Ce
Yield 91%, yellow crystals, m.p. 88e92 ꢃC, IR nmax (KBr) cmꢀ1
:
1618.8 cmꢀ1 (C]N). 1H NMR (400 MHz, DMSO):
d
0.21 (s, 6H,
(CH3)3), 1.17e1.21 (m, 2H, eCH3), 1.26e1.30 (m, 1H, eCH3),
3.05e3.12 (m, 0.4H, H-3), 3.55e3.58 (m, 0.6H, H-3), 3.68 (s, 6H, 2ꢂ
OeCH3), 3.73 (s, 3H, OeCH3), 3.78 (s, 3H, OeCH3), 4.44 (bs, 0.4H, H-
4), 5.05 (d, 0.6H, J ¼ 5.5 Hz, H-4), 6.51 (s, 1H, Ar-H), 6.52 (s, 1H, Ar-
H), 6.54 (s (br), 1H, Ar-H), 6.67e6.82 (m, 2H, Ar-H). 13C NMR
CH3eSieCH3), 1.04 (s, 9H, SieCe(CH3)3), 3.74 (s, 3H, OeCH3), 3.87 (s,
6H, 2ꢂ OeCH3), 3.93 (s, 3H, OeCH3), 6.58 (s, 2H, Ar-H), 7.12 (d, 1H,
J ¼ 8.2 Hz, Ar-H), 7.48e7.55 (m, 2H, Ar-H), 8.51 (s,1H, CH]N).13C NMR
(100 MHz, DMSO):
d
ꢀ5.78 (CH3eSieCH3),17.71 (CH3eCeCH3), 24.74
(Ce(CH3)3), 55.00 (OeCH3), 59.25 (OeCH3), 98.04, 111.22, 121.27,
125.43, 129.50, 135.95, 144.68, 147.73, 153.32 (Aromatic C), 158.27
(CH]N). HRMS: Calculated for C23H34O5Si: 432.2216; Found:
432.2213 (Mþ þ H).
(100 MHz, CDCl3):
12.52 (CH3), 17.87 (Ce(CH3)3), 28.73 (Ce(CH3)3), 48.61 (CH), 54.50
(OeCH3), 54.87 (OeCH3), 54.93 (OeCH3), 55.36 (CH), 55.39
d
ꢀ5.38 to ꢀ5.30 (CH3eSieCH3), 9.05 (CH3),
d
(OeCH3), 60.32 (CH), 62.10 (CH), 94.10, 94.32, 111.48, 111.76, 110.17,
120.08, 127.78, 133.53, 133.61, 144.55, 150.39, 152.90 (Aromatic C),
167.80 (C]O), 167.94 (C]O).
4.1.9. 3-(tert-Butyldimethylsilanyloxy)-(4-methoxyphenyl)-(3,4,5-
trimethoxybenzylidene)amine (11d)
Preparation as described above from 2-methoxy-5-[(3,4,5-tri-
methoxybenzylidene)amino]phenol (11c) (0.02 mol, 6.34 g). Yield
4.1.12. 4-([3-tert-Butyldimethylsilanyloxy]-4-methoxyphenyl)-3,3-
dimethyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (12c)
71%, yellow crystals, m.p.108e109 ꢃC. IR nmax (KBr) cmꢀ1
:
Preparation as described above from [3-(tert-butyldimethylsi-
lanyloxy)-(4-methoxyphenyl]-(3,4,5-trimethoxybenzylidene)
amine (11a) (10 mmol, 1.831 g) and ethyl-2-bromoisobutyrate
(12 mmol, 1.77 mL). Yield 80%, orange solid, which was used
without further purification. IR nmax (film) cmꢀ1: 1751.2 cmꢀ1 (C]
1614.7 cmꢀ1 (C]N). 1H NMR (400 MHz, DMSO):
d 0.20 (s, 6H,
CH3eSieCH3), 1.03 (s, 9H, SieCe(CH3)3), 3.84 (s, 3H, OeCH3), 3.93
(s, 3H, OeCH3), 3.96 (s, 6H, 2ꢂ OeCH3), 6.84e6.87 (m, 3H, Ar-H),
7.15 (2H, s, Ar-H), 8.36 (s, 1H, CH]N). 13C NMR (100 MHz, DMSO):
d
ꢀ5.05 (CH3eSieCH3), 18.02 (CH3eCeCH3), 25.30 (Ce(CH3)3),
O, b d 0.03 (s, 3H, SieCH3), 0.07
-lactam). 1H NMR (400 MHz, CDCl3):
55.24 (OeCH3), 55.78 (OeCH3), 60.53 (OeCH3), 105.04, 111.72,
113.62, 113.82, 131.52, 140.16, 144.65, 144.95, 149.33, 153.02
(Aromatic C), 157.53 (CH]N). HRMS: Calculated for C23H34NO5Si:
432.2206; Found: 432.2216, (Mþ þ H).
(s, 3H, SieCH3), 0.88 (s, 3H, eCH3), 0.93 (s, 9H, Ce(CH3)3), 1.50 (s,
3H, eCH3), 3.72 (s, 6H, 2ꢂ OeCH3), 3.77 (s, 3H, OeCH3), 3.88 (s, 3H,
OeCH3), 4.69 (s, 1H, H-4), 6.57 (s, 2H, Ar-H), 6.67 (d, 1H, J ¼ 8.5 Hz,
Ar-H), 6.77e6.80 (m, 1H, Ar-H), 6.83 (d, 1H, J ¼ 2.5 Hz, Ar-H). 13C
NMR (100 MHz, CDCl3):
d
ꢀ4.97 (CH3eSieCH3), 17.60 (CeCH3)3),
4.1.10. 4-[3-(tert-Butyldimethylsilanyloxy)-4-methoxyphenyl]-1-
(3,4,5-trimethoxyphenyl)azetidin-2-one (12a)
To a suspension of zinc dust (0.9 g, 13.8 mmol) in benzene
(20 mL) under nitrogen was added trimethylchlorosilane (0.65 mL,
18.28 (Ce(C)eC), 22.64 (eCH3), 25.46 (eCH3), 55.19 (CH3eCeCH3,
C3), 55.30 (OeCH3), 55.82 (OeCH3), 60.78 (OeCH3), 66.25 (CH),
94.84, 111.85, 119.28, 120.10, 127.47, 133.87, 134.01, 144.97, 150.74,
153.30 (Aromatic C), 171.34 (C]O).

5762
M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
4.1.13. 1-[3-(tert-Butyldimethylsilanyloxy)-4-methoxyphenyl]-4-
(3,4,5-trimethoxyphenyl)azetidin-2-one (12g)
Preparation as described above from [3-(tert-butyldimethylsi-
lanyloxy)-4-methoxyphenyl]-(3,4,5-trimethoxybenzylidene)amine
(11d) (10 mmol, 4.310 g). Yield 47%, orange crystals, m.p. 112 ꢃC. IR
nmax (KBr) cmꢀ1: 1747.6 cmꢀ1 (C]O). 1H NMR (400 MHz, CDCl3):
(s, 6H, 2ꢂ OeCH3), 3.54 (s, 3H, OeCH3), 4.78 (dd, 1H, J ¼ 2.0 Hz,
5.5 Hz, H-4), 6.34 (s, 2H, Ar-H), 6.83e6.87 (m, 2H, Ar-H), 7.17e7.21
(m, 2H, Ar-H). 13C NMR (100 MHz, CDCl3):
d 46.42 (CH2), 53.23 (CH),
55.45 (OeCH3), 60.44 (OeCH3), 90.90,115.58,127.22,133.36,133.52,
133.84, 153.00 (Aromatic C), 160.94 (CeF), 163.40 (C]O). HRMS:
Calculated for C18H18FNO4Na : 354.1118; Found 354.1132 (Mþ þ Na).
d
0.21 (s, 6H, CH3eSieCH3), 0.87 (s, 9H, Ce(CH3)3), 2.84 (dd, 1H,
J ¼ 2.0 Hz, 15.0 Hz, H-3), 3.40 (dd, 1H, J ¼ 5.4 Hz, 15.0 Hz, H-3), 3.82
(s, 3H, OeCH3), 3.85 (s, 9H, 3ꢂ OeCH3), 4.79 (dd, 1H, J ¼ 2.4 Hz,
J ¼ 5.0 Hz H-4), 6.53 (s, 2H, Ar-H), 6.57 (d, 1H, J ¼ 3.2 Hz, Ar-H), 6.62
(d, 1H, J ¼ 3.4 Hz, Ar-H), 6.72 (d, 1H, J ¼ 8.8 Hz, Ar-H). 13C NMR
4.1.17. 4-(2,4,5-Trimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (12n)
Preparation as described above from (2,4,5-trimethox-
ybenzylidene)-3,4,5-trimethoxyphenylamine (11f) (10 mmol,
3.614 g). Yield 28%, orange gel, IR nmax (film) cmꢀ1: 1746.9 cmꢀ1
(100 MHz, CDCl3):
d
ꢀ5.14 (CH3eSieCH3), 17.97 (CH3eCeCH3),
25.24 (Ce(CH3)3), 43.10 (C3, CH2), 55.34 (OeCH3), 60.35 (OeCH3),
70.04 (CH), 102.03, 111.82, 113.36, 136.76, 138.00, 152.80 (Aromatic
C), 167.08 (C]O). HRMS: Calculated for C25H35NO6SiNa: 496.2131;
Found 496.2131 (Mþ þ Na).
