Development of 3-(4-chlorophenyl)-1-(phenethyl)urea analogs as allosteric modulators of the Cannabinoid Type-1 receptor: RTICBM-189 is brain Penetrant and attenuates reinstatement of cocaine seeking behavior

Abstract

We have shown CB₁ receptor negative allosteric modulators (NAMs) attenuated the reinstatement of cocaine seeking behaviors in rats. In an effort to further define the structure-activity relationships and assess the drug-like properties of the 3-(4-chlorophenyl)-1-(phenethyl)urea-based CB₁ NAMs that we recently reported, we introduced substituents of different electronic properties and size to the phenethyl group and evaluated their potency in CB₁ calcium mobilization, cAMP and GTPγS assays. We found that 3-position substitutions such as Cl, F, Me afforded enhanced CB₁ potency, whereas 4-position analogs were generally less potent. The 3-chloro analog (31, RTICBM-189) showed no activity at >50 protein targets and excellent brain permeation but relatively low metabolic stability in rat liver microsomes. Pharmacokinetic studies in rats confirmed the excellent brain exposure of 31 with a brain/plasma ratio K_p of 2.0. Importantly, intraperitoneal administration of 31 significantly and selectively attenuated the reinstatement of cocaine-seeking behavior in rats without affecting locomotion.

Graphical Abstract

Introduction

Cocaine addiction is one of the major public health issues worldwide, especially among young adults.¹ Currently there are no medications approved by the U.S. Food and Drug Administration to treat cocaine addiction. Therefore, there is an urgent need to develop novel effective therapeutics to treat cocaine addiction and abuse. The cannabinoid type-1 receptor (CB₁), a key member of the endocannabinoid system, is implicated in the neurobiology of cocaine addiction and represents one such promising target. One of the most abundantly expressed G protein-coupled receptors (GPCR) in the human brain, CB₁ is involved in the regulation of many important physiological processes including appetite control, cardiovascular regulation, learning and memory, pain regulation, and in particular, drug reward and dependence.^2-4

CB₁ is activated by endogenous cannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), as well as compounds found in Cannabis such as 9-tetrahydrocannabinol (Δ⁹-THC). Cocaine has been found to stimulate the release of 2-AG in the rat ventral midbrain to suppress GABAergic inhibition of dopamine neurons via activation of presynaptic CB₁ receptors.⁵ In the brains of cocaine addicts and cocaine-treated animals, CB₁ and its associated signaling networks are dysregulated.⁶ In rodents, CB₁ antagonists/inverse agonists SR141716 (rimonabant) and AM251 reduced self-administration of cocaine, as well as several other drugs of abuse including heroin, nicotine, and alcohol.^7-9 AM251 inhibited the acquisition and/or expression of cocaine-induced sensitization and conditioned place preference (CPP) in mice.¹⁰ In general, CB₁ is involved in the association of cocaine reward with environmental cues and reinstatement of cocaine self-administration, implicating CB₁ blockade as a potential anti-craving strategy in the treatment of cocaine addiction.

In addition to the numerous orthosteric agonists and antagonists,^11-13 several allosteric modulators of CB₁ have been reported (for recent reviews, see^14-16) which bind an allosteric site of the receptor and modulate the binding and/or signaling of the orthosteric ligands. These allosteric modulators offer a much needed alternative approach to modulate CB₁ signaling after the psychiatric side effects associated with orthosteric CB₁ antagonists/inverse agonists resulted in the withdrawal of rimonabant from the European market. Compared to orthosteric ligands, GPCR allosteric modulators may exhibit greater subtype selectivity because of the higher sequence divergence at extracellular allosteric binding sites than the more conserved orthosteric sites.^17-20 They may also demonstrate tissue selectivity by exerting effects only where endogenous ligands are present, and may have better safety profiles because of saturability of effect due to their dependence on endogenous ligands for signaling (effect “ceiling”).^{21, 22} In particular, many of these CB₁ allosteric modulators such as Org27569 (1) and PSNCBAM-1 (2) behaved as negative allosteric modulators (NAMs) by reducing orthosteric agonist efficacy (β<1), but also enhanced agonist binding (α>1),^{23, 24} as seen with positive allosteric modulators (PAMs), although 1 also appeared to be an allosteric agonist activating ERK signaling alone.^{23, 25} The interesting characteristic of these molecules (termed PAM-antagonists) may offer a unique advantage as it can allow for targeting of highly active receptor systems because the modulator’s affinity will increase as the concentration of endogenous agonist proximal to the receptor increases.²⁶ For example, palonosetron (trade name Aloxi), a 5-HT3 PAM antagonist for chemotherapy-induced nausea, showed increased affinity in the present of 5-HT and notably a longer duration of action in vivo presumably due to the presence of 5-HT in the brain.^{27, 28}

We have previously reported that, similar to rimonabant, 1, 2 and RTICBM-74 (3) attenuated drug- and/or cue-induced reinstatement of extinguished cocaine- and/or methamphetamine-seeking behavior.^29-31 As part of our effort in the development of potent and selective CB₁ NAMs,^32-36 we recently described the design and synthesis of a series of 3-(4-chlorophenyl)-1-(phenethyl)ureas as hybrids of 1 (Org27569) and 2 (PSNCBAM-1).³³ In the study, our molecular modeling revealed that 2 bound in the same binding pocket as that of 1 and made the same interactions with the CB₁ receptor, and therefore a hybrid scaffold of 1 and 2 was developed (e.g. 4).³³ The new alkyl-aryl-urea core does not have the planarity of the diphenylureas 2 and 3 and has increased sp³ characteristics, which are commonly associated with improved drug-like properties.^37-41 The substitution patterns on the phenethyl group of 4, which was suggested to be important for activity by our computational modeling studies,³³ were briefly examined with analogs such as 4-amino groups (e.g. 5 and 6, Figure 1). To further explore the structure-activity relationships (SAR) at the phenyl ring and start to assess the drug-like properties of these compounds, herein we investigated a series of substituents of different sizes and electronic properties (compounds 7-41) and evaluated their in vitro potency in CB₁ calcium mobilization, GTPγS and cAMP assays. Lastly, we examined the target selectivity, ADME and pharmacokinetic properties, and in vivo effects of a potent analog (compound 31, RTICBM-189) in the reinstatement of cocaine seeking behavior and locomotion assays.

Figure 1.

Representative CB₁ NAMs

Results and Discussion

Chemistry.

Compounds 7 - 41 were obtained by coupling the corresponding aliphatic primary amines to 4-chlorophenyl isocyanate as shown in Scheme 1. All amines were obtained from commercial vendors except amines 42 - 45 and 48. Amines 42 - 45 were prepared from the corresponding phenylacetonitriles by reduction with LiAlH₄/AlCl₃ in THF at room temperature or borane dimethyl sulfide under reflux. The amine group of 45 was protected with a Boc group and the intermediate 46 underwent palladium-catalyzed halide exchange-cyanation⁴² to afford 47 which was Boc-deprotected to provide amine 48 for the preparation of compound 23.

Scheme 1.

Synthesis of compounds 7 - 41.

Reagents and conditions: a) corresponding amine, CHCl₃, 60 °C, 16 h, 12-91% and b) LiAlH₄, AlCl₃, THF, 0 °C to rt, 16 h, 81-90% c) BH₃.Me₂S, THF, reflux, 16 h, 60% -quant. d) Boc₂O, aq. K₂CO₃, 1,4-dioxane, rt, 16 h, quant. e) K₃[Fe(CN)₆], tBuXPhos Pd G3, t-BuXPhos, KOAc, H₂O, 1,4-dioxane, 100 °C, 16 h, 10% f) 4N HCl, 1,4-dioxane, rt, 16 h, quant.

Biological evaluations.

All compounds were first evaluated in FLIPR-based calcium mobilization assays using CHO cells that overexpress the promiscuous Gα₁₆ proteins (RD-HGA16 cells, Molecular Devices) engineered to stably express the human CB₁ or CB₂, as described previously.^{32, 34, 35, 43} Their potency (pIC₅₀) was determined by measuring inhibition of the mobilization of intracellular calcium levels stimulated by the orthosteric agonist CP55,940. Select compounds were subsequently evaluated in the [³⁵S]GTPγS binding assay using hCB₁ HEK293 membranes as well as mouse cerebellar membranes to determine the inhibitory activity against G protein activation following the agonist occupation at both the human receptor and in mouse tissue expressing native receptor/G protein stoichiometry. These compounds were also tested in the BRET CAMYEL cAMP assay to assess effects downstream of the CB₁ receptors, i.e. the ability of compounds to reverse the inhibitory effect of CP55,940 on forskolin-mediated cAMP production.^{44, 45}

Potency in calcium mobilization assays.

We have previously reported analogs with an unsubstituted phenyl group (4, pIC₅₀ = 6.73) or alkylamino substitutions at the 4-position of the phenyl ring such as N-piperidinyl (5, pIC₅₀ = 6.04) or N,N-dimethylamino (6, pIC₅₀ = 6.59).³³ We therefore first examined other electron-donating groups at the 4-position on the phenyl ring. As shown in Table 1, bulkier electron-donating substituents 4-tBu (7, pIC₅₀ = 6.27) and 4-Ph (8, pIC₅₀ = 6.06) demonstrated lower potency than the unsubstituted analog 4, suggesting a limit on size at this position. Smaller electron-donating groups at other positions of the phenyl ring was then investigated. When the N,N-dimethylamino was moved from the 4-position (6) to the 3-position on the phenyl ring, the potency was reduced (9, pIC₅₀ = 5.79). Similarly, 4-methoxy (12, pIC₅₀ = 6.67) had better potency than the 2- and 3-position methoxy isomers (10, pIC₅₀ = 5.80, and 11, pIC₅₀ = 6.35), suggesting 4-position substitutions are preferred for strong electron donating groups. The 4-methoxy analog (12) was similar in potency as both the 4-dimethylamino (6) and the unsubstituted phenyl analog (4). However, the 3,4- and 3,5-dimethoxy analogs (13 and 24) were both less potent, suggesting increased electron density or size is not desired. Biological activity in the calcium paradigm was abolished when the 4-methoxy was replaced with a 4-hydroxy (15) and similarly, this phenomenon was also seen with the 3-methoxy-4-hydroxy analogue (16) whose activity was also completely obliterated compared to the 3-methoxy (11) or 4-methoxy (12) analogs. These results imply that the presence of a hydrogen bond donating group at the 4-position on the phenyl ring was detrimental to CB₁ allosteric modulatory activity. Interestingly, the methyl analogs displayed the opposite trend with the methoxy and amino series. The 3-methyl (17, pIC₅₀ = 7.38) was more potent than the 4-methyl analog (18, pIC₅₀ = 6.87), although both were more potent than the unsubstituted 4. The 3,4-dimethyl (19, pIC₅₀ = 7.32) and 3,5-dimethyl (20, pIC₅₀ = 7.54) both exhibited good potency comparable to the 3-methyl analog (17). Overall, the calcium mobilization results imply that strong electron-donating groups generally preferred the 4-position on the phenyl ring, but provided no further improvement on potency at CB₁ as compared to the unsubstituted analog (4), whereas smaller and weaker electron-donating groups such as methyl showed better potency at the 3-position.

