AMD3100: A Versatile Platform for CXCR4 Targeting 68Ga-Based

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AMD3100: a versatile platform for CXCR4 targeting 68Ga-based radiopharmaceuticals Sophie Poty, Eleni Gourni, Pauline Désogère, Frédéric Boschetti, Christine Goze, Helmut R. Maecke, and Franck Denat Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00689 • Publication Date (Web): 17 Feb 2016 Downloaded from http://pubs.acs.org on February 18, 2016

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Bioconjugate Chemistry

AMD3100: a versatile platform for CXCR4 targeting 68Ga-based radiopharmaceuticals

Sophie Poty,† Eleni Gourni,‡,§,# Pauline Désogère,† Frédéric Boschetti, Christine Goze,*† Helmut R. Maecke*§ and Franck Denat*† †

Institut de Chimie Moléculaire de l’Université de Bourgogne, UMR6302, CNRS, Univ.

Bourgogne Franche-Comté, F-21000 Dijon, France. ‡

German Cancer Consortium (DKTK), Heidelberg 69120, Germany.

§

Department of Nuclear medicine, University Hospital Freiburg, Freiburg 79106, Germany.

#

German Cancer Research Center (DKFZ), Heidelberg 69120, Germany.

CheMatech, 9 Av. Alain Savary, BP 47870 21000 Dijon cedex, France.

Abstract CXCR4 is a G protein-coupled receptor (GPCR), which is overexpressed in numerous diseases, particularly in multiple cancers. Therefore, this receptor represents a valuable target for imaging and therapeutic purposes. Among the different approaches, which were developed for CXCR4 imaging, a CXCR4 antagonist biscyclam system (AMD3100, also called Mozobil), currently used in clinic for the mobilisation of hematopoietic stem cells, was radiolabelled with different radiometals such as 62Zn, 64Cu, 67Ga or 99mTc. However, cyclam is not an ideal chelator for most of these radiometals, and could lead to the release of the radionuclide in-vivo. In the current study, a new family of CXCR4 imaging agents is presented, in which AMD3100 is used as a carrier for specific delivery of an imaging reporter, i.e. a

68

Ga complex for PET imaging. AMD3100 was functionalised on the phenyl

moiety with different linkers, either ethylenediamine or diamino-polyethylene glycol 3 (PEG3). The resulting AMD3100 analogues were further coupled with two different chelators,

ACS Paragon Plus Environment


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1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane1-glutaric acid-4,7-acetic acid (NODAGA). Five potential CXCR4 targeting agents were obtained. The derived AMD3100-based ligands were labelled with influence of the spacer nature on the

68

68

Ga, highlighting the

Ga-labelling yield. The lipophilic character of the

different systems was also investigated, as well as their affinity for the CXCR4 receptor. The most promising compound was further evaluated in-vivo in H69 tumor xenografts by biodistribution and PET imaging studies, validating the proof of principle of our concept. AMD3100 NH HN NH

N N

HN

NH HN

NH HN Ni2+ NH N

HO O

S

O

NH

N 6 8 Ga

N

HO

N H

N

O

NH NH

O

HO

O

O O 68Ga-11

2

N HN Ni2+ NH HN

Introduction Nuclear imaging has gained an important role in the broad field of molecular imaging, since it allows non-invasive, early detection and potential therapy of several types of cancers.1 The molecular/cellular processes imaged can be the location of a specific population of cells or the determination of levels of a given protein receptor on the surface of the cells.2 The overexpression of the C-X-C chemokine receptor 4 (CXCR4) plays an important role in oncology since it is involved in tumor development, growth and organ specific metastasis. CXCR4 is a seven-transmembrane domain G protein-coupled receptor (GPCR)3 and one of the most studied chemokine receptors mainly due to its early found role as a co-receptor for HIV-entry.4 The activation of CXCR4 by its sole known natural ligand, the stromal cellderived factor-1α (SDF-1α, also known as CXCL12)5 results in a variety of physiological


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responses, such as chemotaxis, cell survival and proliferation, intracellular calcium flux, and gene transcription. However, CXCR4 is also involved in the pathogeneses of a wide range of diseases.6 Upregulation of CXCR4 has been reported in at least 23 different types of cancer7,8,9,10,11 and is correlated with poor prognosis, tumor aggressiveness, increased risk of metastasis and a higher probability of recurrence.12,13,14,15,16 The evaluation of CXCR4 expression is currently clinically assessed by the immunohistochemistry of tumorous tissue samples,17 which involves invasive patient biopsies and may not be representative of the entire tumor volume. Considering the critical role of the SDF-1α/CXCR4 axis in various disease states, there is currently significant interest in the discovery and the development of CXCR4 inhibitors and imaging probes for non-invasive monitoring of CXCR4 expression.17,18,19 CXCR4-specific radiotracers for nuclear imaging have been developed based on CXCR4 antagonists functionalised on their scaffold by a prosthetic group or a chelating agent. 17,18,19 For the development of highly-specific CXCR4 radiotracers, one could start with SDF-1α, the CXCR4 endogenous ligand, but the high molecular weight in combination with a low in vivo stability prevent its in vivo applicability.20,21 In an attempt to image CXCR4 expression using specific antibodies, 12G5 was labelled with

125

I but did not show receptor-mediated tumor

uptake.22,23 The most promiment downsized group of CXCR4 nuclear imaging agents are peptide-based ligands. The development of CXCR4-targeted peptides resulted in the discovery of a 14 amino acid cyclic peptide named T140 (Arg1-Arg2-Nal3-cyclo(Cys4-Tyr5Arg6-Lys7-D-Lys8-Pro9-Tyr10-Arg11-Cit12-Cys13)-Arg14),24 which was functionalised with different

prosthetic

groups

and

chelators

and

widely

studied

with

different

radionuclides.25,26,27,28 To improve the in vivo stability of the T140 derivatives, Fujii et al., developed a library of cyclic pentapeptides such as FC131 (cyclo(D-Tyr1-Arg2-Arg3-2-Nal4Gly5)) based on structure-activity relationship studies.29 FC131 displays improved metabolic



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stability with potent CXCR4 antagonistic profile. Furthermore, a cyclic CXCR4-binding pentapeptide,

CPCR4-2

((cyclo(D-Tyr1-D-[NMe]Orn2-[4-(aminomethyl)

benzoic

acid

[AMBS], DOTA]-Arg3-2-Nal4-Gly5) with improved pharmacokinetics and persistent tumor uptake was developed by Demmer et al..30,31

