Design, Synthesis, Radiosynthesis and Biological Evaluation of Fenretinide Analogues as Anticancer and Metabolic Syndrome-Preventive Agents

Article abstract of DOI:10.1002/cmdc.202000143

Fenretinide (4-HPR) is a synthetic derivative of all-trans-retinoic acid (ATRA) characterised by improved therapeutic properties and toxicological profile relative to ATRA. 4-HPR has been mostly investigated as an anti-cancer agent, but recent studies sho

Full text of DOI:10.1002/cmdc.202000143

Accepted Article
Title: Design, synthesis, radiosynthesis and biological evaluation of
Fenretinide analogues as anticancer and metabolic syndrome-
preventive agents
Authors: Matteo Zanda, Ilaria Patruno, Dawn Thompson, Sergio
Dall’Angelo, Albert D. Windhorst, Danielle J. Vugts, Alex J.
Poot, and Nimesh Mody
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000143
Link to VoR: https://doi.org/10.1002/cmdc.202000143
01/2020

10.1002/cmdc.202000143
ChemMedChem
FULL PAPER
Design, synthesis, radiosynthesis and biological evaluation of
Fenretinide analogues as anticancer and metabolic syndrome-
preventive agents
Ilaria Patruno,[a] Dawn Thompson,[a] Sergio Dall’Angelo,[a] Albert D. Windhorst,[b] Danielle J. Vugts,[b] Alex
J. Poot,[b] Nimesh Mody,*[a] Matteo Zanda*[a],[c],[§]
[a]
[b]
Ilaria Patruno, Dr. Dawn Thompson, Dr. Sergio Dall’Angelo, Dr. Nimesh Mody, Prof. Dr. Matteo Zanda
Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, AB25 2ZD, UK
E-mail: n.mody@abdn.ac.uk, m.zanda@lboro.ac.uk
Prof. Dr. Albert D. Windhorst, Dr. Danielle J.Vugts, Dr Alex J. Poot
Amsterdam UMC, VU University Medical Center, Cancer Center Amsterdam, dept. Radiology and Nuclear Medicine, De Boelelaan 1117, 1081 HV
Amsterdam, The Netherlands
[c]
[§]
Prof. Dr. Matteo Zanda
C.N.R.-SCITEC, via Mancinelli 7, 20131 Milan (Italy)
Prof. Dr. Matteo Zanda
Current address: Loughborough University, School of Science, Centre for Sensing and Imaging Science, Sir David Davies Building, Loughborough LE11
3TU, UK.
Supporting information for this article can be found under XXX.
Abstract: Fenretinide (4-HPR) is a synthetic derivative of All-Trans-
Retinoic Acid (ATRA) characterised by improved therapeutic
properties and toxicological profile relative to ATRA.ꢀ4-HPR has been
mostly investigated as an anti-cancer agent, but recent studies
evidenced its promising therapeutic potential for preventing metabolic
syndrome. Several biological targets are involved in 4-HPR’s activity,
leading to the potential use of this molecule for treating different
pathologies. However, although 4-HPR displays quite well-
understood multi-target promiscuity with regards to pharmacology,
interpreting its precise physiological role remains challenging. In
addition, despite promising results in vitro, the clinical efficacy of 4-
HPR as a chemotherapeutic agent has not been satisfactory so far.
Here we describe the preparation of a library of 4-HPR analogues,
followed by the biological evaluation of their anti-cancer and anti-
obesity/diabetic properties. The click-type analogue 3b showed good
capacity to reduce the amount of lipid accumulation in 3T3-L1
molecules consists of an alicyclic end group, a polyene spacer
chain and a polar end group.3 Conversely, in the structure of 4-
HPR the polar carboxylate end group is replaced by a 4-
hydroxyphenyl carboxamide. Thanks to this chemical modification,
4-HPR preferentially accumulates in fat tissue over liver,
displaying an improved toxicological profile. Therefore, 4-HPR
has been investigated for decades as an anti-cancer agent,
showing to be more potent than ATRA in inhibiting cancer cell
proliferation.4 Moreover, recent studies reported the potential
therapeutic properties of 4-HPR for the treatment of metabolic
syndrome, showing a leaner phenotype and improved glucose
handling in mouse models of obesity treated with 4-HPR.5–7
Various mechanisms of action are involved in the beneficial
effects of 4-HPR; these include for example the capacity of 4-HPR
to induce apoptosis through generation of radical oxygen species
(ROS) or its ability to reduce ceramides (Cer) levels via the
inhibition of the de novo synthesis pathway.8 In addition, 4-HPR
may act via a mechanism ATRA-dependent in the inhibition of
adipocyte differentiation.9 Specifically, ATRA blocks adipogenesis
when introduced at early stage of differentiation by inhibiting the
transcription of CAAT/enhancer binding protein (C/EBP) which
regulates adipogenesis and adipocyte biology.5,10
Earlier studies reported some 4-HPR derivatives with a reduced
IC50 in the inhibition of cancer cell proliferation compared to 4-
HPR.11-18 However, none of the 4-HPR derivatives previously
synthesized showed a marked increase in potency relative to 4-
HPR, with IC50 values still in the micromolar range. Importantly, to
the best of our knowledge, the only reported structure activity
relationship (SAR) study focused on the use of 4-HPR derivatives
in metabolic syndrome relies on 4-HPR derivatives designed as
disrupters of the protein-protein interactions between Serum
Retinol Binding Protein and Transthyretin in order to prevent
insulin resistance and diabetes.19 Lastly, a comprehensive study
on 4-HPR’s SAR investigating both the anti-cancer and the anti-
obesity/diabetic properties has not been reported to date.
adipocytes during differentiation. Furthermore, it showed an IC50
=
0.53±0.8M in cell viability tests on breast cancer cell line MCF-7,
together with a good selectivity (SI = 121) over non-cancerous
HEK293 cells. Thus, 3b was selected as a potential PET tracer to
study retinoids in vivo and the radiosynthesis of [18F]3b was
successfully developed. Unfortunately, the stability of [18F]3b turned
out to be insufficient for pursuing imaging studies.
Introduction
Fenretinide, or N-(4-hydroxyphenyl)retinamide (4-HPR), is a
synthetic derivative of All-Trans-Retinoic Acid (ATRA), which is
the most active metabolite of vitamin A (Figure 1).1
ATRA has important physiological functions including roles in
vision, regulation of cell proliferation and differentiation, growth of
bone tissue, immune function and activation of tumor suppressor
genes.2 However, the chronic administration of therapeutic
concentrations of ATRA leads to hepatic toxicity, limiting its use
in therapy. The basic structure of ATRA and other retinoid
1
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10.1002/cmdc.202000143
ChemMedChem
FULL PAPER
In this study we synthesized a library of 4-HPR analogues using
a SAR approach to determine which chemical groups are
important for the biological activity of 4-HPR, in order to shed
further light on its pharmacological mechanism in vitro. Our final
goal was to identify 4-HPR analogues with improved anti-cancer
activity and/or anti-obesity and anti-diabetic properties relative to
the parent molecule. Furthermore, we designed new 4-HPR-click-
type analogues to be used as candidate PET imaging tracers for
studying retinoids in vivo. This was deemed to be important,
because despite the promising results obtained in vitro on several
cancer cell lines,20,21 the efficacy of 4-HPR was shown to be
reduced in vivo. This is likely due to its low bioavailability, which
decreases the amount of drug that effectively reaches the tumour
in vivo.22 The use of radiolabeled 4-HPR analogues might
Scheme 1. Synthesis of derivatives 1a-j and 2a-d. Reagents and conditions: ia)
HATU, DIPEA, DCM, r.t., overnight. ib) HATU, 2,4,6-collidine, DCM, r.t.,
overnight.
Table 1. Derivatives 1a-j and 2a-d.
Compound
L
R1
R2
R3
yield
represent
a
powerful tool to further investigate the
1a
1b
1c
1d
1e
1f
none
none
none
none
none
-CH2-
none
-CH2-
none
none
-CH2-
none
none
none
H
H
F
60%
67%
63%
60%
65%
89%
70%
88%
50%
85%
60%
60%
60%
62%
pharmacological profile of 4-HPR and analogues in vivo, thus
facilitating the development of new retinoid drug candidates.
Herein, we report (1) the design and synthesis of a library of 4-
HPR analogues (1a-j, 2a-d, 3a-b); (2) the capacity of analogues
2b and 3b to selectively inhibit MCF-7 cell viability with a reduced
IC50 relative to 4-HPR and their biological characterization; (3) the
capacity of analogues 2b, 2d and 3b to reduce lipid accumulation
during 3T3-L1 adipocytes differentiation; (4) the radiosynthesis of
the candidate PET tracer [18F]3b.
H
CF3
t-Bu
H
H
H
H
H
Ph
Cl
H
H
H
H
H
1g
1h
1i
H
Cl
H
H
Cl
H
Cl
H
NO2
H
1j
OCH3
H
H
2a
2b
2c
2d
H
OH
OH
OH
OH
Figure 1. Chemical structure of ATRA and 4-HPR.
H
F
CH3
H
H
Results and Discussion
Cl
CHEMISTRY
Literature reports have shown that retinoids – due to the presence
of the all-trans conjugated double bonds system – are rather
unstable and can be readily oxidised and/or isomerised to related
compounds, especially in the presence of oxidants including air,
light, and excessive heat.24 This was confirmed for most of the
newly synthesised 4-HPR derivatives. We observed that a faster
degradation occurred at temperatures higher than 40 °C or when
0.1% TFA was used as an additive for preparative RP-HPLC. In
this specific case the molecules were fully decomposed after
solvent evaporation. In order to preserve their stability, the 4-HPR
analogues were therefore handled in dim light, using labware
wrapped with aluminium foil and avoiding any excess of heating
or low pH conditions at each stage of synthesis and purification.
4-HPR analogues 1a-j and 2a-b
To perform a SAR study on the phenol ring of 4-HPR, the ATRA
scaffold was maintained intact. To this end, different functions (R1,
R2, R3) such as halogens, polar groups and non-polar groups
were added to the aromatic ring. Furthermore, in analogues 1f,
1h and 2a a -CH2- spacer between the amide group and the
aromatic ring was introduced. The synthesis of 4-HPR analogues
1a-j and 2a-d relied on an amide coupling between ATRA
(commercially available) and benzylamine or aniline derivatives.
