Developing tamoxifen-based chemical probes for use with a dual-modality fluorescence and optical coherence tomography imaging needle

Article abstract of DOI:10.1071/CH19364

Fluorescent small molecules based on the chemotherapeutic tamoxifen have been synthesised for use with an imaging needle capable of acquiring simultaneous fluorescence and optical coherence tomography (OCT) images. The chemical probes are based on the active metabolite of the drug, 4-hydroxytamoxifen that is coupled with a diamine linker to commercially available Alexa Fluor or 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene (BODIPY) dyes. The tamoxifen derivatives were then added to cultures of live oestrogen receptor positive MCF-7 human breast cancer cells and imaged using the miniaturised fibre-optic device enclosed within a 23-gauge needle (outer diameter 640 μm). The OCT images showed the micro-architecture of the cell culture, while the fluorescence identified oestrogen receptor positive cells. Both dyes were found to have suitable excitation and emission properties and are good candidates to further develop as probes for fluorescence-guided surgery.

Full text of DOI:10.1071/CH19364

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
https://doi.org/10.1071/CH19364
Developing Tamoxifen-Based Chemical Probes for Use with
a Dual-Modality Fluorescence and Optical Coherence
Tomography Imaging Needle
A
B C D
, ,
E
Louisa A. Ho, Loretta Scolaro,
Elizabeth Thomas,
B C D
B C D
, ,
,
,
Bryden C. Quirk,
Rodney W. Kirk,
B C D
, ,
A F G H
, , ,
Robert A. McLaughlin,
and Rebecca O. Fuller
A
School of Molecular Sciences M310, The University of Western Australia,
35 Stirling Hwy, Crawley, WA 6009, Australia.
B
Australian Research Council Centre of Excellence for Nanoscale Biophotonics,
Adelaide Medical School, The University of Adelaide, North Terrace, Adelaide,
SA 5005, Australia.
C
Institute for Photonics and Advanced Sensing, The University of Adelaide,
North Terrace, Adelaide, SA 5005, Australia.
D
E
School of Engineering, The University of Western Australia, 35 Stirling Hwy, Crawley,
WA 6009, Australia.
School of Surgery M507, The University of Western Australia, QEII Medical Center,
Monash Ave, Nedlands, WA 6009, Australia.
F
School of Molecular and Life Sciences, Curtin University, Bentley, WA 6102, Australia.
G
Current address: School of Natural Sciences – Chemistry, University of Tasmania,
Hobart, Tas. 7001, Australia.
H
Corresponding author. Email: rebecca.fuller@utas.edu.au
Fluorescent small molecules based on the chemotherapeutic tamoxifen have been synthesised for use with an imaging
needle capable of acquiring simultaneous fluorescence and optical coherence tomography (OCT) images. The chemical
probes are based on the active metabolite of the drug, 4-hydroxytamoxifen that is coupled with a diamine linker to
commercially available Alexa Fluor or 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes. The tamoxifen
derivatives were then added to cultures of live oestrogen receptor positive MCF-7 human breast cancer cells and imaged
using the miniaturised fibre-optic device enclosed within a 23-gauge needle (outer diameter 640 mm). The OCT images
showed the micro-architecture of the cell culture, while the fluorescence identified oestrogen receptor positive cells. Both
dyes were found to have suitable excitation and emission properties and are good candidates to further develop as probes
for fluorescence-guided surgery.
Manuscript received: 5 August 2019.
Manuscript accepted: 1 October 2019.
Published online: 12 December 2019.
receptor (ER) positive tumours.[4] Being a selective drug, it only
modulates the oestrogen receptors.[5] It is one of the success
stories in medical oncology, finding widespread use in a variety
Introduction
The development of small molecule fluorescent probes is a
significant area of current interest.[1–3] Molecules that have the
ability to bind to specific target receptors in cells provide the
promise of imaging at a molecular level. Research is often
focussed on the development of new probes that are selective to
specific cellular processes and properties, providing a means for
scientists to understand the mechanisms behind key biological
processes through the visualisation and quantification of cellular
features such as nucleic acids, proteins, and ions.[1] Some of the
most highly anticipated fluorescent probes are for the detection
of cancer and its associated processes.
N
O
R
Tamoxifen is a small molecule (Fig. 1) that has become an
essential component in the prevention and treatment of breast
cancer. The drug was the first therapeutic that targeted oestrogen
Fig. 1. The chemical structureof the drug tamoxifen (R ¼ H) and the active
metabolite 4-hydroxytamoxifen (R ¼ OH).