68

Ga-CPCR4-2 also called

68

Ga-pentixafor

reached clinical trials and its initial evaluation is now completed.32,33 Small molecules are also an interesting group of potent imaging agents; biscyclams have been identified as potent and selective inhibitors of HIV and were recognised to target CXCR4.34 Among

them,

1,1’-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane],

AMD3100 (Mozobil, Genzyme),35,36 mobilises hematopoietic stem cells and has also been approved by the U.S. Food and Drug Administration for use in non-Hodgkin’s lymphoma and multiple myeloma patients.37,38 In parallel, since macrocyclic polyamines such as cyclam are known to strongly coordinate metal ions, AMD3100 was metallated with ZnII, NiII and CuII.39 The resulting highly stable metal complexes demonstrated enhanced affinity for CXCR4.39 This paves the way for the direct labelling of AMD3100 ligand with

62

Zn,

64

Cu,

67

Ga and

99m

Tc, thus leading to radiotracers suitable for nuclear imaging (Figure 1).40,41,42,43 The first in

vivo study of [64Cu]-AMD3100 in non-tumor bearing mice demonstrated high activity accumulation in immune-related organs such as spleen, lymph nodes and bone marrow, thus validating the CXCR4 targeting ability of the tracer.40 Later on, CXCR4-specific uptake in tumors was observed on tumor bearing mice but the highest accumulation of radioactivity was noted in liver.44 This small molecule presents high potential for CXCR4 imaging and is of particular interest for us. 64Cu-AMD3100

AMD3100

NH HN

NH HN NH

N

N N

NH HN

HN

N

64Cu

N

N

HN

N

Figure 1: Schematic representation of AMD3100 and 64Cu-AMD3100


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In the frame of this study, we aimed at developing a new class of CXCR4 targeting radiotracers based on the AMD3100 moiety. Our group recently demonstrated that the functionalisation of the central bridging phenyl group of AMD3100 could be carried out without significant loss in the affinity toward the CXCR4 receptor. This offers an interesting platform for bioconjugation of molecular imaging probes.45 AMD3100 was functionalised with two linkers of different lengths –either ethylenediamine (first generation) or PEG3 (second generation)- and coupled to DOTA and NODAGA chelators. This study particularly highlights the importance of the linker and chelator in the design of such radiotracers.46,47 Considering the wide range of radionuclides available for PET imaging,48 we focused on a radionuclide of growing interest with numerous advantages for PET,

68

Ga.49,50,51 We report,

herein, an alternative to the direct labelling of the cyclam cavities, which is enabled by the coupling of different chelators suitable for

68

Ga coordination. Finally, the most promising

candidate was selected for further biodistribution study and PET imaging studies, validating the proof of principle of our concept.

Results Synthesis of a first and second generation of AMD3100 analogues The ethylenediamine spacer of compound 145 enables the coupling of the two widely used macrocyclic chelators, DOTA and NODAGA,52 for labelling with

68

Ga, using N-hydroxy-

succinimidyl (NHS) activated esters. Both coupling reactions resulted in the first generation ligands 2 and 3 in 20 % yield after purification on reverse phase flash chromatography (Scheme 1). The second generation of AMD3100 analogues was synthesised starting from compound 445, which was first hydrolysed with a yield of 95 %. Compound 5 enables then the introduction of a Boc protected diamino-PEG3 spacer leading to 6 in a 50 % yield. The deprotection of Boc



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results in compound 7 in a yield of 75 %. Ni2+ metallation gives the complex 8 with a yield of 60 %. Compound 8 was further functionalized with the chelators DOTA, NODAGA and a pNCS-Benzyl-NODAGA derivative resulting in compounds 9, 10 and 11. The latter were prepared in yields of 84, 63 and 80 % respectively, with purity > 99 % confirmed by RPHPLC (Table 1). The ligands were also characterized by HRMS. Synthesis of the natGa complexes The complexes

nat

Ga-9,

nat

Ga-10 and natGa-11 were prepared using 1.2 to 1.8 equivalents of

Ga(NO3)3. The latter were obtained in yields ranging from 17 to 30 % after purification on semi-preparative HPLC. All the final complexes were > 99 % pure and were characterized by high-resolution mass spectrometry (HRMS) (Table 1) together with liquid chromatographyHRMS (see supporting information). Table 1: Analytical data of the second generation of AMD3100 analogues. HRMS

tR (min)a,b

Compound

calcd MW

obsvd MW

9

624.3169

624.3199 [M + 2H]2+

6.24

609.8063

609.8079 [M + 2H]

2+

6.43

2+

6.83

10 11

691.8267

691.8278 [M + 2H]

Ga-9

657.2707

657.2704 [M + 2H]2+

6.37

nat

Ga-10

642.7573

642.7568 [M + 2H]2+

6.32

nat

Ga-11

724.7778

724.7784 [M + 2H]2+

6.62

nat

a

HPLC

Using an HPLC gradient system as described in the Supporting information, tR = retention time. bPurity was >

99 % in all cases.


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Scheme 1: Synthesis of first and second generation of AMD3100 analogues. Reagents and conditions: (a) DOTA-NHS ester (1 eq.), NEt3 (23 eq.), DMF, 5h, R.T.; (b) NODAGA-NHS ester (1 eq.), NEt3 (8 eq.), DMF, 5h, R.T.; (c) NaOH (1M, 4 eq.), MeOH, 6 d, R.T.; (d) BocTOTA (1.1 eq.), HBtu (1.05 eq.), HOBt (1.1 eq.), DIPEA (1.5 eq.), DMF/CH2Cl2, overnight, 40°C; (e) 1) HCl (5M), overnight, R.T., 2) NaOH (16M), CHCl3; (f) Ni(NO3)3.6H2O (2 eq.), H2O/MeOH (1:2). (g) DOTA-NHS ester (1.2 eq.), NEt3 (8 eq.), DMF, 5 h, R.T.; (h) NODAGA-NHS ester (1.2 eq.), NEt3 (8 eq.), DMF, 5 h, R.T.; (i) p-NCS-Benzyl-NODAGA (1.1 eq.), NEt3 (8 eq.), DMF, 5 h, R.T..



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Radiochemistry 68

Ga labelling gives [68Ga]-2 with a radiolabelling yield of 70 % and a specific activity (S.A.)

of 0.2 MBq/nmol (Table 2). In the case of ligand 3, no radiolabelling was observed whatever the labelling conditions and the amount of ligand involved. 68

Ga labellings with the second generation of ligands gives [68Ga]-9 and [68Ga]-11 in 97 %

and 99 % radiochemical yields with specific activities of about 16 MBq/nmol. The NODAGA derivative shows lower radiolabelling performances with a radiolabelling yield of only 40 % Table 2: Radiolabelling of ligands of first and second generation. Yield (%)

S.A.

