Synthesis and In Vitro Autoradiographic Evaluation of a Novel High-affinity Radioiodinated Ligand for Imaging Brain Cannabinoid Subtype-1 Receptors

Abstract

There is strong interest to study the involvement of brain cannabinoid subtype-1 (CB₁₎ receptors in neuropsychiatric disorders with single photon emission computed tomography (SPECT) and a suitable radioligand. Here we report the synthesis of a novel high-affinity radioiodinated CB₁ receptor ligand ([¹²⁵I]8, [¹²⁵I]1-(2-iodophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxylate, [¹²⁵I]SD7015). By autoradiography in vitro, [¹²⁵I]8 showed selective binding to CB₁ receptors on human brain postmortem cryosections and now merits labeling with iodine-123 for further evaluation as a SPECT radioligand in non-human primate.

Cannabis sativa (marijuana) is one of the oldest known plant derived-therapeutics, owing mainly to its anti-nociceptive properties.¹ Other beneficial effects of cannabis intake may include anti-emesis and appetite stimulation. Conversely, the accompanying psychological “high” and memory impairment have limited its therapeutic value. The most abundant and psychoactive cannabinoid, Δ⁹-tetrahydrocannabinol (Δ⁹-THC, 1), binds with high affinity to two known body receptor systems. These receptors have been named cannabinoid subtype-1 (CB₁) and cannabinoid subtype-2 (CB₂).^2–4 CB₁ receptors have high-densities in brain and are implicated in a number of neuropsychiatric disorders⁵ such as, schizophrenia^6,7 and depression^8,9. CB₂ receptors are located mainly in the periphery and are of less interest to neuropsychiatric research.^10,11

Ability to image and measure brain CB₁ receptors non-invasively with radiation computed tomography would assist neuropsychiatric research and drug development. The development of radioligands suitable for imaging brain CB₁ receptors is therefore of strong importance. Recently, there has been a great deal of progress in the development of useable radioligands for positron emission tomography (PET). Some of the more notable PET radioligands (Figure 1) are [¹¹C]MePPEP ([¹¹C]2)^12,13, [¹⁸F]PipISB ([¹⁸F]3)^14,15, [¹⁸F]MK-9470 ([¹⁸F]4)^16,17, (−)-[¹¹C]SD5014 ((−)-[¹¹C]5)¹⁸, [¹¹C]OMAR ([¹¹C]6)^19,20, and [¹¹C]JHU75575 ([¹¹C]7)^20,21. In comparison, the development of radioligands for more widespread single-photon computed tomography (SPECT) is much less advanced. Due to the longer half-life of radioiodine for SPECT imaging (e.g., ¹²³I, t_1/2 = 13.2 h), an on-site cyclotron is not required for radiolabeling, as with PET, which allows for more widespread use in pharmaceutical, academic and hospital settings. Here we report the synthesis, receptor screening, radioiodination and in vitro autoradiographic evaluation of a novel radioiodinated CB₁ receptor radioligand ([¹²⁵I]4-cyano-1-(2-iodophenyl)-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide, [¹²⁵I]8, [¹²⁵I]SD7015). Our results strongly suggest that [¹²³I]8 would merit evaluation as a SPECT radioligand in vivo.

Figure 1.

Structures of [¹¹C]2, [¹⁸F]3, [¹⁸F]4, [¹¹C]5– 7, and [¹²⁵I]8. Asterisks denote positions of radiolabels.

Compound 8 was synthesized according to the procedure depicted in Scheme 1. Conversion of 2-iodoaniline into a diazonium salt, followed by treatment with ethyl 2-chloroacetoacetate in ethanol-water solution under basic conditions gave chloro[(2-iodophenyl)hydrazono]ethyl acetate (9). Heating of a solution of 9, 4-methoxybenzoylacetonitrile and N,N-diisopropylethylamine in tert-butanol to reflux gave 10 in low but adequate yield. Hydrolysis of 10 with LiOH in aq-tetrahydrofuran gave the corresponding carboxylic acid, which was then converted into the acyl chloride with oxalyl chloride and a catalytic amount of N,N-dimethylformamide. The synthesis of 8 was completed by coupling the acyl chloride with 1-aminopiperidine under basic conditions.

Scheme 1.

Synthesis of 8. Reagent, conditions and yields: a) concentrated HCl, NaNO₂, ethyl 2-chloroacetoacetate, NaOAc, EtOH– H₂O, 16 h, 72%; b) 4-methoxybenzoylacetonitrile, DIPEA, tert-BuOH, reflux, 16 h, 6%; c) aq-LiOH, THF, 65 °C, 4 h; d) DMF_(cat), (COCl)₂, DCM; e) 1-aminopiperidine, DIPEA, DCM, 2 h, 73%.

The trimethylstannylated precursor, 11, required for radiolabeling, was synthesized by refluxing a solution of the known bromo compound 7²⁰ and hexamethylditin in the presence of palladium catalyst (Scheme 2)

Scheme 2.

Radiosynthesis of [¹²⁵I]8. Reagents, conditions and yields: a) hexamethylditin, Pd(PPh₃)₄, toluene, 20 h, 32%; b) [¹²⁵I]NaI, chloramine- T, aq-HCl, 5 min, 48– 59%.