(C]O, b d 3.01 (dd, 1H,
-lactam). 1H NMR (400 MHz, CDCl3):
J ¼ 2.0 Hz, 15.0 Hz, H-3), 3.51 (dd, 1H, J ¼ 6.0 Hz, 15.1 Hz, H-3), 3.73
(s, 6H, 2ꢂ OeCH3), 3.74 (s, 3H, OeCH3), 3.75 (s, 3H, OeCH3), 3.77 (s,
6H, 2ꢂ OeCH3), 5.24 (dd, 1H, J ¼ 2.5, 5.7 Hz, H-4), 5.91 (s, 2H, Ar-H),
6.59 (s, 1H, Ar-H), 6.78 (s,1H, Ar-H). 13C NMR (100 MHz, CDCl3):
4.1.14. 1-[(3-tert-Butyldimethylsilanyloxy)-4-methoxyphenyl]-3-
methyl-4-(3,4,5-trimethoxyphenyl)azetidin-2-one (12h)
d 44.88 (CH2), 48.02 (CH), 51.70 (OeCH3), 55.49 (OeCH3), 56.24
(OeCH3), 60.45 (OeCH3), 60.58 (OeCH3), 92.05, 109.79, 118.39,
133.64, 133.71, 142.68, 145.07, 149.39, 151.32, 152.96, (Aromatic C),
164.68 (C]O). HRMS: Calculated for C21H25NO7Na: 426.1529;
Found 426.1534 (Mþ þ Na).
Preparation as described above from [(3-tert-butyldimethylsi-
lanyloxy)-4-methoxyphenyl]-(3,4,5-trimethoxybenzylidene)amine
(11d) (5 mmol, 2.158 g) and ethyl-2-bromopropionate (6 mmol,
0.78 mL) and isolated as mixture of diastereomers. Yield 10%,
orange gel. IR nmax (film) cmꢀ1: 1749.1 cmꢀ1 (C]O,
b
-lactam). 1H
4.1.18. 4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-
2-one (12o)
NMR (400 MHz, CDCl3):
d 0.01e0.03 (m, 3H, SieCH3), 0.05e0.06 (m,
3H, SieCH3), 0.90 (s, 9H, Ce(CH3)3), 0.96 (d, 2H, J ¼ 7.6 Hz, CH3),1.46
(d, 1H, J ¼ 7.0 Hz, CH3), 3.14 (m, 0.4H, H-3), 3.65e3.68 (m, 0.6H, H-
3), 3.75e3.78 (m, 6H, 2ꢂ OeCH3), 3.81e3.84 (m, 6H, 2ꢂ OeCH3),
4.42 (bs, 0.4H, H-4), 5.03 (d, 0.6H, J ¼ 5.5 Hz, H-4), 6.42 (s, 1H, Ar-H),
6.55 (s, 1H, Ar-H), 6.65e6.66 (m, 1H, Ar-H), 6.74e6.76 (m, 1H, Ar-H),
Preparation as described above from (4-methoxybenzylidene)-
3,4,5-trimethoxyphenylamine (11g) (5 mmol, 1.506 g). Yield 43%,
green crystals, m.p. 70e71 ꢃC. IR nmax (film): 1747.5 cmꢀ1 (C]O,
b-
lactam). 1H NMR (400 MHz, CDCl3):
d
2.85 (dd, 1H, J ¼ 2.5 Hz,
15.0 Hz, H-3), 3.48 (dd, 1H, J ¼ 5.5 Hz, J ¼ 15.1 Hz, H-3), 3.65 (s, 6H,
2ꢂ OeCH3), 3.70 (s, 3H, OeCH3), 3.73 (s, 3H, OeCH3), 4.88 (dd,
J ¼ 2.6 Hz, 5.5 Hz, 1H, H-4), 6.50 (s, 2H, Ar-H), 6.86 (d, 2H, J ¼ 8.6 Hz,
Ar-H), 7.26 (d, 2H, J ¼ 8.6 Hz, Ar-H). 13C NMR (100 MHz, CDCl3):
6.99e7.02 (m, 1H, Ar-H). 13C NMR (100 MHz, CDCl3):
d
ꢀ5.32
(CH3eSieCH3), 9.17 (eCH3), 12.63 (eCH3), 17.93 (Ce(CH3)3), 25.14
(Ce(CH3)3), 25.28 (Ce(CH3)3), 48.80 (C3,CH), 54.73 (C3,CH), 55.38
(OeCH3), 55.42 (OeCH3), 55.59 (OeCH3), 55.64 (OeCH3), 55.69
(OeCH3), 60.33 (CH), 60.37 (OeCH3), 62.81 (CH), 102.13, 103.44,
109.71, 110.71, 110.15, 110.18, 112.13, 112.23, 130.27, 130.88, 131.04,
131.04, 137.05, 137.38, 147.19, 147.23, 153.03, 153.42 (Aromatic C),
167.38 (C]O), 167.51 (C]O). HRMS: Calculated for C26H37NO6SiNa:
510.2288; Found: 510.2281 (Mþ þ Na).
d
46.36 (CH2), 53.56 (CH), 54.78 (OeCH3), 55.49 (OeCH3), 60.36
(OeCH3), 93.92, 113.58, 126.83, 129.48, 133.62, 133.68, 152.94,
159.29 (Aromatic C), 164.14 (C]O). HRMS: Calculated for
C19H21NO5Na: 366.1317; Found: 366.1330 (Mþ þ Na).
4.1.19. 4-(4-Methoxyphenyl)-3-methyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (12p)
4.1.15. 1-[(3-tert-Butyldimethylsilanyloxy)-4-methoxyphenyl]-3,3-
dimethyl-4-(3,4,5-trimethoxyphenyl)azetidin-2-one (12i)
Preparation as described above from (4-methoxybenzylidene)-
(3,4,5-trimethoxyphenyl)amine (11g) (5 mmol, 1.506 g) and ethyl-
2-bromopropionate (6 mmol, 0.78 mL) and isolated as mixture of
diastereomers. Yield 83%, dark orange gel. IR nmax (film):
Preparation as described above from [(3-tert-butyldimethylsi-
lanyloxy)-4-methoxyphenyl]-(3,4,5-trimethoxybenzylidene)amine
(11d) (5 mmol, 2.158 g) and ethyl-2-bromoisobutyrate (6 mmol,
1725.5 cmꢀ1 (C]O, -lactam). 1H NMR (400 MHz, CDCl3):
b d 0.75 (d,
0.88 mL). Yield 18%, orange solid, m.p. 95 ꢃC. IR nmax (film) cmꢀ1
:
2H, J ¼ 7.5 Hz, CH3), 1.33 (d, 1H, J ¼ 7.5 Hz, CH3), 3.54 (q(br),
0.66H, J ¼ 7.5 Hz, H-3), 3.94 (q(br), 0.34H, J ¼ 7.5 Hz, H-3), 3.57 (3H,
s, OeCH3), 3.58 (3H, s, OeCH3), 3.62 (s, 1.2H, OeCH3), 3.64 (s, 2.8H,
OeCH3), 4.42 (bs, 0.34H, H-4), 5.01 (d, 0.66H, J ¼ 6.0 Hz, H-4), 6.44
(s, 1.3H, Ar-H), 6.46 (s, 0.8H, Ar-H), 6.76 (m, 2H, Ar-H), 7.06 (d, 1.33H,
J ¼ 8.0 Hz, Ar-H), 7.20 (d, 0.67H, J ¼ 8.5 Hz, Ar-H). 13C NMR
1747.5 cmꢀ1 (C]O,
b
-lactam). 1H NMR (400 MHz, CDCl3):
d
ꢀ0.17 (s,
3H, SieCH3), ꢀ0.12 (s, 3H, SieCH3), 0.72 (s 9H, Ce(CH3)3), 0.74 (s,
3H, eCH3), 1.32 (s, 3H, eCH3), 3.57 (s, 3H, OeCH3), 3.58 (s, 6H, 2ꢂ
OeCH3), 3.65 (s, 3H, OeCH3), 4.46 (s, 1H, H-4), 6.21 (s, 2H, Ar-H),
6.57 (m, 2H, Ar-H), 6.81 (dd, 1H, J ¼ 2.5 Hz, 8.5 Hz, Ar-H). 13C NMR
(100 MHz, CDCl3):
d
ꢀ5.37 (CH3eSieCH3), 17.23 (Ce(C)eC), 22.36
(100 MHz, CDCl3): d 9.16 (eCH3), 12.43(CH3),48.65 (CH), 55.38 (CH),
(Ce(CH3)3), 25.11 (eCH3), 25.25 (eCH3), 54.92 (CH3eCeCH3), 55.60
(OeCH3), 55.69 (OeCH3), 60.32 (OeCH3), 66.47 (CH), 94.84, 111.85,
119.28, 120.10, 127.47, 133.87, 134.01, 144.96, 150.74, 153.30
(Aromatic C), 171.33 (C]O). HRMS: Calculated for C27H39NO6SiNa :
524.2444; Found 524.2427 (Mþ þ Na).
