Micro-flow photosynthesis of new dienophiles for inverse-electron-demand Diels-Alder reactions. Potential applications for pretargeted in vivo PET imaging

Article abstract of DOI:10.1039/c6sc02933g

Pretargeted PET imaging has emerged as an effective two-step in vivo approach that combines the superior affinity and selectivity of antibodies with the rapid pharmacokinetics and favorable dosimetry of smaller molecules radiolabeled with short-lived radionuclides. This approach can be based on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between tetrazines and trans-cyclooctene (TCO) derivatives. We aimed to develop new [¹⁸F]TCO-dienophiles with high reactivity for IEDDA reactions, and favorable in vivo stability and pharmacokinetics. New dienophiles were synthesized using an innovative micro-flow photochemistry process, and their reaction kinetics with a tetrazine were determined. In vivo stability and biodistribution of the most promising ¹⁸F-radiolabeled-TCO-derivative ([¹⁸F]3) was investigated, and its potential for in vivo pretargeted PET imaging was assessed in tumor-bearing mice. We demonstrated that [¹⁸F]3 is a suitable dienophile for IEDDA reactions and for pretargeting applications.

Full text of DOI:10.1039/c6sc02933g

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Shahbazali, M. Ahamed, F. Cleeren, T. Noel, M. Koole, A. Verbruggen, V. Hessel and G. Bormans, Chem.
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Journal Name
ARTICLE
Micro-Flow Photosynthesis of New Dienophiles for Inverse-
Electron-Demand Diels-Alder Reactions. Potential applications for
pretargeted in vivo PET imaging.
Received 00th January 20xx,
Accepted 00th January 20xx
E. M.F. Billauda, E. Shahbazalib, M. Ahameda, F. Cleerena, T. Noëlb, M. Koolec, A. Verbruggena, V.
DOI: 10.1039/x0xx00000x
Hesselb and G. Bormansa
www.rsc.org/
Pretargeted PET imaging has emerged as an effective two-step in vivo approach that combines the superior affinity and
selectivity of antibodies with the rapid pharmacokinetics and favorable dosimetry of smaller molecules radiolabeled with
short-lived radionuclides. This approach can be based on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA)
reaction between tetrazines and trans-cyclooctene (TCO) derivatives. We aimed to develop new [18F]TCO-dienophiles with
high reactivity for IEDDA reactions, and favorable in vivo stability and pharmacokinetics. New dienophiles were
synthesized using an innovative micro-flow photochemistry process, and their reaction kinetics with a tetrazine were
determined. In vivo stability and biodistribution of the most promising 18F-radiolabeled-TCO-derivative ([18F]3) was
investigated, and its potential for in vivo pretargeted PET imaging was assessed in tumor-bearing mice. We demonstrated
that [18F]3 is a suitable dienophile for IEDDA reactions and for pretargeting applications.
patients. Moreover, it can be produced in large quantities
(>400 GBq/batch) with a cyclotron.4
Introduction
Combination of antibodies with fluorine-18 is challenging,
In the field of cancer research, radiolabeled monoclonal
antibodies directed against tumor-associated antigens have
emerged as promising vectors to visualize or treat cancer
lesions, due to their high affinity and specificity.1,2 However,
because of their high molecular weight (~150 kDa), antibodies
usually have long biological half-lives, thus requiring multiple
days to clear from blood and non-target tissues, and to reach
an optimal accumulation in tumor. Therefore, radionuclides
with relatively long physical half-lives must be employed to
radiolabel antibodies for direct in vivo imaging. For instance,
zirconium-89 (t1/2 = 78.4 h) can be used for positron emission
tomography (PET) imaging.3 This however leads to high
radiation doses in healthy tissues for patients.