Journal compilation ꢀ CSIRO 2019
www.publish.csiro.au/journals/ajc

B
L. A. Ho et al.
of treatment and preventative measures.[6] This molecule is an
ideal candidate for further development, both in terms of
improving the therapeutic potential as well as for use as a
chemical probe. Indeed, many new examples are being devel-
oped for a variety of uses.[7,8] Despite this potential, there are
very few examples of fluorescent analogues that are reported in
the literature.[9–11]
The development of small molecule fluorescent probes for
cancer detection is not a new idea. A large body of research into
the synthesis of these materials already exists.[12,13] Probes are
developed for use both in laboratories as well as in clinical
applications.[3,14] One under-represented area of this research is
the use of molecular probes in fluorescence-guided surgery.[15]
We aim to build on this through use of a fluorescent probe paired
with an intra-operative imaging device to improve the accuracy
of breast cancer surgery.
Fig. 2. Imaging needle (diameter 640 mm).
brightest and most stable fluorophores (FL) that minimises
self-quenching, an Alexa Fluor 488 dye (AF488). This dye
was chosen to maximise our likelihood of observing the cells
during imaging with the imaging needle. We have also prepared
a previously reported[9] analogue using the more economic
and widely used 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
(BODIPY) FL. While both dyes have similar excitation and
emission profiles, they differ in cell permeability. The Alexa
Fluor dyes are cell impermeable, and unlike the BODIPY FL
may lack the ability to enter the cell and interact with the ER. The
two analogues provide scope to determine which FL is more
suitable for use with an imaging needle.
In particular, our goal is to produce a conjugate of tamoxifen
that targets the oestrogen receptors, and demonstrate that it may
be imaged with a dual-modality fluorescence þ optical coher-
ence tomography (OCT) device. OCT is an imaging modality
commonly used in ophthalmology[16] and cardiology.[17,18]
It provides label-free images of tissue micro-architecture,
typically at a spatial resolution of 5 to 20 mm. OCT is based on
detection of back-scattered near-infrared light using low-
coherence interferometry.[19] While its use has been explored
in breast cancer,[20] it suffers poor tissue differentiation because
several tissues present in breast cancer have similar optical
scattering and absorption properties. The simultaneous detec-
tion of a cancer-specific fluorescent probe would significantly
improve tissue differentiation, while complementing the ability
of OCT to image the structural organisation of the tissues.
Utilising optical imaging in a solid tissue cancer such as
breast cancer is limited by the poor image penetration of optical
techniques in turbid tissue. Techniques such as fluorescence and
OCT are typically limited to a few hundred micrometres up to
1.5 mm.[21,22] To address this, we make use of an imaging needle
(Fig. 2).[23,24] The device consists of an optical fibre with a
miniaturised lens fabricated at the distal end.[25] The optical
fibre transmits and detects the imaging light (both OCT and
fluorescence). This is enclosed within a 23 gauge needle (outer
diameter 640 mm). By incorporating the imaging device into a
small, rigid needle, we are able to insert it deep within tissue,
well beyond the image penetration depth that typically limits
optical imaging techniques. This provides a technical pathway
to utilise such imaging techniques intra-operatively for the
detection of residual malignant tissue following surgical exci-
sion of the tumour.[26] One application of such a tool is to reduce
the incidence of involved margins in breast cancer surgery, and
thus reduce the proportion of patients who are required to
undergo additional surgery.
Results and Discussion
Synthesis of the new compound, 4 is based on two previously
reported methods (Scheme 1).[9,27] Briefly the oestrogen mod-
ulator, 4-hydroxytamoxifen was made through a McMurry
coupling reaction. The amine-containing basic side chain is
known to extend out of the binding pocket of the oestrogen
receptor a, hence a useful group to modify for conjugation.[28]
In this case, triphenylethylethylene (1) is monoalklylated with
dibromoethane (2). A mixture of the E and Z isomers is produced
from the reaction. It is the Z isomer which predominantly is
found to bind to the receptor both in vivo and in vitro.[29] It has
been shown in pure (99 %) hydroxytamoxifen isomers that they
undergo facile isomerisation in tissue culture medium at
378C.[29] Despite the mixture of isomers produced in this work,
there should be sufficient quantities of the active trans form of
the metabolite for binding. Nucleophilic substitution of the
haloalkane with diaminohexane is then used to provide a spacer
group 3 for conjugation of the fluorophore AF488 to the small
molecule 4. A second analogue 5 was also prepared, only dif-
fering from complex 4 in the final conjugation step, which
involved an alternative dye BODIPY FL. Cell lines were incu-
bated with 4 and 5 using conditions developed previously.[11]
The cellular localisation of 4 with ER-positive MCF7 breast
cell lines was initially visualised by fluorescent confocal micros-
copy (Fig. 3). A brightfield confocal image for the control MCF7
cell lines has been included in the Supplementary Material
(Fig. S1). Following incubation of the cells with the conjugate,
uptake studies found the molecule was associated with the cells.