54.52 (OeCH3), 54.58 (OeCH3), 54.65 (OeCH3), 55.32 (OeCH3),
57.76 (CH), 60.25 (CH), 62.09 (OeCH3), 93.98, 94.24, 113.51, 113.93,
126.05, 126.77, 127.67, 129.14, 133.47, 152.90, 158.90 (Aromatic C),
167.78 (C]O), 167.93 (C]O). HRMS: Calculated for C20H23NO5Na:
380.1473; Found 380.1473 (Mþ þ Na).
4.1.16. 4-(4-Fluorophenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-
one (12m)
4.1.20. 4-(4-Methoxyphenyl)-3,3-dimethyl-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (12q)
Preparation as described above from (4-fluorobenzylidene)-
3,4,5-trimethoxyphenylamine (11e) (10 mmol, 2.89 g). Yield 53%,
pale orange crystals, m.p. 96 ꢃC. IR nmax (KBr) cmꢀ1: 1739.8 cmꢀ1
Preparation as described above from (4-methoxybenzylidene)-
(3,4,5-trimethoxyphenyl)amine (11g) (5 mmol, 1.507 g) and ethyl-
2-bromoisobutyrate (6 mmol, 0.88 mL). Yield 68%, yellow powder,
(C]O,
b
-lactam). 1H NMR (400 MHz, CDCl3):
d
2.72 (dd, 1H,
m.p. 110 ꢃC. IR nmax (KBr): 1747.6 cmꢀ1 (C]O, -lactam). 1H NMR
b
J ¼ 2.5 Hz, 15.5 Hz, H-3), 3.34 (dd, 1H, J ¼ 5.5 Hz, 15.0 Hz, H-3), 3.48
(400 MHz, CDCl3):
d 0.86 (s, 3H, eCH3), 1.52 (s, 3H, eCH3), 3.72 (s,

M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
5763
6H, 2ꢂ OeCH3), 3.78 (s, 3H, OeCH3), 3.81 (s, 3H, OeCH3), 4.73 (s,
1H, H-4), 6.57 (s, 2H, Ar-H), 6.87 (d, 2H, J ¼ 8.5 Hz, Ar-H), 7.14 (d, 2H,
(d, 1H, J ¼ 8.0 Hz, Ar-H). 13C NMR (100 MHz, CDCl3):
d
9.18 (eCH3),
12.62 (eCH3), 48.84 (CH), 54.59 (CH), 55.40 (OeCH3), 55.49
(OeCH3), 55.51 (OeCH3), 55.54 (OeCH3), 55.58 (OeCH3), 58.18
(CH), 60.49(OeCH3), 60.60 (OeCH3), 62.65 (CH), 92.06, 94.64,
107.90, 109.31, 110.67, 110.89, 118.28, 119.04, 126.68, 130.08, 133.55,
142.61, 152.98, 153.36 (Aromatic C), 168.00 (C]O), 168.13 (C]O).
HRMS: Calculated for C21H25NO6Na: 410.1580; Found: 410.1567
(Mþ þ Na).
J ¼ 8.5 Hz, Ar-H). 13C NMR (100 MHz, CDCl3):
d 17.43 (eCH3), 22.31
(eCH3), 54.79 (OeCH3), 54.93 (C-3), 55.57 (OeCH3), 60.51 (OeCH3),
66.11 (CH), 94.50, 113.61, 126.79, 127.39, 133.68, 153.00, 158.92
(Aromatic C), 171.11 (C]O). HRMS: Calculated for C21H25NO5Na:
394.1631; Found: 394.1620 (Mþ þ Na).
4.1.21. 1-(3,4,5-Trimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)
azetidin-2-one (12r)
Preparation as described above from (3,4,5-trimethoxybenzy-
lidene)-(3,4,5-trimethoxyphenyl)amine (11h) (5 mmol, 1.807 g).
Yield 68%, orange gel. IR nmax (film): 1746.9 cmꢀ1 (C]O). 1H NMR
4.1.25. 4-(3,4-Dimethoxyphenyl)-3,3-dimethyl-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (12v)
Preparation as described above using method 2 from (3,4-dime-
thoxybenzylidene)-(3,4,5-trimethoxyphenyl)amine (11j) (5 mmol,
1.657 g) and ethyl-2-bromoisobutyrate (6 mmol, 0.88 mL) Yield 10%,
greenpowder, m.p.102 ꢃC. IR nmax (film): 1745.9 cmꢀ1 (C]O).1H NMR
(400 MHz, CDCl3):
d
2.88 (dd, 1H, J ¼ 2.5 Hz, 15.5 Hz, H-3), 3.49 (dd,
1H, J ¼ 5.5 Hz, J ¼ 15.1 Hz, H-3), 3.64 (s, 3H, OeCH3), 3.66 (s, 6H, 2ꢂ
OeCH3), 3.76 (s, 9H, 3ꢂ OeCH3), 4.82 (dd, J ¼ 5.5 Hz, 2.5 Hz, 1H, H-
4), 6.55 (s, 2H, Ar-H), 6.59 (s, 2H, Ar-H). 13C NMR (100 MHz, CDCl3):
(400 MHz, CDCl3): d 0.90 (s, 3H, eCH3),1.52 (s, 3H, eCH3), 3.73 (s, 6H,
2ꢂ OeCH3), 3.79 (s, 3H, OeCH3), 3.84 (s, 3H, OeCH3), 3.89 (s, 3H,
OeCH3), 4.73 (s,1H, H-4), 6.60 (s, 2H, Ar-H), 6.70 (d,1H, J ¼ 2.0 Hz, Ar-
H), 6.81 (dd,1H, J ¼ 2.0 Hz, 8.5 Hz, Ar-H), 6.88 (d,1H, J ¼ 8.5 Hz, Ar-H).
d
46.25 (CH2), 55.19 (CH), 55.53 (OeCH3), 55.68 (OeCH3), 60.22
(OeCH3), 60.41 (OeCH3), 90.86, 102.22, 129.58, 133.41, 137.46,
138.00, 152.93, 153.36 (Aromatic C), 164.14 (C]O). HRMS: Calcu-
lated for C21H25NO7Na: 426.1529; Found 426.1530 (Mþ þ Na).
13C NMR (100 MHz, CDCl3):
d 17.29 (eCH3), 22.35 (eCH3), 30.48 (C-3),
54.99 (OeCH3), 55.39 (OeCH3), 55.56 (OeCH3), 55.58 (OeCH3), 66.39
(CH), 94.47, 109.01, 110.67, 118.69, 127.33, 133.71, 133.77, 148.32,
148.65, 152.99 (Aromatic C), 171.12 (C]O). HRMS: Calculated for
C22H27NO6Na: 424.1736; Found 424.1726 (Mþ þ Na).
4.1.22. 4-(2,3,4-Trimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (12s)
Preparation as described above from (2,3,4-trimethoxybenzy-
lidene)-(3,4,5-trimethoxyphenyl)amine (11i) (5 mmol, 1.807 g).