Different coupling agents such as 2-Ethoxy-1-ethoxycarbonyl-1,2-
dihydroquinoline (EEDQ), N,N'-dicyclohexylcarbodiimide (DCC)
and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were
screened. However, we obtained the best yields – along with the
most effective work-up and purification – with the widely used
coupling reagent 1-[Bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU).23
The base used was generally DIPEA, but 2,4,6-collidine was used
for compounds 2a-d in order to avoid phenolic deprotonation
(Scheme 1 and Table 1).
4-HPR click-type analogues 3a and 3b
New 4-HPR click-type analogues were designed as potential
fluorine-18 PET tracers. In doing so, we firstly considered the
stability of the ATRA scaffold which is amenable to degradation
particularly when subjected to heating. Radiofluorination is
usually performed at high temperature, thus the direct introduction
of fluorine-18 onto 4-HPR scaffold was not deemed to be a viable
strategy. Conversely, the synthetic strategy based on the Cu(I)-
catalyzed Azide-Alkyne Cycloaddition (CuAAC) (Figure 2) which
is widely used for the labelling of sensitive molecules with
radioactive or fluorescent probes,25 provided a more promising
The preparation of these new 4-HPR derivatives was
straightforward, leading to compounds 1a-j and 2a-d in good yield,
whereas the main issue was the stability of these derivatives.
2
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10.1002/cmdc.202000143
ChemMedChem
FULL PAPER
alternative to deliver 4-HPR scaffolds compatible with PET
imaging applications. In addition, 1,2,3-triazoles are often used in
medicinal chemistry because of their advantageous
physicochemical features such as polarity, rigidity, ability to act as
both hydrogen bond donors and acceptors, as well as marked
stability under hydrolytic, oxidative and reductive conditions.26
dramatically reduced (IC50 > 50 µM). Interestingly, we observed
that replacement of the 4-HPR phenyl ring with a triazole ring
preserved – or increased, as in compound 3b – the capacity to
inhibit MCF-7 cell viability. Specifically, we recorded for
compounds 3a and 3b IC50 values of 22.6 µM and 0.53 µM,
respectively. In addition, our data indicated that 4-HPR and some
4-HPR derivatives exhibited higher capacity to reduce cancer cell
viability than ATRA itself (IC50 = 82.9 µM), implying an ATRA-
independent mechanism as previously reported.4 From these
results it was concluded that the addition of the bulky 4-
hydroxyphenyl group to the ATRA scaffold is not only responsible
for the improved toxicological profile, but also for the increased
anticancer activity of the molecule. Compounds 1d, 2b, 2c, 2d
and 3b were subsequently tested for their selectivity towards non-
cancerous cells HEK293. The MTT assay was repeated on
HEK293 cells after treatment with selected compounds. It was
found that – as for 4-HPR – compounds 2b, 2c, 2d and 3b had a
reduced capacity to inhibit HEK293 cell viability relative to the
treatment at the same concentrations on MCF-7 cells (Figure 3).
Interestingly, while 2c and 2d showed the same selectivity when
compared to 4-HPR, 2b and 3b were more selective, displaying a
Selectivity Index (SI) of 666 and 121 respectively (Table 2). On
the contrary, compound 1d had a non-specific cytotoxic activity.
Figure 2. Retrosynthetic CuAAC strategy for generating 4-HPR analogues.
The click-type derivatives 3a and 3b were successfully
synthesised starting from ATRA, which was coupled with
propargylamine using HATU and DIPEA. The propargylamide
derivative 1k was then reacted with an azide (obtained in turn
from benzyl bromide or 4-fluoro-bromobutane and sodium azide)
at room temperature via the CuAAC to give the triazole derivatives
3a and 3b in high yield after Flash Chromatography (FC)
purification (80-90%) (Scheme 2).
Table 2. IC50 of ATRA, 4-HPR and compounds 1a-j, 2a-d, 3a-b concerning the
inhibition of cell viability measured on MCF-7 and HEK293 cells and relative SI.
Compound
IC50 MCF-7[a]
IC50 HEK293[a]
SI[b]
[µM]
[µM]
ATRA
4-HPR
1a
82.9±3.8
10.4±3.0
9.3±1.7
60.1±43
20.8±11
0.096±0.05
4.1±5.2
100
-
-
49.3±17
5
-
-
1b
-
-
Scheme 2. Synthesis of 4-HPR analogues 3a and 3b. Reagents and conditions:
i) propargylamine, HATU, DIPEA, DCM, r.t., overnight. iia) benzylazide, CuSO4,
Sodium Ascorbate in t-BuOH/H2O, r.t., 3h. iib) 1-azido-4 fluorobutane, CuSO4,
sodium ascorbate in t-BuOH/H2O, r.t., overnight.
1c
-
-
1d
0.047±0.03
0.5
1e
-
-
1f
-
-
IN VITRO STUDY AND SAR
1g
100
-
-
Cell viability assays to evaluate the anti-cancer activity
The capacity of compounds 1a-j, 2a-d and 3a-b to inhibit cell
viability after treatment was screened on the breast cancer cell
line MCF-7 using the MTT assay. It was found that several
compounds inhibited cell viability of MCF-7 cells by 50% when
incubated for 24 hours at micro to nanomolar concentrations
(Table 2). Specifically, it was observed that the introduction of a
methyl group or a chlorine atom on the 4-HPR phenol ring (2c,
2d) did not influence the activity of the parent molecule (the IC50
values were comparable). Conversely, the meta substitution with
a fluorine atom (2b) reduced the IC50 from 10.4 µM to 56 nM,
strongly increasing the capacity of inhibiting MCF-7 cells viability.
The absence of a group in para position (1b, 1c, 1g, 1j) instead
reduced the potency of the new compounds. Replacement of the
OH with a halogen (1a, 1e) did not alter the activity, while the
replacement with a non-polar group – such as a phenyl ring, 1d –
reduced the IC50 to 96 nM. Lastly, when the OH was replaced by
a nitro group (1i) or a spacer (-CH2-) was introduced between the
amide group and the aromatic ring (1f, 1h, 2a) the cytotoxicity was
1h
100
-
-
1i
100
-
-
1j
70.4±29
50.3±26
0.056±0.09
7.4±5.6
11.0±1.7
22.5±22
0.53±0.8
-
-
-
2a
-
2b
37.3±24
32.1±21
68.7±4
-
666
4
2c
2d
6
3a
-
3b
64.6±31
121
[a] IC50 values were calculated as the concentration of compound required for
50% cell viability inhibition as estimated by GraphPad Prism software. Values
are the mean±SD of at least three independent experiments.
[b] SI: IC50HEK293/IC50MCF-7.
3
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FULL PAPER
4-HPR
1d
MCF-7
150
150
100
50
MCF-7
HEK293
HEK293
100
50
0
**
0
0
0
0
DMSO 1nM 10nM100nM 1µM 10µM
0
0
0
DMSO 1nM 10nM100nM 1µM 10µM
conc
conc
2b
2c
150
100
50
150
100
50
MCF-7
MCF-7
HEK293
HEK293
*
****
**** ****
****
****
0
0
DMSO 1nM 10nM100nM 1µM 10µM
DMSO 1nM 10nM100nM 1µM 10µM
conc
conc
3b
2d
150
100
50
150
100
50
MCF-7
MCF-7
HEK293
HEK293
**
**
*
***
*
*
****
****
0
0
DMSO 1nM 10nM100nM 1µM 10µM
DMSO 1nM 10nM100nM 1µM 10µM
conc
conc
Figure 3. Cell viability curves of MCF-7 or HEK293 cells after treatment with 4-HPR and compounds 1d, 2b, 2c, 2d and 3b at different concentrations. Untreated
cells (0) or VEH cells (DMSO) were used as negative control. Each data point is the meanSEM of three independent experiments performed in triplicate.
Significance *P0.05, **P0.01, ***P0.001, ****P0.0001 calculated by two-way ANOVA with Bonferroni post-hoc test.
In vitro characterisation of the anti-cancer mechanism of the
lead compounds 2b and 3b.
Interestingly, both 2b and 3b – as 4-HPR – induced PARP-1
cleavage in MCF-7 cells when compared to the untreated (0) or
vehicle control (DMSO), confirming the involvement of caspases
in their apoptotic mechanism (Figure 4A, panel 1). Furthermore,
the capacity of selected compounds to induce apoptosis via ER
stress was evaluated through western blot analyses of the
immunoglobulin Binding Protein (BiP) GRP-78. The synthesis of
BiP is markedly induced under stress conditions leading to the
accumulation of unfolded polypeptides in the ER, which in
particular circumstances promotes the cells apoptotic death via
the Unfolded Protein Response (UPR) signalling network.29
Interestingly, we observed that BiP was significantly increased in
MCF-7 cells after treatment with concentrations ≥ 5 µM of
compound 2b, as well as with 4-HPR. Conversely, compound 3b
was capable to significantly increase BiP only at 10 µM
concentration (Figure 4A, panel 2, 4B). Finally, we observed
compound 2b to significantly induce p38 phosphorylation at
concentrations ≥ 5 µM, while no significant increase was
observed in cells treated with 3b (Figure 4A, panel 3, 4C).
Compounds 2b and 3b – showing the most promising results in
term of efficacy and selectivity – were therefore tested through
signalling assays in order to characterize the biological pathways
involved in their anticancer activity. Although the anti-cancer
mechanism of 4-HPR is not yet completely elucidated, the most
credited hypothesis is based on the capacity of 4-HPR to generate
ROS which might induce apoptosis of cancer cells. Specifically,
the pro-apoptotic activity induced by 4-HPR is mediated by the
activation of mitogen-activated proteins serine/threonine kinases
(MAPKs) such as JNKs, p38, ERK and PKC.21 In addition,
apoptosis is triggered by the Endoplasmic Reticulum (ER) stress
which in turn is induced by ROS generation.27 Also caspases play
a role in 4-HPR-induced apoptosis and their activity can be
assessed detecting the cleavage of Poly(ADP-Ribose)
Polymerase 1 (PARP-1), which is a cellular substrate of
caspases.28 Therefore, these downstream signalling pathways
were investigated in MCF-7 cells after 24 hours treatment with
compounds 2b and 3b.
