FOXM1 Inhibitors as Potential Diagnostic Agents: First Generation of a PET Probe Targeting FOXM1 To Detect Triple-Negative Breast Cancer in vitro and in vivo

Article abstract of DOI:10.1002/cmdc.202100279

The FOXM1 protein controls the expression of essential genes related to cancer cell cycle progression, metastasis, and chemoresistance. We hypothesize that FOXM1 inhibitors could represent a novel approach to develop ¹⁸F-based radiotracers for Positron Emission Tomography (PET). Therefore, in this report we describe the first attempt to use ¹⁸F-labeled FOXM1 inhibitors to detect triple-negative breast cancer (TNBC). Briefly, we replaced the original amide group in the parent drug FDI-6 for a ketone group in the novel AF-FDI molecule, to carry out an aromatic nucleophilic (¹⁸F)-fluorination. AF-FDI dissociated the FOXM1-DNA complex, decreased FOXM1 levels, and inhibited cell proliferation in a TNBC cell line (MDA-MB-231). [¹⁸F]AF-FDI was internalized in MDA-MB-231 cells. Cell uptake inhibition experiments showed that AF-FDI and FDI-6 significantly decreased the maximum uptake of [¹⁸F]AF-FDI, suggesting specificity towards FOXM1. [¹⁸F]AF-FDI reached a tumor uptake of SUV=0.31 in MDA-MB-231 tumor-bearing mice and was metabolically stable 60 min post-injection. These results provide preliminary evidence supporting the potential role of FOXM1 to develop PET radiotracers.

Full text of DOI:10.1002/cmdc.202100279

Accepted Article
Title: FOXM1 inhibitors as potential diagnostic agents: 1st generation
of a PET probe targeting FOXM1 to detect triple negative-breast
cancer in vitro and in vivo
Authors: David J Perez, Seyed Amirhossein Tabatabaei Dakhili,
Cody Bergman, Jennifer Dufour, Melinda Wuest, Freimut
D. Juengling, Frank Wuest, and Carlos Alberto Velazquez-
Martinez
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202100279
Link to VoR: https://doi.org/10.1002/cmdc.202100279
01/2020

10.1002/cmdc.202100279
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FOXM1 inhibitors as potential diagnostic agents: 1st generation
of a PET probe targeting FOXM1 to detect triple negative-breast
cancer in vitro and in vivo
[b]
[b]
David J. Pérez, [a] [c] Seyed Amirhossein Tabatabaei Dakhili, [a] Cody Bergman,
Jennifer Dufour,
Melinda Wuest, [b] Freimut D. Juengling, [b] Frank Wuest, [b] Carlos A. Velázquez-Martínez, * [a]
[a]
Dr. D. J. Pérez, Dr. S. A. Tabatabaei Dakhili, Prof. C. A. Velázquez-Martínez*
Faculty of Pharmacy and Pharmaceutical Sciences
University of Alberta
Edmonton, AB. Canada T6E 2E1.
E-mail: velazque@ualberta.ca
[b]
[c]
C. Bergman, J. Dufour, Dr. M. Wuest, Prof. F. D. Juengling, Prof. F. Wuest
Department of Oncology, Faculty of Medicine and Dentistry
University of Alberta.
Edmonton, AB. Canada T6E 2E1.
Dr. D. J. Pérez (Present Address)
Facultad de Medicina, Unidad de Radiofarmacia/ciclotrón
Universidad Nacional Autónoma de México, Ciudad de México
C.P. 04510, CDMX, MEXICO
Supporting information for this article is given via a link at the end of the document.
Abstract: The FOXM1 protein controls the expression of essential Introduction
genes related to cancer cell cycle progression, metastasis, and
chemoresistance. We hypothesize that FOXM1 inhibitors could
represent a novel approach to develop 18F-based radiotracers for
Positron Emission Tomography (PET). Therefore, in this report we
describe the first attempt to use 18F-labeled FOXM1 inhibitors to
detect triple-negative breast cancer (TNBC). Briefly, we replaced the
original amide group in the parent drug FDI-6 for a ketone group in
the novel AF-FDI molecule, to carry out an aromatic nucleophilic (18F)-
fluorination. AF-FDI dissociated the FOXM1-DNA complex,
decreased FOXM1 levels, and inhibited cell proliferation in a TNBC
cell line (MDA-MB-231). [18F]AF-FDI was internalized in MDA-MB-231
cells. Cell uptake inhibition experiments showed that AF-FDI and FDI-
6 significantly decreased the maximum uptake of [18F]AF-FDI,
suggesting specificity towards FOXM1. [18F]AF-FDI reached a tumor
uptake of SUV = 0.31 in MDA-MB-231 tumor-bearing mice and was
metabolically stable 60 min post-injection. These results provide
preliminary evidence supporting the potential role of FOXM1 to
develop PET radiotracers.
The Fork head box transcription factor (FOXM1) is an essential
protein that controls the expression of proteins involved in cell
replication. In normal cells, the expression level of FOXM1 is
almost undetectable in quiescent or fully developed cells.[1-2]
However, it has been observed that the expression of FOXM1 in
a wide variety of cancer cells is significantly higher than that
detected in normal cell counterparts,[3] suggesting that FOXM1
can be considered as an oncogenic and carcinogenic protein.[1-2,
4]
In this regard, the FOXM1 protein has gained increasing
attention in medicinal chemistry, becoming a potential drug target
for cancer treatment.[1-4] Nevertheless, unlike enzymes, cell
receptors, or ion channels, transcription factors such as FOXM1
do not have classical drug binding sites, which reduces the
options to develop small molecule drugs capable of binding to,
and inhibiting, this oncogenic protein. However, despite this
challenging situation, a few experimental FOXM1 “inhibitors” have
been described in the scientific literature, possessing several
structural differences.[5-7] Based on that, Gormally et al. reported
in 2014 a series of “forkhead domain inhibitors”, or FDIs, among
which the heterocyclic molecule labelled as FDI-6. This molecule
was the most potent and relatively selective forkhead domain
inhibitor in the series.[5]
Recently, we have carried out a series of simulations of molecular
modelling dynamics and identified a possible binding site, along
with key amino acid residues that could be essential for drugs to
exert binding interactions at the interface of the protein and its
DNA domain (DBD).[8] Furthermore, our group conducted a series
of experiments aimed to validate the drug binding site mentioned
above, in which we subsequently reported the chemical synthesis
and FOXM1 inhibitory activity of a novel group of FDI-6
1
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derivatives. The drug recognition process required the presence
of sulfur-containing heterocycles that bind to His287 in the
protein.[9]
Herein we report the synthesis of AF-FDI, its in vitro screening as
FOXM1 inhibitor, its radiolabeling with 18F, its cell uptake and
blocking characteristics in MDA-MB-231 cells, as well as its in vivo
metabolic stability and PET imaging profile using a MDA-MB-231
tumor-bearing rodent model. These experiments constitute the
first steps towards the validation of FOXM1 as a potential target
for theranostic agents.