Yield 16%, yellow gel. IR nmax (film): 1751.7 cmꢀ1 (C]O). 1H NMR
4.1.26. 4-Thiophen-2-yl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one
(12w)
(400 MHz, CDCl3):
d
2.99 (dd, 1H, J ¼ 2.5 Hz, 15.1 Hz, H-3), 3.50 (dd,
Preparation as described above from thiophen-2-ylmethylene-
(3,4,5-trimethoxyphenyl)amine (11k) (5 mmol, 1.387 g). Yield 27%,
dark brown gel. IR nmax (film): 1735.5 cmꢀ1 (C]O). 1H NMR
1H, J ¼ 5.5 Hz, J ¼ 15.1 Hz, H-3), 3.73 (s, 6H, 2ꢂ OeCH3), 3.75 (s, 3H,
OeCH3), 3.83 (s, 3H, OeCH3), 3.86 (s, 3H, OeCH3), 3.92 (s, 3H,
OeCH3), 5.21 (dd,1H, J ¼ 2.5 Hz, 6.0 Hz, H-4), 6.57 (s, 2H, Ar-H), 6.64
(d, 1H, J ¼ 8.5 Hz, Ar-H), 6.97 (d, 1H, J ¼ 8.5 Hz, Ar-H). 13C NMR
(400 MHz, CDCl3):
d
2.95 (dd, 1H, J ¼ 15.1 Hz, J ¼ 2.9 Hz, H-3), 3.61
(dd, 1H, J ¼ 15.1 Hz, J ¼ 5.8 Hz, H-3), 3.74 (s, 6H, 2ꢂ OeCH3), 3.76 (s,
3H, OeCH3), 5.05, (dd, 1H, J ¼ 2.9 Hz, 5.5 Hz, H-4), 6.61 (s, 2H, Ar-H),
7.00 (m, 1H, thiophene H), 7.17 (d, 1H, J ¼ 4.1 Hz, thiophene H), 7.31
(100 MHz, CDCl3):
d 45.34 (CH2), 48.66 (CH), 55.50 (OeCH3), 55.56
(OeCH3), 59.80 (OeCH3), 60.33 (OeCH3), 60.95 (OeCH3), 93.84,
107.10, 120.99, 122.69, 133.64, 139.03, 141.66, 151.42, 153.38
(Aromatic C), 164.38 (C]O). HRMS: Calculated for C21H25NO7Na:
426.1529; Found: 426.1549 (Mþ þ Na).
(d, 1H, J ¼ 5.2 Hz, thiophene H). 13C NMR (100 MHz, CDCl3):
d 47.52
(CH2), 51.40 (CH), 55.68 (OeCH3), 55.84 (OeCH3), 60.85 (OeCH3),
94.26, 123.82, 125.81, 127.05, 133.67, 134.24, 141.93, 153.31
(Aromatic C), 163.86 (C]O). HRMS: Calculated for C16H18NO4S:
320.0957; Found 320.1003 (Mþ þ H).
4.1.23. 4-(3,4-Dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (12t)
Preparation as described above from (3,4-dimethoxybenzy-
lidene)-(3,4,5-trimethoxyphenyl)amine (11j) (5 mmol, 1.655 g).
Yield 15%, orange crystals, m.p. 99 ꢃC. IR nmax (KBr): 1745.9 cmꢀ1
4.1.27. 4-(7-Methoxynaphthalen-2-yl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (12x)
Preparation as described above from (6-methoxynaphthalen-2-
ylmethylene)-(3,4,5-trimethoxyphenyl)amine (11l) (5 mmol,
1.757 g). Yield 20%, orange gel. IR nmax (film): 1736.4 cmꢀ1 (C]O).
(C]O). 1H NMR (400 MHz, CDCl3):
d
2.86 (dd,1H, J ¼ 2.5 Hz,15.1 Hz,
H-3), 3.48 (dd, 1H, J ¼ 5.5 Hz, J ¼ 15.1 Hz, H-3), 3.64 (s, 6H, 2ꢂ
OeCH3), 3.67 (s, 3H, OeCH3), 3.69 (s, 6H, 2ꢂ OeCH3), 4.83 (dd, 1H,
J ¼ 2.5 Hz, 5.5 Hz, H-4), 5.84 (s(br), 2H, Ar-H), 6.50 (s, 2H, Ar-H), 6.91
1H NMR (400 MHz, CDCl3):
d
3.02 (dd, 1H, J ¼ 2.5 Hz, 12.1 Hz, H-3),
3.61 (dd, 1H, J ¼ 5.5 Hz, J ¼ 15.3 Hz, H-3), 3.69 (s, 6H, 2ꢂ OeCH3),
3.76 (s, 3H, OeCH3), 3.89 (s, 3H, OeCH3), 5.09, (dd, 1H, J ¼ 2.5 Hz,
J ¼ 5.5 Hz, H-4), 5.89 (s, 2H, Ar-H), 6.62 (s, 1H, Ar-H), 7.10e7.14 (m,
2H, Ar-H), 7.46e7.48 (dd, 1H, Ar-H), 7.69e7.78 (m, 2H, Ar-H). 13C
(dd, 1H, J ¼ 2 Hz, 8.0 Hz, Ar-H). 13C NMR (100 MHz, CDCl3):
d 46.37
(CH2), 54.16 (OeCH3), 55.27 (OeCH3), 55.48 (OeCH3), 60.52 (CH),
90.91, 105.19, 118.60, 124.46, 127.70, 128.28, 128.63, 133.59, 153.25,
157.90 (Aromatic C), 164.16 (C]O). HRMS: Calculated for
C20H23NO6Na: 396.1423; Found: 396.1408 (Mþ þ Na).
NMR (100 MHz, CDCl3):
d 47.52 (CH2), 51.40 (CH), 55.68 (OeCH3),
55.84 (OeCH3), 60.85 (OeCH3), 90.90, 94.26, 105.31, 118.60, 123.82,
125.81, 127.05, 128.43, 128.85, 129.62, 133.67, 134.24, 141.93, 153.31
(Aromatic C), 163.86 (C]O).
4.1.24. 4-(3,4-Dimethoxyphenyl)-3-methyl-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (12u)
Preparation as described above from (3,4-dimethoxybenzy-
lidene)-(3,4,5-trimethoxyphenyl)amine (11j) (5 mmol, 1.657 g) and
ethyl-2-bromopropionate (6 mmol, 0.78 mL) and isolated as
mixture of diastereomers. Yield 10%, brown gel. IR nmax (film):
4.1.28. 4-(3-Hydroxy-4-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (12d)
To
a suspension of 4-[3-(tert-butyldimethylsilanyloxy)-4-
methoxyphenyl]-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (12a)
(0.634 mmol, 0.30 g) in THF (20 mL) was added of 1 M tetra-n-
butylammonium fluoride (1.5 equiv). The solution was stirred in an
1744.6 cmꢀ1 (C]O). 1H NMR (400 MHz, CDCl3):
d 0.94 (d, 2H,
J ¼ 7.5 Hz, eCH3), 1.48 (d, 1H, J ¼ 7.5 Hz, eCH3), 3.16 (m, 0.34H, H-3),
3.69 (m, 0.66H, H-3), 3.72 (s, 3H, OeCH3), 3.76 (s, 3H, OeCH3), 3.80
(s, 9H, 3ꢂ OeCH3), 4.50 (d, 0.34H, J ¼ 2.0 Hz, H-4), 5.11 (d, 0.66H,
J ¼ 6.0 Hz, H-4), 6.56 (s, 0.6H, Ar-H), 6.59 (s, 1.4H, Ar-H), 6.72 (d, 1H,
J ¼ 1.5 Hz, Ar-H), 6.72e6.85 (dd, 1H, J ¼ 1.65 Hz, 8.1 Hz, Ar-H), 6.88
ice bath for 15 min to avoid decomposition of the b-lactam ring. The
reaction mixture was then diluted with ethyl acetate (100 mL) and
quenched with 10% HCl (100 mL). The layers are separated and the
aqueous layer was extracted with ethyl acetate (2 ꢂ 50 mL). The

5764
M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
organic layer was then washed with water (100 mL) and brine
(100 mL) and dried with sodium sulphate. The crude product was
purified by flash chromatography over silica gel (eluent: dichloro-
methane). Yield 62%, yellow gel. IR nmax (film): 1746.0 (C]O),
4.1.32. 1-(3-Hydroxy-4-methoxyphenyl)]-3-methyl-4-(3,4,5-
trimethoxyphenyl)azetidin-2-one (12k)
Preparation as described above from 1-[3-(tert-butyldime-
thylsilanyloxy)-4-methoxyphenyl]-3-methyl-4-(3,4,5-trimethox-
yphenyl)azetidin-2-one (12h) (0.149 mmol, 0.072 g) and isolated as
mixture of diastereomers. Yield 82%, brown solid, m.p. 165 ꢃC; nmax
(film): 1739.6 (C]O), 3409.0 cmꢀ1 (OH). 1H NMR (400 MHz, CDCl3):
3404.2 cmꢀ1 (OH). 1H NMR (400 MHz, CDCl3):
d 2.91 (dd, 1H,
J ¼ 2.4 Hz, 15.6 Hz, H-3), 3.48 (dd, 1H, J ¼ 5.5 Hz, 15.0 Hz, H-3), 3.70
(s, 6H, 2ꢂ OeCH3), 3.74 (s, 3H, OeCH3), 3.85 (s, 3H, OeCH3), 4.86
(dd, 1H, J ¼ 2.5 Hz, J ¼ 5.5 Hz, H-4), 6.54 (s, 2H, Ar-H), 6.81e6.94 (m,
d
0.93 (d, 2H, J ¼ 7.6 Hz, eCH3),1.40 (d,1H, J ¼ 7.8 Hz, eCH3), 3.15 (dq,
3H, Ar-H). 13C NMR (100 MHz, CDCl3):