Radiochemical

(MBq/nmol)

purity (%)

logPoctanol/PBS

[68Ga]-2

70

0.2

70

-

[68Ga]-3

-

-

-

-

[68Ga]-9

97

15

81

-3.10 ± 0.18

[68Ga]-10

40

-

39

-

[68Ga]-11

99

17

92

-1.50 ± 0.18

LogPoctanol/PBS The logPoctanol/PBS values of [68Ga]-9 and [68Ga]-11 are -3.10 ± 0.18 and -1.50 ± 0.18 respectively (Table 2). (Lipophilicity = the logarithm of the partition coefficient P, where P is the ratio of the distribution of a compound in two solvents, here octanol and PBS).

Competition binding / Internalisation studies – Flow cytometry assay The IC50 values of

nat

Ga-9,

nat

Ga-10 and

nat

Ga-11 along with the reference compound

AMD3100 are summarised in Table 3. The IC50 tendency was confirmed by flow cytometry and the percentages of antibody inhibition are also summarised in Table 3. All compounds showed lower binding affinities as compared to AMD3100 and the affinities are highly dependent on the chelator. Among the three gallium complexes, p-NCS-Benzyl-NODAGA derivative

nat

Ga-11 has the highest binding affinity with an IC50 of 121 nM and an inhibition


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percentage of 89.7 %, which is lower than AMD3100 (94.7 %). On the other hand,

nat

Ga-10

has the lowest affinity, with an IC50 of 1485 nM and a percentage of antibody inhibition of 33.6 %.

nat

Ga-9 has a moderate affinity with an IC50 of 516 nM and a percentage of mAb

inhibition of 40.6 %. Table 3: IC50 of CXCR4 ligands for competition binding of 125I-SDF1α and inhibition percentages obtained by flow cytometry. IC50 (nM)

pIC50

% mAb inhibition

14

7.86 ± 0.16

94.7

AMD3100 nat

Ga-9

516

6.29 ± 0.06

40.6

nat

Ga-10

1485

5.82 ± 0.16

33.6

nat

Ga-11

121

6.92 ± 0.33

89.7

The two most promising radiotracers, [68Ga]-9 and [68Ga]-11, were then incubated with H69 cells to determine the total cell uptake and the internalised radioactivity. After 30 min of incubation with the cells, [68Ga]-11 shows higher total cell uptake (1.77 ± 0.10 %) compared to [68Ga]-9 (0.61 ± 0.22 %) (Table 4). About one third of the cell-associated activity for both radiotracers was internalised into the cells at the same time point (0.59 ± 0.13 % for [68Ga]-11 and 0.28 ± 0.09 % for [68Ga]-9).

Table 4: Binding and internalisation of [68Ga]-9 and [68Ga]-11 on H69 CXCR4 expressing cells.

[68Ga]-9

[68Ga]-11

Total cell uptake (%)

Surface Bound (%)

Internalised (%)

30 min

0.61 ± 0.22

0.33 ± 0.17

0.27 ± 0.09

1h

0.28 ± 0.05

0.13 ± 0.03

0.15 ± 0.01

30 min

1.77 ± 0.10

1.18 ± 0.03

0.59 ± 0.13

1h

1.67 ± 0.27

1.05 ± 0.05

0.46 ± 0.11

Biodistribution study and PET imaging of [68Ga]-11



Biodistribution study upon the injection of 10 pmol of [68Ga]-11 in H69 xenografted nude mice was performed 1 h post-injection (Figure 2). High accumulation of radioactivity was observed in organs such as liver, spleen, lung and bone marrow with up to 67.97 ± 7.93 %IA/g in the liver. Tumor uptake was 1.27 ± 0.38 %IA/g at 1 h p.i. The co-injection of an excess of AMD3100 (20 nmol) resulted in a clear decrease of activity uptake in organs such as liver, spleen, lung and bone marrow and in the tumor with 0.60 ± 0.03 %IA/g, confirming the receptor-mediated uptake. PET images were obtained in H69 xenografted nude mice upon injection of 100 pmol, one hour post-injection (supplementary information).

80

Unblocked Blocked

60

40

% AI/g

20 6

4

2

0

Bl oo d H ea rt Li ve Sp r le en Lu n K g id S t n ey om I n a ch te st A ine dr e Pa nal nc re a M s us cle Bo ne Bo M ne H arr 69 ow -tu m or

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Figure 2: Biodistribution of [68Ga]-11 on H69 xenografted nude mice.

Discussion The purpose of this study was to develop new

68

Ga-based PET imaging agents for CXCR4

imaging, using a novel approach consisting in functionalising a small CXCR4 antagonist (AMD3100) with a specific radiochelatant. The in vivo evaluation of the previously studied 64

Cu-AMD3100 demonstrated high liver uptake that could be due to the high lipophilicity of

the radiotracer (logPoctanol/PBS = 0.52) and/or the in-vivo instability of the copper complex resulting in a dissociation phenomenon and metal release in the organism.40,44 A first


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approach to determine the influence of the Cu-AMD3100 complex stability was recently investigated, using an AMD3100 analogue named AMD3465,53 where the cyclam moiety was replaced by a cross-bridged cyclam known to be six to eight times more stable than their nonbridged counterparts.54 However, the configurationally restricted macrocycle did not result in a significant decrease of the liver uptake.55 In an attempt to overcome these undesired in vivo properties, AMD3100 was functionalised and coupled to two different chelators, which present favourable chelating properties for labelling with

68

Ga, namely DOTA and

NODAGA. Our goal was indeed to use these specific chelators for the coordination of the radionuclide instead of using the cyclam cavities of AMD3100 and to figure out if this approach prevents transchelation phenomenon and results in an improved pharmacokinetic performance of the derived radiotracers. Two different spacers were used to link the chelators to AMD3100 (ethylenediamine and diamino-polyethylene glycol 3 (PEG3)), in order to study the influence of the spacer on the properties (chelation, hydrophilicity) of the resulting systems. We herein describe the synthesis and the preclinical evaluation of

68

Ga-labeled AMD3100-

based radiotracers for the diagnostic imaging of CXCR4 receptor, with a particular focus on the radiolabelling, lipophilicity, affinity and binding studies, biodistribution study, blocking study and PET study in mice bearing CXCR4-overexpressing human small cell lung cancer H69 tumors. A bis-nickel AMD3100 analogue functionalised on the phenyl moiety with an ethylenediamine spacer45 was chosen as a starting material for the synthesis of the first generation of specific CXCR4-based radiotracers. Indeed, NiII-cyclam complexes are known to be particularly inert due to their stability constant indicative of high thermodynamic stability (log K = 19.9-20.3)56 and their slow dissociation kinetics.57 Complexes persist almost indefinitely even in strong acidic solutions and the only reported method to remove Ni2+ from



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cyclam involves cyanide at high temperature. The latter should then persist during radiolabelling with 68Ga. This starting material enables the introduction of a specific chelator suitable for labelling with

68

Ga, while the cyclam cavities have been “blocked” with Ni(II).