4
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10.1002/cmdc.202000143
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4-HPR
µM
2b
µM
3b
µM
O
A
S
M
D
0
1
5
10
1
5
10
1
5
10
Cleavage
(89 kDa)
PARP-1
BiP/GRP78
p-p38
Thr 180/Tyr 182
total
p38
GAPDH
B
C
*
*
*
*
*
*
4
*
*
2.5
2.0
1.5
1.0
0.5
0.0
*
*
*
*
*
*
3
2
1
0
*
0
O
M
M
M
M
M
M
M
M
M
0
O
M
M
M
M
M
M
M
M
M
S
µ
µ
µ
µ
µ
µ
µ
µ
µ
S
µ
µ
µ
µ
µ
µ
µ
µ
µ
1
0
1
5
0
1
5
0
M
5
1
1
1
1
5
0
1
5
0
5
0
M
1
D
1
1
1
D
4-HPR
2b
3b
4-HPR
2b
3b
Figure 4. A) Western blot analyses of PARP-1 cleavage, BiP/GRP-78 and pp38 in MCF-7 cells treated with 4-HPR or 2b or 3b at different concentrations of 1, 5
and 10 µM. Untreated cells (0) and VEH cells (DMSO) were used as negative control. Protein levels of GAPDH were used as confirmation of equal loading of
samples. Representative images are shown. B) Graphical representation of BiP/GAPDH. C) Graphical representation of pp38/GAPDH. Data are normalised to
GAPDH and are the meanSEM of three independent results. Significance calculated by one-way ANOVA with Dunnett’s multiple comparison test. *P<0.05,
**P<0.01, ***P <0.001 vs 0 or DMSO.
Studies on 3T3-L1 adipocytes to evaluate the anti-
obesity/diabetic properties
glucose uptake from the bloodstream.7 We sought therefore to
investigate if these pathways could also be involved in the
mechanism of action of our novel compounds.
In addition to its anti-cancer role, 4-HPR has been suggested to
exhibit anti-obesity/diabetic properties. Previous studies have
attributed these properties to the ability of 4-HPR to inhibit
adipogenesis via an ATRA-dependent mechanism by activation
of ATRA receptors (RAR) and genes involved in retinoids
metabolism.7 However, it is also known that 4-HPR acts via
different ATRA independent pathways. For example, 4-HPR –
unlike ATRA30 – can inhibit the enzyme Desaturase1 (Des1).1,31
This enzyme is able to catalyse the final step in the de novo Cer
biosynthesis, introducing a double bond in dihydroceramide
(dhCer) to form Cer.8 Interestingly, the Des1 gene has been
identified as a hot spot in a genome-wide association study of
obesity traits and excessive amount of Cer might be responsible
for lipotoxicity.32 Furthermore, accumulation of Cer is responsible
for inducing the activation of phosphatase 2A which in turn
promotes the dephosphorylation of Akt/PKB. Importantly,
activation of the Akt pathway is involved in glucose homeostasis
and insulin signalling, promoting the translocation of the glucose
transporter to the plasma membrane and hereby enabling
The potential anti-obesogenic activity was assessed by testing
the capacity of compounds 1a-j, 2a-d and 3a-b to reduce the
amount of lipid accumulation in 3T3-L1 adipocytes during
differentiation using the Oil Red O stain. We observed that both
4-HPR and ATRA significantly inhibited adipogenesis, as
previously reported7 (Figure 5A-B). However, treatment with most
of the newly synthesised analogues did not reduce the
accumulation of lipids in 3T3-L1 adipocytes. Interestingly, we
found a significant decrease in lipid accumulation only when 3T3-
L1 adipocytes were treated with compounds 1d, 2b and 2d but to
a lesser degree if compared to 4-HPR and ATRA. Although the
reduced lipid accumulation observed for 1d was presumably
ascribed to its unspecific cytotoxicity, the anti-adipogenesis effect
of 2b and 2d might be correlated with the presence of the
characteristic 4-HPR phenol ring in both structures. Importantly,
we observed that also the click-type analogue 3b was able to
significantly reduce lipid accumulation although to a lesser extent
if compared to 4-HPR and ATRA (Figure 5A-B).
5
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Our results suggest that modifications on the 4-HPR’s scaffold
might affect the interactions with the RAR signalling pathway, thus
decreasing the anti-adipogenesis activity of the new analogues.
150
100
50
A
B
0
DMSO
nd*
ATRA
4-HPR
a
b
a
b
a
b
a
b
a
b
a
a
a
2b
2d
0
Compounds
C
3b
0
p-Akt
Ser 473
total
Akt
SKI II
GAPDH
*nd: non differentiated cells
Figure 5. A) Images of lipid stained with Oil Red O and B) quantification of the absorbance at 520 nm after treatment of 3T3-L1 adipocytes during differentiation
with ATRA, 4-HPR, SKI II and compounds 1a-j, 2a-d and 3a-b. Data are the average of three independent results. Significance calculated by one-way ANOVA with
Dunnett’s multiple comparison test. a = P < 0.05 vs 0 or DMSO, b = P < 0.05 vs 4-HPR. C) Representative images of western blot analyses of p-Akt in 3T3-L1
adipocytes after treatment with 4-HPR, SKI II and compounds 2b, 2d and 3b. Untreated cells (0) and VEH cells (DMSO) were used as negative control. Protein
levels of GAPDH were used as confirmation of equal loading of samples.
In order to investigate whether the decrease in lipid accumulation
was also correlated with Des1 inhibition, the Des1 inhibitor SKI II
was screened. We observed that SKI II slightly inhibited
adipogenesis compared with 4-HPR (Figure 5A-B). SKI II has
been reported to be a more potent Des1 inhibitor (Ki = 0.3 µM)33,34
than 4-HPR (Ki = 8 µM)31, therefore we concluded that the
mechanism utilised by 4-HPR – and presumably by compounds
2b, 2d and 3b – in reducing lipid accumulation might be due to a
combined involvement of the ATRA-dependent and -independent
pathways.
Furthermore, we also measured the increase of phospho-
Akt/PKB (p-Akt) in 3T3-L1 cells during differentiation (Figure 5C).
Western blotting for p-Akt determined a comparable increase in
p-Akt and suggested a comparable activity of compounds 2b, 2d
and 3b with 4-HPR (Figure 5C), thus reflecting the results
obtained with the Oil Red O stain. Therefore, the regulation of this
downstream signalling pathway represents a promising target to
prevent insulin resistance and type-2 diabetes.
Based on the positive in vitro results, compound 3b was
developed as a PET tracer. Importantly, the CuAAC using a
fluorine-18 labeled azide is
a common approach for the
radiolabeling of bioactive molecules which – as in our case – lack
stability under conventional radiofluorination reaction conditions
(especially pH and temperature).35 Our strategy consisted of
reacting the radiolabeled azide [18F]5 – obtained in turn from
precursor 4 and K[18F]F-K222 at high temperature – with the
sensitive alkyne 1k (Scheme 3). [18F]5 was reacted with 1k either
after purification via distillation or directly in a one-pot strategy.
Although we observed the formation of the product [18F]3b using
both strategies, the purification of the one-pot mixture was quite
challenging and no pure [18F]3b could be obtained. Conversely,
the purification of the tracer [18F]3b was possible using HPLC,
when the azide [18F]5 was distilled. This resulted in [18F]3b with a
satisfactory UV purity, a radiochemical purity of 95% immediately
after purification and a radiochemical yield of 17% (decay
corrected). A progressive degradation of the tracer was observed
over time in HPLC eluent and the degradation was even faster in
the formulation buffer. After HPLC purification the tracer was
collected in a mixture containing a reduced percentage of water,
while in the formulation buffer the organic solvent was reduced to
10%, therefore we ascribed the fast degradation of the tracer
RADIOCHEMISTRY
Radiosynthesis of [18F]3b
6
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10.1002/cmdc.202000143
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FULL PAPER
during this stage to a low stability in water. Since the degradation
process of the corresponding non-radioactive compound was
much slower – an aqueous solution of [19F]3b was stable for
several days in similar conditions – we attributed this fast
decomposition to radiolysis of water and subsequent ROS
generation which may be responsible for the degradation of the
tracer [18F]3b. The addition of anti-oxidant stabilizers was also
tested, but the stability of the tracer was not improved. Radiolytic
decomposition is a common issue in radiopharmaceuticals
development – mostly when the product is prepared in high doses
– and considerable efforts are required to limit this phenomenon.36
There is evidence in literature that the structural characteristics of
the tracer as well as the ingredients present in the aqueous
solution can affect the stability of PET tracer formulations.37–42
Several factors should be therefore considered when a new PET
tracer is formulated. This was also confirmed by our experience
and further attempts are warranted to improve the stability of
[18F]3b.
1,2,3-triazole group increases the hydrophilicity of 3b (calculated
LogP = 5.34-5.89 vs. experimental LogP = 6.31 for 4-HPR,43 see
Supporting Information for more details) without dramatically
affecting the in vitro behaviour, as confirmed by our preliminary in
vitro results. Importantly, the low bioavailability of 4-HPR is mostly
related to the hydrophobic nature of the molecule. Hence, the
more hydrophilic derivative 3b may represent a valid alternative
for developing 4-HPR derivatives with increased hydrophilicity
and improved bioavailability.
Experimental Section
General Information
Dry solvents were obtained from commercial sources and used without
further purification. Reactions performed under nitrogen atmosphere were
carried out in dry solvents. Reactions were monitored by thin-layer
chromatography (TLC), unless otherwise noted. TLCs were performed on
Merck silica gel glass plates (60 F254). Visualisation was accomplished by
UV light (254 nm) or staining with ceric ammonium molybdate or KMnO4
solution. Flash chromatography (FC) was performed manually using Silica
gel (60 Å, particle size 40–63 μm) purchased from Merck. Lab handling of
the molecules was performed in dim light and using vessels wrapped with
aluminium foil. 1H NMR and 13C NMR spectra were recorded on a Bruker
AVANCE III 400 NMR spectrometer and calibrated using residual
undeuterated solvent as internal reference. 19F NMR spectra were
recorded on a Bruker AVANCE III 400 NMR spectrometer and were
referenced to CFCl3. 13C NMR spectra were recorded with complete proton
decoupling. Chemical shifts (δ) are reported in parts per million (ppm) and
coupling constants (J) are given in Hertz (Hz), multiplicity is described
using the following abbreviations: s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad, or combinations thereof. Mass analyses
were performed using Agilent 1200 HPLC system coupled to Agilent
G6120 single quadrupole detector equipped with an electrospray
ionization (ESI) source in direct infusion modality. ESI-MS spectra were
recorded in positive mode. RP-HPLC-MS analyses were performed with
an Agilent 1200 HPLC system equipped with a DAD and an ESI-MS
detector. HPLC conditions for analytical analyses: Phenomenex Luna C18
column, 5 μm, 100 Å, 250 × 4.6 mm (L × ID) injected volume 10 μL, flow
rate 1 mL/min, unless otherwise specified. HPLC preparative purification
was performed using an Agilent 1260 HPLC system equipped with DAD
detector, HPLC conditions for preparative purification: Phenomenex Luna
C18 column, 5 μm, 100 Å, 250 × 21.2 mm (L × ID), flow rate 20 mL/min.
