Synthesis of a DOTA-C-glyco bifunctional chelating agent and preliminaryin vitroandin vivostudy of [68Ga]Ga-DOTA-C-glyco-RGD

Article abstract of DOI:10.1039/d0ra09274f

The design of bifunctional chelating agents (BFCA) allowing straightforward radiometal labelling of biomolecules is a current challenge. We report herein the development of a bifunctional chelating agent based on a DOTA chelator linked to aC-glycosyl compound, taking advantage of the robustness and hydrophilicity of this type of carbohydrate derivative. This new BFCA was coupled with success by CuAAC with c(RGDfK) for α_vβ₃integrin targeting. As attested byin vitroevaluation, the conjugate DOTA-C-glyco-c(RGDfC) demonstrated high affinity for α_vβ₃integrins (IC₅₀of 42 nM). [⁶⁸Ga]Ga-DOTA-C-glyco-c(RGDfK) was radiosynthesized straightforwardly and showed high hydrophilic property (log?D_7.4= ?3.71) andin vitrostability (>120 min). Preliminaryin vivoPET study of U87MG engrafted mice gave evidence of an interesting tumor-to-non-target area ratio. All these data indicate that [⁶⁸Ga]Ga-DOTA-C-glyco-c(RGDfK) allows monitoring of α_vβ₃expression and could thus be used for cancer diagnosis. The DOTA-C-glycoside BFCA reported here could also be used with various ligands and chelating other (radio)metals opening a broad scope of applications in imaging modalities and therapy.

Full text of DOI:10.1039/d0ra09274f

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PAPER
Synthesis of a DOTA-C-glyco bifunctional chelating
agent and preliminary in vitro and in vivo study of
[68Ga]Ga-DOTA-C-glyco-RGD†
bc
Cite this: RSC Adv., 2021, 11, 7672
Floriane Mangin,a Charlotte Collet,
Valerie Jouan-Hureaux,d Fatiha Maskali,be
´
a
Emilie Roeder,b Julien Pierson,d Katalin Selmeczi,
Pierre-Yves Marie,bef
a
a
Cedric Boura,d Nadia Pellegrini-Moıse
and Sandrine Lamande-Langle
´
*
*
´
¨
The design of bifunctional chelating agents (BFCA) allowing straightforward radiometal labelling of
biomolecules is a current challenge. We report herein the development of a bifunctional chelating agent
based on a DOTA chelator linked to a C-glycosyl compound, taking advantage of the robustness and
hydrophilicity of this type of carbohydrate derivative. This new BFCA was coupled with success by
CuAAC with c(RGDfK) for avb3 integrin targeting. As attested by in vitro evaluation, the conjugate DOTA-
C-glyco-c(RGDfC) demonstrated high affinity for avb3 integrins (IC50 of 42 nM). [68Ga]Ga-DOTA-C-
glyco-c(RGDfK) was radiosynthesized straightforwardly and showed high hydrophilic property (log D7.4
¼
ꢀ3.71) and in vitro stability (>120 min). Preliminary in vivo PET study of U87MG engrafted mice gave
evidence of an interesting tumor-to-non-target area ratio. All these data indicate that [68Ga]Ga-DOTA-
C-glyco-c(RGDfK) allows monitoring of avb3 expression and could thus be used for cancer diagnosis.
The DOTA-C-glycoside BFCA reported here could also be used with various ligands and chelating other
(radio)metals opening a broad scope of applications in imaging modalities and therapy.
Received 31st October 2020
Accepted 9th February 2021
DOI: 10.1039/d0ra09274f
rsc.li/rsc-advances
molecular imaging. In light of this, it is worth noting that the
introduction of a carbohydrate moiety on biomolecules, such as
Introduction
peptides, has oen been shown to enhance their pharmacokinetic
and in vivo clearance properties.5,11–13
Peptides are an important class of ligands and have emerged in
molecular imaging and nuclear medicine due to their advan-
tageous properties, which include high target specicity and
selectivity, low toxicity, biocompatibility and biodegrad-
ability.1–3 In particular, radiolabelled peptides or proteins are
suitable for positron emission tomography (PET)1,4–8 as new
diagnostic agents for preclinical and clinical research and are
today considered as fully “druggable” moieties.2
18F-glycopeptides, obtained by attachment of a radio-
uorinated carbohydrate entity on peptides for an application
in PET imaging have been a subject of interest in our group for
several years. In particular, RGD peptide derivatives are selective
for avb3 integrins, which are important cell adhesion receptors
involved in angiogenic processes allowing cancer diag-
nosis.4,14,15 Various 18F-glyco-RGD containing an O- or a C-
glycosidic moiety conjugated with a RGD peptide derivative
have been designed, synthetized and their automated radio-
synthesis has been developed.4,5,16,17 However, 18F-radiolabelling
of biomolecules remains a challenge because it requires time
consuming multistep automated radiosyntheses.
Peptides can be radiolabelled with uorine-18 (18F) by addi-
tion of a uorinated prosthetic group or with radiometals (gallium-
68, copper-64 or zirconium-89) through a bifunctional chelating
agent (BFCA).1,9,10 However, this structural modication can disrupt
the metabolic prole and/or decrease the affinity of the biomolecule
for its biological target, which is a major drawback, in particular for
The use of radiometals allows straightforward peptides
radiolabelling by simple metal coordination via a chelating
agent previously introduced on the peptide. Furthermore, 68Ga-
labelled peptides are attracting increasing interest in PET diagnos-
tics because 68Ga is easily produced with a [68Ge]/[68Ga] generator
thus not requiring a biomedical cyclotron, and its short half-life of
68 min reduces the radiation burden to the patient.
DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid) is one of the most important chelator in the eld of
medical imaging and molecular radiotherapy.1,18–20 Undoubt-
edly, this azamacrocycle forms very stable complexes with
a
´
Universite de Lorraine, CNRS, L2CM, F-5400 Nancy, France. E-mail: Sandrine.
