Targeting telomerase with radiolabeled inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.09.028

The expression of telomerase in approximately 85% of cancers and its absence in the majority of normal cells makes it an attractive target for cancer therapy. However the lag period between initiation of telomerase inhibition and growth arrest makes direct inhibition alone an insufficient method of treatment. However, telomerase inhibition has been shown to enhance cancer cell radiosensitivity. To investigate the strategy of simultaneously inhibiting telomerase while delivering targeted radionuclide therapy to cancer cells,¹²³I-radiolabeled inhibitors of telomerase were synthesized and their effects on cancer cell survival studied. An¹²³I-labeled analogue of the telomerase inhibitor MST-312 inhibited telomerase with an IC₅₀of 1.58?μM (MST-312 IC₅₀: 0.23?μM). Clonogenic assays showed a dose dependant effect of¹²³I-MST-312 on cell survival in a telomerase positive cell line, MDA-MB-435.

Full text of DOI:10.1016/j.ejmech.2016.09.028

Accepted Manuscript
Targeting telomerase with radiolabeled inhibitors
Philip A. Waghorn, Mark R. Jackson, Veronique Gouverneur, Katherine A. Vallis
PII:
S0223-5234(16)30758-9
10.1016/j.ejmech.2016.09.028
EJMECH 8894
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 June 2016
Revised Date: 8 September 2016
Accepted Date: 9 September 2016
Please cite this article as: P.A. Waghorn, M.R. Jackson, V. Gouverneur, K.A. Vallis, Targeting
telomerase with radiolabeled inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.09.028.
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For Table of Contents
Targeting Telomerase with Radiolabeled Inhibitors
Philip A. Waghorn, Mark R. Jackson, Veronique Gouverneur and Katherine A.Vallis
MDA-MB-435
U2OS
1.0
0.8
0.6
0.4
0.2
0.0
IC50: 1.58 μM
Treatment

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Targeting Telomerase with Radiolabeled Inhibitors
Philip A. Waghorn,a Mark R. Jackson,a Veronique Gouverneur,b and
Katherine A.Vallis*,a
a CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford,
Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ, UK
b Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA,
UK
Email addresses of authors: pawaghorn@gmail.com
mark.jackson.2@glasgow.ac.uk
veronique.gouverneur@chem.ox.ac.uk
katherine.vallis@oncology.ox.ac.uk
Corresponding author: Professor K.A. Vallis
CR-UK/MRC Oxford Institute for Radiation Oncology
University of Oxford
Old Road Campus Research Building
Off Roosevelt Drive
Oxford, OX3 7DQ
Tel: +44 (0)1865 255209
Fax: +44 (0)1865 857533
Email: katherine.vallis@oncology.ox.ac.uk
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ABSTRACT
The expression of telomerase in approximately 85% of cancers and its absence in the majority of
normal cells makes it an attractive target for cancer therapy. However the lag period between
initiation of telomerase inhibition and growth arrest makes direct inhibition alone an insufficient
method of treatment. However, telomerase inhibition has been shown to enhance cancer cell
radiosensitivity. To investigate the strategy of simultaneously inhibiting telomerase while
delivering targeted radionuclide therapy to cancer cells, 123I-radiolabeled inhibitors of telomerase
were synthesized and their effects on cancer cell survival studied. An 123I-labelled analogue of
the telomerase inhibitor MST-312 inhibited telomerase with an IC50 of 1.58ꢀM (MST-312 IC50:
0.23ꢀM). Clonogenic assays showed a dose dependant effect of 123I-MST-312 on cell survival in
a telomerase positive cell line, MDA-MB-435.
Keywords: telomerase; telomerase inhibitors; molecularly targeted radiotherapy; Iodine-123
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1. INTRODUCTION
Preclinical and clinical evidence supports the inhibition of telomerase as a promising form of
cancer therapy [1, 2]. Telomerase is present in the majority (85-90%) of cancers cell lines, and is
responsible for rescuing them from crisis, and promoting uncontrolled and continued tumor
growth [1, 3, 4]. Telomerase inhibition disrupts the replicative capacity of cancer cells by
preventing maintainance of the telomeric sequences at the chromosome terminus, while leaving
most normal somatic cells largely unaffected. Telomerase activity is minimally reconstituted in
vitro by combining the human telomerase RNA template component (hTR) [5] and the catalytic
protein unit, human telomerase reverse transcriptase (hTERT) [6].
The hTR template region provides an accessible substrate-binding site allowing for direct
enzyme inhibition using antisense oligonucleotides, peptide nucleic acids (PNAs) and chemically
modified PNAs as competitive inhibitors, preventing active complex formation with the hTERT
component or binding to the telomere substrate [7]. GRN163L (Imetelstat) is a lipid-conjugated
N3’-P5’ thio-phosphoramidate 13-mer oligonucleotide that has been shown to inhibit telomerase
and cause telomere shortening in cells from brain, bladder, liver, lung, prostate and stomach
cancers [8-14].
Although hTR is expressed ubiquitously, telomerase activity is restricted by the
expression of the hTERT component [15, 16]. Numerous small molecule inhibitors of telomerase
have been identified [17-20]. Most notably BIBR-1532 [21, 22], where dose-dependent
inhibition of telomerase with increasing concentrations of BIBR-1532 has been shown to be
without significant effects on normal cells [23]. Other inhibitors include azidothymidine [24, 25],
the epicatechin derivatives, EGCG and MST-312 that strongly and directly inhibit telomerase
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[26-28], isothiazolone and bis-indole derivatives [29, 30], and several G-quadruplex stabilizing
molecules [31-33].