d
30.47 (CH2), 46.31 (CH),
0.36H, J ¼ 2.0 Hz, 7.6 Hz, H-3), 3.64 (dq, 0.64H, J ¼ 7.5 Hz, 5.2 Hz, H-3),
3.79 (s, 3H, OeCH3), 3.83 (m, 9H, 3ꢂ OeCH3), 4.43 (d, 0.36H,
J ¼ 1.9 Hz, H-4), 5.05 (d, 0.64H, J ¼ 5.5 Hz, H-4), 5.81 (bs,1H, OH), 6.41
(s, 0.89H, Ar-H), 6.53 (s, 1.10H, Ar-H), 6.74 (2ꢂ d, overlapping, 1H,
J¼ 8.5 Hz, 8.6 Hz, Ar-H), 6.84e6.90 (2ꢂ ddoverlapping,1H, J ¼ 2.5 Hz,
8.8 Hz, Ar-H), 6.92 (d, 0.5H, J ¼ 2.5 Hz), 6.95 (d, 0.5H, J ¼ 2.3 Hz, Ar-H).
53.66 (OeCH3), 55.53 (OeCH3), 60.44 (OeCH3), 94.01,110.13,111.60,
117.35, 130.64, 133.57, 143.21, 145.88, 146.44, 152.94, 164.21 (C]O).
HRMS: Calculated for C19H22NO6: 360.1447; Found 360.1454
(Mþ þ H).
4.1.29. 4-(3-Hydroxy-4-methoxyphenyl)-3-methyle1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (12e)
13C NMR (100 MHz, CDCl3):
d 9.16 (eCH3), 12.66 (eCH3), 48.86 (CH),
54.81 (CH), 55.70 (OeCH3), 55.73 (OeCH3), 58.41 (CH), 60.42
(OeCH3), 62.80 (CH), 102.01, 103.34, 103.81, 103.90, 108.28, 108.39,
110.51, 110.51, 130.23, 131.46, 133.22, 142.73, 142.79, 145.41,
153.02e153.39 (Aromatic C), 167.69 (C]O). HRMS: Calculated for
C20H23NO6Na: 396.1423; Found: 396.1443 (Mþ þ Na).
Preparation as described above from 4-([3-tert-butyldime-
thylsilanyloxy]-4-methoxyphenyl)-3-methyle1-(3,4,5-trimethox-
yphenyl)azetidin-2-one (12b) (8 mmol, 3.901 g) and isolated as
mixture of diastereomers. Yield 45%, brown gel. IR nmax (KBr):
1724.2, (C]O), 3240.2 cmꢀ1 (OH). 1H NMR (400 MHz, CDCl3):
d 0.91
(d, 2H, J ¼ 7.5 Hz, eCH3), 1.44 (d, 1H, J ¼ 7.5 Hz, eCH3), 3.09e3.14
(dq, 0.33H, J ¼ 2.2 Hz, 7.5 Hz, H-3), 3.59e3.67 (dq, 0.67H, J ¼ 7.5 Hz,
6.0 Hz, H-3), 3.72 (s(br), 6H, 2ꢂ OeCH3), 3.76 (s,1H, OeCH3), 3.78 (s,
2H, OeCH3), 3.89 (s, 3H, OeCH3), 4.44 (d, 0.33H, J ¼ 2.0 Hz, H-4),
5.07 (d, 0.67H, J ¼ 5.6 Hz, H-4), 5.81 (bs, 1H, OH), 6.55 (s, 0.67H, Ar-
H), 6.57 (s, 1.33H, Ar-H), 6.70e6.85 (m, 3H, Ar-H). 13C NMR
4.1.33. 1-(3-Hydroxy-4-methoxyphenyl)]-3,3-dimethyl-4-(3,4,5-
trimethoxyphenyl)-azetidin-2-one (12l)
Preparation as described above from 1-[3-(tert-butyldime-
thylsilanyloxy)-4-methoxyphenyl]-3,3-dimethyl-4-(3,4,5-trime-
thoxyphenyl)azetidin-2-one (12i) (0.7 mmol, 0.352 g). Yield 59%,
colourless powder, m.p. 174e178 ꢃC. IR nmax (film): 1724.5 (C]O),
(100 MHz, CDCl3):
d
9.15 (eCH3), 12.62 (eCH3), 45.51 (CH), 54.61
3367.7 cmꢀ1 (OH). 1H NMR (400 MHz, CDCl3):
d 0.91 (s, 3H, eCH3),
(CH), 55.47 (OeCH3), 55.56 (OeCH3), 56.62 (OeCH3), 57.90 (CH),
62.31 (CH), 94.14, 94.37, 110.17, 110.49, 112.71, 118.24, 127.36, 130.48,
133.64, 145.33, 146.29, 152.98 (Aromatic C), 167.94 (C]O), 168.10
(C]O). HRMS: Calculated for C20H23NO6Na : 396.1423; Found:
396.1419 (Mþ þ Na).
1.51 (s, 3H, eCH3), 3.78 (s, 6H, 2ꢂ OeCH3), 3.83 (s, 3H, OeCH3), 3.85
(s, 3H, OeCH3), 4.67 (s, 1H, H-4), 6.39 (s, 2H, Ar-H), 6.74 (d, 1H,
J ¼ 8.5 Hz, Ar-H), 6.80 (dd, 1H, J ¼ 2.5 Hz, 8.8 Hz, Ar-H), 7.08 (d, 1H,
J ¼ 2.5 Hz, Ar-H). 13C NMR (100 MHz, CDCl3):
d 17.20 (eCH3), 22.34
(eCH3), 54.91 (C-3), 55.66 (OeCH3), 55.69 (OeCH3), 60.38 (OeCH3),
66.60 (C-4), 103.06, 104.37, 108.27, 110.62, 130.79, 131.44, 137.05,
143.00, 145.60, 153.00, 170.98 (C]O). HRMS: Calculated for
C21H25NO6Na: 410.1580; Found 410.1591 (Mþ þ Na).
4.1.30. 4-(3-Hydroxy-4-methoxyphenyl)-3,3-dimethyle1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (12f)
Preparation as described above from 4-([3-tert-butyldime-
thylsilanyloxy]-4-methoxyphenyl)-3,3-dimethyl-1-(3,4,5-trime-
thoxyphenyl)azetidin-2-one (12c) (8 mmol, 4.01 g). Yield 53%, pale
brown powder. IR nmax (KBr): 1747.4 (C]O), 3460.1 cmꢀ1 (OH). 1H
4.1.34. 4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-
2-thione (13a)
4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-
one (12o) (0.206 g, 0.6 mmol) and Lawesson’s reagent (0.4 mmol,
0.162 g) (0.68 equiv) were refluxed in toluene (5 mL) at 135 ꢃC
under N2 for 3 h. The solvent was evaporated and the brown solid
residue was purified by flash column chromatography (eluent:
dichloromethane) to afford the product as a yellow solid. Yield
NMR (400 MHz, CDCl3): d 0.79 (s, 3H, eCH3),1.40 (s, 3H, eCH3), 3.63
(s, 6H, 2ꢂ OeCH3), 3.69 (s, 3H, OeCH3), 3.79 (s, 3H, OeCH3), 4.60 (s,
1H, H-4), 6.50 (s, 2H, Ar-H), 6.61 (dd, 1H, J ¼ 2.0 Hz, 8.5 Hz, Ar-H),
6.71 (d, 1H, J ¼ 2.0 Hz, Ar-H), 6.76 (d, 1H, J ¼ 8.5 Hz, Ar-H). 13C NMR
(100 MHz, CDCl3):
d 17.24 (eCH3), 22.27 (eCH3), 54.89 (C-3), 55.53
(OeCH3), 55.56 (OeCH3), 60.46 (OeCH3), 66.06 (CH), 94.54, 110.24,
112.45, 117.80, 127.87, 133.63, 133.73, 145.38, 146.12, 152.96, 153.17
(Aromatic C), 171.56 (C]O). HRMS: Calculated for C21H25NO6Na:
410.1580; Found: 410.1588 (Mþ þ Na).