langle@univ-lorraine.fr; Nadia.pellegrini@univ-lorraine.fr
b
´
NancycloTEP, Molecular Imaging Platform, CHRU-Nancy, Universite de Lorraine,
Nancy, F-54000, France
c
´
Universite de Lorraine, INSERM, U1254 IADI, F-54000 Nancy, France
d
´
Universite de Lorraine, CNRS, CRAN, F-54000 Nancy, France
e
´
Universite de Lorraine, INSERM, DCAC, UMR 1116, F-54000 Nancy, France
fDepartment of Nuclear Medicine, CHRU-Nancy, F-54000 Nancy, France
† Electronic supplementary information (ESI) available: Experimental details and
physico-chemical characterization of new compounds and quality control
protocols. See DOI: 10.1039/d0ra09274f
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a variety of metal ions, such as lanthanides and radionuclides, uoro-C-glyco-RGD.4 Interestingly, the robust C–C link at the
explaining its wide range of applications in the main imaging anomeric position displays a high resistance to acidic and
modalities i.e. Magnetic Resonance (MRI), Single Photon enzymatic hydrolysis, conferring an improved in vivo stability to
Emission Computed Tomography (SPECT) and Positron Emis- C-glycosyl compounds compared to O-glycosides.4,33 A C-glyco-
sion Tomography (PET), and also for therapeutic purposes.21–26 side moiety with a 3-carbon spacer-arm bearing an azido group
This prompted research into the chemistry of DOTA, in partic- for the CuAAC reaction was thus investigated. The pseudo-
ular on the modication of the side arm of the azamacro- anomeric position was chosen for the azido propyl arm intro-
cycle.27,28 For example, the side arm has been derivatized with duction given the high reactivity of this position. A protection/
different polyethylene glycol moieties (PEG), amino acids or deprotection strategy allowed us to generate
a reactive
aliphatic chains displaying functional group like amine, primary hydroxyl group and its tosylated counterpart in position
carboxylic acid or azide for subsequent coupling with targeting 6 (sugar numbering) for DOTA coupling. Azido-C-glyco deriva-
molecules.27,29–31
tive 7 was synthesized as previously described (Scheme 2).4
In close link with our interests in PET radiotracers, we have Briey, 1-O-acetyl-2,3,4,6-tetra-O-benzyl-D-glucopyranose 5 was
designed an original strategy for 68Ga-labelling of peptides and stereoselectively converted with allyl bromide into b-C-allylglu-
we have implemented this method for the preparation of 68Ga- copyranoside subsequently treated with 9-borabicyclo(3.3.1)
labelled glyco-RGD. A new bifunctional chelating agent named nonane (9-BBN) to furnish the resulting alcohol. The latter was
DOTA-C-glyco was elaborated by linking the common poly- then converted to bromide derivative 6 via a bromination
azamacrocycle DOTA and a functional carbohydrate moiety. reaction followed by hydrogenolysis of the benzyl ether pro-
This latter bears an azido-arm which enables the conjugation of tecting groups under H2 atmosphere. Compound 6 was reacted
biological vectors of interest, by Copper-catalyzed Azide-Alkyne with sodium azide, underwent a selective tritylation of the
Cycloaddition (CuAAC) with a propargylated peptide such as primary alcohol, then peracetylation and trityl group removal
propargylated c(RGDfK). The ability of the synthesized DOTA-C- provided compound 7.4 Lastly, tosylated compound 8 was ob-
glyco-c(RGDfK) to target integrins was evaluated in vitro by tained in 52% yield aer reaction of 7 with tosyl chloride, trie-
a solid-phase binding assay. 68Ga-labelling was carried out and thylamine and a catalytic amount of dimethylaminopyridine
PET imaging was implemented to assess the performance of (DMAP).
this new 68Ga-labelled radiotracer for cancer diagnosis.
Having in hand DOTA derivative 4 and C-glycoside
compounds 7 and 8, their coupling was examined. We rst
attempted a N-alkylation of 4 with tosylated compound 8.
Unfortunately, the reaction did not occur and the starting
materials were recovered even aer modication of various
parameters such as the nature of the solvent (acetonitrile or
DMF), temperature (30 or 80 ꢁC), reaction time and stoichi-
ometry. A second strategy based on the formation of a carba-
mate bond has been explored (Scheme 3). Reaction of 7 with
N,N-carbonyldiimidazol (CDI) was performed in dioxane to give
intermediate 9 (62%). The imidazolylcarbonyl derivative 9 was
treated with compound 4 in the presence of triethylamine to
afford the protected DOTA-glycoconjugate 10 in 53% yield.
Deprotection of tert-butyl ester and acetyl protecting groups
with an aqueous solution of 4N HCl at room temperature during
4 hours led to compound 11 quantitatively.
Results
Chemistry
The construction of DOTA-C-glycopeptide requires the prelim-
inary synthesis of the DOTA-C-glyco bifunctional chelating
agent. We rst investigated the preparation of a DOTA moiety
bearing an amino functional arm for the conjugation with the C-
glycoside. To this end, N-(2-benzyloxycarbonylaminoethyl)bromoa-
cetamide 1 was chosen (Scheme 1).32 The commercially available
protected
tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7-
triacetate (DO3A(t-BuO)) 2 was N-alkylated with 1 using K2CO3 and
few drops of dimethylformamide in dry acetonitrile. The removal of
the Cbz protecting group by catalytic hydrogenolysis was carried out
under H2 (atmospheric pressure) with Pd/C-10% (10% w/w) in
methanol to furnish compound 4 in quantitative yield.32
The introduction of the peptide on DOTA-C-glyco tool 11 was
performed site-specically by CuAAC with alkyne-modied
c(RGDfK) 12.34 The reaction proceeded efficiently under very
The synthesis of the glycosidic derivative was then carried
out taking advantage of our precedent work on the synthesis of
Scheme 1 DOTA derivative synthesis. Reagents and conditions: (i) K2CO3, DMF, dry acetonitrile, rt, 12 h, 52%; (ii) H2, Pd/C-10% (10% w/w), MeOH,
rt, 5 h, quantitative.
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Scheme 2 C-glycoside derivative synthesis. Reagents and conditions: (i) TMSI, CH2Cl2, 0 ꢁC, 1 h, then AllylMgBr, 0 ꢁC to rt, 3 h, 90%; (ii) (1) 9-BBN,
THF, D, 4 h; (2) EtOH, NaOH, H2O2, 12 h, 89%; (iii) PPh3, CBr4, CH2Cl2, 0 ꢁC, 1 h, 90%; (iv) Pd(OH)2/C (20% w/w), THF/H2O (5/1 v/v), H2, 33 psi, 13 h,
93%; (v) NaN3, acetone/water (5/1 v/v), reflux, 12 h, 95%; (vi) (1) TrCl, DMAP, pyridine, 48 h, 45 ꢁC. (2) Ac2O, 0 ꢁC to rt, 2 h, 62%; (vi) TFA (20% v) in
CH2Cl2, 0 ꢁC, 10 min, 73%; (vii) tosyl chloride, DMAP cat., triethylamine, CH2Cl2, 12 h, 30 ꢁC, 52%.
mild conditions in a water/tert-butanol solution (1/1 v/v) using calculated for C60H93N18O21Ga [M + Ga]2+ (calcd. 735.3006,
copper(II) acetate (1.5 equivalents) and sodium ascorbate (3 found 735.3004) and for C60H93N18O21GaNa [M + Ga + Na]3+
equivalents) as reducing agent (Scheme 3). A stoichiometric (calcd. 497.8637, found 497.8633) (see ESI†).
