Synthesis and evaluation of azetidinone analogues of combretastatin A-4 as tubulin targeting agents

Article abstract of DOI:10.1021/jm101115u

The synthesis and antiproliferative activity of a new 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 natural combretastatin stilbene products. These novel compounds are also substituted at position 3 of the β-lactam ring with an aryl ring. A number of analogues showed potent nanomolar activity in human MCF-7 and MDA-MB-231 breast cancer cell lines, displayed in vitro inhibition of tubulin polymerization, and did not cause significant cytotoxicity in normal murine breast epithelial cells. 4-(4-Methoxyaryl)-substituted compound 32, 4-(3-hydroxy-4-methoxyaryl)-substituted compounds 35 and 41, and the 3-(4-aminoaryl)-substituted compounds 46 and 47 displayed the most potent antiproliferative activity of the series. β-Lactam 41 in particular showed subnanomolar activity in MCF-7 breast cancer cells (IC₅₀ = 0.8 nM) together with significant in vitro inhibition of tubulin polymerization and has been selected for further biochemical assessment. These novel β-lactam compounds are identified as potentially useful scaffolds for the further development of antitumor agents that target tubulin.

Full text of DOI:10.1021/jm101115u

J. Med. Chem. 2010, 53, 8569–8584 8569
DOI: 10.1021/jm101115u
Synthesis and Evaluation of Azetidinone Analogues of Combretastatin A-4 as Tubulin Targeting Agents
Niamh M. O’Boyle,† Miriam Carr,† Lisa M. Greene,‡ Orla Bergin,§ Seema M. Nathwani,‡ Thomas McCabe,
David G. Lloyd,^ Daniela M Zisterer,‡ and Mary J. Meegan*,†
†School of Pharmacy and Pharmaceutical Sciences, Centre for Synthesis and Chemical Biology, Trinity College Dublin, Dublin 2, Ireland,
‡School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland, §UCD Conway Institute and School of Biomolecular and
Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland, School of Chemistry, Trinity College Dublin, Dublin 2, Ireland, and
^Molecular Design Group, School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland
Received August 27, 2010
The synthesis and antiproliferative activity of a new 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 natural combretastatin stilbene products. These novel compounds are also substituted at
position 3 of the β-lactam ring with an aryl ring. A number of analogues showed potent nanomolar
activity in human MCF-7 and MDA-MB-231 breast cancer cell lines, displayed in vitro inhibition of
tubulin polymerization, and did not cause significant cytotoxicity in normal murine breast epithelial cells.
4-(4-Methoxyaryl)-substituted compound 32, 4-(3-hydroxy-4-methoxyaryl)-substituted compounds 35
and 41, and the 3-(4-aminoaryl)-substituted compounds 46 and 47 displayed the most potent anti-
proliferative activity of the series. β-Lactam 41 in particular showed subnanomolar activity in MCF-7
breast cancer cells (IC50=0.8 nM) together with significant in vitro inhibition of tubulin polymerization and
has been selected for further biochemical assessment. These novel β-lactam compounds are identified as
potentially useful scaffolds for the further development of antitumor agents that target tubulin.
Introduction
site of tubulin.10 A water-soluble prodrug, combretastatin
A-4-phosphate (2b, Figure 1), is now in clinical trials for
thyroid cancer11-13 and in patients with advancedcancer.14 2b
induces vascular shutdown within tumors at doses less than
one-tenth of the maximum tolerated dose and without detect-
able morbidity, assuming a MTDa of 1000 mg/kg.7 Hydrolysis
in vivo by endogenous nonspecific phosphatases under physio-
logical conditions affords 2a.15,16 The amino derivative of 2a
(compound 3a) is also in clinical trials as a water-soluble
amino acid prodrug (3b, Figure 1).17 In contrast to colchicine,
the antivascular effects of 2a in vivo are apparent well below
the maximum tolerated dose, offering a wide therapeutic
window. 2a as well as being a potent inhibitor of colchicine
binding is also shown to inhibit the growth and development of
blood vessels, angiogenesis.5,18-21 The cis configuration only of
2a is biologically active, with the trans form showing little or no
activity.22 The active cis double bond in 2a is readily converted to
the more stable trans isomer during storage or metabolism,
resulting in a dramatic decrease in antitumor activity.23,24
Various structural modifications to 2a have been reported
including variation of the A- and B-ring substituents.25-27 Many
modifications of the B-ring result in decreased bioactivity;
however, substitution of the 30-OH with an amino group
Microtubules are cytoskeletal structures that are formed
by self-assembly of R and β tubulin heterodimers and are
involved in many cellular functions.1 Their most important
role is in the formation of the mitotic spindle, and they are
essential to the mitotic division of cells. Tubulin is an R,β hetero-
dimeric protein that is the main constituent of microtubules.
Tubulin is the target of numerous small molecule antiprolif-
erative ligands that act by interfering with microtubule
dynamics.2 These ligands can be broadly divided into two
categories: those that inhibit the formation of the mitotic
spindle such as colchicine (1, Figure 1)3,4 and vinblastine5 and
those that inhibit the disassembly of the mitotic spindle once
it has formed, such as paclitaxel.6 The three characterized
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. Many tubulin binding com-
pounds, such as paclitaxel and vinblastine, are in clinical
use for various types of cancer.2 Antimitotic agents are one
of the major classes of cytotoxic drug for cancer treatment,
and microtubules are a significant target for many natural
product anticancer agents such as combretastatin A-4 (2a,
Figure 1)7 and podophyllotoxin (4, Figure 1).2,8
The combretastatins are a group of diarylstilbenes isolated
from the stem wood of the South African tree Combretum
caffrum.9 2a was found to have potent anticancer activity
against a number of human cancer cell lines including multidrug
resistant cancer cell lines and binds to the colchicine-binding
a Abbreviations: CA-4, combretastatin A-4; EI, electron impact; ER,
estrogen receptor; GTP, guanidine triphosphate; HRMS, high resolution
molecular ion determination; IC, inhibitory concentration; MTD, max-
imum tolerated dose; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide; NCI, National Cancer Institute; NMR, nuclear magnetic
resonance; PBS, phosphate buffered saline; SAR, structure-activity rela-
tionship; TBAF, tetrabutylammonium fluoride; TBDMS, tert-butyldi-
methylchlorosilane; THF, tetrahydrofuran; TMCS, trimethylchlorosilane;
TMS, tetramethylsilane.
*To whom correspondence should be addressed. Phone: þ353-1-
8962798. Fax: þ353-1-8962793. E-mail: mmeegan@tcd.ie.
r
2010 American Chemical Society
Published on Web 11/16/2010
pubs.acs.org/jmc

8570 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
O’Boyle et al.
Figure 1. Colchicine (1), combretastatin A-4 (2a), related compounds 2b, 3a, 3b, and podophyllotoxin (4).
results in potent bioactivity and good water solubility.28 The
3,4,5-trimethoxy substituted pattern in ring A, resembling the
trimethoxyaryl ring of colchicine, is optimal for bioactivity of
2a.24 Many conformationally restricted analogues of 2a are
known. Most of these compounds replace the cis-double bond
in 2a with a heterocyclic rigid ring scaffold structure that pre-
vents isomerization of the cis-double bond. These analogues
include the heteroarylcoumarin,29 aroylindole,27 imidazole,30
1,3-dioxolane,31 pyrazole,23 furazan (1,2,5-oxadiazole),25 and
benzoxepin32 ring systems. Such nonisomerizable compounds
inhibit cell growth of several human cancer cell lines, and
many have been shown to be significant tubulin binding and
depolymerizing effects. We now describe the synthesis and
tubulin targeting activity of nonisomerizable β-lactam-con-
taining analogues of 2a. The anticancer activity of some
β-lactam-containing compounds has been reported,33 including
the nonisomerizable combretastatin analogues containing OH
and OMe substituents at C-3.34 We have recently reported the
potent antiproliferative activity of β-lactam-containing com-
pounds that are unsubstituted at C-3 or contain methyl
substituent(s) at C-3.35 We have also reported antiprolifera-
tive and antiestrogenic activity of compounds containing the
β-lactam scaffold in MCF-7 cells.36 In continuation of our
earlier work, a further panel of compounds containing the
β-lactam ring were examined as potential tubulin targeting
agents. These novel compounds contain an aryl-type substi-
tuent at C-3, while the rigid β-lactam ring scaffold structure
allows a similar spatial arrangement between the two phenyl
rings as observed in the cis conformation of 2a.
Scheme 1. Synthesis of Imines 5a-fa
a Reagents and conditions: (a) EtOH, reflux, 3 h.
afford (3-benzyloxyphenyl)acetic acid.40 The acid chloride 6d
is prepared from (4-aminophenyl)acetic acid on first reaction
with benzyl chloroformate to afford the (4-benzyloxycarbo-
nylaminophenyl)acetic acid,41 which was then converted to
the unstable acid chloride 6d on reaction with thionyl chloride
(Scheme 2, step a). The chlorination reactions were monitored
by IR spectroscopy until acid chloride carbonyl absorption
was observed (ν=1790-1815 cm-1).