Several clinical trials are currently underway, targeting both the telomeres and telomerase
function. Clinical trials with Imetelstat for haematological malignancies (essential
thrombocythemia (ET), myelodysplastic syndrome, acute myelogenous leukaemia) and
myelofibrosis (MF) are planned, underway or completed [34]. So far phase II trials for ET and
MF have found no correlation between clinical response and telomere length [35]. Currently
phase I/II clinical trials with the oncolytic virus, OBP-301, are underway in patients with
hepatocellular carcinoma. In phase I testing OBP-301 was well tolerated with no serious adverse
effects [36]. The cancer vaccine, GV1001, a TERT derived peptide for telomerase driven
immunotherapy is involved in several clinical trials in NSCLC, pancreatic cancer, hepatocellular
carcinoma and malignant melanoma, where few side effects have been reported [37]. In phase
I/II NSCLC studies a GV1001-specific immune response was observed [38]. In a phase III trial
in pancreatic cancer, however, no improvement in overall survival was observed [39]. Although
BIBR-1532, MST-312 and several G-quadruplex inhibitors have had success in preclinical
testing they have not yet entered into clinical trials. The G-quadruplex stabilizer Quarfloxin/CX-
3543 has entered phase I and II trials but is thought to induce apoptosis through inhibition of
ribosomal RNA (rRNA) [40]. Several tankyrase inhibitors such as XAV939, which disrupt
telomere length regulation are being tested as treatment strategies but have not yet entered
clinical trials [41]. Despite significant insights into the role of telomerase in disease there is still
no agent yet approved for clinical use [42].
The relationship between cellular radiosensitivity and telomere length is one that has
been investigated extensively [43-47]. Goytisolo et al reported the connection between
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shortenened telomeres in late generation mTR-/- mice and radiation response, evident as
organism hypersensitivity to IR and increased DNA damage after irradiation [48]. Similarly
Wong et al have shown that telomerase inhibition and telomere dysfunction in fibroblasts from
late generation Terc-/- mice imparts an enhanced radiosensitivity associated with increased
mortality [49-51]. Similar studies have shown enhanced radiosensitivity in mice where telomeres
have been shortened by mutant hTERT expression [44, 45, 52, 53]. Increased telomerase
expression has been associated with enhanced genome stability and DNA repair mechanisms,
providing a protective mechanism against DNA damage [54, 55].
Radiolabeled agents that specifically inhibit telomerase activity would be expected,
therefore, to selectively increase radiosensitivity and so increase tumor cell kill [56]. We report
here the synthesis of a series of small molecule telomerase inhibitors, the protocols for
radiolabeling them with the Auger electron-emitting isotope, 123I, and their effect on telomerase
inhibition and cancer cell survival.
2. RESULTS AND DISCUSSION
The telomerase inhibitory capabilities of BIBR-1532, MST-312 and the flavonoid species 2-(3,4-
dihydroxyphenyl)-7,8-dihydroxy-4H-chromen-4-one (Figure 1) have been directly compared
under the same experimental conditions obtaining IC50 values of 3.6, 12.1 and 0.23 µM,
respectively [57]. Structure activity relationship studies with the BIBR-1532 and flavonoid
species have shown that certain site specific structural modifications to these parent structures
have only minimal effects on telomerase inhibition, suggesting inclusion of an Iodine-123
radiolabel modification to allow for combined targeted therapy would have limited effect on
telomerase inhibition in these species. As well as decaying by release of high energy, short
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pathlength Auger electrons, 123I was selected for site-specific inclusion in these molecules to
minimize the structural alterations that would be encountered with metal radioisotopes.
2.1 Chemistry
In Figure 1 the chemical structures of the iodinated analogues and their parent precursors,
tested for in vitro telomerase inhibition, are reported. Schemes 1-3 show the synthetic pathways
employed in the preparation of the iodinated derivatives 1-6. The iodinated BIBR-1532
analogues (Scheme 1) were prepared using an adapted literature protocol [58]. Briefly, the
condensation reaction of the acid chloride of (E)-3-(naphthalen-2-yl)but-2-enoic acid with either
the commercially available 5-iodo-2-aminobenzoate or with 4-iodo-2- aminobenzoate prepared
from N-(4-iodo-2-methyl-phenyl)-acetamide in 2 steps gave 1b and 2b in 72% and 67% yield
respectively.
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Figure 1: Structures of telomerase inhibitors; BIBR-1532, Flavonoid (FLAV) and MST-312,
and iodinated derivatives (1-6)
Base hydrolysis of 1b and 2b gave 1 and 2 in 93% and 95% yield respectively. The trimethyltin
analogue was prepared by reaction of 1b with hexamethyldistannane and bis-
(triphenylphosphine)-palladium(II)-dichloride in 1,4-dioxane to afford 1c after column
chromatography. Base hydrolysis of 1c gave 1d in 87% yield.
The iodinated flavonoids (Scheme 2) were prepared using the Baker-Venkataraman
transformation following an adapted literature protocol. An appropriate hydroxyacetophenone
was reacted with an appropriate analogue of 3,4-dimethoxybenzoyl chloride to yield a benzoyl
ester that, after treatment with base, yields a 1,3-diketone. Treatment of the diketone in acid
affords the protected flavone and demethylation achieved using 1M boron tribromide in the
protected flavone and demethylation achieved using 1M boron tribromide in dichloromethane at
0°C yields the flavone product. In the case of 3, iodinated hydroxyacetophenone 3a was prepared
by reaction of 2,3-dimethoxy-2-hydroxyacetophenone with N-iodosuccinimide and p-
toluenesulphonic acid in acetonitrile [59].
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O
O
R=I, R'=H (1a)
R=H, R'=I (2a)
H2N
i)
R
O
O
R'
H
N
OH
R=I, R'=H (1b)
R=H, R'=I (2b)
O
O
R
R'
iii)
ii)
O
OH
O
O
H
N
H
N
O
O
R
SnMe3
R'
R=I, R'=H (1)
R=H, R'=I (2)
(1c)
iv)
O
OH
O
OH
v)
H
N
H
N
O
O
SnMe3
I123
(1d)
[
123I]-(1)
Scheme 1: i) a) oxalyl chloride, DMF, DCM, 5h 0°C, b) 1a/2a, pyridine, DMAP, THF, 2h, r.t;
ii) LiOH, THF, H2O, 12h, r.t.; iii) Sn2Me6, PdCl2(PPh3)2, 1,4-dioxane, 2h, 60°C; iv) LiOH, THF,
H2O, 12h, r.t.; v) [123I]NaI, Chloramine T, MeCN, 15min, r.t.
