Development of the First Potential Nonpeptidic Positron Emission Tomography Tracer for the Imaging of CCR2 Receptors

Abstract Herein we report the design and synthesis of a series of highly selective CCR2 antagonists as 18F‐labeled PET tracers. The derivatives were evaluated extensively for their off‐target profile at 48 different targets. The most potent and selective candidate was applied in vivo in a biodistribution study, demonstrating a promising profile for further preclinical development. This compound represents the first potential nonpeptidic PET tracer for the imaging of CCR2 receptors.


Introduction
The CÀ C chemokine receptor type 2 (CCR2) is a key player in the trafficking of lymphocytes and monocytes/macrophages leading to the development of various pathophysiological processes like inflammatory and autoimmune diseases, [1] tumor growth and metastasis formation. [2] CCR2 receptor is increasingly gaining attention in the field of positron emission tomography (PET) imaging as a promising diagnostic target for lung inflammation, [3] injured heart [4] or pancreatic ductal adenocarcinoma (phase 1; NCT03851237, 1R01CA235672-01, 201807099). So far only a peptidic ligand that binds to the first extracellular loop of the CCR2 receptor ECL1i was applied in PET imaging either as 64 Cu-DOTA-ECL1i or 68 Ga-DOTA-ECL1i conjugate. [3][4] There are no small-molecule, nonpeptidic PET tracers for the imaging of CCR2 receptors reported thus far.
The CCR2 receptors share 71 % sequence identity and an overlapping expression pattern with the CÀ C chemokine type 5 (CCR5) receptors. [5] The CCR5 receptor is expressed on a variety of cells and tissues such as monocytes, macrophages, Tlymphocytes, microglia, dendritic cells, the endothelium, and vascular smooth muscle. The CCR2 expression is more restricted to certain cell types such as monocytes, NK (natural killer) cells, and T lymphocytes. [6] Many potent CCR2 ligands demonstrate affinity to both receptors. [7] In the past, we have reported the design and synthesis of novel, selective as well as dual-targeting CCR2 and CCR5 receptor antagonists, [8] as well as the positive allosteric modulators (PAMs) for the CCR5 receptors. [9] Based on this work we envisaged to evaluate fluorine-18 radiolabeled CCR2 targeting ligands as PET radiotracers for molecular imaging of inflammation and cancer.
Our previous structure-activity and structure-affinity studies of novel CCR2 and CCR5 receptors targeting compounds derived from TAK-779 (1) revealed a first strategy for the introduction of high CCR2 receptor selectivity. While TAK-779 (1) is more or less equipotent at CCR2 and CCR5 receptors introduction of a bulky isopropoxy residue at the 6-position of a pyridine ring as seen in compound 2 ( Figure 1) yielded different properties of binding to the active site residues within the CCR2 and CCR5 receptors, leading to higher CCR2 selectivity (Table 1