47.5%, Rf 0.89 (CH2Cl2: MeOH; 19:1), m.p. 84 ꢃC. IR: NaCl film nmax
:
1595.7 cmꢀ1 (C]S). 1H NMR (400 MHz, CDCl3):
d
3.00 (dd, 1H,
J ¼ 15.6 Hz, J ¼ 2.0 Hz, H-3), 3.50 (dd, 1H, J ¼ 15.6 Hz, J ¼ 5.0 Hz, H-
3), 3.72 (s, 6H, 2ꢂ OeCH3), 3.78 (s, 3H, OeCH3), 3.80 (s, 3H, OeCH3),
5.43 (dd, J ¼ 2.0 Hz, J ¼ 5.0 Hz, 1H, H-4), 6.92 (d, 2H, J ¼ 8.5 Hz, Ar-
H), 7.19 (s, 2H, Ar-H), 7.30e7.32 (d, 2H, J ¼ 9.0 Hz, Ar-H). 13C NMR
4.1.31. 1-(3-Hydroxy-4-methoxyphenyl)]-4-(3,4,5-
trimethoxyphenyl)azetidin-2-one (12j)
(100 MHz, CDCl3): d 49.30 (CH2), 54.90 (OeCH3), 55.58 (OeCH3),
Preparation as described above from 1-[3-(tert-butyldime-
thylsilanyloxy)-4-methoxyphenyl]-4-(3,4,5-trimethoxyphenyl)
azetidin-2-one (12g) (1.05 mmol, 0.497 g). Yield 39%, orange gel. IR
nmax (film): 1731.9 (C]O), 3500.0 cmꢀ1 (OH). 1H NMR (400 MHz,
60.43 (OeCH3), 62.13 (CH), 95.26, 114.21, 127.04, 128.29, 134.11,
135.17, 152.63, 159.65 (Aromatic C), 196.01 (C]S). HRMS, Calculated
for C19H21NO4SNa: 382.1089; Found: 382.1076 (Mþ þ Na).
CDCl3):
d
2.90 (dd, 1H, J ¼ 2.7 Hz, 15.0 Hz, H-3), 3.50 (dd, 1H,
4.1.35. 4-(4-Methoxyphenyl)-3-methyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-thione (13b)
J ¼ 5.5 Hz, 15.3 Hz, H-3), 3.82 (s, 3H, OeCH3), 3.85 (s, 9H, 3ꢂ
OeCH3), 4.86 (dd,1H, J ¼ 2.7 Hz, 5.4 Hz, H-4), 6.55 (s, 2H, Ar-H), 6.73
(d, 1H, J ¼ 8.9 Hz, Ar-H), 6.82 (dd, 1H, J ¼ 2.1 Hz, 8.8 Hz, Ar-H), 6.95
Preparation as described above from 4-(4-methoxyphenyl)-3-
methyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (12p) (0.214 g,
0.6 mmol). Evaporation of the solvent yielded a brown solid residue
which was purified using by flash column chromatography over
silica gel (eluent: dichloromethane) to afford the diastereomeric
product as a yellow gel. Yield 63.8%, Rf 0.86 (CH2Cl2: MeOH; 19:1).
IR: NaCl film nmax: 1591.11 cmꢀ1 (C]S). 1H NMR (400 MHz, CDCl3):
(d, 1H, J ¼ 2.7 Hz, Ar-H). 13C NMR (100 MHz, CDCl3):
d 46.43 (CH2),
54.07 (CH), 55.56 (OeCH3), 55.63 (OeCH3), 60.34 (OeCH3), 102.05,
103.92, 107.98, 110.64, 131.38, 133.49, 143.05, 145.56, 152.76, 153.33
(Aromatic C), 164.01 (C]O). HRMS: Calculated for C19H22NO6:
360.1447; Found: 360.1449 (MþþH).

M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
5765
d
0.96 (d, 2H, J ¼ 7.5 Hz, CH3), 1.47 (d, 1H, J ¼ 7.5 Hz, eCH3),
flask). After 24 h, cells were treated with either 50
m
L of ethanol (1%
2.99e3.04 (m, 0.33H, H-3), 3.46e3.49 (m, 0.67H, H-3), 3.71 (s, 6H,
2ꢂ OeCH3), 3.77 (s, 1H, OeCH3), 3.80 (s, 5H, OeCH3), 5.02 (d,
J ¼ 1.8 Hz, 0.33H, H-4), 5.67 (d, 0.67H, J ¼ 5.5 Hz, H-4), 6.88e6.90
(m, 2H, Ar-H), 6.92e7.31 (m, 4H, Ar-H). 13C NMR (100 MHz, CDCl3):
v/v) as vehicle control or selected compound ranging from 10 nM to
10 M (final concentration). They were incubated for 72 h.
m
Following incubation, the cells were removed from the bottom of
the flask by scraping and the medium placed in a 20 mL sterilin.
Cells were then centrifuged for 10 min at 600 ꢂ g. The supernatant
was decanted and the pellet resuspended in 1 mL of ice-cold
phosphate buffered saline (PBS); cells were again centrifuged for
10 min at 600 ꢂ g. The supernatant was decanted and the pellet
d
11.31 (eCH3), 14.59 (eCH3), 50.66 (CH), 54.80 (OeCH3), 54.89
(OeCH3), 55.56 (OeCH3), 55.61 (OeCH3), 56.06 (CH), 60.43
(OeCH3), 66.31 (CH), 70.38 (CH), 95.50, 95.61, 113.73, 114.16, 125.21,
127.06, 127.77, 133.93, 135.07, 152.61, 158.19, 159.59 (Aromatic C),
201.72, 202.21 (C]S). HRMS: Calculated for C20H23NO4SNa:
396.1245; Found: 396.1235 (Mþ þ Na).
resuspended in 200 mL of ice-cold (PBS). Subsequently ice-cold 70%
ethanol (2 mL) was slowly added to the tube as it was gently vor-
texed. The cells were kept at ꢀ20 ꢃC for at least 1 h. After the
4.1.36. 4-(4-Methoxyphenyl)-3,3-dimethyl-1-(3,4,5-
trimethoxyphenyl)azetidin-2-thione (13c)
fixation 5 mL of FBS was added to the samples. The cells were har-
vested by centrifugation at 600 ꢂ g for 10 min. The ethanol was
carefully removed and the pellet resuspended in 400 L of PBS and
transferred to FACS microtubes. A 25 L aliquot of RNase A (1 mg/
mL) and 75 L of propidium iodide (PI) 1 mg/mL, a DNA binding
Preparation as described above from 4-(4-methoxyphenyl)-3,3-
m
dimethyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one
(12q)
m
(1.065 g, 2.868 mmol). Evaporation of the solvent yielded a brown
solid residue which was purified using flash column chromatog-
raphy over silica gel (eluent: dichloromethane) to afford the
product as a yellow solid. Yield 46.7%, Rf 0.85 (CH2Cl2: MeOH; 19:1),
m.p. 94 ꢃC. IR: NaCl film nmax: 1595.2 cmꢀ1 (C]S). 1H NMR
m
fluorescent dye, was added to each tube. The samples were wrap-
ped in aluminium foil and incubated for a minimum of 30 min at
37 ꢃC. The samples were read at 488 nM using a FACscalibur flow
cytometer from Becton Dickinson. The FACS data for 10,000 cells
was analysed using the Macintosh-based application Cellquest and
the data was stored as frequency histograms.
(400 MHz, CDCl3):
d 0.88 (s, 3H, eCH3), 1.48 (s, 3H, eCH3), 3.71 (s,
6H, 2ꢂ OeCH3), 3.79 (s, 3H, OeCH3), 3.795 (s, 3H, OeCH3), 5.29 (s,
1H, H-4), 6.89 (d, 2H, J ¼ 8.6 Hz, Ar-H), 7.1 (d, 1H, J ¼ 8.5 Hz, Ar-H),
7.23 (s, 2H, Ar-H). 13C NMR (100 MHz, CDCl3):
d
19.37 (eCH3), 24.34
4.2.3. Tubulin polymerisation assay
(eCH3), 54.79 (OeCH3), 51.14 (C-3), 56.60 (OeCH3), 60.42 (OeCH3),
73.85 (C-4), 95.91, 113.69, 125.85, 127.46, 133.99, 135.03, 152.62,
159.11 (Aromatic C), 206.80 (C]S). HRMS: Calculated for
C21H26NO4S: 388.1583; Found: 388.1586 (Mþ þ Na).