Considering that it might be possible to use a drug binding site on
the DBD of the FOXM1 protein, and that the levels of this
transcription factor are significantly elevated in most (if not all)
cancer cell types, we hypothesize that drug binding interactions
may serve not only to inhibit its transcriptional activity but also to
detect its expression in vivo, serving as biomarker. This
hypothesis is supported by a recent work published by Nandi et
al., in which the authors propose the use of FOXM1 as a
biomarker to predict prognosis and patient response to
chemotherapy.[2] In this regard, among the different types of
human breast cancer, the highest levels of FOXM1 have been
detected in triple negative-breast cancers (TNBC).[10-11] Thus, we
chose to use a triple negative-breast cancer cell line (MDA-MB-
231), as well as mice xenografts from the same cell model.
Positron Emission Tomography (PET) is one of the most widely
used imaging techniques for cancer diagnosis and staging using
a positron emitting radionuclide.[12] Fluorine-18 (18F), with a
radioactive half-life of 109.8 min, has proven to be an ideal
radionuclide for PET imaging and is one of the working horses for
developing PET imaging probes, as demonstrated with the
glucose derivative [18F]FDG, the “golden standard” PET
radiotracer in oncology. Furthermore, with a positron emission
decay energy providing excellent spatial resolution in imaging,
and a shelf live allowing for transportation from radio–pharmacies
to imaging centers, 18F today is the most commonly used PET
radionuclide, and thus research to develop novel 18F radiotracers
for PET imaging is an ongoing field.[12-13]
Figure 1. Chemical structures of the parent forkhead box inhibitor 6 (FDI-6) and
its “activated” analogue AF-FDI possessing a 4-ketone electron withdrawing
group.
Results and Discussion
Synthesis of AF-FDI.
As shown in Scheme 1, to prepare the target molecule we have
modified the protocol reported for the synthesis of FDI-6 and
some of its derivatives before.[5, 16-17] Briefly, the reaction between
intermediates 3 and 4 under microwave irradiation yielded AF-FDI
as a yellow solid in 80% yield and about 95% purity after a simple
filtration from the reaction mixture (assessed by NMR, HPLC and
LC-MS).
We hypothesize that binding interactions exerted by FOXM1
inhibitors could not only inhibit its transcriptional activity but serve
as PET-based imaging probes, specifically, for 18F-based imaging.
Following this idea, we attempted to replace the fluorine atom
present in FDI-6 for an 18F atom, using copper-assisted
fluorination[14-15] but were unsuccessful even after several
attempts under different experimental conditions. The main
reason behind the lack of reactivity towards the attempted (and
failed) aromatic nucleophilic substitution on FDI-6 may be the lack
of an electron-withdrawing group para to the fluorine atom.
Consequently, and prompted by these initial results, we designed
the derivative AF-FDI possessing a 4-ketone group, which unlike
the amide moiety, is supposed to activate the radiolabeling with
18F (Figure 1).
Scheme 1. Chemical synthesis of the target molecule AF-FDI.
The design of an “activated” molecule was mainly based on the
chemical structure of the lead forkhead box inhibitor-6 (FDI-6)
reported by Gormally et al. in 2014.[5] In the present work, we
report the first attempt to use a FOXM1 inhibitor as a PET-based
imaging agent to detect TNBC. The use of a highly relevant
oncoprotein involved in essentially all stages of cancer initiation,
cancer progression, metastasis and chemoresistance bears a
significant clinical potential, because it could form the basis for the
development of a new class of theranostic agents to diagnose and
treat not only TNBC but any other human cancer that
overexpresses the FOXM1 transcription factor.
The overall yield from intermediates 1 and 2 was 80% following a two-step
reaction.
Electrophoretic Mobility Shift Assay
To evaluate the binding affinity of the novel AF-FDI molecule, we
determined the ability of this drug to bind to, and dissociate, the
2
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FOXM1-DNA complex using the cell-free electrophoretic mobility
shift assay (EMSA). As reported previously, this assay represents
a fast and reliable way to assess direct FOXM1 inhibitory activity,
and to differentiate active from inactive compounds.[5],[9] In this
regard, we observed that AF-FDI exerts a direct inhibitory activity
by dissociating the FOXM1-DBD with an IC50 = 46.38 ± 1.20 M
and Ki = 22.2 ± 0.56 M (n = 3; Figure 2). The IC50 and Ki values
are slightly higher, but still comparable to those determined in our
lab for the parent drug FDI-6 (IC50 = 39.57 ± 1.15 M; Ki = 16.8 ±
1.15 M; n = 3).[9]
FOXM1 in TNBC at 40, 60 and 80 M (24 h incubation). However,
the effect of AF-FDI was lower than that observed with the parent
drug FDI-6 at the same test drug concentrations (Figure 3).