On the other hand, fluorine-18 (t1/2 = 110 min) is the PET
radionuclide of choice given its favorable properties including
its decay mode (97% β+ emission), low positron energy
(634 keV maximum), and short β+ trajectory in tissues
(<2.3 mm). Its half-life is long enough to allow multistep
syntheses but is short enough to avoid extended irradiation of
due to the incompatibility between the long plasma half-life of
the antibody and the short physical half-life of the
radionuclide. Pretargeting addresses this issue in two steps.5
First, a non-radiolabeled antibody modified with a tag is
administered. Few days later, when the antibody has reached
a maximum uptake in the tumor and a sufficient clearance
from non-target tissues,
a relatively small radiolabeled
molecule is injected. The latter has the property to selectively
bind to the antibody via the tag, while the non-bound
radiotracer is rapidly cleared. Then, high contrast images can
be acquired few hours after the injection of the radiolabeled
molecule. The overall radiation dose for patients is thus
reduced, compared to the use of an antibody directly labeled
with 89Zr for instance.
Pretargeting approaches can be based on an inverse-
electron-demand Diels-Alder (IEDDA) click reaction between
1,2,4,5-tetrazines and trans-cyclooctene (TCO) derivatives.6
Indeed, this fast, selective, high-yield, biocompatible, and
bioorthogonal reaction has already proven to be suitable for
this kind of applications, both in vitro and in vivo,7 even using
18F-labeled tetrazines.8 However, to the best of our knowledge,
no in vivo pretargeting PET imaging results have been reported
on using a 18F-labeled small TCO compound, although this
approach may have specific advantages with regard to
pharmacokinetics and stability. Some radiolabeling procedures
were developed for TCO [18F]1 (Figure 1)9 but it was applied as
a prosthetic group for subsequent 18F-labeling of biologically-
active molecules.10
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4 waVsiew uArstieclde Onflioner
other hydroxyl group. Then, synthon
nucleophilic substitution with cis-cyclooctenol to
DOI: 10.1039/C6SC02y9i3e3lGd
derivative . The choice of TIPS as the hydroxyl protecting
group was important to obtain good yield. After
deprotection using tetrabutylammonium fluoride (TBAF), the
O
F
5
1
a
O
O
O
O
F
F
O
O
O
hydroxyl group of compound
a sulfonate intermediate.
6 was replaced by fluorine (9) via
H
H
H
2a
For the synthesis of 18, PEG synthon 13 was first prepared
in two steps starting from tetraethylene glycol: after
protection of one hydroxyl group using benzoyl chloride (BzCl),
the other hydroxyl was oxidized to an aldehyde in the
presence of Dess-Martin periodinane reagent. In parallel,
oxidation of 1,5-cyclooctadiene into diol 12 was carried out
using cetyltrimethylammonium permanganate. Then, PEG
synthon 13 and diol 12 were involved in an acetalization,
leading to dioxolane 14. The stereochemistry of 14 was
O
H
2b
3
H
O
F
O
O
O
H
O
H
Figure 1. Chemical structures of previously reported TCO derivative 19 determined according to Darko et al.12 After deprotection
and newly developed dienophiles 2a
,
2b, and
3
using LiOH, the hydroxyl group of compound 15 was replaced
by fluorine (18) via a sulfonate intermediate.
Wyffels et al. explored biodistribution of [18F]1 in healthy
Trans-for-cis isomerization of hydroxy-derivatives
mice, from 5 to 240 min p.i.9b Results demonstrated renal and sulfonate precursors for radiofluorination
16 17, and
hepatobiliary clearance of radioactivity, slow blood clearance, fluoro-derivatives 18 was performed using an innovative
6, 15,
7
,
8
,
,
9
,
as well as increasing bone uptake values (from 60 min p.i.). The micro-flow photochemistry process. Basic design of the setup
bone uptake is due to defluorination, as [18F]F- is known to was based on the work of Royzen et al.13 This group devised an
have a high affinity for bone, and is an indication of tracer apparatus where the reaction mixture, containing a cis-
instability. Therefore, we aimed to develop new TCO cyclooctene derivative and methyl benzoate (a singlet
derivatives, with improved in vivo stability, favorable sensitizer), is photoirradiated at 254 nm, and continuously
pharmacokinetics, and high reactivity for IEDDA reactions.