The effect of compounds on the assembly of purified bovine
brain tubulin was determined spectrophotometrically by moni-
toring the change in turbidity. This assay used a 96-well plate
d
format with 300
well. Lyophilised tubulin (1 mg, Cytoskeleton, Denver, CO) was
resuspended on ice in 300 L in ice-cold G-PEM buffer (80 mM
mg of >99% purified bovine brain tubulin in each
4.2. Biochemical evaluation of activity
m
PIPES pH 6.9, 0.5 mM MgCl2, 1 mM EGTA, 1 mM guanidine
triphosphate (GTP), 10.2% (v/v glycerol)) and was left on ice for
4.2.1. Antiproliferation studies
All assays were performed in triplicate for the determination of
mean values reported. Compounds were assayed as the free bases
isolated from reaction. The human breast tumour cell line MCF-7 was
cultured in Eagles minimum essential medium in a 95% O2/5% CO2
1 min to allow for complete resuspension. 10
each compound tested was pipetted into a half area 96-well plate
prewarmed to 37 ꢃC. A 100
L volume of tubulin was then pipetted
into the prewarmed plate. Samples were mixed well and tubulin
assembly was monitored at an absorbance of A340 nm at 30 s
intervals for 60 min at 37 ꢃC in a Spectromax 340PC spectropho-
tometer (Molecular Devices).
m
L of 10ꢂ strength of
m
atmosphere with 10% fetal bovine serum, 2 mM
L-glutamine and
100 g/mL penicillin/streptomycin. The medium was supplemented
m
with 1% non-essential amino acids. MDA-MB-231 cells were main-
tained in Dulbecco’s Modified Eagle’s medium (DMEM), supple-
mented with 10% (v/v) Fetal bovine serum, 2 mM
L-glutamine and
4.3. Computational procedure
100 mg/mL penicillin/streptomycin (complete medium). Cells were
trypsinised and seeded at a density of 2.5 ꢂ 104 cells/mL in a 96-well
For ligand preparation, all compounds were drawn using ACD/
Chemsketch v10 and SMILES strings generated. A single conformer
was generated using Corinav3.4 and ensuring Omegav2.2.1 was
subsequently employed to generate a maximum of 1000 confor-
mations of each compound. For the receptor preparation, the PDB
entries 1SA0 and 1SA1 were downloaded from the Protein Data Bank
(PDB). All waters were retained in both isoforms. Addition and
optimization of hydrogen positions for these waters was carried out
using MOE 2007.09 ensuring all other atom positions remained
fixed. Using the reported X-ray structure of tubulin co-crystallised
with a colchicine derivative, DAMA-colchicine (PDB entry e 1SA0)
[42], possible binding orientations ligands were probed with the
docking program FREDv2.2.3 (Openeye Scientific Software) [43].
Docking was carried out using FREDv2.2.3 in conjunction with
Chemgauss3. 3-D ligand conformations of compound 12d were
enumerated using CORINAv3.4 (Molecular Networks GMBH) [44] for
ligands followed by generation of multiple conformations using
OMEGAv2.2.1 (Openeye Scientific Software) [45]. Each conformation
was subsequently docked and scored with Chemgauss3 as outlined
previously [46]. The top binding poses were refined using the LigX
procedure (MOE e Chemical Computing Group) [47] together with
Postdock analysis (SVL script; MOE) of the docked ligand poses.
plateandincubatedat37 ꢃC,95% O2/5%CO2 atmospherefor24h. After
this time they were treated with 2
which had been pre-prepared as stock solutions in ethanol to furnish
the concentration range of study,1 nMe100 M, and re-incubated for
mL volumes of test compound
m
a further 72 h. Control wells contained the equivalent volume of the
vehicle ethanol (1% v/v). The culture medium was then removed and
the cells washed with 100
50 L MTT added, to reach a final concentration of 1 mg/mL MTT
added. Cells were incubated for 2 h in darkness at 37 ꢃC. At this point
solubilization was begun through the addition of 200 L DMSO and
mL phosphate buffered saline (PBS) and
m
m
the cells maintained at room temperature in darkness for 20 min to
ensure thorough colour diffusion before reading the absorbance. The
absorbance value of control cells (no added compound) was set to
100% cell viability and from this graphs of absorbance versus cell
density per well were prepared to assess cell viability and from these,
graphs of percentage cell viability versus concentration of subject
compound added were drawn.
4.2.2. Cell cycle analysis
Flow cytometry : The MDA-MB-231 cells were seeded at
a density of 18 ꢂ 104 cells/mL in 5 mL of medium (900,000 cells per

5766
M. Carr et al. / European Journal of Medicinal Chemistry 45 (2010) 5752e5766
[25] L. Wang, K.W. Woods, Q. Li, K.J. Barr, R.W. McCroskey, S.M. Hannick, L. Gherke,
Acknowledgements
R.B. Credo, Y.H. Hui, K. Marsh, R. Warner, J.Y. Lee, N. Zielinski-Mozng, D. Frost,
S.H. Rosenberg, H.L. Sham, Potent, orally active heterocycle-based com-
bretastatin A-4 analogues: synthesis, structure-activity relationship, phar-
macokinetics, and in vivo antitumor activity evaluation, J. Med. Chem. 45
(2002) 1697e1711.
This work was supported through funding from the Trinity
College IITAC research initiative (HEA PRTLI), Enterprise Ireland
(EI), Science Foundation Ireland (SFI), and the Health Research
Board (HRB), with additional support for computational facilities
from the Wellcome Trust. A postgraduate research award from
Trinity College is gratefully acknowledged.
[26] R. Shirai, H. Takayama, A. Nishikawa, Y. Koiso, Y. Hashimoto, Asymmetric
synthesis of antimitotic combretadioxolane with potent antitumor activity
against multi-drug resistant cells, Bioorg. Med. Chem. Lett. 8 (1998) 1997e2000.
[27] G.C. Tron, F. Pagliai, E. Del Grosso, A.A. Genazzani, G. Sorba, Synthesis and
cytotoxic evaluation of combretafurazans, J. Med. Chem. 48 (2005)
3260e3268.
References
[28] O.G. Ganina, E. Daras, V. Bourgarel-Rey, V. Peyrot, A.N. Andresyuk, J.P. Finet,
A.Y. Fedorov, I.P. Beletskaya, S. Combes, Synthesis and biological evaluation of
polymethoxylated 4-heteroarylcoumarins as tubulin assembly inhibitor, Bio-
org. Med. Chem. 16 (2008) 8806e8812.
[1] K.H. Downing, E. Nogales, Tubulin and microtubule structure, Curr. Opin. Cell
Biol. 10 (1998) 16e22.
[29] J.P. Liou, Y.L. Chang, F.M. Kuo, C.W. Chang, H.Y. Tseng, C.C. Wang, Y.N. Yang,
J.Y. Chang, S.J. Lee, H.P. Hsieh, Concise synthesis and structure-activity relation-
ships of combretastatin A-4 analogues, 1-aroylindoles and 3-aroylindoles, as
novel classes of potent antitubulin agents, J. Med. Chem. 47 (2004) 4247e4257.
[30] I. Banik, F.F. Becker, B.K. Banik, Stereoselective synthesis of beta-lactams with
polyaromatic imines: entry to new and novel anticancer agents, J. Med. Chem.
46 (2003) 12e15.
[31] L. Sun, N.I. Vasilevich, J.A. Fuselier, S.J. Hocart, D.H. Coy, Examination of the
1,4-disubstituted azetidinone ring system as a template for combretastatin A-
4 conformationally restricted analogue design, Bioorg. Med. Chem. Lett. 14
(2004) 2041e2046.
[32] M.J. Meegan, M. Carr, A.J. Knox, D.M. Zisterer, D.G. Lloyd, Beta-lactam type
molecular scaffolds for antiproliferative activity: synthesis and cytotoxic
effects in breast cancer cells, J. Enzyme Inhib. Med. Chem. 23 (2008) 668e685.
[33] C.C. Palomo, P. Fernando, Ana Arrieta, Jose M. Odriozola, Mikel Oiarbide, Jesus
M. Ontoria, The Reformatskii type reaction of Gilman and Speeter in the
[2] M.A. Jordan, Mechanism of action of antitumor drugs that interact with
microtubules and tubulin, Curr. Med. Chem. Anticancer Agents 2 (2002) 1e17.
[3] M.A. Jordan, L. Wilson, Microtubules as a target for anticancer drugs, Nat. Rev.
Cancer 4 (2004) 253e265.
[4] J.A. Hadfield, S. Ducki, N. Hirst, A.T. McGown, Tubulin and microtubules as
targets for anticancer drugs, Prog. Cell Cycle Res. 5 (2003) 309e325.
