Discovery and Labeling of High Affinity 3,4-Diarylpyrazolines as Candidate Radioligands for In Vivo Imaging of Cannabinoid Subtype-1 (CB₁) Receptors

Abstract

Imaging of cannabinoid subtype-1 (CB₁) receptors in vivo with positron emission tomography (PET) is likely to be important for understanding their role in neuropsychiatric disorders and for drug development. Radioligands for imaging with PET are required for this purpose. We synthesized new ligands from a 3,4-diarylpyrazoline platform of which (-)-12a ((-)-3-(4-chlorophenyl)-N’-[(4-cyanophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine) was found to have high-affinity and selectivity for binding to CB₁ receptors. (-)-12a and its lower affinity enantiomer ((+)-12a) were labeled with carbon-11 (t_1/2 = 20.4 min) using [¹¹C]cyanide ion as labeling agent and evaluated as PET radioligands in cynomolgus monkey. After injection of [¹¹C](-)-12a there was high uptake and retention of radioactivity across brain according to the rank order of CB₁ receptor densities. The distomer, [¹¹C](+)-12a, failed to give a sustained CB₁ receptor-specific distribution. Polar radiometabolites of [¹¹C](-)-12a appeared moderately slowly in plasma. Radioligand [¹¹C](-)-12a is promising for the study of brain CB₁ receptors and merits further investigation in human subjects.

Introduction

The psychotropic, analgesic and healing properties of Cannabis sativa (marijuana) have been known throughout documented history.¹ As for some other plant-derived medications (e.g., opium), there has been significant abuse of marijuana mainly because of an accompanying feeling of relaxation and psychological “high”.^2,3 Nevertheless, legitimate medical use of marijuana may extend to the treatment of chemotherapy-induced emesis,^4,5 appetite stimulation in acquired immune deficiency syndrome (AIDS)^6,7 and movement disorders caused by multiple sclerosis^8,9.

Efforts to elucidate the biological response to marijuana intake have identified (-)-(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol, 1 (Δ⁹-THC, Figure 1)^10,11, as its most abundant active compound. 1 interacts with two main receptor types, namely cannabinoid subtype-1 (CB₁) and cannabinoid subtype-2 (CB₂) receptors.^12,13 Two spliced variants of the CB₁ receptor have also been identified, CB_1A and CB_1B.^14,15 CB₁ receptors are located throughout the body and have high densities in regions of the brain, such as the hippocampus, striatum and basal ganglia.^16,17 By contrast, CB₂ receptors are located mainly in peripheral tissues and are associated with the immune system.^13,18,19

Figure 1.

Structures of CB₁ receptor ligands (1-3).

In 1994 Sanofi-Synthlabo introduced 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide, 2 (SR141716A, rimonabant, Figure 1), as a high-affinity inverse agonist at CB₁ receptors.²⁰ 2 has recently gained approval for use in the European Union as a treatment for morbid obesity. The therapeutic use of 2 may extend to addiction and neurodegenerative disorders. Consequently, there has been a considerable effort by pharmaceutical industry to develop novel CB₁ receptor inverse agonist platforms. Solvay AB succeeded with the development of (4S)-3-(4-chlorophenyl)-N-methyl-N’-[(4-chlorophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine, 3 (SLV319, Figure 1).^21-23

Brain CB₁ receptors may be involved in several neuropsychiatric disorders. Currently, there is a need for suitable ligands that are amenable to labeling with positron-emitters for non-invasively imaging CB₁ receptors in vivo with PET under control and diseased states. Previous attempts at radioligand development have focused on the modification of the 1,5-diarylpyrazole CB₁ receptor class of 2 to allow for labeling with carbon-11 (t_1/2 = 20.4 min), fluorine-18 (t_1/2 = 109.7 min) or iodine-124 (t_1/2 = 4.15 d). Some success has been achieved with this approach (Figure 2), namely through the development of [¹¹C]4 ([¹¹C]JHU75528)^24,25 and [¹¹C]5 ([¹¹C]JHU75575)²⁵. Promising PET radioligands from other structural platforms have recently been reported (Figure 2), such as [¹¹C]6 ([¹¹C]PipISB),²⁶ [¹⁸F]6 ([¹⁸F]PipISB),²⁶ [¹⁸F]7 ([¹⁸F]MK-9760)^27,28 and [¹¹C]8 ([¹¹C]MePPEP)²⁹.

Figure 2.

Structures of [¹¹C]4-6, [¹⁸F]6, [¹⁸F]7 and [¹¹C]8.

A 3,4-diarylpyrazoline class of CB₁ receptor ligand also presents favorable physiological and pharmacological attributes for PET radioligand development. The lead structure (3) shows high selectivity and potency for CB₁ receptors with little to no substrate behavior for P-glycoprotein (P-gp) efflux pumps.²² Nevertheless, this structural class has remained largely unexplored for PET radioligand development. With this purpose in mind, we have synthesized novel analogs of 3 that are amenable to radiolabeling. The CB₁ receptor affinities (K_i values) and selectivities were determined for the enantiomers of two ligands that were considered amenable to labeling with carbon-11 or radioiodine (e.g., iodine-123, t_1/2 = 13.13 h, or iodine-124). Additionally, one racemic ligand (12a), along with its eutomer and distomer, were labeled in high-specific radioactivity with [¹¹C]cyanide ion and investigated in monkey with PET imaging. The emergence of radiometabolites in monkey plasma was measured with HPLC.

Results and Discussion

Chemistry

Ligands (12a-c) were synthesized by modifications of known general procedures (Scheme 1).²² Briefly, the appropriate 4-substituted benzenesulfonamides (9a-c) were treated with methyl chloroformate plus triethylamine in acetonitrile to give the corresponding carbamic acid methyl esters (10a-c), which were then treated with 3-(4-chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole in toluene to give 11a-c in good yields. Treatment of 11a-c with PCl₅ in chlorobenzene gave the crude imino chlorides, which were readily converted with methanolic NH₃ into the target ligands, 12a-c. These ligands were then resolved into their enantiomers with chiral HPLC.

Scheme 1.