circulated through a bed of AgNO3-impregnated silica gel. The
We designed compounds 2a 2b and
,
3
derivatised with trans-cyclooctene derivative forms a complex with Ag+ and is
polyethylene glycol (PEG) chains (Figure 1) with the aim to selectively retained on the bed, while the corresponding cis-
increase their hydrophilicity and their stability towards cyclooctene binds weakly to Ag+ and elutes back to the
enzymatic degradation.11,7l We developed a conformationally- reaction flask, where it is photoirradiated again. For trans-for-
strained dioxolane-fused trans-cyclooctene (3), encouraged by cis isomerization of our compounds, we used a micro-flow
the results reported by Darko et al.12 Indeed, it was setup, since the short characteristic inner diameter of the
demonstrated that this strained trans-cyclooctenes react microreactor allows a high overall absorption even at larger
faster with 3,6-diphenyl-s-tetrazine than non strained analogs, concentration which increases the gross conversion rate
and display excellent chemical stability in aqueous solutions largely and reduces the reaction time from hours to minutes
and plasma. Moreover, dioxolane-fused trans-cyclooctenes for typical photo-flow processes.14 In addition, process scale-
can be prepared easily through diastereoselective synthesis.
up is facilitated by the numbering-up of several flow
Herein we report (i) the syntheses of new TCO derivatives, microcapillaries with almost identical performance.15 Two
via a trans-for-cis photoisomerization step using an innovative microreactors coiled around the UV lamp were used in
micro-flow process; (ii) reaction kinetics of these new parallel, and flow was adjusted to result in 2 to 3 min
dienophiles with a tetrazine, as well as their stability in irradiation time (Scheme 2, Supporting Information). Although
aqueous solution; (iii) 18F-radiolabeling of the most promising Royzen et al. used
8 lamps of 35 W (light intensity:
TCO derivative 3
; (iv) in vitro stability of [18F]3 and in vivo 12800 µW.cm-2),13 a single UV lamp of 10 W (light intensity:
biodistribution studies after injection of [18F]3; (v) proof of 21-24 µW.cm-2) provided sufficient power for the
principle in vitro and in vivo pretargeting experiments using isomerization reaction in our micro-flow setup. In-flow
[18F]3
.
separation process based on Ag+ complexation was also
optimized, by using several packed beds made of AgNO3-
impregnated silica gel and glass beads. During experiment,
flow was switched after 30-90 min from one packed bed to the
next, in order to avoid saturation. After experiment, a NH4OH
solution was used to liberate the trans-compound. With this
optimized method using microreactors, 85% conversion can be
achieved for trans-for-cis isomerization of cyclooctenol in 3 h
(Supporting Information), compared to a reported 73% in 8 h
or 70% in 3 h for non-microfluidic productions.13,16 For the new
Results and discussion
Syntheses New dienophiles 2a, 2b and 3 were prepared as
shown in Scheme 1. First, the corresponding cis-derivatives
and 18 were synthesized, in 6 and 7 steps respectively.
9
PEG synthon
4 was obtained in two steps starting from
tetraethylene glycol: protection of one hydroxyl group using
triisopropylsilyl (TIPS) chloride followed by mesylation of the
2 | J. Name., 2012, 00, 1-3
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functionalized cyclooctene derivatives, photoisomerization For sulfonate precursors, only mesylate 11b andVi2ew0AcrtoiculelOdnlbinee
yields reached 76% for a 6 h experiment, with fluoro- isolated after isomerization, but 11b waDsOqI:u1i0te.10u3n9s/Cta6bSCle02(9d3a3tGa
compound
3
.
not shown).
O
MsO
OTIPS
O
O
OH
3
TBAF, THF
RT, 2 h
4
OTIPS
4
OH
4
4
4
NaH, THF
45 °C, 17 h
5
64%
6
89%
96%
TsCl or MsCl, NEt3,
DMAP, CH2Cl2
from 7:
O
6
O
O
TBAF, THF
OH
OR1
4
F
4
4
RT, 1-2 h
80 °C, 3 h
7: R1 = OTs
8: R1 = OMs
96%
94%
9
h, Et2O/hexane
methyl benzoate
O
O
O
R2
R2
R2
H
H
H
+
H
4
microfluidic
RT, 6-10 h
6: R2 = OH
8: R2 = OMs
9: R2 = F
10a: R2 = OH
11a: R2 = OMs
2a: R2 = F
10b: R2 = OH
11b: R2 = OMs
2b: R2 = F
9%
n.d.