quantity of copper(II) acetate was required instead of the cata-
lytic amounts usually employed, because copper ions were
Integrin avb3 binding assay
chelated by the DOTA macrocycle. Furthermore, the removal of
The affinity of compounds 13 and (Ga)13 for avb3 integrin was
complexed Cu2+ from the cage proved tricky. Indeed, the clas-
evaluated by a solid-phase binding assay (Fig. 1). The results
sical strategy using a chelating resin (Chelex®) has failed. In our
showed that the IC50 values of 13 and (Ga)13 (77 and 45 nM
case, the best solution was the addition of 6 equivalents of
respectively), although slightly lower than the reference positive
sodium sulde nonahydrate leading to the precipitation of
controls Echistatin (4 nM) and c(RGDfK) (16 nM), remain in the
copper(II) as monosulde.35 Aer ltration on a Celite pad and
nanomolar range. These data demonstrate a high affinity of 13
freeze-drying, purication by steric exclusion gel ltration was
and (Ga)13 for the avb3 integrin.
performed affording DOTA-C-glyco-c(RGDfK) 13 in a high yield
of 72%. High resolution mass spectrometry as well as 1D and 2D
NMR data conrmed the structure of the conjugate.
Radiochemistry
The gallium(III) complex of compound 13, (Ga)13, was also
synthetized in order to evaluate its in vitro affinity for avb3
integrin. For the complexation reaction, 1 equivalent of galliu-
m(III) nitrate was added to a solution of compound 13 in water
and the mixture was heated for 20 min at 110 C. Aer freeze-
drying, Ga-DOTA-C-glyco-c(RGDfK) (Ga)13 was obtained as
DOTA-C-glyco-c(RGDfK) 13 was radiolabelled with gallium-68
on a MiniAllInOne (Trasis®) synthesiser (Scheme 4). Different
parameters such as amount of 13, reaction time and tempera-
ture were screened. The optimal amount of compound 13 was
found to be 50 mg in 1 mL of 0.7 M acetate solution (pH ¼ 7.9).
As commonly described for 68Ga-DOTA radiolabelling,
a temperature of 110 ꢁC was required to reach maximum
gallium-68 chelation for a reaction time of 10 min (86% deter-
mined by HPLC analyses). Formulation of the resulting [68Ga]
ꢁ
a
white powder. The gallium(III) ion complexation was
conrmed by mass spectrometry, the measured m/z values and
the observed isotopic patterns are in good agreement with those
Scheme 3 DOTA-C-glyco and DOTA-C-glyco-c(RGDfK) synthesis. Reagents and conditions: (i) N,N-carbonyldiimidazole, dioxane, rt, 62%. (ii) 4,
triethylamine, dry THF, rt, 12 h, 53%. (iii) H2O, 4N HCl, rt, 4 h, quantitative. (iv) (1) H2O/tert-butanol 1/1 v/v, sodium ascorbate, Cu(OAc)2, rt, 5 h. (2)
Na2S$9H2O, rt, 15 min, 72%. (v) Ga(NO3)3, H2O, 110 ꢁC, 20 min.
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are targeted by RGD-containing tracers, are highly expressed in
high-grade gliomas in which the magnitude of this expression
has been associated with the potential for tumor invasion,
angiogenesis, and metastasis.36,37
Corresponding time–activity curves for tumor and non-target
area (contralateral area) showed that the uptake of [68Ga]13
increased faster in the tumor than in the non-target area
(Fig. 2b), resulting in an enhancement of the tumor-to-non-
target area ratio during 120 min of dynamic analysis (Fig. 2c).
Otherwise, [68Ga]13, showed a fast renal clearance resulting in
particularly high activities in the kidneys and bladder.
Whole-body image of the second hour of PET recordings is
depicted in Fig. 3a, providing an illustration of the potential of
Fig. 1 Solid-phase binding assay. Effect of 13 (dark) and (Ga)13 (grey)
on the binding of avb3 integrin onto vitronectin compared with two
reference molecules: c(RGDfK) (red) and Echistatin (orange). IC50
values (nM) were calculated using one-site fit log-IC50 non-linear
analysis regression (graphpad prism).
[
68Ga]13 for tumor imaging. The Fig. 3b shows the mean stan-
dard value uptakes within various organs of reference (heart,
brain, kidney, liver), within the tumor and within a non-target
area (contralateral area). The Fig. 3c displays the uptake ratios
of the tumor versus the area cited above. These data were
computed for the second hour of PET recording and showed the
radiotracer prole. High radioactivity accumulation was
observed in the kidneys, showing a raised clearance of the tracer
during the second hour. Moreover, the tumor activity from [68Ga]13,
which likely corresponds to integrin binding, was found two-fold
higher than that of the non-target region (0.21 ꢂ 0.04 vs. 0.10 ꢂ
0.01; p <0.05 for SUVmean of tumor and non-target area respec-
tively), and approximatively four times higher than that of normal
brain (0.06 ꢂ 0.01), giving high tumor-to-non-target area (2.20 ꢂ
0.45) and tumor-to-brain (3.90 ꢂ 0.10) ratios.
Ga-DOTA-C-glyco-c(RGDfK) was performed on a HLB Oasis
cartridge, eluted with 0.6 mL of ethanol and washed with 4.5 mL
of 0.9% NaCl. [68Ga]Ga-DOTA-C-glyco-c(RGDfK) was straight-
forwardly radiosynthesised in 78 ꢂ 3% (n ¼ 20) decay corrected
radiochemical yield. Typical preparation run produced in
average 175 MBq of 5 mL of [68Ga]13 solution in approximately
30 minutes starting from activity elution.
Quality control of radiotracer [68Ga]13 was carried out on an
aliquot of the nal batch. The radiotracer solution was clear and
colorless with a pH around 5.5–6. Radiochemical and chemical
purities were evaluated by analytical HPLC on a C18 column
using UV and radioactive detection. UV chromatogram showed
no by-products demonstrating high chemical purity. The
radiochemical purity was determined to be over 95% with
a molar activity average of 5 MBq per nmol. In vitro plasma
stability of [68Ga]13 was also determined at 37 ꢁC in rodent
plasma by radioactive HPLC detection. Analyses of acetonitrile
plasma extracts at selected time points (0, 30, 60 and 120 min)
demonstrated an in vitro stability >120 min for compound [68Ga]13.
Experimental log D7.4 of ꢀ3.71 (n-octanol/PBS pH 7.4 partition
coefficient) showed excellent hydrophilic property for [68Ga]13.