Synthesis of 3,4-diarylpyrazoline derivatives. Conditions: a) methyl chloroformate, TEA, MeCN; b) 3-(4-chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole, toluene, reflux; c) chlorobenzene, PCl₅; d) methanolic NH₃; e) ChiralPak AD, MeCN, 8 mL/min for 12a and 12b and 6 mL/min for 12c.

CB₁ and CB₂ in vitro binding assays

High-affinity is a prerequisite in candidate radioligands for PET imaging of neuroreceptors.³⁰ Generally, the more sparse the receptor the higher the affinity must be to permit successful imaging. As a guide, binding potentials represented by B_max/K_d should well exceed unity, when B_max and K_d (or as surrogate, K_i or IC₅₀) are expressed in nM.³⁰ CB₁ receptors are amongst the most abundant receptors in brain^16,17, and hence moderately high-affinity (K_i < 10 nM) may be acceptable. The IC₅₀ and K_i values of rimonabant, (-)-12a, (+)-12a, (-)-12c and (+)-12c are shown in Table 1. The (-)-enantiomers exhibited high affinities for CB₁ receptors with IC₅₀ values and K_is in low or sub nM range, respectively. These values compare well with other successful radioligands targeting CB₁ receptors, which generally have potencies or affinities in the nM range (e.g., [¹⁸F]7 and [¹¹C]8). The (+)-enantiomers of 12a and 12c exhibited lower CB₁ receptor affinities than their (-)-enantiomers; eudismic ratios were found to be about 35 for 12a and 56 for 12c. These ratios are similar to those of similar CB₁ receptor ligands from the 3,4-diarylpyrazoline class.²² The eutomer of one such ligand has been shown to have the S configuration by X-ray crystallography.²² Hence, the eutomers of 12a and 12c are also predicted to have S configuration.

Table 1.

IC₅₀ and K_i values for the CBL₁ and CB₂ receptors and cLogP data for ligands 2, (-)-12a, (+)-12a, (-)-12c and (+)-12c.

Ligand	R1	CB1 IC50 (nM)a	CB2 IC50 (nM)a	CB1 Ki (nM)a	CB2 Ki (nM)a	cLogPb
2		2.2 ± 0.5	4,570 ± 410	0.4 ± 0.1	697 ± 63	6.95
(-)-12a	CN	2.8 ± 0.3	> 33,000	0.5 ± 0.1	> 5,000	3.85
(+)-12a	CN	100 ± 10	> 33,000	16.9 ± 2.0	> 5,000	3.85
(-)-12c	I	1.9 ± 0.7	> 33,000	0.3 ± 0.1	> 5,000	5.07
(+)-12c	I	103.5 ± 9.0	> 33,000	17.4 ± 2.0	> 5000	5.07

^aValues are represent the mean ± SD of three determinations.

^bcLogP values were calculated by using the Pallas 3.0 software (Compudrug, USA).

Receptor screening

Ligand (-)-12a showed < 50% inhibition (n = 4) for the following receptors and binding sites: 5-HT_1A-E, 5-HT_2B-C, 5-HT₃, 5-HT_5A, 5-HT₆, 5-HT₇, α_1A,B, α_2A-C, β_1-3, D_1-5, DAT, DOR, H_1,4, KOR, M_1-5, MOR, NET, SERT, σ_1,2. K_i values (n = 3) of > 7,710 ± 1,110 nM for the 5-HT_2A and > 10,000 nM for H_2,3 receptors were found. Hence, (-)-12a was found to have excellent CB₁ receptor selectivity for development as a PET radioligand.

Lipophilicities

The lipophilicity of a radioligand may critically influence its ability to penetrate the blood-brain barrier. Generally, a LogP value in the range 2.0 to 3.5 is considered desirable for adequate brain entry without excessive non-specific binding to brain tissue (i.e., fats, proteins).³¹ cLogP is a useful tool for predicting lipophilicity trends among compounds of the same structural class.^30,31 cLogP was computed for (-)-12a, (+)-12a, (-)-12c and (+)-12c (Table 1). Ligand 3 has previously been shown to penetrate the blood-brain barrier, despite its very high cLogP value (5.01).²² The cLogP values of 12a and its enantiomers are substantially lower (3.85) than that of 3 (Table 1), and hence they may be expected to enter brain readily. The values for 12c and its enantiomers are similar to 3 and hence they may also be expected to enter brain adequately.

Radiosynthesis

Initially, we set out to find an effective and rapid method for labeling 12 as its racemate. [¹¹C]Cyanide ion is a useful precursor for ¹¹C-labeling molecules with an aryl nitrile group.³² The incorporation of [¹¹C]cyanide ion into an aryl ring is best achieved with copper^33,34 or palladium³² catalyzed reactions. We considered each method for labeling (±)-12a. At first glance, [¹¹C]Cu(I)CN appears attractive for labeling PET radiopharmaceuticals. In general, use of this labeling agent requires one-pot and is insensitive to H₂O or NH₃ accompanying the production of [¹¹C]HCN. However, the overall radiochemical yields can be very low (e.g., 2.5%), and inferior to those from the palladium-catalyzed method.³⁴ Hence, the latter method was selected for labeling (±)-12a.

[¹¹C]HCN, which itself was prepared from cyclotron produced [¹¹C]methane, was trapped in a DMSO solution of KOH, and 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K 2.2.2) yielding a [¹¹C]CN^--K⁺-K 2.2.2. complex, which was then added to the bromo precursor ((±)-12b) and Pd(PPh₃)₄ in DMSO and heated (Scheme 2). [¹¹C](±)-12a was separated from the crude product with reverse phase HPLC. The fraction containing [¹¹C](±)-12a was evaporated to dryness and formulated for safe intravenous injection. The overall radiosynthesis time was about 30 min. The non-optimized decay-corrected yield was 36% (n = 2). There was no great improvement in yield when using the iodo compound (-)-12c as precursor. [¹¹C](±)-12a was obtained in high radiochemical purity (> 98%) and was free of labeling precursors. Specific radioactivities were ≥ 56 GBq/μmol at time of injection. Product identity was confirmed by liguid chromatography-mass spectrometry (LC-MS) of associated carrier and by co-injection with 12 in HPLC analysis and observation of co-elution.

Scheme 2.

