Design, synthesis and biochemical evaluation of estrogen receptor ligand conjugates as tumour targeting agents

Article abstract of DOI:10.2174/157018012799129864

The estrogen receptors (ERα and ERβ), are ligand inducible nuclear receptors which play a key role in many cellular functions through specific gene expression regulation. The estrogen receptor is regarded as an attractive therapeutic target for hormone-dependent breast cancers. The antiestrogen drug tamoxifen is useful in the treatment of breast cancer. To develop new ER targeting agents as probes of estrogen receptor action, the synthesis and preliminary biochemical evaluation of five structurally varied estrogen receptor ligand conjugates containing the tamoxifen metabolite endoxifen are now reported. These structurally varied conjugates bind to the estrogen receptor (commonly overexpressed in breast cancer cells) and contain DNA alkylating, aromatase inhibitor and COX2 inhibitor moieties. The ER targeting group endoxifen (E/Z 4-hydroxy-N-desmethyltamoxifen) was selected for its ability to bind to the estrogen receptor. Compound 11 exhibited moderate antiproliferative activity IC₅₀ = 1.64 μM in MCF-7 breast cancer cells, while compound 9b demonstrated the most potent ER binding effects with IC₅₀ values of 35.6 nM(ERα), 19.5 nM(ERβ).

Full text of DOI:10.2174/157018012799129864

Letters in Drug Design & Discovery, 2012, 9, 295-304
295
Design, Synthesis and Biochemical Evaluation of Estrogen Receptor
Ligand Conjugates as Tumour Targeting Agents
Niall O. Keely1, Daniela M. Zisterer2 and Mary J. Meegan*,1
1School of Pharmacy and Pharmaceutical Sciences, Centre for Synthesis and Chemical Biology, Trinity College Dublin,
Dublin 2, Ireland
2School of Biochemistry & Immunology, Trinity College Dublin, Dublin 2, Ireland
Received July 29, 2011: Revised October 07, 2011: Accepted October 24, 2011
Abstract: The estrogen receptors (ERꢀ and ERꢁ), are ligand inducible nuclear receptors which play a key role in many
cellular functions through specific gene expression regulation. The estrogen receptor is regarded as an attractive
therapeutic target for hormone-dependent breast cancers. The antiestrogen drug tamoxifen is useful in the treatment of
breast cancer. To develop new ER targeting agents as probes of estrogen receptor action, the synthesis and preliminary
biochemical evaluation of five structurally varied estrogen receptor ligand conjugates containing the tamoxifen metabolite
endoxifen are now reported. These structurally varied conjugates bind to the estrogen receptor (commonly overexpressed
in breast cancer cells) and contain DNA alkylating, aromatase inhibitor and COX2 inhibitor moieties. The ER targeting
group endoxifen (E/Z 4-hydroxy-N-desmethyltamoxifen) was selected for its ability to bind to the estrogen receptor.
Compound 11 exhibited moderate antiproliferative activity IC50 = 1.64 ꢀM in MCF-7 breast cancer cells, while compound
9b demonstrated the most potent ER binding effects with IC50 values of 35.6 nM(ERꢀ), 19.5 nM(ERꢁ).
Keywords: Estrogen receptor, Conjugates, Tamoxifen, Tumour targeting, Antiproliferative activity, Breast cancer.
INTRODUCTION
doxorubicin [14, 15] and 5-fluorouracil [16] have been found
to show lower ER binding affinities and variable cytotoxicity
effects in ER positive breast tumour cells. Doxorubicin–
formaldehyde conjugates linked to the antiestrogen 4-
hydroxytamoxifen incorporating a base labile N-Mannich
linking group have been reported to inhibit the growth of
breast cancer cell lines with enhanced activity relative to
doxorubicin [17, 18]. 17ꢁ-Estradiol platinum (II) complexes
have demonstrated antiproliferative enhanced activity (when
compared to cisplatinum) in ER positive and ER negative
breast cancer cell lines [19, 20].
Nuclear receptors play a key role in many cellular
functions through specific gene expression regulation and
are targeted by a large number of both endogenous and
exogenous ligands [1]. The majority of early stage breast
cancers, in both pre- and postmenopausal women, are
hormone-dependent [2]. Estradiol, the endogenous hormonal
ligand, has a key role in the development and progression of
the tumour and the estrogen receptor is regarded as an
attractive target for hormone-dependent breast cancers [3-5].
Tamoxifen 1a is widely used as an estrogen receptor
antagonist in the treatment of breast cancer. However, many
cancer chemotherapies lack specificity which can lead to
toxicity and undesirable side effects and more selective
chemotherapeutic approaches have been investigated to
target tumours. For example, agents targeting nuclear
receptors over-expressed in tumours can be directed to
malignant tissue and result in improved chemotherapeutic
treatments. In hormone-dependent cancers, such as certain
breast cancers, a number of structurally varied estrogen
receptor ligand conjugates have previously been
investigated, attempting to take advantage of the presence of
over-expressed estrogen receptor [6]. Conjugate compounds
containing multiple ligands, separated by covalent linking
groups, can exert a synergistic and improved selective action
on the target disease [7]. Many examples of the coupling of
cytotoxic agents to the steroid estradiol and related scaffolds,
hexestrol, diethylstilbestrol and tamoxifen have been
reported [8-10]. Conjugates of estradiol with geldanamycin
[11], the indole alkaloid ellipticine [12], mitomycin C [13],
To develop new ER targeting agents as probes of
estrogen receptor action, the synthesis and ER binding
effects of novel conjugates containing components with
known activity in the prevention of breast cancer
proliferation (e.g. chlorambucil and indomethacin), linked to
the tamoxifen metabolite endoxifen are now reported. The
ER targeting group endoxifen (E/Z 4-hydroxy-N-
desmethyltamoxifen, 1c) was selected for its ability to bind
to the estrogen receptor. Endoxifen has been shown to be the
major metabolite of tamoxifen 1a, and is present at greater
concentrations than 4-hydroxytamoxifen 1b in human
plasma in breast cancer patients treated with tamoxifen [21].
Chlorambucil 2 is a nitrogen mustard alkylating agent
used in the treatment of select chronic lymphocytic
leukaemia and advanced ovarian and breast carcinomas.