Discussion
The development of new diagnostic/therapeutic tools for
molecular imaging like PET or molecular radiotherapy,
combining high affinity toward receptor, high in vivo stability
and optimal pharmaco-kinetic prole remains a daunting
challenge. Bioavailability is another crucial characteristic,
especially in the context of PET imaging relying on radioiso-
topes with short half-lives. In this paper, we have designed an
original DOTA-C-glyco-c(RGDfK) containing a C-glycosyl moiety
PET imaging
A series of dynamic images (coronal sections) were obtained up bearing an azido spacer-arm. Having a stable and non-
to 120 min aer the injection of [68Ga]13 on whole-body PET hydrolysable C–C link at the pseudo-anomeric position, C-
images of U87MG engraed mice (Fig. 2a). avb3 integrins, which glycosyl compounds are key components for the construction of
Scheme 4 Radiosynthesis of [68Ga]13. Reagents and conditions: (i) [68Ga]GaCl3, AcONa (0.7 M) 110 ꢁC, 10 min, 78 ꢂ 3% dc.
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glycosyl moiety with a long spacer arm afforded a distance between
DOTA and the peptide, which is obviously favourable for binding.
The gallium(III) complexed counterpart of this DOTA-glycopeptide
was also successfully synthesized.
In vitro evaluation and comparison of derivative DOTA-C-
glyco-c(RGDfK) 13 were carried out with the gold standard
integrin inhibitor Echistatin and with native c(RGDfK). To be as
close as possible to the 68Ga-radiolabelled form injected for PET
imaging, Ga-DOTA-C-glyco-c(RGDfK) (Ga)13 was also evaluated
in the same experimental protocol. In many cases, the addition
of reporting moieties like uorophores or chelating agents
affects the binding affinity of the modied biomolecules. In our
case, compounds (Ga)13 and 13 presented a slight decrease of
affinity compared to Echistatin and native RGD, however,
remaining in the same nanomolar range. These data showed
that the Ga-DOTA complex and the carbohydrate linker did not
signicantly impede the affinity of c(RGDfK) peptide for avb3
integrin. The binding affinity of (Ga)13 is slightly greater
compared to compound 13. However, both IC50 are in the same
range (42 nM vs. 75 nM for (Ga)13 and 13 respectively).
[68Ga]13 was synthesized straightforwardly in high radio-
chemical yields and purity. Incorporation of gallium-68 into the
DOTA requires heating to 110 ꢁC, however, despite this weakness,
this chelator was chosen for its versatility towards other metal ions.
Some other chelator like the acyclic HBED-CC or the NOTA require
lower energy for complex formation. However, HBED-CC forms
different diastereomers upon complexation of gallium, which is
undesirable because different isomers may have different
pharmacological proles. Whereas NOTA does not present the
optimal number of coordination site for the chelation of other
metals used in medical imaging and molecular radio-
therapy.41,42 Experimental partition coefficient (ꢀ3.71) demon-
strated excellent hydrophilic property for [68Ga]13. This value is
slightly lower than those of commonly used [68Ga]Ga-DOTA-
c(RGDfK) (around ꢀ3.43)40 indicating an improved hydrophi-
licity which could be attributed to the carbohydrate moiety.
A 120 min dynamic PET imaging with [68Ga]13 showed
a slow increasing uptake of the tracer in the tumor and non-
target area up to 60 min post injection of [68Ga]13. This obser-
vation is in accordance with previous literature on RGD-
containing tracers, leading to the current recommendation of
delaying PET recording for 30 to 60 min aer tracer injec-
tion.43–46 This avoids an early high level of nonspecic activity,
possibly due to the circulating blood tracers, and thus prevents
overestimation of integrin density. The 120 min time activity
curves of biodistribution of [68Ga]13 showed a higher uptake of
the tracer in the tumor than in the non-target area, suggesting
a specic uptake of [68Ga]13 by avb3 integrin in glioblastoma.
These integrins, highly expressed in high-grade gliomas, are
known to be poorly expressed within the different tissue compo-
nents of normal brain. These considerations likely explain the high
tumor-to-normal brain ratio documented herein for [68Ga]13, i.e.,
3.90 ꢂ 0.10 for this mouse model. These data combined with high in
vitro stability and affinity toward avb3 integrin makes [68Ga]13
a potential radiotracer for cancer diagnosis.
Fig. 2 (a) Dynamic PET images displayed through coronal sections
and recorded during 120 min following [68Ga]13 injection in U87MG
engrafted mice (n ¼ 3). Color scale is expressed in SUV values. The
tumors are indicated with white arrows, the kidneys with yellow arrows
and the bladder with red arrows. (b) Corresponding time–activity
curves for mean SUV from tumor (black square) and from non-target
area (white circle), as well as for tumor-to-non-target area ratio (black
triangles) (c).
stable glycoconjugates for various in vivo applications.4,33,38
Besides, the DOTA-C-glycoside tool bears an azide group to
ensure CuAAC conjugation with biological ligands. This
strategy allows a site-specic conjugation to a large panel of
ligands previously derivatized with an alkyne function. Previous
works on C-glyco-RGD derivatives showed adequate exibility
and ideal steric arrangement with a three-carbon spacer-arm,
conferring them similar affinities toward avb3 integrin than
the native RGD.4 Given the importance of maintaining the highest
affinity, an analogous C-glycosyl compound was chosen for the
design of the tracer. We conjugated with success the DOTA-C-
glycosyl derivative on alkyne-modied c(RGDfK) peptide for avb3
integrin targeting. Compared to common DOTA-c(RGDfK),39,40 the C-
The synthesized DOTA-C-glycosyl bifunctional chelating
agent is a versatile tool. In this study, it has been studied and
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conjugation to c(RGDfK) peptide using CuAAC. The design of
the DOTA-C-glyco BFCA exploited the hydrophilicity and the
robustness of C-glycosyl moiety. In vitro binding of DOTA-C-
glyco-c(RGDfK) and its gallium(III) complexed counterpart, to
avb3 integrin showed that conjugation of DOTA-C-glycoside to
c(RGDfK) did not affect its affinity. [68Ga]Ga-DOTA-C-glyco-
c(RGDfK) [68Ga]13 was radiosynthesized straightforwardly and
showed high hydrophilic property (log D7.4 ¼ ꢀ3.71) and in vitro
stability (>120 min). Tumor-to-non-target area ratio obtained by
in vivo PET study in U87MG engraed mice, demonstrated that
[68Ga]Ga-DOTA-C-glyco-c(RGDfK) could be a potential radio-
tracer for cancer diagnosis by PET imaging. As reported in this
study, the DOTA-C-glycoside BFCA is a versatile tool, successfully
employed for 68Ga-radiolabelling of c(RGDfK). Furthermore, the
possibility of using others ligands and (radio)metals opens a broad
scope of applications in imaging modalities and therapy.