Test molecule AF-FDI showed a modest dose-dependent activity
on inhibition of cell proliferation of MDA-MB-231 cells with an IC50
= 41.9 ± 1.2 M (see Figure S5 in supporting information), which
is relatively weak from a drug development standpoint, but still
similar to that observed with the parent drug molecule FDI-6 (31.1
± 8.7 M).[9]
Despite the relatively high concentration of AF-FDI required to
dissociate the FOXM1-DNA complex, this is in agreement with
experimental results published by our group in which small-
molecule FOXM1 inhibitors showed activity without affecting the
levels of the SP1 transcription factor, reported to activate the
expression of FOXM1 in concentrations between 30-60 M in
EMSA assay and in vitro.[18]
Figure 3. Concentration-dependent inhibition of FOXM1 protein levels in MDA-
MB-231 triple negative-breast cancer cells. The activity of the test molecule AF-
FDI was compared to that of the parent drug FDI-6 at 40, 60 and 80 M after a
24-hour incubation period; *p < 0.05, **p < 0.01, ***p < 0.0001 compared to
DMSO. Data are shown as mean + SEM calculated from 3 different experiments.
Radiochemistry
The next step involved the radiolabeling of the parent drug FDI-6
using 18F to produce [18F]FDI-6. We first attempted the “amide
inversion” in FDI-6 to activate the ring towards the nucleophilic
aromatic substitution, although we encountered chemical
challenges that precluded this approach (data not shown). Then,
Figure 2. Electromobility shift assay (upper part) and the dose-response curve
used to determine the IC50 and Ki values for the drug-induced dissociation of
the FOXM1-DNA binding domain (lower part). Data are shown as mean + SEM
calculated from 3 different experiments.
we
decided
to
attempt
different
copper-assisted
In vitro FOXM1 inhibition in MDA-MB-231 cells
fluorodeborylation reactions using the boronic ester FDI-BPin as
a precursor as a path to prepare [18F]FDI-6 [14-15, 20] (Scheme 2).
After determining the binding affinity of AF-FDI, we needed to
establish if this property could exert a decrease in the protein
levels of FOXM1 in MDA-MB-231 cells. According to a data
analysis from breast cancer patients from the Canadian Breast
Cancer Foundation Tumor Bank, provided by the Cross Cancer
Institute (Edmonton, AB, Canada) tissue samples obtained from
patients diagnosed with breast cancer showed a 19-fold increase
in FOXM1 mRNA expression compared to normal breast tissue,
which was consistent with previous reports in the literature.[10]
Based on the observation that FOXM1 controls its own
expression,[19] we have used western blot experiments to
determine the concentration-dependent effect of AF-FDI on
FOXM1 protein levels. AF-FDI decreased the expression of
Scheme 2. Attempted synthesis of radiolabeled [18F]FDI-6.
Reagents and conditions: (a) K2CO3, [K/K222]+ 18F-, DMF, 150˚C,
[Cu(OTf)2(Py)4]; 20 min (b) Et4NHCO3 in n-BuOH, 18F-, 110˚C, DMA,
[Cu(OTf)2(Py)4], 20 min. Neither of the two reaction conditions yielded the target
molecule.
3
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which cancer cells MBA-MD-231 were incubated with increasing
concentrations of the non-radioactive reference compound AF-
FDI, or parent compound FDI-6, before radiotracer [18F]AF-FDI
was added. A significant dose-dependent decrease in the
maximum control cell uptake for [18F]AF-FDI was observed as the
concentration of AF-FDI or FDI-6 increased, reaching about 50%
inhibition of the maximum cell uptake (Figure 4C). For AF-FDI an
IC50 value of 56 M and for FDI-6 34 M was determined. To
distinguish between compound effects and effects of percent
DMSO in the inhibition solutions, control experiments with
different DMSO amounts were performed as high molar stock
solutions of compounds FDI-6 or AF-FDI at 1 mM contained 10%
DMSO. Only the difference between inhibitory compound effects
and effects of DMSO concentrations would give the real inhibition
effects for FDI-6 or AF-FDI. It is also worth mentioning that the
cell uptake for [18F]AF-FDI could not be completely inhibited by
the cold reference or FDI-6, which suggests some degree of
unspecific binding (off-target binding) of the radiotracer.
Nevertheless, none of the experimental conditions yielded the
desired radiolabeled product (See Scheme S1 in the supporting
information). We hypothesized that the presence of the
unprotected amino moiety in the FDI-BPin molecule might be the
cause for the lack of reactivity; in the presence of a copper
catalyst the unprotected amino group may have undergone a
Chan-Evan-Lan reaction side-reaction.[21-22] Prompted by these
unsuccessful radiolabeling results, we considered two alternative
and potentially more suitable routes to prepare the radiolabeled
target molecule [18F]AF-FDI. First, precursor 7 was synthesized
using triflate as the leaving group during the radiolabeling
nucleophilic substitution reaction by adapting a previously
reported method.[23] Using this method [18F]AF-FDI was obtained
in 73 ± 3% radiochemical yield decay (corrected), within a 2-h time
frame, a molar activity = 5.5 Gbq/mol and a radiochemical purity
(RCP) of 95% as shown in Scheme 3. After the synthesis, a log P
value of 2.5 + 0.1 was determined for [18F]AF-FDI, showing a
moderate lipophilic profile.
Taken together, these results suggest that the novel drug AF-FDI
not only has a good cell membrane permeability, but it also binds
to FOXM1 inside MBA-MD-231 cells as shown by both the cell
uptake and blocking experiments as well as during the western
blot experiments (see Figure 3). This profile was promising for a
further evaluation as a PET imaging probe.
Alternatively, we also prepared the precursor 9 possessing a nitro
group as leaving group, yielding [18F]AF-FDI in 20 ± 5%
radiochemical yield, RCP = 95%, and a molar activity of 16
Gbq/mol (See Scheme S2 in the supporting information file).
Scheme 3. Synthesis of radiolabeled [18F]AF-FDI.