Radiosynthesis of [¹¹C](±)-12a. Conditions, reagents and decay-corrected yield: a) Pt, NH₃ (20-30 mL/min), 990 °C. b) 50% H₂SO₄, 90 °C; c) Pd(PPh₃)₄, KOH, K2.2.2, DMSO, 110 °C, 36% (n = 2).

We attempted to prepare [¹¹C](-)-12a from precursor (-)-12b under the reaction conditions used to prepare [¹¹C](±)-12a. However, chiral HPLC analyses of the collected radioactive products revealed that complete racemization had occurred during the reactions (Table 2). Racemization was likely promoted by the strong base (KOH plus K 2.2.2) (Scheme 3).

Table 2.

Enantiomer composition (%) of [¹¹C]12a after treating (-)-12b, (-)-12c or (+)-12c with [¹¹C]cyanide ion in DMSO the presence of various bases

Precursor	Base (a)	[11C](-)-12a (%)	[11C](+)-12a (%)
(-)-12b	KOH, K2.2.2	50	50
	NaHCO3	90	10
(-)-12c	NaOAc	50	50
	NaHCO3	90	10
	KH2PO4	> 97 (n = 4)	< 3 (n = 4)
(+)-12c	KH2PO4	< 3 (n = 1)	> 97 (n = 1)

Scheme 3.

Proposed mechanism for the epimerization of [¹¹C](-)-12a under labeling conditions.

To try to avoid racemization, several weaker bases were used in place of the KOH plus K 2.2.2. Interestingly, the use of NaHCO₃ as base gave, [¹¹C](-)-12a to [¹¹C](+)-12a in a 9: 1 ratio. Serendipitously, we found that the use of KH₂PO₄ (Table 2) gave [¹¹C](-)-12a in > 94% ee (n = 4). These conditions were also used with (+)-12c as precursor and the resulting product, [¹¹C](+)-12a, was obtained in > 94% ee. The chemical identities of [¹¹C](-)-12a and [¹¹C](+)-12a were confirmed with LC-MS of associated carrier. Thus, [¹¹C](-)-12a and [¹¹C](+)-12a were obtained in high-chiral purity for evaluation as radioligands in monkeys with PET.

PET measurements

After intravenous injection of [¹¹C](±)-12a into cynomolgus monkey, the brain radioactivity distributed according to the rank order of regional CB₁ receptor densities (Panel A, Figure 3). The highest radioactivity uptake was in CB₁ receptor-rich striatum, reaching 220% SUV at 30 min after injection. This slowly diminished to 180% SUV at 90 min after injection. The lowest maximal brain uptake was in pons reaching 150% SUV at 24 min after injection. The concentration of radioactivity in this region diminished to 124% SUV at 90 min after injection. In an experiment in which the CB₁ receptor-selective ligand 6 was given in high dose (1 mg/kg, i.v.) at 25 min after injection of [¹¹C](±)-12a, the regional brain radioactivity became homogeneous and diminished to about 95% SUV at 90 min after injection (Panel B, Figure 3). When 6 (1 mg/kg, i.v.) was given at 20 min before injection of [¹¹C](±)-12a, brain radioactivity became homogenous and was characterized by a lower maximal uptake and fast washout, reaching 175% SUV at 15 min after injection and declining to 85% SUV at 90 min after injection (Panel C, Figure. 3). These results demonstrated that a high proportion of brain radioactivity in the baseline experiment was reversibly bound to CB₁ receptors. The higher affinity enantiomer in this racemic radioligand was expected to be responsible for the majority of receptor-specific binding, while the lower affinity enantiomer was expected to bind mostly non-specifically. We therefore set out to inject the homochiral radioligands, [¹¹C](-)-12a and [¹¹C](+)-12a, to test these expectations.

Figure 3.

Regional time-radioactivity curves after i.v. injection of [¹¹C](±)-12a in cynomolgus monkey under baseline condition (Panel A), with 6 (1 mg/kg, i.v.) administered as a displacing agent at 25 min (Panel B), or pretreatment condition with 6 (1 mg/kg, i.v.) (Panel C). Key: ×, striatum; △, cerebellum; ◇, frontal cortex; □, lateral temporal cortex; +, thalamus; ○, medial temporal cortex; ▲, pons.

After intravenous injection of the higher affinity enantiomer, [¹¹C](-)-12a, into monkey, the brain radioactivity again distributed according to the regional rank order of CB₁ receptor densities. The highest uptake of radioactivity was in striatum, reaching 200% SUV at 48 min after injection. The lowest maximal brain uptake was in pons reaching and maintaining ∼ 125% SUV from 24 min after injection (Panel A, Figure 4). These time-activity curves are consistent with a high proportion of receptor-specific binding in all examined brain regions, as also seen in the experiment with racemic radioligand (Panel A, Figure 3).

Figure 4.

Regional time-radioactivity curves after i.v. injection of [¹¹C](-)-12a (100 MBq) (Panel A) or [¹¹C](+)-12a (98 MBq) (Panel B) in cynomolgus monkey. Key: ×, striatum; △, cerebellum; ◇, frontal cortex; □, lateral temporal cortex; +, thalamus; ○, medial temporal cortex; ▲, pons.

In these experiments, as in PET imaging studies of other CB₁ receptor radioligands,^27,29 no region could be identified to represent non-specific binding only. Pons does not serve this purpose, since it contains some CB₁ receptors^16,17,27 and measurements of its radioactivity concentration are contaminated from other nearby regions (e.g., CB₁ receptor-rich cerebellum) through the partial volume effect. In the absence of a reference region, ratios of specific to non-specific binding cannot be estimated at all accurately by visual inspection of time-activity curves. Bolus plus constant infusion³⁵ or full kinetic compartmental model utilizing an arterial input function would be required to extract this information.²⁹

By contrast with results from [¹¹C](-)-12a, after intravenous injection of the lower affinity enantiomer, [¹¹C](+)-12a, into monkey, maximal brain radioactivity concentration reached 280% SUV at 1.5 min, but then rapidly declined in all regions to about 95% SUV at 90 min (Panel B, Figure 4). These features of the time-activity curves are consistent with a high proportion of non-specific binding in all examined regions, and are consistent with the lower affinity of the radioligand.