DOTA and NODAGA were conjugated to compound 1 but even though these chelators can efficiently be radiolabeled under the conditions used (pH 3.0-4.0, 95 °C), especially NODAGA, ligands 2 and 3 showed poor labelling capabilities. Therefore, a second generation of AMD3100 analogues was synthesised with the combination of a longer spacer (PEG3) and a wider range of chelators, to obtain compounds 9, 10 and 11. The introduction of a longer PEG3 derivative spacer had a marked influence on the labelling properties of the ligands. The DOTA derivative 9 demonstrated good labelling properties, with a 18-fold improved specific activity compared to compound 2. p-NCS-Benzyl-NODAGA was also conjugated to study the influence of the introduction of a lipophilic phenyl group in the spacer and resulted in compound 11. The latter showed similar labelling performances as compound 9 but the NODAGA derivative 10, was still difficult to radiolabel. No explanation can be given at this stage; a hypothesis may be that the flexible PEG spacer allows the folding of the chelator over one of the NiII-cyclam complexes, one of the free carboxylic acid of NODAGA could then complete the coordination sphere of the complex therefore making the labelling difficult. Nonradioactive gallium complexes of ligands 9, 10 and 11 were then synthesised successfully to evaluate the affinity of these potential radiotracers toward the CXCR4 receptor. Significant difference in natGa-9, natGa-10 and natGa-11 IC50 values can be observed. The overall charge of the DOTA and NODAGA-gallium complex is neutral for both compounds; the difference in affinity is therefore not due to this parameter. NODAGA and DOTA chelators differ in size, the bulkier DOTA derivative, NODAGA derivative,

nat

Ga-9, has a three times higher affinity than the

nat

Ga-10. The introduction of a bulky chelator may therefore help the

chelating moiety of the radiotracer to be distanced from the CXCR4 binding pocket and thus


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enhance the interaction of the AMD motif with the receptor. In the case of

nat

Ga-11, the

introduction of a lipophilic phenyl moiety and by the same time, the increase of the length of the linker leads to a clear increase of the affinity by a factor of 4 compared to the DOTA derivative natGa-9, and by a factor of 12 compared to the NODAGA derivative natGa-10. The affinity trend of our radiotracers was also confirmed by flow cytometry. Once again the NODAGA derivative

nat

Ga-10 has the lowest affinity with the lowest percentage of antibody

inhibition (33.6 %), the DOTA derivative has a moderate affinity (40.6 %) and the highest percentage of antibody inhibition was observed with the p-NCS-Benzyl-NODAGAconjugated AMD moiety,

nat

Ga-11 (89.7 %). Both in vitro binding assays demonstrate the

same affinity trend and a clear influence of the chelator and the linker. The lipophilicity of the 68

Ga-complexes of compounds 9 and 11, -3.10 ± 0.18 and -1.50 ± 0.18 respectively,

highlights the significant lipophilic character brought to the compound 11 by the phenyl moiety. Furthermore, all the aforementioned modifications on the AMD3100 moiety resulted in a clear increase of the hydrophilic character of the newly developed radiotracers compared to the previously reported

64

Cu-AMD3100 (logPoctanol/PBS = 0.52).44 The two best candidates

for further evaluation are therefore [68Ga]-9 and [68Ga]-11 and were studied in binding and internalisation assays on H69 cells. This cell line was chosen as a tumor model, as flow cytometry experiments demonstrated high expression of the CXCR4 receptor (see supporting information). The total cell associated uptake is low for each radiotracer but correlates with the previously determined IC50 values. For each radiotracer, about one third of the compound is internalised. This phenomenon translates the partial agonist character of our AMD3100 analogues. Internalisation of the CXCR4 receptor induced by AMD3100 was already previously reported. 58,59 [68Ga]-11, having the highest affinity, was chosen for further in vivo studies. The in vivo pharmacokinetic of [68Ga]-11 is characterised by a high accumulation in immune-related organs with high expression of the CXCR4 messenger RNA, liver, spleen,



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lung and bone marrow at one hour post-injection and is comparable to the biodistribution of 64

Cu-AMD3100.40 However, the accumulation of the radioactivity in the tumor was low with

a value of 1.27 ± 0.38 %IA/g. This is due to the lower IC50 value of our radiotracer compared to 64Cu-AMD3100. In vivo competition by co-injection of a blocking dose of AMD3100 (20 nmol) led to the significant reduction of the radioactivity accumulation in CXCR4 expressing organs and in the tumor, thus demonstrating the specificity of [68Ga]-11 for the CXCR4 receptor. Activity accumulation in other organs was not affected except for the kidney uptake, which increased two-fold and can be explained by the faster excretion of the radiotracer when the receptors are blocked. Even though [68Ga]-11 has a low accumulation in the tumor, its specificity for the CXCR4 receptor supports our proof of principle. At this stage, no improvement of the in vivo behaviour was observed compared to the previously described 64

Cu-AMD3100. Nevertheless, our approach offers the possibility to modify fundamental

parameters of radiopharmaceuticals such as the linker, the chelator or the radionuclide and therefore pave the way toward the development of newly designed AMD-based radiopharmaceuticals with better pharmacokinetics.

Conclusions The functionalisation on the phenyl moiety of AMD3100 via the introduction of different spacers and chelators results in newly developed CXCR4 receptor PET imaging agents. The introduction of a chelator does increase the hydrophilicity of the final radiotracer when radiolabelled with 68Ga but did not result in lower liver uptake compared to 64Cu-AMD3100. Spacer and chelator proved to be important pharmacokinetic modifiers. In the case of the functionalisation of the small CXCR4 antagonist, AMD3100, a PEG3 spacer and a lipophilic moiety like a phenyl group are the best combinations found yet. The importance of the spacer for the labelling performance of our radiotracers was especially highlighted. The modification of the AMD3100 core leads to a decrease in the affinity toward the receptors but we are


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confident that improvement of the pharmacokinetics can be obtained by choosing a right combination of linker and chelator. Compound [68Ga]-11 proved to be CXCR4 specific in vivo and supports the proof of principle that AMD3100 could be an efficient platform for the introduction of imaging probes for CXCR4 targeting molecular imaging.