20%
6%
n.d.
5%
H
OBz
O
OBz
OH
3
O
O
O
O
OH
OH
O
O
3
O
LiOH, EtOH
RT, 2 h
13
3
O
O
p-toluenesulfonic acid
THF, 50 °C, 24 h
12
14
60%
15
92%
TsCl or MsCl, NEt3,
DMAP, CH2Cl2
OH
OR1
F
3
O
O
from 16:
TBAF, THF
O
O
O
3
3
O
O
O
RT, 1-3 h
80 °C, 2 h
16: R1 = OTs
17: R1 = OMs
83%
99%
15
18
81%
h, Et2O/hexane
or TBME
methyl benzoate
Trans-for-cis isomerisations:
H
O
R2
R2
O
O
O
micro-photoreactor:
capillary tubing (FEP), length: 1-5 m,
internal diameter: 0.5 mm
3
3
O
H
microfluidic
RT, 6-10 h
H
19: R2 = OH
20: R2 = OMs
3: R2 = F
15: R2 = OH
17: R2 = OMs
18: R2 = F
21%
44%
76%
lamp: UV low pressure lamp,
10 W, 254 nm
THF tetrahydrofuran, MsCl mesyl chloride, TsCl tosyl chloride, DMAP 4-(dimethylamino)pyridine, TBME tert-butyl methyl ether, n.d. not determined
Scheme 1. Syntheses of dienophiles 2a, 2b and 3
Reactivity and stability of new dienophiles Rate constants
for reaction between new dienophiles and 3,6-di(pyridin-2-yl)-
1,2,4,5-tetrazine in MeOH at 25°C were determined by UV-
spectrophotometry at 290 nm under pseudo-first order
conditions (Figure S2). Compounds 10a and 10b react with the
tetrazine with rate constants of 476 ± 33 M-1.s-1 and
1913 ± 196 M-1.s-1 respectively. Compared to trans-
cyclooctenol rate constants (392 ± 6 M-1.s-1 for major isomer
and 300 ± 22 M-1.s-1 for minor isomer), the presence of the
PEG chain on 10a and 10b does not reduce the reactivity.
Reaction rate of 19 with the tetrazine is also fast, with a rate
constant of 1620 ± 149 M-1.s-1.These rate constants are
comparable to the ones reported in literature for this type of
dienophiles.6a,12 While the new dienophiles are reactive
Scheme 2. Microfluidic setup optimized for trans-for-cis isomerization
of functionalized cyclooctene derivatives
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toward a tetrazine, they also display excellent stability. Indeed, Interestingly, brain uptake was observed (1.3 ± 0.2 to
View Article Online
DOI: 10.1039/C6SC02933G
fluoro-trans-derivatives 2a, 2b and 3 are stable in phosphate- 0.6 ± 0.0 SUVw from 2 to 60 min p.i.). Analysis of brain and
buffered saline (PBS, pH 7.4) at 37 °C for 19 h at least, as biofluids by HPLC after 15 min p.i. revealed 20.8 ± 1.1% of
determined by HPLC (Tables S3, S5, S7).
intact [18F]3 in brain, 5.9 ± 0.6% in plasma and 0.1 ± 0.0% in
Radiolabeling with fluorine-18 In view of the limited urine (Figure S6), indicating fast metabolism.
stability of precursors for radiofluorination 11a and 11b, we
In vitro pretargeting experiments using [18F]3 To check the
decided to continue the project with dioxolane-fused trans- usefulness of [18F]3 as a new dienophile for IEDDA reactions
cyclooctene compounds only. Semi-automated radiosynthesis and for pretargeted PET imaging, in vitro and in vivo
of [18F]3 was performed on
a home-made module. experiments were performed using a pseudo peptide with
Nucleophilic substitution on mesylate precursor 20 by reaction affinity for prostate-specific membrane antigen (PSMA)17
with dry K[18F]F,K222 complex was achieved in MeCN at 90 °C conjugated to 3-(4-(trifluoromethyl)phenyl)-6-phenyl-1,2,4,5-
for 15 min. During radiosynthesis of [18F]3, less than 1% of cis- tetrazine10f
(
21, Scheme 3, synthesis described in Supporting
compound [18F]18 was generated. After purification by HPLC, Information).