Alkylating agents have limited selectivity towards malignant
cells over normal tissue and as a result can be quite toxic. By
coupling alkylating agents such as chlorambucil, to a carrier
agent such as endoxifen which targets tumour cells, the
selectivity of the agents would be increased, their toxicity
diminished and their efficacy improved. The synthesis of an
endoxifen conjugate with indomethacin 3 was of interest as
there is considerable evidence showing that prostaglandins
*Address correspondence to this author at the School of Pharmacy and
Pharmaceutical Sciences, Centre for Synthesis and Chemical Biology,
Trinity College Dublin, Dublin 2, Ireland; Tel: +353-1-8962798; Fax: +353-
1-8962793; E-mail: mmeegan@tcd.ie
1875-628X/12 $58.00+.00
© 2012 Bentham Science Publishers

296 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3
Keely et al.
R1
N
Cl
H3CO
CO2H
O
N
Cl
N
O
O
Cl
3 Indomethacin
OH
R2
1a R1 = CH3, R2 = H, Tamoxifen
2 Chlorambucil
1b R1 = CH3, R2 = OH, 4-Hydroxytamoxifen
1c R1 = H, R2 = OH, Endoxifen
1d R1 = H, R2 = OTBDMS
CO2H
NC
NH2
O
OMe
N
MeO
OMe
OH
OMe
RO
6
5
4a R = OH
4b R = OMe
Fig. (1). Structures of estrogen receptor antagonists 1a-d and compounds 2-6.
(PGs) play an important role in breast cancer development
and growth [22] and there has been interest in a possible role
of COX inhibitors such as indomethacin in the prevention of
malignancy. Cyclooxygenase-2 (COX-2) is overexpressed in
several epithelial tumours, including breast cancer. The
COX-2 inhibitor indomethacin 3 has been shown to induce
apoptosis and reduce the proliferation rate of HT-29 colon
cancer [23], and MCF-7 cancer cell lines [24] and also exerts
estrogen hexestrol have been reported by Bergmann [29].
We wished to investigate any antagonistic effect that the
presence of one ER-ligand covalently linked to a second ER-
ligand would have on antiproliferative activity through the
displacement of Helix-12 in the ER binding site.
The acrylonitrile combretastatin analogue 5 was chosen
for conjugation with endoxifen 1c as an example of a potent
antitubulin drug with cytotoxic effects in against murine
Colon 26 adenocarcinoma [30]. A conjugate incorporating
the tetralone aromatase inhibitor 6 [31] was also examined.
Inhibitors of aromatase enzyme reduce the production of
estrogen thus decreasing the stimulation of breast tumours,
hence developing multitargeting agents may result in more
selective drug delivery to the tumour target site.
a
synergistic effect when combined with other
chemotherapeutic drugs such as doxorubicin and vincristine
[25]. We wished to determine if the indomethacin fragment
of the conjugate, covalently bound via an amide linkage to
endoxifen, would act as a bulky side chain to displace the
helix-12 in the ER binding site resulting in increased (or
pure) antagonist activity.
A dual ER-ligand conjugate was also synthesised
comprising of the fragments endoxifen 1c and GW7604, 4a,
a selective estrogen receptor downregulator [26], in which
the dimethylaminoethoxy group of tamoxifen is replaced by
an acrylate side chain. A secondary binding site exists on the
ER, also known as the antiestrogen binding site (AEBS) that
demonstrates high affinity binding of triarylethylene
antiestrogens (i.e. specifically tamoxifen and its hydroxy
derivatives) which is not in competition with the binding of
estrogens [27, 28]. By synthesising a bivalent ER-ligand
conjugate we wish to probe the possibility of binding in both
the ER and AEBS sites concurrently. The binding properties
of bivalent ER targeting ligands based on the nonsteroidal
CHEMISTRY
Endoxifen 1c was synthesised [17], and used as the
protected tert-butyldimethylsilyl ether 1d in the subsequent
coupling reactions. The chlorambucil conjugate 7 was
obtained by the reaction of chlorambucil 2 with the silyl-
protected endoxifen ligand 1d via a DCC/DMAP coupling
reaction, immediately followed by the TBAF deprotection
step (Scheme 1) to afford a 1:1 E/Z isomeric mixture of the
desired product 7 (60 % yield). Previous work has
demonstrated that p-hydroxy substituted triarylbutenes may
isomerise under cell culture conditions, having little impact
on ER binding activity [32, 33]. In the 13C-NMR spectrum,

Design and Evaluation of Estrogen Receptor Ligand Conjugates
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3 297
1d
(i)
(ii)
(i)
Cl
Cl
OR1
Cl
N
OCH3
O
N
O
N
O
O
N
N
O
O
O
R2O
HO
HO
9a R1 = TBDMS, R2 = OMe (76%)
9b R1 = R2 = OH (88%)
7 (60%)
8 (73%)
(iii)
Scheme 1. Synthesis of conjugates 7, 8, 9a and 9b.
Reagents and conditions: (i) (a) DCC, DMAP, 2 or 3, CH2Cl2, rt, 24h (b) TBAF, THF, rt, 24h (c) CH2Cl2, 10 % HCl, rt, 5min; (ii) DCC,
DMAP, 4b, CH2Cl2, rt, 24h (iii) (a) BBr3, CH2Cl2, -78o, 45min, then rt, 3h (b) TBAF, THF, rt, 24h (c) 10 % HCl, CH2Cl2, rt, 5min.
the amide carbon is observed at 173.2 ppm while in the IR
spectrum, the amide signal is observed at 1723.9 cm-1 and
the broad hydroxyl signal at 3401.5 cm-1. The conjugate 8
was synthesised by reaction of indomethacin 3 with the silyl-
protected endoxifen ligand 1d via a DCC/DMAP coupling
reaction, immediately followed by the TBAF deprotection
step (Scheme 1) to afford a 1:1 E/Z isomeric mixture of the
desired product 8 in 73 % yield. The main diagnostic signal
of the coupled conjugate is observed in the 13C-NMR
spectra, where three amide carbons (due to the E- and Z-
isomers of conjugate and also the indomethacin moiety) are
observed at 171.82 ppm, 171.88 ppm, 172.22 ppm. In the IR
spectrum, the amide signal is observed at 1724.5 cm-1and the
broad hydroxyl signal at 3327.2 cm-1. The GW7604
analogue 4b [34] was coupled with 1d using a DCC/DMAP
reaction (Scheme 1) to afford the protected-conjugate
product 9a (76 %). Following demethylation with boron
tribromide and subsequent silyl deprotection steps, the
product 9b was afforded in a 1:1 E/Z isomeric mixture (88 %
yield).