Table 1.

Allosteric Antagonist Potencies of Compounds in the hCB₁ Calcium Mobilization Assay

Compound	R	pIC50 ± SEMa	Compound	R	pIC50 ± SEMa
4 33	H	6.73 ± 0.09	23	3-CN	6.84 ± 0.08
5 33		6.04 ± 0.05	24	4-CN	6.36 ± 0.05
6 33	4-NMe2	6.59 ± 0.14	25	3-Br	7.49 ± 0.07
7	4-tBu	6.27 ± 0.10	26	4-Br	6.83 ± 0.05
8	4-Ph	6.06 ± 0.08	27	2-F	6.77 ± 0.07
9	3-NMe2	5.79 ± 0.08	28	3-F	7.29 ± 0.08
10	2-OMe	5.80 ± 0.07	29	4-F	6.98 ± 0.08
11	3-OMe	6.35 ± 0.08	30	2-Cl	6.64 ± 0.07
12	4-OMe	6.67 ± 0.10	31	3-Cl	7.54 ± 0.06
13	3,4-diOMe	5.75 ± 0.07	32	4-Cl	6.79 ± 0.04
14	3,5-diOMe	5.97 ± 0.04	33	2-CF3	6.90 ± 0.10
15	4-OH	< 5	34	3-CF3	7.54 ± 0.10
16	3-OMe,4-OH	< 5	35	4-CF3	6.75 ± 0.10
17	3-Me	7.38 ± 0.08	36	3-OCF3	7.03 ± 0.08
18	4-Me	6.87 ± 0.06	37	3,5-diCl	7.62 ± 0.08
19	3,4-diMe	7.32 ± 0.15	38	2,4-diCl	6.78 ± 0.08
20	3,5-diMe	7.54 ± 0.06	39	2-Cl,6-F	6.40 ± 0.07
21	4-NO2	7.07 ± 0.06	40	2,4,6-triF	7.10 ± 0.05
22	4-SO2Me	< 5	41	2,3,4,5,6-pentaF	7.49 ± 0.09

^aCompounds were tested against EC₈₀ (100 nM) of CP55,940. Values are the mean pIC₅₀ ± SEM of at least three independent experiments performed in duplicate.

Next, the effects of electron-withdrawing groups on the phenyl ring were examined. We started with several substituents at the 4-position, which was preferred in the strong electron-donating series discussed above. While the 4-nitro analog (21, pIC₅₀ = 7.07) displayed good potency, the 4-methylsulfonyl analog (22, pIC₅₀ < 5) showed no activity, possibly due to the larger size of this substituent as observed with 7 and 8 in the electron donating series. The 3-cyano analog exhibited a modest enhancement over the 4-position analog (23, pIC₅₀ = 6.84 vs. 24, pIC₅₀ = 6.36), similar to the methyl series. Introduction of halogen atoms to the phenyl ring was then investigated. The 4-bromo analog (26, pIC₅₀ = 6.83) had comparable potency to the unsubstituted 4, whereas the 3-bromo analog (25) showed better potency (pIC₅₀ = 7.49). Introduction of a fluoro group at the 4-position, which has a similar size as hydrogen, displayed increased potency as compared to the unsubstituted 4 and the 4-bromo analog 26. Further potency enhancement was observed with the 3-fluoro analog (28, pIC₅₀ = 7.29), while the 2-fluoro analog (27, pIC₅₀ = 6.77) had slightly lower potency than 28 but similar to 4. The chloro-substituted series saw a similar trend in which the 3-chloro analog displayed the best potency (31, pIC₅₀ = 7.54), whereas the 2 and 4-chloro analogs (30 and 32) both demonstrated reduced potency. Consistent with the earlier series, the 3-trifluoromethyl analog (34, pIC₅₀ = 7.54 nM) had similar potency as the 3-fluoro (28) and 3-chloro (31) analogs, and higher than both the 2 and 4-substitued analogs (34 and 36). Lastly, 36 with the electron-withdrawing 3-trifluoromethoxy substituent was more potent than 11 with the electron-donating 3-methoxy group, supporting a preference for electron-withdrawing groups at the 3-position. Introduction of additional halogen atoms were then examined. In general, good potency was obtained with these analogs, but no further improvement was observed compared to the 3-chloro analogs (31, pIC₅₀ = 7.54). The 3,5-dichloro (37, pIC50 = 7.62), 2,4,6-trifluoro (40, pIC₅₀ = 7.10) and 2,3,4,5,6-pentafluoro (41, pIC₅₀ = 7.49) all had good potency comparable to 31, whereas, the 2,4-dichloro (38) and 2-chloro-6-fluoro (39) analogs were both less potent than 31. Together, these results indicate that an electron-withdrawing substituent at the 3-position on the phenyl ring was preferred for CB₁ allosteric modulation and introduction of additional electron-withdrawing groups did not significantly affect potency.

All compounds were screened alone at 10 μM in the CB1 calcium mobilization assay and no significant agonist effects (<30% of CP55,940 E_max) were observed for any of the compounds. All these compounds were also screened for agonist and antagonist activity at the CB₂ to determine receptor subtype selectivity using our previously established CB₂ calcium mobilization assay.^{35, 46} None of the compounds had significant CB₂ agonist activity at 10 μM (<20% of CP55,940 E_max) or CB₂ antagonist activity at 10 μM (<35% inhibition of CP55,940 EC₈₀ concentration).

Evaluation of select compounds in cAMP and [³⁵S]GTPγS assays.

Representative compounds and those that showed good potency in the CB₁ calcium mobilization assays were then tested in hCB₁ and mCB₁ [³⁵S]GTPγS, and cAMP assays. In general, similar SAR trends were observed in these cAMP and [³⁵S]GTPγS assays as in the Ca²⁺ mobilization assays, although the potency was generally lower (Table 2). For instance, the 4-tert-butyl analog (7) was completely inactive at concentrations up to 31.6 μM in the cAMP and both hCB₁ and mCB₁ [³⁵S]GTPγS assays (pIC₅₀ < 5), in agreement with its weak inhibitory activity in the calcium assay. Like the calcium results, the 4-methoxy analog (12) was slightly more potent than the 3-methoxy analogue (11) in both the cAMP and hCB1 [³⁵S]GTPγS assays. The 3,4-dimethoxy analog (13) was inactive in the cAMP assay at concentrations up to 31.6 μM and only partially inhibited in the hCB₁ [³⁵S]GTPγS assay with an pIC₅₀ < 5 , consistent with its low potency in Ca²⁺ mobilization. A similar reversal of trend was observed with the methyl series, where the potency for the 3-methyl (17, pIC₅₀ = 6.04) was slightly greater than that of the 4-methyl analog (18, pIC₅₀ = 5.69) in the cAMP assay, as well as the GTPγS assays. The 3,5-dimethyl analog (20) exhibited similar potency to the 3-methyl analog (17) and better than the 3,4-dimethyl analog (19) in the GTPγS and cAMP assays. For electron-withdrawing groups, the 4-nitro (21), 3-cyano (23), and 3-bromo (25) analogs had similar potency across all three assays and imparted higher potency than the unsubstituted phenyl 4 in the cAMP and hCB₁ [³⁵S]GTPγS assays. The 3-fluoro (28) had greater potency than the 4-fluoro analog (29) in cAMP, consistent with the characterization in the calcium assay. Similarly, the 3-CF₃ analog (34) was able to induce greater inhibition of the agonist (CP55,940) signal than the 2-CF₃ analog (33) in cAMP and hCB₁ [³⁵S]GTPγS. The 3-chloro analog 31 displayed similar potency in cAMP and hCB₁ [³⁵S]GTPγS as the other potent 3-position substituted analogs (17, 28 and 34), but was slightly more potent in mCB₁ [³⁵S]GTPγS. The 3,5-dichloro (37) analog had slightly lower potency in hCB₁ [³⁵S]GTPγS but lower potency in cAMP assay compared to 31. In contrast to other analogs with electron-withdrawing groups, (36) with the 3-trifluoromethoxy substituent had lower potency in both hCB₁ [³⁵S]GTPγS and cAMP assays compared to the unsubstituted analog 4. In general, these CB₁ NAMs showed similar potency in cAMP and [³⁵S]GTPγS assays but lower than the Ca²⁺ assays, and the trends in these in vitro functional assays are consistent with each other.

Table 2.

Allosteric Antagonist Potencies of Compounds in cAMP and [³⁵S]GTPγS Binding Assays

Compound	R	pIC50 ± SEMa
		[35S]GTPγS		cAMP
		hCB1	mCB1
4 33	H	5.17 ± 0.20	N.D.	5.46 ± 0.03
7	4-tBu	< 5	< 5	< 5
11	3-OMe	4.91± 0.10	N.D.	5.11 ± 0.07
12	4-OMe	5.25 ± 0.09	5.43 ± 0.28	5.38 ± 0.06
13	3,4-diOMe	< 5	N.D.	< 5
17	3-Me	5.52 ± 0.03	5.38 ± 0.10	6.04 ± 0.10
18	4-Me	4.86 ± 0.09	N.D.	5.69 ± 0.14
19	3,4-diMe	5.59 ± 0.24	N.D.	5.94 ± 0.11
20	3,5-diMe	5.18 ± 0.20	N.D.	5.18 ± 0.32
21	4-NO2	5.53 ± 0.08	6.11 ± 0.17	5.75 ± 0.15
23	3-CN	6.08 ± 0.06	N.D.	6.06 ± 0.11
25	3-Br	5.46 ± 0.70	N.D.	6.14 ± 0.24
28	3-F	5.17 ± 0.23	5.82 ± 0.10	5.87 ± 0.08
29	4-F	5.36 ± 0.13	6.05 ± 0.11	5.77 ± 0.13
31	3-Cl	5.29 ± 0.07	6.25 ± 0.09	5.82 ± 0.13
33	2-CF3	5.37 ± 0.09	N.D.	5.49 ± 0.21
34	3-CF3	5.89 ± 0.07	6.12 ± 0.14	6.17 ± 0.03
36	OCF3	5.10 ± 0.17	N.D.	4.97 ± 0.12
37	3,5-diCl	5.76 ± 0.08	N.D.	5.36 ± 0.19

^aValues are the mean pIC₅₀ ± SEM from at least three independent experiments performed in duplicate.