[18F]3 was obtained in 60 min, with 12% radiochemical yield
For in vitro experiments, prostate tumor slices (LNCaP and
(decay-corrected), a radiochemical purity >99% (Figure S4), PC-3 cells) were incubated with 21, washed, and [18F]3 was
and a specific activity of 70-188 GBq.µmol-1. added. Direct incubation with ”preclicked”-compound [18F]22
In vitro stability of [18/19F]3 In PBS (pH 7.4), [18F]3 was (Scheme 3, Figure S7) was also performed. To check the
rather stable, with 94% of intact compound after 2 h specificity of the approach, blocking experiments with the non-
incubation at 37 °C. In rat plasma, [18F]3 slowly isomerized into structural related inhibitor 2-(phosphonomethyl)pentane-1,5-
the corresponding cis-derivative [18F]18, with 52% and 34% of dioic acid18 and incubation with [18F]3 only were also carried
intact trans-compound after 1 h and 2 h incubation at 37 °C out. The fraction of bound activity was determined after
respectively (Figure S5). For pretargeted PET imaging, this slow autoradiography (Figures 3 and S9). Significant PSMA-specific
degradation is not an issue, as the IEDDA reaction takes place binding to LNCaP tumor slices (expressing PSMA receptors)
in a few seconds and plasma clearance is expected to be was observed in the pretargeting experiment, but it was lower
relatively fast (<30 min). Additional experiments were than the specific binding of ”preclicked”-compound [18F]22. In
performed to investigate the cause(s) of the isomerization,7b,12 PC-3 cells (negative control), no significant binding was
and the presence of
temperature were found to promote cis-for-trans
isomerization of (Table S8, Figure S3).
a thiol (2-mercaptoethanol) or observed.
O
3
Biodistribution of radioactivity after injection of [18F]3
Pharmacokinetic profile of [18F]3 was evaluated in vivo, in
healthy NMRI mice, from 2 to 60 min p.i. (Figure 2, Table S11).
Results demonstrate absence of in vivo defluorination, as no
significant bone uptake was observed at 60 min p.i.
(0.3 ± 0.0 SUVw). Radioactivity was cleared via urinary and
hepatobiliary systems.
HN
O
OH
N
N
O
O
N
H
N
HO
OH
N
N
N
H
21
H
O
O
CF3
H
18F
O
O
3
H
O
[
18F]3
H
18F
3
IEDDA reaction
in aqueous buffer/ethanol
O
O
O
HN
O
O
OH
O
O
N
H
HO
OH
N
N
N
N
H
[
18F]22
H
H
Figure 2. Biodistribution of radioactivity after injection of [18F]3 in
healthy NMRI mice in selected organs and fluids. Data are expressed as
standardized uptake value (SUVw).
O
O
CF3
Scheme 3. Reaction between dienophile [18F]3 and tetrazine 21
leads to [18F]22 (mixture of isomers) quantitatively in a few seconds.
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DOI: 10.1039/C6SC02933G
Figure 4. A. In vivo PET images 2 h after i.v. injection of
bioorthogonal [18F]3 (11 MBq), in a mouse bearing an LNCaP tumor
treated with tetrazine 21 (50 µg). B. Time-activity curves from 0 to
60 min after i.v. injection of [18F]3, in LNCaP tumor-bearing mice with
(N=3) or without (= control, N=3) intratumoral injection of 21
.
Figure 3. Percentage of binding of radioactivity to LNCaP tumor
slices after a pretargeting experiment with successive addition of 21
and [18F]3 or after incubation with [18F]22 was significantly higher than
in control experiments.
Proof of principle in vivo PET imaging On the basis of these
promising results, proof of principle PET imaging experiments
were conducted in LNCaP prostate tumor-bearing mice.