Radioactivity levels were measured using a Veenstra VDC-405 dose
calibrator. Radiochemistry was carried out in house assembled remotely
controlled device.44 RP-HPLC analyses of the radiotracer and precursors
were performed using a Shimadzu SPD-20A system equipped with a PDA
UV detector and HERM LB500 activity detector and LabSolutions 5.85
software (Shimadzu Corporation, Japan). HPLC semi-preparative
purification of the radiotracer was performed using Jasco (Jasco Benelux,
de Meern, the Netherlands) HPLC system.
Scheme 3. Radiosynthesis of [18F]3b. Reagents and conditions: i) K[18F]F-K222
,
DMF, 90 °C, 10 min; ii) 1k, CuSO4, sodium ascorbate, DMF/H2O, 35 °C, 15 min.
Conclusion
In the present study, a library of 4-HPR analogues was
synthesised by using a single step amide coupling (1a-j and 2a-
d) or through CuAAC (3a-b). Subsequently, these compounds
were evaluated in vitro to assess their anti-cancer activity and
their capacity to potentially prevent metabolic syndrome. Our
results indicated that certain modifications on the 4-HPR scaffold,
specifically on the aromatic ring (1d, 2b and 3b), improved the
capacity to inhibit MCF-7 cells viability as well as their selectivity
(2b and 3b). Taken together, our preliminary results suggest that
2b and 3b might represent promising anti-cancer agents to be
tested in vivo. The in vivo chemotherapeutic activity of 4-HPR is
compromised by its low bioavailability which results in insufficient
concentrations of drug in the blood – as well as in cancer tissues
– which would be required to induce apoptosis of cancer cells.
Since compounds 2b and 3b are more potent in the inhibition of
cancer cell viability in vitro, even low concentrations might be
sufficient to promote the same effect in cancer tissues in vivo. On
the other hand, 4-HPR analogues with improved anti-
adipogenesis activity in 3T3-L1 adipocytes – relative to ATRA and
4-HPR – could not be identified. Finally, we successfully
radiosynthesised [18F]3b as a candidate PET imaging tracer.
Although we could not use [18F]3b in pre-clinical PET imaging
studies, owing to its insufficient chemical stability, we showed that
the non-radioactive click-type derivative 3b has interesting
biological properties. We observed that the introduction of the
General procedure for the synthesis of compounds 1a-k and 2a-d
(General procedure A)
To a stirred solution of ATRA (1.0 equiv,100.0 mg, 0.33 mmol), HATU (1.5
equiv, 188.2 mg, 0.49 mmol) and DIPEA (3.0 equiv, 0.17 mL, 0.99 mmol)
or 2,4,6-collidine (3.0 equiv, 0.13 mL, 0.99 mmol) in DCM (4 mL), the
appropriate benzylamine or aniline (1.5 equiv, 0.49 mmol) was added. The
mixture was stirred overnight at room temperature under nitrogen
atmosphere. The reaction was quenched with saturated NH4Cl (10 mL),
extracted with DCM (3x30 mL), washed with brine (10 mL), dried over
Na2SO4 and concentrated under vacuum at low temperature. After a first
purification by FC, the compounds synthesised were subjected to RP-
HPLC analyses and then, depending on the HPLC profile and separation,
7
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10.1002/cmdc.202000143
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FULL PAPER
a further preparative RP-HPLC purification was required in order to
achieve a higher degree of purity.
((CD3)2SO, 400 MHz) δ: 10.11 (s, 1H), 7.76 (d, J = 8.7 Hz, 2H), 7.71 – 7.58
(m, 4H), 7.46-7.42 (m, 2H), 7.33 (tt, J = 1.2, 7.2 Hz, 1H), 7.03 (dd, J = 15.1,
11.4 Hz, 1H), 6.46 – 6.13 (m, 4H), 6.07 (s, 1H), 2.38 (s, 3H), 2.11 – 1.96
(m, 5H), 1.71 (s, 3H), 1.63-1.55 (m, 2H), 1.49-1.41 (m, 2H), 1.03 (s, 6H).
13C ((CD3)2SO, 101 MHz) δ: 165.3, 149.1, 140.2, 138.8, 137.8, 136.4,
135.1, 130.6, 130.4, 129.9, 129.4, 128.0, 127.3, 126.7, 123.0, 119.8, 39.6
(under solvent signal), 34.3, 33.1, 29.3, 22.0, 19.2, 13.8, 13.1.
(2E,4E,6E,8E)-N-(4-fluorophenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (1a)
ATRA was reacted with 4-fluoro-aniline (55.0 mg, 0.49 mmol), HATU and
DIPEA according to general procedure A. The crude was purified by FC
using n-Hex/EtOAc (8:2) to obtain 1a in 60% yield (78.0 mg) as a yellow
solid. Rf = 0.5 (n-Hex/EtOAc 9:1). Analytical RP-HPLC: solvent A: H2O,
solvent B: MeOH, gradient from 90% to 95% of B in 30 min, rt = 14.9 min.
MS (ESI, m/z): C26H33FNO [M+H]+ calc. 394.25, found 394.3,
C26H32FNNaO [M+Na]+ calc. 416.25, found 416.3. 1H NMR (CDCl3, 400
MHz), δ: 7.53 (br, 2H), 7.42 (br,1H), 7.04-6.97 (m, 3H), 6.32-6.14 (m, 4H),
5.82 (s, 1H), 2.43 (s, 3H), 2.06-2.02 (m, 5H), 1.74 (s, 3H), 1.66-1.62 (m,
2H), 1.51-1.48 (m, 2H), 1.06 (s, 6H). 19F NMR (CDCl3, 376 MHz), δ: -
(2E,4E,6E,8E)-N-(4-chlorophenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (1e)
ATRA was reacted with 4-Chloroaniline (68.0 mg, 0.49 mmol), HATU and
DIPEA according to general procedure A. The crude was purified by FC
using n-Hex/EtOAc (9:1) to obtain 1e in 65% yield (90.0 mg) as an orange
solid. Rf = 0.6 (n-Hex/EtOAc 7:3). Analytical RP-HPLC: solvent A: H2O,
solvent B: CH3CN, gradient from 95% to 100% of B in 15 min, rt = 12.68
min. MS (ESI, m/z): C26H33ClNO calc. [M+H]+ 410.22, found 410.2. 1H
NMR (CDCl3, 400 MHz) δ: 7.43 (d, J = 8.3 Hz, 2H), 7.25 (br, 1H), 7.22 –
7.15 (m, 2H), 6.91 (dd, J = 14.99, 11.37 Hz, 1H), 6.23-6.16 (m, 2H), 6.11
– 6.04 (m, 2H), 5.71 (s, 1H), 2.34 (s, 3H), 2.01 – 1.89 (m, 5H), 1.65 (s, 3H),
1.58 – 1.51 (m, 2H), 1.44 – 1.37 (m, 2H), 0.96 (s, 6H).13C NMR (CDCl3,
101 MHz) δ: 165.1, 151.3, 139.6, 137.7, 137.2, 136.9, 135.1, 130.8, 130.0,
129.4, 129.0, 128.7, 120.9, 120.7, 39.6, 34.3, 33.1, 29.0, 21.8, 19.2, 13.8,
12.9.
119.64 (m, 1F). 13C NMR (CDCl3, 101 MHz), δ: 165.2, 159.2 (d, JC-F
=
244.8 Hz), 150.8, 139.4, 137.7, 137.3, 135.2, 134.3, 130.6, 130.0, 129.5,
128.6, 121.5 (d, JC-F = 6.3 Hz), 121.0, 115.6 (d, JC-F = 22.2 Hz), 39.6, 34.3,
33.1, 29.0, 21.8, 19.2, 13.7, 12.9.
(2E,4E,6E,8E)-N-(3-trifluoromethyl-phenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (1b)
ATRA was reacted with 3-trifluoromethyl aniline (80.0 mg, 0.49 mmol),
HATU and DIPEA according to general procedure A. The crude was
purified by FC using n-Hex/EtOAc (9:1) to obtain 1b in 67% yield as an
orange solid (90.0 mg). Rf = 0.35 (n-Hex/EtOAc 9:1). Analytical RP-HPLC:
solvent A: H2O, solvent B: CH3CN, gradient from 87% to 100% of B in 15
min, then 100% of B, rt = 16.04 min. MS (ESI, m/z): C27H33F3NO [M+H]+
calc. 444.24, found 444.3. 1H NMR (CDCl3, 400 MHz), δ: 7.90 (br, 1H),
7.76 (d, J = 8.0 Hz, 1H), 7.63 (br, 1H), 7.44 – 7.31 (m, 2H), 7.03 (dd, J =
15.0, 11.5 Hz, 1H), 6.35 – 6.12 (m, 4H), 5.83 (s, 1H), 2.45 (s, 3H), 2.09 –
1.99 (m, 5H), 1.74 (s, 3H), 1.67-1.61 (m, 2H), 1.51-1.48 (m, 2H), 1.06 (s,
6H). 19F NMR (CDCl3, 376 MHz) δ: -62.72 (m, 3F). 13C NMR (CDCl3, 101
(2E,4E,6E,8E)-N-(benzyl)-3,7dimethyl-9-(2,6,6-trimethylcyclohex-1-
en-1yl)nona-2,4,6,8-tetraenamide (1f)
ATRA was reacted with benzylamine (53.8 mg, 0.49 mmol), HATU and
DIPEA according to general procedure A. The crude was purified by FC
using n-Hex/EtOAc (9:1). The product was then submitted to an additional
preparative RP-HPLC purification: solvent A: H2O, solvent B: CH3CN,
isocratic 93% B to obtain the final product 1f as a yellow solid in 89% yield
(117.0 mg). Rf = 0.35 (n-Hex/EtOAc 8:2). Analytical RP-HPLC: gradient
from 93% to 100% of B in 20 min, rt = 9.86 min. MS (ESI, m/z): C27H36NO
[M+H]+ calc. 390.27, found 390.3, C27H35NNaO [M+Na]+ calc. 412.27,
found 412.2. 1H NMR (CDCl3, 400 MHz) δ: 7.37 – 7.29 (m, 5H), 6.95 (dd,
J = 15.0, 11.4 Hz, 1H), 6.31 – 6.10 (m, 4H), 5.92 (t, J = 5.6 Hz, 1H), 5.71
(s, 1H), 4.50 (d, J = 5.6 Hz, 2H), 2.41 (s, 3H), 2.09 – 1.99 (m, 5H), 1.74 (s,
3H), 1.69 – 1.60 (m, 2H), 1.53 – 1.47 (m, 2H), 1.06 (s, 6H). 13C NMR
(CDCl3, 101 MHz) δ: 166.9, 149.0, 138.8, 138.6, 137.8, 137.4, 135.6,
129.84, 129.81, 128.7, 128.2, 127.8, 127.4, 121.2, 43.4, 39.6, 34.3, 33.1,
29.0, 21.8, 19.3, 13.7, 12.9.
