Lead identification of β-lactam and related imine inhibitors of the molecular chaperone heat shock protein 90

Article abstract of DOI:10.1016/j.bmc.2011.08.048

Heat shock protein 90 is an emerging target for oncology therapeutics. Inhibitors of this molecular chaperone, which is responsible for the maintenance of a number of oncogenic proteins, have shown promise in clinical trials and represent a new and exciting area in the treatment of cancer. Heat shock protein 90 inhibitors have huge structural diversity, and here we present the lead identification of novel inhibitors based on β-lactam and imine templates. β-Lactam 5 and imines 12 and 18 exhibit binding to heat shock protein 90-α with IC₅₀ values of 5.6 μM, 14.5 μM, and 22.1 μM, respectively. The binding affinity displayed by these compounds positions them as lead compounds for the design of future inhibitors of heat shock protein 90 based on the β-lactam and imine templates.

Full text of DOI:10.1016/j.bmc.2011.08.048

Bioorganic & Medicinal Chemistry 19 (2011) 6055–6068
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Lead identification of b-lactam and related imine inhibitors
of the molecular chaperone heat shock protein 90
Niamh M. O’Boyle a, , Andrew J.S. Knox b, Trevor T. Price b, D. Clive Williams b,
⇑
Daniela M. Zisterer b, David G. Lloyd c, 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 and Immunology, Trinity College Dublin, Dublin 2, Ireland
c Molecular Design Group, School of Biochemistry and 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:
Heat shock protein 90 is an emerging target for oncology therapeutics. Inhibitors of this molecular chap-
erone, which is responsible for the maintenance of a number of oncogenic proteins, have shown promise
in clinical trials and represent a new and exciting area in the treatment of cancer. Heat shock protein 90
inhibitors have huge structural diversity, and here we present the lead identification of novel inhibitors
based on b-lactam and imine templates. b-Lactam 5 and imines 12 and 18 exhibit binding to heat shock
Received 18 February 2011
Revised 18 August 2011
Accepted 19 August 2011
Available online 25 August 2011
protein 90-
a with IC50 values of 5.6 lM, 14.5 lM, and 22.1 lM, respectively. The binding affinity dis-
Keywords:
Heat shock protein 90 (Hsp90)
b-Lactam
played by these compounds positions them as lead compounds for the design of future inhibitors of heat
shock protein 90 based on the b-lactam and imine templates.
Ó 2011 Elsevier Ltd. All rights reserved.
Azetidinone
Imine
Structure–activity relationship
Anti-cancer
1. Introduction
Fig. 1),4 a related derivative, 7-allylaminogeldanamycin (17-AAG,
1b)5 and radicicol (2, Fig. 1)6,7 bind at this ATP-site, leading to dimin-
Heat shock protein 90 (Hsp90) is a molecular chaperone that
has a diverse ‘clientele’ of proteins, many of which are signal trans-
ducers that have roles in cellular proliferation and survival path-
ways.1 A significant number of these proteins are oncogenic in
nature and include protein kinases ERBB2 and BRAF, mutant p53
and steroid hormone receptors (estrogen and androgen).1,2 The
ability of Hsp90 inhibition to affect many oncogenic signaling cas-
cades simultaneously through inhibition of a single target is highly
desirable and unique and marks it as an attractive target for cancer
therapy.1
ished ATPase activity and preventing dissociation of client proteins
from the Hsp90 complex.8 Although radicicol has higher affinity
for full-length homodimeric Hsp90 than geldanamycin, it has not
progressed as far in terms of drug development. In vitro all three
mediate the characteristic response to Hsp90 inhibition–depletion
of client proteins and upregulation of heat shock proteins,6 but the
presence of epoxy and unsaturated carbonyl groups in radicicol
leads to inactivation in vivo by 1,6-Michael addition with thiol-de-
rived nucleophiles.9
Many small molecule inhibitors of Hsp90 have been reported.10
High-throughput screening of a chemical library of 60,000 com-
pounds identified the pyrazole based structure 3a (Fig. 1) as a
Hsp90 inhibitor.11 This compound causes depletion of Hsp90 client
proteins, induction of Hsp70, upregulation of heat shock proteins,
growth arrest and apoptosis in cancer cells, although in vivo activity
and toxicity have yet to be reported.1,12 The resorcinol ring mimics
the substitution on the aromatic ring of radicicol (2, Fig. 1). Exten-
sive structure–activity relationships have been determined for the
pyrazole based Hsp90 ligands.13–15 Other Hsp90 small-molecule
inhibitors include many purine-based analogues including 3c16
and indazole 4 as the first reported compound to target both
Hsp90 and tubulin (Fig. 1).17 At the end of 2010, 13 Hsp90 inhibitors
were reported to be in clinical trials, including 17-AAG 1b and a re-
lated geldanamycin derivative, 17-DMAG (1c, Fig. 1).18,19
Hsp90 consists of three flexibly linked domains. The N-terminal
domain contains an unusual adenine-nucleotide-binding pocket
known as the Bergerat fold.3 An ATPase cycle is central to the chap-
eroning activity of Hsp90. Hsp90 inhibitors geldanamycin (1a,
Abbreviations: 17-AAG, 17-allylamino-17-demethoxygeldanamycin; 17-DMAG,
17-dimethylaminoethylamino-17-demethoxygeldanamycin; DMF, dimethylform-
amide; DMSO, dimethylsulfoxide; GA, geldanamycin; HIF, hypoxia-inducable
factor; HRMS, high resolution molecular ion determination; Hsp90, heat shock
protein 90; HTMA, hexamethylenetetramine (hexamine); IR, infra red; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMR, nuclear magnetic
resonance; PBS, phosphate buffered saline; TBDMS, tertbutyldimethylchlorosilane;
TMCS, trimethylchlorosilane; TLC, thin layer chromatography.
⇑
Corresponding authors. Tel.: +353 1 8962798; fax: +353 1 8962793.
E-mail addresses: oboyleni@tcd.ie (N.M. O’Boyle), mmeegan@tcd.ie (M.J. Meegan).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.048

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N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
O
H
O
O
O
HN
O
O
R
O
O
Me
O
H2N
H
O
HO
OH
Cl
O
OH
1a: R=OCH3
2
1b
: R=NHCH2CHCH2
1c: R=NHCH2CH2N(CH3)2
OH
OH
H3CO
Cl
OCH3
OCH3
Cl
HO
HO
NH2
N
O
N
N
HN
N
HN
N
O
OCH3
N
O
N
H
3a
3b
3c
OH
O
HO
H3CO
H3CO
N
N
N
OCH3
O
O
O
NH2
5
4
Figure 1. Hsp90 binding compounds including geldanamycin 1a, 17-AAG 1b, 17-DMAG 1c, radicicol 2, pyrazole-based inhibitors 3a and 3b, purine-based inhibitor 3c, dual
tubulin-Hsp90 inhibitor 4 and proposed b-lactam based inhibitor 5.
Additional Hsp90 inhibitors for which structures have since been
disclosed and which are not previously reported in reviews include
Debio 0932 (formerly CUDC-305),20 AT1338721 and KW-2478.22
The number of structurally diverse Hsp90 inhibitors in clinical
trials indicates the ability of the Hsp90 ATP-binding site to accom-
modate a large variety of chemical structures. The pyrazole scaffold
of 3a is a small nitrogen-containing heterocyclic core, similar to a
b-lactam nucleus, and we were interested in determining if the
b-lactam scaffold was capable of acting as a template for Hsp90
inhibitors. The b-lactam ring scaffold is an established template
for antibiotics,23 cholesterol absorption inhibitors24 selective estro-
gen receptor modulators17 and antiproliferative tubulin-binding
antiproliferative agents.25,26 Herein, we report the synthesis and
biochemical evaluation of a b-lactam based Hsp90 inhibitor 5
(Fig. 1) and related compounds, which was designed to contain
similarly substituted aryl rings positioned at C-4 and N-1 to the
aryl substituents at C-3 and C-4 of pyrazole 3a (Figs. 1 and 2).
The proposed compounds would add further structural diversity
to the rapidly expanding field of small molecule Hsp90 inhibitors,
with potential applications in the area of Hsp90 based therapeu-
tics, and provide additional insights into the chemical scaffolds
that can be accommodated in the Hsp 90N-terminal ATP-binding
site.
2. Molecular modelling
Figure 2. Flexible alignment of pyrazole 3a (green) and b-lactam 5 (coloured by
atom; grey = carbon; red = oxygen; blue = nitrogen).
Molecular modelling studies on the proposed Hsp90-binding b-
lactam 5 were carried out to explore potential binding interactions
with the N-terminal ATP-binding site of Hsp90. In addition, the
synthetic imine precursors to b-lactam preparation were examined
as they possess the necessary pharmacophore required for binding