Experimental section
General information
Solvents and liquid reagents were puried and dried according
to recommended procedures. Thin layer chromatography (TLC)
analyses were performed using standard procedures on Kie-
selgel 60F254 plates (Merck, Kenilworth, NJ, USA). Compounds
were visualized using UV light (254 nm), ninhydrin and/or
a methanolic solution of sulfuric acid and charred. Column
chromatography was performed on silica gel SI 60 (63–200 mm)
(Merck). c(RGDfK) was purchased from Bachem (Germany) with
>95% purity. Purication of RGD-conjugate was achieved by gel
ltration on LH20 using water as eluent. Melting points were
determined with a Tottoli apparatus and are uncorrected. FTIR
spectra were recorded on a Shimadzu IRAffinity-1, ATR PIKE
Technologies model GladiAT (cmꢀ1). Optical rotations were
measured on an Anton-Paar MCP 300 polarimeter. Analytical
Fig. 3 (a) Whole-body image of the 2nd hour of PET recordings after
[
68Ga]13 injection. (b) Results of SUVmean values obtained at the 2nd
hour of PET recording for tumors and various organs, after the injec-
tion of [68Ga]13. (c) Comparison of tumor-to-organ ratio for activities
measured during the 2nd hour of PET recording. *: p < 0.05 The tumor High Performance Liquid Chromatography (HPLC) analyses
is indicated with white arrows and the color scale is expressed in SUV
values (n ¼ 3).
were run on a Waters system (2695eb pump, auto sampler
injector, 2998 PDA detector, 2424 ELSD detector, and NaI
detector from Berthold [Bad Wildbad, Germany]) controlled by
the Empower Soware (Orlando, FL, USA). Analyses were per-
illustrated with gallium-68 labelling of an RGD peptide deriva-
tive, however this BFCA could be used with others biological
ligands like peptides, proteins or antibodies and for other
formed on a Vydac 218 TP C18 (5 mm, 250 ꢃ 4.6 mm) from Grace
with ACN/H2O/TFA mixture (proportions given in brackets) at 1
mL minꢀ1 1H, 13C, and 19F NMR spectra were recorded on
.
medical applications. Indeed, DOTA is widely used in the eld
of medical imaging and molecular radiotherapy because it
chelates efficiently a large panel of (radio)metals. Hydrophilic
BFCA 11 could be potentially suitable for different imaging
modalities and therapy, such as PET (68Ga, 64Cu), SPECT (99mTc,
111In) and MRI (Gd). Besides, targeted cancer therapy might also
be possible with DOTA-C-glycoside 11 labelled with 177Lu, 90Y or
186Re. DOTA-C-glyco could also be employed in theranostics
enabling concomitant diagnostic and radiotherapy by labelling
with diagnostic and therapeutic radioisotopes.
a Bruker DPX250 (250 MHz, 62.9 MHz and 235 MHz, respec-
tively) or DRX400 (400 MHz and 100.6 MHz, respectively)
spectrometers on the NMR Platform of the Jean Barriol Institute
´
(Universite de Lorraine, Nancy, France). For complete assign-
ment of 1H and 13C signals, two-dimensional 1H, 1H COSY and
1H, 13C correlation spectra were recorded. Chemical shis (d)
are given in parts per million relative to the solvent residual
peak. For clarity, atom numbering starts from the end of the
anomeric arm rather than resulting from the IUPAC naming of
compounds (see ESI Fig S1†). The following abbreviations are
used for multiplicity of NMR signals: s ¼ singlet, d ¼ doublet, t
¼ triplet, q ¼ quadruplet, m ¼ multiplet, b ¼ broad signal and
app ¼ apparent multiplicity. The given J values refer to apparent
Conclusion
In this study, we reported for the rst time the synthesis of multiplicities and do not represent the true coupling constants.
DOTA-C-glycosyl bifunctional chelating agent (BFCA) and its High resolution ESI-MS spectra were recorded on a Bruker
© 2021 The Author(s). Published by the Royal Society of Chemistry
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Daltonics microTOFQ apparatus provided by the MassLor afford compound 10 (64.5 mg). Yield 53%. Yellow solid. Mp: 99–
Platform (Universite de Lorraine, Nancy, France). 68GaCl3 was 101 ꢁC; [a]D: + 3.6 (c 0.05; EtOH); IR lm (n, cmꢀ1): 2976, 2816,
´
1
eluted from a 68Ge/68Ga generator (Galliapharm; Eckert & Zie- 2093, 1722, 1668, 1520, 1368, 1221, 1155, 1105, 1030. H NMR
gler Europe) with 5 mL of a 0.1 N HCl solution. Radiosynthesis (400 MHz, DMSO-d6): d 1.36–1.43 (m, 1H, H3a), 1.45 (s, 18H,
was carried out on a miniAllInOne (miniAIO) module from tBu), 1.46 (s, 9H, tBu), 1.54–1.64 (m, 2H, H2), 1.65–1.74 (m, 1H,
Trasis®. Female immunodecient (nu/nu) NMRI mice were H3b), 1.93 (s, 3H, CH3), 1.98 (s, 3H, CH3), 2.00 (s, 3H, CH3),
purchased from Janvier Laboratories. PET recordings were ob- 2.24–2.41 (m, 8H, CH2 cyclen), 2.54–2.70 (m, 8H, CH2 cyclen),
tained with a camera dedicated to small animal studies (Inveon, 3.00–3.21 (m, 12H, H11, H12, H14, H17, H20, H23), 3.33 (t, 2H,
Siemens Preclinical Solutions, Knoxville, USA).
J1,2a ¼ J1,2b ¼ 6.5 Hz, H1), 3.64 (b ddd, 1H, J8,7 ¼ 10.0 Hz, J8,9b
7.5 Hz, J8,9a ¼ 2.5 Hz, H4), 3.80 (b ddd, 1H, J8,7 ¼ 8.0 Hz, J8,9b
5.0 Hz, J8,9a ¼ 3.0 Hz, H8), 4.02 (dd, 1H, J9a,9b ¼ 12.0 Hz, J9a,8
¼
¼
¼
Experimental procedures and physico-chemical
characterizations
3.0 Hz, H9a), 4.07 (dd, 1H, J9b,8 ¼ 5.0 Hz, H9b), 4.71 (app t, 1H,
J7,6 ¼ 9.5 Hz, H5), 4.88 (dd, 1H, J5,6 ¼ 9.5 Hz, H7), 5.17 (app t,
Chemistry. Compounds 1,32 2,32 4,32 6,4 7 (ref. 4) and 12 1H, H6), 6.87 (bs, 1H, NH), 7.93 (bs, 1H, NH). 13C NMR (100.6
(ref. 34) were prepared according to previously described MHz, DMSO-d6): d ¼ 20.7 (CH3), 20.7 (CH3), 20.8 (CH3), 24.6
methods (see ESI†).