This was subsequently reacted with the commercially available 3,4-dimethoxybenzoyl chloride
to give 3b which was converted to the 1,3-diketone, 3c. Cyclodehyration was achieved in acetic
acid to give 3d and demethylation gave 3. For 4 and 5, 3,4-dimethoxy-2-hydroxyacetophenone
was reacted with the acid chloride of either the commercially available 2-iodo-4,5-
dimethoxybenzoic acid to give 4b or with 3-iodo-4,5- dimethoxybenzoic acid prepared from 3-
iodo-4-hydroxy-5-methoxybenzaldehyde in 2 steps to give the benzoyl ester 5b. Subsequent
steps as for 3 afforded 4 and 5.
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O
O
R'
O
O
O
i)
ii)
I
R
O
R
O
HO
O
O
OH
O
O
O
O
R'
R''
(3a)
R=I, R'=R''=H (3b),
R'=I, R=R''=H (4b),
R''=I, R=R'=H (5b)
R=I, R'=H (5a),
R'=I, R'=H (4a),
iii)
O
OH R'
O
O
R'
R
O
R''
O
R
O
R''
OH
OH
O
O
O
O
O
R=I, R'=R''=H (3c),
R'=I, R=R''=H (4c),
R''=I, R=R'=H (5c)
iv)
O
O
O
O
O
v)
Sn
O
R
R
vi)
R'
O
R'
R''
O
R''
O
O
HO
O
O
OH
O
OH
O
OH
(3e)
R=I, R'=R''=H (3d),
R'=I, R=R''=H (4d),
R''=I, R=R'=H (5d)
R=I, R'=R''=H (3),
R'=I, R=R''=H (4),
R''=I, R=R'=H (5)
vii)
O
O
O
R
R
O
R'
viii)
O
R'
R''
O
R''
O
O
O
O
O
O
O
O
O
O
O
O
O
O
R=I, R'=R''=H (3f),
R'=I, R=R''=H (4f),
R''=I, R=R'=H (5f)
R=SnMe3, R'=R''=H (3g),
R'=SnMe3, R=R''=H (4g),
R''=SnMe3, R=R'=H (5g)
Scheme 2: i) 3,4-dimethoxybenzoyl chloride, pyridine, 2h, r.t.; ii) a) oxalyl chloride, DMF,
DCM, 2h 0°C, b) 3a, pyridine, DCM, 2h, r.t; iii) KOH, pyridine, 1h, 50°C; iv) NaOAc, AcOH,
2h, 120°C; v) Sn2Me6, PdCl2(PPh3)2, 1,4-dioxane, 1.5h, 60°C; vi) BBr3, DCM, 2h, 0°C – r.t.; vii)
Ac2O, pyridine, 20h, 45°C; viii) Sn2Me6, PdCl2(PPh3)2, 1,4-dioxane, 1.5h, 60°C.
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The trimethyltin analogue of 3 was prepared by first synthesizing the acetyl protected analogue
by reaction of 3 with acetic anhydride in pyridine. Then 3g was prepared by reaction of 3f with
hexamethylditin and bis-(triphenylphosphine)-palladium(II)-dichloride to give 3g in 58% yield.
4g and 5g were prepared in identical fashion.
O
I
O
O
O
O
i)
O
O
O
iii)
HO
HO
H2N
NH2
(6a)
(6d)
(6b)
O
O
O
I
O
O
O
I
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
H
H
H
H
(6c)
(6e)
iv)
ii)
I
Sn
OH
O
OH
O
O
O
HO
OH
O
O
O
O
N
H
N
H
N
H
N
H
(6f)
(6)
v)
I123
I123
OH
O
OH
O
O
O
HO
OH
O
O
O
O
N
H
N
H
N
H
N
H
vi)
123I]-(6)
[
123I]-(6e)
[
Scheme 3: i) a) 6b, oxalyl chloride, DMF, DCM, 2h 0°C, b) 6a, pyridine, DCM, 2h, r.t.; ii)
BBr3, DCM, 2h, 0°C – r.t.; iii) 6c, oxalyl chloride, DMF, DCM, 2h 0°C; c) 6a, pyridine, DCM,
2h, r.t; iv) Sn2Me6, PdCl2(PPh3)2, 1,4-dioxane, 12h, 75°C; v) [123I]NaI, H2O2:HOAc, MeCN,
15min, r.t.; vi) 3M HCl, MeOH, 0.5h, 80°C.
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The iodinated MST-312 analogue 6 (Scheme 3) was prepared by the condensation of 6a
with the acid chloride of 6b, followed by demethylation with boron tribromide. The stannane
analogue was prepared by the condensation of 6a with the acid chloride of 6d, followed by
reaction with hexamethylditin and bis-(triphenylphosphine)-palladium(II)-dichloride to give 6f in
26% yield.
2.2 Telomerase inhibition
Telomerase Repeat Amplification Protocol (TRAP) measurements have demonstrated
that the iodinated compounds were able to directly inhibit telomerase activity in a cell free
system with minimally reduced inhibitory potencies compared to their respective parent
compounds as a result of inclusion of the iodide substituent. Compounds 1 and 2 were shown to
have IC50 values of 30.09 µM and 28.08 µM respectively, corresponding to a ~2.5 fold reduction
in telomerase inhibitory activity with respect to the parent BIBR-1532 compound (IC50: 11.57
µM). The iodinated flavonoid isomers 3 and 5 displayed telomerase inhibitory capabilities with
IC50 values of 1.65 and 1.73 µM respectively, similarly corresponding to a 2.0-2.2 fold reduction
in telomerase inhibitory activity with respect to the parent flavonoid compound (IC50: 0.74 µM).