For the synthesis of the endoxifen conjugates with 5 and
6, the protected endoxifen was first reacted with succinic
acid to afford the endoxifen-succinimide 10, (Scheme 2).
The cis-acrylonitrile 6 was prepared following the literature
method [30], and was reacted with endoxifen analogue 10
via a DCC/HOBt coupling, immediately followed by the
TBAF deprotection step (Scheme 2) to afford a 1:1 E/Z
isomeric mixture of the product 11 (26 % yield). The two
amide carbons of the linker fragment are observed at 171.48
and 173.27 ppm in the 13C-NMR spectrum of the coupled
conjugate 11 with the nitrile, amide and hydroxyl signals
observed at 2208.6, 1734.8 and 3401.5 cm-1 in the IR
spectrum. The conjugate 12 was obtained by reaction of the
aromatase inhibitor 5 and the protected endoxifen ligand 10
via a DCC/HOBt coupling reaction, followed by the TBAF
deprotection step to afford a 1:1 E/Z isomeric mixture of the
desired product 12 in 63 % yield, (Scheme 2). The coupled
conjugate is observed in the 13C-NMR spectrum, where the
ester and amide carbons are identified at 171.45 and 172.00
ppm and the carbonyl group of the aromatase inhibitor
fragment at 198.52 ppm. In the IR spectrum, the ester signal

298 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3
Keely et al.
CN
MeO
MeO
OMe
NH
MeO
O
O
OH
N
O
(ii)
O
O
N
CO2H
HO2C
(i)
O
(ii)
1d
HO
11 (26%)
O
N
O
TBDMSO
O
O
10 (92%)
N
O
HO
12 (63%)
Scheme 2. Synthesis of conjugates 11 and 12.
Reagents and conditions:(i) CH2Cl2, rt, 16h (ii) (a) DCC, HOBt, 5 or 6, CH2Cl2, rt, 24h (b) TBAF, THF, rt, 24h (c) 10 % HCl, CH2Cl2, rt,
5min.
is observed at ꢀ1738.0 cm-1, the amide signal at ꢀ1631.2
cm-1 and the broad hydroxyl signal at ꢀ3435.0 cm-1.
in combination with the DNA intercalating drugs
doxorubicin). Therefore, some structural comparison may
highlight possible mechanistic features regarding the
functionality of the conjugates.
BIOLOGICAL RESULTS AND DISCUSSION
The antiproliferative and cytotoxicity data for the
conjugates evaluated in the ER positive MCF-7 breast cancer
cells are shown in Table 1. The data obtained for this series
of conjugates ranged from low micromolar antiproliferative
IC50 values to IC50 values greater than 50 ꢀM. All the
conjugates displayed low cytotoxicity levels at 10 ꢀM in the
lactate dehydrogenase assay [35]. The endoxifen-
acrylonitrile conjugate 11 displays the most potent
antiproliferative activity of the series, with IC50 = 1.64 ꢀM,
The conjugates in this series contain an ER-ligand
component (i.e. endoxifen) linked via amide and/or ester
linkages, with or without a succinic acid linker group, to
another compound with bioactivity towards cancers and
tumours. The subset of bioactive compounds include an
aromatase inhibitor, an alkylating agent (chlorambucil), a
tubulin targeting agent, an ER antagonist and the NSAID
indomethacin (shown to have synergistic effects when used

Design and Evaluation of Estrogen Receptor Ligand Conjugates
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3 299
Table 1. Antiproliferative and ER Binding Affinities for Conjugates 7, 8, 9b, 11 and 12
ER ꢁ
IC50 (nM)d
ER ꢂ
IC50 (nM) d
Selectivity
ꢁ/ ꢂ ratio
RBAf
RBAf
MCF-7 IC50
(ꢀM) a
Cytotoxicity 10
ꢀMc
Compound
(ERꢀ )
(ERꢁ )
7
30.3
11.6
>50
0
0
490
78.9
35.6
524
51.4
70 e
415
200
0.85
2.5
1
7
1
3
8
9b
11
0
19.5
269
0.55
0.51
1.00
2.42
16
1
29
2
1.64
12.6
4.12b
1.7
0
12
51.6
170 e
11
11
Tamoxifen
13.4
8.14
2.97
aIC50 values are half maximal inhibitory concentrations required to block the growth stimulation of MCF-7 cells. Values represent the mean S.E.M (error values x 10-6) for three
experiments performed in triplicate.
bThe IC50 value obtained for Tamoxifen using the MTT assay is 4.12 0.038 ꢁM, with cytotoxicity value 13.4 % (10 ꢁM) is in good agreement with the reported IC50 value for
tamoxifen on human MCF-7 cells, (reference 36).
cLactate Dehydrogenase assay: Following treatment of the cells, the amount of LDH was determined using LDH assay kit from Promega. Data is presented as % cell lysis at
compound concentration of 10 ꢂM (reference 35).
dValues are an average of at least nine replicate experiments, for ERꢀ with typical standard errors below 15%, and six replicate experiments for ERꢁ, with typical standard errors
below 15%.
eThe ER binding values obtained are in agreement with the reported ER IC50 binding data for tamoxifen (ERꢀ 60.9 nM ERꢁ188 nM, Panvera/Invitrogen).
fThe RBA (Relative Binding Affinity value, %) is calculated as the ratio of the binding IC50 value for the reference ligand (estradiol) divided by the binding IC50 value for the
selected ligand, multiplied by 100.
comparable to activity of the acid 6 (IC50 = 0.83 ꢂM) in this
cell line, with low cytotoxicity in the LDH assay. The
endoxifen conjugates of chlorambucil 7, indomethacin 8 and
conjugate 12, which did not demonstrate potent
antiproliferative activity, display impressive binding
affinities towards ERꢂ and ERꢃ with competitive binding
IC50 values in the range 50 – 200 nM. The acrylonitrile
conjugate 11 displayed the lowest ER binding of the series
with IC50 of 524 nM(ERꢀ) and 269 nM(ERꢁ). The bivalent
ER-ligand conjugate 9b which also did not display
significant antiproliferative activity, demonstrated potent
competitive binding with IC50 values of 35.6 nM (ERꢂ) and
19.5 nM (ERꢃ). This may suggest that these conjugates bind
in orientations or sites which as a result do not displace
helix-12 in the ER, hence, do not display a significant
antagonistic activity in vitro. The phenolic hydroxy group
present on the endoxifen fragment can interact favourably
with residues Glu353, Arg394 and His524 in the ER binding
site through hydrogen bonding which can explain the high
binding affinity for all conjugates which make them useful
probes for the estrogen receptor action.
the aromatase inhibitor 12 displayed
moderate
antiproliferative activity when compared to tamoxifen [36].