(C2), 27.9 (3CH3, tBu), 28.0 (6CH3, tBu), 28.3 (C3), 39.7 (C12), 40.4
4,8-Anhydro-1-azido-1,2,3-trideoxy-5,6,7-tri-O-acetyl-9-O-tosyl-
(C11), 51.3 (C1), 55.7 (8C, C-cyclen), 56.1 (4C, C14, C17, C20, C23),
D-glycero-D-gulo-nonitol (8). To a solution of 4,8-anhydro-1-azido- 63.6 (C9), 69.6 (C7), 72.0 (C5), 74.6 (C6), 76.0 (C8), 76.9 (C4), 82.0
1,2,3-tridesoxy-5,6,7-tri-O-acetyl-D-glycero-D-gulo-nonitol (424 mg, (6CqtBu), 82.0 (3CqtBu), 156.4 (C]Ocarb), 169.8 (C]O), 169.8 (C]O),
1.14 mmol) in dry DCM (10 mL) tosyl chloride (658.4 mg, 3.42 170.3 (C]O), 172.0 (C]O), 172.3 (3C]O). HRMS (ESI,
mmol), DMAP (66 mg, 0.57 mmol) and triethylamine (0.63 mL, C46H80N9O16: [M + H]+) calcd 1014.5723, found 1014.5741.
4.54 mmol) were added under argon. The reaction was stirred
Compound 11. To a suspension of compound 10 (73 mg; 0.072
overnight at 30 ꢁC. The mixture was concentrated under mmol) in water (1.13 mL) was added HCl 37% (375 mL). The
reduced pressure and puried on by ash column chromatog- reaction mixture was stirred at room temperature for 4 h and
raphy (silica gel, Cyclohexane/EtOAc 6/4) to afford 8 (607 mg). evaporated under reduce pressure using MeOH as co-solvent to
Yield 52%. White solid. Mp: 78–80 ꢁC; [a]D: +12.0 (c 0.09; EtOH); afford 11 (51.7 mg). Yield quantitative. Yellow solid. Mp: 93–
IR lm (n, cmꢀ1): 2102, 1738, 1599, 1368, 1256, 1227, 1188, 1173, 95 C; [a]D: ꢀ7.8 (c 0.03; EtOH); IR lm (n, cmꢀ1): 3260, 3082,
ꢁ
1
1
1119, 1098, 1032; H NMR (400 MHz, CDCl3): d 1.36–1.46 (m, 2932, 2864, 2095, 1722, 1674, 1539, 1385, 1352, 1211, 1084; H
1H, H3a), 1.51–1.63 (m, 2H, H2a and H3b), 1.68–1.78 (m, 1H, NMR (400 MHz, D2O): d 1.44–1.57 (m, 1H, H3a), 1.64–1.76 (m,
H2b), 1.98 (s, 3H, CH3), 1.99 (s, 3H, CH3), 2.03 (s, 3H, CH3), 2.46 1H, H2a), 1.76–1.86 (m, 1H, H2a), 1.88–1.98 (m, 1H, H3b), 3.22
(s, 3H, CH3), 3.20–3.31 (m, 2H, J1,2a ¼ J1,2b ¼ 6.5 Hz, H1), 3.38 (appt, 1H, J5,4 ¼ J5,6 ¼ 8.5 Hz, H5), 3.24–3.60 (m, 25H, H4, H7,
(bddd, 1H, J4,5 ¼ 9.5 Hz, J4,3b ¼ 9.0 Hz, J4,3a ¼ 2.5 Hz, H4), 3.66 H12, H11, H6, H14, 16Hcyclen), 3.37 (t, 2H, J1,2a ¼ J1,2b ¼ 7.0 Hz,
(ddd, 1H, J8,7 ¼ 9.0 Hz, J8,9b ¼ 6.0 Hz, J8,9a ¼ 3.0 Hz, H8), 4.02 H1), 3.53–3.61 (m, 1H, H8), 3.73–4.00 (m, 6H, H17, H20, H23),
(dd, 1H, J9a,9b ¼ 11.0 Hz, H9a), 4.09 (dd, 1H, H9b), 4.80 (app t, 4.24 (dd, 1H, J9a,9b ¼ 11.5 Hz, J9a,8 ¼ 6.5 Hz, H9a), 4.37 (bd, 1H,
1H, J4,5 ¼ J5,6 ¼ 9.5 Hz, H5), 4.89 (app t, 1H, J7,6 ¼ J8,7 ¼ 9.0 Hz, H9b). 13C NMR (100.6 MHz, D2O): d 24.2 (C2), 27.8 (C3), 39.3
H7), 5.13 (app t, 1H, H6), 7.35 (d, 2H, J ¼ 8.0 Hz, Ar), 7.78 (d, 2H, (C12), 39.6 (C11), 44.4–50.4 (8C, C-cyclen), 50.9 (C1), 55.0 (4C,
J ¼ 8.0 Hz, Ar). 13C NMR (100.6 MHz, CDCl3): d 20.7 (CH3), 20.7 C14, C17, C20, C23), 64.0 (C9), 69.9 (C7), 73.4 (C5), 77.2 (C6),
(CH3), 20.8 (CH3), 21.8 (CH3), 24.6 (C2), 28.4 (C3), 51.2 (C1), 68.1 77.4 (C8), 79.1 (C4), 158.4 (C]Ocarb), 175.3 (C]O), 175.6 (3C]
(C9), 69.0 (C7), 71.7 (C5), 74.1 (C6), 75.5 (C8), 77.4 (C4), 128.2 O). HRMS (ESI, C28H49N9O13Na: [M + Na]+) calcd 742.3348,
(2CAr), 130.0 (2CAr), 132.7 (Cq), 145.3 (Cq), 169.6 (C]O), 169.7 found 742.3252; (ESI, C28H48N9O13Na2: [M ꢀ H + 2Na]+) calcd
(C]O), 170.4 (C]O). HRMS (ESI, C22H29N3O10SNa: [M + Na]+) 764.3167, found 764.3066.
calcd 550.1471, found 550.1493.