Compound 31 was one of the most potent compounds as determined by Ca²⁺ mobilization, cAMP and GTPγS assays. As can be seen in Figure 2, 31 inhibited CP55,940-stimulated calcium mobilization or GTPγS binding (Figure 2A, 2C-D), as expected with NAMs. In the cAMP assay, CP55,940 inhibited 5 μM forskolin-induced cAMP signaling, which was fully reversed by compound 31 and resulted in a small stimulation, reflecting some but modest inverse agonist activity (Figure 2B).

Figure 2.

Concentration-response curves showing modulation CP55,940’s (CP) effects by 31 as assessed by (A) calcium mobilization in CHO cells expressing hCB₁ and Gα16 in the presence of 100 nM of the agonist CP55,940; (B) mobilization of forskolin (F)-stimulated cAMP in hCB₁ expressing HEK293 cells in the presence of 1 μM of the agonist CP55,940; (C) [³⁵S]GTPγS binding in hCB₁ expressing HEK293 membranes in the presence of 100 nM of the agonist CP55,940; and (D) [³⁵S]GTPγS binding in mouse cerebellar membranes in the presence of 100 nM of the agonist CP55,940. Data are from a representative experiment and show mean ± SEM of conditions performed in technical duplicate or triplicate.

ADME studies.

Compounds 17, 31 and 34 were assessed for their preliminary ADME (absorption, distribution, metabolism and excretion) properties in vitro. As shown in Table 3, all three compounds had relatively low stability against rat liver microsomes (RLM) in vitro, with an apparent half-life (t_1/2) ~15 min, clearance (C_L) of ~100 μL/min/mg and percentage of hepatic blood flow (%Qh) of ~70%. These results were similar to 2 (t_1/2 = 13.4 min, C_L = 113.7 μL/min/mg, %Qh = 72.6%).^{32, 35} Given the similar in vitro potency and metabolic stability, only 31 was further evaluated in additional ADME assays. In the MDCK-MDR1 Transwell assay measuring CNS permeability, 31 had apparent permeability P_app(A-to-B) of 19.2 x 10⁻⁶ cm/s and P_app(B-to-A) of 21.3 x 10⁻⁶ cm/s. These values are significantly higher than those of 2, which had P_app values of 2.6 and 1.6 x 10⁻⁶ cm/s, respectively.³² A P_app value of 15 x 10 ⁻⁶ cm/s is generally considered highly CNS permeable,⁴⁷ and these results suggest excellent passive BBB permeability of 31. Compound 31 was not a P-glycoprotein substrate with an efflux ratio of 1.1 [P_app(B-to-A)/P_app(A-to-B)]. Finally, 31 is highly protein bound when tested against rat plasma, showing protein binding of 99.6%, and had low aqueous solubility (< 1 μM).

Table 3.

Metabolic stability and BBB permeability of select CB₁ allosteric modulators.

Cmpd	2c	17	31	34
Rat liver microsome t1/2 (min)a	13.4 ± 4.1	13.7 ± 1.3	14.9 ± 3.5	15.5 ± 0.8
Rat liver microsome Clearance (μL/min/mg)a	113.7 ± 34.4	102.0 ± 11.0	99.3 ± 23.8	90.0 ± 4.6
%Qhb	72.6	72.2	70.6	69.8
Papp (A-to-B) (x10−6 cm/s) a	2.6 ± 0	N.D.	19.2 ± 0.4	N.D.
Papp (B-to-A) (x10−6 cm/s) a	2.2 ± 0.1	N.D.	21.3 ± 1.1	N.D.
Efflux BA/AB	0.8	N.D.	1.1	N.D.
Rat Plasma Protein Binding	99.9%	N.D.	99.6%	N.D.
Solubility (μM)	< 0.5	N.D.	< 1.0	N.D.

^aValues are expressed as mean ± SD from two independent experiments.

^bPercentage of hepatic blood flow.

^cPart of data have been previous reported.^{32, 35} N.D. Not determined.

Receptor Selectivity of 31.

The target selectivity of 31 for the CB1 receptor was determined by binding to a panel of ~50 GPCRs offered by the NIMH Psychoactive Drug Screening Program (PDSP). These GPCRs include adrenergic (α_1A, α_1B, α_1D, α_2A, α_2B, α_2C, β1, β2, β3), dopamine (D₁ - D₅), GABA_A, histamine (H1 - H4), muscarinic (M1 - M5), opioid (μ, κ, δ), serotonin (5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_1E, 5-HT_2A, 5-HT_2B, 5-HT_2C, 5-HT₃, 5-HT_5A, 5-HT₆, 5-HT_7A), sigma (σ1, σ2), dopamine transporter (DAT), norepinephrine transporter (NET), serotonin transporter (SERT), and Benzodiazepine Rat Brain Site. No significant affinity was detected for any of the receptors at 10 μM (< 50% inhibition), suggesting excellent target selectivity.

PK studies of 31.

The in vivo PK profile of 31 was determined in Sprague-Dawley rats using intraperitoneal (i.p.) administration at a dose of 10 mg/kg. Upon injection, 31 was rapidly absorbed into systemic circulation, with peak plasma concentration (C_{max, plasma} = 288.4 ng/mL) observed at t_max,plasma of 0.4 h post-dose (Table 4). Peak brain levels were also reached at t_max,brain of 0.4 h with a significantly higher C_{max, brain} value of 594.6 ng/mL in the brain. Brain concentration was approximately twice that of plasma concentration at all data points (Figure 3). The brain/plasma ratio (K_p) of 31, as determined by the AUC_inf,brain and AUC_inf,plasma, was 2.0, confirming its high BBB permeability. These results also corroborated the MDCK-mdr1 results with excellent P_app values. Compound 31 was eliminated from both the plasma and the brain at a relatively high rate, although more slowly in the brain, with a clearance of 120.7 mL/min/kg and 240.6 mL/min/kg from the brain and plasma, respectively. The estimated half-life of 31 was 0.63 h and 0.90 h in the plasma and brain, respectively. These results are consistent with the relatively low stability of 31 in RLM.

Table 4.

In vivo PK profile of 31 following a single i.p. dose at 10 mg/kg to male Sprague-Dawley rats

	Cmax (ng/mL)	tmax (h)	CL_F (mL/min/kg)	AUCinf (ng/mL*h)	Half-life t1/2 (h)
Plasma	288.4	0.4	240.6	715.2	0.63
Brain	594.6	0.4	120.7	1438.2	0.90
Kp (Brain/plasma ratio)				2.0

Figure 3.

Brain and plasma pharmacokinetic profiles of 31 following a single i.p. dose at 10 mg/kg to male Sprague-Dawley rats.

Compound 31 selectively attenuated reinstatement of cocaine-seeking behavior.

Blockade of CB₁ in vivo with the antagonist/inverse agonist SR141716 has been demonstrated to reduce intake of palatable food, self-administration of several drugs of abuse, and reinstatement of food and drug-seeking behaviors. Rats pretreated with 1 and 2 have been previously shown to reduce seeking of drugs of abuse, including cocaine or methamphetamine, when given the drug non-contingently after a period of extinction.^{35, 48} Therefore, we examined the effect of compound 31 on drug-induced reinstatement of cocaine self-administration. As shown in Figure 4A, a cocaine priming injection significantly reinstated the extinguished active lever response (t test: t[18] = 3.754, p < 0.01; data from last extinction session not shown). Pretreatment with 31 (10 mg/kg, i.p.) significantly attenuated drug-induced reinstatement of cocaine-seeking behavior (t test: t[18] = 3.381 , p < 0.01). The effect of 31 on cocaine reinstatement is similar to that induced by 3 at 10 mg/kg and 2 at 30 mg/kg,³⁵ suggesting enhanced in vivo potency compared to 2. This enhancement on in vivo potency may have resulted from the improved CNS permeability of 31 as shown in the MDCK assay. At this dose of 10 mg/kg i.p., 31 did not significantly affect locomotion (two-way ANOVA: main effect of treatment: F(1, 8)= 0.57; p > 0.05; Fig. 4C) or inactive lever response (t test: t[18] = 1.701 , p > 0.05; Fig. 4B).

Figure 4.

Compound 31 (10 mg/kg, i.p.) attenuated the reinstatement of extinguished cocaine-seeking behavior as compared to saline treatment without any effects (A) on inactive lever responding responses (B) and locomotion (C) in rats (n = 10 male, p > 0.05 by Student’s t test).

CONCLUSIONS

CB₁ plays important roles in addiction of a number of drugs including cocaine. We have previously reported that several CB₁ NAMs successfully attenuated the reinstatement of cocaine seeking behaviors. We have developed several series of CB₁ NAMs based on the scaffold of PSNCBAM-1.^{32-35, 46} The present study describes a novel series of substituted 3-(4-chlorophenyl)-1-(phenethyl)ureas bearing different substituents on the phenethyl group. Our SAR studies suggest that strong electron donating group at the 4-position of the phenyl ring provided good potency, but no improvement over the unsubstituted phenyl analog 4, whereas a small and weak electron-donating methyl provide better potency at the 3-position. Among electron withdrawing groups at different positions, the 3-positon appeared to offer the best potency, with similar high potency between the Cl, F and CF₃ analogs. Notably, compound 31 with a 3-chloro group displayed good in vitro potencies in calcium mobilization, cAMP and [³⁵S]GTPγS assays. Compound 31 showed no significant affinity for >50 other GPCRs, confirming its good CB1 selectivity. ADME and PK assessment suggests that 31 had relatively low metabolic stability but excellent brain penetration, as determined in the MDCK-mdr1 and PK studies. Importantly, 31 (10 mg/kg, i.p.) significantly and selectively attenuated reinstatement of cocaine-seeking behavior in rats. These results support the potential of CB₁ NAMs such as 31 (RTICBM-189) as a therapeutic candidate for the treatment of cocaine addiction.