The IC50 values for these conjugates were in the range of
11.6 - 30.3 ꢁM and displayed virtually no cytotoxicity at 10
ꢁM in the lactate dehydrogenase assay [35]. The aromatase
inhibitor conjugate 12 contains an ester linkage between the
tetralone and the succinate linker fragment which may be
enzymatically hydrolysed in vivo, thus possibly exerting a
dual-effect through aromatase inhibition and ER-antagonism.
The dual ER-ligand conjugate 9b containing the amide
linking groups, did not display significant antiproliferative
activity, with an IC50 value greater than 50 ꢁM, resulting in
negligible cytotoxicity. The presence of the amide linkage
may restrict the conformation of this conjugates
unfavourably. This suggests that presence of the bulky
ligand side chain in this case has a detrimental effect on the
antiproliferative activity of either the endoxifen or GW7604
ER-ligand fragment at the ER site. As the conjugate 9b
contains an amide linkage in the scaffold, it is possible that
9b may not be easily hydrolysed in vivo and could act as a
possible pure ER antagonist due to the large bulky side chain
group.
The relative binding affinity (RBA) of the estrogen
receptor ligands is reported. Estradiol is the reference ligand,
with 100 % binding value. Using the IC50 binding values
obtained for estradiol for ERꢂ (5.7 nM) and ERꢃ (5.6 nM),
the relative binding affinities of the selected conjugates were
calculated (Table 1). As the estrogen receptor has between
1000 and 10000 molecules per cell and calculations on the
basis of the number of receptors per cell and the possible
drug concentration show that the relative binding affinity
(RBA) value should be at least 1% of that of estradiol for
effective activity [12]. All of the conjugates investigated in
the ER competitor binding assays had RBA values greater
than or equal to 1%. The bivalent ER conjugate 9b
demonstrates impressive RBA values of 16 for ERꢀ and 29
for ERꢁ.
The conjugates were investigated for their ERꢂ and ERꢃ
binding abilities in a fluorescence polarization based-
competitor assay. The ER-binding data for these selected
conjugates is reported in Table 1. In order to function as ER-
targeting agents, the conjugates should display some binding
affinity towards the receptor target. All the conjugates
display binding affinities for the ERꢂ and ERꢃ with IC50
values below 1 ꢂM, confirming their ability to act as ER-
targeting agents. The acrylonitrile conjugate 11 and
chlorambucil conjugate 7 showed the least affinity for either
ER isoform yet still demonstrated sub-micromolar
competitive binding IC50 values towards ERꢂ (96 and 490
nM respectively) and ERꢃ (415 and 61 nM respectively).
The indomethacin conjugate 8 and aromatase inhibitor
CONCLUSION
We have synthesised a new series of estrogen receptor
ligand conjugates, which contain components with known
activity in the prevention of breast cancer proliferation

300 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3
Keely et al.
covalently linked to the tamoxifen metabolite endoxifen.
Preliminary in vitro evaluation of the antiproliferative and
cytotoxic activities of the compounds in MCF-7 breast
cancer cells, together with determination of the ER binding
indicated that some of these estrogen receptor ligand
conjugate molecules may be useful as probes of estrogen
receptor action and may also result in a more selective and
targeted delivery of non-specific agents such as chlorambucil
to breast tumour cells. The bivalent ER conjugate 9b
demonstrates potent ER binding with IC50 values of 35.6
nM(ERꢀ), 19.5 nM(ERꢁ) and RBA values of 16 (ERꢀ) and
29 (ERꢁ) and provides a template scaffold for the
development of further ER ligands.
4.80, 13.21, 13.24, 17.75, 25.24, 25.26, 28.47, 28.60, 35.39,
35.26, 49.47, 50.03, 50.12, 65.73, 65.99, 112.80, 113.54,
118.54, 119.12, 125.44, 125.48, 127.33, 127.41, 129.25,
130.12, 130.20, 131.43, 131.57, 135.68, 136.02, 136.06,
136.35, 137.44, 137.53, 140.59, 140.65, 142.11, 142.19,
153.06, 153.84, 156.10, 156.94. IR: ꢁmax (KBr) cm-1: 3340.9,
2956.9, 2930.0, 2857.0, 1605.5. HRMS (EI): Found
488.2980 (M++H), C31H42NO2Si requires 488.2985.
4-{4-[Bis-(2-chloroethyl)amino]phenyl}-N-(2-{4-[(E/Z)-1-
(4-hydroxyphenyl)-2-phenylbut-1-enyl]phenoxy}ethyl)-N-
methylbutyramide (7)
Chlorambucil 7 (0.06 g, 0.21 mmol), DCC (0.04 g, 0.21
mmol) and DMAP (0.03, 0.21 mmol) were stirred for 10
mins in anhydrous CH2Cl2 (4 mL). A solution of the amine
1d (0.10 g, 0.21 mmol) in dry CH2Cl2 (4 mL) was added to
the reaction mixture and then allowed stir for 24 h under
nitrogen at room temperature. Reaction was monitored via
TLC (CH2Cl2:MeOH, 4:1) until no more starting materials
were visible. The reaction mixture was diluted to 15 mL with
anhydrous CH2Cl2 and filtered to remove DCU. The filtrate
was evaporated to dryness under reduced pressure. The
residue was dissolved in 3 mL anhydrous THF and stirred
under a nitrogen atmosphere. A solution of 0.1 M TBAF
(0.21 mL, 0.21 mmol) was added to the mixture and allowed
stir for 24 h. The mixture was evaporated to dryness under
reduced pressure. The residue was dissolved in CH2Cl2 and
washed with 10 % HCl solution. The resulting organic phase
was dried over sodium sulfate and evaporated to dryness
under vacuum. The residue was purified via flash
chromatography (CH2Cl2:MeOH, 20:1) to yield an isomeric
mixture of the product 7 as a brown oil (81.0 mg, 60 %, E/Z
EXPERIMENTAL SECTION
General
Uncorrected melting points were measured on a Stuart
SMP11 apparatus. Infra-red spectra were recorded on a
Perkin Elmer Paragon 100 Spectrometer. Band positions are
given as cm-1. Solid and resin samples were analysed by KBr
disc, while oils were analysed as neat films on NaCl plates.