MDA-MB-231 tumor bearing mice
[18F]AF-FDI was further analyzed in vivo with a dynamic PET
experiments using MDA-MB-231 tumor-bearing mice. After 60
min post injection radiotracer [18F]AF-FDI reached a tumor uptake
SUV60min value of (0.31 ± 0.05 (n=4; Figure 5). From the dynamic
PET experiments also time-activity curves (TAC) were generated
for selective organ uptake and clearance profiles for [18F]AF-FDI.
The muscle uptake was reaching an SUV60min value of 0.41 ± 0.03
(n=4; (Figure 6A) resulting in no difference between tumor and
muscle uptake. It is interesting to note that the radioactivity after
injection of the radiotracer in both muscle and tumor tissues was
still increasing after 60 min post injection, suggesting that no
equilibrium was reached yet. These observations let us conclude
that [18F]AF-FDI is structurally not optimized yet as it still shows
non-specific behavior in vivo. However, longer observations
beyond 60 min post injections as well as fine tuning of the FOXM1
inhibitor molecule structure will be necessary to generate a
second-generation radiotracer targeting FOXM1 in vivo. It is worth
contrasting the tumor uptake of [18F]AF-FDI with that of [18F]FDG
(SUV60min ≈ 1), reported in our previous work using the MDA-MB-
231 tumor-bearing mice model, which is well validated in our
lab.[24-25]
Regarding the clearance profile we observed that [18F]AF-FDI is
mainly cleared by the hepatobiliary path (Figure 6B), reaching its
highest value at the liver right after injection, decreasing to
SUV60min of 8. Only a fraction of the radiotracer is cleared through
the kidneys reaching a peak immediately after injection and then
decreasing after a 10 min-period to a steady level from 20 to 60
min (SUV60min 1.5; Figure 6C). We think that the high hepatobiliary
clearance might be due to the relative lipophilic character of
[18F]AF-FDI (LogP = 2.58 ± 0.17), which will be considered for the
design and chemical optimization of the next generation of 18F-
FOXM1 inhibitors. On this note, we have recently designed and
published a series of troglitazone-based FOXM1 inhibitors
(TFIs),[18] containing a thiazolidinedione moiety bearing an acidic
Reagents and conditions: (a) K2CO3, EtOH, 90˚C, 3h, MW; (b) DCM, 25˚C,
CF3SO3CH3, 18 h; (c) K2CO3, [K/K222]+ 18F-, 120-130˚C, CH3CN, 15 min. The
overall radiochemical yield obtained for compound[18F]AF-FDI = 73 + 3%.
Cell uptake
As a next step radiolabeled [18F]AF-FDI was analyzed for its in
vitro profile in MDA-MB-231 cells. Uptake levels were high
(~500% of radioactivity per mg of protein after 60 min incubation
Figure 4A) in comparison to other experimental tracers,[20]
suggesting that [18F]AF-FDI is considerably taken up by TNBC
cells in vitro. Furthermore, to know exactly how much of the
compound would bind to the cell membrane and how much would
be internalized by the cells, we also determined the percentage of
the internalized fraction (cell membrane permeability). About 55%
of the radiotracer was found to be internalized in MDA-MB231
cells, while ~45% seemed to be membrane-bound (Figure 4B).
Finally, to assess the specificity of [18F]AF-FDI towards the target
protein FOXM1, in vitro blocking experiments were performed in
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proton which, upon dissociation, should form (to some extent) a
negatively charged anion at physiological pH conferring a more
polar (hydrophilic) character. We hypothesize that by increasing
the polarity of second generation of radiolabeled FOXM1
inhibitors, the ratio between renal and hepatobiliary clearance
may improve the profile of the tracer. In this regard, we have
identified one fluorine-containing lead that can be considered a
scaffold to develop a 2nd generation of 18F-tracers to target
FOXM1 for PET imaging, which will be reported shortly.
resulting from a potential enzymatic degradation of [18F]AF-FDI.
Therefore, to assess the in vivo stability of [18F]AF-FDI, metabolic
stability in vivo was analyzed by administering [18F]AF-FDI to
normal female BALB/c mice and collecting blood samples at 5, 15,
30 and 60 min post injection. In addition, radiotracer distribution
in different blood compartments was also measured. After 5 min
post injection ~80% of the radiotracer was bound to red blood
cells and ~20% was freely present in plasma. It is interesting to
note that within 60 min, the concentration of the radiotracer
detected in red blood cells oscillated ~60%, which was significant
and suggests that this may be an important factor limiting the low
distribution of the radiotracer towards the tumor tissue and its
subsequent uptake into the tumor cells in vivo. As [18F]AF-FDI is
strongly bound to plasma and red cell proteins, it is possible that
this might also be a contributor to the observed low tumor uptake.
It merits a more detailed structural analysis to determine why the
radiolabeled compound had such high affinity for red blood cells
(Figure 7A). Nevertheless, by sampling the blood fractions at
different time points (5, 15, 30 and 60 min) and using radio-TLC
(shown in the supporting information file), after 60 min ~90% of
intact [18F]AF-FDI was detected, demonstrating that this molecule
is relatively stable and withstands metabolic breakdown in mice
(Figure 7B).
Interestingly, we observed significant brain uptake was detected
over 60 minutes suggesting that [18F]AF-FDI can cross the blood
brain barrier (Figure 6D), and therefore, it may be useful to reach
brain tumors, opening interesting research opportunities for brain
cancer.
[18F]AF-FDI distributes in blood following a somewhat different
profile in which the maximum drug concentration decreased
immediately after the initial peak (SUV = 16) to a blood
concentration that remained essentially constant from 5 to 60
minutes (SUV60min = 2; Figure 6E). The comparison of blood
clearance after 5 minutes with the high accumulation in the liver
at the same time could also be related to the low tumor uptake
since there is less tracer available in the bloodstream to reach the
tumor.
These results suggest that [18F]AF-FDI is metabolically stable and
therefore, suitable for longer in vivo studies in which the tissue
distribution of the radiotracer can be measured at periods of time
> 60 min.