A radioligand for imaging brain CB₁ receptors would ideally possess adequately high affinity and moderate lipophilicity to promote rapid development of a high ratio of receptor-specific binding to non-specific binding in brain in vivo, that might then allow accurate computation of output measures, such as binding potential.^22,23 CB₁ receptors are one of the most abundant G protein-coupled receptors in brain, reaching a concentration (B_max) of 1752 fmol/mg protein (175 nM) in rat cerebellum.²⁴ Generally, a SPECT radioligand should show a substantial B_max/K_d value (> 5) to be successful. Hence, an effective CB₁ receptor radioligand might require only a moderately high affinity (K_i or K_d < 35 nM). Ligand 8 was found to have about ten-fold higher affinity (3.4 nM; Table 1) than perhaps needed. Calculated ligand lipophilicity (cLogD_7.4) can be an important predictor of blood–brain barrier penetration and brain non-specific binding. Moderate lipophilicity (cLogD_7.4 in the range 2.0 – 3.5) is usually preferred for adequate brain entry without incurring excessive non-specific binding in brain.²³ However, when target binding sites exist in high concentration, higher lipophilicity may be tolerated. For example, [¹¹C]MePPEP is a successful PET radioligand for brain CB₁ receptors despite having a high cLogD_7.4 value of 5.42.^12,13 The cLogD_7.4 value of 8 is 4.14 and appears more favorable for a radioligand than that of [¹¹C]MePPEP. Futhermore, the physiochemical and pharmacological properties of 8 compare well with other successful PET radioligands.^20,21 Therefore, ligand 8 presents acceptable CB₁ receptor affinity and lipophilicity for development as a SPECT radioligand (Table 1).

Table 1.

K_i values for CB₁ and CB₂ receptors, selectivities for CB₁ versus CB₂ receptors and calculated lipophilicities.

Ligand	CB1Ki (nM)a	CB2Ki (nM)a	CB1 versus CB2 selectivitya	cLogD7.4b
rimonabant	1.38 ± 0.17	927 ± 66	672 ± 94	6.01
8	3.40 ± 0.43	548 ± 23	161 ± 47	4.14

^aValues represent mean ± SD of three determinations.

^bcLogD_7.4 values were calculated using Advanced Chemistry Development (ACD) 9.2.

In addition to acceptable binding affinity and lipophilicity a candidate SPECT radioligand should be selective for binding to the target protein. Ligand 8, at 10 μM concentration, showed < 50% inhibition (n = 4) of radioligand binding to the sites: 5-HT_{1B –E}, 5-HT_{2A– C}, 5-HT₃, 5-HT_5A, 5-HT₆, 5-HT₇, α_1A,B, α_2A,B,β_1–3, D_{1– 4}, DOR, H_{1– 4}, M_1–5, NET, SERT,σ_1,2, V_1A,1B,2. K_i values (n = 3) of 93.5 ± 20.4 nM (5-HT_1A), > 10,000 nM (α_2C), 1,477 ± 148 nM (KOR), 1,496 ± 216 nM (MOR) and 3,166 ± 586 nM (TSPO) were found. Details of the employed binding assays may be found at the NIMH PDSP web site: http://pdsp.med.unc.edu. Hence, 8 was found to have excellent CB₁ receptor selectivity for development as a SPECT radioligand.

[¹²⁵I]8 was prepared by [¹²⁵I]iodo-destannylation of the corresponding trimethylstannyl precursor (11) with [¹²⁵I]NaI, aq-HCl and chloramine-T (oxidizer) in methanol (Scheme 2). The crude product was purified with high-performance liquid chromatography (HPLC) as described in Supplementary Information. The decay-corrected radiochemical yield of [¹²⁵I]8 ranged from 48 to 59%. The specific radioactivity of [¹²⁵I]8 was 81.4 GBq/μmol and the radiochemical purity > 98%. [¹²⁵I]8 was thus obtained in adequate yield and purity for further evaluation with sensitive post mortem autoradiography in vitro. Furthermore, these conditions would be applicable to labeling 8 with iodine-123 for evaluation in SPECT imaging.

CB₁ receptors are spread heterogeneously in brain, with high-densities appearing in substantia nigra, globus pallidus, amygdala, cortical regions and striatum.^25,26 Brain regions with low CB₁ receptor densities are thalamus, pons and white matter. In post mortem autoradiography in vitro, [¹²⁵I]8 bound substantially to human brain regions with high CB₁ receptor densities (Figure 2; Panels A, C, E & G) with highest binding in globus pallidus and substantia nigra (Figure 2; Panels A & E). Additionally [¹²⁵I]8 showed much lower binding in brain regions with low CB₁ receptor density, including thalamus (Figure 2; Panels C & E). Under conditions in which the CB₁ receptors were blocked with the selective CB₁ ligand, rimonabant (10 μM), the binding of [¹²⁵I]8 in CB₁ rich regions was greatly reduced and the distribution of radioligand became more homogeneous (Figure 2, Panels B, D, F & H). Therefore, the regional selectivity of [¹²⁵I]8 indicated that ¹²³I-labeled 8 would be promising for imaging brain CB₁ receptors with SPECT.

Figure 2.

Autoradiographs from whole-hemisphere cryosections (Panels A– D; 100 m thickness) and cryosections (20 μm thickness) covering thalamus and brainstem (Panels E & F) and temporal cortex (Panels G & H) incubated with [¹²⁵I]8 under baseline (Panels A, C, E & G) and blocked (rimonabant, 10 μM; Panels B, D, F & H) conditions. Abbreviations: Amg, amygdala; Ca, caudate nucleus; CG, cingulate gyrus; FC, frontal cortex; Hi, hippocampus; IGP, globus pallidus, internal segment; PC, parietal cortex; Pu, putamen; SN, substantia nigra; TC, temporal cortex; Th, thalamus.

Ligand 8 demonstrated high affinity and good selectivity for CB₁ receptors. [¹²⁵I]8 was obtained in acceptable radiochemical yield, specific radioactivity and purity for evaluation in vitro. Autoradiographs of human brain obtained with [¹²⁵I]8 showed radioactivity distribution according to known regional CB₁ receptor densities. Future research evaluating [¹²³I]8 with SPECT imaging is therefore warranted.