EXPERIMENTAL SECTION

Chemistry.

All solvents and chemicals were reagent grade. Unless otherwise mentioned, all reagents and solvents were purchased from commercial vendors and used as received. Flash column chromatography was carried out on a Teledyne ISCO CombiFlash Rf system using prepacked columns. Solvents used include hexane, ethyl acetate (EtOAc), dichloromethane, methanol, and chloroform/methanol/ammonium hydroxide (80:18:2) (CMA-80). Purity and characterization of compounds were established by a combination of HPLC, TLC, mass spectrometry, and NMR analyses. Melting point was recorded by the Mel-Temp II instrument (Laboratory Devices Inc. , U.S.). ¹H and ¹³C NMR spectra were recorded on a Bruker Avance DPX-300 (300 MHz) spectrometer and were determined in chloroform-d, DMSO-d6, or methanol-d4 with tetramethylsilane (TMS) (0.00 ppm) or solvent peaks as the internal reference. Chemical shifts are reported in ppm relative to the reference signal, and coupling constant (J) values are reported in hertz (Hz). Thin layer chromatography (TLC) was performed on EMD precoated silica gel 60 F254 plates, and spots were visualized with UV light or iodine staining. Low resolution mass spectra were obtained using a Waters Alliance HT/Micromass ZQ system (ESI). All test compounds were greater than 95% pure as determined by HPLC on an Agilent 1100 system using an Agilent Zorbax SB-Phenyl, 2.1 mm × 150 mm, 5 μm column using a 15 minute gradient elution of 5-95% solvent B at 1 mL/min followed by 10 minutes at 95% solvent B (solvent A, water with 0.1% TFA; solvent B, acetonitrile with 0.1% TFA and 5% water; absorbance monitored at 220 and 280 nm).

General Procedure A: To a solution of amine (1 eq) in anhydrous chloroform (0.04 M) was added 4-chlorophenyl isocyanate (1 eq) at room temperature. The reaction mixture was then heated at 60 °C for 16 h. The precipitated product was filtered and thoroughly washed with dichloromethane.

1- [2- (4- tert- Butylphenyl)ethyl]- 3- (4- chlorophenyl)urea (7) was prepared from 4-tert-butylphenethylamine (0.05 g, 0.32 mmol) following the general procedure A as white solid (0.03 g, 29%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.63 (s, 1H), 7.41 (d, J = 8.85 Hz, 2H), 7.33 (d, J = 8.29 Hz, 2H), 7.25 (d, J = 8.85 Hz, 2H), 7.16 (d, J = 8.10 Hz, 2H), 6.15 (t, J = 5.46 Hz, 1H), 3.27 - 3.32 (m, 2H), 2.70 (t, J = 7.16 Hz, 2H), 1.27 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 148.3, 139.5, 136.4, 128.4, 128.3, 125.1, 124.4, 119.0, 35.2, 34.0, 31.2. MS (ESI) m/z [M+H]⁺ calcd: 331.1; found: 331.2.

3- (4- Chlorophenyl)- 1- [2- (4- phenylphenyl)ethyl]urea (8) was prepared from 4-phenylphenethylamine (0.05 g, 0.32 mmol) following the general procedure A as white solid (0.09 g, 76%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.64 (s, 1H), 7.63 (dd, J = 8.10, 10.55 Hz, 4H), 7.31 - 7.50 (m, 7H), 7.25 (d, J = 8.85 Hz, 2H), 6.19 (t, J = 5.56 Hz, 1H), 3.36 - 3.42 (m, 2H), 2.80 (t, J = 6.97 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 155.0, 140.0, 139.5, 138.8, 138.0, 129.2, 128.9, 128.4, 127.2, 126.6, 126.5, 124.4, 119.0, 40.5, 35.3. MS (ESI) m/z [M+H]⁺ calcd: 351.1; found: 351.2.

3- (4- Chlorophenyl)- 1- {2- [3- (dimethylamino)phenyl]ethyl}urea (9) was prepared from 3-dimethylaminophenethylamine (0.07 g, 0.33 mmol) following the general procedure A as white solid (0.05 g, 50%). ¹H NMR (300 MHz, CDCl₃) δ 7.13 - 7.24 (m, 5H), 6.59 - 6.64 (m, 1H), 6.53 - 6.58 (m, 2H), 6.14 (s, 1H), 4.60 - 4.67 (m, 1H), 3.53 (q, J = 6.53 Hz, 2H), 2.93 (s, 6H), 2.80 (t, J = 6.69 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 155.2, 151.0, 139.7, 137.2, 129.5, 129.2, 128.8, 122.0, 116.9, 113.0, 110.9, 41.6, 40.6, 36.4. MS (ESI) m/z [M+H]⁺ calcd: 318.1; found: 318.2.

3- (4- Chlorophenyl)- 1- [2- (2- methoxyphenyl)ethyl]urea (10) was prepared from 2-methoxyphenethylamine (0.05 ml, 0.32 mmol) following the general procedure A as white solid (0.05 g, 46%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.58 (br. s., 1H), 7.41 (d, J = 8.67 Hz, 2H), 7.25 (d, J = 8.48 Hz, 2H), 7.11 - 7.22 (m, 2H), 6.97 (d, J = 7.54 Hz, 1H), 6.89 (t, J = 7.16 Hz, 1H), 6.12 (br. s., 1H), 3.78 (s, 3H), 3.29 (d, J = 5.65 Hz, 2H), 2.68 - 2.77 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 157.3, 154.9, 139.5, 130.0, 128.4, 127.5, 127.2, 124.3, 120.2, 119.0, 110.7, 55.3, 40.4, 30.3. MS (ESI) m/z [M+H]⁺ calcd: 305.1; found: 305.4.

3- (4- Chlorophenyl)- 1- [2- (3- methoxyphenyl)ethyl]urea (11) was prepared from 3-methoxyphenethylamine (0.05 ml, 0.32 mmol) following the general procedure A as white solid (0.05 g, 47%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.63 (s, 1H), 7.41 (d, J = 8.85 Hz, 2H), 7.23 - 7.29 (m, 2H), 7.19 - 7.23 (m, 1H), 6.75 - 6.84 (m, 3H), 6.13 (t, J = 5.65 Hz, 1H), 3.74 (s, 3H), 3.29 - 3.34 (m, 2H), 2.72 (t, J = 7.06 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 159.3, 154.9, 141.0, 139.5, 129.3, 128.4, 124.4, 120.9, 119.0, 114.2, 111.5, 54.9, 40.4, 35.7. MS (ESI) m/z [M+H]⁺ calcd: 305.1; found: 305.4.

3- (4- Chlorophenyl)- 1- [2- (4- methoxyphenyl)ethyl]urea (12) was prepared from 4-methoxyphenethylamine (0.05 ml, 0.32 mmol) following the general procedure A as white solid (0.07 g, 66%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.62 (s, 1H), 7.41 (d, J = 9.04 Hz, 2H), 7.25 (d, J = 8.85 Hz, 2H), 7.15 (d, J = 8.48 Hz, 2H), 6.87 (d, J = 8.48 Hz, 2H), 6.11 (t, J = 5.56 Hz, 1H), 3.72 (s, 3H), 3.24 - 3.31 (m, 2H), 2.67 (t, J = 7.06 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 157.7, 154.9, 139.5, 131.3, 129.6, 128.4, 124.3, 119.0, 113.8, 55.0, 34.8. MS (ESI) m/z [M+H]⁺ calcd: 305.1; found: 305.4.

3- (4- Chlorophenyl)- 1- [2- (3,4- dimethoxyphenyl)ethyl]urea (13) was prepared from 3,4-dimethoxyphenethylamine (0.05 ml, 0.32 mmol) following the general procedure A as white solid (0.04 g, 38%). ¹H NMR (300 MHz, CDCl₃) δ 7.16 - 7.24 (m, 4H), 6.75 - 6.81 (m, 1H), 6.67 - 6.74 (m, 2H), 6.52 (s, 1H), 4.83 (t, J = 5.18 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.49 (q, J = 6.66 Hz, 2H), 2.77 (t, J = 6.69 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 155.3, 149.0, 147.7, 137.3, 131.4, 129.2, 128.6, 121.7, 120.7, 112.0, 111.4, 55.9, 55.8, 41.4, 35.6. MS (ESI) m/z [M+H]⁺ calcd: 335.1; found: 335.3.

3- (4- Chlorophenyl)- 1- [2- (3,5- dimethoxyphenyl)ethyl]urea (14) was prepared from 3,5-dimethoxyphenethylamine (0.06 ml, 0.32 mmol) following the general procedure A as white solid (0.08 g, 70%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.64 (s, 1H), 7.41 (d, J = 8.67 Hz, 2H), 7.25 (d, J = 8.67 Hz, 2H), 6.40 (s, 2H), 6.35 (br. s., 1H), 6.11 (t, J = 5.09 Hz, 1H), 3.72 (s, 6H), 3.31 - 3.36 (m, 2H), 2.68 (t, J = 6.78 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 160.4, 154.9, 141.8, 139.5, 128.4, 124.4, 119.0, 106.6, 98.0, 55.0, 40.3, 36.0. MS (ESI) m/z [M+H]⁺ calcd: 335.1; found: 335.3.

3- (4- Chlorophenyl)- 1- [2- (4- hydroxyphenyl)ethyl]urea (15) was prepared from 4-hydroxyphenethylamine (0.04 g, 0.32 mmol) following the general procedure A as white solid (0.07 g, 78%). ¹H NMR (300 MHz, DMSO-d₆) δ 9.19 (br. s., 1H), 8.62 (s, 1H), 7.41 (d, J = 8.67 Hz, 2H), 7.25 (d, J = 8.85 Hz, 2H), 7.02 (d, J = 8.29 Hz, 2H), 6.69 (d, J = 8.29 Hz, 2H), 6.10 (t, J = 5.46 Hz, 1H), 3.26 (q, J = 6.66 Hz, 2H), 2.62 (t, J = 7.16 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 155.6, 154.9, 139.5, 129.5, 128.4, 124.3, 119.0, 115.1, 34.9. MS (ESI) m/z [M-H]^- calcd: 289.1; found: 289.2.