Compound 4 uses a cell-impermeable fluorescent dye, AF488,
meaning it is unlikely to be internalised. Indeed, localisation
appears to be consistent with a similar literature example involv-
ing the Alexa Fluor 546 dye.[9] Cellular uptake of conjugate 5 was
consistent with previous literature reports,[9] it maintains the
ability to bind to ER alpha cells with similar localisation to 4.
Experiments were also conducted with ER-negative (MDA231)
This proof of concept study involves using theimaging needle
to detect small molecule fluorescent probes in cell lines. The
chemical probe is based on the active metabolite of the drug
tamoxifen, 4-hydroxytamoxifen (Fig. 1) that is known to work by
inhibiting the growth of ER cells that account for 75 % of breast
cancer cases. To date, several tamoxifen analogues that have
the ability to conjugate to fluorophores have been developed by
the wider scientific community to be used to study oestrogen
receptor mediated processes both inside and outside of the cells
using confocal microscopy.[9–11] We have aimed to develop a
new analogue to test the suitability of this small molecule as a
contrast agent for use with the dual-modality imaging needle. In
particular, we have conjugated 4-hydroxytamoxifen to one of the

Tamoxifen-Based Chemical Probes
C
O
Br
Br
Zn(s), THF,
TiCI4, –10°C to reflux,
O
CsCO3, CH3CN,
60°C, 24 h, 20 %
4 h, 71 %
HO
HO
OH
Br
OH
HO
O
1
2
NH2(CH2)6NH2,
THF, 18 h,
65 %
–
–
SO3
SO3
+
NH2
H2N
O
Alexa Fluor 488
succinimidyl ester,
–
COO
DMSO, rt, 24 h
H
N
H
N
H
O
HO
(CH2)6N
HO
(CH2)6NH2
O
O
4
3
Scheme 1. Synthesis of the AF488 conjugate of 4-hydroxytamoxifen (4).
Fig. 3. Wide field fluorescence images of ER-positive MCF7 cell lines following incubation with conjugate for 1 h of dosing with
10 mM of 4. Scale bar 0.5 mm.
cell lines (Figs S2 and S3, Supplementary Material). Others have
seen that there is no specificity for compound 5 nor an alternative
Alexa Fluor analogue.[9] We also find there is no selectivity using
either compound 4 or 5. This is likely the result of the hydropho-
bic linker used to conjugate the fluorophore to the tamoxifen and
in the case of 4, the impermeable nature of AF488. Nonetheless,
both compounds still demonstrate that fluorescent analogues of
tamoxifen maybe visualised with the highly miniaturised optics
present in an imaging needle.
corner of each image). The OCT shows that both control and
labelled cells were successfully cultured. In the unlabelled
experiment, fluorescence is not observed, consistent with the
nature of the sample. For the cell lines that have been incubated
in 4 the areas of fluorescence are consistent with the OCT image.
The gradual variation in the level of fluorescence is due to a
slight experimental misalignment of the imaging needle with the
cell plate, such that some areas were located further from the
imaging needle.
Fig. 4 shows images acquired by positioning and translating
the imaging needle a few hundred micrometres above cell plates
containing ER-positive MCF7 cell lines which were either
unlabelled (control, top row of Fig. 4) or had been incubated
with 4 (bottom row of Fig. 4). Both OCT and fluorescence
images were acquired simultaneously through the same focus-
ing optics and so are intrinsically co-registered. In the OCT
image, cells appear as localised areas of high optical backscatter
(light grey) and show the structure of the cell culture. Individual
cells can be seen in areas of low concentration (bottom left
Further experiments were conducted using the imaging
needle to image ER-positive MCF7 cell lines incubated with
the BODIPY based conjugate, 5. Fig. 5 shows unlabelled,
control samples (top row) and cells that have been incubated
with 5. Similar results to those from the AF488 conjugate 4 are
shown, with no fluorescence detected in the control samples,
and a clear correspondence in the labelled sample between the
location of cells in the OCT and the fluorescence signal. We find
that the conjugated BODIPY FL is sufficiently emissive enough
to be detected with the imaging needle.

D
L. A. Ho et al.
(a)
(b)
(c)
(d)
Fig. 4. (a, c) Structural OCT images and (b, d) corresponding fluorescence signal in the same sample. (top row) Control
ER-positive MCF7 cell lines. (Bottom row) ER-positive MCF7 cell lines that have been incubated with 4. Scale bar is
0.5 mm.