MHz), δ: 165.5, 151.7, 139.7, 138.9, 137.7, 137.2, 135.0, 131.3 (q, JC-F
=
29.2Hz), 131.0, 130.1, 129.4, 128.8, 123.9 (q, JC-F = 273.3 Hz), 122.8,
120.56, 120.49, 120.45, 39.6, 34.3, 33.1, 29.0, 21.8, 19.2, 13.8, 12.9.
(2E,4E,6E,8E)-N-(3-tert-Butyl-phenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (1c)
ATRA was reacted with 3-(t-butyl)aniline (74.0 mg, 0.49 mmol), HATU and
DIPEA according to general procedure A. The crude was purified by FC
using n-Hex/EtOAc (9:1) to obtain 1c in 63% yield as a yellow solid (90.0
mg). Rf = 0.4 (n-Hex/EtOAc 9:1). Analytical RP-HPLC: solvent A: H2O,
solvent B: CH3CN, gradient from 93% to 100% of B in 15 min, then 100%
of B, rt = 14.98 min. MS (ESI, m/z): C30H42NO [M+H]+ calc. 432.32, found
432.3. 1H NMR (CDCl3, 400 MHz) δ: 7.92 (s, 1H), 7.64 (br, 1H), 7.50 (d, J
= 7.2 Hz, 1H), 7.25 (t, J = 7.2 Hz, 1H), 7.14 (d, J = 7.2 Hz, 1H), 6.99 (dd, J
= 15.0, 11.4 Hz, 1H), 6.33 – 6.15 (m, 4H), 5.93 (s, 1H), 2.46 (s, 3H), 2.09–
2.03 (m, 5H), 1.77 (s, 3H), 1.70-1.64 (m, 2H), 1.53-1.50 (m, 2H), 1.33 (s,
9H), 1.08 (s, 6H).13C NMR (CDCl3, 101 MHz) δ: 165.5, 152.1, 150.1, 139.0,
138.2, 137.8, 137.4, 135.6, 130.2, 129.9, 129.7, 128.6, 128.4 , 121.9,
121.1, 117.2, 117.0, 39.6, 34.7, 34.3, 33.1, 31.3, 29.0, 21.8, 19.3, 13.8,
12.9.
(2E,4E,6E,8E)-N-(3-chlorophenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (1g)
ATRA was reacted with 3-chloroaniline (68.0 mg, 0.49 mmol), HATU and
DIPEA according to general procedure A. The crude was purified by FC
using n-Hex/EtOAc (9:1) and then submitted to an additional preparative
RP-HPLC purification: solvent A: H2O, solvent B: CH3CN from 96% to
100% of B in 12 min. The final product 1g was obtained as a yellow solid
in 70% yield (130.0 mg). Rf = 0.6 (n-Hex/EtOAc 8:2). Analytical RP-HPLC:
gradient from 96% to 100% of B in 12 min, rt = 10.51 min. MS (ESI, m/z):
C26H33ClNO calc. [M+H]+ 410.22, , found 410.2. 1H NMR (CDCl3, 400 MHz)
δ: 7.70 (br, 1H), 7.42 (br, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz,
1H), 7.06 – 6.94 (m, 2H), 6.31 – 6.10 (m, 4H), 5.78 (s, 1H), 2.41 (s, 3H),
2.05 – 1.97 (m, 5H), 1.71 (s, 3H), 1.65 – 1.57 (m, 2H), 1.48-1.45 (m, 2H),
1.02 (s, 6H). 13C NMR (CDCl3, 101 MHz) δ: 165.6, 151.4, 139.6, 139.6,
137.8, 137.3, 135.2, 134.6, 130.8, 130.1, 129.9, 129.6, 128.7, 124.0, 120.9,
119.9, 117.9, 39.7, 34.3, 33.2, 29.0, 21.8, 19.3, 13.8, 13.0.
(2E,4E,6E,8E)-N-(diphenyl)-3,7dimethyl-9-(2,6,6-trimethylcyclohex-1-
en-1yl)nona-2,4,6,8-tetraenamide (1d)
ATRA was reacted with 4-Aminobiphenyl (83.7 mg, 0.49 mmol), HATU and
DIPEA according to general procedure A. The crude was purified by FC
using n-Hex/EtOAc (9:1) to obtain 1d in 60% yield as a white solid (89.0
mg). Rf = 0.5 (n-Hex/EtOAc 8:2). Analytical RP-HPLC: solvent A: H2O,
solvent B: CH3CN, gradient from 93% to 100% of B in 15 min, then 100%
of B, rt = 14.15 min. MS (ESI, m/z): C32H38NO [M+H]+ calc. 452.29, found
452.3, C32H37NNaO [M+Na]+ calc. 474.29, found 474.2. 1H NMR
(2E,4E,6E,8E)-N-(2,4-dichlorobenzyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (1h)
ATRA was reacted with 2,4-chlorobenzylamine (87.0 mg, 0.49 mmol),
HATU and DIPEA according to general procedure A. The crude was
8
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10.1002/cmdc.202000143
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FULL PAPER
purified by FC using n-Hex/EtOAc (9:1) to obtain the final product 1h as a
yellow solid in 88% yield (135.0 mg). Rf = 0.4 (n-Hex/EtOAc 9:1). Analytical
RP-HPLC: solvent A: H2O, solvent B: CH3CN, gradient from 96% to 100%
of B in 12 min, rt = 10.70 min. MS (ESI, m/z): C27H34Cl2NO [M+H]+ calc.
ATRA was reacted with 4-hydroxybenzylamine (61.0 mg, 0.49 mmol),
HATU and 2,4,6-collidine according to general procedure A. The crude
was purified by FC using n-Hex/EtOAc (7:3). The product was then
submitted to an additional preparative RP-HPLC purification using solvent
A: H2O, solvent B: CH3OH, gradient from 85% to 95% of B in 20 min to
obtain the final product 2a as a yellow solid in 60% yield (80.0 mg). Rf =
0.5 (n-Hex/EtOAc 5:5). Analytical RP HPLC: 85% to 95% of solvent B in
20 min, rt = 13.48. MS (ESI, m/z): C27H36NO2 [M+H]+ calc. 406.27, found
406.3. 1H NMR (CDCl3, 400 MHz) δ: 7.16 (d, J = 8.5 Hz, 2H), 6.93 (dd, J
= 15.0, 11.3 Hz, 1H), 6.79 (d, J = 8.5 Hz, 2H), 6.24 – 6.10 (m, 4H), 5.70 (t,
J = 5.6 Hz, 1H), 5.66 (s, 1H), 4.41 (d, J = 5.6 Hz, 2H), 2.38 (s, 3H), 2.03 –
1.99 (m, 5H), 1.64 (s, 3H), 1.64 - 1.58 (m, 2H), 1.48 – 1.45 (m, 2H), 1.02
(s, 6H). 13C NMR (CDCl3, 101 MHz) δ: 167.4, 155.8, 149.3, 139.0, 137.7,
137.3, 135.4, 130.1, 129.9, 129.6, 129.5, 129.2, 128.4, 121.0, 115.7, 43.2,
39.6, 34.3, 33.1, 29.0, 21.8, 19.2, 13.8, 12.9.
1
458.19, found 458.3. H NMR (CDCl3, 400 MHz) δ: 7.34 (d, J = 2.0 Hz,
1H), 7.30 (d, J = 8.0 Hz, 1H), 7.17 (dd, J = 8.0, 2.0 Hz, 1H), 6.90 (dd, J =
15.0, 11.4 Hz, 1H ), 6.29 – 6.09 (m, 5H), 5.70 (s, 1H), 4.48 (d, J = 6.1 Hz,
2H), 2.33 (s, 3H), 2.03 – 1.98 (m, 5H), 1.71 (s, 3H), 1.64 – 1.58 (m, 2H),
1.47 – 1.44 (m, 2H), 1.02 (s, 6H). 13C NMR (CDCl3, 101 MHz) δ: 167.1,
149.5, 139.0, 137.7, 137.3, 135.4, 134.7, 134.1, 133.8, 130.8, 130.1, 129.9,
129.5, 129.2, 128.4, 127.3, 120.7, 40.7, 39.6, 34.3, 33.1, 29.0, 21.8, 19.2,
13.7, 12.9.
(2E,4E,6E,8E)-N-(4-nitrophenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (1i)
(2E,4E,6E,8E)-N-(3-fluoro-4-hydroxy-phenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (2b)
ATRA was reacted with 4-nitroaniline (70.0 mg, 0.49 mmol), HATU and
DIPEA according to general procedure A. The crude was purified by FC
using n-Hex/EtOAc (9:1) to obtain the final product 1i as an orange solid
in 50% yield (70.0 mg). Rf = 0.5 (n-Hex/EtOAc 8:2). Analytical RP-HPLC:
solvent A: H2O, solvent B: CH3CN, gradient from 96% to 100% of B in 15
min, rt = 9.55 min. MS (ESI, m/z): C26H33N2O3 [M+H]+ calc.421.24, found
421.3. 1H NMR (CDCl3, 400 MHz) δ: 8.19 (d, J = 9.2 Hz, 2H), 7.75 (d, J =
9.2 Hz, 2H), 7.70 (s, 1H), 7.05 (dd, J = 15.0, 11.4 Hz, 1H ), 6.33 – 6.13 (m,
4H), 5.82 (s, 1H), 2.44 (s, 3H), 2.04 – 2.00 (m, 5H), 1.72 (s, 3H), 1.65 –
1.60 (m, 2H), 1.49 – 1.46 (m, 2H), 1.03 (s, 6H). 13C NMR (CDCl3, 101 MHz)
δ: 165.2, 153.2, 144.5, 143.1, 140.3, 137.7, 137.1, 134.7, 131.7, 130.2,
129.3, 129.1, 125.1, 119.9, 118.8, 39.6, 34.3, 33.1, 29.0, 21.8, 19.2, 13.9,
13.0.