1H and 13C Nuclear magnetic resonance (NMR) spectra were
recorded at 20°C on a Bruker DPX 400 spectrometer (400.13
MHz, 1H; 100.61 MHz, 13C) in CDCl3, with internal standard
1
trimethylsilane (TMS), or CD3OD. H-NMR spectra were
assigned relative to the TMS peak at 0.00. 13C NMR spectra
were assigned relative to the centre peak of the CDCl3 triplet
at 77.00 ppm. Coupling constants are reported in Hertz (Hz).
High resolution molecular ion determinations (HRMS) were
acquired on a Micro mass spectrometer (E.I. Mode) at the
Department of Chemistry, Trinity College Dublin and a
Micro mass spectrometer (E.I. Mode) at the Centre for
Synthesis and Chemical Biology, University College Dublin.
Thin layer chromatography (TLC) was carried out using
Merck F-254 silica plates. Flash chromatography was carried
out on Merck Kieselgel 60F254 silica. Solvents were dried
according to the standard protocols.
1
= 1:1). H-NMR (400 MHz, CDCl3): ꢀ 0.91- 0.96 (m, 3H,
CH3), 1.29 - 2.63 (m, 8H, 4xCH2), 2.97 - 3.15 (m, 3H,
NCH3), 3.60 - 4.16 (m, 12H, 3xCH2N, CH2O, 2xCH2Cl),
6.45 – 7.18 (m, 17H, ArH), OH not observed. 13C-NMR (100
MHz, CDCl3), (Z/E mixture): ꢀ 13.30, 24.45, 25.12, 25.22,
16.15, 26.49, 28.48, 29.26, 29.26, 29.88, 32.27, 32.76, 33.42,
33.66, 37.25, 40.13, 44.94, 47.61, 48.73, 53.12, 111.68,
112.69, 113.37, 113.99, 114.51, 114.65, 118.16, 128.65,
129.24, 130.21, 130.34, 131.59, 133.71, 136.64, 133.71,
136.64, 143.81, 154.11, 154.67, 156.85, 173.23. IR: ꢁmax
(KBr) cm-1: 3401.5, 2929.6, 2850.2, 1723.9, 1609.5. HRMS
(EI): Found 659.2803 (M++H), C39H45 Cl2N2O3 requires
659.2807.
[2-(4-{(E/Z)-1-[4-(tert-Butyldimethylsilanyloxy)phenyl]-2-
phenylbut-1-enyl}phenoxy)ethyl] methylamine (1d)
(4-{(E/Z)-1-[4-(2-Bromoethoxy)phenyl]-2-phenylbut-1-
enyl}phenoxy)-tert-butyldimethylsilane (0.54 g, 1.00 mmol)
was reacted with methylamine in anhydrous tetrahydrofuran
(20 mL) in a sealed tube at 60 °C under pressure while
stirring for 48 – 72 h, [17]. After this time the reaction
container was allowed sufficient time to cool, and then
sodium carbonate/sodium hydrogen carbonate pH 10 buffer
solution (50 mL) was added. The mixture was then extracted
with CH2Cl2 (3 x 50 mL). The organic phases were
combined, dried over sodium sulfate and the solvent
evaporated in vacuo to afford a crude product which was
then purified via flash chromatography over silica gel
(CH2Cl2:MeOH, 4:1) to afford the product as a brown oil
2-[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]-N-(2-{4-[(E/Z)-1-(4-hydroxyphenyl)-2-phenylbut-1-
enyl]phenoxy}ethyl)-N-methylacetamide (8)
Indomethacin 3 (0.33 g, 0.91 mmol), DCC (0.19 g, 0.91
mmol) and DMAP (0.11, 0.91 mmol) were stirred for 10
mins in anhydryous CH2Cl2 (7 mL). A solution of the amine
1d (0.45 g, 0.91 mmol) in dry CH2Cl2 (8 mL) was added to
the reaction mixture and then allowed stir for 24 h under
nitrogen at room temperature. Reaction was monitored via
TLC (CH2Cl2:MeOH, 4:1) until no more starting materials
were visible. The reaction mixture was diluted to 15 mL with
anhydrous CH2Cl2 and filtered to remove DCU. The filtrate
was evaporated to dryness under reduced pressure. The
residue was dissolved in 3 mL anhydrous THF and stirred
under a nitrogen atmosphere. A solution of 0.1 M TBAF
(0.91 mL, 0.91 mmol) was added to the mixture and allowed
1
(0.38 g, 78 %, Z/E = 1:1.3). H-NMR (400 MHz, CDCl3): ꢀ
0.13 (s, 0.57 x 6H, Si(CH3)2), 0.26 (s, 0.43 x 6H, Si(CH3)2),
0.95 – 1.03 (m, 12H, (CH3)3, CH3), 2.45 – 2.53 (m, 5H,
NCH3, CH2), 2.89 (s, 0.43 x 2H, CH2), 2.99 (s, 0.57 x 2H,
CH2), 3.36 (s, 1H, NH), 3.95 (t, 0.43 x 2H, J = 5.0 Hz, CH2),
4.11 (t, 0.57 x 2H, J = 5.0 Hz, CH2), 6.50 – 7.20 (m, 13H,
ArH). 13C-NMR (100 MHz, CDCl3): (Z/E mixture) ꢀ -4.91, -

Design and Evaluation of Estrogen Receptor Ligand Conjugates
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3 301
stir for 24 h. The mixture was evaporated to dryness under
reduced pressure. The residue was dissolved in CH2Cl2 and
washed with 10 % HCl solution. The resulting organic phase
was dried over sodium sulfate and evaporated to dryness
under vacuum. The residue was purified via flash
chromatography (CH2Cl2:MeOH, 20:1) to yield an isomeric
mixture of the product 8 as a brown oil (472 mg, 73 %, E/Z =
residue was dissolved in 5 mL of anhydrous THF. To the
solution, 1.0M TBAF in THF (0.50 mL, 0.50 mmol, 2.5eq.)