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
6057
to the Hsp90 protein. Existing X-ray co-crystal structures of the
Hsp90 N-terminal ATP-binding site with ADP,3 geldanamycin
(1a),8 radicicol (2),7 17-DMAG (1c)27 and small-molecule Hsp90
inhibitors pyrazole 3a, 3b11,28 and purine 3c16 (Fig. 1) provide in-
sight into the requirements for binding to Hsp90. The binding
pocket is of mixed hydrophobic and polar character, with approx-
imately half of the 17 amino acids lining its interior being hydro-
phobic, a quarter polar and a quarter charged. Mutation of the
Asp93 residue to asparagine abolishes Hsp90 function in vivo.7
As the binding pocket becomes increasingly hydrophobic towards
the bottom, Asp93 is the only charged residue in the deepest part
of the binding pocket, along with one polar residue (Thr 184).8
Asp93 is conserved in all known Hsp90 homologs from 35 species.8
These interactions are considered critical for binding of small
molecule Hsp90 inhibitors.
Imine 12, the synthetic precursor to b-lactam 5, was also
docked in the ATP-binding site on the N-terminal of Hsp90 as it
also contains the required pharmacophore for Hsp90 binding
(Figs. 5 and 6). The molecule is predicted to adopt a similar orien-
tation to radicicol, with the two hydroxyl groups and the ethyl
group penetrating deep into the pocket and interacting with
Asp93 and a conserved water molecule. The dioxane ring points to-
wards the top of the binding pocket and binding is reinforced by
strong hydrogen bonding interactions with Lys58 and Asn106
(Figs. 5 and 6). Interactions with Met98, Phe138, Lys58, Asp102,
Ala55 and Ser52 are also predicted for the imine. These interac-
tions are present for geldanamycin and other Hsp90 ligands and
are identified as a common requirement of Hsp90 activity in a
number of co-crystallised structures. On the basis of this molecular
docking study, it would be expected that imine 12 would display
Hsp90 binding affinity.
Molecular docking studies show that b-lactam 5 (Figs. 1–4) is
predicted to interact with the ATP-binding site of Hsp90 in a sim-
ilar manner to both radicicol and the pyrazole class of small mole-
cule inhibitors.7,11 Flexible alignment of 5 with pyrazole 3a reveals
a large degree of overlap between the resorcinol and benzodioxan
rings but a slight offset of the nitrogen heterocycle itself (Fig. 2).
When docked in the ATP-binding site of Hsp90, b-lactam 5 is seen
to be orientated with the ethylresorcinol ring extended towards
the bottom of the ATP-binding pocket, and the benzodioxane ring
towards the top of the pocket and into solvent (Fig. 3). This is a
similar binding conformation to radicicol and could be expected
from the similar substitution pattern on the aromatic rings of the
two compounds. The crucial interaction at the bottom of the bind-
ing pocket between a phenolic group on the b-lactam with Asp93 is
present (Fig. 3). Interactions with Thr184 are also seen for 5, mim-
icking key interactions of the endogenous adenine base and also
the natural product ligands geldanamycin and radicicol. Nearing
the top of the binding cavity, interactions with Lys112 are present
amongst others. A 2D representation of these interactions is illus-
trated (Fig. 4).29 Interactions with Asp93 are common to all Hsp90
ligands and can be considered to be necessary for binding to
Hsp90. b-Lactam compounds without the resorcinol hydroxyl
groups, such as methoxy-containing derivatives 19 and 20, are
not predicted to interact with Asp93 and this is likely to account
for the lack of binding affinity observed in vitro.
3. Chemistry
The 4-ethylresorcinol and 4-chlororesorcinol moieties are two
of the most commonly seen ring systems in a broad range of
Hsp90 inhibitors, including radicicol (2) and pyrazole 3a.10,12,30 In
order to design b-lactams containing these aryl substitution pat-
terns, the synthesis of appropriate aldehydes as precursors for
the required imines was first carried out. It was also necessary to
protect both hydroxyl groups of the resorcinol molecule prior to
the b-lactam forming reactions. 4-Ethylresorcinol and 4-chlorores-
orcinol are commercially available. The formylation of 4-ethylres-
orcinol (6b) was achieved using a Vilsmeier–Haack reaction,
utilising N,N-dimethylformamide and phosphorus oxychloride
(Scheme 1).31 The formylated product 7b was obtained from 6b
in yields of up to 36%. 1H NMR analysis shows an additional chem-
ical shift at d 9.91 ppm attributable to the aldehyde proton, there is
disappearance of one aromatic signal and the remaining two aro-
matic protons appear as singlets at d 6.39 and d 7.34 ppm. IR spec-
troscopy analysis shows absorption at
m
1645.8 cmÀ1 due to the
carbonyl group. The Vilsmeier–Haack reaction using phosphorous
oxychloride and anhydrous dimethylformamide was unsuccessful
for the formylation of 4-chlororesorcinol. The Duff reaction,32–34
using hexamethylenetetramine (hexamine) in acidic solution at
Figure 3. Docking of b-lactam 5 in the N-terminal of Hsp90
blue = nitrogen; hydrogen bonds shown as dashed blue lines and
a
(PBD code: 1OSF27). Key interactions for binding are depicted. Colour key: green/grey = carbon; red = oxygen;
interactions as dashed green lines.
p

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N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
Figure 4. 2D representation of proposed binding interactions of b-lactam 5 with Hsp90.
Figure 5. Imine 12 in the ATP-binding site of Hsp90 with selected residues for binding shown (PDB code: 1OSF27); Colour key: green/grey = carbon; red = oxygen;
blue = nitrogen; hydrogen bonds shown as dashed blue lines and interactions as dashed green lines.
p
room temperature did not result in formylation, whilst heating at
100 °C led to formylation at both the 2 and 6 positions of 4-chlor-
oresorcinol (6c) to afford 7c (Scheme 1).
Benzyl protection of the resorcinol phenolic groups was
achieved using benzyl bromide and potassium hydroxide to give
the desired product (Scheme 1). The phenolic groups of
commercially available 2,4-dihydroxybenzaldehyde (7a) were
protected by this method to prepare 8a (Scheme 1). The dibenzyl
protected product 8b was obtained in 90% yield from 5-ethyl-
2,4-dihydroxybenzaldehyde 7b. Use of the TBDMS group was also
investigated. This protecting group did not remain intact during
Vilsmeier–Haack formylation. Silylation of aldehyde 7b was

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
6059
Figure 6. 2D representation of proposed binding interactions of imine 12 with the ATP-binding site of Hsp90.
attempted, resulting in protection of only one of the phenolic
groups. This is thought to be due to intramolecular H-bonding be-
tween the aldehyde and phenolic groups on adjacent positions of
the aromatic ring.
Hsp90 imine precursors 9–18 were obtained by condensation of
the appropriately substituted aldehydes and amines (Scheme 1).
Yields for all products were over 85% with the exception of 18
(59%). The characteristic signal at approximately d 8.70–8.85 ppm
in the 1H NMR spectra attributable to the imine proton was ob-
served for all products.
b-lactam compound 5 mimics the substitution pattern of the pyr-
azole 3a. A number of analogues were designed to confirm that the
two hydroxyl groups and the ethyl group of the resorcinol ring are
essential for Hsp90 activity. b-Lactam 27 lacks an ethyl group, and
compounds 19 and 20 substitute methoxy groups for the hydrox-
yls. Pyrazoles with either a 4-methoxyphenyl ring28 or methylene-
dioxane11 ring in place of the benzodioxane ring has been shown to
have improved activity and the corresponding b-lactam analogues
28 and 29 were also synthesised. Finally, b-lactam containing the
trimethoxyphenyl ring found in the dual-acting inhibitor 4 was
also prepared.
The initial Hsp90-targeting b-lactams chosen for synthesis are
unsubstituted at the 3-position of the azetidinone ring (com-
pounds 19–25). The Reformatsky reaction using microwave tech-
nology was employed to synthesise these compounds in low
yields, by reaction of imines 9–18 with ethyl bromoacetate
(Scheme 2). All intermediate compounds 19–25 showed IR absorp-
4. Biochemical evaluation
The Hsp90 binding affinities of b-lactams 5, 27–31 (Scheme 3)
were first evaluated using a fluorescent displacement assay and
Hsp90 recombinant human protein, based on their ability to com-
pete with geldanamycin for Hsp90 binding.35 Two imines, 12 and
18, were also screened for activity as they possess the necessary
pharmacophore for binding to Hsp90. Of the series of b-lactams
evaluated, azetidinone 5 was the only compound to show a signif-
tions at approximately
m
1740 cmÀ1 confirming formation of the
azetidin-2-one. The synthesis of an alternative structural example,
compound 26, was achieved containing a phenyl substituent at the
3-position of the b-lactam ring, as we wished to investigate the ef-
fect of the introduction of a larger substituent at the C-3 position.
This analogue was obtained with exclusively trans geometry as evi-
denced by the coupling constant of 2.0 Hz between the protons at
positions 3 and 4 of the b-lactam ring.
Benzyl protecting groups were removed from compounds 21–
26 subsequent to the Reformatsky reaction by hydrogenation over
a palladium catalyst leaving the b-lactam ring intact to form six
different final products 5 and 27–31 (Scheme 3). The substitutions
on the N-1 aryl ring reflect the aryl substitution pattern observed
for pyrazole analogues with the greatest Hsp90 activity previously
reported in literature.11,17,28 As discussed previously, the lead
icant effect in the Hsp90
5.63 M (Fig. 7). The other seven analogues showed disappointing
activity with IC50 values greater than 200 M (Table 1). Low
a binding assay with an IC50 value of
l
l
activity was expected for b-lactams 19, 20 and 27 as they were
synthesised to confirm that the hydroxy and ethyl substituents of
the resorcinol ring were necessary for activity. Analogues 19 and
20 replace the hydroxy groups of 5 with methoxy groups, and this
substitution leads to marked decrease of Hsp90 binding ability.
This is consistent with literature reports that the hydroxyl groups