Experimental Procedures The supplier information for all reagents and details of instruments used are provided in the supplemental data (available at …). Synthesis of AMD derivatives of first generation. Compound 2 and 3 were synthesised starting from compound 1, an AMD3100 derivative functionalised on the phenyl moiety with an ethylenediamine spacer which synthesis was previously described by our group in a recent paper.45 Compound 2: Triethylamine (155.6 µL, 1.12 mmol) and DOTA NHS ester + HPF6 + TFA (107.5 mg, 0.05 mmol) were added subsequently to a stirred solution of 1 + 4 NO3 + 6 H2O (150.0 mg, 0.05 mmol) in DMF (15 mL). The reaction mixture was stirred for 5 hours and the solvent was evaporated in vacuo. Inverse phase flash chromatography (A: H2O, B: CH3CN, B 41 %) gave 2 + HPF6 + TFA + NEt3 + 10 H2O (50.4 mg, yield = 20 %) as a light purple powder. HRMS-ESI: m/z = calculated for [C47H86N14O8Ni2 + 2H]2+: 544.2646, obtained 544.2650. Elemental analysis for C47H83N14O8Ni2 + HPF6 + TFA + NEt3 + 10 H2O: Calculated: C (40.60 %), H (7.19 %), N (12.91 %); Obtained: C (40.91 %), H (7.23 %), N (12.67 %). HPLC (System 1): tr = 5.75 min, purity 95 %. Compound 3: Triethylamine (155.6 µL, 1.12 mmol) and NODA-GA NHS ester + HPF6 + TFA (104.0 mg, 0.14 mmol) were added subsequently to a stirred solution of 1 + 4 NO3 + 6 H2O (150.0 mg, 0.14 mmol) in DMF (15 mL). The reaction mixture was stirred for 5 hours and the solvent was evaporated in vacuo. Inverse phase flash chromatography (A: H2O, B: CH3CN, B 12 %) gave 2 + HPF6 + TFA + NEt3 + 9 H2O (44.3 mg, yield = 20 %) as a purple



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powder. MALDI-TOF: m/z = 1058.527 [M+H]+. HRMS-ESI: m/z = calculated for [C46H79N13O8Ni2

+ 2H]2+: 529.7514, obtained

529.7491. Elemental analysis for

C46H80N13O8Ni2 + HPF6 + TFA + NEt3 + 9 H2O: Calculated: C (40.95 %), H (7.32 %), N (12.38 %); Obtained: C (41.15 %), H (7.17 %), N (12.05 %). HPLC (System 1): tr = 5.83 min, purity 91 %. Synthesis of AMD derivatives of second generation. Compound 5: Compound 5 was synthesised starting from compound 4, an AMD3100 derivative functionalised on the phenyl moiety with an ester moiety which synthesis was previously described by our group in a recent paper.45 Compound 4 (300.0 mg, 0.26 mmol) was dissolved in methanol (2.0 mL) and a solution of NaOH (1M) (1.04 mL, 1.04 mmol) was added. The reaction mixture was stirred during six days. After evaporation of methanol, the resulting product was dissolved in water (50 mL). The aqueous layer was acidified progressively until pH 1 and the desired product was extracted with diethylether (3*50 mL). 5 + Et2O (284.0 mg, yield = 95 %) was obtained as a white foam. 1H NMR (300 MHz, CDCl3, 300 K) δ (ppm): 1.25-1.52 (m, 54H; CH3Boc), 1.60-1.70 (m, 2H), 1.73-1.97 (m, 6H), 2.252.41 (m, 2H), 2.50-2.75 (m, 4H), 2.82-2.99 (m, 2H), 3.08-3.51 (m, 24H), 3.54 (br.s, 2H), 3.69-3.91 (m, 2H), 7.03-7.18 (m, 1H), 7.29-7.45 (m, 1H), 7.96-8.11 (m, 1H). HRMS-ESI: m/z = calculated for C59H102N8O14 + Na+: 1169.7408, obtained 1169.7396. Elemental analysis for C59H102N8O14 + Et2O: Calculated: C (61.18 %), H (9.43 %), N (9.36 %); Obtained: C (61.00 %), H (9.35 %), N (9.65 %). HPLC (System 1): tr = 6.48 min, purity 94%. Compound 6: HBtu (372 mg, 0.98 mmol), HOBt (133 mg, 1.03 mmol) dissolved in DMF (2 mL) and N,N-diisopropylethylamine (245 µL, 1.41 mmol) were added to a solution of 5 (1.08 mg, 0.94 mmol) in dichloromethane (45 mL) and the solution was stirred at 40 °C for 20 min. 1-(t-Butyloxycarbonyl-amino)-4,7,10-trioxa-13-tridecanamine


(331

mg,

1.03

mmol)

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dissolved in 5 mL of dichloromethane was added to the previous reaction mixture and the solution was stirred at 40 °C for one night. Dichloromethane, dimethylformamide and N,Ndiisopropylethylamine were evaporated in vacuo. The resulting product was dissolved in dichloromethane (200 mL) and washed with a citric acid solution (2.2 M) (50 mL), a saturated solution of sodium bicarbonate (50 mL) and finally water (50 mL). The organic layer was dried on magnesium sulfate and evaporated in vacuo. Inverse phase flash chromatography (A: H2O, B: CH3CN + 0.1 % TFA, B 50 %) and normal phase flash chromatography (A: CH2Cl2, B: CH3OH, B 8 %) gave compound 6 (675 mg, yield = 50 %) as a white foam. 1H NMR (500 MHz, CDCl3, 330 K) δ (ppm): 1.38 (s, 9H; Boc), 1.39 (s, 9H; Boc), 1.41 (s, 9H; Boc), 1.43 (s, 18H; 2*Boc), 1.45 (s, 9H; Boc), 1.46 (s, 9H; Boc), 1.72 (quint, 2H, 3J(H,H) = 6.3 Hz), 1.83 (sext, 4H, 3J(H,H) = 6.8 Hz), 1.92 (quint, 4H, 3J(H,H) = 6.6 Hz), 1.99 (quint, 2H, 3J(H,H) = 7.1 Hz), 2.87-2.97 (m, 2H), 3.01-3.10 (m, 2H), 3.17 (quad, 4H, 3J(H,H) = 6.2 Hz), 3.26-3.43 (m, 22H), 3.43-3.66 (m, 19H), 4.05 (s, 2H), 4.42 (s, 2H), 7.56 (d, 1H, 3J(H,H) = 7.6 Hz), 7.76 (d, 1H, 3J(H,H) = 7.8 Hz), 7.83 (s, 1H), 8.10-8.30 (br.s, 1H, NHamide).