ATRA was reacted with 4-amino-2-Fluorophenol (63.5 mg, 0.49 mmol),
HATU and 2,4,6-collidine according to general procedure A. The crude
was purified by FC using n-Hex/EtOAc (8:2). The product was then
submitted to an additional preparative RP-HPLC purification using: solvent
A: H2O, solvent B: CH3CN, gradient from 93% to 100% of B in 8 min, then
100% of B to obtain the final product 2b as a yellow solid in 60% yield (81.0
mg). Rf = 0.5 (n-Hex/EtOAc 7:3). Analytical RP-HPLC: 93% to 100% of B
in 8 min, then 100% of B, rt = 7.36 min. MS (ESI, m/z): C26H33FNO2 [M+H]+
calc. 410.24, found 410.3. 1H NMR (CD3OD, 400 MHz) δ: 7.53 (dd, J =
13.1, 2.3 Hz, 1H), 7.16 – 6.98 (m, 2H), 6.92 – 6.81 (m, 1H), 6.26 (m, 4H),
5.96 (s, 1H), 2.39 (s, 3H), 2.07-2.02 (m, 5H), 1.73 (s, 3H), 1.57 – 1.44 (m,
2H), 1.52 – 1.49 (m, 2H), 1.05 (s, 6H). 19F NMR (CD3OD, 376 MHz) δ: -
(2E,4E,6E,8E)-N-(2-methoxyphenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (1j)
137.84 (m, 1F). 13C NMR (CD3OD, 101 MHz) δ: 166.1, 150.9 (d, JC-F
=
240.0 Hz), 149.6, 141.0 (d, JC-F = 15.9 Hz), 138.5, 137.7, 137.6, 135.6,
131.2 (d, JC-F = 7.9 Hz), 129.8, 129.7, 129.2, 127.8, 121.4, 117.1, (d, JC-F
= 3.6 Hz), 115.8 (d, JC-F = 3.2 Hz), 108.2 (d, JC-F = 23.70 ), 39.4, 33.8, 32.6,
28.0, 20.6, 18.9, 12.5, 11.0.
ATRA was reacted with o-Anisidine (62.5 mg, 0.49 mmol), HATU and
DIPEA according to general procedure A. The crude was purified by FC
using n-Hex/EtOAc (9:1) to obtain the final product 1j as an orange solid
in 85% yield (113.0 mg). Rf = 0.6 (n-Hex/EtOAc 8:2). Analytical RP-HPLC:
solvent A: H2O, solvent B: CH3CN, gradient: from 96% to 100% of B in 15
min, rt = 10.48 min. MS (ESI, m/z): C27H36NO2 [M+H]+ calc.406.27, found
(2E,4E,6E,8E)-N-(2-methyl-4-hydroxy-phenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (2c)
1
406.1, H NMR (CDCl3, 400 MHz) δ: 8.49 (br, 1H), 7.85 (br, 1H), 7.06 –
6.95 (m, 3H), 6.88 (dd, J = 8.0, 1.3 Hz, 1H), 6.34 – 6.14 (m, 4H), 5.88 (s,
1H), 3.70 (s, 3H), 2.44 (s, 3H), 2.06 – 2.03 (m, 5H), 1.75 (s, 1H), 1.68 –
1.60 (m, 2H), 1.52 – 1.47 (m, 2H), 1.06 (s, 6H). 13C NMR (CDCl3, 101 MHz)
δ: 165.0, 150.2, 147.8, 139.1, 137.8, 137.4, 135.5, 130.2, 129.9, 129.6,
128.4, 128.1, 123.4, 121.9, 121.1, 119.6, 109.8, 55.7, 39.6, 34.3, 33.1,
29.1, 21.8, 19.2, 13.6, 12.9.
ATRA was reacted with 4-amino-3-methylphenol (61.5 mg, 0.49 mmol),
HATU and 2,4,6-collidine according to general procedure A. The crude
was purified by FC using n-Hex/EtOAc (9:1) to obtain the final product 2c
as a yellow solid in 60% yield (80.00 mg). Rf = 0.5 (n-Hex/EtOAc 8:2).
Analytical RP-HPLC: solvent A: H2O, solvent B: CH3CN, gradient from
93% to 100% of B in 8 min, then 100% of B, rt = 7.08 min. MS (ESI, m/z):
C27H36NO2 [M+H]+ calc. 406.27, found 406.2. 1H NMR ((CD3)2SO, 400
MHz) δ: 9.18 (s, 1H), 9.13 (s, 1H), 7.12 (d, J = 8.5 Hz, 1H), 6.95 (dd, J =
15.0, 11.5, 1H), 6.60 (d, J = 2.5 Hz, 1H), 6.53-6.56 (dd, J = 8.5, 2.5 Hz,
1H), 6.15-6.38 (m, 4H), 6.08 (s, 1H), 2.32 (s, 3H), 2.10 (s, 3H), 2.03-1.98
(m, 5H), 1.70 (s, 3H), 1.60 -1.56 (m, 2H), 1.46-1.44 (m,2H), 1.03 (s, 6H).
13C NMR ((CD3)2SO, 400 MHz) δ: 165.2, 155.2, 147.6, 138.3, 137.8, 137.5,
136.6, 134.1, 130.6, 129.8, 128.3, 127.8, 127.1, 123.4, 121.3, 117.0, 112.9,
49.06, 34.3, 33.1, 29.3, 22.0, 19.2, 18.6, 13.7, 13.1.
(2E,4E,6E,8E)-N-(propargyl)-3,7dimethyl-9-(2,6,6-trimethylcyclohex-
1-en-1yl)nona-2,4,6,8-tetraenamide (1k)
ATRA was reacted with propargylamine (0.03 mL, 0.49 mmol), HATU and
DIPEA according to general procedure A. The crude was purified by FC
using n-Hex/EtOAc (8:2) to obtain 1k as a yellow solid in 90% yield (100.0
mg). Rf = 0.5 (n-Hex/EtOAc 7:3). Analytical RP-HPLC: solvent A: H2O,
solvent B: CH3CN, 85% of B isocratic, rt = 12.09. MS (ESI, m/z): C23H32NO
[M+H]+ calc.338.24, found 338.2. 1H NMR (CDCl3, 400 MHz) δ: 6.92 (dd,
J = 15.0, 11.5 Hz, 1H), 6.26 – 6.09 (m, 4H), 6.04 (br, 1H), 5.68 (s, 1H),
4.08 (dd, J = 4.9, 2.2 Hz, 2H), 2.35 (s, 3H), 2.21 (br, 1H), 1.99 (m, 5H),
1.69 (s, 3H), 1.61 – 1.58 (m, 2H), 1.46-1.43 (m, 2H), 1.01 (s, 6H). 13C NMR
(CDCl3, 101 MHz) δ: 166.7, 149.6, 138.9, 137.7, 137.3, 135.4, 130.1,
129.8, 129.5, 128.3, 120.4, 79.9, 71.3, 39.6, 34.2, 33.1, 29.1, 28.9, 21.7,
19.2, 13.6, 12.8.
(2E,4E,6E,8E)-N-(3-chloro-4-hydroxy-phenyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (2d)
ATRA was reacted with 4-amino-2-chlorophenol (72.0 mg, 0.495), HATU
and 2,4,6-collidine according to general procedure A. The crude was
purified by FC using n-Hex/EtOAc (8:2). The product was then submitted
to a further preparative RP-HPLC purification: solvent A: H2O and solvent
B: CH3CN, gradient from 93% to 100% of B in 8 min to obtain the final
product 2d as a yellow solid in 62% yield (88.0 mg). Rf = 0.5 (n-Hex/EtOAc
7:3). Analytical RP-HPLC: 93% to 100% of B in 8 min then 100% of B, rt =
8.31 min. MS (ESI, m/z): C26H33ClNO2 calc. [M+H]+ 426.21, found 426.2.
1H NMR (CD3OD, 400 MHz) δ: 7.57 (d, J = 2.5 Hz, 1H), 7.17 (dd, J = 8.8,
2.5 Hz, 1H), 6.95 (dd, J = 15.0, 11.3 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.30
(2E,4E,6E,8E)-N-(4-hydroxybenzyl)-3,7dimethyl-9-(2,6,6-
trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (2a)
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000143
ChemMedChem
FULL PAPER
– 6.01 (m, 4H), 5.85 (s, 1H), 2.27 (s, 2H), 1.97 – 1.88 (m, 5H), 1.61 (s, 3H),
1.58 – 1.51 (m, 2H), 1.43 – 1.37 (m, 2H), 0.94 (s, 6H). 13C NMR (CD3OD,
101 MHz) δ: 166.2, 149.6, 149.4, 138.5, 137.7, 137.6, 135.6, 131.6, 129.8,
129.6, 129.2, 127.8, 121.5, 121.4, 120.0, 119.6, 116.0, 39.4, 33.8, 32.6,
28.0, 20.5, 18.9, 12.5, 11.4.
4-bromo-1-butanol (300.0 mg, 1.96 mmol, 1.0 equiv) was dissolved in DMF
(2 mL) and reacted overnight with NaN3 (380.0 mg, 5.88 mmol, 3.0 equiv)
heating at 70 °C. The reaction mixture was diluted with 5 mL of H2O and
the azide was extracted with Et2O (3x30 mL). The organic phase was
washed with 20 mL of brine and dried over Na2SO4. The volume of the
solvent was reduced using a flow of nitrogen and the residue was directly
reacted with tosyl chloride (747.0 mg, 3.92 mmol, 2.0 equiv) and Et3N (0.55
mL, 3.92 mmol, 2.0 equiv) in 5 mL of DCM overnight. The mixture was
diluted with 5 mL of H2O and the product extracted with DCM (3x30 mL).
The organic phase was washed with brine (10 mL), dried over Na2SO4 and
concentrated under vacuum. The crude was purified by FC using n-
Hex/EtOAc (9:1) to obtain compound 4 as a clear oil in 50% yield (260.0
mg). Rf = 0.4 (n-Hex/EtOAc 8:2). Analytical RP solvent A = H2O, solvent B
= CH3CN; 0-3 min 5% B, gradient from 5% to 90% of B until 20 min, 20-25
min 90% B, rt = 22.06 min. MS (ESI, m/z): C11H16N3O3S [M+H]+ calc.
270.08, found 270.1, C11H16N1O3S [M+H-N2]+ calc. 242.09, found 242.1.