A co-registered multi-modal image, showing an overlay of
In this work we have taken the first steps towards the goal of
developing a fluorescent probe for use with an imaging needle
with the potential to visualise the borders of tumours and normal
tissue. We have synthesised two fluorescent tamoxifen analo-
gues based on the conjugation of 4-hydroxytamoxifen with
either the AF488 or BODIPY FL dye. These molecular probes
where used to label MCF-7 human breast cells and were then
imaged with a dual-modality fluorescence þ OCT imaging
needle. Our results showed that the miniaturised optics in the
imaging needle were able to successfully detect both fluor-
ophores and the florescence signal corresponded closely to the
structural OCT images of the cell cultures.
the structural OCT and fluorescence images is shown in Fig. 6.
This image was obtained from ER positive cell lines incubated
with 5 and shows the powerful nature of multi-modal imaging
techniques. These experiments provide some of the first steps to
developing a contrast medium that could be used in conjunction
with an imaging needle for the detection of trace amounts of
breast cancer following surgical excision of a tumour.
Previous work with dual-modality imaging needles involved
detecting fluorescently labelled antibodies in human and mouse
liver samples.[24] However, there has been no demonstration of
the effectiveness of imaging needles to detect small molecules
targeting specific receptors, as is commonly used in cancer
diagnostics and therapeutics. Uncertainty in the detectability of
cells incubated with a fluorescent small molecule probe war-
ranted the synthesis of a molecular system conjugated to the
most emissive dye (AF488) suitable with the needle probe. From
the results presented in Figs 4 and 5, we can conclude that the use
of the more cost effective and cell permeable BODIPY FL will
be suitable for future work. Future work will involve experi-
ments with an imaging needle using a selective small molecule
in a wider range of cells.
Experimental
General Experimental
All reactions were carried out under an argon gas atmosphere in
flame-dried glassware with magnetic stirring, unless otherwise
stated. All reaction temperatures refer to bath temperatures.
All reactions involving heating were performed in a preheated
oil bath at the specified temperature, unless otherwise stated.
Solvents were used dry, unless otherwise stated. Solvents were
dried and purified according to the methods described by
Armarego and Chai.[30] All reagents were purchased from
Sigma–Aldrich, Fluka, Merck, or Boron Molecular and used
without further purification, unless otherwise stated. Thin layer
chromatography (TLC) was performed on Merck silica gel
Conclusion
Small molecule fluorescent probes have significant potential for
intra-operative identification of tumour margins during surgery.

Tamoxifen-Based Chemical Probes
E
(a)
(b)
(c)
(d)
Fig. 5. (a, c) Structural OCT images and (b, d) corresponding projected fluorescence signal in the same sample. (Top row)
Control ER-positive MCF7 cell lines. (Bottom row) ER-positive MCF7 cell lines that have been incubated with 5. Scale bar
is 0.5 mm.
OCT
Fluorescence
OCT + Fluorescence
ER + cells
ER + cells
ER + cells
500 µm
500 µm
500 µm
Fig. 6. ER-positive MCF7 cell lines that have been incubated with 5. The OCT, fluorescence, and combined images are shown. Labelled
cells provide a point of reference for the images. Note that the other areas of fluorescence also indicate the presence of MCF7 cells.
60 F254 pre-coated aluminium sheets. Visualisation of devel-
oped plates was achieved through the use of a 254 or 365 nm UV
lamp or staining with phosphomolybdic acid stain solution.
Column chromatography was performed using silica gel 60
(0.063–0.200 nm) as supplied by Merck, unless otherwise
stated. HPLC was conducted using an Agilent 1200 with a
photodiode array detector (PDA). Separation was achieved
using a 250 ꢀ 10 mm i.d., 5 mm, Apollo C18 reversed phase
column (Grace-Division) with a 33 mm ꢀ 7 mm guard column
of the same material. The detection wavelength was set at
260 and 485 nm. 1H and 13C NMR spectra were acquired in the
specified deuterated solvent using either a Bruker AV600
(600.13 MHz for 1H and 150.9 MHz for 13C), a Bruker AV500
(500.13 MHz for 1H and 125.8 MHz for 13C), or a Varian
Gemini-400 (399.85 MHz for 1H and 100.5 MHz for 13C)
spectrometer at 258C. Chemical shifts are reported in parts per
million downfield from tetramethylsilane using the solvent
resonance as an internal standard.[31] Data are reported as fol-
lows: chemical shift, multiplicity (app ¼ apparent, s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, br ¼ broad,

F
L. A. Ho et al.
BODIPY FL-X
succinimidyl ester,
F
F
DMSO, rt, 24 h
O
N
B
N
H
N
H
N
+
HO
O
(CH2)6NH2
HO
O
(CH2)6N
H
3
5
Scheme 2.
sept ¼ septet), coupling constant, integration, and assignment.