Horizontal PET images obtained at the level of the striatum from data acquired between 9 and 93 min after injection of [¹¹C](-)-12a showed a distribution of radioactivity consistent with a large proportion of specific binding to CB₁ receptors, whereas corresponding images obtained with [¹¹C](+)-12a were strikingly homogeneous indicating little receptor-specific binding.

Emergence of radiometabolites of [¹¹C](-)-12a in plasma

Analysis of venous samples showed that after injection of [¹¹C](-)-12a into cynomolgus monkey, three less lipophilic radiometabolite fractions (t_Rs = 2.3, 5.8 and 8 min, cf. t_R = 8.5 min for [¹¹C](-)-12a) emerged in plasma (Panel A, Figure 6). Unchanged radioligand had declined to 50% of radioactivity in plasma at 45 min after injection (Panel B, Figure 6). The presence of the three radiometabolite fractions slowly increased as a percentage of total radioactivity in plasma throughout the scan. In this study, we did not determine the identities of any of these radiometabolite fractions nor whether they crossed the blood-brain barrier.

Figure 6.

Radio-HPLC of plasma of [¹¹C](-)-12a in cynomolgus monkey (15 min after injection, Panel A), and time course of radioactivity in plasma represented by parent radioligand and radiometabolite fractions (Panel B). Key: □, [¹¹C](-)-12a; △, metabolite a; ▽, metabolite b; ○, metabolite c.

Finally, (-)-12c was not labeled with radioiodine in this study, but its properties (high-affinity, high-selectivity and lipophilicity) suggest it has potential for development as a radioligand for imaging brain CB₁ receptors, either for PET or SPECT.

Conclusions

3,4-Diarylpyrazoline CB₁ ligands with high-affinity and selectivity for CB₁ receptors were discovered. One racemic ligand ((±)-12a), its eutomer ((-)-12a) and its distomer ((+)-12a) were successfully labeled with carbon-11 in high specific radioactivity. [¹¹C](-)-12a was found to be a promising radioligand for PET receptor imaging and merits further exploration in humans.

Experimental Section

Materials

All reagents were of ACS or HPLC quality and purchased from commercial sources, and were used as received. 4-Cyanophenylsulfonamide, 4-bromophenylsulfonamide and 4-iodophenylsulfonamide were synthesized by known procedures.³⁶ 3-(4-Chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole was also synthesized as reported.³⁷ 6 was provided by Eli Lilly and Co.

General methods

¹H (400 MHz) and ¹³C (100 MHz) NMR spectra were recorded at room temperature on an Avance-400 spectrometer (Brucker; Billerica, MA). Chemical shifts are reported in δ units (ppm) downfield relative to the chemical shift for tetramethylsilane. Signals are quoted as s (singlet), d (doublet), dd (double doublet), dt (double triplet), t (triplet), q (quartet) or m (multiplet). High-resolution mass spectra (HRMS) were determined using a time-of-flight electrospray instrument (University of Illinois at Urbana, Champaign, IL, USA). Melting points (mp) were determined using a Mel-temp melting point apparatus (Electrothermal, Fisher Scientific, USA) and were uncorrected. Chiral HPLC, for the preparative resolution of racemates to enantiomers, was performed on a chiral column (ChiralPak AD, 20 × 250 mm) eluted with acetonitrile at 6 or 8 mL/min, as later specified. The enantiomeric excess (ee) of each resolved compound was measured by HPLC with the same method as used for resolution. Optical rotations $\left({\left[\alpha \right]}_D^{22}\right)$ were measured with a P-1010 polarimeter (JASCO; Easton, MD). Specific rotations were obtained at room temperature. Mass spectra (MS) were acquired using a LCQ^DECA LC-MS instrument (Thermo Finnigan; San Jose, CA, USA) fitted with a reverse phase LC column (Luna, C18; 5 μm, 2 × 150 mm; Phenomenex). Radiosyntheses were performed in a custom-made remotely-controlled apparatus.³⁸ Radioligand separations were performed with HPLC on a reverse phase column (μ-Bondapak C-18; 7.8 × 300 mm, 10 μm; Waters). The column outlet was connected to an absorbance detector (λ = 254 nm) in series with a GM-tube for radiation detection. [¹¹C](±)-12a, [¹¹C](-)-12a and [¹¹C](+)-12a were purified in this system using MeCN-0.01 M H₃PO₄ (55: 45, v/v) as mobile phase at 6 mL/min. The radiochemical purities and specific radioactivities of each product were determined with reverse phase HPLC on a μ-Bondapak C-18 column (3.9 × 300 mm, 10 μm; Waters) eluted at 3 mL/min with MeCN-H₃PO₄ (0.01 M; 55: 45 v/v) as mobile phase. Eluate was monitored with an absorbance detector (λ = 254 nm) in series with a β-flow detector (Beckman) for radiation detection. The enantiomeric excess of each labeled product was measured by chiral HPLC, as described above; eluate was monitored for absorbance and radioactivity. Specific radioactivities (GBq/μmol) were determined with analytical HPLC calibrated for absorbance (λ = 254 nm) response per mass of ligand. The specific radioactivity was calculated as the radioactivity of the radioligand peak (decay-corrected) (GBq) divided by the mass of the associated carrier peak (μmol). The metabolism of [¹¹C](-)-12a was assessed with HPLC on a reverse phase (μ-Bondapak C-18 column; 7.8 × 300 mm, 10 μm; Waters) eluted at 6 mL/min with a gradient of MeCN (A) and aq-H₃PO₄ (0.01 M) (B), with A increasing linearly from 35 to 65% v/v for 6 min and then to 35% v/v over the next 2 min and then held for 4 min. The column outlet was connected to an absorbance detector (λ = 270 nm) in series with a GM-tube for radiation detection.

N-[(4-Cyanophenyl)sulfonyl]carbamic acid methyl ester (10a)

Methyl chloroformate (6.34 mL, 82.4 mmol) was slowly added to a stirred solution of 4-cyanobenzenesulfonamide (10 g, 54.9 mmol) and triethylamine (23 mL, 165 mmol) in acetonitrile (75 mL). The reaction was stirred at room temperature for 16 h and then evaporated to dryness in vacuo. After addition of ethyl acetate and aq. NaHCO₃ to the crude residue, the aqueous layer was separated and acidified. The oily precipitate crystallized on standing and was filtered off, washed with water and dried to give 10a (6.9 g, 52% yield); mp 130-132 °C; ¹H NMR (400 MHz, CDCl₃): δ 3.72 (s, 3H), 7.87 (2H, dt, J = 9.0, 2.0 Hz), 8.19 (2H, dt, J = 8.8, 2.0 Hz), NH proton invisible.