3- (4- Chlorophenyl)- 1- [2- (4- hydroxy- 3- methoxyphenyl)ethyl]urea (16) was prepared from 4-hydroxy-3-methoxyphenethylamine (0.07 g, 0.32 mmol) following the general procedure A as white solid (0.03 g, 27%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.73 (s, 1H), 8.63 (s, 1H), 7.41 (d, J = 8.85 Hz, 2H), 7.25 (d, J = 8.67 Hz, 2H), 6.77 (s, 1H), 6.67 - 6.72 (m, 1H), 6.58 - 6.64 (m, 1H), 6.08 (t, J = 5.46 Hz, 1H), 3.75 (s, 3H), 3.24 - 3.30 (m, 2H), 2.63 (t, J = 6.97 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 147.4, 144.8, 139.5, 130.1, 128.4, 124.3, 120.7, 119.0, 115.4, 112.8, 55.5, 40.8, 35.3. MS (ESI) m/z [M-H]^- calcd: 319.1; found: 319.4.

3- (4- Chlorophenyl)- 1- [2- (3- methylphenyl)ethyl]urea (17) was prepared from 3-methylphenethylamine (0.05 g, 0.32 mmol) following the general procedure A as white solid (0.03 g, 62%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.62 (s, 1H), 7.41 (d, J = 8.85 Hz, 2H), 7.25 (d, J = 8.85 Hz, 2H), 7.16 - 7.22 (m, 1H), 7.04 (d, J = 3.96 Hz, 2H), 7.01 (s, 1H), 6.14 (t, J = 5.65 Hz, 1H), 3.27 - 3.33 (m, 2H), 2.70 (t, J = 7.16 Hz, 2H), 2.29 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.5, 139.3, 137.3, 129.3, 128.4, 128.2, 126.7, 125.6, 124.4, 119.0, 35.7, 21.0. MS (ESI) m/z [M+H]⁺ calcd: 289.1; found: 289.1.

3- (4- Chlorophenyl)- 1- [2- (4- methylphenyl)ethyl]urea (18) was prepared from 4-methylphenethylamine (0.05 ml, 0.32 mmol) following the general procedure A as white solid (0.03 g, 32%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.62 (s, 1H), 7.41 (d, J = 8.85 Hz, 2H), 7.25 (d, J = 8.85 Hz, 2H), 7.08 - 7.15 (m, 4H), 6.11 (t, J = 5.46 Hz, 1H), 3.26 - 3.32 (m, 2H), 2.69 (t, J = 7.16 Hz, 2H), 2.27 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.5, 136.3, 135.0, 128.9, 128.5, 128.4, 124.3, 119.0, 35.3, 20.6. MS (ESI) m/z [M+H]⁺ calcd: 289.1; found: 289.2.

3- (4- Chlorophenyl)- 1- [2- (3,4-dimethylphenyl)ethyl]urea (19) was prepared from 3,4-dimethylphenethylamine (0.06 ml, 0.4 mmol) following the general procedure A as white solid (0.05 g, 40%). ¹H NMR (300 MHz, CD₃OD) δ 7.32 (d, J = 9.61 Hz, 2H), 7.21 (d, J = 9.23 Hz, 2H), 6.98 - 7.06 (m, 2H), 6.91 - 6.96 (m, 1H), 3.39 (t, J = 7.06 Hz, 2H), 2.73 (t, J = 7.16 Hz, 2H), 2.23 (s, 3H), 2.21 (s, 3H). ¹³C NMR (75 MHz, CD₃OD) δ 158.0, 139.9, 137.9, 137.6, 135.4, 131.1, 130.7, 129.7, 128.1, 127.2, 121.3, 42.4, 36.8, 19.8, 19.3. MS (ESI) m/z [M+H]⁺ calcd: 303.1; found: 303.0.

3- (4- Chlorophenyl)- 1- [2- (3,5-dimethylphenyl)ethyl]urea (20) was prepared from 3,5-dimethylphenethylamine hydrochloride 44 (0.12 ml, 0.6 mmol) following the general procedure A as white solid (0.18 g, 91%). ¹H NMR (300 MHz, CD₃OD) δ 7.29 - 7.35 (m, 2H), 7.18 - 7.24 (m, 2H), 6.78 - 6.87 (m, 3H), 3.36 - 3.44 (m, 2H), 2.73 (t, J = 7.16 Hz, 2H), 2.26 (s, 6H). ¹³C NMR (75 MHz, CD₃OD) δ 158.0, 140.4, 139.9, 139.1, 129.7, 128.8, 128.1, 127.6, 121.3, 42.4, 37.1, 21.3. MS (ESI) m/z [M+H]⁺ calcd: 303.1; found: 303.0.

3- (4- Chlorophenyl)- 1- [2- (4- nitrophenyl)ethyl]urea (21) was prepared from 4-nitrophenethylamine hydrochloride (0.07 g, 0.32 mmol) following the general procedure A as white solid (0.06 g, 54%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.64 (s, 1H), 8.18 (d, J = 8.67 Hz, 2H), 7.53 (d, J = 8.67 Hz, 2H), 7.40 (d, J = 9.04 Hz, 2H), 7.25 (d, J = 9.04 Hz, 2H), 6.22 (t, J = 5.75 Hz, 1H), 3.37 - 3.43 (m, 2H), 2.90 (t, J = 6.88 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 148.0, 146.1, 139.4, 130.0, 128.4, 124.4, 123.4, 119.1, 40.4, 35.5. MS (ESI) m/z [M+H]⁺ calcd: 320.1; found: 320.2.

3- (4- Chlorophenyl)- 1- [2- (4- methanesulfonylphenyl)ethyl]urea (22) was prepared from 2-(4-methylsulfonyl-phenyl)ethylamine hydrochloride (0.08 g, 0.32 mmol) following the general procedure A as white solid (0.04 g, 35%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.64 (s, 1H), 7.87 (d, J = 8.29 Hz, 2H), 7.52 (d, J = 8.29 Hz, 2H), 7.41 (d, J = 8.85 Hz, 2H), 7.25 (d, J = 8.85 Hz, 2H), 6.21 (t, J = 5.56 Hz, 1H), 3.36 - 3.43 (m, 2H), 3.20 (s, 3H), 2.87 (t, J = 6.88 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 155.0, 145.8, 139.4, 138.7, 129.6, 128.4, 127.0, 124.4, 119.1, 43.6, 35.5, 30.6. MS (ESI) m/z [M+H]⁺ calcd: 353.1; found: 353.2.

3- (4- Chlorophenyl)- 1- [2- (3-cyanophenyl)ethyl]urea (23) was prepared from 3-cyanophenethylamine hydrochloride 48 (0.02 g, 0.1 mmol) following the general procedure A as white solid (0.3 g, 95%). ¹H NMR (300 MHz, CD₃OD) δ 7.63 (br. s., 1H), 7.55 - 7.60 (m, 2H), 7.44 - 7.52 (m, 1H), 7.29 - 7.35 (m, 2H), 7.19 - 7.24 (m, 2H), 3.41 - 3.48 (m, 2H), 2.89 (t, J = 6.97 Hz, 2H). ¹³C NMR (75 MHz, CD₃OD) δ 157.9, 142.6, 139.8, 134.9, 133.5, 131.2, 130.6, 129.7, 128.2, 121.4, 119.8, 113.5, 41.8, 36.8. MS (ESI) m/z [M+H]⁺ calcd: 300.1; found: 300.0.

3- (4- Chlorophenyl)- 1- [2- (4-cyanophenyl)ethyl]urea (24) was prepared from 4-cyanophenethylamine (0.10 g, 0.64 mmol) following the general procedure A as white solid (0.04 g, 22%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.73 (s, 1H), 7.78 (d, J = 8.29 Hz, 2H), 7.45 (d, J = 8.10 Hz, 2H), 7.40 (d, J = 9.04 Hz, 2H), 7.24 (d, J = 8.85 Hz, 2H), 6.26 (t, J = 5.65 Hz, 1H), 3.35 - 3.42 (m, 2H), 2.84 (t, J = 6.97 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 145.7, 139.4, 132.2, 129.8, 128.4, 124.4, 119.1, 118.9, 109.0, 40.4, 35.8. MS (ESI) m/z [M+H]⁺ calcd: 300.1; found: 300.3.

3- (4- Chlorophenyl)- 1- [2- (3-bromophenyl)ethyl]urea (25) was prepared from 3-bromophenethylamine hydrochloride 45 (0.15 g, 0.6 mmol) following the general procedure A as white solid (0.12 g, 54%). ¹H NMR (300 MHz, CD₃OD) δ 7.44 (s, 1H), 7.34 - 7.39 (m, 1H), 7.29 - 7.34 (m, 2H), 7.19 - 7.25 (m, 4H), 3.42 (t, J = 7.06 Hz, 2H), 2.81 (t, J = 7.06 Hz, 2H). ¹³C NMR (75 MHz, CD₃OD) δ 158.0, 143.4, 139.9, 132.9, 131.3, 130.5, 129.7, 128.8, 128.2, 123.4, 121.4, 42.1, 36.9. MS (ESI) m/z [M+H]⁺ calcd: 353.0; found: 353.0.

3- (4- Chlorophenyl)- 1- [2- (4-bromophenyl)ethyl]urea (26) was prepared from 4-bromophenethylamine (0.08 g, 0.32 mmol) following the general procedure A as white solid (0.06 g, 50%). ¹H NMR (300 MHz, CDCl₃) δ 7.43 (d, J = 8.29 Hz, 2H), 7.18 - 7.25 (m, 4H), 7.08 (d, J = 8.29 Hz, 2H), 6.23 (s, 1H), 4.61 (br. s., 1H), 3.45 - 3.55 (m, 2H), 2.74 - 2.85 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.5, 138.9, 131.1, 131.0, 128.4, 124.4, 119.1, 119.0, 40.4, 35.0. MS (ESI) m/z [M-H]^- calcd: 353.1; found: 353.1.

3- (4- Chlorophenyl)- 1- [2- (2- fluorophenyl)ethyl]urea (27) was prepared from 2-fluorophenethylamine (0.04 ml, 0.32 mmol) following the general procedure A as white solid (0.02 g, 21%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.62 (s, 1H), 7.41 (d, J = 8.67 Hz, 2H), 7.21 - 7.35 (m, 4H), 7.11 - 7.20 (m, 2H), 6.22 (t, J = 5.27 Hz, 1H), 3.26 - 3.33 (m, 2H), 2.79 (t, J = 7.06 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.4, 137.8, 132.5, 131.6, 128.4, 126.7, 125.8, 125.7, 124.5, 119.1, 40.4, 32.6. MS (ESI) m/z [M+H]⁺ calcd: 293.1; found: 293.3.