Mass spectra were acquired on a Waters liquid chromatograph
premier (LCT) mass spectrometer through atmospheric pressure
chemical ionisation (APCI) or electrospray ionisation (ES).
tube, and heated with stirring for 18h. After this time, the reaction
mixture was cooled to room temperature and then fused to silica
gel. The crude reaction mixture was then subjected to flash col-
umn chromatography (80 : 15 : 5 CHCl3/MeOH/NH4OH) to give
3 as an oil (132mg, 65%). The spectroscopic data for compound
3 matched that reported previously in the literature.[32]
1,1-Bis(4-hydroxyphenyl)-2-phenylbut-1-ene (1)
Zinc powder (6.12 g, 0.094 mol) and dry THF (60 mL) were
transferred to a flame-dried flask fitted with a magnetic stirrer
bar, reflux condenser, and dropping funnel, under argon gas. The
reaction mixture was then cooled to ꢁ108C in a carefully main-
tained dry ice/acetone bath. To the cooled mixture was added
TiCl4 (5.0 mL, 8.65 g, 0.046 mol) drop-wise. After the addition
was complete, the reaction mixture was heated at reflux for 2 h.
After this time, the reaction mixture was cooled in an ice bath,
and a solution of ice bath cooled 4,40-hydroxybenzophenone
(1.5 g, 0.007mol), propiophenone (3.0 mL, 3.0 g, 0.022mol), and
dry THF (120 mL) was added using a cannula. The resulting
mixture was then refluxed for a further 2 h in the dark. After being
cooled to room temperature the reaction mixture was quenched
with 10 % aqueous potassium carbonate (90 mL) and then
extracted with ethyl acetate (2ꢀ 90 mL). The combined organic
layers were washed with brine (90 mL), dried over MgSO4,
and concentrated under reduced pressure. The crude reaction
mixture was then subjected to flash column chromatography
(5 : 95 - 20 : 80 ethyl acetate/hexanes) to give 1 as a white solid
(1.50 g, 71 %). The spectroscopic data for compound 1 matched
that reported previously in the literature.[27]
Conjugation of 4-(1-(4-(2-(6-Aminohexylamino)ethoxy)-
phenyl)-2-phenylbut-1-enyl)phenol with Alexa Fluor 488
Fluorophore (4)
Commercially available Alexa Fluor 488 succinimidyl ester
(2 mg) was dissolved in dry DMSO (0.25 mL) and transferred to
a flask containing 3 (10 mg) and dry DMSO (0.25 mL). The
resulting mixture was left to stir at room temperature under an
atmosphere of argon gas overnight in the dark. After this time,
the reaction mixture was purified via preparative HPLC (60 : 40
methanol/water, 55 min). The eluent also contained 0.1 % tri-
fluoroacetic acid. Analytical purity . 99 % by HPLC (retention
time: 27.57 min). The fractions containing the appropriate
product were concentrated under reduced pressure to remove
acetonitrile and then freeze-dried using a lyophiliser to give a
red solid (0.2 mg). Insufficient material was recovered to
acquire a fully resolved NMR spectra. Details can be found in
the Supplementary Material. HRMS m/z (APCI) 973.2805; calc.
for C51H49N4O12S2 [M þ H]þ 973.2788.
Conjugation of 4-(1-(4-(2-(6-Aminohexylamino)ethoxy)-
phenyl)-2-phenylbut-1-enyl)phenol with BODIPY
Fluorophore (5)
E and Z 4-{1-[4-(2-Bromo-ethoxy)-phenyl]-2-phenyl-but-1-
enyl}-phenol (2)
A solution of 1 (1.5 g, 0.005 mol), caesium carbonate (9.48 g,
0.029 mol), and acetonitrile (A.R. grade, 60 mL) was heated at
608C for 15 min under an atmosphere of argon gas. After this
time, 1,2-dibromoethane (3 mL, 6.54g, 0.035 mol) was added to
the reaction mixture and it was left to stir at 608C overnight. After
being cooled to room temperature, the reaction mixture was
concentrated under reduced pressure to remove acetonitrile,