The racemic β-lactam products 7-14 were obtained in low
yields on treatment of the imines 5a-f at reflux in dichloro-
methane with the appropriate acid chloride in the presence
of triethylamine (Scheme 2, route I; one enantiomer only
shown). β-Lactam compounds 15-17 were prepared using a
modified procedure at room temperature, as they were not
successfully obtained by the usual conditions of the Staudinger
reaction (Scheme 2, route II). Failure to form β-lactams with
4-nitrophenylacetyl chloride under standard Staudinger con-
ditions has previously been reported.42 The stereochemistry of
the β-lactam products obtained in Staudinger reactions depends
on numerous factors, including the reaction conditions, the
order of addition of the reagents, and the substituents present
on both the imine and the acid chloride.33,43 In the present
reactions, the trans products were isolated exclusively in all
cases, as evident from the 1H NMR spectrum of compound 7
where the H-3 and H-4 were identified at δ 4.23 and δ 4.79,
respectively, as a pair of coupled doublets, J3,4 =2.0 Hz.
The β-lactams 18-31 were obtained directly from the ap-
propriate phenylacetic acid using an acid-activating agent in a
one-stepreaction(Scheme2, routeIII), whichwasthereaction
of choice for the majority of the products. Many acid-activating
agents have been reported for this reaction, e.g., Mukaiyama’s
reagent (2-chloro-N-methylpyridinium iodide), ethyl chloro-
formate, trifluoroacetic anhydride, p-toluenesulfonyl chloride,
and various phosphorus derived reagents including triphos-
gene, which was successfully used in the present study.44
Chemistry
The synthesis of the target β-lactams is illustrated in
Schemes 2-4. The compounds that were initially chosen for
synthesis contained the 3,4,5-trimethoxyphenyl (ring A) as the
β-lactam N-1 and 4-methoxyphenyl as the β-lactam C-4 substi-
tuents, which are present in 2a.β-Lactam synthesis was achieved
with a Staudinger cycloaddition reaction between a ketene
(generated from an acid chloride) and an imine under basic
conditions.33,37 The required imines 5a-f were obtained by
condensation reaction of the appropriately substituted benz-
aldehydes and anilines (Scheme 1). In the case of 5e, the
phenolic group present on the starting 3-hydroxy-4-methoxy-
benzaldehyde was first protected as the tert-butyldimethyl-
chlorosilane (TBDMS) ether38 which can be removed later
under mild conditions with tetrabutylammonium flouride
which is suitable for the stability of the β-lactam functional
group.39 Acid chlorides 6a-d(Scheme 2) were prepared by reac-
tion of the appropriately substituted acetic acids with thionyl
chloride. For the preparation of the acid chloride 6c, (3-hydroxyl-
phenyl)acetic acid was first treated with benzyl bromide to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8571
Scheme 2. Synthesis of Azetidinones 7-31: Routes I, II, and IIIa
a Reagents and conditions: (a) SOCl2, CHCl3, reflux, 3 h; (b) (route I) triethylamine, anhydrous CH2Cl2, reflux, 3 h; (c) (route II) triethylamine,
anhydrous CH2Cl2, 20 ꢀC, 18 h; (d) (route III) triphosgene, triethylamine, anhydrous CH2Cl2, reflux, 5 h; 20 ꢀC, stirred 18 h.
Scheme 3. Synthesis of Azetidinones 32-34: Route IVa
Scheme 4. Synthesis of Azetidinones 35-38: Route Va
a Reagents and conditions: (a) zinc, trimethylchlorosilane, benzene,
microwave.
a Reagents and conditions: TBAF, THF, 0 ꢀC, 15 min.
Trans stereochemistry was also observed for these products,
e.g., for compound 18 where the H-3 and H-4 were identified
atδ 4.20and δ 4.89, respectively, asa pair of coupled doublets,
J3,4=2.5 Hz. With selected phenylacetic acid derivatives, this
reaction was unsuccessful and the acids were converted to acid
chlorides 6a-d.β-Lactams were then obtained by the Staudinger
route (route I) as described above.
In some cases, a Reformatsky reaction was used for the
synthesis of the required β-lactam compounds (Scheme 3).45,46
We have recently reported the application of the Reformatsky
reaction to obtain 1,3-diarylazetidinones, which are unsubstituted
at C-3 or contain methyl substituent(s) at C-3.35 We now
report the preparation of 1,3,4-triarylazetidinones using a
Reformatsky reaction that has been adapted for microwave
conditions. Previous investigations found TMCS to be super-
ior for zinc activation47 in Reformatsky reactions than either
iodine or zinc washed with 10% nitric acid. We examined the
use of zinc powder that was preactivated with trimethylchloro-
silane in microwave conditions in the Reformatsky reactions,
resulting in slightly increased yield of the desired β-lactam
products 32-34 and a significant reduction in the reaction
time from 8 h to 30 min (Scheme 3). In the present reactions,

8572 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
O’Boyle et al.
Figure 2. ORTEP representation of the X-ray crystal structure of azetidinone 32 with 50% thermal ellipsoids.
Scheme 5. Synthesis of Azetidinones 39-42: Route VIa
Scheme 6. Synthesis of Amino Substituted Azetidinones 43-45:
Route VIIa
a Reagents and conditions: (a) H2, Pd/C, EtOH/EtOAc (1:1).
the trans products were isolated exclusively in all cases, as
evident from the 1H NMR spectrum of compound 32 where
the H-3 and H-4 were identified at δ 4.29 and δ 4.87 ppm,
respectively, as a pair of coupled doublets, J3,4 =2.5 Hz. The
trans configuration can be seen in the X-ray crystal structure
of β-lactam 32 in Figure 2. The enantiomeric composition of
compound 32 was clearly demonstrated on analysis by chiral
HPLC (tR of 11.93 and 12.53 min).
The phenolic products 35, 36, 37, and 38 were obtained on
treatment of the silyl ethers 13, 29, 30, and 34, respectively,
with tetrabutylammonium fluoride at 0 ꢀC (Scheme 4). Phe-
nolic products 39, 40, 41, and 42 were obtained by careful
hydrogenolysis of the corresponding benzyl ethers 12, 28, 37,
and 31 respectively (Scheme 5). Reduction of the nitro group
in compounds 15, 14, and 17 to the corresponding amines 43,
44, and 45, respectively, was achieved by treatment with zinc
dust and glacial acetic acid (Scheme 6). Treatment of the
carbamate protected compounds 38 and 16 with hydrogen
and palladium afforded the amines 46 and 47, respectively
(Scheme 7).
a Reagents and conditions: (a) Zn, CH3CO2H, 7 days.
To further investigate the contribution of the β-lactam
carbonyl group to the activity of this compound class, a novel
thione analogue 48 was prepared in good yield by reaction of
the compound 32 with Lawesson’s reagent (2,4-bis-(4-methoxy-
phenyl)-1,2,3,4-dithiaphosphetane 2,4-disulfide) (Scheme 8).
The characteristic CdS absorption band was observed at
ν=1592.5 cm-1 for this product. A broad variety of β-lactams
have been subjected to reductive ring-opening with metal
hydrides to yield the corresponding γ-aminoalcohols.48 To
investigate the contribution of the intact β-lactam ring structure
to the activity of this series of compounds, 35 was subjec-
ted to treatment with LiAlH4 to afford the amino alcohol
product 49 which was isolated by an acid-base extraction to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8573
Scheme 7. Synthesis of Azetidinones 46 and 47: Route VIIIa
two isomers. There are also two signals for each of the R and β
protons (formerly at positions 3 and 4 of the azetidinone ring)
between δ 3 ppm and δ 5 ppm. In the 13C NMR spectrum
of 50, there are two carbonyl signals at δ 171.55 ppm and
δ 172.70 ppm.
The structures of the various β-lactams synthesized are
illustrated in Tables 1, 2, and 3, together with their routes of
synthesis. Table 1 lists the initial β-lactams with the methoxy
functionalities at different positions of the β-lactam scaffold.
The comprehensive series of β-lactams prepared with struc-
tural modification of the aryl ring at the C-3 position of the
β-lactam heterocycle are listed in Table 2. Finally, Table 3
shows further phenolic and amino modifications to the initial
series of compounds with the aim of improving activity. Com-
pounds 32 and 35 were selected for chemical stability analysis
and further development based on the analysis of their drug-
like (Lipinski) properties together with predictions of perme-
ability, metabolic stability, Pgp substrate status, blood-brain
barrier partition, plasma protein binding, and human intestinal
absorption properties. The stability of the target compounds
32 and 35 was evaluated in phosphate buffer at pH 4, 7.4,
and 9, and the half-life was determined to be greater than 24 h
for each compound at these pH values.
a Reagents and conditions: (a) H2, Pd/C, EtOH/EtOAc (1:1).
Scheme 8. Synthesis of Azetidinthione 48a
Antiproliferative Activity
These analogues were first evaluated for their antiprolifera-
tive activity in the MCF-7 (ER-positive) human breast cancer
cell line, and the most potent compounds were then screened
against the MDA-MB-231 cell line (ER-negative). The activ-
ities of the azetidin-2-ones are presented in Tables 4 and 5. In
addition, compounds 32 and 35 were evaluated in the NCI-60
cell line screen (Table 9, Supporting Information). Initially,
the azetidin-2-ones prepared containing a 4-methoxy substi-
tuent on the C-4 phenyl ring were examined (compounds
7-12, 14, 15, 17-27, 32). From these early lead compounds, a
more extensive series of active derivatives were synthesized
containing the 3-hydroxy-4-methoxy and 3-amino-4-methoxy
substitution pattern on the C-4 phenyl ring, (compounds 35,
36, 39-47). This latter series of compounds was designed to
contain structural features similar to those of 1, 2a, and the
CA-4 amino analogue (3a).
a Reagents and conditions: (a) Lawesson’s reagent, toluene, reflux, 3 h.