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N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
HO
OH
R
HO
OH
R
BnO
O
OBn
R
a
b
O
H
H
6a: R = H
7a
: R = H
8a R = H
8b
6b
6c
: R = CH2CH3
: R = Cl
7b
: R = CH2CH3
R = CH2CH3
c
O
H
HO
OH
Cl
O
H
7c
R3
R2
R1
O
NH2
R1
R2
CHO
R3
d
O
O
N
+
O
R=H unless otherwise indicated
9
: R2=R3=OCH3
10
: R1=R3=OCH3
11: R2=R3=OH
12
13
: R1=C2H5; R2=R3=OH
: R2=R3=OCH2C6H5
14: R1=C2H5; R2=R3=OCH2C6H5
R4O
OR4
R1
R2
NH2
CHO
OR4
d
R1
R2
N
+
R4O
R3
R3
R=H unless otherwise indicated
15
: R1=R2= -OCH2O-; R4=CH2C6H5
16: R1=R2=R3=OCH3; R4=CH2C6H5
17
18
: R2=OCH3; R4=CH2C6H5
: R1=R2= -OCH2O-
Scheme 1. Synthesis of imines 9–18. Reagents and conditions: (a) DMF, POCl3, 80 °C; (b) C6H5CH2Br, K2CO3, CH3CN; (c) HTMA, CF3COOH, 100 °C, 30 min; (d) ethanol, reflux,
3 h.
of the resorcinol ring are essential for hydrogen bonding interac-
tions with the Hsp90 protein.13,14 Analogue 27, with the hydroxyl
groups of 5 intact but without the ethyl group, shows decreased
activity compared to 5, indicating that the ethyl group of 5 is also
crucial for to Hsp90 binding activity. This pattern of activity was
seen for radicicol and analogues, where analogues that lack the
chloro substituent of radicicol at this position have substantially
lower affinity for Hsp90.36 The lack of binding affinity was not
anticipated for b-lactam 29, in which the benzodioxane ring of 5
dose–response curve for 12 is shown alongside that of azetidinone
5 and 17-AAG in Figure 7. These are the first reported imines with
Hsp90 binding activity.
The antiproliferative activity of Hsp90 binding compounds has
been evaluated in various cell lines including HCT116 colon
cells,15,28,37 MCF-7 breast cancer cells,38–42 SKBr3 breast cancer
cells38,41,43,44 and BT474 breast cancer cells.45 In the present work
the antiproliferative effects of the imines and azetidinones
synthesised was evaluated in human MCF-7 breast cancer cells
(Table 1). The most potent antiproliferative compound was dihy-
is replaced with
a 3,4-methylenedioxyphenyl moiety. This
substitution leads to an over 40-fold reduction in activity. Similar
substitution has been made in a purine series of compounds and
did not result in decreased activity.11 The monomethoxy ring at
the N-1 position of b-lactam 28 and trimethoxy ring at the N-1
position of b-lactam 30 also did not result in any Hsp90 binding
affinity. The substitutions at the N-1 position of the b-lactam ring
are likely to be orientated out of the ATP-binding pocket and into
solvent, not providing optimal binding compared to the benzodiox-
droxy analogue 27 with an IC50 value of 23.50
The only b-lactam that significantly inhibited Hsp90, 5, displayed
an IC50 value of 48.22 M in MCF-cells. The two imines that
showed inhibition of Hsp90, 12 and 18, did not show antiprolifer-
ative activity in MCF-7 cells at concentrations up to 100 M. The
lack of correlation between the binding affinity of these com-
pounds for Hsp90 and their antiproliferative activity warrants
future evaluation of these compounds in a cell line such as the
K562 chronic myelogenous leukaemia (CML), as a client protein
Bcr-Abl is readily degraded in response to Hsp90 inhibition.46
lM in MCF-7 cells.
l
l
ane ring of 5. Imines 12 and 18 exhibited IC50 values of 14.49
lM
and 22.08 M, respectively in the Hsp90 binding assay.
l
A

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
6061
R2
R1
R3
R3
R2
R1
O
a
O
O
N
+
O
N
Br
O
O
9 10, 13 14
,
,
O
R=H unless otherwise indicated
19
20
: R1=R3=OCH3
: R2=R3=OCH3
21: R2=R3=OCH2C6H5
22
: R1=C2H5; R2=R3=OCH2C6H5
OBn
OBn
+
O
a
R1
R2
N
OBn
N
O
O
OBn
Br
R3
R3
R2
R1
15 17
-
R=H unless otherwise indicated
23: R2=OCH3
24
25
: R1=R2= -OCH2O-
: R1=R2=R3=OCH3
OBn
OBn
O
a
O
O
N
+
O
OBn
N
OBn
Br
O
O
26
O
14
Scheme 2. Synthesis of azetidin-2-ones 19–26. Reagents and conditions: (a) zinc, TMCS, anhydrous benzene, microwave. Only one enantiomer is illustrated.
The ‘On Target’ activity of the b-lactam Hsp90 inhibitor 5 as well
as imine-scaffold compounds 12 and 15 was analysed by investigat-
ing their ability to bind to the Hsp90 ATP binding site and subse-
quently induce proteosomal degradation of the Hsp90 client
substituted aryl rings positioned at C-4 and N-1 to the aryl substit-
uents at C-3 and C-4 of pyrazole 3a, a known Hsp90 inhibitor as we
wished to determine if the b-lactam scaffold was capable of acting
as a template for providing the necessary interactions with the
Hsp90 ATP-binding site. Molecular modelling studies were used
prospectively to examine proposed binding interactions for both
b-lactam compound 5 and imine 12 in the ATP binding site of
protein, estrogen receptor alpha (ER-a). Human ER-apositive breast
cancer cells, MCF-7, were treated with vehicle, compound 5, 12, 18
or a known Hsp90 inhibitor, 17-AAG, at indicated concentrations
(Fig. 8). The positive control, 17-AAG, potently induces degradation
Hsp90. b-Lactam 5 displayed significant inhibition of Hsp90
a with
of ER-
ports.17,47 Test compounds 5, 12 and 18 were treated at concentra-
tions between 50 M and 200 M, comparable to IC50 values for
cell death induced by these compounds in MCF-7 cells (Table 1).
Results indicate that treatment with all three compounds induced
a
at concentrations as low as 1
l
M, in line with previous re-
an IC50 of 5.63 M and a moderate IC50 value of 48.22 M in an
l
l
antiproliferative assay using MCF-7 human breast cancer cells.
Two imines, 12 and 18 (synthetic precursors of the b-lactams 5
and 29 respectively), were identified as having the required phar-
macophore for Hsp90 binding and were also evaluated for Hsp90
binding activity. They displayed promising results with low micro-
l
l
degradation of ER-
binding displacement assay and cytotoxicity data, 5 was the most
potent compound inducing partial degradation at 50 M and com-
plete loss of ER- expression with 100 M and 150 M incubations.
a in MCF-7 cells (Fig. 8A and B). In line with the
molar inhibition of Hsp90
18, were demonstrated to induce proteosomal degradation of the
Hsp90 client protein ER . From our results showing the displace-
a. b-Lactam 5, as well as imines 12 and
l
a
l
l
a
These results indicate that the lead b-lactam compound 5 as well as
imines 12 and 18 act as Hsp90 inhibitors.
ment of the N-terminal binding agent geldanamycin, together with
the structural similarity of the compounds to the previously
reported N-terminal binding agents, we can conclude that these
b-lactams and imines bind at the N-terminal domain of Hsp90
rather than acting at the C-terminal domain in a manner that is
similar to the novobiocin analogues. These results expand the
range of known structural types accommodated by the ATP-
binding site of Hsp90 and may contribute to the future use of
5. Conclusion
Two novel templates for Hsp90 inhibitor design were identified.
The first reported b-lactam and imine inhibitors of Hsp90 are de-
scribed. b-Lactam compound 5 was designed to contain similarly

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N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
Table 1
OBn
OH
Antiproliferative and Hsp90-binding effects of b-lactams and imines
BnO
HO
Compound
IC50 MCF-7a
(l
M)
IC50 Hsp90ab
(lM)
a
5
12
18
19
48
>100
>100
54 26
2
5.6 3.6
14.5 2.6
22.1 1.4
>200
N
N
O
O
O
O
O
20
27
28
29
53
23 11
52
31 18
31
3
>200
>200
>200
>200
O
27
21
6
30
8
>200
31
17-AAG
>100
>200
1.19
0.0940
OBn
OH
a
MCF-7 IC50 values are half maximal concentrations required to inhibit the
growth stimulation of MCF-7 cells. Values represent the mean SEM (error values
Â10À6) for three independent experiments performed in triplicate.
a
b
OBn
OH
Hsp90
a values after 24 h using isolated human Hsp90a; 17-AAG is used as a
N
N
positive control in the Hsp90 fluorescent displacement assay and the value
O
O
R1
R1
obtained agrees with the reported IC50 value for binding of 17-AAG in Hsp90 of
1.27 l
M.28
R2
R2
R3
R3
antiproliferative activity and the physiochemical properties of
the identified lead compounds.
5
: R1=R2= -OCH2CH2O-
22 - 25
28: R2=OCH3
29
30
: R1=R2= -OCH2O-
: R1=R2=R3=OCH3
6. Experimental section
6.1. Experimental note
OBn
OH
All reagents were commercially available and were used with-
out further purification unless otherwise indicated. IR spectra were
recorded as thin films on NaCl plates or as KBr discs on a Perkin-El-
mer 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
or DMSO-d6 (internal standard tetramethylsilane) by Dr. John
O’Brien and Dr. Manuel Ruether in the School of Chemistry, Trinity
College Dublin. High resolution accurate mass determinations for
all final target compounds were obtained on a Micromass Time
of Flight mass spectrometer (TOF) equipped with electrospray ion-
isation (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 chroma-
tography 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 (230–400 mesh) obtained from Merck. Analytical
high-performance liquid chromatography (HPLC) to determine
the purity of the final compounds was performed using a Waters
2487 Dual Wavelength Absorbance detector, a Waters 1525 binary
HPLC pump, a Waters In-Line Degasser AF and a Waters 717plus
Autosampler. The column used was a Varian Pursuit XRs C18 re-
verse phase 150 Â 4.6 mm chromatography column. Samples were
detected using a wavelength of 254 nm. All samples were analysed
using a mobile phase consisting of acetonitrile (70%): water (30%)
over 10 min and a flow rate of 1 mL/min.
a
OBn
OH
N
N
O
O
O
O
O
O
26
31
Scheme 3. Synthesis of azetidin-2-ones 5, 27–31. Reagents and conditions: (a) H2,
Pd/C, ethanol/ethyl acetate (1:1). Only one enantiomer is illustrated.
Figure 7. Dose response graph for b-lactam 5, imine 12 and 17-AAG (1b) for
binding to Hsp90
a
. MCF-7 cells were seeded at a density of 2.5 Â 104 cells per well
in 96 well plates. The plates were left for 24 h to allow the cells to adhere to the
6.2. Procedure for Vilsmeir–Haack formylation of
4-ethylresorcinol
surface of the wells. A range of concentrations (0.01 nM–100 lM) 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 anti-proliferation. The values represent the mean SEM
(error values) for three experiments performed in triplicate.
6.2.1. 5-Ethyl-2,4-dihydroxybenzaldehyde (7b)
Dimethylformamide (38.2 mmol) and phosphorous oxychloride
(43.1 mmol) were mixed at 0 °C and stirred for 15 min before addi-
tion of 4-ethylbenzene-1,3-diol 6b (14.5 mmol) dissolved in
dimethylformamide (10 mL). The mixture was heated to 80 °C for
8 h. The reaction was quenched by the slow and careful addition
Hsp90 directed therapeutics for the treatment of cancer. Future
SAR work will aim to extend the SAR and improve the