3- (4- Chlorophenyl)- 1- [2- (3- fluorophenyl)ethyl]urea (28) was prepared from 3-fluorophenethylamine (0.04 ml, 0.32 mmol) following the general procedure A as white solid (0.02 g, 17%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.62 (s, 1H), 7.38 - 7.44 (m, 2H), 7.30 - 7.37 (m, 1H), 7.25 (d, J = 9.04 Hz, 2H), 7.08 (d, J = 8.67 Hz, 2H), 6.99 - 7.05 (m, 1H), 6.17 (t, J = 5.65 Hz, 1H), 3.32 - 3.40 (m, 2H), 2.77 (t, J = 7.06 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 142.5, 139.5, 130.2, 130.1, 128.4, 124.8, 124.4, 119.1, 115.5, 115.2, 112.9, 112.7, 40.4, 35.3. MS (ESI) m/z [M-H]^- calcd: 293.1; found: 293.1.

3- (4- Chlorophenyl)- 1- [2- (4- fluorophenyl)ethyl]urea (29) was prepared from 4-fluorophenethylamine (0.04 ml, 0.32 mmol) following the general procedure A as white solid (0.05 g, 48%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.61 (s, 1H), 7.41 (d, J = 8.85 Hz, 2H), 7.21 - 7.31 (m, 4H), 7.09 - 7.17 (m, 2H), 6.15 (t, J = 5.56 Hz, 1H), 3.27 - 3.33 (m, 2H), 2.74 (t, J = 7.06 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.5, 135.6, 135.5, 130.4, 130.3, 128.4, 124.4, 119.0, 115.1, 114.8, 40.6, 34.8. MS (ESI) m/z [M-H]^- calcd: 293.1; found: 293.0.

3- (4- Chlorophenyl)- 1- [2- (2- chlorophenyl)ethyl]urea (30) was prepared from 2-chlorophenethylamine (0.05 ml, 0.32 mmol) following the general procedure A as white solid (0.07 g, 17%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.61 (s, 1H), 7.39 - 7.46 (m, 3H), 7.30 - 7.38 (m, 2H), 7.28 (d, J = 1.70 Hz, 4H), 6.24 (t, J = 5.56 Hz, 1H), 3.34 - 3.40 (m, 2H), 2.88 (t, J = 7.06 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.5, 136.8, 133.1, 131.0, 129.2, 128.4, 128.1, 127.2, 124.4, 119.1, 40.4, 33.5. MS (ESI) m/z [M+H]⁺ calcd: 309.1; found: 309.3.

3- (4- Chlorophenyl)- 1- [2- (3- chlorophenyl)ethyl]urea (31) was prepared from 3-chlorophenethylamine (0.05 ml, 0.32 mmol) following the general procedure A as white solid (0.09 g, 92%). ¹H NMR (300 MHz, CD₃OD) δ 7.30 - 7.35 (m, 2H), 7.25 - 7.29 (m, 2H), 7.21 - 7.25 (m, 2H), 7.15 - 7.21 (m, 2H), 3.43 (t, J = 7.06 Hz, 2H), 2.82 (t, J = 7.06 Hz, 2H). ¹³C NMR (75 MHz, CD₃OD) δ 156.4, 141.6, 138.3, 133.8, 129.5, 128.4, 128.2, 126.8, 126.6, 126.0, 119.9, 40.5, 35.4. MS (ESI) m/z [M+H]⁺ calcd: 309.1; found: 309.0.

3- (4- Chlorophenyl)- 1- [2- (4- chlorophenyl)ethyl]urea (32) was prepared from 4-chlorophenethylamine (0.05 g, 0.32 mmol) following the general procedure A as white solid (0.07 g, 66%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.61 (s, 1H), 7.38 (dd, J = 8.57, 11.59 Hz, 4H), 7.22 - 7.29 (m, 4H), 6.14 (t, J = 5.56 Hz, 1H), 3.28 - 3.33 (m, 2H), 2.74 (t, J = 6.97 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.5, 138.5, 130.7, 130.5, 128.4, 128.2, 124.4, 119.0, 40.4, 35.0. MS (ESI) m/z [M+H]⁺ calcd: 309.1; found: 309.1.

3- (4- Chlorophenyl)- 1- {2- [2- (trifluoromethyl)phenyl]ethyl}urea (33) was prepared from 2-trifluoromethylphenethylamine (0.06 ml, 0.32 mmol) following the general procedure A as white solid (0.07 g, 15%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.62 (s, 1H), 7.70 (d, J = 7.91 Hz, 1H), 7.61 - 7.67 (m, 1H), 7.51 (d, J = 7.72 Hz, 1H), 7.44 - 7.48 (m, 1H), 7.39 - 7.43 (m, 2H), 7.25 (d, J = 9.04 Hz, 2H), 6.32 (t, J = 5.75 Hz, 1H), 3.36 - 3.43 (m, 1H), 2.93 (t, J = 7.16 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.4, 137.8, 132.5, 131.6, 128.4, 127.5, 127.2, 126.7, 125.7, 124.5, 119.1, 40.4, 32.6. MS (ESI) m/z [M+H]⁺ calcd: 343.1; found: 343.3.

3- (4- Chlorophenyl)- 1- {2- [3- (trifluoromethyl)phenyl]ethyl}urea (34) was prepared from 3-trifluoromethylphenethylamine (0.06 ml, 0.32 mmol) following the general procedure A as white solid (0.08 g, 76%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.62 (s, 1H), 7.52 - 7.62 (m, 4H), 7.42 (d, J = 8.85 Hz, 2H), 7.24 (d, J = 8.85 Hz, 2H), 6.21 (t, J = 5.56 Hz, 1H), 3.34 - 3.44 (m, 2H), 2.86 (t, J = 6.97 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 155.0, 141.0, 139.4, 132.8, 129.2, 128.4, 125.2, 125.1, 124.5, 122.8, 122.8, 119.1, 40.2, 35.4. MS (ESI) m/z [M+H]⁺ calcd: 343.1; found: 343.3.

3- (4- Chlorophenyl)- 1- {2- [4- (trifluoromethyl)phenyl]ethyl}urea (35) was prepared from 4-trifluoromethylphenethylamine (0.05 ml, 0.32 mmol) following the general procedure A as white solid (0.07 g, 64%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.61 (s, 1H), 7.67 (d, J = 7.91 Hz, 2H), 7.47 (d, J = 7.91 Hz, 2H), 7.40 (d, J = 8.85 Hz, 2H), 7.25 (d, J = 8.85 Hz, 2H), 6.18 (t, J = 5.65 Hz, 1H), 3.37 - 3.42 (m, 2H), 2.85 (t, J = 6.97 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 144.5, 139.4, 129.5, 128.4, 125.1, 125.1, 125.0, 124.4, 119.1, 40.4, 35.5. MS (ESI) m/z [M+H]⁺ calcd: 343.1; found: 343.3.

1-(4-chlorophenyl)-3-{2-[3-(trifluoromethoxy)phenyl]ethyl}urea (36) was prepared from 2-(3-trifluoromethoxy-phenyl)-ethylamine (0.09 g, 0.4 mmol) following the general procedure A as white solid (0.11 g, 71%). ¹H NMR (300 MHz, CD₃OD) δ 7.36 - 7.42 (m, 1H), 7.30 - 7.34 (m, 2H), 7.25 (d, J = 8.67 Hz, 1H), 7.19 - 7.23 (m, 2H), 7.17 (br. s., 1H), 7.12 (d, J = 8.10 Hz, 1H), 3.44 (t, J = 7.16 Hz, 2H), 2.87 (t, J = 7.16 Hz, 2H). ¹³C NMR (75 MHz, CD₃OD) δ 158.0, 150.7, 143.5, 139.9, 131.1, 129.7, 128.8, 128.2, 122.5, 121.4, 119.8, 42.0, 37.0, 30.7. MS (ESI) m/z [M+H]⁺ calcd: 359.1; found: 359.0.

3- (4- Chlorophenyl)- 1- [2- (3,5-dichlorophenyl)ethyl]urea (37) was prepared from 3,5-dichlorophenethylamine (0.08 g, 0.4 mmol) following the general procedure A as white solid (0.11 g, 77%). ¹H NMR (300 MHz, CD₃OD) δ 7.30 - 7.35 (m, 2H), 7.29 (s, 1H), 7.19 - 7.25 (m, 4H), 3.42 (t, J = 7.06 Hz, 2H), 2.82 (t, J = 6.97 Hz, 2H). ¹³C NMR (75 MHz, CD₃OD) δ 158.0, 144.9, 139.8, 136.0, 129.7, 128.7, 128.2, 127.4, 121.5, 41.8, 36.7. MS (ESI) m/z [M+H]⁺ calcd: 343.1; found: 343.0.

3- (4- Chlorophenyl)- 1- [2- (2,4- dichlorophenyl)ethyl]urea (38) was prepared from 2,4-dichlorophenethylamine (0.05 ml, 0.32 mmol) following the general procedure A as white solid (0.02 g, 19%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.60 (s, 1H), 7.59 (d, J = 1.32 Hz, 1H), 7.37 - 7.43 (m, 4H), 7.25 (d, J = 9.04 Hz, 2H), 6.22 (t, J = 5.75 Hz, 1H), 3.28 - 3.34 (m, 2H), 2.86 (t, J = 6.97 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.4, 136.1, 134.1, 132.3, 131.6, 128.6, 128.4, 127.3, 124.4, 119.1, 40.4, 32.9. MS (ESI) m/z [M-H]^- calcd: 341.1; found: 341.4.

3- (4- Chlorophenyl)- 1- [2- (2-chloro-6- fluorophenyl)ethyl]urea (39) was prepared from 2-chloro-6-fluorophenethylamine (0.06 g, 0.32 mmol) following the general procedure A as white solid (0.02 g, 23%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.60 (br. s., 1H), 7.39 (s, 2H), 7.29 (d, J = 17.52 Hz, 5H), 6.29 (br. s., 1H), 3.29 - 3.37 (m, 2H), 2.87 - 2.99 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 154.9, 139.5, 134.5, 128.8, 128.4, 125.4, 125.3, 124.4, 119.1, 114.4, 114.1, 38.1, 27.0. MS (ESI) m/z [M+H]⁺ calcd: 327.1; found: 327.3.