The IC50 values measured for BIBR-1532 and the parent flavonoid, 7,8,3’,4’-
tetrahydroxyflavone, differ from those previously reported in the literature, which we ascribe to
variations in method and the cell line that was used. Compound 4 was found not to inhibit
telomerase activity to the same extent as 3 and 5 (See Supporting Information) and as such
further radiolabeling studies with 4 were not conducted. Compound 6 displayed no telomerase
inhibitory activity under the described protocol, however under these experimental conditions the
parent MST-312 compound similarly failed to inhibit telomerase in the TRAP setup that was
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used in this study. TRAP experiments were repeated with compound 6 and parent MST-312
compound using a range of alternative telomerase positive cell lysates but no positive inhibition
with MST-312 was observed, suggesting that direct inhibition of telomerase is not the method of
inhibition in these cell lines. Indirect effects such as signaling or changes in expression levels, or
the need of metabolic activation of MST-312 in cells may be required for telomerase inhibition.
To address this, cells were plated in six well plates and treated with different concentrations of
either parent MST-312 or 6 over a 24 h incubation period. Cell lysates for each treated sample
were prepared as described in the Supporting Information and the TRAP assay performed on the
individually prepared lysate samples. Results indicate a concentration dependent inhibition of
telomerase activity after in vitro treatment of whole cells with the MST compounds, with the
iodinated species 6 having an IC50 of 1.58 µM compared with 0.23 µM for the parent MST-312
compound. In all cases the inclusion of an iodide substituent led to only a slight reduction in IC50
values, maintaining the possibility for potential telomerase inhibition and targeting.
The lag period required after telomerase inhibition to observe critical telomere shortening
and cell crisis suggests that telomerase inhibition alone may be insufficient as a stand-alone
treatment. The presence of components of the DNA damage response pathways at the telomeres
together with a reported telomere-independent role for telomerase in DNA repair suggests that a
combination of telomere inhibition and sensitivity to ionizing radiation (IR) may be potentially
interacting [60]. We therefore prepared the radiolabelled analogues of the iodinated inhibitors to
investigate further the effect of a combined radiotherapeutic with telomerase inhibition.
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Compound
IC50 (µM)
11.57
30.09
28.08
0.74
BIBR-1532
1
2
Flavonoid
1.65
3
N/D
4
1.73
5
MST-312
6
0.23
1.58
Table 1: Telomerase Inhibition Data for Compounds 1-6
N/D: Not determined
2.3 Radioiodination
To regioselectively prepare radioiodinated agents trimethyltin precursors were first
prepared. In the case of the BIBR-1532 derivative the trimethyltin complex 1d could not be
isolated as a pure species by direct stannylation of the iodinated analogue, (1). Instead the
trimethyltin complex of 1b was prepared which was stable to column chromatography on neutral
silica and 1d prepared subsequently by basic hydrolysis of 1c, following workup with careful
control of pH to avoid proto-destannylation. [123I]-(1) was subsequently radiolabeled using
Chloramine T (N-chloro-p-toluenesulfonamide sodium salt) as an oxidant with a decay corrected
isolated end of synthesis yield of 76% ± 6% (n = 10) and a radiochemical purity as assessed by
analytical radio-HPLC of >97%, with a specific activity of 6.08 ± 0.28 Ci/µmol (Figure 2).
Attempts to prepare a trimethyltin derivative directly from the parent compound, 3, met with
poor yields and difficulties in purification. Accordingly a trimethyltin analogue 3e was prepared
from the methoxy protected precursors necessitating the inclusion of a subsequent deprotection
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step following successful radiolabeling. Although radiolabeling was possible in good yields
(72%), subsequent deprotection attempts of the four methoxy groups using HI or BBr3 as per the
cold synthesis, met with limited success resulting in low yields and with multiple side products
seen by radio-HPLC analysis. To improve labeling yields alternative protecting group strategies
were sought and the acetylated analogue, 3g, was prepared accordingly. Again the initial
radiolabeling step was performed in good to high yields, and following treatment with 3M HCl at
elevated temperatures deprotection of the four acetyl groups was achieved in good yield.
Attempts to deprotect the acetyl groups under basic conditions led to complete deiodination of
the flavonoid species. [123I]-(3) was radiolabeled in a yield of 86 ± 8% (n = 10) and following
deprotection was prepared with a decay corrected end of synthesis yield of 64 ± 4% (n = 10) and
with a radiochemical purity as assessed by analytical radio-HPLC of >98%, with a specific
activity of 2.60 ± 0.17 Ci/µmol (Figure 2).
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Figure 2: Radiotrace and UVtrace for a) [123I]-(1), b) [123I]-(3) and c) [123I]-(6)
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[123I]-(5) was labeled in a similar fashion with an end of synthesis yield of 67% ± 6% (n = 10)
and with a radiochemical purity of >98%. The trimethyltin derivative, 6f was prepared in similar
fashion to the flavonoid species. A range of oxidants was explored and labeling optimized with
hydrogen peroxide in acetic acid giving a radiolabeling yield of 55 ± 3% (n=6). Following
deprotection and subsequent HPLC and Seppak cartridge purification steps a decay corrected end
of synthesis yield of 22 ± 5% was obtained, with a radiochemical purity > 97%, with a specific
activity of 2.89 ± 0.24 Ci/µmol.
The lipophilicity of compounds can affect their tissue permeability properties, which can
impact their localization in target tissues. Lipophilicity may also affect binding to low affinity
nonspecific sites that can compromise target tissue to background tissue ratios. The
octanol/water and octanol/PBS partition coefficients were measured using a standard protocol.
[123I]-(1) displayed a Log P: 1.84 ± 0.06 and Log D: 1.57 ± 0.14, indicative of a lipophillic
species possessing aromatic functionality as for the naphthyl group present. [123I]-(3) and [123I]-
(5) were more polar with Log P: 1.14 ± 0.07 and Log D: 0.99 ± 0.06, and Log P: 1.18 ± 0.08 and
Log D: 0.98 ± 0.07 respectively. [123I]-(6) displays a Log P: 1.57 ± 0.11 and Log D: 1.23 ± 0.21.