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
6063
Figure 8. b-Lactam compound 5 and imines 12 and 18 induce Hsp90 client protein degradation. b-Lactam scaffold compound 5 and imine scaffold compounds 12 and 18
induced proteasomal degradation of Hsp90 client protein, Estrogen Receptor Alpha (ERa), in the ERa+ breast cancer cell line, MCF-7. MCF-7 cells were treated with indicated
concentrations of 5, 12 and 18 in figure (A) and (B). Cells were seeded at a density of 1.0 Â 106 cells/25 cm2 tissue culture flasks (5 mL Media). Cells were treated with Vehicle
control (C—DMSO (0.5%)), test compounds 5, 12, 18 and positive control 17-AAG (1, 5 and 10 lM) and left to incubate for 24 h. Whole cell lysates were prepared and ERa
protein expression was analysed by SDS-PAGE and Western Blot analysis. Beta-actin was used as a protein loading control. Results are representative of three independent
experiments.
of saturated aqueous sodium bicarbonate solution (150 mL) and
was stirred overnight. The solution was extracted with CH2Cl2
(50 mL three times) and the combined organic layers were dried
over Na2SO4. The pure product was isolated by flash column chro-
matography over silica gel (eluent: hexane/ethyl acetate gradient)
and isolated as a white powder (yield 36.0%); Mp: 132 °C (lit. mp:
6.3. General procedure for dibenzyl protection of resorcinol
derivatives
Benzyl bromide (0.11 mol) was added to a mixture of resorcinol
derivative (0.045 mol) and potassium carbonate (0.11 mol) in ace-
tonitrile (200 mL). The mixture was heated at reflux for 5 h and
stirred overnight at room temperature. The mixture was filtered
and the solid filter cake was washed with CH2Cl2 (200 mL). The
combined organic fractions were evaporated in vacuo to leave
the product. The crude product was triturated with hexane and fil-
tered to give the pure product.
130–131 °C48); IR (KBr)
m
max: 1645.83 (–C@O), 3193.79 (broad,
–OH) cmÀ1
;
1H NMR (400 MHz, DMSO-d6) d 1.12 (t, 3H, CH3),
2.49 (q, 2H, CH2), 6.39 (s, 1H, ArH), 7.34 (s, 1H, ArH), 9.91 (s, 1H,
CHO); 13C NMR (400 MHz, DMSO-d6) d 13.92 (CH3), 21.76 (CH2),
101.79, 114.80, 122.98, 130.63, 161.44, 163.08 (ArC), 190.78
(C@O); HRMS: C9H10O3 requires 167.0708; found 167.0711; Ele-
mental Anal. C9H10O3 requires C, 65.05; H, 6.07. Found: C, 64.99;
H, 6.07.
6.3.1. 2,4-Bisbenzyloxybenzaldehyde (8a)
Compound 8a was prepared from 2,4-dihydroxybenzaldehyde
7a and isolated as a white solid (98.0% yield); Mp: 85 °C (lit. mp:
6.2.2. 5-Chloro-2,4-dihydroxyisophthalaldehyde (7c)
85–86 °C49); IR (KBr) mmax 1677.88 cmÀ1 (–C@O); 1H NMR
:
To 4-chlorobenzene-1,3-diol (3 mmol) in trifluoroacetic acid
(40 mL) was added HTMA (30 mmol, 10 equiv). The mixture was
heated to 100 °C for 30 min after which it was left to cool to room
temperature. Water (60 mL) was carefully added followed by so-
dium bicarbonate (with vigorous stirring) until neutralised. Dichlo-
romethane (100 mL) was added and the mixture was stirred for 2 h
at room temperature. The layers were separated, the aqueous layer
was extracted with dichloromethane/methanol (9:1, 100 mL) and
the combined organic fractions were dried with anhydrous Na2SO4
and the solvent was removed in vacuo. The product was isolated as
a white solid in 56.2% yield; 1H NMR (400 MHz, CDCl3) d 7.29 (s,
1H, ArH), 7.79 (s, 1H, ArH), 9.73 (s, 1H, CHO), 10.41 (s, 1H, CHO),
12.42 (s, 1H, OH), 13.26 (s, 1H, OH); 13C NMR (400 MHz, CDCl3) d
109.45, 113.15, 139.96, 164.44, 164.87 (ArC), 192.79 (C@O),
193.34 (C@O).
(400 MHz, DMSO-d6) d 5.23 (s, 2H, CH2), 5.29 (s, 2H, CH2), 6.76
(d, 1H, J = 8.8 Hz, ArH), 6.93 (s, 1H, ArH), 7.36–7.68 (m, 10H,
ArH), 7.70 (d, 1H, J = 8.8 Hz, ArH), 10.25 (s, 1H, CHO); 13C NMR
(100 MHz, DMSO-d6) d 70.34 (CH2), 70.38 (CH2), 100.93, 108.15,
119.11, 128.03, 128.47, 128.63, 129.01, 129.04, 130.37, 136.67,
136.84, 162.87, 165.44 (ArC), 187.77 (C@O); HRMS: C21H18O3Na
requires 341.1154; found: 341.1155 (M++Na); Elemental Anal.
C21H18O3 requires C, 79.22; H, 5.70. Found: C, 78.89; H, 5.73.
6.3.2. 2,4-Bisbenzyloxy-5-ethylbenzaldehyde (8b)
Compound 8b was prepared from 5-ethyl-2,4-dihydroxybenzal-
dehyde (7b) according to the procedure above. The product was
obtained as a white powder (yield 89.5%); Mp: 123 °C; IR (KBr)
m
max: 1663.00 cmÀ1 (–C@O); 1H NMR (400 MHz, DMSO-d6) d 1.12
(t, 3H, CH3), 2.54 (q, 2H, CH2), 5.28 (s, 2H, CH2), 5.30 (s, 2H, CH2),