N-[(4-Bromophenyl)sulfonyl]carbamic acid methyl ester (10b)

10b was prepared from 4-bromobenzenesulfonamide in 48% yield by the method described for 10a; mp 120-122 °C; ¹H NMR (400 MHz, CDCl₃): δ 3.72 (s, 3H), 7.71 (2H, dt, J = 8.8, 2.0 Hz), 7.93 (2H, dt, J = 8.8, 2.0 Hz), NH proton invisible.

N-[(4-Iodophenyl)sulfonyl]carbamic acid methyl ester (10c)

10c was prepared from 4-iodoobenzenesulfonamide in 56% yield by the method described for 10a; mp 116-118 °C; ¹H NMR (400.13 MHz, CDCl₃): δ 3.72 (s, 3H), 7.77 (2H, dt, J = 8.8, 2.0 Hz), 7.93 (2H, dt, J = 8.8, 2.0 Hz), NH proton invisible.

3-(4-Chlorophenyl)-N-[(4-cyanophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (11a)

To a solution of 3-(4-chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole (6.4 g, 25.4 mmol) in toluene (100 mL) was added 10a (6.1 g, 25.4 mmol) and the resulting solution was heated to reflux for 2 h. After cooling to room temperature, 11a began to crystallize slowly from solution. The crystals were filtered off and washed twice with MTBE to give pure 11a (9.2 g, 78% yield); mp 208-210 °C; ¹H NMR (400 MHz, CDCl₃): δ 3.92 (1H, dd, J = 6.2, 5.2 Hz), 4.34 (1H, t, J = 4.3 Hz), 4.75 (1H, dd, J = 6.2, 5.2 Hz), 7.12 (2H, dt, J = 6.6, 1.6 Hz), 7.33-7.24 (5H, m), 7.55 (2H, dt, J = 8.6, 1.8 Hz), 7.86 (2H, dt, J = 8.6, 1.8 Hz), 8.30 (2H, dt, J = 8.6, 1.8 Hz), 8.8 (1H, bs); ¹³C NMR (100 MHz, CDCl₃): δ 51.54, 54.02, 117.39, 127.21, 127.92, 128.25, 128.74, 129.08, 129.23, 129.61, 132.72, 136.85, 138.93, 143.06, 147.55, 156.82; LC-MS m/z (M⁺ + H) = 464.9; HRMS calcd for C₂₃H₁₈N₄O₃SCl (M⁺ + H), 465.0788; found, 465.0774; error (ppm): - 3.0.

3-(4-Chlorophenyl)-N-[(4-bromophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (11b)

11b was prepared from 3-(4-chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole and 10b in 82% yield by the method described for 11a; mp 214-216 °C; ¹H NMR (400 MHz, CDCl₃): δ 3.92 (1H, dd, J = 6.2, 5.2 Hz), 4.34 (1H, t, J = 4.3 Hz), 4.75 (1H, dd, J = 6.2, 5.2 Hz), 7.12 (2H, dt, J = 6.6, 1.6 Hz), 7.33-7.24 (5H, m), 7.55 (2H, dt, J = 8.6, 1.8 Hz), 7.71 (2H, dt, J = 8.6, 1.8 Hz), 8.04 (2H, dt, J = 8.6, 1.8 Hz), 8.76 (1H, s); ¹³C NMR (100.62 MHz, DMSO-d₆): δ 21.02, 49.43, 54.53, 101.86, 125.28, 127.25, 127.48, 128.17, 128.56, 128.87, 129.06, 129.17, 129.26, 134.69, 137.31, 137.92, 138.16, 139.59, 140.15, 148.52, 155.89; LC-MS m/z (M⁺ + H) = 519.9; HRMS calcd for C₂₂H₁₈N₃O₃SClBr (M⁺ + H), 517.9941; found 517.9929; error (ppm): - 2.3.

3-(4-Chlorophenyl)-N-[(4-iodophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (11c)

11c was prepared from 3-(4-chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazole and 10c in 74.5% yield by the method described for 11a; mp 212-214 °C; ¹H NMR (400 MHz, CDCl₃): δ 3.92 (1H, dd, J = 6.2, 5.2 Hz), 4.34 (1H, t, J = 4.3 Hz), 4.73 (1H, dd, J = 6.1, 5.2 Hz), 7.16 (2H, dt, J = 6.6, 1.6 Hz), 7.33-7.24 (5H, m), 7.55 (2H, dt, J = 8.7, 2.0 Hz), 7.71 (4H, qt, J = 9.8, 8.8, 1.8 Hz), 8.8 (1H, s); ¹³C NMR (100.62 MHz, CDCl₃): δ 51.5, 54.0, 127.2, 128.1, 128.2, 128.3, 128.7, 129.1, 129.6, 129.9, 132.3, 136.7, 138.1, 139.1, 147.8, 156.4; LC-MS m/z (M⁺ + H) = 565.8; HRMS calcd for C₂₂H₁₈N₃O₃SClI (M⁺ + H), 565.9802; found: 565.9801; error (ppm): - 0.2.