In vitro stability experiments were conducted in PBS, and in serum complete medium
(10% FBS). At regular intervals over a 24 h period HPLC analysis was performed to evaluate
complex stability. Incubation of [123I]-(1) and [123I]-(6) gave a single peak by radio-HPLC at all
time points with an integrated area of >98% and a retention time that correlated with 1 and 6
respectively. No signs of deiodination or decomposition were observed in PBS buffer. In
contrast, HPLC analysis of [123I]-(3) showed signs of complex decomposition with a loss of
radio-iodide label within 4 h incubation in PBS. After 24 h in PBS, only 48% of intact compound
remained. The only peaks in the radiotrace corresponded to intact complex and a peak with the
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same retention time as free iodide, suggesting direct loss of iodide, possibly as a result of a
radical anion pathway mediated by the oxidation of the flavonoid species in solution to give
reactive radical intermediates [61, 62]. The instability of [123I]-(3) and [123I]-(5) over extended
incubation periods rendered these compounds unsuitable for further in vitro development.
Similar complex instability was seen for [123I]-(5) in PBS, with only 53% intact compound
remaining after 24 h respectively. In keeping with their lipophillic nature both 1 and 6 after
incubation in serum containing medium, remained localized to the protein pellet after protein
precipitation. Sephadex size exclusion separation of the solutions reinforced that all activity was
associated with large protein biomolecules, with the radioactive fraction eluting in early solvent
volumes. Free iodide controls eluted later suggesting that there is no loss of free iodide after 24 h
incubation of 1 or 6 in serum containing medium.
2.4 In Vitro Evaluation
Cell uptake studies were performed in the MDA-MB-435 melanoma cell line as a
telomerase expressing cell line. Internalization studies with [123I]-(1) showed limited cell uptake,
with <0.1% of the radiolabeled drug internalized over a 24 h incubation period, despite favorable
partition coefficient data. Repeat internalization studies with alternative cancer cell lines
including MDA-231/H2N breast and U2OS osteosarcoma cancer cell lines all showed/confirmed
low cell uptake. No loss in survival was subsequently seen on incubation with the MDA-MB-435
cell line as determined by colony formation, and as such attention was focused thereafter on
[123I]-(3) and the iodinated MST-312 compound.
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After 24 h incubation, the iodinated MST-312 compound, [123I]-(6), displayed uptake in
MDA-MB-435 cells, with 15% of the total incubated activity being cell-associated after 24 h, of
which 60% was shown to be localized within the nucleus.
The effects of combined irradiation with telomerase inhibition have so far only been
investigated using external beam sources. To date no approach has pursued the use of
intrinsically targeted small molecule radiotherapeutics to study if radiosensitization effects can
be achieved, by retention of a radioactive probe in cancer cells that over-express telomerase.
Localization of a radionuclide in proximity to the enzyme active-site may further enhance
catalytic inhibition, following radiolytic disruption. Binding to the active-site of this intranuclear
enzyme is also likely to promote the retention of small molecule inhibitors in the nuclei (of
cancer cells), potentiating effects mediated via radioactive decay, rendering telomerase an
attractive target for dual therapy with short-range electron-emitting isotopes. Given the nuclear
uptake and telomerase inhibition of 6, clonogenic studies were perfomed with increasing
radioactivity concentrations of the radiolabeled [123I]-(6) species. Concentrations between 0-20
MBq/mL were incubated with the MDA-MB-435 cell line for either 4 or 24 h before being
plated out at a concentration of 1000 cells/mL in DME medium and left to form colonies over a
10 day period. Reduced survival with increasing concentration of radiolabeled drug was
observed, with lower surviving fraction (SF) after incubation for 24 h. Control clonogenic assays
with MST-312 and non-radioactive 6 run in parallel show that for the radiolabeled species even
at the highest radioactivity concentrations used (20 MBq/mL), the concentration of compound
present (0.18 µM, based on specific activity measurements) was not sufficient to illicit a
significant clonogenic effect (survival fraction: 0.97). As such the low SF observed is as a direct
result of a response to radiation-induced cell damage.
18

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For comparison with a telomerase negative cell line, the U2OS osteosarcoma cell line
was studied in parallel to the MDA-MB-435 cell line (Figure 3). Comparable uptake and
retention to the MDA-MB-435 cell line was seen in the U2OS cell line. Clonogenic studies (n=8)
in U2OS and MDA-MB-435 cells lines run in parallel show that although at the highest activity-
concentrations comparable loss in survival is observed for both cell lines (SF at 20 MBq/mL;
0.11 vs 0.11 for MDA-MB-435 vs U2OS, P = 0.76), at lower activity concentrations the SF for
the MDA-MB-435 cell line is lower than that for U2OS (SF at 5 MBq/mL: 0.55 vs 0.72 for
MDA-MB-435 vs U2OS,
= 0.001)), which it is hypothesised may result from the presence of telomerase facilitating
nuclear retention of the iodinated MST-312 species.
P = 0.004; SF at 7 MBq/mL: 0.32 vs 0.46 for MDA-MB-435 vs U2OS,
P
19

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a)
MDA-MB-435
U2OS
1.0
0.8
0.6
0.4
0.2
0.0
Treatment
b)
MST-312
(6)
Figure 3: Clonogenic survival data for MDA-MB-435 and U2OS cell lines after 24 h treatment
with increasing activity concentrations of [123I]-(6); B) Clonogenic survival data for MDA-MB-
435 cell line after 24h treatment with increasing activity concentrations of (6) or MST-312
Cell studies with [123I]-(3) in the MDA-MB-435 cell line showed uptake of 2.9 % of the total
activity to be cell associated after 24 h. Clonogenic studies between 0-16 MBq/mL of [123I]-(3)
20

ACCEPTED MANUSCRIPT
with MDA-MB-435 cell line for 24 h showed statistically significantly reduced survival with
increasing concentration of radiolabelled drug compared to free iodide alone (Figure 4). Cell
survival was reduced to 36 ± 2 % following incubation with 16 MBq/mL of [123I]-(3), compared
to 84 ± 4 % with the Na123I control, suggesting that the combination of telomerase inhibition and
irradiation is cytotoxic. However, given the instability of [123I]-(3) (and [123I]-(5)) over extended
incubation periods such class of compounds were deemed not suitable for further development.
Na123
123I]-(3)
I
1.0
0.8
0.6
0.4
0.2
0.0
[
Treatment
Figure 4: Clonogenic survival data for MDA-MB-435 cell line after 24h treatment with
increasing activity concentrations of [123I]-(3) or Na123I.