Compound 21 (50 µg) was administered intratumorally 10 min
before intravenous (i.v.) injection of [18F]3, following a similar
protocol reported by Emmetiere et al.,7f to avoid variability
due to tetrazine concentration in target tissues. Dynamic (0-
60 min p.i.) and static (120 min p.i.) microPET scans were
acquired. PET imaging with [18F]3 allowed visualization of the
tumor, as shown in Figure 4. Moreover, tumor uptake was
significantly higher than muscle uptake, as early as 30 min p.i.
and up to 2 h p.i. In order to ensure that the tumor
accumulation was due to the 18F-labeled conjugate formed by
the IEDDA reaction, control experiments were also performed
in mice that received only i.v. injection of [18F]3. Uptake of
radioactivity in the tumor was significantly lower than in
tetrazine-21-preinjected tumors, from 30 min to 2 h p.i.
(Figures 4 and S10). No significant difference was observed
between muscle uptake and tumor uptake in control
experiments. According to another PET imaging experiment
(Figures 5 and S11), the accumulation of radioactivity in
tetrazine-enriched tissues is not an effect due to the injection
of the tetrazine, as the injection of the same volume of vehicle
alone (saline with 10% dimethyl sulfoxide) does not lead to a
significant uptake of radioactivity. These proof of principle
experiments demonstrate the usefulness of [18F]3 for the in
vivo IEDDA reaction. In order to successfully apply this new
dienophile to pretargeted immunoPET, stability will be favored
over reactivity for the choice of the tetrazine, as a tetrazine-
derivatised antibody will be injected several days before the
radiolabeled dienophile. A tetrazine such as reported by
Selvaraj et al.,10f or by Karver et al.,7c possibly modified with a
PEG linker, might be a good choice.
Figure 5. A. In vivo PET images 60 min after i.v. injection of [18F]3
(11 MBq) in an healthy NMRI mouse which received an intramuscular
injection of tetrazine 21 (50 µg) in the muscle on the right shoulder
(arrows), and saline with 10% dimethyl sulfoxide in the muscle on the
left shoulder. B. Time-activity curves from 0 to 60 min after i.v.
injection of [18F]3, in healthy NMRI mice (N=3). The uptake of
radioactivity in the tetrazine-enriched tissue is significantly higher than
in the tissue which received only the vehicle, from 3 min to 47 min p.i.
of [18F]3
.
Conclusion
In summary, we developed three new dienophiles for
IEDDA reactions, and compound was selected for
pretargeting applications. Trans- has been prepared via
diastereoselective synthesis, and the trans-for-cis
3
3
isomerization step has been performed by micro-flow
photochemistry with 76% yield. The new microfluidic setup
reported here can be applied as continuous process which is
promising for process scale-up. 18F-Radiolabeling of
3 can be
carried out by nucleophilic substitution at high specific activity,
in 60 min, with 12% radiochemical yield, and >99%
radiochemical purity. In vivo, [18F]3 demonstrated a suitable
pharmacokinetic profile and no defluorination was observed.
Proof of principle PET imaging experiments with [18F]3, on a
prostate tumor model injected with a tetrazine-coupled PSMA
antagonist 10 min before radiotracer injection, allowed clear
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visualization of the tumor tissue, due to the 18F-labeled
conjugate formed by the IEDDA reaction. In conclusion, new
dienophile [18F]3 seems suitable for pretargeted PET imaging,
although further structural modification can still be done to
favor urinary excretion. In the future, [18F]3 will be
investigated for pretargeted immunoPET, by using a tetrazine-
derivatised antibody.
Sawada, W. W. Scholz and J. S. Lewis, J. Nucl. Med. 2016, 57
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The authors thank Julie Cornelis, Pieter Haspeslagh, Ivan
Sannen, Michiel Nuyts, Jana Hemelaers from the Laboratory of
Radiopharmacy, and Ann Van Santvoort, Tinne Buelens from
Nuclear Medicine and Molecular Imaging, for their assistance.
This research project received support from IWT Flanders (SBO
130065 MIRIAD) and the European Research Council
(Advanced Grant “Novel Process Windows – Boosted Micro
Process Technology” 267443).
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