Scheme 9. Azetidinone Reduction and Methanolysisa
The positions of the 3,4,5-trimethoxyphenyl and 4-methoxy-
phenyl ring substitution were initially examined at different
locations on the azetidinone ring (compounds 7, 18, 32, and
33) (Table 4). 2a was used as the positive control. The optimal
positioning was found to be in compound 32 (IC50(MCF-7)=
34 nM), where the 3,4,5-trimethoxyphenyl ring is located at
the N-1 position and the 4-methoxyphenyl ring is at the C-4
position, as previously observed by Sun et al. for other β-lactam
compounds.34 Any other arrangement of these two rings was
significantly less active than 32 by a factor of over 1000. The
effect of the introduction of substituents onto the C3-aryl ring
of compound 32 was next investigated. In the 3-aryl substi-
tuted series (compounds 7-31), larger substituents such as
3,4,5-trimethoxyphenyl (20), 4-trifluoromethylphenyl (27),
and 4-benzyloxyphenyl (12) were not tolerated and led to a
significant decrease in activity against MCF-7 cells. The most
active analogues in this series were found to be those with
small, polar substituents including examples 24 (4-fluoro-
phenyl), 39 (4-hydroxyphenyl), 40 (3-hydroxyphenyl), and
43 (4-aminophenyl) with IC50 values in MCF-7 cells of 42, 59,
68, and 50 nM, respectively.
a Reagents and conditions: (a) LiAlH4, dry THF, reflux 3 h; (b) NaOCH3,
MeOH, 20 ꢀC, 8 days.
give the desired γ-amino alcohol (Scheme 9). Cleavage of the β-
lactam N1-C2 bond usually takes place with nucleophilic
reagents such as alkoxide ion.49-52 The ease of hydrolysis is
apparently a function of increasing size and number of sub-
stituents at the 3-position; with increased size and number
hydrolysis becomes increasingly difficult.53 A methoxide ring-
opening reaction was carried out on 35 with 4 equivalents
of sodium methoxide at room temperature over 8 days until
1
reaction was complete on TLC. H NMR analysis of the
product 50 indicated formation of a diastereomeric product
mixture (Scheme 9).51,52 For methyl ester 50, the presence of
two amino signals at δ 5.75 ppm and δ 6.10 ppm is indicative of
The activity of the series was further optimized by modi-
fication of the C-4 aryl substituent of the azetidinone ring to

8574 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
O’Boyle et al.
Table 1. Azetidinones with Methoxy Substitution at N-1, C-3, and C-4 Aryl Rings
compd
R1
R2
R3
R4
R5
R6
R7
R8
R9
route for synthesis
7
H
OCH3
OCH3
H
H
H
H
H
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
I
18
32
33
OCH3
H
OCH3
H
H
H
H
III
IV
IV
OCH3
H
OCH3
OCH3
OCH3
H
H
OCH3
H
OCH3
H
H
H
Table 2. Azetidinones with C-3 Aryl Substitution
Table 3. Azetidinones with Hydroxyl and Amine-Type Substituents on
C-3 and C-4 Aryl Rings and Related Compounds
compd
R1
R2
OCH3
R3
R4
route for synthesis
route for
synthesis
8
H
H
H
I
compd
R1
R2
9
H
H
OCH3
OCH3
OCH3
Cl
H
I
13
14
16
17
29
30
31
34
35
36
37
38
41
42
44
45
46
47
OSi(CH3)2C(CH3)3
NO2
F
I
I
10
11
12
15
19
20
21
22
23
24
25
26
27
28
32
39
40
43
H
H
I
H
NHC(O)OCH2C6H5
H
H
H
I
NO2
NO2
II
H
H
OCH2C6H5
NO2
H
H
I
F
OCH2C6H5
II
III
H
H
H
II
OSi(CH3)2C(CH3)3
OSi(CH3)2C(CH3)3
OCH3
H
H
OCH3
H
III
III
III
III
III
III
III
III
III
III
IV
II, VI
III, VI
II, VII
NHC(O)OCH2C6H5
OCH2C6H5
III
III
OCH3
SCH3
CH3
Br
OCH3
H
NO2
OSi(CH3)2C(CH3)3
H
H
H
IV
IV, V
I, V
H
H
H
OH
OH
H
H
H
H
F
OCH2C6H5
NHC(O)OCH2C6H5
H
H
Cl
F
Cl
H
OH
OH
III, V
III, V
III, VI, VI
III, VI
I, VII
II, VII
III, V, VIII
II, VIII
H
H
H
F
F
H
OH
OH
OH
H
H
H
OCH2C6H5
CF3
H
H
NH2
NH2
NH2
OH
H
H
H
H
H
H
F
H
H
OH
H
H
NH2
NH2
H
OH
H
H
NH2
H
NH2
H
in MCF-7 cells). The activity of nitro-substituted compound
17 is included for comparison (IC50 of 1.4 μM), indicating the
critical importance of a polar H-bonding substituent on the
C-3 phenyl ring. The phenolic hydroxyl group is the optimal
substituent at the 3-position of the 4-phenyl ring. The most
potent compound in the series, 41, displayed subnanomolar
antiproliferative activity in the MCF-7 cell line with an IC50 of
0.8 nM. Figure 3 shows a dose-response result for the anti-
proliferativeeffect of β-lactam compounds 32and 41inMCF-
7 cells. Compound 46, containing a 3-(4-aminophenyl) sub-
stituent, also showed greater activity than the lead compound
32, with an IC50 of 4 nM in MCF-7 cell line.
include either a hydroxy or an amino group as in compounds
35-38 and 41-47 (Schemes 4-7, Table 4). Analogues of the
three most active compounds from Table 2 were synthesized
with both 4-(3-hydroxy-4-methoxyphenyl) and 4-(3-amino-
4-methoxyphenyl) substitution (Table 3). With one exception,
the 3-hydroxy-4-methoxy substituted analogues were over 10-
fold moreactivethan theircorresponding 3-amino-4-methoxy
derivatives (compare 41 to 42, for example, with IC50 values of
0.8 and 39.5 nM, respectively, in MCF-7 cells). The exception
was the fluorinated analogues, in which the introduction of an
amino substituent to the C-4 phenyl ring of 24 resulted in a
slight increase in potency (compound 45) when compared to
the hydroxyl analogue 36, and both were less active than
compound 24 (IC50 values of 55, 66, and 42 nM, respectively,
The replacement of the β-lactam carbonyl group with the
thione in compound 48 resulted in a reduction in the observed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8575
Table 4. Antiproliferative Effects of Azetidinones in MCF-7 Human
Breast Cancer Cells
antiproliferative activitya
MCF-7 cells
antiproliferative activitya
MCF-7 cells
compd
IC50 (μM)
compd
IC50 (μM)
7
37.75 ( 23.35
27
32
33
35
36
39
40
41
42
43
44
45
46
47
48
49
50
2ab
1.6577 ( 0.4283
0.0344 ( 0.0198
43.12 ( 16.47
8
0.3263 ( 0.1565
0.1820 ( 0.0262
0.2228 ( 0.1585
0.3052 ( 0.1380
7.9857 ( 1.8590
0.4663 ( 0.0361
0.4856 ( 0.2815
1.3642 ( 0.74
9
10
11
12
14
15
17
18
19
20
21
22
23
24
25
26
0.0096 ( 0.0026
0.0657 ( 0.0056
0.0591 ( 0.0198
0.0689 ( 0.0592
0.0008 ( 0.0004
0.0395 ( 0.0216
0.0508 ( 0.0194
0.0653 ( 0.0111
0.0553 ( 0.0087
0.0045 ( 0.0032
0.0309 ( 0.0357
0.72 ( 0.47
Figure 3. Antiproliferative effect of β-lactam compounds 32 and 41
alongside 2a in MCF-7 human breast cancer cells. MCF-7 cells were
seeded at a density of 2.5 ꢀ 104 cells per well in 96-well plates. The
plates were left for 24 h to allow the cells to adhere to the surface of
the wells. A range of concentrations (0.01 nM to 50 μM) of the
compound were added in triplicate and the cells left for another 72 h.
Control wells contained the equivalent volume of the vehicle
ethanol (1% v/v). An MTT assay was performed to determine the
level of antiproliferation. The values represent the mean ( SEM
(error values) for three experiments performed in triplicate.
43.13 ( 20.44
0.6995 ( 0.1874
7.1193 ( 3.9135
0.4480 ( 0.2304
0.3152 ( 0.0714
0.4196 ( 0.0817
0.0423 ( 0.0420
3.3607 ( 1.0377
0.3139 ( 0.1792
24.5600 ( 2.6196
>100
0.0052 ( 0.002
the results are displayed in Table 5. Compound 35 was found
to be the most effective of the series with IC50 value of 28 nM;
compounds 32 and 45 are also seen to be effective, with IC50
values of 78 and 80 nM, respectively. Compound 41 was also
evaluated in the leukemia cell lines HL-60 and K562 and
was found to be extremely potent with IC50 values of 0.34 and
0.89 nM, respectively, which compared favorably with the con-
trol 2a [IC50 values of 1.99 nM (HL-60) and 3.68 nM (K562)].