6064
N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
7.00 (s, 1H, ArH), 7.35–7.51 (m, 11H, ArH), 10.24 (s, 1H, CHO); 13C
NMR (100 MHz, DMSO-d6) d 13.90 (CH3), 22.05 (CH2), 69.86 (CH2),
70.21 (CH2), 98.33, 117.76, 125.06, 127.50, 127.55, 127.68, 128.06,
128.59, 128.63, 128.69, 129.29, 136.45, 161.34, 162.56 (ArC),
187.26 (C@O); HRMS: C23H22O3Na requires 369.1467; found:
369.1465 (M++Na); Elemental Anal. C23H22O3 requires C, 78.83;
H, 6.31. Found: C, 79.74; H, 6.40.
1H NMR (400 MHz, DMSO-d6) d 1.13 (t, 3H, CH3), 2.46–2.51 (m,
2H, CH2), 4.25 (s, 4H, OCH2CH2O), 6.34 (s, 1H, ArH), 6.84–6.92
(m, 3H, ArH), 7.26 (s, 1H, ArH), 8.71 (s, 1H, CH@N), 10.14 (broad
s, 1H, OH), 13.35 (broad s, 1H, OH); 13C NMR (100 MHz, DMSO-
d6) d 14.20 (CH3), 21.90 (CH2), 64.07 (OCH2CH2O), 64.12 (OCH2-
CH2O), 102.03, 109.02, 111.72, 114.56, 117.46, 121.97, 132.61,
141.87, 142.04, 143.79 (ArC), 159.85 (C@N), 160.81, 161.17 (ArC);
HRMS: C17H18NO4 requires 300.1236; found 300.1237 (M++H); Ele-
mental Anal. C17H17NO4 requires C, 68.21; H, 5.72; N, 4.68. Found:
C, 67.85; H, 5.85; N, 4.50.
6.4. General method for imine preparation
The appropriate amine (10 mmol) was heated at reflux with the
appropriate aldehyde (10 mmol) in ethanol (50 mL) for 3 h. The
reaction mixture was cooled and then the solvent evaporated in
vacuo. The resulting solid product was recrystallised from ethanol.
6.4.5. (2,4-Bisbenzyloxybenzylidene)(2,3-dihydrobenzo [1,4]
dioxin-6-yl)amine (13)
Compound 13 was prepared from 2,3-dihydrobenzo[1,4]dioxin-
6-ylamine and 2,4-bisbenzyloxybenzaldehyde (8a) as a yellow
6.4.1. (2,3-Dihydrobenzo[1,4]dioxin-6-yl)(2,4-dimethoxybenzy
lidene)amine (9)
powder in 90.5% yield; Mp: 144 °C; IR (KBr) m
max: 1608.90 cmÀ1
(–N@C–); 1H NMR (400 MHz, CDCl3) d 4.29 (s, 4H, OCH2CH2O),
5.12–5.14 (s, 4H, 2 Â CH2), 6.63 (d, 1H, J = 2.04 Hz, ArH), 6.68–6.71
(dd, 1H, ArH), 6.78–6.82 (m, 2H, ArH), 6.88 (d, 1H, J = 8.56 Hz,
ArH), 7.36–7.44 (m, 10H, ArH), 8.15 (d, 1H, J = 8.52 Hz, ArH), 8.87
(s, 1H, CH@N); 13C NMR (100 MHz, CDCl3) d 63.91 (CH2), 63.98
(CH2), 69.77 (CH2), 70.01 (CH2), 99.85, 106.52, 109.29, 114.28,
116.94, 126.90, 127.14, 127.67, 127.76, 127.87, 128.24, 128.28,
128.32, 128.50, 135.91, 141.30, 143.18 (ArC), 154.03 (C@N),
159.47, 162.18 (ArC); HRMS: C29H26NO4 requires 452.1862; found
452.1865 (M++H); Elemental Anal. C29H25NO4 requires: C, 77.14;
H, 5.58; N, 3.10. Found: C, 76.89; H, 5.52; N, 2.99.
Compound 9 was prepared from 2,3-dihydrobenzo[1,4]dioxin-
6-ylamine and 2,4-dimethoxybenzaldehyde and isolated as a
brown oil in 76.8% yield and was used in the subsequent reaction
without further purification; IR (KBr) m
max: 1609.48 cmÀ1 (–N@C–);
1H NMR (400 MHz, CDCl3) d 3.86 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 4.26 (s, 4H, OCH2CH2O), 6.46 (s, 1H, ArH), 6.58 (d, 1H,
J = 8.80 Hz, ArH), 6.81–6.89 (m, 3H, ArH), 8.15 (d, 1H, J = 8.76 Hz,
ArH), 8.82 (s, 1H, HC@N); 13C NMR (100 MHz, CDCl3) d 55.05,
55.09, 63.10, 64.24, 97.55, 97.92, 103.68, 105.16, 108.22, 109.25,
114.33, 116.89, 128.30, 143.18, 146.30, 154.30, 160.37, 163.13
(ArC); HRMS: C17H18NO4 requires 300.1236; found 300.1239;
(M++H).
6.4.6. (2,4-Bisbenzyloxy-5-ethylbenzylidene)(2,3-dihydrobenzo
[1,4]dioxin-6-yl)amine (14)
6.4.2. (2,3-Dihydrobenzo[1,4]dioxin-6-yl)(2,5-dimethoxybenzy
lidene)amine (10)
Compound 14 was prepared from 2,3-dihydrobenzo[1,4]dioxin-
6-ylamine and 2,4-bisbenzyloxy-5-ethylbenzaldehyde (8b) as a
Compound 10 was prepared from 2,3-dihydrobenzo[1,4]dioxin-
6-ylamine and 2,5-dimethoxybenzaldehyde as a yellow solid in
yellow powder in 86.9% yield; Mp: 158 °C; IR (KBr) mmax:
1625.71 cmÀ1 (–N@C–); 1H NMR (400 MHz, CDCl3) d 1.23–1.27 (t,
3H, CH3), 2.64–2.70 (m, 2H, CH2), 4.30 (s, 4H, OCH2CH2O), 5.14
(s, 4H, 2 Â CH2), 6.58 (s, 1H, ArH), 6.82–6.86 (m, 2H, ArH), 6.91
(d, 1H, J = 8.52 Hz, ArH), 7.13 (s, 1H, ArH), 7.35–7.39 (m, 10H,
ArH), 8.50 (s, 1H, CH@N); 13C NMR (100 MHz, CDCl3) d 13.80
(CH3), 22.18 (CH2), 63.94 (CH3), 63.97 (CH3), 69.45 (CH3), 99.73,
109.03, 111.89, 113.99, 117.26, 123.80, 126.71, 127.51, 128.16,
131.27, 136.22, 141.91, 143.49 (ArC), 159.64 (C@N), 160.19,
25.0% yield; Mp: 69 °C; IR (KBr) m
max: 1621.25 cmÀ1 (–N@C–); 1H
NMR (400 MHz, DMSO-d6) d 3.77 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3), 4.26 (s, 4H, OCH2CH2O), 6.78–6.90 (m, 3H, ArH), 7.10 (s,
2H, ArH), 7.49 (s, 1H, ArH), 8.78 (s, 1H, CH@N); 13C NMR
(100 MHz, DMSO-d6) d 55.93 (OCH3), 57.74 (OCH3), 64.52 (CH2),
64.55 (CH2), 110.01, 110.36, 114.04, 114.75, 117.85, 119.67,
124.88, 142.50, 144.06, 145.90 (ArC), 153.64 (C@N), 154.14,
154.21 (ArC); HRMS:
C
17H18NO4 requires 300.123; found
161.73 (ArC); HRMS: C31H30NO4 requires 480.2175; found
300.1235, (M++H); Elemental Anal. C17H17NO4 requires C, 68.21;
H, 5.72; N, 4.68. Found: C, 68.17; H, 5.72; N, 4.74.
480.2185 (M++H); Elemental Anal. C31H29NO4 requires C, 77.64;
H, 6.10; N, 2.92. Found: C, 77.36; H, 6.11; N, 2.96.
6.4.3. 4-[(2,3-Dihydrobenzo[1,4]dioxin-6-yli mino)methy l]ben
zene-1,3-diol (11)
6.4.7. Benzo[1,3]dioxol-5-yl(2,4-bisbenzyloxy-5-ethylbenzylid
ene)amine (15)
Compound 11 was prepared from 2,3-dihydrobenzo[1,4]dioxin-
6-ylamine and 2,4-dihydroxybenzaldehyde as an orange solid in
Compound 15 was prepared from benzo[1,3]dioxol-5-ylamine
and 2,4-bisbenzyloxy-5-ethylbenzaldehyde (8b) as a brown pow-
95.4% yield; Mp: 144 °C; IR (KBr)
m
max: 1625.03 cmÀ1 (–N@C–);
der in 87.8% yield; Mp: 129 °C; IR (KBr) m
max: 1611.89 cmÀ1
1H NMR (400 MHz, DMSO-d6) d 4.27 (s, 4H, OCH2CH2O), 6.28 (s,
1H, ArH), 6.40 (m, 1H, ArH), 6.85–6.95 (m, 3H, ArH), 7.40 (s, 1H,
ArH), 8.75 (s, 1H, CH@N), 10.22 (broad s, 1H, OH), 13.62 (broad s,
1H, OH); 13C NMR (100 MHz, DMSO-d6) d 64.54 (CH2), 64.58
(CH2), 102.81, 108.17, 109.56, 112.53, 115.10, 117.94, 134.67,
142.08, 142.61, 144.26 (ArC), 161.63 (C@N), 162.57, 163.27 (ArC);
HRMS: C15H12NO4 requires 270.0766; found 270.0776 (M++H); Ele-
mental Anal. C15H13NO4 requires C, 66.41; H, 4.83; N, 5.16. Found:
C, 66.14; H, 4.84; N, 5.24.
(–N@C–); 1H NMR (400 MHz, CDCl3) d 1.26 (t, 3H, CH3), 2.68–
2.73 (m, 2H, CH2), 5.11 (s, 4H, 2xCH2), 5.99 (s, 2H, OCH2O), 6.55
(s, 1H, ArH), 6.81 (s, 1H, ArH), 6.81–6.84 (m, 2H, ArH), 7.42–7.44
(m, 10H, ArH), 7.98 (s, 1H, ArH), 8.85 (s, 1H, CH@N); 13C NMR
(100 MHz, CDCl3) dd 13.94 (CH3), 22.45 (CH2), 69.54 (CH2), 70.57
(CH2), 97.22, 100.76 (OCH2O), 101.60, 107.82, 114.27, 117.37,
125.94, 126.60, 126.81, 126.88, 127.22, 127.56, 127.68, 128.22,
128.27, 136.22, 136.26, 145.02, 147.64 (ArC), 154.21 (C@N),
157.97, 159.64 (ArC); HRMS: C30H28NO4 requires 466.2018; found
466.2018 (M++H); Elemental Anal. C30H27NO4 requires C, 77.40; H,
5.85; N, 3.01. Found: C, 76.38; H, 5.83; N, 2.93.
6.4.4. 4-[(2,3-Dihydrobenzo[1,4]dioxin-6-ylimino)methyl]-6-
ethylbenzene-1,3-diol (12)
Compound 12 was prepared from 5-ethyl-2,4-dihydroxybenzal-
dehyde (7b) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine as or-
6.4.8. (2,4-Bisbenzyloxy-5-ethylbenzylidene)(3,4,5-trimethoxy
phenyl)amine (16)
ange powder in 87.0% yield; Mp: 181 °C; IR (KBr) mmax
:
Compound 16 was prepared from 3,4,5-trimethoxyaniline and
2,4-bisbenzyloxy-5-ethylbenzaldehyde (8b) as pale yellow flakes
1628.34 cmÀ1, 1611.89 cm-1 (–N@C–), 3439.61 cmÀ1 (broad, OH);