13

C{1H} NMR (125 MHz, CDCl3, 330 K) δ (ppm): 24.7, 25.3, 25.6,

28.60, 28.63, 28.74, 28.77, 28.78, 28.91, 29.06, 29.57, 30.1, 44.6, 46.8, 46.9, 47.08, 47.12, 47.5, 47.7, 48.2, 48.3, 48.8, 49.1, 51.6, 52.0, 52.4, 53.5, 57.1, 58.7, 69.5, 69.7, 70.41, 70.46, 70.77, 70.82, 80.28, 80.3, 80.5, 80.7, 81.1, 81.2, 115.7, 118.1, 130.7, 133.5, 134.1, 137.4, 155.83, 155.85, 155.9, 156.0, 156.2, 156.4, 169.2. HRMS-ESI: m/z = calculated for C74H132N10O18 + H+: 1449.9838, obtained 1449.9794 and calculated for C74H132N10O18 + Na+: 1471.9557, obtained 1471.9613. Elemental analysis for C74H132N10O18 + 3 CH2Cl2: Calculated: C (54.25 %), H (8.16 %), N (8.22 %); Obtained: C (54.21 %), H (8.12 %), N (8.11 %). HPLC (System 2): tr = 10.58 min, purity 92%. Compound 7: Compound 6 (645.0 mg, 0.44 mmol) was dissolved in a solution of 5 M HCl (10 mL). The mixture was stirred overnight at room temperature and lyophilized. Compound



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7 + 3 HCl + 15 H2O was obtained as a light yellow solid (437 mg, yield = 88 %). HRMS-ESI: m/z = calculated for C39H76N10O4 + H+: 749.6124, obtained 749.6112. Elemental analysis for C39H76N10O4 + 3 HCl + 15 H2O: Calculated: C (41.50 %), H (9.73 %), N (12.41 %); Obtained: C (41.39 %), H (10.20 %), N (12.85 %). Few drops of NaOH (16 mol.L-1) were added on compound 6 + 3 HCl + 15 H2O in order to obtain a viscous solution and chloroform (100 mL) was added quickly. The two layers were separated and the organic layer was dried over magnesium sulfate. After evaporation of the solvent, 7 + 0.7 CHCl3 (275 mg, yield = 75 %) was obtained as a light yellow oil. 1H NMR (500 MHz, CDCl3, 300 K) δ (ppm): 1.55-1.63 (m, 4H; CH2β), 1.66 (q, 2H, 3J(H,H) = 6.5 Hz; CH2CH2CH2 PEG), 1.69-1.76 (m, 2H; CH2β), 1.78-1.83 (m, 2H), 1.86 (q, 2H, 3J(H,H) = 6.5 Hz; CH2CH2CH2 PEG), 2.29-2.84 (m, 42H), 3.40-3.63 (m, 18H), 7.22 (d, 1H, 3J(H,H) = 7.8 Hz; CHar meta), 7.40 (s, 1H; CHar ortho), 7.51 (d, 1H, 3J(H,H) = 7.8 Hz; CHar para), 8.46 (t, 1H; NHamide). 13C{1H} NMR (125 MHz, CDCl3, 330 K) δ (ppm): 26.5, 26.8, 28.3, 28.5, 30.1, 33.9, 37.9, 39.87, 39.90, 47.7, 47.9, 48.1, 48.17, 48.21, 48.4, 48.8, 48.9, 49.1, 49.4, 50.3, 50.8, 53.3, 53.5, 53.9, 54.3, 54.7, 56.4, 58.3, 69.8, 70.5, 70.6, 70.9, 128.7, 130.6, 136.0, 138.0, 138.2, 170.1. HRMS-ESI: m/z = calculated for C39H76N10O4 + H+: 749.6124, obtained 749.6110. Elemental analysis for C39H76N10O4 + 0.7 CHCl3: Calculated: C (57.27 %), H (9.28 %), N (16.82 %); Obtained: C (57.05 %), H (9.54 %), N (17.18 %). HPLC (system 2): tr = 5.69 min, purity 96%. Compound 8: A titrated solution of Ni(NO3)2, 6 H2O (5.76 mL, 0.59 mmol, c = 0.096 mol.L-1) in water was added to a solution of 7 + 0.7 CHCl3 (216 mg, 0.29 mmol) in 10 mL of MeOH. The reaction was stirred at 50 °C overnight. After completion, reverse phase C18 semipreparative chromatography (System A, tr = 23 min) was performed to furnish 8 + 5 TFA + 4 H2O (258 mg, 60 %) as a light purple foam. ESI-MS: m/z = 463.71 [M + NO3 - H]2+, 494.21 [M+ 2 NO3]2+. MALDI-TOF: m/z = 861.365 [M+H]+. HRMS-ESI: m/z = calculated for [C39H72N10Ni2O4