On the basis of their potency, compounds 9 and 35 were
evaluated in the National Cancer Institute (NCI)/Division of
Cancer Treatment and Diagnosis (DCTD)/Developmental
Therapeutics Program (DTP),54 in which the activity of each
compound was determined using approximately 60 different
cancer cell lines of diverse tumor origins. These studies were
performed at the NCIas part oftheir drug-screening program.
Compounds 9 and 35 were tested for inhibition of growth
(GI50) and for cytotoxicity (LC50) in the NCI panel of 56 cell
lines and showed broad-spectrum antiproliferative activity
against tumor cell lines derived from leukemia, breast cancer,
non-small-cell lung cancer, colon cancer, CNS cancer, mela-
noma, ovarian cancer, renal cancer, and prostate cancer (see
Table 9, Supporting Information). β-Lactam 9 showed sub-
micromolar GI50 values in all but five cell lines (the GI50 value
is the concentration of drug required for 50% inhibition of
cellular growth). The mean GI50 value for compound 9 across
all cell lines is 0.21 μM [log GI50 =-6.67 M]. For compound
35, the GI50 values obtained were below 10 nM for 38 of the
cell lines investigated, including breast cancers MCF-7, MDA-
MB-231, and the adriamycin resistant NCI/ADR-RES cell line,
with the mean GI50 value across all cell lines being 23.99 nM,
[log GI50=-7.62 M]. LC50 values are a measure of the cyto-
toxicity of these compounds and were greater than 100 μM for
compound 9 in all but 3 cell lines and greater than 100 μM
for compound 35 in all but 10 cell lines (Table 9, Supporting
Information). These results confirm the requirement for the
3-hydroxy-4-methoxy substitution pattern on ring B for opti-
mum antiproliferative activity in these analogues.
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 ( SEM (error values ꢀ 10-6) for three experiments
performed in triplicate. b The IC50 value obtained for 2a in this assay is
0.0052 μM for MCF-7 and is in good agreement with the reported values
for 2a using the MTT assay on human MCF-7 breast cancer cell lines,
(see refs 7, 73, and 74).
Table 5. Antiproliferative Effects of Selected β-Lactam Compounds in
MDA-MB-231 Human Breast Cancer Cells
antiproliferative activitya
MDA-MB-231 cells
IC50 (μM)
antiproliferative activitya
MDA-MB-231 cells
IC50 (μM)
compd
compd
24
32
35
36
39
0.2284 ( 0.1755
0.0782 ( 0.0348
0.0288 ( 0.0199
0.1072 ( 0.0558
0.1577 ( 0.0352
41
42
43
44
45
2ab
0.1764 ( 0.1578
1.6839 ( 1.9756
0.2245 ( 0.1778
0.4288 ( 0.0670
0.0808 ( 0.0738
0.043
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 ( SEM (error values ꢀ 10-6) for three experiments
performed in triplicate. b The IC50 values obtained for 2a in this assay are
0.043 μM for MDA-MB-231 and is in good agreement with the reported
values for 2a using the MTT assay on MDA-MB-231 breast cancer cell
lines (see refs 7, 73, and 74).
antiproliferative activity (IC50=0.72 μM for MCF-7 cells and
IC50 = 0.89 μM for MDA-MB 231 breast cancer cells). The
β-lactam ring is strained and can undergo a number of ring-
opening reactions. In the present work, two ring-opened pro-
ducts werealso synthesizedand evaluated for antiproliferative
activity to assess the lactam scaffold dependence of biological
activity. Alcohol49isthe result oftreatment of35withlithium
aluminum hydride, while ester 50 is the product from the nucleo-
philic ring-opening reaction of 35 with sodium methoxide.
These compounds are more flexible than the rigid β-lactam
parent compound 35. They show over 2000-fold decrease in
antiproliferative activity, indicating the critical importance of
the rigid β-lactam scaffold and the relative orientations of the two
methoxy-containing aromatic rings for preservation of antipro-
liferative activity. The importance of the relative orientations of
the two aromatic rings has also been noted previously for CA-4.22
The more potent compounds in the MCF-7 cell line were
next examined in an ER-negative MDA-MB-231 cell line, and
COMPARE Analysis of β-Lactams 9 and 35
Matrix COMPARE analysis55,56 (measuring the correla-
tion between two compounds with respect to their differential
antiproliferative activity) demonstrated good correlation between
35 and 2a (r = 0.62) and with other tubulin binding drugs

8576 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
O’Boyle et al.
Figure 4. Dose-response curve for β-lactams 35, 41, and 2a in murine mammary epithelial cells at 25 000 cells/mL. Mouse mammary epithelial
cells were harvested from mid- to late-pregnant CD-1 mice and cultured. The isolated mammary epithelial cells were seeded at two
concentrations. After 24 h, they were treated with 2 μL volumes of test compound which had been preprepared as stock solutions in ethanol
to furnish the concentration range of study, 1 nM to 100 μM, and reincubated for a further 72 h. Control wells contained the equivalent volume
of the vehicle ethanol (1% v/v). The cytotoxicity was assessed using alamarBlue dye.
Table 6. Inhibition of Tubulin Polymerization for Compounds 32, 35,
41, and 2aa
vincristine, vinblastine, paclitaxel, maytansine, and rhizoxin
(Table 10, Supporting Information). However, this algorithm
does not distinguish between different tubulin-based mecha-
nisms of action.57 In vitro tubulin-binding studies confirmed
that 35 is acting as a tubulin-binding agent. The antiproliferative
activity observed for these compounds indicated that there is a
significant therapeutic window between the concentration
required for cancer cell growth inhibition and the concentra-
tion that is toxic to cells.
compd
fold-reduction in Vmax at 10 μM
2a
32
35
41
6.0 ( 1.4
6.1 ( 0.9
8.3 ( 2.6
11.76 ( 3.85
a Effects of 2a, 32, 35 and 41 on in vitro tubulin polymerization.
Purified bovine tubulin and GTP were mixed in a 96-well plate. The
reaction was started by warming the solution from 4 to 37 ꢀC. 2a (10 μM)
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 polymerization was calculated using the Vmax
value for each reaction. The results represent the mean ( standard error
of the mean for three separate experiments.
Evaluation of Toxicity in Normal Murine Mammary Epithelial
Cells
Two of the most potent β-lactam compounds from anti-
proliferative studies in the MCF-7 cell line, 35 and 41, were
evaluated further for cytoxicity in murine mammary epithelial
cells. 2a was used as the positive control. Mouse mammary
epithelial cells were harvested from mid- to late-pregnant CD-1
mice and cultured as described previously.58,59 Two different
cell concentrations were used in the toxicity assay: 25000 and
50000 cells/mL. The results are presented in Figure 4 and in
the Supporting Information (Figures 10-12). The IC50 value
is greater than 10 mM for the three compounds evaluated.
This is further evidence that these β-lactam compounds are
minimally toxic to nonproliferating cells.
polymerization curves of each test compound with reference
to ethanol control were calculated and are detailed in Table 6.
As anticipated, the active β-lactam CA-4 analogues 32, 35,
and 41 inhibited the polymerization of tubulin (Table 9,
Supporting Information). In more detail, the active β-lactams
when evaluated at 10 μM, reduced the Vmax value for the rate
of tubulin polymerization from 6-fold for 2a to 11.7-fold for
compound 41 and to 8.3-fold for compound 35, while com-
pound 32showed a6-fold reductionin the value for Vmax. This
value is comparable if not superior to the rate of inhibition
of tubulin assembly (6-fold) observed with 2a. An IC50 value
of 3.65 μM was calculated for compound 41 for the reduction
in Vmax, while an IC50 value of 4.89 μM was obtained for the
effect in overall polymer mass (calculated from area under the
polymerization curve). Inhibition of tubulin polymerization
forcompound41isillustratedin Figure5. Thetubulin binding
studies clearly demonstrated that tubulin is the target of these
new compounds; however, the specific binding site on tubulin
was not investigated, for example, by use of a radiolabeled
colchicine displacement assay. On the basis of the very close
3D structural similarity between the ligands 1, 2a, and the
Tubulin Binding Studies
The effects of representative β-lactam CA-4 analogues on
the assembly of purified bovine tubulin were evaluated.
Compounds 32, 35, and 41 which demonstrated potent anti-
proliferative effects in vitro were assessed. The ability of 2a to
effectively inhibit the assembly of tubulin was assessed as a
positive control. Tubulin polymerization was determined by
measuring the increase in absorbance over time at 340 nm.
The Vmax value offers the most sensitive indicator of tubulin/
ligand interactions, and hence, Vmax values were calculated
for each test compound. Fold changes in Vmax values for

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8577
Figure 5. Effects of compound 41 on in vitro tubulin polymerization. Purified bovine tubulin and GTP were mixed in a 96-well plate. The
reaction was started by warming the solution from 4 to 37 ꢀC. 2a (10 μM) 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 polymerization was calculated using the Vmax value for each reaction. The results represent the mean (standarderror
of the mean for three separate experiments.