Metabolism experiments
At this point it was unknown whether the radioactivity detected in
the animals was due to the tissue distribution of the intact
radiotracer, or the signal was detected from metabolic products
Figure 4. In vitro experiments for radiotracer [18F]AF-FDI using MDA-MB-231 triple negative-breast cancer cells. A) Cell uptake; B) cell internalization, and C) uptake
inhibition (blocking) using the non-radiolabeled compound FDI-AF or the parent compound FDI-6. Results are shown as mean ± SEM from n data points determined
from x experiments (shown as n/x in each figure).
5
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Figure 5. Representative PET images at 60 min post injection of [18F]AF-FDI. From left to right: Coronal; Saggital; and Transaxial. (SUV = standardized uptake
value).
Figure 6. Time-activity curves (TACs) of the uptake and clearance profile for radiotracer [18F]AF-FDI. A) Tumor and muscle uptake; B) liver tissue clearance; C)
kidney clearance; D) brain uptake; and E) blood clearance. All data are presented as mean ± SEM from n=4 experiments.
6
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Figure 7. A) Blood compartment distribution (blood cells and free plasma fraction. B) In vivo stability of radiotracer [18F]AF-FDI; in mice; the values represent
mean ± SEM calculated from three experiments (n = 3).
Solvents, reagents and the building blocks: 2-thenoyltrifluoroacetone (1),
2-cyanothioacetamide (2), 2-bromo-4′-fluoroacetophenone (4), 2-bromo-
Conclusions
1-(4-dimethylamino-phenyl)-ethanone (5). For the microwave-assisted
synthesis, we used an Initiator Reactor (Biotage). All reactions were
In summary, to the best of our knowledge this is the first study in
which the FOXM1 transcription factor is used as a potential PET
monitored by thin-layer chromatography (RediSep® TLC plates), and
imaging target to detect TNBC in vivo. The replacement of an
visualized using UV light. Melting points were measured with an
amide group, as present in the parent drug FDI-6, for a ketone
moiety in AF-FDI had only a minor effect on the activity profile of
the FOXM1 inhibitor. As it was shown in the EMSA, the drug AF-
FDI concentration-dependently dissociated the FOXM1-DNA
complex, suggesting that the amide functionality is important but
not essential. It is significant to note that during the western blot
experiments some precipitation of the drug AF-FDI was observed
and that this may affected the results compared to FDI-6. This
observation was consistent to previous results reported by our
group in which FDI-6 derivatives possessing lipophilic functional
groups decreased the potency of these molecules in the western
blot assay but not in the EMSA.[9]
Finally, we had circumvented the lack of reactivity toward
nucleophilic aromatic substitution observed with the parent drug
FDI-6, and successfully developed a nucleophilic aromatic
substitution for the radiofluorination of the novel FOXM1 inhibitor
AF-FDI adopted on previously reported procedures.[23] We still
have to optimize the labeling conditions for future 18F-labeled
FOXM1 inhibitors to improve their molar activity.
The in vivo analysis of AF-FDI clearly showed that the biological
profile of novel FOXM1-targeting probes will require further fine-
tuning regarding optimization of tumor uptake and longer
detection times beyond 60 min post injection. However, the in vivo
metabolic stability of [18F]AF-FDI was already superior with ~90%
intact after 60 min. On the other hand, the affinity of FOXM1
targeting molecules derived from the FDI-6 structure or other
scaffolds will also require further improvements for optimized
structure-activity relationship to decrease its binding to blood cells
and to increase the uptake into tumor cells in vivo. Also, using
knock-out FOXM1 mice models, we could compare tumor uptake
when FOXM1 is expressed and when not.
electrothermal melting point apparatus (Thermofisher, USA) and were
uncorrected. 1H-NMR, 13C-NMR and 19F-NMR spectra were determined
on a Bruker FT-600 MHz instrument (600, 150 and 565 MHz respectively)
using DMSO-d6 as the solvent and TMS as a reference. Chemical shifts
(δ) and coupling constants (J) are expressed in parts per million and Hertz,
respectively. Signal multiplicity is expressed as s (singlet), d (doublet), t
(triplet),
q (quartet), m (multiplet) and br (broad singlet). Liquid
Chromatography and ESI-Mass Spectrometry (LC-MS) were performed on
an Agilent Technologies 1260 HPLC with C6130B MSD. The synthesis of
6-(thiophen-2-yl)-2-thioxo-4-(trifluoromethyl)-1,2-dihydropyridine-3-
carbonitrile (3),[17] and FDI-6 [5, 9], was carried out following the method
reported, and confirmed by 1H, 13C and 19F NMR. Radio TLC was
performed on a radio-TLC reader and Winscan analysis software. High-
performance liquid chromatography (HPLC) radiochemical purifications
were performed using a Phenomenex LUNA C18 column (100 Å, 250 × 10
mm, 10 mm) on a Gilson 322 pump module fitted with a 171 Diode Array
and a radio detector.
Synthesis of AF-FDI and precursors 6 and 7.
We adapted the method that we reported previously for the synthesis of
FDI-6 and its derivatives.[9] Briefly, a mixture of the corresponding phenone
(1 equiv.), 3 (1 equiv.), and K2CO3 (1 equiv.) in 5mL of EtOH, was
irradiated under microwave for 3 h at 90 °C in a sealed vial. After 3 h, the
reaction mixture was allowed to cool down to RT to yield a precipitated that
was filtered off and washed with water and hexane to yield the sought
compound in an overall 70 % yield. The purity of the compound AF-FDI,
and for precursors 7 and 9 used for the radiolabeling was assessed by
HPLC and LC-MS.
Synthesis of [3-amino-6-(thiophen-2-yl)-4-(trifluoromethyl)thieno[2,3-
b]pyridin-2-yl](4-fluorophenyl)methanone (AF-FDI).