diluted with ethyl acetate (60 mL), and then poured into a solu-
tion of saturated ammonium chloride (60 mL). The aqueous and
organic layers were separated and the aqueous layer was then
extracted further with ethyl acetate (2 ꢀ 60 mL). The combined
organic layers were washed with brine (60 mL), dried over
MgSO4, and concentrated under reduced pressure. The crude
reaction mixture was then subjected to flash column chroma-
tography (5 : 95 - 10 : 90 ethyl acetate/hexanes) to give 2 as a
white solid (409 mg, 20 %). The spectroscopic data for com-
pound 2 matched that reported previously in the literature.[32]
Commercially available BODIPY FL-X succinimidyl ester
(5 mg) was dissolved in dry DMSO (0.25 mL) and transferred to
a flask containing OHT-6C (10 mg) and dry DMSO (0.25 mL)
(Scheme 2). The resulting mixture was left to stir at room
temperature under an atmosphere of argon gas overnight in the
dark. After this time, the reaction mixture was purified via
preparative HPLC (20 : 80 acetonitrile/water, 45 min - 100 %
acetonitrile, 15 min). The eluent also contained 0.1 % tri-
fluoroacetic acid. The fractions containing the appropriate
product were concentrated under reduced pressure to remove
acetonitrile and then freeze-dried using a lyophiliser to give a
red solid (3 mg). The spectroscopic data for compound 5 mat-
ched that reported previously in the literature.[9] Briefly: dH
(600.13 MHz, CD3OD) 7.43 (1H, s), 7.18–6.99 (10H, m), 6.78
(1H, dd, J 8.7, 3.2), 6.40 (1H, d, J 8.1), 6.32 (1H, t, J 4.2), 6.21
(1H, s), 3.46 (4H, br, m), 3.43–3.37 (2H, m), 3.21–3.17 (3H, m),
3.09 (1H, t, J 7.9), 3.02 (1H, t, J 7.9), 2.62–2.58 (2H, m), 2.51–
2.42 (5H, m), 2.27 (3H, s), 1.77–1.65 (2H, m), 1.56–1.32
(7H, m), 0.88 (3H, t, J 7.7). Insufficient material was generated
to obtain a 13C NMR spectrum. HRMS m/z (ES) 733.4080; calc.
for C44H52BF2N4O3 [M þ H]þ 733.4022: 755.3928; calc. for
C44H51BF2N4O3Na [M þ Na]þ 755.3920.
4-(1-(4-(2-(6-Aminohexylamino)ethoxy)phenyl)-2-
phenylbut-1-enyl)phenol (3)
Compound 2 (188 mg, 0.44 mmol), 1,6-diaminohexane (1.88 g,
0.016 mol), and dry THF (7.5mL) were combined in a sealed

Tamoxifen-Based Chemical Probes
G
Cell Culture and Bioconjugate Incubation
Acknowledgements
R.O.F. thanks Dr Anthony Reeder (CMCA-UWA) for help with mass
spectra acquisition and Dr Gavin Flematti for assistance with HPLC, and is
grateful for financial support from the Australian Research Council
(DE180100112), Cancer Council Western Australia through a Susan
Cavanagh ECI grant, and the University of Western Australia ReEntry
Fellowship. R.A.M. is supported by the Australian Research Council
(CE140100003 and DP150104660), Breast Cancer Research Centre WA,
and a Premier’s Research and Industry Fund grant provided by the South
Australian Government Department for Industry and Skills.
Breast cancer cells (ER positive MCF7 and ER negative
MDA231) were cultured in RPMI media containing 10 % fetal
bovine serum (Life Technologies). Cells were grown at 378C
in a humidifying incubator and then were sub-cultured onto
glass coverslips in sterile six-well plates at a concentration of
4 ꢀ 105 cells mLꢁ1. Sub-confluent cultures were incubated for
one hour in either 4 or 5 diluted in culture media to 10 mg mLꢁ1
.
Prior to imaging, cultures were rinsed and replaced into normal
growth media. Live stained cells were mounted under buffered
saline and visualised using a Nikon A1Si fluorescent confocal
microscope. Sequential fields were captured in the z-plane to
confirm internal cellular localisation.