β-lactam analogues reported in this study (Figures 6B and
7D), we propose that the binding site for these compounds is
most likely to be the colchicine site, since it has been demon-
stratedthat2a and many reported examples of the structurally
related conformationally constrained 2a analogues bind atthe
colchicine site.27,60,61
2a62 and 2b22 suggest that the conformation of this stilbene is
not planar. These crystal structures reveal that the planes of
the two phenyl rings are inclined to each other, suggesting
a low-energy conformation that may be the one involved in
binding at the tubulin receptor site.22
To rationalize the potential binding modes of these β-lactam
compounds in tubulin, docking studies were carried out with
two of the most potent compounds in the 3-aryl β-lactam
series, 32 and 41. By use of the reported X-ray structure of
tubulin cocrystallized with a colchicine derivative, N-deacetyl-
N-(2-mercaptoacetyl)colchicine (DAMA-colchicine, PDB
entry 1SA0), possible binding orientations for 32 and 41 were
probed with the docking program FRED, version 2.2.3 (Openeye
Scientific Software).64 Because of the structural similarity
between the β-lactams and colchicine site ligands such as 2a,
it was proposed that 32 and 41 bind to tubulin at the colchicine-
binding site. The colchicine-binding site in tubulin is mainly
buried in the β-subunit while maintaining few interactions
with the R-subunit; there is one such site on each tubulin
heterodimer. The H7 and H8 R-helices, the T7 loop, and the
S8 and S9 β-strands contribute to the binding site and interact
with the ligand. Two important residues for binding of colchicine-
type ligands to tubulin have been identified as Val β318 and
Cys β241.63 Val β318 tubulin variants have reduced sensitivity
to 1, and colchicine substituted with more reactive groups
instead of the methoxys can be cross-linked with Cys β241.63
Molecular modeling studies were carried out with the β-lactam
compounds synthesized and evaluated to determine if these
interactions were predicted to be present. Figure 6A illustrates
β-lactam 32 docked in the colchicine-binding site of tubulin,
while Figure 7A and Figure 7B show similar graphics with
β-lactam 41 docked. β-Lactam 41 was the most active compound
in antiproliferative assays and also in the tubulin polymerization
Structural and Molecular Modeling Studies of Selected Azeti-
dinones
The molecular structure of a representative example of the
active series, compound 32, was determined by single-crystal
X-ray crystallography. The ORTEP diagram is presented in
Figure 2, where the trans configuration at positions 3 and 4
of the β-lactam ring is clearly seen (note that in the X-ray
numbering scheme in Figure 2, the carbon at the 3-position
of the β-lactam ring is denoted as C2 and the carbon the
4-position of the β-lactam ring is denoted as C3). The structure
revealed a conformation for the azetidinone 32 in which the
two aromatic rings located at N-1 and C-4 are not coplanar.
The rigid azetidinone ring provides a scaffold that can accom-
modate the steric and geometric requirements of the aromatic
pharmacophores for tubulin binding. Ligands that bind at the
colchicine-binding site of tubulin have the common feature of
a trimethoxy aromatic ring, noted for such ligands as 1, 2a, and4.
The azetidinone 32 is structurally similar to 1 and 2a, with
the common structural features of a trimethoxyphenyl ring
and a second aromatic ring substituted with methoxy groups
in a noncoplanar diaryl system, with the observed tortional
angle for ring A-ring B of 46.9ꢀ compared to 55ꢀ and 53ꢀ for
the corresponding rings in 2a62 and 1,63 respectively. These
structural similarities support the observed antiproliferative and
tubulin polymerization inhibitory activity for these compounds
as tubulin-targeting agents. The X-ray crystal structures of

8578 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
O’Boyle et al.
Figure 6. (A) Docked pose of β-lactam 32 in the colchicine binding site of tubulin (PDB entry 1SA0). Significant binding residues Cys 241 and
Val 318 are indicated. Hydrogens are not shown for clarity. Coloring is by atom: gray (carbon); red (oxygen); blue (nitrogen); yellow (sulfur).
Residue numbers are those used by Ravelli et al.63 (B) Docked pose of 32 (colored by atom) overlaid with N-deacetyl-N-(2-mercaptoacetyl)-
colchicine (DAMA-colchicine) (yellow) in the tubulin binding site (PDB entry 1SA0). Residues are not shown for clarity. (C) 2D representation
of the binding of β-lactam 32 in the colchicine-binding site of tubulin: 2-D rendering of ligand-protein interactions using LigX module of MOE
used to create docked structures of 32 in the colchicine-binding site of tubulin.
inhibition assay. The two important residues for binding in
the colchicine-binding site of tubulin are identified as Val β318
and Cys β241. Figures 6A, 7A, and 7B show that the trimethoxy
rings of both 32 and 41 are well positioned in proximity to the
Cys241 residue (residue numbers are those used by Ravelli
et al63), and the compounds adopt an orientation very similar
to that of the trimethoxy ring of DAMA-colchicine in the
cocrystallized structure. In addition, for compound 41, Thr
179 is seen to establish a strong H-bond to the 3-OH sub-
stituent of ring B (Figure 7A, Figure 7B). This residue has
previously been identified as interacting with a number of
colchicine siteligands.32,65,66 This interaction mayalsoexplain
the binding of the related compound in the series with an OH
substituent in similar position (e.g., compounds 35, 36, 46)
and also the related analogues with NH2 substituent at C-3 of
the aryl B-Ring. Activity is optimized by additional substituents
on the C-3 aryl such as 4-F, 4-NH2, 4-OH, 3-OH (compounds
24, 39, 40, 43) which preserve the key interactions with active
residues for ring A and ring B. The H-bonding interaction
between the 4-hydroxyaryl group atthe 3-position ofβ-lactam
41 and the Lys 254 residue (Figure 7A, 7B), in addition to
hydrophobic contacts with the 3-phenyl ring (not shown for
clarity), could contribute to the observed higher antiprolifera-
tive activity observed for this analogue. The hydrophobic
interactions are illustrated in Figure 7C and include interac-
tions of the 3-phenyl ring with Leu248, Ala250, and Leu255.
Activity is significantly reduced for β-lactam analogues of 32
that have multiple methoxy substitutions on the C-3 aryl ring
as in compound 20 or increased steric hindrance with the
benzyloxy ether (compound 28).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8579
Figure 7. (A) Proposed binding and docked pose of β-lactam 41 (green) in colchicine binding site of tubulin (PDB entry 1SA0). Hydrogens are
not shown for clarity. Significant binding residues Thr179, Cys241, Val318, and Lys254 are indicated. Residue numbers are those used by
Ravelli et al.63 (B) Docked pose of β-lactam 41 in colchicine binding site of tubulin (PDB entry 1SA0) with isolated important residues.
Hydrogens are not shown for clarity. Significant binding residues Cys241, Val318, and Lys254 are indicated. Coloring is by atom: gray
(carbon); red (oxygen); blue (nitrogen); yellow (sulfur). Residue numbers are those used by Ravelli et al.63 (C) 2D representation of the binding
of β-lactam 41 in the colchicine-binding site of tubulin: 2-D rendering of ligand-protein interactions using LigX module of MOE used to create
docked structures of 41 in the colchicine-binding site of tubulin. (D) Docked pose of 41 (colored by atom) overlaid with N-deacetyl-
N-(2-mercaptoacetyl)colchicine (DAMA-colchicine) (yellow) in the tubulin binding site (PDB entry 1SA0). Residues are not shown for clarity.
Several other hydrophobic contacts are predicted to stabi-
lize the binding of 32 and 41 to tubulin. Figures 6C and 7C
depict the predicted binding interactions of docked β-lactams
32and 41in2Dformat.67 From theseresultsit canbeseen that
the important interactions discussed above are predicted to be
present for these compounds. The docked pose of 32 (colored
by atom) overlaid with colchicine (yellow) in the tubulin bind-
ing site is illustrated in Figure 6B. It can be seen that the
methoxy substituent of ring B is in proximity in the binding site
to the corresponding methoxy group on ring C of colchicine.
A similar binding orientation is observed for colchicine and 41
(Figure 7D). These binding parallels may rationalize the
potency observed for 32 and 41 in their inhibition of tubulin
polymerization.
Conclusion
We have synthesized a comprehensive series of β-lactam
compounds that show potent antiproliferative activity in a
range of tumor cell lines, notably in human breast cancer
MCF-7 and MDA-MB-231 cell lines. The most potent com-
pound in the series 41 displayed subnanomolar activity in
the MCF-7 cell line with an IC50 of 0.8 nM. Compound 46

8580 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
O’Boyle et al.
containing a 3-(4-aminophenyl) substituent also showed greater
activity than the lead compound 32, with an IC50 of 4 nM in
MCF-7 cell line, with the 3-phenylsubstituted β-lactam 32
having an IC50 of 34 nM. The compounds did not cause sig-
nificant cytotoxicity in normal murine breast epithelial cells.
Compounds 32, 35, and 41 were shown to inhibit the poly-
merization of tubulin with improved efficacy when compared
with 2a.
X-ray crystal structure of 32 revealed that the compound
provides a scaffold structure with a similar spatial arrange-
ment between the two phenyl rings as observed in cis config-
uration of 2a. Preliminary modeling studies indicate a possible
binding mode for these potent inhibitors of tubulin polymer-
ization. These conformationally restricted β-lactam structures
are not easily isomerized, unlike the cis-stilbene 2a, and are
identified as promising lead compounds in the development
of new anticancer agents. Further studies are in progress to
further rationalize SAR for this series of azetidinones and to
determine the antiangiogenic effects of these compounds.