A mixture of 4 (1 equiv., 100 mg, 0.46 mmol), 3 (1 equiv., 131 mg, 0.46
mmol) and K2CO3 (1 equiv., 65 mg, 0.46 mmol) in EtOH (5 mL), was
irradiated under microwave for 3 h, to yield AF-FDI in a 80 % yield, as a
yellowish flocculent solid; m.p. 165 °C; 1H NMR (600 MHz, DMSO) δ 8.32
(s, 1H), 8.26 (dd, J = 3.8, 1.1 Hz, 1H), 7.91 – 7.85 (m, 3H), 7.76 (br, 2H),
7.40 (t, J = 8.9 Hz, 2H), 7.27 (dd, J = 5.0, 3.8 Hz, 1H); 13C NMR (151 MHz,
DMSO) δ 188.20, 164.63, 162.98, 162.79, 153.88, 148.31, 141.93, 137.00,
Experimental Section
Experimental Details.
7
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10.1002/cmdc.202100279
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136.98, 133.13, 132.91, 132.09, 130.37, 130.31, 129.92, 129.22, 123.45,
121.63, 116.51, 115.73, 115.58, 112.96, 112.92, 105.07; 19F NMR (565
MHz, DMSO) δ -58.53, -108.13; LCMS: found [M+H]+: 423.0 m/z
(calculated: 422.0 m/z for C19H10F4N2OS2); Purity ≥ 95, as judged by NMR,
HPLC and LC-MS.
and DNA in each reaction was 715 nM and 12.8 nM, respectively. The Ki
values were calculated for each AF-FDI and FDI-6 using
Equation. 1:[26]
[퐼]50
퐾푖 =
Synthesis of [3-amino-6-(thiophen-2-yl)-4-(trifluoromethyl)thieno[2,3-
b]pyridin-2-yl][4-(dimethylamino)phenyl]methanone (6).
[ ]
퐾푑
퐿
[ ]
푃
⁄
50
0
⁄
(
+
+ 1)
퐾푑
A mixture of 5 (1 equiv., 100 mg, 0.413 mmol), 3 (1 equiv., 118 mg, 0.413
mmol) and K2CO3 (1 equiv., 60 mg, 0.413 mmol) in EtOH (5 mL), was
irradiated under microwave for 3 h at 90 °C, to yield 6 in a 90 % yield, (172
mg, 0.38 mmol), which was used for the next step without any further
purification; m.p. 245-247 °C; 1H NMR (600 MHz, DMSO) δ 8.33 (s, 1H),
8.26 (dd, J = 3.8, 1.1 Hz, 1H), 7.87 (d, J = 1.0 Hz, 1H), 7.80 (d, J = 9.0 Hz,
2H), 7.51 (s, 2H), 7.28 (dd, J = 5.0, 3.8 Hz, 1H), 6.81 (d, J = 9.0 Hz, 2H),
3.05 (s, 6H); 13C NMR (151 MHz, DMSO) δ 187.94, 162.27, 153.21,
152.60, 147.10, 142.11, 132.75, 132.52, 131.71, 130.05, 129.55, 129.20,
127.06, 123.57, 121.75, 117.02, 112.73, 110.86, 105.85; 19F NMR (565
MHz, DMSO) δ -58.15; LCMS: found [M+H]+: 448.1 m/z (calculated: 447.1
m/z for C21H16F3N3OS2 %).
Where: [I]50 = IC50 of the inhibitor; [L]50 = concentration of IR-labelled DNA
at 50% inhibition; [P]0 = concentration of the FOXM1 protein; and Kd
dissociation constant calculated from the initial titration curve.
=
Western blot.
We employed our reported protocol for the screening of FDI-6 and its
derivatives, and used a FOXM1 mouse monoclonal antibody (Santa Cruz
Biotechnology) and IRDye® 800CW Goat anti-Mouse IgM (Li-Cor
Biosciences). MDA-MB-231 Cell was seeded in a density of 2 x 105 cells
per well in 6-well plates. After treatment with AF-FDI and FDI-6 at 40, 60
and 80 µM for 24 hours, the cells were lysed with RIPA lysis and extraction
buffer (Thermo Fisher) according to the manufacturer's protocol to yield
the whole-cell extracts, which were then centrifuged to remove any cell
debris. The protein levels in the supernatant were measured using
Bradford’s assay, then the protein (40 μg/lane) was loaded into a 4-20%
SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis).
After completion of the run, the protein was transferred from the gel to a
nitrocellulose membrane (Thermofisher), and stained with REVERT (Li-
Cor Biosciences) total protein stain, to determine total protein for
normalization. Alternatively, we also used -actin to normalize FOXM1
levels after treatment. The membrane was then detected in the 700 nm
channel using Odyssey scanner (LI-COR Biosicences). The REVERT was
then reversed and the membrane was blocked with 10% fat-free milk in
TBST for 1 h. The membrane was incubated with the primary antibody
(1:1000 dilution) at 4 °C overnight. Then, the membrane was washed three
times with TBST, incubated with the corresponding Li-Cor secondary
antibody and incubated again at room temperature for 1 h. The membrane
was washed three times (15 minutes total) with TBST. The blots were
visualized using Odyssey scanner (LI-COR Biosciences). The
quantification was carried out for all proteins relative to total protein
(REVERT) using ImageJ for each lane.
Synthesis of 4-{[3-amino-6-(thiophen-2-yl)-4-(trifluoromethyl)thieno[2,3-
b]pyridin-2-yl]carbonyl}-N,N,N-trimethylanilinium
trifluoromethanesulfonate (7).