+ 2H]2+: 431.2295, obtained

431.2297. Elemental analysis for


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C39H72N10Ni2O4 + 5 TFA + 4 H2O: Calculated: C (39.11 %), H (5.69 %), N (9.31 %); Obtained: C (39.07 %), H (5.25 %), N (9.09 %). HPLC (System 2): tr = 6.13 min, purity 100 %. Compound 9: Triethylamine (9.5 µL, 68 µmol) and DOTA NHS ester + HPF6 + TFA (7.8 mg, 10.3 µmol) were added subsequently to a stirred solution of 8 + 5 TFA + 4 H2O (13.2 mg, 8.6 µmol) in DMF (400 µL). The reaction mixture was stirred for 5 hours on a mixer at room temperature. Reverse phase C8 semi-preparative chromatography (System C, tr = 25 min) gave 8 (10.93 mg, yield = 84 %) as a light purple powder. ESI-MS: m/z = 624.32 [M + H]2+. MALDI-TOF: m/z = 1247.426 [M+H]+, 1269.406 [M+Na]+, 1285.370 [M+K]+. HRMSESI: m/z = calculated for [C55H98N14O11Ni2 + 2H]2+: 624.3196, obtained 624.3199. HPLC (System 1): tr = 6.24 min, purity 100 %. Compound 10: Triethylamine (7.3 µL, 52 µmol) and NODAGA NHS ester + TFA+ HPF6 (5.8 mg, 7.9 µmol) were added subsequently to a stirred solution of 8 + 5 TFA + 4 H2O (10.1 mg, 6.5 µmol) in DMF (400 µL). The reaction mixture was stirred for 5 hours on a mixer at room temperature. Reverse phase C8 semi-preparative chromatography (System B, tret = 27 min) gave 10 (6.0 mg, yield = 63 %) as a light purple powder. ESI-MS: m/z = 610.77 [M + H]2+. MALDI-TOF: m/z = 1218.500 [M+H]+, 1256.448 [M+K]+. HRMS-ESI: m/z = calculated for [C54H95N13O11Ni2 + 2H]2+: 609.8063, obtained 609.8079. HPLC (System 2): tr = 6.43 min, purity 100 %. Compound 11: Triethylamine (6.9 µL, 50 µmol) and p-NCS-Benzyl-NODAGA (3.6 mg, 6.9 µmol) were added subsequently to a stirred solution of 8 + 5 TFA + 6 H2O (9.6 mg, 6.3 µmol) in DMF (400 µL). The reaction mixture was stirred for 5 hours on a mixer at room temperature. Reverse phase C18 semi-preparative chromatography (System C, tr = 26 min) gave 11 (8.6 mg, yield = 80 %) as a light purple powder. ESI-MS: m/z = 692.81 [M + 2H]2+.



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HRMS-ESI: m/z = calculated for [C62H103N15O11Ni2S + 2H]2+ :691.8267, obtained 691.8278. HPLC (System 2): tr = 6.83 min, purity 100 %. Preparation of nonradioactive gallium complexes. Compound natGa-9: Compound 9 (4.62 mg, 3.7 µmol) and a solution of Ga(NO3)3, x.H2O in water (17.99 µL, 4.43 µmol, 0.25 M) were dissolved in HEPES buffer (300 µL, pH = 3.5, 0.1 M). The reaction mixture was stirred for one night at 35 °C on a thermomixer. Compound was purified by reverse phase C8 semipreparative chromatography (System B, tr = 26 min) to give

nat

Ga-9 (0.81 mg, yield = 17 %)

as a light purple powder. MALDI-TOF: m/z = 1315.552 [M+H]+. HRMS-ESI: m/z = calculated for [C55H95GaN14Ni2O11S + 2H]2+: 657.2707, obtained 657.2704. HPLC (System 4): tr = 6.37 min, purity 100 %. LC-HRMS: tr = 1.61 min, purity 100 %, m/z = calculated for [C55H95GaN14Ni2O11S + 3H]3+: 438.5162, obtained 438.5170. Compound

nat

Ga-10: Compound 10 (3.41 mg, 2.7 µmol) and a solution of Ga(NO3)3, x.H2O

in water (13.52 µL, 3.41 µmol, 0.25 M) were dissolved in HEPES buffer (300 µL, pH = 3.5, 0.1 M). The reaction mixture was stirred for one night at 35 °C on a thermomixer. Compound was purified by reverse phase C8 semi-preparative chromatography (System B, tret = 26 min) to give

nat

Ga-10 (0.81 mg, yield = 17 %) as a light purple powder. MALDI-TOF: m/z =

1286.581 [M+H]+. HRMS-ESI: m/z = calculated for [C54H92GaN13Ni2O11 + 2H]2+: 642.7573, obtained 642.7568. HPLC (System 2): tr = 6.32 min, purity 100 %. LC-HRMS: tr = 1.61 min, purity 100 %, m/z = calculated for [C54H92GaN13Ni2O11 + 3H]3+: 428.8407, obtained 428.8421. Compound

nat

Ga-11: Compound 11 (4.68 mg, 2.2 µmol) and a solution of Ga(NO3)3, x.H2O

in water (26.0 µL, 4.1 µmol, 0.15 M) were dissolved in HEPES buffer (300 µL, pH = 3.5, 0.1 M). The reaction mixture was stirred for one night at 35 °C on a thermomixer. Compound was purified by reverse phase C18 semi-preparative chromatography (System C, tret = 27 min)


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to give

nat

Ga-11 (1.49 mg, yield = 30 %) as a light purple powder. MALDI-TOF: m/z =

1450.610 [M+H]+. HRMS-ESI: m/z = calculated for [C62H100GaN15Ni2O11S + 2H]2+: 724.7778, obtained 724.7784. HPLC (System 2): tr = 6.62 min, purity 100 %. LC-HRMS: tr = 1.61 min, purity 100 %, m/z = calculated for [C62H100GaN15Ni2O11S + 3H]3+: 483.5210, obtained 483.5223. Radiochemistry. The 68Ge/68Ga-generator was eluted with 7 mL HCl 0.1 N and the eluate (~ 350 MBq) was loaded onto a cation exchange column (Strata-XC, Phenomenex). 68Ga was eluted with 800 µL of a mixture of NaCl sol./HCl (5 N/5.5 N) directly in a vial containing 400 µL of Na acetate buffer (0.1 M, pH 4.0), 2 mL of water and the corresponding ligand (17-180 nmol). The reaction mixture was heated for 6-20 min at room temperature or 90 °C and purified afterward on a C18 light cartridge with 700 µL EtOH/H2O (50:50). The radiotracer solutions were prepared by dilution with 2 mL of 0.9% NaCl. All radiolabelled compounds were analyzed with (analytic) RP-HPLC. LogPoctanol/PBS. The lipophilicity (log D, pH 7.4) was estimated by the “shake-flask” method: The labeled conjugates (10 µL) were added to a solution of 1-octanol (500 µL) and PBS (500µL, pH 7.4). The mixture was vortexed for 30 min to reach the equilibrium and then centrifuged (300 rpm) for 10 minutes. For each phase, an aliquot (10 µL) was pipetted out and measured in a gamma-counter. Each measurement was repeated five times. Care was taken to avoid-cross contamination between the phases. The partition coefficient was calculated as the average log ratio of the radioactivity in the organic fraction and the PBS fraction. Cell cultures. Human leukemia T cell lymphoblasts (Jurkat) and human small cell lung cancer cells (h-SCLC) H69 were cultured in RPMI 1640 medium treated with sterile filtered fetal bovine serum (FBS) (10 %) and penicillin and streptomycin (100 units/ 5 mL) antibiotics. The cells cultures were maintained at 37 °C in a humidified, CO2 (5 %) controlled