Excluding the germ line, telomerase expression in stem-cell compartments is rarely
sufficient to maintain telomere length, with shortening observed with increasing age [63].
Though anti-telomerase therapy could possibly affect stem cells of renewal tissues (such as crypt
cells of the intestine, basal cells of the skin, and certain hematopoietic cells of the blood) [64],
21

ACCEPTED MANUSCRIPT
the telomeres of such cells are generally much longer than cancer cell telomeres [5, 65-66]. This
differential telomerase expression in cancer versus stem cells provides a means to deliver the
telomerase-targeting radiotherapeutic [123I]-(6) to neoplastic cells, with minimal damage to
normal stem-cell populations.
3. Conclusion
To investigate the possible use of telomerase as a target for molecularly targeted radiotherapy,
we synthesized and characterized a panel of radioiodinated analogues of three known telomerase
inhibitors: BIBR-1532, a flavonoid (2-(3,4-dihydroxyphenyl)-7,8-dihydroxy-4H-chromen-4-one)
and MST-312. The radioiodinated derivatives all retained telomerase inhibitory capacity either in
cell-free (lysate) or in whole cell experiments. 123I-BIBR-1532 internalized into cancer cells to
only a modest extent. 123I-labeled flavonoid was markedly cytotoxic in clonogenic assays but
was moderately unstable in serum. 123I-MST-312, however, showed uptake in two cancer cell
lines, was stable and elicited radioactivity concentration-dependent cancer cell death. The
reduction in cell survival was greater in a telomerase positive versus telomerase negative cell
line. Further studies will look at a greater range of telomerase positive and negative cell lines to
establish the broad applicability of (6) as a targeted radiotherapy agent against telomerase-
positive cancers.
22

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4. Experimental Section
4.1 General Methods
All reactions were carried out in oven-dried glassware under a nitrogen atmosphere.
Nitrogen gas was dried by passage through silica gel. All solvents were dried according to
standard procedures. All reagents were purchased from Aldrich and used without further
purification. Column chromatography was performed on silica gel 60 M (mesh 230-400)
1
Macherey-Nagel. H and 13C NMR spectra were recorded on a Bruker DPX400 (400 MHz)
spectrometer or Bruker AVC500 (500 MHz) spectrometer at 298 K and referenced to residual
non-deuterated solvent peaks. Mass spectrometry was performed using a Bruker Micromass LCT
time-of-flight mass spectrometer under conditions of electrospray ionization (ESI-MS). Accurate
masses are reported to four decimal places using tetraoctylammonium bromide (466.5352 Da) as
an internal reference. HPLC characterization (analytical HPLC) of compounds was performed
using a Waters C-18 column (4.6 x 250 mm) with UV/Vis detection at λobs = 254 nm with a 1.0
mL/min gradient elution method; Method A (Solvent A: acetonitrile with 0.1 % TFA v/v, Solvent
B: water with 0.1% TFA v/v): start 50 % A, gradient over 7 min reaching 80 % A, then gradient
until 13 min reaching 95 % A, reverse gradient till 14 min reaching 50 % A, then hold to 15 min
at 50 % A, Method B (Solvent A: acetonitrile with 0.1 % TFA v/v, Solvent B: water with 0.1%
TFA v/v): start 5 % A, gradient over 15 min reaching 95 % A, hold to 16 min at 95 % A, reverse
gradient till 18 min reaching 5 % A, then hold to 20 min at 5 % A.
Elemental analyses were performed by Mr. S. Boyer, at London Metropolitan University.
No-carrier added radiochemical sodium iodide I-123 was supplied by GE Healthcare. Radio-
HPLC was carried out under identical conditions to analytical HPLC, on an Agilent 1200 series
module equipped with a LabLogic Gamma-Ram-4 radiodetector.
23

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Compounds (
E
)-3-(naphthalen-2-yl)but-2-enoic acid [68], 4-iodo-2-aminobenzoate [69],
3-Iodo-4,5-dimethoxybenzoic acid [70] and 1-iodo-3,5-diaminobenzene [71] were prepared as
previously described. All data was in accord with literature values. Full experimental data and
spectra for all compounds prepared in this work may be found in the Supporting Information.
4.1.1. I-BIBR-1532 compound data
4.1.1.1 (E)-Methyl 5-iodo-2-(3-(naphthalen-2-yl)but-2-enamido)benzoate (1b)
(E)-3-(Naphthalen-2-yl)but-2-enoic acid (1.00 g, 4.71 mmol) was suspended in dry
dichloromethane (15 mL) in a flame dried flask under an argon atmosphere and cooled to 0°C.
Oxalyl chloride (0.848 g, 0.447 mL, 9.42 mmol) and a catalytic amount of DMF (2 drops) was
added to the suspension and allowed to warm to room temperature. The solution was stirred at
room temperature for 5h after which all volatiles were removed under reduced pressure. The
crude product was redissolved in dry dichloromethane (15 mL) and solvent removed under
reduced pressure three times before drying under high vac. The crude product was redissolved in
dry THF (20 mL) in a flame-dried flask under an argon atmosphere. To this was added 5-iodo-2-
aminobenzoate (1.86 g, 7.07 mmol), pyridine (1.12 g, 1.14 mL, 14.1 mmol) and a catalytic
amount of DMAP (0.01 g, 0.082 mmol). After stirring at 60°C for 16h, the reaction was allowed
to cool to room temperature, poured onto 1N HCl and extracted into diethyl ether. The combined
organic layers were dried over anhydrous magnesium sulphate and concentrated under vacuum
to give the crude product. Purification by flash chromatography (8% methanol in
1
dichloromethane) afforded the analytically pure product. (Yield: 1.60 g, 3.39 mmol, 72 %). H
NMR (CDCl3, 500 MHz, 20°C): δ = 11.18 (s, 1H, NH), 8.70 (d, J = 9.1 Hz, 1H, ArH), 8.36 (d, J
24

ACCEPTED MANUSCRIPT
= 2.2 Hz, 1H, Ar
Hz, 1H, Np ), 7.64 (dd,
COC ), 3.95 (s, 3H, COOCH3), 2.77 (d,
20°C):
H
), 7.98 (d,
J
= 1.3 Hz, 1H, Np
H
), 7.89 (m, 3H, Ar
H
+ Np
H
), 7.83 (d,
J = 2.2
H
J
= 8.5, 1.9 Hz, 1H, Np
H
), 7.53 (m, 2H, Np
H
), 6.37 (q, J
= 0.9 Hz, 1H,
13
H
J
= 1.0 Hz, 3H, CCH3). C NMR (CDCl3, 125 MHz,
δ
= 167.6, 165.3, 154.0, 143.1, 141.7, 139.6, 139.3, 133.4, 133.1, 128.5, 128.2, 127.6,
126.6, 126.5, 125.8, 124.0, 122.2, 120.8, 116.5, 84.7, 52.6, 18.0. ESMS calcd for C22H18INNaO3
[M+Na]+ : 494.0224, found 494.0221. HPLC (method A): Rt =14.14 min.