Methyl trifluoromethanesulfonate (1.6 equiv., 72 µL, 0.63 mmol) was
added dropwise to a solution of 6 (1 equiv., 172 mg, 0.38 mmol), in dry
CH2Cl2 (5 mL) at room temperature, this mixture was stirred vigorously for
18 h to yield an orange precipitated that was filtered off and then washed
with water and hexane to give precursor 7 in an 80 % yield (200 mg, 0.3
1
mmol), as orange flocculent crystals; m.p. > 250 °C; H NMR (600 MHz,
DMSO) δ 8.35 (s, 1H), 8.29 (dd, J = 3.8, 1.1 Hz, 1H), 8.16 (d, J = 8.9 Hz,
2H), 8.04 (d, J = 8.9 Hz, 2H), 7.89 (m, 1H), 7.88 (br, 1H), 7.29-7.28 (dd, J
= 4.4 Hz, 1H), 3.68 (s, 9H); 13C NMR (151 MHz, DMSO) δ 187.56, 163.01,
154.17, 148.87, 141.83, 141.70, 133.30, 133.08, 132.26, 130.15, 129.29,
129.07, 123.40, 121.75, 121.59, 121.13, 119.61, 116.35, 113.08, 104.83,
56.42; 19F NMR (565 MHz, DMSO) δ -58.66, -77.76; LCMS: found: [M+H]+
462.1 m/z (calculated 462.1 m/z for C22H19F3N3OS2+); Purity ≥ 95, as
judged by NMR, HPLC and LC-MS.
Electrophoretic Mobility Shift Assay (EMSA).
We employed our “in house” EMSA protocol previously reported.[9] For the
protein expression and purification we used the PEX-N-GST-FOXM1-DBD
plasmid (OriGene Technologies, USA) transformed into BL21(DE3) E. Coli
cells; positive colonies were selected on LB agar plates with ampicillin (100
μg/mL). Then, these cells were grown in LB media with ampicillin (100
μg/ml) at 37ꢀ°C with orbital shaking at 220 rpm until reaching an optical
density (OD600) of 0.8; protein expression was induced by adding 1ꢀmM
isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6ꢀhr at 37ꢀ°C. The GST
protein and GST-FOXM1 protein from soluble fractions were purified using
glutathione resin (GenScript, USA), following the manufacturer’s
instructions.
Radiosynthesis of [18F]AF-FDI
18F was produced via 18O(p, n)18F from [18O]H2O on an ACSI TR19/9
cyclotron. [18F-] fluoride was trapped on a light QMA anion exchange and
was eluted with Kryptofix K222 and K2CO3 in CH3CN and dried under
azeotropic conditions (nitrogen and 95 °C while adding 3 mL of CH3CN).
Compound 7 (0.0050 mmol, 2.5 mg) was dissolved in 500 µL of dry MeCN,
and added to a solution of dried [18F-]Fluoride in 300 µL CH3CN (typically
0.6 to 0.9 GBq). The mixture was heated to 130 °C for 15 min in a sealed
vial. [18F]AF-FDI was purified using HPLC (Rt= 17.5 min) linked to a radio
detector (conditions: gradient CH3CN: H2O, starting from 80:20 ratio) and
obtained in 65% decay corrected RCY, in a 95% radio purity with a molar
activity 5 GBq/µmol. The octanol-water partition coefficient (LogP value) of
All values of a titration (binding) curve of recombinant FOXM1-DBD with
its target double-strand DNA oligo (Forward strand: 5′-/IRD700/-
AAACAAACAAACAATCAAACAAACAAACAATC-3′), were recorded using
EMSA by the method previously reported by Gormally et al.[5] Briefly,
dsDNA and an increasing concentration of the FOXM1 protein were
incubated at RT for 30 minutes in a buffer solution containing 20 mM Tris
(pH 7.5), 100 mM KCl, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol, before
running the samples on 6% native gel for 30 min at 120 V. The dissociation
constant of protein DNA complex (Kd) was calculated using Graph pad
Prism 6.2. The displacement EMSA experiments were carried out by
incubating each test compound with the FOXM1 peptide, for 1.5 h, at room
temperature, followed by a second incubation with DNA, for 20 minutes,
before conducting the electrophoresis. The concentration of FOXM1-DBD
[
18F]AF-FDI was measured by adapting our method previously reported
method.
Cell uptake.
In vitro of cell uptake of [18F]AF-FDI.
[20, 27]
We adapted our previously reported protocol:
MDA-MB231 cells
were grown in 12-wellthe medium was removed 1 h before the experiment,
cells were washed 2X with PBS and starved of glucose in glucose-free
Krebs-Ringer solution (120mM NaCl, 4mM KCl, 1.2 mM KH2PO4, 2.5 mM
8
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10.1002/cmdc.202100279
ChemMedChem
FULL PAPER
MgSO4, 25 mM NaHCO3, 70mM CaCl2, pH = 7.4) for 1 h at 37°C. Next,
300 ml of Krebs-Ringer solution with 0.7 – 1 [18F]AF-FDI was added to
each well. Plates were incubated at 37°C for specific time points (5, 10, 15,
30, 60, 120 min). Radiotracer uptake was stopped with 1ml ice-cold PBS,
and cells were washed 2x with PBS and lysed in 0.4 ml lysis buffer (50 mM
Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 0.5%TritonX).
Radioactivity in cell lysates was measured using Wizard2 Automatic g
Counter (PerkinElmer; Waltham, MA, USA). Total protein concentration in
the samples was determined using a Pierce Bicinchoninic Acid–Based
Protein Assay (ThermoFisher Scientific). Data were calculated as the
percentage of total added radioactivity per milligram of protein.
intravenously with a delay of 15 s through a tail vein catheter. Dynamic
PET data acquisition was performed in a 3-dimensional list mode for 60
min. Dynamic list mode data were sorted into sonograms with 54 time
frames (10 × 2, 8 × 5, 6 × 10, 6 × 20, 8 × 60, 120,5 × 300 s).
Frames were reconstructed using Ordered Subset Expectation
Maximization or maximum
a posteriori reconstruction modes. No
correction for partial volume effects was performed. Image files were
further processed using the Rover v.2.0.51 software (ABX). Masks
defining 3-dimensional regions of interest were set and defined by 50%
thresholding. Mean standardized uptake values [SUVmean = (activity/ml
tissue)/(injected activity/body weight)] (ml/kg), were calculated for each
region of interest. Time-activity curves (TACs) were generated from the
dynamic scans.