The organic mobile phase was 2-propanol, andthe aqueousphase
was a sodium phosphate buffer. The sodium phosphate buffer,
consisting of 10 mM sodium dihydrogen orthophosphate di-
hydrate (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. Details of the synthesis of inter-
mediate compounds and characterizations of target β-lactams are
contained in the Supporting Information.
General Method for Imine Preparation. The appropriate
amine (10 mmol) was refluxed with the appropriate aldehyde
(10 mmol) in ethanol (50 mL) for 3 h. The reaction mixture was
reduced in vacuo, and the resulting solution was allowed to
stand until solid product crystallized from solution. The result-
ing imine was recrystallized from ethanol.
[3-(tert-Butyldimethylsilanyloxy)-4-methoxybenzylidene]-(3,4,5-
trimethoxyphenyl)amine (5e). 5e was prepared by reacting 3-(tert-
butyldimethylsilanyloxy)-4-methoxybenzaldehyde with 3,4,5-tri-
methoxybenzenamine following the general method above. The
product was obtained as a yellow solid. Yield 64.3%. Mp 105 ꢀC.
IR (KBr) υ 1619.8, 1579.73 cm-1 (-CdN-); 1H NMR (400 MHz,
CDCl3) δ 0.20 (s, 6H, 2xCH3), 1.03 (s, 9H, C(CH3) 3), 3.87-3.91
(m, 12H, 4ꢀOCH3), 6.48 (s, 2H, ArH), 6.93 (d, 2H, J=8.04 Hz,
ArH), 7.43-7.47 (m, 1H, ArH), 8.35 (s, 1H, CHdN); 13C NMR
(100 MHz, CDCl3) δ -5.04 (CH3-Si-CH3), 18.03(CH3-C-
CH3), 25.27 (C(CH3)3), 54.98 (-OCH3), 55.63 (-OCH3), 97.62,
110.94, 119.71, 123.48, 128.95, 135.53, 144.87, 147.94, 153.05, 153.59
(ArC), 158.84 (-CdN-). Elemental analysis: found C, 63.99; H,
7.65; N, 3.21; C23H33NO5Si requires C, 64.01; H, 7.71; N, 3.25%.
General Method for β-Lactam Synthesis I: Staudinger Reac-
tion (Compounds 7-14). The appropriate imine (5 mmol) and
triethylamine (15 mmol) were added to dry CH2Cl2 (50 mL), and
the mixture was brought to reflux at 60 ꢀC. The appropriate acid
chloride (7.5 mmol) was then added dropwise to the mixture via
a septum. The reaction mixture was then refluxed for 3 h, then
cooled and washed first with distilled water (2ꢀ50 mL) and then
with saturated aqueous sodium bicarbonate solution (50 mL).
The organic layer was dried by filtration through anhydrous
sodium sulfate. The organic layer containing the product was
collected and reduced in vacuo to afford the crude product
which was purified by flash column chromatography over silica
gel (eluent: hexane/ethyl acetate gradient).
General Method for β-Lactam Synthesis II: Staudinger Reac-
tion Modified (Compounds 15-17). A solution of the appro-
priate imine (10 mmol) and acid chloride (10 mmol) in CH2Cl2
(50 mL) under nitrogen was stirred for 2 h. Triethylamine
(10 mmol) was added dropwise, and the mixture was left to stir
overnight. The mixture was washed first with distilled water
(2ꢀ50 mL) and then with saturated aqueous sodium bicarbonate
solution (50 mL). The organic layer was dried by filtration through
anhydrous sodium sulfate. The organic layer containing the
product was collected and reduced in vacuo to afford the crude
product which was purified by flash column chromatography
over silica gel (eluent: hexane/ethyl acetate gradient 10:1 to 2:1).
General Method for β-Lactam Formation III: Acid Activation
with Triphosgene (Compounds 18-31). A mixture of the appropriate
acetic acid (15 mmol) and triphosgene [bis(trichloromethyl) car-
bonate] (5 mmol) in dry CH2Cl2 (50 mL) was heated at refluxed for
30 min. A solution of the appropriately substituted imine (10 mmol)
in dry CH2Cl2 (10 mL) was added dropwise to the refluxing solution
followed by triethylamine (30 mmol). The reaction mixture was
heated at reflux for 5 h and stirred at room temperature overnight.
The mixture was washed first with distilled water (2 ꢀ 50 mL) and
then with saturated aqueous sodium bicarbonate solution (50 mL).
The organic layer was dried over anhydrous sodium sulfate to afford
the crude product, which was purified by flash column chromatog-
raphy over silica gel (eluent: hexane/ethyl acetate gradient).
Experimental Section
All reagents were commercially available and were used without
further purification unless otherwise indicated. Tetrahydrofuran
(THF) was distilled immediately prior to use from Na/benzophenone
under a slight positive pressure of nitrogen. Toluene was dried by
distillation from sodium and stored on activated molecular sieves
˚
(4 A), and dichloromethane was dried by distillation from calcium
hydride prior to use. IR spectra were recorded as thin films on
NaCl plates or as KBr discs on a Perkin-Elmer Paragon 100 FT-IR
spectrometer. 1H and 13C NMR spectra were obtained on a Bruker
Avance DPX 400 instrument at 20 ꢀC, 400.13 MHz for 1H spectra,
100.61 MHz for 13C spectra, in CDCl3, CD3COCD3, or CD3OD
(internal standard tetramethylsilane) by Dr. John O’Brien and
Dr. Manuel Ruether in the School of Chemistry, Trinity College
Dublin. Low resolution mass spectra were run on a Hewlett-
Packard 5973 MSD GC-MS 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 School
of Chemistry, Trinity College Dublin. Thin layer chromatography
was performed using Merck silica gel 60 TLC aluminum sheets
with fluorescent indicator visualizing with UV light at 254 nm. Flash
chromatography was carried out using standard silica gel 60
(230-400 mesh) obtained from Merck. All products isolated were
homogeneous on TLC. The purity of the tested compounds was
determined by HPLC or combustion analysis, and unless otherwise
stated, the purity level was g95%. Elemental analyses were per-
formed on an Exetor Analytical CE4400 CHN analyzer in the
Microanalysis Laboratory, Department of Chemistry, University
College Dublin, Ireland. Analytical high-performance liquid
chromatography (HPLC) was performed using a Waters 2487
dualwavelengthabsorbancedetector,aWaters1525binaryHPLC
pump, and a Waters 717Plus autosampler. The column used was a
Varian Pursuit XRs C18 reverse phase 150 mm ꢀ 4.6 mm chro-
matography column. Samples were detected using a wavelength of
254 nm. All samples were analyzed using acetonitrile (70%)/
water (30%) over 10 min and a flow rate of 1 mL/min. Chiral
liquid chromatography was carried out on selected compounds
using a Chiral-AGP 150 mm ꢀ 4.0 mm column supplied by
ChromTech Ltd. (now supplied by Chiral Technologies Europe)
with a Chiral- AGP 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 degasserAF, anda Waters717plus autosampler. Gradient
elution was used beginning with 10% of organic phase and
finishing with 90% of organic phase over a period of 20 min.
3-(4-(Benzyloxy)phenyl)-4-(3-((tert-butyldimethylsilyl)oxy)-
4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one 29.
29 was obtained from (4-benzyloxyphenyl)acetic acid and imine
5e following general method III as a brown oil, yield 6.8%, and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8581
was deprotected immediately without further purification to
afford 37 (see below).
3-(4-(Benzyloxy)phenyl)-4-(3-hydroxy-4-methoxyphenyl)-
1-(3,4,5-trimethoxyphenyl)azetidin-2-one 37. 37 was obtained
from azetidinone 29 following general method V as a brown oil,
yield 96.8%, which was further deprotected to form 41 without
purification.
{4-[2-(3-Hydroxy-4-methoxyphenyl)-4-oxo-1-(3,4,5-trimethoxy-
phenyl)-azetidin-3-yl]phenyl}carbamic Acid Benzyl Ester 38. 38
was obtained from carbamate 30 following general method V, yield
33.7%, and was further deprotected to 46 without purification.
Debenzylation of β-Lactams (General Method VI) (Compounds
39-42). The appropriate benzyl ether compound (2 mmol) was
dissolved in ethanol/ethyl acetate (50 mL, 1:1 mixture) and
hydrogenated over palladium on carbon (1.2 g, 10%) at atmo-
spheric pressure for 2 h. The catalyst was filtered, the solvent was
removed under vacuum, and the product was purified by flash
column chromatography over silica gel (eluent: hexane/ethyl
acetate gradient) to afford the phenolic product.