4.1.1.2. (E)-5-Iodo-2-(3-(naphthalen-2-yl)but-2-enamido)benzoic acid (1)
LiOH (27.8 mg, 0.663 mmol, 1 M aq. solution) was added to a solution of (E)-methyl 5-
iodo-2-(3-(naphthalen-2-yl)but-2-enamido)benzoate (0.250 g, 0.530 mmol) in THF/H2O (5:1, 10
mL) and stirred at room temperature. After 12 h, the reaction was acidified to pH 4 using 1 M aq.
oxalic acid solution and extracted with ethyl acetate. The combined organic layers were dried
over anhydrous magnesium sulphate and concentrated under vacuum to give the final product.
1
(Yield: 0.226 g, 0.493 mmol, 93 %). H NMR (d6-DMSO, 500 MHz, 20°C):
δ
= 13.87 (br s,
= 2.2 Hz, 1H, Ar ),
), 7.75 (dd, = 8.8,
= 1.1 Hz, 3H,
= 168.0, 164.5, 151.7, 142.0, 140.4, 139.0,
1H, COO
H
), 11.16 (s, 1H, N
= 1.3 Hz, 1H, Np ), 7.99 (m, 1H, Np
), 7.54 (m, 2H, Np ), 6.51 (d,
H
), 8.38 (d,
J
= 8.8 Hz, 1H, Ar
), 7.92 (m, 3H, Ar
= 0.9 Hz, 1H, COC
H
), 8.23 (d,
J
H
8.14 (d,
J
H
H
H
+ Np
H
J
1.9 Hz, 1H, Np
H
H
J
H), 2.66 (d,
J
13
CCH3). C NMR (d6-DMSO, 125 MHz, 20°C):
δ
138.8, 133.0, 132.8, 128.5, 128.1, 127.4, 126.8, 126.6, 125.7, 123.9, 122.5, 120.8, 119.2, 85.9,
17.2. ESMS calcd for C21H16INNaO3 [M+Na]+ : 480.0067, found 480.0071. HPLC (method A):
Rt = 9.89 min. Elemental analysis for C21H16INO3, calc: C 55.2 %, H 3.5 %, N 3.1 %, found: C
55.3 %, H 3.6 %, N 2.9 %.
25

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4.1.1.3.Methyl (E)-2-(3-(naphthalen-2-yl)but-2-enamido)-5-(trimethylstannyl)benzoate (1c)
To a solution of (E)-methyl 5-iodo-2-(3-(naphthalen-2-yl)but-2-enamido)benzoate (0.100
g, 0. 212 mmol) in degassed anhydrous 1,4-dioxane (5.00 mL) was added hexamethyldistannane
(0.167 g, 106 µL, 0.509 mmol) and bis-(triphenylphosphine)-palladium(II)-dichloride (1.50 mg,
2.17 µmol), and the solution stirred at 60 °C for 1.5 h under argon. The solution was cooled to
room temperature and filtered through a plug of celite with dichloromethane washing. The
solvent was removed in vacuo and purification by flash chromatography on neutral silica (2 %
methanol in dichloromethane) afforded the analytically pure product as a white solid. (Yield:
1
69.0 mg, 0.136 mmol, 64 %). H NMR (CD2Cl2, 500 MHz, 20°C):
δ
= 11.20 (s, 1H,
H9), 8.81
(d,
J
= 8.2 Hz, 1H,
H7), 8.18 (d,
J
= 1.2 Hz (84%), with Sn satellites (J2Sn-H = 47 and 44 Hz
(16%)), 1H,
H
4), 8.01 (d,
J
= 1.6 Hz, 1H,
H
15), 7.84 - 7.95 (m, 3H,
H
17+20+22), 7.71 (dd,
6), 7.68 (dd, = 8.8, 1.8 Hz,
11), 3.93 (s, 3H, 1), 2.75 (d,
= 54 and 56 Hz (16%)), 9H,
J =
8.1, 1.3 Hz (84%), with Sn satellites (J2Sn-H = 48 Hz (16%)), 1H,
H
J
1H,
H23), 7.53 (m, 2H,
H
18+19), 6.41 (d,
J
= 1.3 Hz, 1H,
H
H
J =
1.3 Hz, 3H,
H
13), 0.34 (s (84%), with Sn satellites (J2
Sn-H
Sn(CH3)3). 13C NMR (CD2Cl2, 125 MHz, 20°C):
δ
= 169.6, 165.7, 153.2, 142.5, 142.4, 140.3,
138.6, 136.2, 134.0, 133.8, 129.0, 128.6, 128.1, 127.1, 127.0, 126.3, 124.6, 121.8, 120.0, 115.2,
52.8, 18.1, -9.2 (with Sn satellites; -7.8, -7.9, -10.6, -10.7), Sn( H3)3. ESMS calcd for
C
C25H27NNaO3Sn [M+Na]+ : 532.0910, found 532.0899. HPLC (method B): Rt = 14.24 min.