Compound 13. To a solution of azido derivative 11 (4.3 mg,
Compound 10. To a solution of 4,8-anhydro-1-azido-1,2,3-tri- 0.006 mmol) diluted in 0.6 mL of water, were added at room
deoxy-5,6,7-tri-O-acetyl-D-glycero-D-gulo-nonitol 7 (75 mg, 0.2 temperature a solution of c(RGDfK) derivative 12 (4.8 mg, 0.006
mmol) in dioxane (1.4 mL), was added under argon, N,N-car- mmol) diluted in 1 mL of tert-butanol, a solution of sodium
bonyldiimidazole (38.9 mg, 0.24 mmol). The mixture was stirred ascorbate (0.018 mmol) in 0.2 mL of water and a solution of
at room temperature until completion of the reaction (TLC Cu(OAc)2 (0.009 mmol) in 0.2 mL of water. The solution was
monitoring), then was concentrated under reduced pressure. stirred 5 h at room temperature, then Na2S$9H2O (8.5 mg, 0.036
The residue was diluted in a cyclohexane/EtOAc 1/1 v/v solution mmol) was added. The mixture was stirred 15 min. Black
(5 mL), ltered on a silica gel pad and evaporated under reduced precipitate appeared immediately, the mixture was ltered on
pressure to obtain intermediate 9 as a colorless oil (57.8 mg, a Celite pad, then freeze-dried. Purication was achieved on
62%). To a solution of 9 in dry THF (2 mL), 4 (110.8 mg, 0.18 Sephadex LH20 resin. Elution with water provided pure
mmol) and triethylamine (167 mL, 1.2 mmol) were added. The compound 13 (6 mg). Yield 72%. White foam. 1H NMR (400
reaction mixture was stirred overnight at room temperature. MHz, D2O): d 0.72–0.86 (m, 2H, H32), 1.17–1.29 (m, 2H, H31),
Aer evaporation under reduced pressure, the crude product 1.38–1.51 (m, 2H, H33a, H2a), 1.51–1.58 (m, 2H, H42), 1.58–1.75
was puried by LH20 chromatography with MeOH as eluant, (m, 2H, H33b, H3a), 1.78–1.95 (m, 2H, H2b, H41a), 1.94–2.15
then by ash chromatography (silica, CH2Cl2/MeOH 9/1) to (m, 2H, H41b, H3b), 2.56 (dd, 1H, J38a,38b ¼ 16.5 Hz, J38a,37
¼
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7.0 Hz, H38a), 2.65 (bt, 2H, J29,28 ¼ 7.0 Hz, H29), 2.70 (dd, 1H, J was added to the eluted solution. The mixture was heated at
¼ 7.0 Hz, H38b), 2.93–3.05 (m, 4H, H28, H30), 3.07–3.36 110 ꢁC during 10 min. The resulting product, [68Ga]13, was
38b,37
(m, 26H, H5, H39, H43, H4, H12, H11, 16H cyclen), 3.37–3.56 trapped on a HLB Oasis cartridge. The product was eluted from
(m, 15H, H1, CH2Bn, H7, H6, H14, H17, H20, H23), 3.61 (dd, 1H, the cartridge with 0.6 mL of ethanol. The cartridge was rinsed
J9a,9b ¼ 11.5 Hz, J9a,8 ¼ 7.5 Hz, H9a), 3.67 (dd, 1H, J9a,8 ¼ 4.5 Hz, with 5 mL of NaCl 0.9%. [68Ga]13 was produced with a decay
H9b), 3.80–3.92 (m, 3H, H8, H34), 4.50 (d, 1H, J ¼ 14.0 Hz, CH2Bn), corrected (dc.) radiochemical yield of 78 ꢂ 3% (n ¼ 20).
4.43 (app t, 1H, J ¼ 7.0 Hz, H40), 4.56–4.63 (m, 1H, H35), 4,67–4.75
(m, 1H, H37), 7.23–7.29 (m, 3H, HAr), 7.31–7.38 (m, 2H, HAr), 7.83
(bs, 1H, H26). 13C NMR (100.6 MHz, D2O): d 21.2 (C28), 22.4 (C32),
24.2 (C2), 27.3, 27.4, 27.6, 27.6 (C41, C42, C31, C3), 30.0 (C33), 35.3
(C29), 37.8 (2C, C39, C30), 38.9 (3C, C38, C12, C11), 40.5 (C43), 43.8
(C36CH2Bn), 47.8–48.7 (8C, C-cyclen), 50.0 (2C, C40, C1), 50.8 (C37),
55.2 (C35 or C34), 55.5 (C34 or C35), 58.5–59.0 (4C, C14, C17, C20,
C23), 63.2 (C9), 72.0 (C7), 72.1 (C8), 73.4 (C5), 77.2 (C6), 78.8 (C4),
123.3 (C26), 127.2 (CAr), 128.8 (2CAr), 129.2 (2CAr), 136.0 (CqAr), 146.3
(C27), 155.0 (C]N), 157.0 (C]Ocarb), 171.3 (C]O), 172.5 (C]O),
172.6 (C]O), 173.4 (C]O), 173.6 (C]O), 174.3 (C]O), 174.4 (C]
O), 175.8 (C]O), 176.2 (C]O), 177.9 (C]O), 178.0 (C]O). HRMS
(ESI, C60H95N18O21K: [M + K]2+) calcd 721.3275, found 721.3256.
Animal models and experimental plan
All protocols were approved by the Lorraine Ethics Committee
No 68 according to Guidelines of Animal Care and Use
(APAFIS#25133-2020041509493813). Tumor xenogras were
obtained in mice anks as described previously.44 Six-week-old
female immunodecient (nu/nu) NMRI mice (n ¼ 3), weigh-
ing 26–27 g, were obtained from Janvier Laboratories (Le Genest
Saint Isle, France) and housed in ventilated cages including
lter tops with an ad libitum access to food and water. Xeno-
gra tumor was induced in all mice through subcutaneous
ank injections of 2 ꢃ 106 of human U87-MG cells. Tumor
growth was assessed twice weekly, according to tumor volume
(in mm3) calculated with the formula d2 ꢃ D/2, where d and D
represent the shortest and longest diameters, respectively,
calculated in millimeter with a caliper (+decrescent PRC, EDVC,
France). The average tumor volume was 107 ꢂ 0.4 mm3.