Cell internalization experiments.
27]
Based on previously reported protocols,[20,
MDA-MB-231 cells were
Metabolism experiments.
seeded 12-well plates 24–48 h before the assay so that cells could reach
95 % confluency. The medium was removed 1 h before the assay, and the
cells were rinsed twice with PBS. After the addition of Krebs buffer (1 mL)
to each well, the cells were incubated at 37 °C. Krebs buffer was aspirated,
and the cells were incubated with 300 μL of [18F]AF-FDI in 0.9 % NaCl (0.1
MBq ) for 60 min at 37 °C. Cellular uptake was stopped by removing
incubation media from the cells and washing the wells twice with ice-cold
PBS buffer (1 mL). Surface bound radioactivity was removed from the cells
by incubating the cells twice with 0.5 mL glycine-HCl in PBS (50 mM, pH
2.5) for 5 min at 37 °C. Cells were washed again with ice-cold PBS before
the addition of radio-immunoprecipitation assay (RIPA) buffer (400 μL) to
lyse the cells. Cells were returned into the incubator for 10 min, and cell
lysates were collected for counting. Radioactivity of surface-bound and
internalized fraction was measured in a WIZARD2 Automatic gamma
counter (Perkin Elmer, Waltham, MA, USA). Total protein concentration in
the samples was determined by the bicinchoninic acid method (BCA;
Pierce, Thermo Scientific 23227) using bovine serum albumin (800, 600,
400, 300, 200, 100, 50 μg/mL, blank) as the protein standard. Data are
expressed as percent of total uptake per 1 mg protein (% of total
uptake/mg protein).
As previously reported,[20] the radiotracer solution containing ~30 MBq
[
18F]AF-FDI in ~7% EtOH/saline was injected intravenously into normal
female BALB/c mice under isoflurane anesthesia. Blood samples from the
tail vein (20-40 μL) were collected at 5, 15, 30 and 60 min p.i.. Plasma was
separated by centrifugation (5 min, 13,000×g) followed by plasma protein
precipitation using methanol (two parts per one part plasma) and a second
centrifugation step (5 min, 13,000×g). Supernatants were analyzed by
radio thin-layer chromatography (radio-TLC) and radioactivity was
determined using a WIZARD2 Automatic gamma counter (Perkin Elmer;
Waltham, MA, USA). TLCs were developed in hexane:ethyl acetate
(80:20) and analyzed using a BAS-5000 reader. [18F]AF-FDI had an Rf
value of 0.5-0.0 in this solvent system.
Statistical Analysis.
All in vitro data and semi quantified PET data are expressed as means
(n=6) ± SEM. Graphs, time-activity curves (TAC), dose-response curves
and IC50 were constructed and calculated using GraphPad Prism 4.0. One
way ANOVA was used to analyze the data from the in vitro screening and
was considered significant when P < 0.05.
In vitro inhibition of [18F]AF-FDI cell uptake.
27]
According to previously reported procedures,[20,
the medium was
removed 1 h before the experiment, and cells were washed 2X with PBS
or sodium-free choline buffer (120 mM C5H14ClNO, 4 mM KCl, 1.2 mM
KH2PO4, 2.5 mM MgSO4, 25 mM C5H14NOHCO3, 70mM CaCl2, pH 7.4)
and starved of glucose in glucose-free Krebs-Ringer solution. MDA-
MB231 cells were incubated with Krebs-Ringer buffer or choline buffer
containing [18F]AF-FDI and increasing concentrations (1, 10, 100 nM and
1, 3, 7, 10mM) of reference FOXM1 inhibitor FDI-6 and non-radioactive
AF-FDI. Control for 100% uptake was determined with Krebs-Ringer buffer
only. After 60 min, cells were rinsed with ice-cold PBS or ice-cold choline
buffer, lysed, and counted for radioactivity as previously described.
Acknowledgements
D. J. P. is grateful for the Postdoctoral Fellowship (287012),
granted by the National Council for Science and Technology,
(CONACYT, México). The authors thank Floyd Baker, David
Clendening, and Blake Lazurko, from the Edmonton
Radiopharmaceutical Center for 18F production on the biomedical
cyclotron. The authors are also grateful to Dan McGinn (Vivarium
of the Cross Cancer Institute, Edmonton, AB, Canada) for
supporting the animal work. The authors also gratefully
acknowledge the Dianne and Irving Kipnes Foundation for
supporting the PET imaging work.
MDA-MB-231 tumor-bearing mice.
27]
All animal experiments were carried out as previously reported,[20,
Keywords: Positron Emission Tomography, PET, diagnostic
following guidelines of the Canadian Council on Animal Care and were
approved by the local animal care committee of the Cross Cancer Institute
under protocol number AC17231. Human TNBC MDA-MB231 cells (5 3
106 cells in 100 ml PBS) were injected subcutaneously and tumors were
grown for 3–4wk, reaching sizes of 200–400mm3, resulting in 300–500-
mm3-sized tumors after 2–3 week. MDA-MB231 tumor-bearing NIH-III
nude mice (Charles River) were anesthetized with isoflurane (40% O2 60%
N2), and their body temperatures were kept constant at 37°C. Mice were
positioned and immobilized in a prone position into the center of the field
of view of an Inveon PET Scanner (Siemens,Munich, Germany), A
transmission scan for attenuation correction was not acquired.
Radioactivity present in the injection solution (0.5-ml syringe) was
determined using a dose calibrator (AtomlabTM 300; Biodex Medical
Systems, New York, NY, USA). After the emission scan was started,
radioactivity (4–8MBq of [18F]AF-FDI in 100–150 ml saline) was injected
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
Targeting FOXM1 for triple-negative breast cancer detection. We report preliminary results to validate FOXM1 transcription factor
as a target for triple-negative breast cancer detection, using a 1st generation 18F-radiolabeled FOXM1 inhibitor as a PET tracer in vitro
and in vivo.
11
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