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
6065
in 84.5% yield; Mp: 129 °C; IR (KBr)
m
max: 1607.16 cmÀ1 (–N@C–);
and ethyl 2-bromoacetate in 13.2% yield as an orange powder;
1H NMR (400 MHz, CDCl3) d 1.25 (t, 3H, CH3), 2.65–2.73 (m, 2H,
CH2), 3.90 (m, 9H, 3 Â OCH3), 5.12 (s, 4H, 2 Â CH2), 5.96 (s, 1H,
ArH), 6.45–6.56 (m, 3H, ArH), 7.36–7.43 (m, 11H, ArH), 8.90 (s,
1H, CH@N); 13C NMR (100 MHz, CDCl3) d 13.90 (CH3), 22.44
(CH2), 55.46 (OCH3), 55.62 (OCH3), 60.59 (OCH3), 69.58 (CH2),
70.57 (CH2), 92.13, 97.17, 97.79, 126.61, 126.81, 127.61, 127.72,
127.85, 128.04, 128.24, 128.31, 136.18, 153.01 (ArC), 155.19
(C@N); HRMS: C32H34NO5 requires 512.2437; found 512.2438
(M++H); Elemental Anal. C32H33NO5 requires C, 75.12; H, 6.50; N,
2.74. Found: C, 74.82; H, 6.44; N, 2.65.
melting point: 164 °C; purity: 98.2%; IR (NaCl film) mmax
:
1746.30 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz, CDCl3) d
2.85–2.90 (dd, 1H, H3), 3.49–3.54 (m, 1H, H3), 3.71 (s, 3H, OCH3),
3.86 (s, 3H, OCH3), 4.23 (s, 4H, OCH2CH2O), 5.28–5.31 (m, 1H,
H4), 6.76–6.88 (m, 6H, ArH); 13C NMR (100 MHz, CDCl3) d 45.27
(C3, CH2), 48.41 (C4), 55.27 (OCH3), 55.53 (OCH3), 63.75 (CH2),
63.99 (CH2), 105.74, 109.90, 111.14, 111.99, 112.81, 116.98,
126.78, 131.60, 139.53, 143.13, 150.75, 153.35 (ArC), 164.17
(C@O); HRMS: C19H20NO5 requires 342.1341; found 342.1356
(M++H).
6.4.9. (2,4-Bisbenzyloxy-5-ethylbenzylidene)(4-methoxyphenyl)
amine (17)
6.5.2. 1-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)-4-(2,4-dimethoxy
phenyl)azetidin-2-one (20)
Compound 17 was prepared from 4-methoxyphenylamine and
2,4-bisbenzyloxy-5-ethylbenzaldehyde (8b) as a yellow powder in
90.5% yield; Mp: 139–140 °C; IR (KBr) mmax
Compound 20 was prepared from (2,3-dihydrobenzo[1,4]diox-
in-6-yl)(2,4-dimethoxy-benzylidene)amine (9) and ethyl 2-bromo-
acetate in 2.0% yield as a yellow oil; purity: 96.1%; IR (NaCl film)
:
1609.00 cmÀ1
(–N@C–); 1H NMR (400 MHz, CDCl3) d 1.27 (t, 3H, CH3), 2.68–2.74
(q, 2H, CH2), 3.87 (s, 3H, OCH3), 5.11–5.13 (s, 4H, 2 Â CH2), 6.55 (s,
1H, ArH), 6.94 (m, 2H, ArH), 7.23–7.44 (m, 13H, ArH), 8.89 (s, 1H,
CH@N); 13C NMR (100 MHz, CDCl3) d 13.93 (CH3), 22.45 (CH2),
55.05 (OCH3), 69.58 (CH2), 70.60 (CH2), 97.21 113.85, 114.34,
115.98, 121.77, 126.60, 126.81, 126.88, 127.58, 127.69, 127.75,
127.85, 128.04, 128.23, 128.26, 128.30, 136.21 (ArC), 153.95
(C@N), 161.12, 162.30 (ArC); HRMS: C30H30NO3 requires 452.2226;
found 452.2217 (M++H); Elemental Anal. C30H29NO3 requires C,
79.80; H, 6.47; N, 3.10. Found: C, 79.72; H, 6.47; N, 3.32.
m
max: 1744.56 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz, CDCl3) d
2.85–2.89 (dd, 1H, H3), 3.45–3.50 (m, 1H, H3), 3.81 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 4.21 (s, 4H, OCH2CH2O), 5.23–5.25 (m, 1H,
H4), 6.44–6.50 (m, 2H, ArH), 6.75 (d, 1H, J = 9.56 Hz, ArH), 6.84–
6.87 (m, 2H, ArH), 7.13 (d, 1H, J = 8.56 Hz, ArH); 13C NMR
(100 MHz, CDCl3) d 45.28 (C3), 48.40 (C4), 54.94 (OCH3), 55.03
(OCH3), 63.75 (CH2), 64.00 (CH2), 98.21, 103.96, 105.73, 109.93,
116.93, 117.85, 126.70, 131.71, 139.43, 143.10, 157.73, 160.27
(ArC), 164.42 (C@O); HRMS: C19H20NO5 requires 342.1341; found
342.1331 (M++H).
6.4.10. 4-((Benzo[d][1,3]dioxol-5-ylimino)methyl)-6-ethylbenz
ene-1,3-diol (18)
6.5.3. 4-(2,4-Bisbenzyloxyphenyl)-1-(2,3-dihydrobenzo [1,4]
dioxin-6-yl)azetidin-2-one (21)
Compound 18 was prepared from 5-ethyl-2,4-dihydroxybenzal-
dehyde (7b) and benzo[d][1,3]dioxol-5-amine as a green powder in
Compound 21 was prepared from (2,4-bisbenzyloxy-benzyli-
dene)(2,3-dihydrobenzo[1,4]dioxin-6-yl)amine (13) and ethyl 2-
bromoacetate in 17.9% yield as a brown gel; purity: 99.3%; IR (NaCl
59.6% yield; Mp: 176 °C; IR (KBr) m
max: 1633.59 cmÀ1, 1610.87 cmÀ1
(–N@C–); 1H NMR (400 MHz, DMSO-d6) d 1.13 (t, 3H, CH3), 2.45–
2.51 (m, 2H, CH2), 6.06 (s, 2H, OCH2O), 6.34 (s, 1H, ArH), 6.83 (d,
1H, J = 2.24 Hz, ArH), 6.94 (d, 1H, J = 8.28 Hz, ArH), 7.08 (d, 1H,
J = 2 Hz, ArH), 7.25 (s, 1H, ArH), 8.73 (s, 1H, CH@N), 10.18 (s, 1H,
OH), 13.28 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) d 14.67
(CH3), 22.35 (CH2), 101.51, 101.89 (CH2), 102.49, 108.91, 112.15,
115.97, 122.46, 133.06, 143.32, 146.17, 148.68, 160.33 (ArC),
film) m
max: 1745.51 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz,
CDCl3) d 2.87–2.92 (dd, 1H, H3), 3.44–3.49 (dd, 1H, H3), 4.22 (s,
4H, OCH2CH2O), 5.03 (s, 2H, CH2), 5.11 (s, 2H, CH2), 5.29–5.31
(m, 1H, H4), 6.54–6.56 (m, 1H, ArH), 6.67 (s, 1H, ArH), 6.76–6.91
(m, 4H, ArH), 7.29–7.47 (m, 10H, ArH); 13C NMR (100 MHz, CDCl3)
d 45.33 (C3), 48.36 (C4), 63.76 (CH3), 63.83 (CH3), 69.82 (CH3),
100.32, 105.46, 105.76, 109.91, 116.95, 118.58, 126.74, 126.93,
126.97, 127.10, 127.12, 127.15, 127.69, 127.72, 128.17, 128.22,
128.25, 131.71, 136.02, 136.20, 139.46, 143.15, 156.74, 159.37
(ArC), 164.28 (C@O); HRMS: C31H27NO5Na requires 516.1787;
found 516.1792 (M++H).
161.15 (C@N), 161.56 (ArC); HRMS:
C16H16NO4 requires
286.1079; found 286.1075 (M++H); Elemental Anal. C16H15NO4 re-
quires C, 67.36; H, 5.30; N, 4.91. Found: C, 66.52; H, 5.32; N, 4.94.
6.5. General method for synthesis of azetidinones 19–26
6.5.4. 4-(2,4-Bisbenzyloxy-5-ethylphenyl)-1-(2,3-dihydrobenzo
[1,4]dioxin-6-yl)azetidin-2-one (22)
Zinc powder (0.927 g, 15 mmol) was activated using trimethyl-
chlorosilane (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 cooling, the appropriately substituted imine
(10 mmol) and substituted ethylbromoacetate (12 mmol) were
added to the reaction vessel and the mixture was refluxed 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 (50 mL). This solution was washed with satu-
rated 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 so-
dium sulfate and the solvent was removed in vacuo. The pure prod-
uct was isolated by flash column chromatography over silica gel
(eluent: hexane/ethyl acetate gradient).
Compound 22 was prepared from (2,4-bisbenzyloxy-5-ethylb-
enzylidene)(2,3-dihydrobenzo[1,4]dioxin-6-yl)amine (14) and
ethyl 2-bromoacetate in 5.6% yield as a brown gel and was depro-
tected to prepare 5 without further characterisation; HRMS:
C
33H31NO5Na requires 544.2100; found 544.2109 (M++Na).
6.5.5. 4-(2,4-Bis(benzyloxy)-5-ethylphenyl)-1-(4-methoxyp
henyl)azetidin-2-one (23)
Compound23 was prepared from (2,4-bisbenzyloxy-5-ethylben-
zylidene)(4-methoxyphenyl)amine (17) and ethyl 2-bromoacetate
in 4.5% yield as a yellow solid; purity: 94.1%; IR (NaCl film) mmax
:
1727.60 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz, DMSO-d6) d
1.02–1.06 (t, 3H, CH3), 2.55–2.61 (m, 2H, CH2), 2.88–2.92 (dd, 1H,
H3), 3.45 (m, 3H, OCH3), 4.36–4.39 (m, 1H, H3), 5.06–5.25 (m, 4H,
2 Â CH2), 5.31 (m, 1H, H4), 6.87 (m, 2H, ArH), 6.92 (s, 1H, ArH),
7.01 (s, 1H, ArH), 7.13 (m, 2H, ArH), 7.40–7.45 (m, 10H, ArH); 13C
NMR (100 MHz, DMSO-d6) d 14.70 (CH3), 22.61 (CH2), 45.21 (C3),
48.73 (C4), (OCH3), 69.86 (CH2), 70.34 (CH2), 99.33, 114.63, 117.76,
117.90, 124.66, 127.31, 127.71, 127.96, 128.12, 128.17, 128.79,
6.5.1. 1-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)-4-(2,5-dimethoxy
phenyl)azetidin-2-one (19)
Compound 19 was prepared by reaction of (2,3-dihydro-
benzo[1,4]dioxin-6-yl)(2,5-dimethoxy-benzylidene)amine
(10)