3- (4- Chlorophenyl)- 1- [2- (2,4,6- trifluorophenyl)ethyl]urea (40) was prepared from 42 (0.11 g, 0.6 mmol) following the general procedure A as white solid (0.11 g, 54%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.67 (s, 1H), 7.45 (d, J = 8.85 Hz, 2H), 7.30 (d, J = 8.85 Hz, 2H), 7.17 - 7.25 (m, 2H), 6.32 (t, J = 5.93 Hz, 1H), 3.33 (q, J = 6.59 Hz, 2H), 2.82 (t, J = 6.69 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 162.6, 159.5, 154.9, 139.4, 128.4, 124.4, 119.1, 111.2, 100.3, 30.6, 22.7. MS (ESI) m/z [M+H]⁺ calcd: 329.1; found: 329.2.

3- (4- Chlorophenyl)- 1- [2- (2,3,4,5,6- pentafluorophenyl)ethyl]urea (41) was prepared from 43 (0.17 g, 0.78 mmol) following the general procedure A as white solid (0.07 g, 23%). ¹H NMR (300 MHz, CDCl₃) d 7.50 (s, 1H), 7.24 - 7.27 (m, 2H), 7.18 - 7.23 (m, 2H), 5.53 (t, J = 5.65 Hz, 1H), 3.46 (q, J = 6.59 Hz, 2H), 2.93 (t, J = 6.69 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 155.0, 146.5, 143.2, 140.5, 139.3, 138.4, 137.3, 135.0, 128.3, 124.6, 119.2, 113.1, 38.2, 23.2. MS (ESI) m/z [M+H]⁺ calcd: 365.1; found: 365.5.

2-(2,4,6-Trifluorophenyl)ethan- 1- amine (42).

To a solution of LiAlH₄ in THF at 0 °C was added anhydrous AlCl₃. After 5 min, 2,4,6-trifluorobenzonitrile (0.13 ml, 1 mmol) was added dropwise slowly. After stirring at room temperature for 1 h, the remaining LiAlH₄ was quenched cautiously with water, and then with 1.6 ml of 6N H₂SO₄. The pH of the solution was adjusted to 11 with KOH pellets and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous MgSO₄, filtered and concentrated in vacuo. The crude yellow liquid product (0.16 g, 90%) was used for the next step without further purification. ¹H NMR (300 MHz, CDCl₃) δ 6.59 - 6.69 (m, 3H), 2.87 - 2.95 (m, 2H), 2.71 - 2.80 (m, 2H). MS (ESI) m/z [M+H]⁺ calcd: 176.1; found: 176.5.

2-(2,3,4,5,6-Pentafluorophenyl)ethan-1-amine (43) was prepared from 2,3,4,5,6-pentafluorobenzonitrile (0.12 ml, 1 mmol) following the same procedure for to obtain the desired product as yellow liquid (0.17 g, 81%). ¹H NMR (300 MHz, CDCl₃) δ 3.53 - 3.86 (m, 1H), 2.77 - 3.08 (m, 1H), 1.57 - 2.17 (m, 2H). MS (ESI) m/z [M+H]⁺ calcd: 212.1; found: 212.1.

2-(3,5-Dimethylphenyl)ethanamine (44).

To a solution of 3,5-dimethylphenylacetonitrile (0.10 g, 0.69 mmol) in anhydrous THF (2.6 mL) was added 2 M BH₃.Me₂S in THF (0.75 mL, 0.31 mmol). The reaction was refluxed for 16 h. Upon cooling to room temperature, the reaction was carefully quenched with methanol and concentrated under reduced pressure. The residue was redissolved in methanol and concentrated under reduced pressure twice to yield the title product as colorless liquid (0.10 g, quant.) which was used in the next step without further purification. MS (ESI) m/z [M+H]⁺ calcd: 150.1; found: 150.0.

2-(3-Bromophenyl)ethanamine (45).

To a solution of 3-bromophenylacetonitrile (1.0 g g, 5.13 mmol) in anhydrous THF (19 mL) was added 2 M BH₃.Me₂S in THF (5.6 mL, 11.28 mmol). The reaction was refluxed for 16 h. Upon cooling to room temperature, the reaction was carefully quenched with methanol and concentrated under reduced pressure. The residue was redissolved in methanol and concentrated under reduced pressure twice to yield the title product as colorless liquid (1.0 g, quant.) which was used in the next step without further purification. MS (ESI) m/z [M+H]+ calcd: 200.0; found: 200.0.

tert-Butyl [2-(3-bromophenyl)ethyl]carbamate (46).

To a solution of 45 (2 g, 10 mmol) in 1,4-dioxane was added 2 M K₂CO₃ (20 mmol, 10 mL) and Boc₂O (2.67 g, 12.24 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was extracted with ethyl acetate three times (50 mL each). The combined organic layers were washed with water, then brine. The organic portion was dried with anhydrous MgSO4, filtered and concentrated. The residue was purified by column chromatography (SiO₂, ethyl acetate/hexanes) to yield the title product was colorless liquid (0.90 g, 90%). ¹H NMR (300 MHz, CDCl₃) δ 7.45 - 7.52 (m, 1H), 7.33 - 7.39 (m, 1H), 7.08 - 7.21 (m, 2H), 3.36 (q, J = 6.53 Hz, 2H), 2.77 (t, J = 6.88 Hz, 2H), 1.44 (s, 9H).

tert-Butyl [2-(3-cyanophenyl)ethyl]carbamate (47).

To a 25 mL round bottom flask was added 46 (0.39 g, 1.3 mmol), potassium ferricyanide (0.28 g, 0.86 mmol), Buldwald t-BuxPhos Pd G3 (0.04 g, 0.04 mmol) and t-BuXPhos (0.02 g, 0.04 mmol). The flask was sealed, evacuated and backfilled with nitrogen three times. 1,4-Dioxane (3.3 mL) and 0.1M potassium acetate (3.3 mL) were added. The reaction mixture was heated at 100 °C for 16 h. Upon cooling to room temperature, the reaction was partitioned between ethyl acetate and water (10 mL each). The aqueous layer was extracted with ethyl acetate three times (50 mL each). The combined organic layers were washed with water and brine. The organic portion was dried with anhydrous MgSO₄ and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (SiO₂, ethyl acetate/hexanes) to afford the title product as white solid (0.03 g, 10%). ¹H NMR (300 MHz, CDCl₃) δ 7.51 - 7.56 (m, 1H), 7.40 - 7.50 (m, 3H), 4.60 (br. s., 1H), 3.38 (q, J = 6.59 Hz, 2H), 2.84 (t, J = 6.97 Hz, 2H), 1.43 (s, 9H).

3-Cyanophenethylamine hydrochloride (48).

47 (0.03 g, 0.1 mmol) was treated with 0.3 mL of 4 N HCl in 1,4-dioxane for 1 h at room temperature. The reaction mixture was concentrated in vacuo to yield the product as white solid (0.02 g, quant.). MS (ESI) m/z [M+H]⁺ calcd: 147.1; found: 147.0.

Calcium Mobilization Assay.

CHO-RD-HGA16 cells (Molecular Devices, CA) stably expressing the human CB₁ receptor were plated into 96-well black-walled assay plates at 25,000 cells/well in 100 μL of Ham’s F12 (supplemented with 10% fetal bovine serum, 100 units of penicillin/streptomycin, and 100 μg/mL Normocin) and incubated overnight at 37 °C, 5% CO₂. Calcium 5 dye (Molecular Devices, CA) was reconstituted according to the manufacturer’s instructions. The reconstituted dye was diluted 1:40 in prewarmed (37 °C) assay buffer (1x HBSS, 20 mM HEPES, 2.5 mM probenecid, pH 7.4 at 37 °C). Growth medium was removed, and the cells were gently washed with 100 μL of prewarmed (37 °C) assay buffer. The cells were incubated for 45 min at 37 °C, 5% CO₂ in 200 μL of the diluted Calcium 5 dye solution. For antagonist assays to determine IC₅₀ values, the EC₈₀ concentration of CP55,940 (100 nM) was prepared at 10x the desired final concentration in 0.25% (w/v) BSA/0.5% DMSO/0.5% EtOH/assay buffer, aliquoted into 96-well polypropylene plates, and warmed to 37 °C. Serial dilutions of the test compounds were prepared at 10x the desired final concentration in 2.25% BSA/4.5% DMSO/4.5% EtOH/assay buffer. After the dye loading incubation period, the cells were pretreated with 25 μL of the test compound serial dilutions and incubated for 15 min at 37 °C. After the pretreatment incubation period, the plate was read with a FLIPR Tetra (Molecular Devices, CA). Calcium-mediated changes in fluorescence were monitored every 1 s over a 90 s time period, with the Tetra adding 25 μL of the CP55,940 EC₈₀ concentration at the 10s time point (excitation/emission: 485/525 nm). Relative fluorescence units (RFU) were plotted against the log of compound concentrations. Data were fit to a three-parameter logistic curve to generate IC₅₀ values (GraphPad Prism 6.0, CA). For agonist screens, the above procedure was followed except that cells were pretreated with 2.25% BSA/4.5% DMSO/4.5% EtOH/assay buffer and the Tetra added single concentration dilutions of the test compounds prepared at 10x the desired final concentration in 0.25% BSA/0.5% DMSO/0.5% EtOH/assay buffer. Test compound RFUs were compared to the CP55,940 E_max RFUs to generate % E_max values. For the CB2 agonist screens, the same procedures were followed except that stable human CB2-CHO-RD-HGA16 cells were used. CB2 antagonist screens were conducted similar to the IC₅₀ experiments except that a single concentration of test compound was used instead of serial dilutions and test compound RFUs were compared to the CP55,940 EC₈₀ RFUs to generate % inhibition values.

cAMP Assay.