4.1.1.4. (E)-2-(3-(Naphthalen-2-yl)but-2-enamido)-5-(trimethylstannyl)benzoic acid (1d)
LiOH (4.71 mg, 0.197 mmol, 1 M aq. solution) was added to a solution of (E)-2-(3-
(naphthalen-2-yl)but-2-enamido)-5-(trimethylstannyl)benzoate (0.050 g, 0.098 mmol) in
THF/H2O (5:1, 10 mL) and stirred at room temperature. After 12 h, the reaction was acidified to
26

ACCEPTED MANUSCRIPT
pH 4 using 1 M aq. oxalic acid solution and extracted with ethyl acetate. The combined organic
layers were dried over anhydrous magnesium sulphate and concentrated under vacuum to give
the final product. (Yield: 42.3 mg, 0.086 mmol, 87 %). 1H NMR (d6-DMSO, 500 MHz, 20°C):
δ
= 13.52 (br s, 1H,
H1), 11.21 (s, 1H,
H9), 8.51 (d, J = 8.2 Hz, 1H, H7), 8.11 (d, J = 1.4 Hz,
1H, 15), 8.08 (d,
H
J
= 1.2 Hz (84%), with Sn satellites (J2Sn-H = 46 and 43 Hz (16%)), 1H,
H4),
7.92 – 8.02 (m, 3H,
(84%), with Sn satellites (J2Sn-H = 47 and 44 Hz (16%)), 1H,
H17+20+22), 7.79 (dd, J=8.7, 1.6 Hz, 1H, H23), 7.72 (dd, J = 8.2, 1.3 Hz
H6), 7.56 (m, 2H,
H18+19), 6.53 (d,
J
= 1.2 Hz, 1H,
H11), 2.67 (d,
J
= 0.7 Hz, 3H, H
13), 0.31 (s (84%), with Sn satellites (J2Sn-H = 53
13
and 56 Hz (16%)), 9H, Sn(CH3)3). C NMR (d6-DMSO, 125 MHz, 20°C):
δ = 170.1, 164.4,
150.6, 140.8, 140.7, 138.9, 138.8, 133.0, 132.8, 131.5, 128.4, 128.1, 127.5, 126.7, 126.6, 125.6,
124.0, 122.4, 121.4, 119.4, 17.3, -9.3 (with Sn satellites; -7.6, -7.7, -10.4, -10.5), Sn( H3)3.
C
ESMS calcd for C24H25NNaO3Sn [M+Na]+ : 518.0753, found 518.0744. HPLC (method B): Rt =
11.97 min. Elemental analysis for C24H25NO3Sn, calc: C 58.3 %, H 5.1 %, N 2.8 %, found: C
57.9 %, H 5.2 %, N 2.9 %.
4.1.2. I-FLAV compound data
4.1.2.1. 1-(5-Iodo-2-hydroxy-3,4-dimethoxyphenyl) ethanone (3a)
To a solution of 3,4-dimethoxy-2-hydroxyacetophenone (0.855 g, 4.36 mmol) in
acetonitrile (10 mL) was added
p-toluenesulphonic acid (0.750 g, 4.36 mmol) and the mixture
stirred for 15 min. -Iodosuccinimide (0.980 g, 4.36 mmol) was added and the mixture stirred
N
for 40 h at room temperature. The reaction was diluted with dichloromethane and washed with
aq. Na2S2O4, aq. NaHCO3 and water. The combined organic layers were dried over anhydrous
magnesium sulphate and concentrated under vacuum to give the crude product. Purification by
27

ACCEPTED MANUSCRIPT
flash chromatography (dichloromethane) afforded the analytically pure product as a white solid.
(Yield: 0.955 g, 2.97 mmol, 68 %). 1H NMR (CDCl3, 400 MHz, 20°C):
), 4.03 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 2.59 (s, 3H, COCH3).13C NMR
δ = 12.58 (s, 1H, OH),
7.90 (s, 1H, Ar
H
(CDCl3, 100 MHz, 20°C):
δ = 202.7, 158.3, 157.8, 140.7, 134.9, 118.6, 78.2, 61.1, 60.9, 26.7.
ESMS calcd for C10H11INaO4 [M+Na]+ : 344.9594, found 344.9597. IR:
ν = 2938, 1637, 1445,
1409, 1358, 1313, 1254, 1069, 1019, 753 cm-1.
4.1.2.2. 6-Acetyl-4-iodo-2,3-dimethoxyphenyl-3,4-dimethoxybenzoate (3b)
To a solution of 1-(5-Iodo-2-hydroxy-3,4-dimethoxyphenyl) ethanone (0.975 g, 3.027
mmol) in anhydrous pyridine (5 mL) under argon, was added 3,4-dimethoxybenzoyl chloride
(1.82 g, 9.08 mmol) over a period of 15 min. The mixture was stirred for 2 h at room temperature
and then acidified with 2 N HCl, extracted with ethyl acetate and washed with water. The
combined organic layers were dried over anhydrous magnesium sulphate and concentrated under
vacuum to give the crude product. Purification by flash chromatography (30 % ethyl acetate in
hexane) afforded the analytically pure product as a white solid. (Yield: 1.40 g, 2.88 mmol, 95
1
%). H NMR (CD2Cl2, 500 MHz, 20°C):
δ = 8.04 (s, 1H, ArH), 7.86 (dd, 1H, J = 8.5, 2.0 Hz,
ArH), 7.65 (d, 1H, J = 2.0 Hz, ArH), 7.00 (d, 1H, J = 8.6 Hz, ArH), 3.95 (s, 3H, OCH3), 3.94 (s,
3H, OCH3), 3.91 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 2.47 (s, 3H, COCH3). 13C NMR (CD2Cl2,
125 MHz, 20°C):
δ = 195.4, 164.5, 157.4, 154.7, 149.7, 146.2, 145.8, 134.5, 130.1, 125.2, 121.4,
113.1, 111.3, 88.9, 61.6, 61.3, 56.6, 56.5, 30.4. ESMS calcd for C19H19INaO7 [M+Na]+ :
509.0068, found 509.0067. IR:
ν = 2941, 1734, 1687, 1598, 1516, 1452, 1401, 1355, 1272,
1209, 1172, 1135, 1076, 1021, 752 cm-1.
28