Integrin avb3 binding assay
The affinity of compounds for integrin protein was evaluated in
terms of the half maximal inhibitory concentration (IC50 values)
through a solid-phase assay as previously described by Tobias
G. Kapp and al.47 Briey, the surface of Maxisorp microplates
(NUNC, Thermocher scientic, France) was coated with 1 mg
mLꢀ1 human vitronectin (Bio-techne, France), overnight at
+4 ꢁC. The non-specic sites were blocked with TSB buffer
(20 mM Tris–HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,
1 mM MnCl2, pH 7,5, 1% BSA) (Sigma-Aldrich, France) for 1 h at
37 ꢁC. Binding of compounds was assessed using 2 mg mLꢀ1
integrin avb3 (Bio-techne, France) in the presence of dilution
series of compounds or two reference molecules: c(RGDfK)
(Bachem, Germany) and echistatin (Bio-techne, France), as
positive control. Aer 1 h incubation at room temperature, the
plates were washed and the amount of bound integrin was stained
by incubation with 2 mg mLꢀ1 mouse anti-human CD51/61 (BD
Pharmingen, France) and 1 mg mLꢀ1 anti-mouse IgG horseradish
peroxidase conjugate antibody (Bio-techne, France). The enzymatic
reaction was allowed in the dark by addition of substrate (Bio-
techne, France) and stopped aer 10 min by the addition of
H2SO4 (Stop Solution, Bio-techne, France). The optical densities
were measured at 450 nm. Values were blank subtracted and results
were expressed as a mean of relative absorbance percentage to wells
containing only integrin avb3 of triplicate measurements. Affinities
were estimated as IC50 values (i.e. the concentration of compounds
that displaced 50% of integrin binding) calculated using one-site t
log-IC50 non-linear analysis regression of GraphPad Prism 6 so-
ware (v 6.05, USA).
Small animal PET studies
Mice were anesthetized with 1.5% isourane, 10 ꢂ 1 MBq of
[
68Ga]13 were injected as a bolus via a lateral tail vein. List-mode
acquisitions of 120 min durations were initiated a few seconds
prior to tracer injection, and the acquired PET data were
subsequently reconstructed in 27 consecutive frames (i.e., 5
frames of 120 s duration followed by 22 frames of 5 min dura-
tion) using the ordered-subsets expectation maximization 3D
algorithm (OSEM3D, 4 iterations, 16 subsets, zoom 1) together
with scatter and attenuation corrections based on transmission
source measurement. The nal voxel size was 0.8 ꢃ 0.8 ꢃ 0.9
mm3. Images were visualized as maximum intensity projections
and from different slices (sagittal, transverse, coronal) to allow
visual and quantitative analysis, for each animal and for each
PET tracer. Tumor and various organ activities were determined
through SUV mean values within 3D regions of interests (ROI),
which were drawn with a dedicated soware (Inveon Research
Workplace 4.1, Siemens®, Knoxville, USA) on the fusion images
encompassing the entire 120 min recording period. Spheroid
ROIs were placed inside the tumor, non-target area, brain,
heart, liver and kidneys regions with the ROI limits approxi-
mating the organ limits as close as possible. Tumor ROIs was
obtained with isocontour ROIs and threshold limits of 50%, of
the maximal voxel value. All data are expressed as mean ꢂ SD.
Unpaired comparison of quantitative variable were performed
with Student's t test. P values less than 0.05 were considered as
statistically signicant.
Radiochemistry
The 68Ga-labelling of 13 was performed using a mAIO synthe-
sizer (Trasis, Ans, Belgium) with removable cassettes. The
68Ge/68Ga generator (Galiapharm; Eckert & Ziegler Europe) was
Author contributions
eluted with 5 mL of a 0.1 M HCl solution. 13 (50 mg, 34 nmol) FlM with the participation of SLL and NPM synthesized all the
previously solubilized in 1 mL of acetate buffer solution (0.7 M), non-radioactive compounds (1 to 13) and completed the NMR
© 2021 The Author(s). Published by the Royal Society of Chemistry
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Paper
analysis of these compounds. KS synthesized (Ga)13, completed 12 R. Haubner, B. Kuhnast, C. Mang, W. A. Weber, H. Kessler,
the mass spectra analysis and co-wrote the manuscript. VJH
with the participation of CB performed in vitro integrin binding
H. J. Wester and M. Schwaiger, Bioconjugate Chem., 2004,
15, 61–69.
assay and they co-wrote the manuscript. CC with the partici- 13 K. Stromgaard and T. Hoeg-Jensen, in Pharmaceutical
pation of SLL and NPM performed the radionuclide-labelling of
13 and wrote radiochemistry section. FM and CB co-wrote the
ethical request for project authorization. JP and CB provided the
animal models. ER with the collaboration of JP and FM performed
Formulation Development of Peptides and Proteins – Peptide
and protein derivatives, ed. L. Hovgaard, S. Frokjaer and M.
Van de Weert, Taylor & Francis Boca Raton, 2nd edn, 2013,
pp. 131–148.
the experimental studies in mice. FM performed images analysis on 14 M. Alipour, M. Baneshi, S. Hosseinkhani, R. Mahmoudi,
the small animal PET. FM and PYM co-wrote the manuscript. SLL
and NPM designed the study and wrote the manuscript. All authors
read and approved the nal manuscript.
A. J. Arabzadeh, M. Akrami, J. Mehrzad and H. Bardania, J.
Biomed. Mater. Res., Part A, 2020, 108, 839–850.
15 S. Asati, V. Pandey and V. Soni, Int. J. Pept. Res. Ther., 2019,
25, 49–65.
´
´
16 C. Collet, F. Maskali, A. Clement, F. Chretien, S. Poussier,
´
Conflicts of interest
G. Karcher, P.-Y. Marie, Y. Chapleur and S. Lamande-
Langle, J. Labelled Compd. Radiopharm., 2016, 59, 54–62.
There are no conicts to declare.
´
´
17 Y. Chapleur, S. Lamande, C. Collet and F. Chretien, PCT Int.,
WO 2014006022 A1 20140109, 2014.
18 N. Alexander, R. Vali, H. Ahmadzadehfar, A. Shammas and
S. Baruchel, Curr. Radiopharm., 2018, 11, 14–21.
19 S. Narayana, M. Chilla, C. Henoumont, L. Vander Elst,
R. N. Muller and S. Laurent, Magn. Reson. Imaging, 2017,
57, 800–808.
Acknowledgements
This work was supported by NancycloTEP. We thank F. Dupire
and S. Adach for technical assistance. We gratefully acknowl-
edge the nancial support of the “Contrat de Plan Etat-Region”
(CPER) from French government and Grand Est Regional
Council.
´
20 K. Tanaka and K. Fukase, Org. Biomol. Chem., 2008, 6, 815–
828.
21 M. Guleria, T. Das, J. Amirdhanayagam, H. D. Sarma and
A. Dash, Nucl. Med. Biol., 2019, 78–79, 31–40.
22 R. Viswas, E. Alizadeh, W. Bernhard, S. V. Hartimath,
W. Hill, R. Chekol, K. M. Barreto, C. R. Geyer and
H. Fonge, Mol. Pharm., 2019, 16, 4807–4816.
23 G. Giannini, F. M. Milazzo, G. Battistuzzi, A. Rosi,
A. M. Anastasi, F. Petronzelli, C. Albertoni, L. Tei, L. Leone,
L. Salvini and R. De Santis, Bioorg. Med. Chem., 2019, 27,
3248–3253.
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