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atmosphere with subculturing done every 2-3 days as appropriate. Trypan-blue exclusion test was used for the counting of the cells. Competition binding studies. For IC50 determination, samples containing 2x105 Jurkat cells (200 µL) in PBS/0.2 % Bovin Serum Albumin were incubated with approximately 60’000 cpm (25 µL)

125

I-SDF1α (Perkin Elmer) in the presence of increasing concentrations (10-9 to

10-3 M in 25 µL) of

nat

Ga-9-10-11 in a total sample volume of 250 µL. AMD3100 was used

as the control compound. Nonspecific binding was determined in the presence of cells (2x105, 200 µL) and the iodinated compound (25 µL, 2.4 kBq, 1.2 nM). All the tubes were gently shaken on a rocker-shaker for 2 hours at room temperature, the incubation was terminated by centrifugation at 1200 rpm for 3 min at 4°C. Cells pellets were washed twice with 400 µL of cold PBS. Experiments were repeated 2-3 times in duplicates. Cell bound radioactivity was determined using a γ-counter. IC50 values of the compounds were calculated by non-linear regression using GraphPad Prism. Flow cytometry assay with CXCR4 specific antibody 12G-5 conjugated to phycoerythrin. Jurkat cells are harvested at about 75% confluency, centrifuged, resuspended in 10 mL PBS and centrifuged again. Cells are kept on ice to prevent receptor internalisation. Cells are resuspended in 1.0 mL PBS/ 0.25 % BSA/ 0.01 M NaN3) and viable cells are counted using trypan blue exclusion test. Cells at density 1-2x105 cells (50 µL) were aliquoted into polypropylene Fluorescence-Activated Cell Sorting (FACS) tubes and preincubated with 50 µM of AMD3100 as a reference or natGa-9-10-11. (10 µL in 18.2 mΩ water) for one hour at 4 °C. A high concentration of compound was used to ensure saturation of the receptors. Thereafter, cells were washed with 1 mL of the buffer (PBS/ 0.25 % BSA/ 0.01 NaN3) to remove the excess of compound that did not bind to cells’ receptors. Cells were then incubated with the 12G-5 mAb conjugated to phycoerythrin for a further 60 min. The cells were washed with 1 mL of the buffer and put in suspension in 300 µL of the buffer. Negative


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control was performed using the same protocol without the preincubation step and with a mouse IgG2A. The potency of compounds is reported as a concentration required to inhibit a specified amount (%) of the mAbs. The Mean Fluorescent Intensity (MFI) was used as a measure of binding and a quantitative way of calculating the inhibition percentage of mAbs. % ℎ = 100 −

ℎ − !100" −

Internalisation studies. For internalization experiments, in tubes containing approx. 1x106 H69 cells (1.3 mL) in PBS/0.2 % Bovin Serum Albumin were added approximately 2.5 pmol/ 40 kBq of the radioligand [68Ga]-11 and the cells were incubated (in triplicates) for 0.5 and 1 h at 37 °C, 5 % CO2. To determine nonspecific membrane binding and internalization, excess of AMD3100 (200 µM, 20 nmol/100 µL) was added to selected tubes (n = 3). At each time point, the internalization was stopped by removing the medium and the subsequent centrifugation at 1200 rpm for 3 min at 4 °C. The cells were washed twice with ice-cold PBS (1 mL). To remove the receptor-bound radioligand, an acid wash was carried out twice with a 0.1 M glycine buffer pH = 2.8 for 5 min on ice. Cells were finally resuspended in 2 mL PBS. The radioactivity of the culture medium, the receptor bound, and the internalized fractions were measured with a γ-counter. Animal model. All animal experiments were conducted according to the regulations of the University Medical Center of Freiburg, Germany, and in accordance with the Guide for the Care and Use of Laboratory Animals.60 Normal female athymic Balb/c nude mice (17-20 g, 4-6 weeks old, n = 5) were obtained from Janvier SAS (St. Berthevin Cedex, France). Mice were provided with food and water ad libitum. For implantation, H69 cells were harvested and 107 cells in 100 µL of phosphate-buffered saline were inoculated subcutaneously into the right shoulder of the mice. After an average age of 3 weeks, tumor size reached



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approximately 200 mg and the animals were used for biodistribution and PET imaging studies. Biodistribution study in H69 xenografts. 10 pmol / 0.2 MBq of [68Ga]-11 in 100 µL NaCl solution (0.9 %, NaCl) were injected intravenously into the tail vein of H69 bearing mice. Animals (n = 2) were sacrificed by isoflurane anesthesia at 1h p.i.. Subsequently, the tissues and organs of interest were weighted and radioactivity counts were determined using a gamma-counter. Biodistribution data are given as percent of injected activity per gram of tissue (% IA/g) of each organ or tissue were then calculated taking into account the injected activity together with tail correction and are mean ± SD (n=2). To demonstrate the specificity of binding of [68Ga]-11, H69 bearing mice were co-injected with AMD3100 (20 nmol/100 µL). Small-animal PET Imaging. PET images were obtained upon injection of 100 pmol [68Ga]11 (1.8-1.9 MBq/100 µL). Static images were acquired for a period of time of 20 to 25 minutes at 1 h and 2 h post-injection.

Author information Corresponding authors: Franck Denat, e-mail: [email protected]; Christine Goze, e-mail: [email protected]; Helmut Maecke, e-mail: [email protected]

Acknowledgements Support was provided by the CNRS, the University of Burgundy and the Conseil Régional de Bourgogne through the 3MIM Project. S.P. and P.D. thank the Ministère de l’Enseignement Supérieur et de la Recherche for PhD grants. COST actions TD 1007 and TD 1004 are also acknowledged for financing STSM of S.P..


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Supporting information 1

H NMR spectra, HPLC and HR-MS analysis of the different compounds; displacement

curves of competition binding assay to CXCR4 of nat

125

I-SDF1α with AMD3100,

nat

Ga-9,

Ga-10 and natGa-11; flow cytometric analysis of the binding of 12G-5 mAb in competition

with compound natGa-9, natGa-10, and natGa-11 (50 µM) using Jurkat cells; PET imaging and biodistribution data of PET image of an athymic nude mice bearing a H69 tumour xenograft with [68Ga]-11. This supporting information is available free of charge on the ACS publications website.

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AMD3100: A Versatile Platform for CXCR4 Targeting 68Ga-Based

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