4-(3-Hydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one 41. 41 was obtained from aze-
tidinone 37 following general method VI as a white powder,
yield 2.9%. Mp 152 ꢀC. IR (NaCl film) υ 1720.6 cm-1 (CdO,
β-lactam); 1H NMR (400 MHz, CDCl3) δ 3.65 (s, 3H, -OCH3),
3.70 (s, 6H, 2ꢀ-OCH3), 3.86 (s, 3H, -OCH3), 4.26 (d, 1H,
J = 2.4 Hz, H-3), 4.98 (d, 1H, J = 2.4 Hz, H-4), 6.71 (s, 2H,
ArH), 6.87 (d, 2H, J = 8.8 Hz, ArH), 7.00 (s, 3H, ArH), 7.22
(d, 2H, J=8.8 Hz, ArH); 13C NMR (100 MHz, CDCl3) δ 54.93
(-OCH3), 59.23 (-OCH3), 63.05 (C-3), 63.76 (C-4), 94.66,
111.31, 112.22, 115.13, 117.48, 125.72, 128.25, 130.39, 133.49,
134.12, 146.70, 147.32, 153.25, 156.50 (ArC), 165.27 (CdO). HRMS
calcd for C25H25NO7Na: 474.1529. Found 474.1548; (Mþ þ Na).
General Method for Reduction of Nitro-Substituted Azetidi-
nones (General Method VII) (Compounds 43-45). To the appro-
priate nitro substituted β-lactam (10 mmol) dissolved in the
minimum amount of glacial AcOH (2-3 mL) was added
metallic zinc dust (10 equiv). The mixture was stirred for 6 days
at room temperature under nitrogen. The residue was filtered
through Celite and was extracted with dichloromethane. The
solvent was evaporated and the residue purified by flash column
chromatography over silica gel (eluent: hexane/ethyl acetate
gradient) to afford the required amine product.
Benzyl (4-(2-(3-((tert-Butyldimethylsilyl)oxy)-4-methoxyphenyl)-
4-oxo-1-(3,4,5-trimethoxyphenyl)azetidin-3-yl)phenyl)carbamate 30.
30 was obtained from (4-benzyloxycarbonylaminophenyl)acetic
acid and imine 5e following general method III as an oil, yield
10.6%, and was deprotected immediately to afford 38 without
further purification.
General Method for β-Lactam Preparation IV: Reformatsky
Reaction Using Microwave (Compounds 32-34). Zinc powder
(0.927 g, 15 mmol) was activated using trimethylchlorosilane
(0.65 mL, 5 mmol) in anhydrous benzene (5 mL) by heating
for 15 min at 40 ꢀC and subsequently for 2 min at 100 ꢀC in a
microwave. After the mixture was cooled, the appropriately
substituted imine (10 mmol) and substituted ethylbromoacetate
(12 mmol) were added to the reaction vessel and the mixture was
placed in the microwave for 30 min at 100 ꢀC. The reaction
mixture was filtered through Celite to remove the zinc catalyst
and then diluted with dichloromethane. This solution was
washed with saturated ammonium chloride solution (20 mL)
and 25% ammonium hydroxide (20 mL) and then with dilute
HCl (40 mL) followed by water (40 mL). The organic phase was
dried over anhydrous sodium sulfate and the solvent was removed
in vacuo to afford the crude product which was purified by flash
column chromatography over silica gel (eluent: hexane/ethyl
acetate gradient) to afford the required β-lactam product.
4-(4-Methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)-
azetidin-2-one 32. 32 was obtained from ethyl 2-bromo-2-phenyl-
acetate and imine 5a following general method IV as a white
crystalline solid, yield 6.8%. Mp 108 ꢀC. IR (NaCl) υ 1753.3 cm-1
1
(CdO, β-lactam); H NMR (400 MHz, CDCl3) δ 3.72 (s, 6H,
2ꢀ-OCH3), 3.77 (s, 3H, -OCH3), 3.83 (s, 3H, -OCH3), 4.29 (d, 1H,
J=2.5 Hz, H-3), 4.87 (d, 1H, J=2.5 Hz, H-4), 6.60 (s, 2H, ArH),
6.95 (d, 2H, J=8.5 Hz, ArH), 7.32-7.40 (m, 7H, ArH); 13C NMR
(100 MHz, CDCl3) δ 54.92 (-OCH3), 55.57 (-OCH3), 60.52
(-OCH3), 63.40 (C-3), 64.59 (C-4), 94.38, 114.24, 126.89, 127.00,
127.45, 128.59, 128.85, 133.28, 134.00, 134.33, 153.05, 159.49 (ArC),
165.21 (CdO). HRMS calcd for C25H25NO5Na: 442.1630. Found:
442.1631 (Mþ þ Na).
4-(3-((tert-Butyldimethylsilyl)oxy)-4-methoxyphenyl)-3-phenyl-
1-(3,4,5-trimethoxyphenyl)azetidin-2-one 34. 34 was obtained
from ethyl 2-bromo-2-phenylacetate and imine 5e following
general method IV as a yellow oil, yield 70%, and was immedi-
ately deprotected to form 35 without further purification.
Desilylation of β-Lactams (General Method V) (Compounds
35-38). To a solution of the appropriately protected phenol
(10 mmol) in THF (50 mL) was added 1 M tetrabutylammonium
fluoride (1.5 equiv). The solution was stirred in an ice bath for
15 min. The reaction mixture was diluted with EtOAc (100 mL)
and quenched with HCl (10%, 100 mL). The layers were separated,
and the aqueous layer was extracted with EtOAc (2 ꢀ 50 mL).
The organic layer was then washed with water (100 mL) and
brine (100 mL), followed by drying over anhydrous sodium
sulfate. The solvent was removed by evaporation under reduced
pressure and the crude product was purified by flash column
chromatography over silica gel (eluent: hexane/ethyl acetate
gradient) to afford the required phenol product.
Deprotection of the Cbz Protected Azetidinones (General Method
VIII) (Compounds 46 and 47). The Cbz-protected compound
(2 mmol) was dissolved in ethanol/ethyl acetate (50 mL; 1:1
mixture) and hydrogenated over palladium on carbon (1.2 g,
10%) for 2 h. The catalyst was filtered, the solvent was removed
under vacuum, and the product was purified by flash column
chromatography over silica gel (eluent: hexane/ethyl acetate
gradient) to afford the amine product.
3-(4-Aminophenyl)-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one 46. 46 was obtained from car-
bamate 38 following general method VIII, as an orange oil, yield
39.4%. IR (NaCl film) υ 1737.4 cm-1 (CdO, β-lactam); 1H
NMR (400 MHz, CDCl3) δ 3.75 (s, 6H, 2ꢀ-OCH3), 3.80 (s,
3H, -OCH3), 3.93 (s, 3H, -OCH3), 4.18 (d, 1H, J=2.5 Hz, H-3),
4.74 (d, 1H, J = 2.5 Hz, H-4), 6.63 (s, 2H, ArH), 6.70 (d, 2H,
J = 8.3 Hz, ArH), 6.89 (m, 2H, ArH), 6.99 (s, 1H, ArH), 7.11
(d, 2H, J = 8.3 Hz, ArH); 13C NMR (100 MHz, CDCl3) δ 55.56
(-OCH3), 55.59 (-OCH3), 60.51 (-OCH3), 63.85 (C-3), 64.19
(C-4), 94.39, 110.54, 111.53, 115.15, 117.33, 124.24, 128.03, 130.32,
133.39, 145.39, 145.8, 146.32, 153.02(ArC), 165.60 (-CdO).
HRMScalcdfor C25H27N2O6: 451.1869. Found: 451.1859 (Mþ).
Antiproliferative MTT Assay. All assays were performed in
triplicate for the determination of mean values reported. Com-
pounds were assayed as the free bases isolated from reaction.
The human breast tumor cell line MCF-7 was cultured in Eagles
minimum essential medium in a 95% O2/5% CO2 atmosphere
with 10% fetal bovine serum, 2 mM L-glutamine, and 100 μg/mL
penicillin/streptomycin. The medium was supplemented with
1% nonessential amino acids. MDA-MB-231 cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM), supplemented
4-(3-Hydroxy-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxy-
phenyl)azetidin-2-one 35. 35 was obtained from azetidinone 34
following general method V as a white solid, yield 97.3%. Mp
1
110 ꢀC. IR (KBr) υ 1718.2 cm-1 (CdO, β-lactam); H NMR
(400 MHz, CDCl3) δ 3.73 (s, 6H, 2ꢀ-OCH3), 3.78 (s, 3H,
OCH3), 3.91 (s, 3H, OCH3), 4.27 (d, 1H, J = 2.5 Hz, H-3), 4.81
(d, 1H, J = 2.5 Hz, H-4), 5.75 (s, 1H, OH), 6.63 (s, 2H, ArH),
6.86-6.93 (m, 2H, ArH), 7.00 (d, 1H, J=2.0 Hz, ArH), 7.31-
7.39 (m, 5H, ArH); 13C NMR (100 MHz, CDCl3) δ 55.58-
(OCH3), 55.60(OCH3), 60.51(OCH3), 63.36(H-3), 64.49(H-4),
94.42, 110.59, 111.56, 117.40, 126.97, 127.43, 128.57, 130.10, 133.27,
134.02, 134.31, 145.91, 146.43, 153.05(ArC), 165.14(CdO). HRMS
calcd for C25H25NO6Na:458.1580. Found: 458.1575 (Mþ þ Na).