was added. The reaction was allowed stir overnight. The
reaction was diluted with 20 mL of THF and washed with 10
% HCl solution. The organic layer was separated, dried over
sodium sulfate and evaporated to dryness in vacuo. The
residue was purified via flash chromatography
(CH2Cl2:MeOH, 20:1) to afford the product 9b as a resin
1
1:1). H-NMR (400 MHz, CDCl3): ꢀ 0.93 - 0.97 (m, 3H,
1
endoxifen CH3), 2.21 – 2.29 (m, 3H, indomethacin CH3),
2.48 - 2.53 (m, 2H, CH2), 2.97 - 3.18 (m, 3H, NCH3), 3.69 -
4.16 (m, 9H, OCH3, CH2N, CH2O, COCH2), 6.44 – 7.19 (m,
20H, ArH), OH not observed. 13C-NMR (100 MHz, CDCl3),
(E/Z mixture): ꢀ 11.42, 11.46, 13.14, 13.25, 28.63, 29.28,
30.53, 30.58, 33.89, 36.07, 37.54, 48.12, 48.82, 55.36, 55.43,
64.50, 64.92, 65.96, 66.21, 99.73, 99.82, 99.89, 103.67,
103.73, 104.20, 110.28, 110.33, 112.77, 113.49, 113.94,
114.62, 125.43, 127.40, 128.37, 129.33, 129.73, 129.80,
130.15, 130.26, 131.51, 131.59, 132.54, 132.69, 132.76,
134.68, 135.08, 136.23, 137.40, 137.51, 140.27, 140.45,
142.29. 142.32, 153.49, 153.51, 153.86, 154.75, 155.61,
155.93, 156.74, 171.82, 171.88, 172.22. IR: ꢁmax (KBr) cm-1:
3327.2, 2929.2, 2851.9, 1724.5, 1626.2. HRMS (EI): Found
735.2620(M++Na), C44H41ClN2O5Na requires 735.2601.
(135 mg, yield = 88 %, E/Z = 1:1). H-NMR (400 MHz,
CDCl3): ꢀ 0.91 – 0.96 (m, 6H, CH3), 2.44 - 2.55 (m, 4H,
CH2), 2.91 – 3.22 (m, 3H, NCH3), 3.65 – 4.41 (6H, NCH2,
OCH2, 2xOH), 6.51 – 7.35 (m, 28H, ArH, CH=CH). 13C-
NMR (100 MHz, CDCl3), (E/Z mixture): ꢀ 13.60, 13.68,
24.90, 25.57, 28.93, 29.03, 30.99, 33.88, 49.26, 65.92, 68.11,
113.98, 114.33, 114.94, 115.06, 115.31, 116.56, 125.82,
126.99, 127.28, 127.81, 127.90, 128.04, 129.52, 129.67,
129.76, 130.02, 130.70, 130.85, 131.31, 132.06, 132.10,
135.54, 135.89, 138.06, 140.63, 142.79, 153.99, 154.95,
157.13. IR: ꢁmax (KBr) cm-1: 3326.6, 2928.9, 2850.8, 1626.2,
1575.2. HRMS (MALDI-TOF): Found 726.3588; C50H48NO4
requires 726.3583(M++H).
N-[2-(4-{(E/Z)-1-[4-(tert-Butyldimethylsilanyloxy)phenyl]-
2-phenylbut-1-enyl}phenoxy)ethyl]-N-methylsuccinamic
acid (10)
(E)-N-[2-(4-{(E/Z)-1-[4-(tert-Butyldimethylsilanyloxy)-
phenyl]-2-phenylbut-1-enyl}-phenoxy)ethyl]-3-{4-[(E/Z)-1-
(4-methoxyphenyl)-2-phenylbut-1-enyl]phenyl}-N-
methylacrylamide (9a)
The amine 1d (0.20 g, 0.41 mmol, E/Z = 1:1) and
succinic anhydride (0.04 g, 0.41 mmol) were dissolved in
5ml of dry CH2Cl2. The reaction was allowed stir at room
temperature for 16 h. Reaction was monitored via TLC
(CH2Cl2:MeOH = 4:1). Reaction mixture was worked up via
the addition of CH2Cl2 (10 mL), washed with 1M NaOH
solution (10 mL). The aqueous phase was extracted with
CH2Cl2 (10 mL x3). The combined organic layers were
acidified with dilute HCl dropwise, washed with water (10
mL) and brine (10 mL), dried over sodium sulfate and then
evaporated to dryness in vacuo to afford an isomeric mixture
(E:Z = 1:1) of the product (222 mg, 92%) as a light brown
The acid 4b [34] (0.13 g, 0.34 mmol, 1 eq.), DCC (0.07
g, 0.34 mmol, 1 eq.), DMAP (0.04 g, 0.34 mmol, 1 eq.) was
stirred in 5 mL of anhydrous CH2Cl2 for 10 mins. 1d (0.166
g, 0.34 mmol, 1 eq.) was added to the reaction mixture and
allowed stir at room temperature for 24 h under nitrogen
atmosphere. After which time the reaction was diluted with
20 mL CH2Cl2 and filtered to remove insoluble solids. The
solvent was removed in vacuo and the residue was purified
via flash chromatography (CH2Cl2:MeOH, 20:1) to afford
the product 9a as a resin (220 mg, yield = 76 %, E/Z = 1:1).
1H-NMR (400 MHz, CDCl3): ꢀ 0.12 – 0.25 (m, 6H,
Si(CH3)2), 0.94 – 1.02 (m, 15H, CH3 ,SiC(CH3)3), 2.49 –
2.53 (m, 4H, CH2), 3.04 – 4.33 (m, 10H, NCH3, NCH2,
OCH2, OCH3), 6.49 – 7.77 (m, 28H, ArH, CH=CH). 13C-
NMR (100 MHz, CDCl3), (E/Z mixture): ꢀ -4.91, -4.80,
13.19, 17.76, 25.25, 28.61, 33.54, 36.06, 48.56, 54.57,
112.39, 112.69, 113.14, 113.47, 116.41, 118.52, 119.10,
125.42, 125.73, 125.86, 126.55, 127.30, 127.48, 129.20,
129.26, 129.54, 130.80, 131.39, 131.51, 134.64, 136.23,
142.12, 145.03, 156.44, 157.16, 162.10. IR: ꢁmax (KBr) cm-1:
3430.0, 3326.2, 2928.7, 2850.8, 1625.3, 1575.8. HRMS (EI):
Found 876.4424, C57H63NO4SiNa requires 876.4424.