1H NMR (CDCl3, 400 MHz) δ: 7.78 (d, J = 8.0 MHz, 2 H), 7.35 (d, J = 8.0
Hz, 2 H), 4.05 (t, J = 6.0 Hz, 2 H), 3.25 (t, J = 6.8 Hz, 2H), 2.44 (s, 3H),
1.76-1.58 (m, 4H). Analytical and spectroscopic data were consistent with
those reported in the literature.45
General procedure for the synthesis of compounds 3a and 3b
(General procedure B)
Benzylazide or n-butylazide was added to a solution of 1k (1 equiv) in a
0.08 M solution of t-BuOH/H2O (2:1). Sodium ascorbate (0.3 equiv) and
CuSO4 (0.1 equiv) were added to the solution and the reaction was stirred
at room temperature. After complete consumption of the starting material,
the reaction was quenched with saturated NH4Cl and the mixture was
extracted with DCM or EtOAc (3x30mL). The organic phase was dried over
Na2SO4, the solvent was removed under reduced pressure at low
temperature and the crude was purified by FC using the indicate solvent.
(2E,4E,6E,8E)-N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-3,7-dimethyl-
9-(2,6,6-trimethylcyclohex-1-en-1yl)nona-2,4,6,8-tetraenamide (3a)
Radiosynthesis of [18F]3b
NaN3 (152.0 mg, 2.34 mmol, 2 equiv) was added to a solution of
benzylbromide (0.14 mL, 1.2 mmol, 1 equiv) in THF (2.5 mL) and the
reaction was stirred at 80 oC overnight. 10 mL of H2O were added to the
reaction and the mixture was extracted with Et2O (3x30 mL), washed with
brine (1x10 mL), dried over Na2SO4 and concentrated under vacuum to
obtain benzylazide as a pale oil. This was directly submitted to the next
step without further purification. Benzylazide (34.0 mg, 0.25 mmol) was
reacted with 1k (86.2 mg, 0.25 mmol) sodium ascorbate (10.0 mg, 0.05
mmol) and CuSO4 (1.60 mg, 0.01 mmol) according to general procedure
B (overnight stirring). The crude was purified by FC (DCM/CH3OH 9.8:0.2)
to obtain compound 3a as a yellow powder in 80% yield (96.0 mg). Rf =
0.2 (DCM/CH3OH 9.8:0.2). Analytical RP HPLC: solvent A: H2O, solvent
B: CH3CN, gradient from 85% to 100% of solvent B in 12 min, rt = 9.78
min. MS (ESI, m/z): C30H39N4O [M+H]+ calc. 471.30, found 471.3. 1H NMR
(CDCl3, 400 MHz) δ: 7.43 (s, 1H), 7 28 – 7.26 (m, 3H), 7.19 – 7.16 (m, 2H),
6.83 (dd, J = 15, 11.5 Hz, 1H), 6.61 (t, J = 5.5 Hz, 1H), 6.19 – 6.02 (m,4H),
5.63 (s, 1H), 5.40 (s, 2H), 4.44 (d, J = 5.5 Hz, 2H), 2.26 (s, 3H), 1.95 – 1.90
(m, 5H), 1.63 (s, 3H), 1.55 – 1.53 (m, 2H), 1.40 – 1.37 (m, 2H), 0.95 (s,
6H). 13C NMR (CDCl3), 400 MHz δ: 167.1, 149.0, 145.4, 138.7, 137.7,
137.3, 135.5, 134.5, 129.8, 129.6, 129.1, 128.8, 128.2, 128.1, 122.3, 121.1,
54.2, 39.6, 34.6, 34.2, 33.1, 29.0, 21.7, 19.2, 13.6, 12.9.
[
18F]fluoride was produced by the 18O(p,n)18F nuclear reaction on an IBA
Cyclone 18/18 cyclotron (IBA, Louvain-la-Neuve, Belgium) and obtained
as aqueous [18F]F-. 10 GBq of [18F]F- was trapped on an ion-exchange
cartridge (PS-HCO3, Chromafix, ABX Radeberg, Germany) and eluted into
a screw cap reaction vessel (vial 1) using 1 mL of a 10 mL solution
containing 20.0 mg of K2CO3 and 130.0 mg of K222 dissolved in a mixture
of CH3CN and water (9:1). Solvent was evaporated under vacuum using a
Helium stream (50 mL/min) and heating the vial at 90 °C. Next, the
temperature was reduced to 60 °C and 500 µL of dry CH3CN (x2) were
added. The temperature was increased again to 90 °C and solvent was
evaporated by azeotropic drying. After complete evaporation of the solvent,
the temperature was increased to 110 °C for 1 min. Subsequently, vial 1
was cooled to room temperature and precursor 4 (6.0 mg, 22.3 µmol) –
previously dissolved in 200 µL of DMF – was added. The reaction was
stirred at 90 °C for 10 min resulting in intermediate [18F]5 which was
reacted with the alkyne precursor 1k either after purification via distillation
(Procedure 1) or directly in a one-pot strategy (Procedure 2). Procedure
1: The temperature of vial 1 was increased to 140 °C, while vial 2 was
cooled to -20 °C. [18F]5 was distilled under a stream of Helium (10 mL/min)
and 5 GBq of [18F]5 was collected in vial 2 containing the alkyne precursor
1k (5.00 mg, 14 µmol) dissolved in 100 µL DMF. A solution of CuSO4 (10
mg, 62 µmol) in water (50 µL) and a solution of sodium ascorbate (30 mg,
180 µmol) in water (50 µL) were mixed and diluted with 100 µL of DMF.
When the solution turned from black to yellow this was added to the
reaction mixture in vial 2. The suspension was stirred for 15 min at 35 °C
and diluted with 1.5 mL of semi-prep HPLC eluent. The mixture was filtered
on hydrophilic PTFE 0.45 µm filter and injected into the semi-prep RP-
HPLC column (Phenomenex Luna C18, 5 µm, 250 mm x 10 mm, 100 Å;
flow rate: 4 mL/min) using 15% H2O/ 85% CH3CN. The fraction
corresponding to the desired product (rt = 16.0 min) was collected from the
HPLC, diluted with at least 60 mL of water to have less than 10% CH3CN
and loaded onto a Sep-Pak t-C18 plus cartridge pre-activated using 10 mL
of EtOH and 10 mL of water. The tracer was trapped on the cartridge, the
Sep-Pak rinsed with water (20 mL), followed by elution of the product using
1.5 mL EtOH. Eventually, the solution of the tracer was diluted with 13.5
mL of a solution of 0.9% NaCl in water (saline) to reduce the content of
EtOH to 10%. Formulated product was obtained in 17% RCY with a
radiochemical purity of 95% at the end of synthesis. The radiochemical
purity of [18F]3b was determined using the following conditions: solvent A:
H2O, solvent B: CH3CN; isocratic 85% of B, HPLC Column Grace
AlltimaTM, C18 5 µm, 250 mm x 4.6 mm (Alltech, The Netherlands),
injected volume 10 µL, flow rate: 1mL/min. The retention time of [18F]3b
was 10.61 min. Procedure 2: Vial 1 was cooled to room temperature and
the alkyne precursor 1k (11.0 mg, 30.0 µmol) dissolved in 100 µL of DMF
was added to the mixture containing [18F]5. CuSO4 and sodium ascorbate
were added as reported in Procedure 1 and the suspension was stirred for
(2E,4E,6E,8E)-N-((1-(4-fluorobutyl-1H-1,2,3-triazol-4-yl)methyl)-3,7-
dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1yl)nona-2,4,6,8-
tetraenamide (3b)
NaN3 (98.00 mg, 1.5 mmol) was added to a solution of 1-Bromo-4-
fluorobutane (155.0 mg, 1.00 mmol) in DMF (0.5 mL) and the reaction was
stirred overnight at room temperature. Et2O (1 mL) and H2O (1 mL) were
added to the mixture and the organic layer was directly transferred to a
suspension of the alkyne 1k (168.0 mg, 0.5 mmol). The mixture was
reacted with CuSO4 (8.0 mg, 0.05 mmol) and sodium ascorbate (30.0 mg,
0.15 mmol) for 4 h according to general procedure B. The crude product
was purified by FC (n-Hex/EtOAc 5:5) to obtain 3b as a yellow solid in 90%
yield (200.0 mg). Rf = 0.5 (n-Hex/EtOAc 3:7). Analytical RP-HPLC: solvent
A: H2O, solvent B: CH3CN, 85% of solvent B isocratic, rt = 10.56 min. MS
(ESI, m/z): C27H40FN4O [M+H]+ calc. 455.31, found 455.3. 1H NMR (CDCl3,
400 MHz) δ: 7.59 (s, 1H), 7.14 (br, 1H), 6.86 (dd, J = 15.0, 12.0 Hz, 1H),
6.21 – 6.04 (m, 4H), 5.74 (s, 1H), 4.48 (d, J = 5.6 Hz, 2H), 4.40 (t, J = 5.6
Hz, 2H), 4.32 (t, J = 6.8 Hz, 2H), 2.30 (s, 3H), 2.00 – 1.93 (m, 7H), 1.68 –
1.62 (m, 5H), 1.56-1.55 (m, 2H), 1.42 – 1.41 (m, 2H), 0.97 (s, 6H). 19F-
NMR (CDCl3, 376 MHz) δ: -218.96 (m, 1F). 13C NMR (CDCl3, 400 MHz) δ:
167.3, 148.8, 145.2, 138.6, 137.7, 137.3, 135.6, 129.74, 129.71, 129.6,
128.1, 122.5, 121.3, 83.1 (d, JC-F = 165.5 Hz), 49.8, 39.6, 34.6, 34.2, 33.0,
28.9, 27.2 (d, JC-F = 20.2 Hz), 26.4 (d, JC-F = 4.0 Hz), 21.7, 19.2, 13.6, 12.8.
4-azidobutyl-4-methylbenzensulfonate (4)
10
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10.1002/cmdc.202000143
ChemMedChem
FULL PAPER
15 min at 35 °C. The mixture was purified and formulated using the
conditions reported in Procedure 1 providing [18F]3b with a radiochemical
purity  95% (5% RCY).
cells. Data were analysed and curves fitted using GraphPad Prism 5.04 to
determine the IC50 of each compound.