8582 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
O’Boyle et al.
with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, and
100 μg/mL penicillin/streptomycin (complete medium). Cells
were trypsinized and seeded at a density of 2.5ꢀ104 cells/mL in a
96-well plate and incubated at 37 ꢀC in 95%O2/5% CO2 atmo-
sphere for 24 h. After this time they were treated with 2 μL
volumes of test compound which had been preprepared as stock
solutions in ethanol to furnish the concentration range of study,
1 nM to 100 μM, and reincubated for a further 72 h. Control
wells contained the equivalent volume of the vehicle ethanol
(1% v/v). The culture medium was then removed. The cells were
washed with 100 μL of phosphate buffered saline (PBS), and
50 μL MTT was added to reach a final concentration of 1 mg/mL
MTT. Cells were incubated for 2 h in darkness at 37 ꢀC. At this
point solubilization was begun through the addition of 200 μL of
DMSO and the cells were maintained at room temperature in
darkness for 20 min to ensure thorough color 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 pre-
pared to assess cell viability using GraphPad Prism software.
Cytotoxicity Assay. Mammary glands from 14 to 18 day preg-
nant CD-1 mice were used as source, and primary mammary
epithelial cell cultures were prepared from these. The first to
third thoracic glands were exposed by pulling the skin back from
the rib cage to the forelimb and extracted in a similar way to the
inguinal glands. Midpregnant glands are large, soft, and pink in
color. Mammary glands that were spongy and pale in color were
not used, as these glands had matured and were producing milk.
The harvested glands were then subjected to mechanical and
enzymatic digestion to release immature alveolar structures and
isolate mammary epithelial cells. The dissected glands were
weighed, and fresh collagenase digestion mixture was prepared.
It contained 480 mg of F10 powdered medium (Sigma), 70 mg of
trypsin (GibcoBrl, Life Technologies), 150 mg of collagenase A
(GibcoBrl, Life Technologies), and 2.5 mL of fetal calf serum
(GibcoBrl, Life Technologies) in a final volume of 50 mL (with
sterile water). The collagenase digestion mixture was subse-
quently adjusted to pH 7.4, filtered through a 0.2 μM sterile
filter, and stored at 4 ꢀC until required. The dissected glands
were minced criss-cross using two sterile scalpel blades. The
minced glands were placed in an autoclaved, sterile 250 mL glass
conical flask with the collagenase digestion mixture (∼4 mL of
digestion mixture per gram of tissue) and digested for 90 min on
a shaking table, 250 rpm at 37 ꢀC. After this step, a stringent
washing/centrifugation protocol was used to isolate epithelial
cells from fibroblasts. Selective centrifugation was carried out as
follows: the digested cell suspension was removed to a 50 mL
tube and centrifuged for 30 s at 100 rpm The supernatant was
transferred to a fresh tube and centrifuged at 800 rpm for 3 min.
The pellet was then subjected to a DNase treatment in order to
achieve separation of single epithelial cells rather than cell
clumps. The DNase mixture contained 480 mg of F10 powdered
medium (Sigma), 250 μL of 10 mg/mL DNase (Roche Diag-
nostics), and 250 μL of 1 M MgCl2 brought up to a final volume
of 50 mL (with water) and passed through a 0.2 μM sterile filter.
The pellet was resuspended in the DNase mixture and incubated
at 37 ꢀC for 30 min on a shaking table at 150 rpm The cell sus-
pension was then transferred to a fresh 50 mL tube and centri-
fuged at 800 rpm for 3 min. The supernatant was discarded. The
pellet was resuspended in 30-50 mL of Ham’s F-12 medium
(with gentamycin, Biowhittaker), depending on the size of the
pellet. Finally, the cell suspension was added to F-12 medium
containing gentamycin (50 μg/mL) with the following: hydro-
cortisone (H) 1 μg/mL (Sigma, stock solution, 1 mg/mL in 100%
ethanol), insulin (I) 5 μg/mL (Sigma, stock solution, 5 mg/mL in
5 mM HCl), and epidermal growth factor (EGF) 5 ng/mL
(Promega, stock solution, 5 μg/mL in F-12 medium). The iso-
lated mammary epithelial cells were seeded at two concentrations
(25 000 cells/mL and 50 000 cells/mL). Initially a third concen-
tration of 100 000 cells/mL was also used, but this proved to be
too high to give meaningful results. After 24 h, they were treated
with 2 μL volumes of test compound which had been prepre-
pared as stock solutions in ethanol to furnish the concentration
range of study, 1 nM to 100 μM, and reincubated for a further
72 h. Control wells contained the equivalent volume of the
vehicle ethanol (1% v/v). The cytotoxicity was assessed using
alamarBlue dye.
Tubulin Polymerization. Tubulin polymerization was carried
out using a kit supplied by Cytoskeleton. It is based on the
principal that light is scattered by microtubules to an extent that
is proportional to the concentration of the microtubule polymer.
Compounds that interact with tubulin will alter the polymeri-
zation of tubulin, and this can be detected using a spectro-
photometer. The absorbance at 340 nm at 37 ꢀC is monitored.
The experimental procedure of the assay was performed as des-
cribed in version 8.2 of the tubulin polymerization assay kit
manual.68
Stability Study for Compounds 32 and 35. Analytical high-
performance liquid chromatography (HPLC) stability studies
were performed using a Symmetry column (C18, 5 μm, 4.6 mmꢀ
150 mm), a Waters 2487 dual wavelength absorbance detector, a
Waters 1525 binary HPLC pump, and a Waters 717plus auto-
sampler. Samples were detected at a wavelength of 254 nm. All
samples were analyzed using acetonitrile (80%)/water (20%) as
the mobile phase over 10 min and a flow rate of 1 mL/min. Stock
solutions are prepared by dissolving 5 mg of compound in 10 mL
of mobile phase. Phosphate buffers at the desired pH values
(4, 7.4, and 9) were prepared in accordance with the British
Pharmacopoeia monograph 2010. Then 30 μL of stock solution
was diluted with 1 mL of appropriate buffer, shaken, and injec-
ted immediately. Samples were withdrawn and analyzed at time
intervals of t = 0 min, 5 min, 30 min, 60 min, 90 min, 120 min,
and 21 h.
Computational Procedure. For ligand preparation, all com-
pounds were built using ACD/Chemsketch, version 10, to gener-
ate SMILES. A single conformer from each string was generated
using Corina, version 3.4, and ensuring that Omega, version
2.2.1, was subsequently employed to generate a maximum of
1000 conformations of each compound. For the receptor pre-
paration, the PDB entry 1SA0 was downloaded from the
Protein Data Bank (PDB). All waters were retained in both
isoforms. Addition and optimization of hydrogen positions for
these waters were carried out using MOE 2007.09, ensuring all
other atom positions remained fixed. By use of the reported
X-ray structure of tubulin cocrystallized with a colchicine
derivative, DAMA-colchicine (PDB entry 1SA0),63 possible
binding orientations ligands were probed with the docking
program FRED, version 2.2.3 (Openeye Scientific Software).64
Docking was carried out using FRED, version 2.2.3, in con-
junction with Chemgauss3 PLP scoring function. The 3D ligand
conformations were enumerated using CORINA, version 3.4
(Molecular Networks GMBH),69 for ligands followed by gen-
eration of multiple conformations using OMEGA, version2.2.1
(Openeye Scientific Software).70 Each conformation was subse-
quently docked and scored with Chemgauss3 PLP as outlined
previously.32 The top binding poses were refined using the LigX
procedure (MOE, Chemical Computing Group)71 together with
Postdock analysis (SVL script, MOE) of the docked ligand poses.
X-ray Crystallography. The X-ray crystallography data for
crystal 32 were collected on a Rigaku Saturn 724 CCD diffracto-
meter. A suitable crystal was selected and mounted on a glass
fiber tip and placed on the goniometer head in a 123K N2 gas
stream. The data set was collected using Crystalclear-SM 1.4.0
software, and 1680 diffraction images of 0.5ꢀ per image were
recorded. Data integration, reduction, and correction for absorp-
tion and polarization effects were all performed using Crystal-
clear-SM 1.4.0 software. Space group determination, structure
solution, and refinement were obtained using Crystalstructure,
version 3.8, and Bruker Shelxtl, version 6.14, software.72 Crystal
data for 32 are as follows: C25H25NO5, MW 419.46, orthorhombic,

Article
space group Pbca, a = 18.876(9) A, b = 9.608(5) A, c =
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8583
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˚
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23.662(12) A, R=β=γ=90ꢀ, U=4291(4) A, Z=8, Dc=1.298
Mg m-3, m = 0.090 mm-1, range for data collection = 1.12-
25.00, reflections collected 17 904, unique reflections 3777 [Rint =
0.1746], data/restraints/parameters 3777/0/284, goodness-of-fit
on F2 1.357, R indices (all data)=R1=0.1908, wR2=0.2609,
final R indices [I > 2σ(I)]=R1=0.2366, wR2=0.2809, CCDC
deposition no. 775568.
Acknowledgment. We thank Dr. Niall Keely for the kind
gift of combretastatin A-4. This work was supported through
funding from the Trinity College Dublin 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 Dublin is gratefully acknowledged.
Supporting Information Available: Experimental procedures
and spectroscopic data for intermediate and final compounds,
additional cytotoxicity data in normal murine epithelial cells for
compounds 35 and 41, results of comparative antitumor evalua-
tions of compounds 9 and 35 in the NCI60 cell line in vitro
primary screen, and results of standard COMPARE analysis of
35. This material is available free of charge via the Internet at
http://pubs.acs.org.
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