3-(4-Chlorophenyl)-N’-[(4-cyanophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (12a)

A mixture of 11a (6 g, 12.9 mmol) and PCl₅ (2.8 g, 13.5 mmol) was dissolved in chlorobenzene (80 mL), refluxed for 1 h and then concentrated in vacuo. The residue was treated with methanolic NH₃ (1 M, 5 mL). The mixture was stirred at room temperature for 1 h and then concentrated in vacuo. The product was recrystallized from MeOH to give 12a (2.2 g, 37%); mp 208-210 °C; ¹H NMR (400.13 MHz, CDCl₃): δ 4.02 (dd, J = 12.0, 4.0 Hz, 1H), 4.42 (t, J = 12.0 Hz, 1H), 4.76 (dd, J = 12.0, 4.0 Hz, 1H), 7.10 (dt, J = 6.4, 2.0 Hz, 2H), 7.34-2.25 (m, 3H), 7.55 (dt, J = 8.7, 2.0 Hz, 2H), 7.6 (d, J = 8.0 Hz, 2H), 7.75 (dt, J = 8.6, 1.4 Hz, 2H), 8.05 (dt, J = 8.6, 1.4 Hz, 2H); ¹³C NMR (100.62 MHz, CDCl₃): δ 51.42, 55.34, 115.29, 117.77, 126.87, 127.18, 128.04, 128.22, 128.69, 129.09, 129.61, 132.58, 136.89, 138.93, 147.60, 152.72, 158.05; LC-MS m/z (M⁺ + H), 464.0; HRMS calcd for C₂₃H₁₉N₅O₂SCl (M⁺ + H), 464.0948; found, 464.0957; error (ppm): 1.9.

(-)-3-(4-Chlorophenyl)-N’-[(4-cyanophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine ((-)-12a)

Resolution of 12a by chiral HPLC (see General Methods) gave (-)-12a (t = 9.8 min at 8 mL/min, > 98% ee); $\left({\left[\alpha \right]}_D^{22}\right)$ = - 69.3°, c = 0.010, CH₂Cl₂; mp 208-210 °C; ¹H-NMR: as found for 12a; MS m/z (M⁺ + H), 464.1; HRMS, calcd for C₂₃H₁₉N₅O₂SCl (M⁺ + H), 464.0948; found, 464.0962; error (ppm): 3.0; Anal. (C₂₃H₁₈ClN₅O₂S) C, H, N.

(+)-3-(4-Chlorophenyl)-N’-[(4-cyanophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine ((+)-12a)

Resolution of 12a by chiral HPLC (see General Methods) gave (+)-12a (t_R = 12.27 min at 8 mL/min, > 98% ee); $\left({\left[\alpha \right]}_D^{22}\right)$ = + 66.3°, c = 0.011, CH₂Cl₂; mp 208-210 °C; ¹H-NMR: as found for 12a; MS m/z (M⁺ + H) 464.1; HRMS calcd for C₂₃H₁₉N₅O₂SCl (M⁺ + H) 464.0948; found, 464.0961, error (ppm): 2.8; Anal. (C₂₃H₁₈ClN₅O₂S) C, H, N.

3-(4-Chlorophenyl)-N’-[(4-bromophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (12b)

12b was prepared from 11b in 43% yield by the method described for 12a; mp 214-216 °C; ¹H NMR (400.13 MHz, DMSO-d₆): δ 3.80 (1H, dd, J = 6.6, 4.7 Hz), 4.37 (1H, t, J = 11.5 Hz), 5.04 (1H, dd, J = 6.6, 4.7 Hz), 7.16 (2H, d, J = 7.1 Hz), 7.25 (1H, t, J = 7.2 Hz), 7.35 (2H, t, J = 7.6 Hz), 7.44 (2H, d, J = 8.6 Hz), 7.76 (2H, d, J = 8.4 Hz), 7.79-7.71 (2H, m); ¹³C NMR (100.62 MHz, DMSO-d₆) δ 49.62, 55.60, 125.22, 127.20, 127.52, 127.85, 128.60, 128.83, 129.12, 129.21, 131.83, 134.92, 140.05, 142.96, 152.76, 157.60; LC-MS m/z [M + H]⁺ 517.0; HRMS calcd for C₂₂H₁₉N₄O₂SClBr (M⁺ + H), 517.0101; found, 517.0117, error (ppm): 3.1.

(-)-3-(4-Chlorophenyl)-N’-[(4-bromophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine ((-)-12b)

Resolution of 12b by chiral HPLC (see General Methods) gave (-)-12b (t_R = 13.78 min at 8 mL/min, > 98% ee); $\left({\left[\alpha \right]}_D^{22}\right)$ = - 65.7°, c = 0.010, CH₂Cl₂; mp 214-216 °C; ¹H NMR: as found for 12b; MS m/z (M⁺ + H) 517.0. HRMS calcd for C₂₂H₁₉N₄O₂SClBr (M⁺ + H) 517.0101, found: 517.0126, error (ppm): 4.8.

(+)-3-(4-Chlorophenyl)-N’-[(4-bromophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine ((+)-12b)

Resolution of 12b by chiral HPLC (see General Methods) gave (+)-12b (t_R = 18.14 min at 8 mL/min, > 98% ee); $\left({\left[\alpha \right]}_D^{22}\right)$ = 65.2°, c = 0.010, CH₂Cl₂; mp 214-216 °C; ¹H NMR: as found for 12b; MS m/z (M⁺ + H) 517.0; HRMS calcd for C₂₂H₁₉N₄O₂SClBr (M⁺ + H) 517.0101, found: 517.0110, error (ppm): 1.7.

3-(4-Chlorophenyl)-N’-[(4-iodophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (12c)

12c was prepared from 11c in 64% yield by the method described for 12a; mp 222-224 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 3.80 (1H, dd, J = 6.6, 4.7 Hz), 4.37 (1H, t, J = 11.5 Hz), 5.04 (1H, dd, J = 6.6, 4.7 Hz), 7.18 (2H, d, J = 7.1 Hz), 7.26 (1H t, J = 7.2 Hz), 7.35 (2H, t, J = 7.6 Hz), 7.44 (2H, d, J = 8.6 Hz), 7.62 (2H, d, J = 8.4 Hz), 7.77 (2H, d, J = 8.6 Hz), 7.90 (2H, d, J = 8.4 Hz); ¹³C NMR (100.62 MHz, DMSO-d₆): δ 49.60, 55.59, 99.28, 127.19, 127.52, 127.59, 128.69, 128.83, 129.11, 129.21, 134.92, 137.65, 140.05, 143.30, 152.74, 157.57; LC-MS m/z (M⁺ + H) 565.1; HRMS calcd for C₂₂H₁₉N₄O₂SClI (M⁺ + H) 564.9962, found: 564.9974, error (ppm): 2.1.