6066
N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
128.82, 131.76, 137.29, 137.52, 155.51, 155.58, 156.83 (ArC), 164.43
(C@O); HRMS: C32H31NO4Na requires 516.2151; found 516.2156
(M++Na).
6.6.1. 1-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)-4-(5-ethyl-2,4-
dihydroxyphenyl)azetidin-2-one (5)
Compound 5 was prepared from 4-(2,4-bisbenzyloxy-5-ethyl-
phenyl)-1-(2,3-dihydrobenzo[1,4]dioxin-6-yl)azetidin-2-one (22)
in 13.2% yield as a brown oil (purity: 97.4%); IR (NaCl film) mmax
:
6.5.6. 1-Benzo[1,3]dioxol-5-yl-4-(2,4-bisbenzyloxy-5-ethylphen
yl)azetidin-2-one (24)
1701.08 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz, DMSO-d6) d
1.02 (t, 3H, CH3), 2.40 (m, 2H, CH2), 2.93–2.96 (dd, 1H, H3), 3.37–
3.41 (dd, 1H, H3), 4.16–4.21 (m, 4H, OCH2CH2O), 5.12–5.13 (m,
1H, H4), 6.37 (s, 1H, ArH), 6.76 (m, 2H, ArH), 6.87 (s, 1H, ArH),
7.32 (s, 1H, ArH), 9.21 (s, 1H, OH), 9.40 (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6) d 14.87 (CH3), 21.07 (CH2), 44.69 (C3), 49.13
(C4), 64.16 (CH2), 64.53 (CH2), 102.83, 105.42, 109.89, 113.63,
117.51, 121.41, 127.80, 132.38, 139.64, 143.55, 154.56, 155.77
(ArC), 164.90 (C@O); HRMS: C19H18NO5 requires 340.1185; found
340.1187 (M++H).
Compound 24 was prepared from benzo[1,3]dioxol-5-yl-(2,4-
bisbenzyloxy-5-ethylbenzylidene)amine (15) and ethyl 2-bromo-
acetate in 6.2% yield as a brown gel; purity: 87.3%; IR (NaCl film)
m
max: 1738.89 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz, CDCl3) d
1.13 (t, 3H, CH3), 2.55–2.65 (m, 2H, CH2), 2.92–2.96 (dd, 1H, H3),
3.43–3.48 (m, 1H, H3), 5.07 (m, 4H, 2xCH2), 5.30–5.32 (m, 1H,
H4), 5.93 (s, 2H, OCH2O), 6.60 (s, 1H, ArH), 6.68 (s, 2H, ArH),
7.03–7.07 (m, 2H, ArH), 7.36–7.44 (m, 10H, ArH); 13C NMR
(100 MHz, CDCl3) d 14.02 (CH3), 22.37 (CH2), 45.23 (C3), 48.64
(C4), 69.75 (CH2), 70.36 (CH2), 97.82, 99.00, 100.64 (OCH2O),
107.75, 109.01, 125.48, 126.43, 126.62, 126.97, 127.47, 127.73,
128.17, 128.25, 132.41, 136.27, 136.63, 143.18, 147.32, 154.73,
6.6.2. 1-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)-4-(2,4-dihydrox
yphenyl)azetidin-2-one (27)
156.54 (ArC), 164.41 (C@O); HRMS:
C
32H29NO5Na requires
Compound 27 was prepared from 4-(2,4-bisbenzyloxyphenyl)-
1-(2,3-dihydrobenzo[1,4]dioxin-6-yl)azetidin-2-one (21) in 17.6%
yield as an yellow powder; melting point: 166 °C; purity: 97.3%;
530.1943; found 530.1953 (M++Na).
IR (NaCl film)
m
max: 1712.93 cmÀ1 (C@O, b-lactam); 1H NMR
6.5.7. 4-(2,4-Bisbenzyloxy-5-ethylphenyl)-1-(3,4,5-trimethox
yphenyl)azetidin-2-one (25)
(400 MHz, DMSO-d6) d 2.88–2.92 (dd, 1H, H3, J = 17.08 Hz,
J = 12.52 Hz), 3.35–3.43 (dd, 1H, H3, J = 20.56 Hz, J = 9.04 Hz),
4.17 (m, 4H, OCH2CH2O), 5.13–5.15 (dd, 1H, H4, J = 8.04 Hz,
J = 2.88 Hz), 6.18–6.21 (m, 1H, ArH), 6.30 (m, 1H), 6.70–6.76 (m,
3H, ArH), 6.96 (d, 1H, J = 8.56 Hz, ArH), 9.35 (s, 1H, OH), 9.68 (s,
1H, OH); 13C NMR (100 MHz, DMSO-d6) d 44.48 (C3), 48.61 (C4),
63.82 (OCH2CH2O), 64.20 (OCH2CH2O), 102.54, 105.07, 106.77,
109.52, 113.89, 117.24, 128.06, 131.91, 143.24, 156.46, 158.13
(ArC), 164.46 (C@O); HRMS: C17H16NO5 requires 314.1028; found
314.1020 (M++H).
Compound25 was prepared from(2,4-bisbenzyloxy-5-ethylben-
zylidene)(3,4,5-trimethoxyphenyl)amine (16) and ethyl 2-bromo-
acetate in 8.7% yield as a brown oil; purity: 98.8%; IR (NaCl film)
m
max: 1746.84 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz, CDCl3) d
1.14 (t, 3H, CH3), 2.56–2.66 (m, 2H, CH2), 3.03–3.07 (d, 1H, H3),
3.44–3.50 (dd, 1H, H3), 3.74 (s, 3H, OCH3), 3.89 (s, 6H, 2 Â OCH3),
5.04–5.07 (m, 4H, 2 Â CH2), 5.32–5.33 (d, 1H, J = 4.48 Hz, H4), 6.59
(d, 3H, J = 11.04 Hz), 7.09 (s, 1H), 7.38–7.42 (m, 10H); 13C NMR
(100 MHz, CDCl3) d 14.14 (CH3), 22.36 (CH2), 44.92 (C3), 48.74 (C4),
55.52 (OCH3), 60.51 (OCH3), 69.70 (CH2), 70.32 (CH2), 93.96, 97.71,
117.10, 125.61, 126.60, 126.80, 126.99, 127.50, 127.76, 128.17,
128.25, 133.93, 136.13, 136.51, 152.94, 154.83 (ArC), 164.72
(C@O); HRMS: C34H35NO6Na requires 576.2362; found 576.2357
(M++Na).
6.6.3. 4-(5-Ethyl-2,4-dihydroxyphenyl)-1-(4-methoxyphenyl)
azetidin-2-one (28)
Compound 28 was prepared from 4-(2,4-bisbenzyloxyphenyl)-
1-(4-methoxyphenyl)azetidin-2-one (23) as a yellow powder in
26.9% yield; purity: 90.4%; IR (KBr) m
max: 1732.59 cmÀ1 (C@O, b-
lactam); 1H NMR (400 MHz, DMSO-d6) d 0.98 (t, 3H, CH3), 2.31–
2.35 (m, 2H, CH2), 2.89–2.94 (m, 1H, H3), 3.36–3.41 (m, 1H, H3),
3.67 (s, 3H, OCH3), 5.13–5.15 (dd, 1H, H4), 6.34 (s, 1H, ArH), 6.84
(d, 3H, ArH), 7.16 (d, 2H, ArH), 9.21 (s, 1H, OH), 9.40 (s, 1H, OH);
13C NMR (100 MHz, DMSO-d6) d 14.55 (CH3), 22.14 (CH2), 44.48
(C3), 48.79 (C4), 55.19 (OCH3), 102.52, 113.46, 114.25, 117.52,
121.00, 127.38, 131.65, 154.21, 155.08, 155.39 (ArC), 164.42
(C@O); HRMS: C18H19NO4Na requires 336.1212; found 336.1207
(M++Na).
6.5.8. 4-(2,4-Bisbenzyloxy-5-ethylphenyl)-1-(2,3-dihydrobenzo
[1,4]dioxin-6-yl)-3-phenyl-azetidin-2-one (26)
Compound 26 was obtained from (2,4-bisbenzyloxy-5-ethylben-
zylidene)(2,3-dihydrobenzo[1,4]dioxin-6-yl)amine (14) and ethyl
2-bromo-2-phenylacetate as an orange oil in 3.3% yield; IR (NaCl
film)
m
max: 1720.90 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz,
CDCl3) d 1.13 (t, 3H, CH3), 2.58–2.63 (m, 2H, CH2), 4.24 (s, 4H, OCH2-
CH2O), 4.29 (d, 1H, J = 2 Hz, H3), 5.03 (s, 2H, CH2), 5.08 (s, 2H, CH2),
5.35 (d, 1H, J = 2 Hz, H4), 6.61 (s, 1H, ArH), 6.78 (s, 1H, ArH), 6.98
(m, 1H, ArH), 7.09 (s, 1H, ArH), 7.16 (s, 1H, ArH), 7.29–7.44 (m,
15H, ArH); 13C NMR (100 MHz, CDCl3) d 13.83 (CH3), 21.90 (CH2),
43.38 (C3), 50.00 (C4) 63.78, 63.92, 63.98, 69.84 (CH2), 100.08,
106.24, 110.35, 116.93, 126.77, 127.17, 128.06, 128.26, 131.33,
134.83, 135.94, 139.61, 143.11, 154.93 (ArC), 165.55 (C@O); HRMS:
6.6.4. 1-Benzo[1,3]dioxol-5-yl-4-(5-ethyl-2,4-dihydroxyphenyl)
azetidin-2-one (29)
Compound 29 was prepared from 1-benzo[1,3]dioxol-5-yl-4-
(2,4-bisbenzyloxy-5-ethylphenyl)azetidin-2-one (24) as a brown
C
39H34NO5 requires 596.2436; found 596.2278 (M+ÀH).
powder in 44.6% yield; purity: 100%; IR (NaCl film) mmax:
1720.37 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz, DMSO-d6) d
1.01 (t, 3H, CH3), 2.34–2.40 (m, 2H, CH2), 2.94–2.98 (dd, 1H, H3),
3.37–3.43 (dd, 1H, H3), 5.13–5.15 (dd, 1H, H4), 5.94–5.97 (m, 2H,
OCH2O), 6.36 (s, 1H, ArH), 6.65–6.68 (m, 1H, ArH), 6.84–6.89 (m,
3H, ArH), 9.26 (s, 1H, OH), 9.46 (s, 1H, OH); 13C NMR (100 MHz,
DMSO-d6) d 15.04 (CH3), 22.62 (CH2), 44.78 (C3), 49.63 (C4),
98.77, 101.49 (CH2), 102.98, 108.87, 109.34, 113.64, 121.57,
128.03, 133.24, 143.34, 147.75, 154.72, 155.96 (ArC), 165.12
(C@O); HRMS: C18H17NO5Na requires 350.1004; found 350.1010
(M++Na).
6.6. General procedure for preparation of b-lactams 5 and 27–31
The benzyl-protected compound (2 mmol) was dissolved in
ethanol: ethyl acetate (50 mL; 1:1 mixture) and hydrogenated over
1.2 g of 10% palladium on carbon until the debenzylation was
complete on TLC. The catalyst was filtered, the solvent was
removed under vacuum and the product was isolated by flash col-
umn chromatography over silica gel (eluent: hexane/ethyl acetate
gradient).