Differentiation of 3T3-L1 pre-adipocytes and treatment
Cell culture
In order to differentiate 3T3-L1 pre-adipocyte cultures into mature
adipocytes, cells were seeded into 6 well plates and grown to 100%
confluency whilst being maintained in growth DMEM media. Once
confluency was reached, cell cultures were left for an additional 2 days in
order to allow for growth arrest to occur. Growth media was removed and
replaced with a differentiation “cocktail” media named MDI (3-isobutyl-1-
methylxanthine (M), dexamethasone (D) and insulin (I)), while one well
containing normal growth media was used as control and was indicated as
non-differentiated (nd) cells. MDI media was comprised of DMEM
containing 4.5 g/L glucose, 10% (v/v) fetal bovine serum (#16170, Gibco
part of Invitrogen/Life Technologies), 1% (v/v) sodium pyruvate, 1 M
dexamethasone, 10 μg/mL human insulin solution and 0.5 mM 3-isobutyl-
1- methylxanthine (all from Sigma-Aldrich, UK). 3T3-L1 cells were either
untreated or stimulated with 1 M concentration of 4-HPR, ATRA, SKI II or
experimental compounds 1a-j, 2a-d and 3a-b and incubated for 48 h at
37 °C. MDI media was then replaced with insulin maintenance media
consisting of all the components of MDI media except for dexamethasone
and 3-isobutyl-1-methylxanthine and cells were once again either treated
or untreated and incubated at 37 °C. After 2 days media was replaced with
fresh insulin maintenance media and cells were for the last time either
treated or untreated and incubated for other 2 days. At this point 3T3-L1
cells were deemed to be fully differentiated into adipocytes, therefore they
were either stained with Oil Red O or overnight serum starved in order to
perform western blot.
The cell lines utilised throughout the present study were: the human breast
adenocarcinoma cell line MCF-7, the human embryonic kidney 293 cells
(HEK293) and the mouse embryonic fibroblast 3T3-L1 pre-adipocyte cell
line. All procedures with MCF-7, HEK293 and 3T3-L1 cell cultures, unless
otherwise stated, were carried out within a Cell Guard ES class II biological
safety cabinet (NuAire, MN, USA) where surfaces had been wiped down
with a 1% (w/v) Virkon disinfectant (DuPont, Stevenage, UK) and then
sprayed with a 70% (v/v) ethanol solution prior to commencing work. Cells
were maintained in media consisting of Dulbecco’s Modified Eagle’s
Medium (DMEM) containing 4.5 g/L glucose (PAA, Somerset, UK), 10%
(v/v) fetal bovine serum (#16170, Gibco (part of Invitrogen/Life
Technologies), Paisley, UK) and 1% (v/v) sodium pyruvate (Lonza, Basel,
Switzerland), termed growth media. During periods of proliferation, cells
had their media replaced every three days with fresh growth media which
was pre-warmed to 37 °C. Cell lines were incubated and maintained in a
HERAcell 150 incubator (Thermo Scientific, UK) with a humidified
environment at 37 °C with a 5% CO2 concentration. Before reaching
complete confluency, cultures were passaged in order to seed new culture
flasks/plates or freeze cells down for storage. To passage cells, growth
media was completely removed and cells were washed once with sterile
phosphate buffered saline (PBS) from Lonza, in order to remove any
remaining media and serum. PBS was removed and a Trypsin-EDTA (PAA,
Somerset, UK) solution was added to cell cultures which were
subsequently incubated for 5 min. Flasks were then gently agitated in order
to assist with detachment of cells. Once detached, cells were diluted with
fresh growth media in order to inactivate the Trypsin-EDTA solution and
seeded to new flasks or cell culture plates.
Oil Red O stain
After 8 days of 3T3-L1 differentiation, media was removed and cells fixed
in formalin (Sigma Aldrich, UK) for 30 min at room temperature. Formalin
was then removed and cells were stained with Oil Red O solution
previously prepared as follows: 60 mg of Oil Red O powder (Sigma Aldrich,
UK) dissolved in 20 mL of propan-2-ol, diluted (60% v/v) with distilled H2O
(dH2O) and filtered through 3MM chromatography paper (Whatman,
Maidstone, UK). Oil Red O staining was carried out for 1 h at room
temperature, after which the stain was removed and fixed cell cultures
were washed with dH2O. Stained cells were then covered with 2 mL dH2O
for visualisation and image acquisition. For quantification purposes, dH2O
was removed from cell cultures and plates were allowed to dry completely.
Oil Red O was eluted from cells by adding 1 mL of propan-2-ol and
incubating at room temperature for 10 min. 100 μL of sample were loaded
in triplicate into a 96 well plate using isopropanol as the blank and the
absorbance read at 520 nm on a Spectramax Plus384 spectrophotometer
(Molecular Devices, CA, USA). Background readings from isopropanol
only wells were subtracted from sample values. To determine the lipid
accumulation, absorbance readings for each drug treatment were
expressed as a percentage of the differentiated untreated control.
Drug preparation
Fenretinide (4-HPR, Cilag AG, Schaffhausen, Switzerland), All-Trans
Retinoic Acid (ATRA, fluorochem, UK), SKI II (Abcam, UK) and all
experimental compounds 1a-j, 2a-d and 3a-b were dissolved in DMSO at
a stock concentration of 10 mM and stored at -20 °C avoiding excessive
exposure to light. Stock solutions of compounds were then diluted to give
the desired final working concentration, as indicated for each individual
experiment. As a control, equivalent volumes of DMSO were always given
to separate cultures in order to determine any effect of the vehicle on the
experiment being undertaken.
MTT Assay
In order to perform the MTT assay, MCF-7 or HEK293 cells were seeded
in 24 well plates at a density of 50.000 cells per well, counted using a
hemocytometer. After one day, cells were stimulated overnight with 4-HPR,
ATRA or experimental compounds 1a-j, 2a-d and 3a-b at concentrations
of 1 nM, 10 nM, 100 nM, 1 μM and 10 μM. Three additional wells served
as controls: an untreated, a DMSO control and a well containing media
only to determine background. Following 24 h of incubation at 37 °C, 250
μL of media were removed from each well and 25 μL of 3-(4,5-
Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) solution (5
mg/mL) were added to the well yielding a final concentration of 0.5 mg/mL.
Plates were incubated at 37 °C for 3 h to allow formation of insoluble
formazan crystals. Subsequently, the media was removed and the crystals
dissolved in 320 μL of DMSO. Plates were wrapped in foil and shaken for
15 min to fully dissolve the coloured MTT formazan. Each well was
transferred to a 96 well plate in triplicate (100 μL per well) and the
absorbance was read at OD = 590 nm on a Spectramax Plus384
spectrophotometer (Molecular Devices, CA, USA). Background
absorbance was determined from the cell-free control well and subtracted
from each value. To determine the inhibition of cell growth for each drug
treatment, data were expressed as percentage of the untreated control
Western blotting
MCF-7 cells were grown to confluency in 6 well plates. MCF-7 cells were
either untreated or treated for 24 h with 1, 5 or 10 μM of 4-HPR or
compounds 2b or 3b. For 3T3-L1 cells, following 8 days differentiation and
treatment with or without 1 μM of compounds as reported above, cells were
serum starved overnight and proteins harvested on the next day. MCF-7
and 3T3-L1 cell cultures were scraped in radioimmunoprecipitation assay
(RIPA) lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA
(pH 8.0), 1 mM NaF, 0.1% (w/v) SDS, 1% (v/v) Triton X- 100, 1% (w/v)
sodium deoxycholate). Sodium orthovanadate (Na3VO4 at 2 mM) and
protease inhibitors (complete protease inhibitor cocktail from Roche, UK,
1 tablet dissolved in 2 mL H2O per 50 mL lysis buffer), were freshly added
to the RIPA lysis buffer prior to start the protein extraction procedure.
Samples were kept on ice at all times during the protein extraction process.
Protein samples were then centrifuged using a refrigerated bench top
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10.1002/cmdc.202000143
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FULL PAPER
micro-centrifuge at 10,000 rpm for 10 min at 4 °C to pellet insoluble
material, the supernatant was collected into new 1.5 mL centrifuge tube.
Protein concentrations of each sample were determined using the BCA
assay (Thermo Scientific, UK), following the manufacturer’s protocols.
Samples were run in duplicate and were compared to bovine serum
albumin (BSA) protein standards ranging from 0.125 mg/mL to 2 mg/mL
along with a RIPA blank set as 0 mg/mL. Protein lysates were combined
with a 5X SDS sample buffer (250 mM Tris-HCl (pH 6.8), 10% (w/v) SDS,
50% glycerol, 0.025% (w/v) bromophenol blue and 5% (v/v) 2-
mercaptoethanol) and RIPA buffer to give each sample the same final
protein concentration. Typically, protein concentrations of 0.5-2 mg/mL
were made for samples to be used for SDS-PAGE. SDS protein samples
were then denatured by heating samples at 95 °C for 5 min and were then
briefly centrifuged. SDS protein samples were stored at -20 °C until
required. Samples were separated on 4-12% NuPAGE Bis-Tris
(Invitrogen) gels by SDS-PAGE and transferred to 0.45 μm nitrocellulose
membranes (BioRad, UK) in order to perform immunoblotting. Membranes
were blocked for 1 h at room temperature with gentle agitation in TBS-T
containing 5% (w/v) skimmed milk powder, cut for the protein of interest
and then incubated overnight at 4 °C with primary antibodies: phospho-
p38 MAPK (Thr180/Tyr182), total p38, PARP, BiP/GRP78, phospho-
Akt/PKB (Ser 473) all from Cell Signalling, total Akt from Santa Cruz and
GAPDH (ThermoFisher) was used as protein loading control. Primary
antibodies were diluted 1:1000 in TBS-T with 5% (w/v) skimmed milk
powder and then washed with TBS-T for 1-1.5 h changing solution every
15-20 min. Membranes were then incubated with secondary antibodies
(goat anti-rabbit HRP-conjugated secondary antibody from Anaspec)
diluted 1:5000 in TBS-T and 5% (w/v) skimmed milk powder at room
temperature for 1 h. Membranes were washed once again in TBS-T for 1-
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h
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Acknowledgements
We thank the European Union’s Horizon 2020 research and
innovation programme under the Marie Sklodowska-Curie grant
agreement N° 675417 (PET3D project) for financial support of the
project and the studentship of I.P. We also thank the British Heart
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Keywords: Fenretinide • Drug discovery •
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Table of Contents
A library of fenretinide analogues has been designed and synthesised by using a single step amide coupling or a click-chemistry
approach. The compounds synthesised were then evaluated in vitro to assess their anti-cancer activity and their anti-obesity/diabetic
properties. Based on the positive in vitro results, the click-type analogue 3b was then selected as potential PET imaging tracer and the
radiosynthesis of [18F]3b was performed. The tracer was insufficiently stable for in vivo imaging, owing to radiolytic decomposition.
14
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