(-)-3-(4-Chlorophenyl)-N’-[(4-iodophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine ((-)-12c)

Resolution of 12c by chiral HPLC (see General Methods) gave (-)-12c (t_R = 18.18 min at 6 mL/min, > 98% ee); $\left({\left[\alpha \right]}_D^{22}\right)$ = - 160°, c = 0.011, CHCl₃; mp 222-224 °C; ¹H NMR: as found for 12c; LC-MS m/z (M⁺ + H) 565.1; HRMS calcd for C₂₂H₁₉N₄O₂SClI (M⁺ + H): 564.9962, found: 564.9960, error (ppm): - 0.4.; Anal.. (C₂₂H₁₈ClIN₄O₂S) C, H, N.

(+)-3-(4-Chlorophenyl)-N’-[(4-iodophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine ((+)-12c)

Resolution of 12c by chiral HPLC (see General Methods) gave (+)-12c (t_R = 24.38 min at 6 mL/min, > 98% ee); $\left({\left[\alpha \right]}_D^{22}\right)$ = + 151°, c = 0.010, CHCl₃; mp 222-224 °C; ¹H NMR: as found for 12c; LC-MS m/z (M⁺ + H) 565.0; HRMS calcd for C₂₂H₁₉N₄O₂SClI (M⁺ + H) 564.9962, found: 564.9960, error (ppm): - 0.4; Anal.. (C₂₂H₁₈ClIN₄O₂S) C, H, N.

CB₁ and CB₂ Binding assays

Frozen cell membranes (recombinant hCB1r or hCB2r; Perkin Elmer) were thawed and diluted (7 μg/well) in membrane buffer (final concentrations: 100 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 50 mM HEPES, 10 μM GDP, 100 μM DTT, 0.01% fatty acid free BSA).

[³⁵S]GTPγS was diluted with distilled water to a final concentration of 0.5 nM. The inhibitory effect of test ligands was measured against a concentration (EC₈₀) of the CB1r/CB2r agonist CP-55,940 (Tocris) added to the solution.

Membrane solution (100 μL) and radioligand solution (100 μL) were added to the assay plates together with 2 μL compound (concentration-response in DMSO) and incubated for 45 min at 30 °C. Bound [³⁵S]GTPγS was separated from free [³⁵S]GTPγS by rapid filtration under vacuum through Whatman GF/B glass fiber filters, followed by washes with cold wash solution (final concentration: 50 mM Tris, 5 mM MgCl₂, 50 mM NaCl). The filters were dried for 60 min at 50 °C. Scintillation film was melted onto the filters, which were then counted in a Microbeta scintillation counter. Non-specific binding was determined in the presence of a saturating concentration of GTPγS (final concentration 20 μM). IC₅₀ values were converted to K_i values according to the Cheng and Prusoff equation.³⁹ The data represent K_i ± SD (nM) from triplicate determinations vs. CB1r or CB2r

Receptor screening

Ligand (-)-12a was screened for binding to a wide range of receptors and transporters by the National Institute of Mental Health Psychoactive Drug Screening Program. Detailed protocols are available on-line for all binding assays at the NIMH-PDSP web site (http://pdsp.med.unc.edu).

[¹¹C]HCN production

[¹¹C]Methane was produced at the Karolinska Hospital with a GEMS PETtrace cyclotron using 16.4 MeV protons in the ¹⁴N(p,α)¹¹C reaction on N₂(g) containing 10% H₂(g).⁴⁰ The target gas was irradiated for 5 min with a beam intensity of 35μA. The [¹¹C]methane was isolated from the target gas in a Porapak Q trap, which was cooled with N₂(l). The Porapak Q trap was warmed and the [¹¹C]methane passed in nitrogen (200 mL/min) with NH₃(g) (20-30 mL/min) over heated (990 °C) Pt wire (1.3 g, 0.127 mm, 99%; Sigma Aldrich) in a quartz tube within a carbolite furnace (MTF 10/15). The resulting [¹¹C]NH₄CN was bubbled through aq. 50% H₂SO₄ (2 mL) at 56 °C to generate [¹¹C]HCN.

Radiosyntheses of [¹¹C](±)-12a, [¹¹C](-)-12a and [¹¹C](+)-12a

The generated [¹¹C]HCN (∼ 12.4 GBq) was trapped in a V-vial (5-mL) containing DMSO (400 μL) base (KOH, 1 mg, 17.8 μmol; K 2.2.2_., 5 mg, 13.3 μmol) for [¹¹C](±)-12a or KH₂PO₄ (1 mg, 7.34 μmol) for [¹¹C](-)-12a or [¹¹C](+)-12a. After trapping of [¹¹C]HCN was complete, the solution was transferred into another V-vial (10 mL) which contained DMSO (400 μL), Pd(PPh₃)₄ (5.5 mg, 4.8 μmol) and the requisite precursor ((±)-12b, (-)-12b, (-)-12c or (+)-12c; 1 mg). The reaction was heated 135 °C for 4 min and then cooled to room temperature. HPLC mobile phase (800 μL) was added to the V-vial and the radioactive product separated with HPLC (see General Methods). The radioactive fraction (t_R = 12.3 min) was collected, evaporated to dryness, and taken up in ethanol-propylene glycol (30: 70 v/v, 3 mL) with sterile phosphate buffer (0.2 M, pH = 7.4, 5 mL) and filtered through a sterile filter (Millex GV, 0.22 μm pore size; Millipore Corp., Corrigtwohill, Co. Cork, Ireland).⁴¹ A sample (100 ∼ μL) was analyzed by HPLC for radiochemical purity and measurement of specific radioactivity (see General methods).

PET measurements in monkey

Cynomolgus monkey (Macaca fascicularis) experiments were performed at the Karolinska Institutet (KI) according to “Guidelines for planning, conducting and documenting experimental research” (Dnr 4820/06-600) of the KI as well as the “Guide for the Care and Use of Laboratory Animals”.⁴² The study was approved by the Animal Ethics Committee of the Swedish Animal Welfare Agency. Two cynomolgus monkeys (3.4 and 4.8 kg) were used in the PET experiments. Anesthesia was induced and maintained with repeated i.m. injections of a mixture of ketamine hydrochloride (3.75 mg/kg^-1 h^-1 Ketalar®, Pfizer) and xylazine hydrochloride (1.5 mg/kg^-1 h^-1 Rompun® Vet., Bayer). The head for each monkey was immobilized in a stereotactic frame for the duration of scans.⁴³ The body temperature was maintained by a Bair Hugger Model 505 (Arizant Healthcare, MN, USA) and monitored by rectal thermometer (Precision Thermometer, Harvard Apparatus, MA, USA).