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
6067
6.6.5. 4-(5-Ethyl-2,4-dihydroxyphenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (30)
trypsinised and seeded at a density of 2.5 Â 104 cells/mL in a 96-
well plate and incubated at 37 °C, 95%O2/5% CO2 atmosphere for
Compound 30 was prepared from 4-(2,4-bisbenzyloxy-5-ethyl-
phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (25) in 6.2%
yield as a white powder; purity: 96.1%; Melting point: 114 °C; IR
24 h. After this time they were treated with 2
compound which had been pre-prepared as stock solutions in eth-
anol to furnish the concentration range of study, 1 nM–200 M,
and re-incubated for a further 72 h. Control wells contained the
equivalent volume of the vehicle ethanol or DMSO (1% v/v). The
culture medium was then removed and the cells washed with
100 lL phosphate buffered saline (PBS) and 50 lL MTT (dissolved
in PBS) added, to give a final concentration of 1 mg/mL MTT. Cells
were incubated for 3 h in darkness at 37 °C. At this point solubili-
zation was begun through the addition of 200 lL DMSO and the
cells maintained at room temperature in darkness for 20 min to en-
sure 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 using Graph-
Pad Prism software.50
lL volumes of test
l
(KBr)
m
max: 1714.00 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz,
DMSO-d6) d 1.03 (t, 3H, CH3), 2.34–2.42 (m, 2H, CH2), 3.10–3.14
(dd, 1H, H3), 3.56 (s, 3H, OCH3), 3.65 (s, 6H, 2 Â OCH3), 3.75 (s,
1H, H3), 5.15–5.18 (m, 1H, H4), 6.37 (s, 1H, ArH), 6.61 (s, 2H,
ArH), 6.98 (s, 1H, ArH), 9.29 (s, 1H, OH), 9.56 (s, 1H, OH); 13C
NMR (100 MHz, DMSO-d6) d 14.64 (CH3), 22.11 (CH2), 43.51 (C3),
49.06 (C4), 55.58 (OCH3), 55.65 (OCH3), 60.08 (OCH3), 93.95,
102.37, 112.87, 121.25, 128.25, 133.21, 134.17, 153.05, 154.53,
155.64 (ArC), 164.98 (C@O); HRMS:
C20H23NO6Na requires
396.1423; found 396.1417 (M++Na).
6.6.6. 1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-4-(5-ethyl-2,4-
dihydroxyphenyl)-3-phenylazetidin-2-one (31)
Compound 31 was obtained from 4-(2,4-bisbenzyloxy-5-ethyl-
phenyl)-1-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-3-phenyl-azetidin-
2-one (26) as a yellow oil in 13.9% yield; purity: 99.4%; IR (KBr)
6.7.3. Hsp90 client protein degradation assay
MCF-7 cells were cultured as described for the antiproliferative
assay. MCF-7 cells were seeded at a density of 1.0 Â 106 cells/
25 cm2 tissue culture flask (5 ml Media) and left to adhere for
18 h prior to treatment. Cells were incubated with Vehicle (DMSO
0.5%) or compounds 17-AAG, 5, 12, and 18 at indicated concentra-
tions for 24 h. At the experimental endpoint, cells are tyrpsinised
and washed twice in ice-cold PBS, before incubation on ice for
1 h with 1 Â Cell Lysis Ripa Buffer (50 mM Tris–HCl (pH 7.4),
150 mM NaCL, 2 mM EDTA, 1% NP-40 (v/v), 0.1% SDS (w/v),
5 mM DTT and protease inhibiters). Cell debris was spun down at
10,000Âg and 4 °C for 10 min and supernatant was taken as whole
cell lysate. Lysate protein concentrations were determined by way
of a BCA protein assay (Thermo Scientific). Equal amount of pro-
m
max: 1716.35 cmÀ1 (C@O, b-lactam); 1H NMR (400 MHz, CDCl3) d
1.15 (t, 3H, CH3), 2.48–2.54 (m, 2H, CH2), 4.20 (s, 4H, OCH2CH2O),
4.46 (d, 1H, J = 2 Hz, H3), 5.12 (d, 1H, J = 2 Hz, H4), 6.28 (s, 1H,
ArH), 6.74 (d, 1H, J = 8.52 Hz, ArH), 6.88 (m, 1H, ArH), 6.99–7.01
(m, 2H, ArH), 7.29–7.37 (m, 5H, ArH); 13C NMR (100 MHz,
DMSO-d6) d 14.54 (CH3), 22.18 (CH2), 42.23 (C3), 52.12 (C4), 63.91
(CH2), 64.17 (CH2), 101.04, 102.46, 103.46, 107.36, 111.48,
111.72, 116.72, 116.95, 118.17, 118.51, 119.99, 121.98, 126.48,
127.53, 127.69, 127.87, 128.12, 128.20, 128.23, 128.52, 128.74,
131.76, 135.21, 138.54, 139.57, 141.15, 142.22, 152.84, 153.08,
153.94, 159.03, 160.15 (ArC), 169.72 (C@O); HRMS: C25H24NO5 re-
quires 418.1654; found 418.1656 (M++H).
teins (25 lg) from each lysate were resolved on an 8% SDS–poly-
6.7. Biochemical evaluation methods
acrylamide gel and transferred to a PVDF membrane (Millipore)
for Western blot analysis.
6.7.1. Hsp90
a
fluorescent displacement assay
Estrogen receptor alpha protein expression levels were deter-
mined by probing with an ER-a mouse monoclonal antibody (Cell
Signalling—62A3). A mouse monoclonal antibody against b-Actin
(Calbiochem—Ab-1) was used as a loading control.
The assay is adapted from the method outlined by Howes.17,35
The components of the Hsp90 assay buffer are as follows: HEPES,
pH 7.3 (20 mM); potassium chloride (50 mM); magnesium chloride
(5 mM); Na2MoO4 (20 nM); 0.01% v/v NP40. The buffer is made up
using distilled water. Directly before each use, 1 mg bovine gamma
globulin (per 10 mL) and 3.085 mg of DL-dithiothreitol (per 10 mL)
6.7.4. Molecular modelling methods
PDB entry 1OSF27 (a co-crystal structure of 17-dimethylamino-
ethylamino-17-demethoxygeldanamycin in complex with human
are added. Hsp90a recombinant human protein (StressgenÓ) was
used at a final protein concentration of 75 nM and is diluted with as-
say buffer. FITC-geldanamycin (FITC-GA) is the fluorescent ligand
used in this displacement assay at a final concentration of 5 nM.
17-AAG is used as a positive control in the Hsp90 fluorescent dis-
placement assay (reported IC50 value for binding in Hsp90 is
Hsp90a) was used in the docking procedure due to correct docking
of 131 Hsp90 actives as demonstrated previously by Knox et al.17
Several binding site waters were retained in the docking process
as they provide key interactions in stabilising the ligand in the active
site. Addition of hydrogens for the receptor and waters was carried
out using MOEv2007.09 and optimization using the Amber99
force-field ensuring all other atom positions remained fixed. Dock-
ing constraints were added such that all docked poses must have a
H-bonding interaction with Asp93 to be considered successfully
docked. Docking was performed using the docking algorithm FRED
(Fast Rigid Exhaustive Docking51) and scored with Chemgauss3.
Subsequent refinement of all side-chains within 5 Å of the docked
pose was carried out using Szybki (http://www.eyesopen.com).
1.27
ligand, 25
of (two of each): buffer (75
(70 L) + receptor (25 L) + FITC-GA (5
GA (5 L) and vehicle controls as necessary. The assay is read on a
l
M28). For the assay, to each well is added: 69
L receptor and 5
l
L buffer, 1
L FITC-GA. The control rows consist
L) + receptor (25 L); buffer
L); buffer (95 L) + FITC-
lL
l
l
l
l
l
l
l
l
l
fluorescent plate reader using excitation of 485/20 nM and emission
535/25 nM with polarisation. IC50 values were calculated using non-
linear regression with a sigmoidal dose-response (variable slope)
curve, using GraphPad Prism.50
Acknowledgments
6.7.2. Antiproliferative MTT assay
All assays were performed in triplicate for the determination of
mean values reported. The human breast tumour cell line MCF-7
was cultured in Eagles minimum essential medium at 37 °C in a
95% O2/5% CO2 atmosphere with 10% fetal bovine serum, 2 mM
This work was supported through funding from the Trinity Col-
lege IITAC research initiative (funded under the Irish Higher Educa-
tion Authority’s Programme for Research in Third Level Institutions
(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
L-glutamine and 100
lg/mL penicillin/streptomycin. The medium
was supplemented with 1% non-essential amino acids. Cells were

6068
N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 6055–6068
26. Carr, M.; Greene, L. M.; Knox, A. J. S.; Lloyd, D. G.; Zisterer, D. M.; Meegan, M. J.
Eur. J. Med. Chem. 2010, 45, 5752.
27. Jez, J. M.; Chen, J. C. H.; Rastelli, G.; Stroud, R. M.; Santi, D. V. Chem. Biol. 2003,
10, 361.
research award from Trinity College is gratefully acknowledged
(NMO’B).
28. Dymock, B. W.; Barril, X.; Brough, P. A.; Cansfield, J. E.; Massey, A.; McDonald,
E.; Hubbard, R. E.; Surgenor, A.; Roughley, S. D.; Webb, P.; Workman, P.;
Wright, L.; Drysdale, M. J. J. Med. Chem. 2005, 48, 4212.
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