Highly Selective, Amine-Derived Cannabinoid Receptor 2 Probes

Abstract

<jats:p>The endocannabinoid (eCB) system is implied in various human diseases ranging from central nervous system to autoimmune disorders. Cannabinoid receptor 2 (CB&lt;sub&gt;2&lt;/sub&gt;R) is an integral component of the eCB system. Yet, the downstream effects elicited by this G protein-coupled receptor upon binding of endogenous or synthetic ligands are insufficiently understood—likely due to the limited arsenal of reliable biological and chemical tools. Herein, we report the design and synthesis of CB&lt;sub&gt;2&lt;/sub&gt;R-selective cannabinoids along with their &lt;i&gt;in vitro&lt;/i&gt; pharmacological characterization (binding and functional studies). They combine structural features of HU-308 and AM841 to give chimeric ligands that emerge as potent CB&lt;sub&gt;2&lt;/sub&gt;R agonists with high selectivity over the closely related cannabinoid receptor 1 (CB&lt;sub&gt;1&lt;/sub&gt;R). The synthesis work includes convenient preparation of substituted resorcinols often found in cannabinoids. The utility of the synthetic cannabinoids in this study is showcased by preparation of the most selective high-affinity fluorescent probe for CB&lt;sub&gt;2&lt;/sub&gt;R to date.</jats:p>

Highly Specific, Fluorescent Cannabinoid Type 2 Receptor Probes Enable Applications in Microscopy, Flow Cytometry and FRET-based Binding Assays

“…Starting from a high-affinity recognition element, such as 1, [38] choice of the exit vector and linker-type for fluorophore attachment is considered the main challenge to ensure high affinity and selectivity. [40,41] We have recently reported that 7-nitrobenzofurazan (NBD) probe 2 shows excellent affinity to hCB2R (hCB2R Ki = 4.2 nM), paired with high selectivity over hCB1R (hKi ratio CB1R/CB2R >2000), suggesting a linker strategy for chemical probes based on amine 1.…”

“…[40,41] We have recently reported that 7-nitrobenzofurazan (NBD) probe 2 shows excellent affinity to hCB2R (hCB2R Ki = 4.2 nM), paired with high selectivity over hCB1R (hKi ratio CB1R/CB2R >2000), suggesting a linker strategy for chemical probes based on amine 1. [38] However, the NBD dye such as in 2 is not ideal for most imaging experiments due to its unfavorable photophysical properties (absorption/excitation maxima <500 nm, causing interference with lipofuscin-dependent tissue auto-fluorescence), low photostability and the pronounced sensitivity of quantum yields to medium polarity. [42] Thus, a series of fluorescent probes 3a-5 was prepared (Fig.…”

“…In addition, the model suggests a beneficial hydrogen-bond between the amide-proton of 3b and the carbonyl oxygen of Ser72. The primary amide in 3c decreased lipophilicity of the probe (for AlogP see SI Table S4), [38] but had detrimental effects on affinity and selectivity (hCB2R Ki = 330 nM, hKi ratio CB1R/CB2R = 8). Probe 5 (incorporating AttoThio12) performed best in terms of both affinity and selectivity, showing sub-nanomolar Ki to hCB2R as well as a 1500-fold selectivity over hCB1R.…”

“…As part of our efforts towards discovery of selective chemical probes for cannabinoid receptors, we have recently disclosed the synthesis and pharmacological evaluation of a series of high-affinity CB2R agonists with excellent selectivity over CB1R. [38] These studies showed that HU-308-derived [39] primary amine 1 can be conjugated to a variety of structures while retaining both high CB2R affinity and selectivity over CB1R. Herein, we report the synthesis and in-depth pharmacological evaluation of a series of CB2R specific, high-affinity fluorescent probes.…”