In baseline experiments, each radioligand was administered by bolus injection over about 5 s, with injected activities of 56, 100, and 98 MBq and specific radioactivities of 78, 65, and 56 GBq/μmol for [¹¹C](±)-12a, [¹¹C](-)-12a and [¹¹C](+)-12a, respectively. The masses of injected carrier ligand were 0.33 μg (0.72 nmol), 0.71 μg (1.53 nmol), and 0.81 μg (1.75 nmol) for [¹¹C](±)-12a, [¹¹C](-)-12a and [¹¹C](+)-12a. In the displacement experiment, 6 (1 mg/kg, i.v.) was infused at 25 min after injection of [¹¹C](±)-12a. For this purpose, compound 6 was formulated in vehicle solution (8 mL) (saline/alcohol/cremophore EL, 9: 1: 0.1 by vol.). The solution was homogenized by vortexing and then passed through a sterile filter (0.2 μm pore size, Millex-GV, Millipore). The injected activity was 56 MBq with a specific radioactivity of 80.3 GBq/μmol. The mass of carrier associated with the injected radioactivity was 0.32 μg (0.70 nmol). The pre-block experiment was performed at 5 h after the baseline experiment. 6 was infused at 20 min before injection of radioligand. The injected activity was 57 MBq with specific radioactivities of 75.4 GBq/μmol. The mass of carrier associated with the injected radioactivity was 0.35 μg (0.76 nmol). In each PET experiment, scans were acquired in 3 frames over 93 min.

PET imaging

Radioactivity in brain was measured with the Siemens ECAT EXACT HR system. The three-ring detector block architecture gives a 15-cm wide field of view. All acquisitions were acquired in 3D-mode.⁴⁴ The transversal resolution in the reconstructed image is about 3.8 mm full-width half-maximum (FWHM) and the axial resolution, 3.125 mm. The data were corrected for attenuation with three rotating ⁶⁸Ge rod sources. Raw PET data were then reconstructed using standard filtered back projection consisting of the following reconstruction parameters: 2-mm Hanning filter, scatter correction, a zoom factor of 2.17, and a 128 × 128 matrix size.⁴⁴ Emission data were collected continuously for 93 min, according to a preprogrammed series of 20 frames starting immediately after i.v. injection of radioligand. The 3 initial frames were 1 min each, followed by 4 frames of 3 min each and 13 frames of 6 min each.

The mean image of the PET measurements (9-93 min) was transformed into a standard anatomical space using the monkey version of the Human Brain Atlas developed at the Karolinska Institutet.⁴⁵ The transformation matrix generated on this image was applied to all frames of the corresponding baseline, displacement and pretreatment experiments. PET data were subsequently re-sliced to a resolution of 1.00, 1.00, 1.00 mm. Volumes of interest (VOIs) were manually defined on the coronal planes of an average monkey MRI. Similar VOIs were applied, as reported by Yasuno et al.,²⁹ including cerebellum (1.9 cm³), frontal cortex (7.4 cm³), lateral temporal cortex (5.0 cm³), medial temporal cortex (2.9 cm³), striatum (2.1 cm³), thalamus (0.9 cm³) and pons (0.7 cm³). Tissue radioactivity concentrations were expressed as % standardized uptake values (%SUV). Tissue radioactivity concentrations were decay-corrected and, in order to normalize for injected dose and body weight, expressed as % standardized uptake values (%SUV), where:

$$\%\mathrm{SUV}=\left[\frac{\%\text{injected activity}}{\text{brain tissue}\left(g\right)}\times \text{body weight}\left(g\right)\right]$$

PET Plasma measurements

For radiometabolite measurements, venous blood (1 mL) was sampled from monkey at 5, 15, 30 and 45 min after injection of each radioligand. Plasma samples were measured as described previously.⁴⁶ Briefly, the supernatant liquid (0.5 mL) obtained after centrifugation at 2000 × g for 1 min was mixed with MeCN (0.7 mL) containing standard (±)-12a. The supernatant liquid (1 mL) after another centrifugation at 2000 × g for 1 min was counted in a well counter and subsequently injected onto HPLC.

Figure 5.

Horizontal PET images, obtained at the level of the striatum from data acquired between 9 and 93 min after injection of [¹¹C](-)-12a (100 MBq, Panel A) or [¹¹C](+)-12a (98 MBq, Panel B).

Abbreviations

ACS

American Chemical Society

AIDS

acquired immune deficiency syndrome

BSA

bovine serum albumin

CB₁

cannabinoid subtype-1

CB₂

cannabinoid subtype-2

CRADA

Cooperative Research and Development Agreement

DAT

dopamine transporter

DMSO

dimethyl sulfoxide

DOR

δ opioid receptor

DTT

dithiothreitol

ee

enantiomeric excess

EDTA

ethylene diamine tetraacetic acid

FWHM

full-width half-maximum

GTPγS

guanosine-5′-(γ-thio)-triphosphate

H

histamine receptor

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectrometry

5-HT

5-hydroxytryptamine

K 2.2.2

4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane

KI

Karolinska Institutet

KOR

κ opioid receptor

LC-MS

liquid chromatography-mass spectrometry

MeCN

acetonitrile

MOR

μ opioid receptor

MRI

magnetic resonance imaging

mp

melting point

MTBE

tert-butyl methyl ether

NET

norepinephrine transporter

NIH

National Institutes of Health

NIMH

National Institute of Mental Health

NMR

nuclear magnetic resonance

PDSP

Psychoactive Drug Screening Program

PET

positron emission tomography

PipISB

N-(4-fluoro-benzyl)-4-(3-(piperidin-1-yl)-indole-1-sulfonyl)benzamide

P-gp

permeability-glycoprotein

ppm

parts per million

SERT

serotonin transporter

SPECT

single-photon emission computed tomography

SUV

standardized uptake value

TEA

triethylamine

Δ⁹-THC

Δ⁹-tetrahydrocannabinol

VOI

volume of interest