1
oil. H-NMR (400 MHz, CDCl3): ꢀ 0.12 – 0.25 (m, 6H,
Si(CH3)2), 0.94 – 1.02 (m, 12H, SiC(CH3)3, CH3), 2.48 –
2.52 (m, 2H, CH2), 2.65 - 2.87 (m, 4H, succinic CH2), 3.00 -
3.22 (m, 3H, NCH3), 3.70 – 4.17 (m, 4H, CH2N, CH2O),
6.49 – 7.20 (m, 13H, ArH), COOH not observed. 13C-NMR
(100 MHz, CDCl3), (E/Z mixture): ꢀ -4.92, -4.80, 13.18,
13.22, 17.75, 25.22, 25.24, 28.46, 28.60, 37.04, 37.10, 47.91,
65.78, 66.12, 112.65, 113.42, 118.53, 119.10, 125.46,
127.31, 127.39, 127.43, 129.26, 130.09, 130.22, 130.30,
131.38, 131.57, 131.65, 135.73, 136.20, 136.27, 137.37,
137.44, 140.65, 142.08, 153.06, 156.68, 172.69, 176.78; IR:
ꢁmax (KBr) cm-1: 3435.7, 2927.5, 1696.5, 1624.0, 1603.6.
HRMS (EI): Found 610.2972 (M+Na)+, C35H45NO5SiNa
requires 610.2965.
(E)-N-(2-{4-[(E/Z)-1-(4-Hydroxyphenyl)-2-phenylbut-1-
enyl]phenoxy}ethyl)-3-{4-[(E/Z)-1-(4-hydroxyphenyl)-2-
phenylbut-1-enyl]phenyl}-N-methylacrylamide (9b)
N-{5-[2-Cyano-2-(3,4,5-trimethoxyphenyl)vinyl]-2-metho-
xyphenyl}-N'-(2-{4-[(E/Z)-1-(4-hydroxyphenyl)-2-phenyl-
but-1-enyl]phenoxy}ethyl)-N'-methylsuccinamide (11)
A solution of 9a (0.18 g, 0.21 mmol, 1 eq.) was stirred in
anhydrous CH2Cl2 (5 mL). The mixture was cooled to -78 °C
and 1.0 M boron tribromide solution (0.84 mL, 0.84 mmol, 4
eq.) was added slowly to the reaction mixture. The reaction
was allowed stir at -78 °C for 45 minutes then allow return to
room temperature while stirred for three hours. The reaction
was quenched through the addition of 3 mL of methanol.
The mixture was evaporated to dryness in vacuo and the
The protected endoxifen succinic acid linker compound
10 (0.10 g, 0.17 mmol, E:Z = 1:1), dicyclohexylcarbodiimide
(0.04 g, 0.17 mmol) and 1-hydroxybenzotriazole hydrate
(0.02 g, 0.17 mmol) were dissolved in dry CH2Cl2 (5 mL)
under a N2 environment. The mixture was allowed to stir for
20 minutes before adding a solution of the cis-acrylonitrile 6
(0.06 g, 0.17 mmol) in dry CH2Cl2 (3 mL). The reaction was

302 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3
Keely et al.
allowed stir at room temperature for 24 h until no starting
material was visible by TLC (CH2Cl2:MeOH, 4:1). The
reaction mixture was filtered to remove the dicyclohexylurea
byproduct. The mixture was evaporated to dryness in vacuo.
The residue was dissolved in 3 mL anhydrous THF and
stirred under a nitrogen atmosphere. A quantity of 0.1 M
TBAF (0.20 mL, 0.02 mmol) was added to the mixture and
allowed stir for 24 h. The mixture was evaporated to dryness
under reduced pressure. The residue was dissolved in CH2Cl2
and washed with 10 % HCl solution. The resulting organic
phase was dried over sodium sulfate and evaporated to
dryness under vacuum. The residue was purified via flash
chromatography on silica gel (CH2Cl2:MeOH, 20:1) to yield
an isomeric mixture (E:Z = 1:1) of the product 12 (35 mg, 26
(E/Z mixture): ꢄ 13.09, 19.53, 21.24, 21.89, 24.26, 24.89,
25.03, 26.17, 26.96, 28.57, 28.86, 29.25, 32.45, 34.81, 47.66,
47.84, 52.38, 112.64, 113.36, 113.43, 113.96, 114.66,
118.40, 119.20, 119.30, 119.55, 125.42, 125.54, 126.91,
127.37, 129.25, 130.04, 130.20, 130.40, 131.59, 132.01,
133.02, 133.71, 139.04, 148.43, 149.02, 153.49, 153.80,
171.45, 172.00, 198.52. IR: ꢅmax (KBr) cm-1: 3435.0, 2930.7,
1738.0, 1631.2, 1605.9. HRMS (MALDI-TOF): Found
709.3290; C44H45N2O6 requires 709.3278(M++H).
Biochemistry
Estrogen Receptor (ER) Competitor Assay
ERꢀ and ERꢆ fluorescence polarization based-competitor
assay kits were obtained from Invitrogen. The recombinant
ER and the fluorescent estrogen ligand were removed from
the -80°C freezer and thawed on ice for one-hour prior to
use. The assay was performed using a protocol described by
the manufacturer. The fluorescent estrogen (2 nM) was
added to the ER (30 nM for ERꢀ and 20 nM for ERꢆ),
screening buffer (100 nM potassium phosphate (pH 7.4), 100
ꢁg/ml BGG, 0.02 M NaN3) was added to make up a final
volume that was dependent on the number of tubes used.
Test compound, 1 ꢁL, in varying concentrations, was added
to 49 ꢁL screening buffer in 96-well black plates, 50 ꢁL of
the fluorescent estrogen/ER complex was added to make up
the total volume to 100 ꢁL. A vehicle control contained 1 %
DMSO (v/v). A negative control contained 50 ꢁL of
screening buffer and 50 ꢁL of fluorescent estrogen/ER
complex. This control was used to determine the polarization
value when no competitor was present (theoretical maximum
polarization). 1 ꢁL of 1 mM estradiol (final concentration 10
ꢁM) was used as complete displacement (minimum
polarization value). The tubes were incubated in the dark at
room temperature for 2 hours and were mixed by shaking on
a plate shaker. The polarization instrument contained 485
nM excitation and 530 nM emission interference filters.