References
[1] E. A. Specht, E. Braselmann, A. E. Palmer, Annu. Rev. Physiol. 2017,
79, 93. doi:10.1146/ANNUREV-PHYSIOL-022516-034055
[2] Y. Fu, N. S. Finney, RSC Adv. 2018, 8, 29051. doi:10.1039/
C8RA02297F
Imaging Experiments
[3] H.-W. Liu, L. Chen, C. Xu, Z. Li, H. Zhang, X.-B. Zhang, W. Tan,
Chem. Soc. Rev. 2018, 47, 7140. doi:10.1039/C7CS00862G
[4] V. C. Jordan, S. Koerner, Eur. J. Cancer 1975, 11, 205. doi:10.1016/
0014-2964(75)90119-X
[5] R. M. O’Regan, V. C. Jordan, Lancet Oncol. 2002, 3, 207. doi:10.1016/
S1470-2045(02)00711-8
[6] V. C. Jordan, Endocr. Relat. Cancer 2014, 21, R235. doi:10.1530/
ERC-14-0092
[7] Ahmad I. Shagufta, Eur. J. Med. Chem. 2018, 143, 515. doi:10.1016/
J.EJMECH.2017.11.056
The imaging needle was fabricated as described by Scolaro
et al.[24] Briefly, the device consisted of a length of double-clad
fibre (SM-9/105/125-20A, Nufern, USA). The distal end was
terminated with focusing optics fabricated by fusion-splicing a
short section of large-core step-index fibre which served as a
beam-expanding spacer, a section of GRIN fibre (100/125
GIMM, Draka Communications, USA) which functioned as a
lens, and an angle-polished section of no-core fibre to redirect the
light beam perpendicular to the fibre. This was enclosed within a
small glass capillary to maintain a fibre–air interface at the angle
polished surface and ensure total-internal reflection of the light
beam. The fibre was inserted into a 23-gauge stainless steel
needle (outer diameter 640 mm) and aligned such that the light
beam was directed out through a small hole that had been elec-
trochemically etched into the side of the needle, and then glued in
place using optical adhesive. The imaging needle was fusion
spliced to a double-clad fibre coupler (Castor Optics, Canada),
which allowed the OCT light and fluorescence excitation to be
inserted into the imaging needle, and the backscattered OCT
light and fluorescence emission tobe separated upon return. OCT
imaging was performed using a custom-built swept-source
OCTsystem, basedon a 50 kHzrepetitionrate wavelength-swept
laser source (Axsun Technologies Inc., USA). Fluorescence
excitation used 488 nm light from a frequency-doubled semi-
conductor laser (Sapphire SF 488, Coherent Inc., USA). The
fluorescence emission was detected using a photo-multiplier
tube (PMT) (9136B, Electron Tubes, United Kingdom).
[8] P. Y. Maximov, R. E. McDaniel, V. C. Jordan, P. Y. Maximov,
R. E. McDaniel, V. C. Jordan, Tamoxifen: Pioneering Medicine in
Breast Cancer 2013 (Springer: Basel).
[9] E. L. Rickert, S. Oriana, C. Hartman-Frey, X. Long, T. T. Webb,
K. P. Nephew, R. V. Weatherman, Bioconjug. Chem. 2010, 21, 903.
doi:10.1021/BC900461H
[10] F. Abendroth, M. Solleder, D. Mangoldt, P. Welker, K. Licha,
M. Weber, O. Seitz, Eur. J. Org. Chem. 2015, 2157. doi:10.1002/
EJOC.201403489
[11] L. A. Ho, E. Thomas, R. A. McLaughlin, G. R. Flematti, R. O. Fuller,
Bioorg. Med. Chem. Lett. 2016, 26, 4879. doi:10.1016/J.BMCL.2016.
09.028
[12] T. Yogo, K. Umezawa, M. Kamiya, R. Hino, Y. Urano, Bioconjug.
Chem. 2017, 28, 2069. doi:10.1021/ACS.BIOCONJCHEM.7B00233
[13] H. Zhu, J. Fan, J. Du, X. Peng, Acc. Chem. Res. 2016, 49, 2115. doi:10.
1021/ACS.ACCOUNTS.6B00292
[14] J. Blagg, P. Workman, Cancer Cell 2017, 32, 9. doi:10.1016/J.CCELL.
2017.06.005
[15] P. A. Valde´s, A. Kim, M. Brantsch, C. Niu, Z. B. Moses, T. D.
Tosteson, B. C. Wilson, K. D. Paulsen, D. W. Roberts, B. T. Harris,
Neuro-Oncology 2011, 13, 846. doi:10.1093/NEUONC/NOR086
[16] M. L. Gabriele, G. Wollstein, H. Ishikawa, L. Kagemann, J. Xu,
L. S. Folio, J. S. Schuman, Invest. Ophthalmol. Vis. Sci. 2011, 52,
2425. doi:10.1167/IOVS.10-6312
During imaging experiments, the imaging needle was
mounted on a two-axis motorised translation stage and posi-
tioned a few hundred micrometres above the coverslips that held
the cell cultures. The needle was scanned parallel to the cover-
slip over a 5 mm ꢀ 5 mm field of view, with 4 mm ꢀ 4 mm
spacing between measurements. OCT and fluorescence mea-
surements were intrinsically co-registered as they were acquired
simultaneously. Data was subsequently processed and visua-
lised using in-house software developed in C11 and Matlab
(MathWorks, USA).
