Synthesis and preliminary evaluation of [ 18F]-labeled 2-oxoquinoline derivatives for PET imaging of cannabinoid CB 2 receptor

INTRODUCTION: The cannabinoid receptor type 2 (CB2) is an important target for development of drugs and imaging agents for diseases, such as neuroinflammation, neurodegeneration, and cancer. Recently we reported synthesis and results of in vitro receptor binding of a focused library of fluorinated 2-oxoquinoline derivatives as CB2 receptor ligands. Some of the compounds demonstrated as good CB2-specific ligands with Ki values in the nanomolar to sub-nanomolar concentrations; therefore, we pursued the development of their (18)F-labeled analogues that should be useful for PET imaging of CB2 receptor expression. Here, we report the radiosynthesis of two (18)F-labeled 2-oxoquinoline derivatives, and preliminary in vitro and ex-vivo evaluation of one compound as a CB2-specific radioligand. METHODS: 4-[(18)F]Fluorobenzyl amine [(18)F]-3 was prepared by radiofluorination of 4-cyano-N,N,N-trimethylanilinium triflate salt followed by reduction with LiAlH(4) and then coupled with acid chlorides 11 and 12 to afford [(18)F]-13 and [(18)F]-14. In vitro CB2 receptor binding assay was performed using U87 cells transduced with CB2- and CB1-receptor. Ex-vivo autoradiography was performed with [(18)F]-14 on spleen, CB2- and CB1-expressing and wild type U87 subcutaneous tumors grown in mice. RESULTS: The radiochemical yields of [(18)F]-13 and [(18)F]-14 were 10%-15.0% with an average of 12% (n=10); radiochemical purity was > 99% with specific activity 1200 mCi/μmole. The dissociation constant Kd for [(18)F]-14 was 3.4 nM. Ex-vivo autoradiography showed accumulation of [(18)F]-14 in the CB2-expressing tumor. CONCLUSION: Two new [(18)F]-labeled CB2 ligands have been synthesized. Compound [(18)F]-14 appears to be a potential PET imaging agent for the assessment of CB2 receptor expression in vivo

Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain

<div><p>Histone deacetylases (HDAC’s) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa–specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([¹⁸F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed <i>in vitro</i>, in a panel of recombinant HDACs, and <i>in vivo</i> using PET/CT imaging in rats. [¹⁸F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [¹⁸F]DFAHA and previously reported [¹⁸F]FAHA. PET imaging with [¹⁸F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues <i>in vivo</i>. Furthermore, PET imaging with [¹⁸F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.</p></div

Synthesis of [¹⁸F]DFAHA and [¹⁸F]TFAHA.

<p>Reaction conditions are as follows: f) [¹⁸F]KF, K_2,2,2; 0.4mL ACN, 105°C 25 min.; g)[¹⁸F]KF, K_2,2,2; 0.4mL ACN, 105°C 25 min.</p

The structures of compounds and docking scores with HDAC8, a HDAC Class I enzyme.

<p>A lower docking score indicates higher affinity.</p

The mechanism of action of HDAC4.

<p>As compared to HDAC class I, HDAC class IIa enzymes exhibit significantly reduced ability to deacetylate. In class IIa enzymes the His976 located in the same position in the catalytic site as Tyr306 in class I HDACs does not serve as a hydrogen bond donor to bind to the carbonyl oxygen of the leaving acetyl group and thus reduces the susceptibility of carbonyl carbon to nucleophilic attack by the water, as in HDAC class I (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133512#pone.0133512.g004" target="_blank">Fig 4</a>).</p

Synthesis of DFAHA and TFAHA.

<p>Reaction conditions are as follows: a) RT overnight; b) 2mL DCM, stirred overnight.</p

The three parts of a HDAC substrate, the cap (red), the linker (green), and the leaving group (blue).

<p>The three parts of a HDAC substrate, the cap (red), the linker (green), and the leaving group (blue).</p

Synthesis of DFAHA and TFAHA precursors.

<p>Reaction conditions are as follows: c) pyridine, acetyl chloride added drop-wise at 0°C, stirred overnight at RT d) 0.9 eq. SOCl₂, 12 hr. stirred under argon at 40°C, catalytic DMF; e) DCM, triethylamine added drop-wise at 0°C, stirred 24 hr. under argon at RT.</p