1
%). H-NMR (400 MHz, CDCl3): ꢄ 0.87 – 0.95 (m, 3H,
CH3), 2.44 – 3.23 (m, 9H, NCH3, CH2, 2xsuccinic-CH2),
3.65 – 4.28 (m, 16H, NCH2, 4xOCH3, OCH2), 6.33 – 7.80
(m, 20H, ArH, C=CH, NH), OH not observed. 13C-NMR
(100 MHz, CDCl3), (E/Z mixture): ꢄ 13.18, 14.24, 17.93,
27.87, 27.92, 28.55, 28.80, 29.25, 31.14, 36.20, 36.96, 47.62,
51.43, 55.70, 58.03, 66.26, 105.43, 106.13, 112.79, 113.40,
114.06, 114.57, 114.73, 115.03, 127.12, 127.37, 128.13,
128.52, 128.66, 129.25, 129.54, 130.21, 130.57, 130.64,
131.24, 131.56, 131.83, 132.14, 134.85, 162.32, 171.48,
173.27. IR: ꢅmax (KBr) cm-1: 3467.8, 2208.6, 1734.8, 1636.3,
1508.9. HRMS (MALDI-TOF):. Found 808.3398;
C47H49N2O8K requires 808.3126.
N-(2-{4-[(E/Z)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl]
phenoxy}ethyl)-N-methyl-succinamic acid 5-oxo-6-pyridin-
4-ylmethyl-5,6,7,8-tetrahydronaphthalen-1-yl ester (12)
The acid 10 (1 equivalent, 0.25 mmol), DCC (1
equivalent, 0.25 mmol, 0.05 g) and HOBt (1 equivalent, 0.25
mmol, 0.03 g) were suspended in 3 mL of anhydrous CH2Cl2
and stirred for 10 minutes under a nitrogen atmosphere. The
phenol 5 was then dissolved in 3 mL of anhydrous DCM and
slowly added to the mixture via syringe. Reaction was
allowed stir for 24 h. Reaction was monitored via TLC
(CH2Cl2:MeOH, 4:1) until no more starting materials were
visible. The reaction mixture was diluted to 15 mL with
anhydrous CH2Cl2 and filtered to remove DCU. The filtrate
was evaporated to dryness under reduced pressure. The
residue was dissolved in 3 mL anhydrous THF and stirred
under a nitrogen atmosphere. A solution of 0.1 M TBAF (2
equivalents) was added to the mixture and allowed stir for 24
h. The mixture was evaporated to dryness under reduced
pressure. The residue was dissolved in CH2Cl2 and washed
with 10 % HCl solution. The resulting organic phase was
dried over sodium sulfate and evaporated to dryness under
vacuum. The residue was purified via flash chromatography
(CH2Cl2:MeOH, 20:1) to yield an isomeric mixture of the
Cell Proliferation Assays
All assays were performed in triplicate for the
determination of mean values reported. Compounds were
assayed as the free bases isolated from reaction. The human
breast tumour cell line MCF-7 was cultured in Eagles
Minimum Essential (MEM) medium in a 95% O2/5% CO2
atmosphere supplemented with 10% foetal bovine serum, 2
mM L-glutamine and 100 ꢁg/mL penicillin/streptomycin
The medium was further supplemented with 1% non-
essential amino acids. Cells were trypsinised and seeded at a
density of 2.5 x 104 cells/mL into a 96-well plate and
incubated for 24 h. After this time they were treated with 2
ꢁl volumes of test compound which had been pre-prepared
as stock solutions in ethanol to furnish the final
concentration range of study, 1nM-100 ꢁM, and re-incubated
for a further 72 h. Control wells contained the equivalent
volume of the vehicle ethanol (1% v/v). The culture medium
was then removed and the cells washed with 100 ꢁL
phosphate buffer saline (PBS) and 50 ꢁL of 1 mg/mL MTT
solution was added. Cells were incubated for 2 h in darkness
at 37°C. At this point solubilisation was begun through the
addition of 200 ꢁL DMSO and the cells maintained at room
temperature in darkness for 20 min to ensure thorough
1
product as a brown oil (112 mg, 63%, E/Z = 1:1). H-NMR
(400 MHz, CDCl3): ꢄ 0.99 (t, J = 7.5Hz, 3H, CH3), 1.27 -
1.43 (m, 2H, CH2), 1.74 (m, 2H, CH2), 2.50 (m, 2H, CH2),
2.64 – 2.87 (m, 4H, CH2), 3.17 – 3.03 (m, 5H, CH2, NCH3),
3.45 (dd, 1H, J = 4 Hz, 13.5 Hz, CH), 3.66 (m, 1H, CH2N),
3.77 (m, 1H, CH2N), 3.97 (m, 1H, CH2O), 4.12 (m, 1H,
CH2O), 6.51 (m, 1H, ArH), 6.70 (m, 1H, ArH), 6.77 (d, 1H,
ArH, J = 8 Hz), 6.85 (m, 2H, ArH), 7.12 (m, 7H, ArH), 7.17
(s, br, 2H, ArH), 7.36 (s, 2H, ArH), 7.62 (d, 1H, ArH, J = 8
Hz), 7.71 (d, 1H, ArH, J = 5.0 Hz), 8.63 (d, 2H, pyridine-H,
J = 5.2 Hz), OH not observed. 13C-NMR (100 MHz, CDCl3),

Design and Evaluation of Estrogen Receptor Ligand Conjugates
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3 303
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[16]
[17]
[18]
CONFLICT OF INTEREST
[19]
[20]
[21]
Declared none.
ACKNOWLEDGEMENTS
This study was supported by the Irish Research Council
for Science, Engineering and Technology (IRCSET).
Nuclear magnetic spectroscopy was carried out by Dr.
Manuel Ruethers and Dr. John O'Brien at the Department of
Chemistry, Trinity College Dublin. High-resolution mass
spectroscopy was carried out by Dr. Martin Feeney at the
Department of Chemistry, Trinity College Dublin.
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