Results and Discussion
Following the idea of addressing different electrostatic properties by the isopropoxy derivative 2, a series of CCR2 selective antagonists 6 a-c bearing a flexible ω-fluoroalkoxy side chain was developed. The phenol derivative 3 served as a starting structure for the introduction of the alkoxy side chain. As the influence of the chain length on the CCR2 and CCR5 receptor activity/affinity should be examined, propyloxy (4 a, n = 3), pentyloxy (4 b, n = 5) and heptyloxy (4 a, n = 7) side chains were considered. The high acidity of the phenol 3 allows deprotonation with weak bases without affecting the primary alcohol of the ω-halogenalkanols used. The reactivity of the ω-halogenalkanols is dependent on the halogen leaving group (I > Br > Cl). However, the reaction with 3-iodopropan-1-ol led to dialkylation of 3 (second alkyl group attached to the tertiary amine).Because we assumed that the dialkylation was due to the high reactivity of 3-iodopropan-1-ol, 3-bromopropan-1-ol with reduced reactivity was employed. Reaction of 3 with 3bromopropan-1-ol and K 2 CO 3 in DMF afforded selectively 4 a in 84 % yield. The homologous alcohols 4 b and 4 c were prepared analogously by alkylation of phenol 3 with 5-bromopentan-1-ol and 7-bromoheptan-1-ol, respectively (Scheme 1). The compounds 5 a-c served as precursors for the development of fluorinated PET ligands [ 18 F]6a-c. The introduction of an 18 Fatom into the molecule requires a good leaving group. Therefore, the primary alcohols 4 a-c were converted into the tosylates 5 a-c. The reaction of the alcohols 4 a-c with tosyl chloride and 4-dimethylaminopyridine (DMAP, Steglich catalyst) provided the tosylates 5 a-c. 9 The in vitro receptor activities and affinities of the 18 F-labeled PET tracers cannot be recorded   directly with the radiolabeled ligands due to the small amount and the short physical half-life of the labeled compounds. Therefore, the in vitro activities and affinities to the CCR2 and other receptors/targets were determined using the non-radioactive 19 F-labeled analogues. The synthesis of the non-radioactive 19 F counterparts 6 a-c can be performed by direct fluorination of the alcohols 4 a-c or by the introduction of an appropriate leaving group and subsequent S N 2 substitution. Tetrabutylammonium fluoride (TBAF) is a common fluorinating reagent. The bulky tetraalkylammonium counterion reduces the ionic bond strength and generates a "naked" fluoride ion with a good solubility in organic solvents. [10] As the tosylates 5 a-c were already prepared for the radiolabeling reaction, they were also employed for the fluorination with TBAF. Tosylates 5 a-c were reacted with TBAF in THF, which afforded the pure fluoro derivatives 6 a-c in 73-84 % yield (Scheme 1). The compounds were evaluated for their in vitro CCR2 activities and affinities as well as their selectivity towards CCR5 receptors in functional as well as in binding assays. The hydroxyalkoxy derivatives 4 a-c display very low CCR2 and CCR5 receptor affinities and moderate to high CCR2 receptor potencies (Table 1). The heptyloxy derivative with a primary alcohol at the end (4 c) shows the highest CCR2 affinity/activity (IC 50 ([ 125 I]CCL2) = 83 nM, IC 50 (Ca 2 + -flux, hCCR2) = 1.1 nM, IC 50 (βarrestin, mCCR2) = 54 nM).
However, the fluoroalkoxy derivatives 6 a-c do not follow the same trend in their SAR. The pentyloxy compound 6 b displays the highest CCR2 binding in the [ 125 I]CCL2 assay with an IC 50 -value of 14 nM and the highest CCR2 activity in the Ca 2 + -flux assay using the human CCR2 receptor, indicating an IC 50value of 0.76 nM. Furthermore, 6 b is highly selective against CCR5 receptors (IC 50 (CCR5; [ 3 H]TAK-779) = 2 000 nM). The fluoroalkoxy derivatives 6 a-c show a probe-dependent CCR5 activity when compared to TAK-779; whereas TAK-779 (1) binding affects CCL5-as well as CCL4-dependent CCR5 receptor activity. The fluoroalkoxy derivatives 6 b and 6 c display only high activity in the cAMP-BRET CCL4-mediated assay with K b values of 65.4 and 27.1 nM, respectively. Only moderate activity is seen in β-arrestin and cAMP BRET CCL5-mediated CCR5 assays, indicating a different binding mode at the CCR5 receptors as compared with that of TAK-779 (1).
PET measurements can be performed with different radionuclides such as 11 C, 13 N, 15 O and 18 F. 13 N and 15 O are very limited due to their short physical half-life (< 10 min) and are mostly used for the imaging of perfusion processes. [11] [ 18 F]fluoride offers several advantages over [ 11 C]carbon, such as longer physical half-life (110 vs. 20 min), maximal β + -particle energy of 650 keV vs. 960 keV, and higher quality images with higher spatial resolution in PET measurements. [11][12] In our efforts to develop an 18 F-labeled CCR2 receptor PET ligand, we selected compounds 6 a-c for radiolabeling and subsequently compound 6 b for the first evaluation in adult C57Bl/6 mice. Besides the high CCR2 receptor affinity and high selectivity over CCR5 and other biological targets, compounds 6 a-c offered the ability to be labeled in an one step nucleophilic substitution (S N 2-type) reaction of the tosyloxy moiety of 5 a-c with [ 18 F] fluoride. Ligands [ 18 F]6a-c were prepared by direct nucleophilic replacement of the tosyloxy moiety with [ 18 F]fluoride in a lead- Table 2. Off-target affinities/activities of fluorinated ligands 6 a-c.

Conclusion
Previously uncovered structure-activity/affinity relationships at CCR2 and CCR5 led the way to the development of highly potent and selective CCR2 receptor antagonists 4 a-c and 6 a-c, which were further converted into potential 18 F-labeled PET

Experimental Section
Complete protocols for both chemical syntheses and biological methods together with characterization data are presented in the Supporting Information.

Author Contributions
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