[17] G. J. Tearney, E. Regar, T. Akasaka, T. Adriaenssens, P. Barlis,
H. G. Bezerra, B. Bouma, N. Bruining, J.-m. Cho, S. Chowdhary,
M. A. Costa, R. de Silva, J. Dijkstra, C. Di Mario, D. Dudeck, E. Falk,
M. D. Feldman, P. Fitzgerald, H. Garcia, N. Gonzalo, J. F. Granada,
G. Guagliumi, N. R. Holm, Y. Honda, F. Ikeno, M. Kawasaki,
J. Kochman, L. Koltowski, T. Kubo, T. Kume, H. Kyono, C. C. S. Lam,
G. Lamouche, D. P. Lee, M. B. Leon, A. Maehara, O. Manfrini,
G. S. Mintz, K. Mizuno, M.-a. Morel, S. Nadkarni, H. Okura, H. Otake,
A. Pietrasik, F. Prati, L. Ra¨ber, M. D. Radu, J. Rieber, M. Riga,
A. Rollins, M. Rosenberg, V. Sirbu, P. W. J. C. Serruys, K. Shimada,
T. Shinke, J. Shite, E. Siegel, S. Sonada, M. Suter, S. Takarada,
A. Tanaka, M. Terashima, T. Troels, S. Uemura, G. J. Ughi,
H. M. M. van Beusekom, A. F. W. van der Steen, G.-A. van Es,
G. van Soest, R. Virmani, S. Waxman, N. J. Weissman, G. Weisz,
J. Am. Coll. Cardiol. 2012, 59, 1058. doi:10.1016/J.JACC.2011.09.079
[18] M. J. Suter, S. K. Nadkarni, G. Weisz, A. Tanaka, F. A. Jaffer, B. E.
Bouma, G. J. Tearney, JACC Cardiovasc. Imaging 2011, 4, 1022.
doi:10.1016/J.JCMG.2011.03.020
Supplementary Material
Additional confocal imaging for both the MCF-7 and MDA-231
cell lines as well as characterisation data, including HRMS,
1
HPLC chromatogram, H NMR for compounds 4 and 5 are
available on the Journal’s website.
Conflicts of Interest
The authors declare no conflicts of interest.

H
L. A. Ho et al.
[19] D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson,
W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, et al.,
Science 1991, 254, 1178. doi:10.1126/SCIENCE.1957169
[20] L. Scolaro, R. A. McLaughlin, B. F. Kennedy, C. M. Saunders,
D. D. Sampson, Photonics Lasers Med. 2014, 3, 225. doi:10.1515/
PLM-2014-0013
[26] M. F. Dillon, A. D. K. Hill, C. M. Quinn, E. W. McDermott, N.
O’Higgins, Ann. Surg. Oncol. 2006, 13, 333. doi:10.1245/ASO.2006.
03.098
[27] D. D. Yu, B. M. Forman, J. Org. Chem. 2003, 68, 9489. doi:10.1021/
JO035164N
[28] A. K. Shiau, D. Barstad, P. M. Loria, L. Cheng, P. J. Kushner, D. A.
Agard, G. L. Greene, Cell 1998, 95, 927. doi:10.1016/S0092-
8674(00)81717-1
[21] R. K. Wang, Phys. Med. Biol. 2002, 47, 2281. doi:10.1088/0031-9155/
47/13/307
[22] J. Welzel, Skin Res. Technol. 2001, 7, 1. doi:10.1034/J.1600-0846.
2001.007001001.X
[23] D. Lorenser, B. C. Quirk, M. Auger, W.-J. Madore, R. W. Kirk, N.
Godbout, D. D. Sampson, C. Boudoux, R. A. McLaughlin, Opt. Lett.
2013, 38, 266. doi:10.1364/OL.38.000266
[24] L. Scolaro, D. Lorenser, W.-J. Madore, R. W. Kirk, A. S. Kramer, G. C.
Yeoh, N. Godbout, D. D. Sampson, C. Boudoux, R. A. McLaughlin,
Biomed. Opt. Express 2015, 6, 1767. doi:10.1364/BOE.6.001767
[25] L. Scolaro, D. Lorenser, R. A. McLaughlin, B. C. Quirk, R. W. Kirk,
D. D. Sampson, Opt. Lett. 2012, 37, 5247. doi:10.1364/OL.37.005247
[29] J. A. Katzenellenbogen, K. E. Carlson, B. S. Katzenellenbogen,
J. Steroid Biochem. 1985, 22, 589. doi:10.1016/0022-4731(85)
90210-9
[30] W. L. F. Armarego, C. Chai, Purification of Laboratory Chemicals,
5th edn 2003 (Butterworth-Heinemann: Cornwall).
[31] H. E. Gottlieb, V. Kotlyar, A. Nudelman, J. Org. Chem. 1997, 62,
7512. doi:10.1021/JO971176V
[32] J. P. Trebley, E. L. Rickert, P. T. Reyes, R. V. Weatherman, in
Chemical Genomics (Eds S. Jaroch, H. Weinmann) 2006, pp. 75–87
(Springer: Berlin)