[5] R.L. Margolis, C.T. Rauch, L. Wilson, Mechanism of colchicine-dimer addition
to microtubule ends: implications for the microtubule polymerization
mechanism, Biochemistry 19 (1980) 5550e5557.
[6] B. Bhattacharyya, D. Panda, S. Gupta, M. Banerjee, Anti-mitotic activity of
colchicine and the structural basis for its interaction with tubulin, Med. Res.
Rev. 28 (2008) 155e183.
[7] G.R. Pettit, S.B. Singh, E. Hamel, C.M. Lin, D.S. Alberts, D. Garcia-Kendall,
Isolation and structure of the strong cell growth and tubulin inhibitor com-
bretastatin A-4, Experientia 45 (1989) 209e211.
[8] J.W. Lippert 3rd, Vascular disrupting agents, Bioorg. Med. Chem. 15 (2007)
605e615.
[9] P. Hinnen, F.A. Eskens, Vascular disrupting agents in clinical development, Br.
J. Cancer 96 (2007) 1159e1165.
[10] K.G. Pinney, C. Jelinek, K. Edvardsen, D.J. Chaplin, G.R. Pettit, The discovery and
development of combretastatins. in: G.M. Cragg, D.G.I. Kingston, D.J. Newman
(Eds.), Anticancer Agents from Natural Products. Taylor & Francis, Boca Raton,
FL, 2005, pp. 23e46.
[11] A. Cirla, J. Mann, Combretastatins: from natural products to drug discovery,
Nat. Prod. Rep. 20 (2003) 558e564.
preparation of valuable
b-lactams in carbapenem synthesis: scope and
synthetic utility, J. Org. Chem. 54 (1989) 5736e5745.
[34] A. Gaudemer, Determination of configurations by NMR spectroscopy. in:
H.B. Kagan (Ed.), Stereochemistry: Fundamentals and Methods, Determina-
tion of Configurations by Spectroscopic Methods, vol. 1. Georg Thieme
Publishers, Stuttgart, 1977, p. 87.
[35] S.B. Rosenblum, T. Huynh, A. Afonso, H.R. Davis Jr., N. Yumibe, J.W. Clader,
D.A. Burnett, Discovery of 1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-
hydroxypropyl]-(4S)-(4 -hydroxyphenyl)-2-azetidinone (SCH 58235):
a designed, potent, orally active inhibitor of cholesterol absorption, J. Med.
Chem. 41 (1998) 973e980.
[36] J.W. Clader, The discovery of ezetimibe: a view from outside the receptor, J.
Med. Chem. 47 (2004) 1e9.
[37] B.L. Flynn, G.P. Flynn, E. Hamel, M.K. Jung, The synthesis and tubulin binding
activity of thiophene-based analogues of combretastatin A-4, Bioorg. Med.
Chem. Lett. 11 (2001) 2341e2343.
[12] G.R. Pettit, C. Temple Jr., V.L. Narayanan, R. Varma, M.J. Simpson, M.R. Boyd,
G.A. Rener, N. Bansal, Antineoplastic agents 322. Synthesis of combretastatin
A-4 prodrugs, Anticancer Drug Des. 10 (1995) 299e309.
[13] http://www.clinicaltrials.gov/ct/gui/show/NCT00060242.
[14] M.M. Cooney, J. Ortiz, R.M. Bukowski, S.C. Remick, Novel vascular targeting/
disrupting agents: combretastatin A4 phosphate and related compounds,
Curr. Oncol. Rep. 7 (2005) 90e95.
[38] G. De Martino, G. La Regina, A. Coluccia, M.C. Edler, M.C. Barbera, A. Brancale,
E. Wilcox, E. Hamel, M. Artico, R. Silvestri, Arylthioindoles, potent inhibitors of
tubulin polymerization, J. Med. Chem. 47 (2004) 6120e6123.
[39] J.M. Bidlack, R.A. Lockshin, Evolution of LDH isozymes during programmed
cell death, Comp. Biochem. Physiol., B 55 (1976) 161e166.
[40] W.t.S. C-QSAR; Biobyte Corp., Suite 204, Claremont, CA.
[41] M. Cushman, H.M. He, C.M. Lin, E. Hamel, Synthesis and evaluation of a series
of benzylaniline hydrochlorides as potential cytotoxic and antimitotic agents
acting by inhibition of tubulin polymerization, J. Med. Chem. 36 (1993)
2817e2821.
[42] R.B. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel, M. Knossow,
Insight into tubulin regulation from a complex with colchicine and a stath-
min-like domain, Nature 428 (2004) 198e202.
[43] T. Schulz-Gasch, M. Stahl, Binding site characteristics in structure-based
virtual screening: evaluation of current docking tools, J. Mol. Model. 9 (2003)
47e57.
[15] S.L. Young, D.J. Chaplin, Combretastatin A4 phosphate: background and
current clinical status, Expert Opin. Investig. Drugs 13 (2004) 1171e1182.
[16] G.R. Pettit, M.R. Rhodes, D.L. Herald, E. Hamel, J.M. Schmidt, R.K. Pettit, Anti-
neoplastic agents. 445. Synthesis and evaluation of structural modifications of
(Z)- and (E)-combretastatin A-41, J. Med. Chem. 48 (2005) 4087e4099.
[17] K. Ohsumi, T. Hatanaka, K. Fujita, R. Nakagawa, Y. Fukuda, Y. Nihei, Y. Suga,
Y. Morinaga, Y. Akiyama, T. Tsuji, Syntheses and antitumor activity of cis-
restricted combretastatins: 5-membered heterocyclic analogues, Bioorg. Med.
Chem. Lett. 8 (1998) 3153e3158.
[18] M. Cushman, D. Nagarathnam, D. Gopal, A.K. Chakraborti, C.M. Lin, E. Hamel,
Synthesis and evaluation of stilbene and dihydrostilbene derivatives as
potential anticancer agents that inhibit tubulin polymerization, J. Med. Chem.
34 (1991) 2579e2588.
[19] G.C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca, A.A. Genazzani, Medicinal
chemistry of combretastatin A4: present and future directions, J. Med. Chem.
49 (2006) 3033e3044.
[20] N.H. Nam, Combretastatin A-4 analogues as antimitotic antitumor agents,
Curr. Med. Chem. 10 (2003) 1697e1722.
[44] M. Feher, C.I. Williams, Effect of input differences on the results of docking
calculations, J. Chem. Inf. Model. (2009).
[45] T. Takagi, M. Amano, M. Tomimoto, Novel method for the evaluation of 3D
conformation generators, J. Chem. Inf. Model. 49 (2009) 1377e1388.
[46] I. Barrett, M. Carr, N. O’Boyle, L.M. Greene, A.J. Knox, D.G. Lloyd, D.M. Zisterer,
M.J. Meegan, Lead identification of conformationally restricted benzoxepin
type combretastatin analogs: synthesis, antiproliferative activity, and tubulin
effects, J. Enzyme Inhib. Med. Chem. 25 (2010) 180e194.
[47] MOEv2008.10, developed and distributed by Chemical Computing Group.
http://www.chemcomp.com.
[48] F. Lara-Ochoa, G. Espinosa-Perez, A new synthesis of combretastatins A-4 and
AVE-8062A, Tetrahedron Lett 48 (2007) 7007e7010.
[21] H.P. Hsieh, J.P. Liou, N. Mahindroo, Pharmaceutical design of antimitotic
agents based on combretastatins, Curr. Pharm. Des. 11 (2005) 1655e1677.
[22] M. Cushman, D. Nagarathnam, D. Gopal, H.M. He, C.M. Lin, E. Hamel, Synthesis
and evaluation of analogues of (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trime-
thoxyphenyl)ethene as potential cytotoxic and antimitotic agents, J. Med.
Chem. 35 (1992) 2293e2306.
[23] K. Ohsumi, R. Nakagawa, Y. Fukuda, T. Hatanaka, Y. Morinaga, Y. Nihei,
K. Ohishi, Y. Suga, Y. Akiyama, T. Tsuji, Novel combretastatin analogues
effective against murine solid tumors: design and structure-activity rela-
tionships, J. Med. Chem. 41 (1998) 3022e3032.
[49] R. Shirai, H. Nobuatsu, T. Okabe, K. Koiso, S. Iwasaki, Preparation of azacom-
bretastatin compounds as antitumor agents; Taisho Pharma Co Ltd; Jpn. Kokai
Tokkyo Koho (1996), JP 08067656 A 19960312.
[24] T. Brown, H. Holt Jr., M. Lee, Series: topics in heterocyclic chemistry. in:
M.E. Lee (Ed.), Heterocyclic Antitumor Antibiotics, vol. 2. Springer, Berlin/
Heidelberg, 2006, pp. 1e51.