Subsets of compounds were tested at either RTI and University of Otago using similar protocols. Forskolin (FSK)-stimulated cyclic adenosine monophosphate (cAMP) production was measured in real-time using a transfected bioluminescence resonance energy transfer (BRET) cAMP sensor, CAMYEL.⁴⁹ The plasmid encodes a cAMP binding domain (Epac1) flanked by yellow fluorescent protein (YFP) and Renilla Luciferase (RLuc) assay, the latter of which can oxidize coelenterazine H and produce a photon as a byproduct. When cAMP is bound to the Epac1 domain, it separates RLuc and YFP so only Rluc emits a photon at a wavelength of 460 nm. When cAMP is not bound, closer proximity means that RLuc can resonate energy to excite YFP, which then emits light at wavelength 535 nm. A plate reader measures both wavelengths and their ratio, 460/535, is calculated to quantify cAMP levels where increases in the ratio indicate increases in cAMP. Human Embryonic Kidney 293 (HEK293) cells stably transfected with either human CB₁ N-terminally tagged with three haemagglutinins (University of Otago; 3HA-hCB₁, cell line first reported in Cawston et al⁴⁵) or untagged human CB₁ (RTI) were maintained at 37°C at 5% CO₂, and seeded in 100 mM dishes for transfection. The next day, cells were either given fresh growth media and transfected with 5 μg of pcDNA3L-His-CAMYEL using linear polyethyleneimine (PEI, 25 kDa, Polysciences, Warrington, PA) in a 1:6 DNA:PEI ratio University of Otago) or media was replaced with Optimem (Gibco, ThermoFisher Scientific) and cells were transfected with CAMYEL using Transit2020 (Mirus Bio, Madison, WI) at a ratio of 1:3 DNA:Transit2020. The next day, cells were lifted with 0.05% trypsin/EDTA, counted and plated on poly-D-lysine (Sigma Aldrich, St. Louis, MO) coated white 96-well CulturPlates (Perkin Elmer, Waltham, MA) at either 60,000 cells per well (University of Otago) or 50,000 cells per well (RTI). The following day, working with wells for one assay run at a time, culture medium was removed by aspirating, cells were rinsed with PBS then serum starved for 30 min in assay medium. Assay medium for experiments conducted at University of Otago was comprised of phenol-free DMEM supplemented with 1 mg/ml BSA (MP Biomedicals, Auckland, New Zealand) and 10 mM HEPES pH 7.4 (Gibco, ThermoFisher Scientific) whereas experiments conducted at RTI were carried out using assay buffer which was comprised of HBSS with calcium and magnesium (Gibco) supplemented with 1 mg/ml BSA (A7030; Sigma Aldrich). Coelenterazine H (Prolume, Pinetop, AZ) constituted in absolute ethanol was prepared in assay medium at 10x concentration and dispensed 5 minutes prior to drug addiction (final concentration in-well was 5 μM). Drug dilutions were prepared in assay medium/buffer (vehicle-controlled) at 10x concentration and pre-mixed in a V-well dispensing plate, then added simultaneously to the assay plate with a multichannel pipette. Drugs conditions included forskolin (in DMSO, to a final concentration of 5 μM), CP55,940 (in absolute ethanol, to a final concentration of 1 μM) and allosteric modulators (in DMSO, and serial dilution in DMSO-controlled assay medium). BRET signal was detected over approximately 20 minutes in a pre-warmed 37°C LUMIStar Omega plate `reader (University of Otago; BMG Labtech, Ortenberg, Germany) or CLARIOstar plate reader (RTI; BMG Labtech), simultaneously, using BRET1 filters (535-30 and 475-30 nm). BRET data was exported into Excel, then inverse BRET ratios (460/535) were calculated for each time point and plotted across time in GraphPad Prism (v8, GraphPad Software, San Diego, CA). Area under the curve analysis was performed for each well, and then normalized to the mean “forskolin alone” (100%) and “vehicle only” (0%) conditions. Concentration-response curves were fit in Prism by 3-parameter non-linear regression. Three independent experiments (biological replicates) were performed, and then the pIC₅₀ values reported in Table 2 were calculated by taking the mean and SEM of each independent experiments’ pIC₅₀. Data shown in Figure 2B are the results of a representative experiment performed in technical duplicate. Importantly, the potencies of CP55,940 and PSNCBAM-1 were consistent between the two protocols carried out at University of Otago and RTI.

Membrane preparation.

Cerebella from adult male CD-1 mice were dissected on ice, snap frozen, and stored at −80°C until the day of the experiment. Cerebella were homogenized by polytron in membrane buffer (50 mM Tris, 3 mM MgCl₂, 0.2 mM EGTA, 100 mM NaCl, pH 7.4) on ice, centrifuged for 10 min at 40,000xg at 4°C. The supernatant was discarded and the pellet was suspended in membrane buffer, homogenized, and centrifuged again for 10 min at 40,000xg. The pellet was resuspended in membrane buffer and protein quantified by Bradford method.

[³⁵S]GTPγS binding assay.

Cerebellar or CB₁-expressing HEK293 cell membranes were preincubated in assay buffer (membrane buffer containing 1 mg/ml bovine serum albumin; BSA) for 10 min with 3 units/ml adenosine deaminase (cerebellar membranes) then incubated for 60 min at 30°C with 30 μM GDP and 0.1 nM [³⁵S]GTPγS. Serial dilutions of test compounds were done in 100% DMSO with final assay DMSO concentration of 0.1%. Non-specific binding was determined by adding 30 μM unlabeled GTPγS. Reactions were terminated by vacuum filtration through GF/C filter plates (Perkin Elmer). GTPγS inhibition curves for test compounds were normalized to CP55,940 (100 nM) stimulation in the absence of test compound (i.e. vehicle = 100%). Curvefits were accomplished using Graphpad Prism 9.0 and where GTPγS data were fit to 3 parameter non-linear regression for IC₅₀ calculation.

Metabolic stability assessment was performed by Paraza Pharma Inc. (Montreal, Canada). Compounds were incubated with rat liver microsomes at 37 °C for a total of 45 minutes. The reaction was performed at pH 7.4 in 100 mM potassium phosphate buffer containing 0.5 mg/mL of rat liver microsomal protein. Phase I metabolism was assessed by adding NADPH to a final concentration of 1 mM and collecting samples at time points 0, 5, 15, 30 and 45 minutes. All collected samples were quenched 1:1 with ice-cold stop solution (1 μM labetalol and 1 μM glyburide in acetonitrile), and centrifuged to remove precipitated protein. Resulting supernatants were further diluted 1:4 with acetonitrile:water (1:1). Samples were analyzed by LC/MS/MS and calculations for half-life, and in vitro clearance were accomplished using Microsoft Excel (2007). Half-life and clearance were determined from two independent experiments in duplicate.

Bidirectional MDCK-MDR1 permeability assay was performed by Paraza Pharma Inc. (Montreal, Canada). MDCK-mdr1 cells at passage 5 were seeded onto permeable polycarbonate supports in 12-well Costar Transwell plates and allowed to grow and differentiate for 3 days. On day 3, culture medium (DMEM supplemented with 10% FBS) was removed from both sides of the transwell inserts and cells were rinsed with warm HBSS. After the rinse step, the chambers were filled with warm transport buffer (HBSS containing 10 mM HEPES, 0.25% BSA, pH 7.4) and the plates were incubated at 37 °C for 30 min prior to TEER (Trans Epithelial Electric Resistance) measurements.

The buffer in the donor chamber (apical side for A-to-B assay, basolateral side for B-to-A assay) was removed and replaced with the working solution (10 μM test article in transport buffer). The plates were then placed at 37 °C under light agitation. At designated time points (30, 60 and 90 min), an aliquot of transport buffer from the receiver chamber was removed and replenished with fresh transport buffer. Samples were quenched with ice-cold ACN containing internal standard and then centrifuged to pellet protein. Resulting supernatants are further diluted with 50/50 ACN/H₂O (H₂O only for Atenolol) and submitted for LC-MS/MS analysis. Reported apparent permeability (Papp) values were calculated from single determination. Atenolol and propranolol were tested as low and moderate permeability references. Bidirectional transport of digoxin was assessed to demonstrate Pgp activity/expression.

The apparent permeability (P_app, measured in cm/s) of a compound is determined according to the following formula from two independent experiments in duplicate.

$$\text{Papp}=\frac{(\text{dQ})∕(\text{dt})}{\mathrm{A}\ast \text{Ci}\ast 60}$$

dQ/dt is the net rate of appearance in the receiver compartment

A is the area of the Transwell measured in cm² (1.12 cm²)

Ci is the initial concentration of compound added to the donor chamber

60 is the conversion factor for minute to second

In vivo pharmacokinetic assay was performed by Paraza Pharma Inc. (Montreal, Canada). On the morning of the PK study, male Sprague-Dawley rats weighing 258-277 g were dosed with either vehicle (5% Cremorphor, 5% ethanol in saline) or 30 (10 mg/kg, i.p.). At selected timepoints (0.25, 0.5, 1, 3, 5, 8 and 24 hours post dose), 2 rats were anesthetized with isoflurane gas to perform a cardiac puncture to collect blood (for plasma analysis), followed by whole body perfusion with phosphate saline buffer (PBS, pH 7.4) to wash out any remaining blood from the organs. Brains were harvested and homogenized by mechanical sheering with a polytron with 1:4 (w/v) 25% isopropanol in water. Brain homogenates were extracted for drug quantification by LC-MS/MS.

Reinstatement of extinguished cocaine-seeking behavior.

Adult male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 280-300 g were used in the study. Animals were housed individually on a 12/12 hr light/dark cycle (behavioral experiments were conducted during the light period) with free access to water and food except during experimental sessions. Animals were maintained and experiments conducted in accordance with the Institutional Animal Care and Use Committee, University at Buffalo, and with the 2011 Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC).

Drug self-administration, extinction and reinstatement:

The reinstatement procedure used has been previously described in detail.^{48, 50} Briefly, rats were surgically implanted with a chronic indwelling jugular catheter. After one-week recovery, rats were trained to press the active lever (left lever) for infusion of cocaine (0.75 mg/kg/inf) under a fixed ratio [FR] schedule (starting FR =1, which was increased to FR 5 within 5 training sessions) schedule during daily 2-hr sessions for 14 days. Reinforcer deliveries were accompanied by the presentation of a stimulus light over the active lever followed by a 30-s time-out period during which lever presses had no programmed consequence. Following acquisition of cocaine self-administration, extinction of drug-seeking behavior took place during 2-hr daily sessions in which lever pressing produced no consequence. All other conditions remained unchanged. After 7 days of extinction, all rats reached the extinction criteria (total responses less than 20% of the training sessions).

Drug-induced reinstatement test was conducted on the day following the last extinction session. Rats were pretreated with vehicle, compound 30 (10 mg/kg) 10 min prior to a priming injection of cocaine (10 mg/kg, i.p.) administered immediately before the start of the reinstatement session.

Locomotor activity:

Locomotor activity was measured using an infrared motion-sensor system (AccuScan Instruments, Inc. Columbus, OH) surrounding plexiglass cages (40 × 40 × 30 cm) ³¹. Versa Max software (Omnitech Electronics, Inc., Columbus, OH) was used to monitor the distance the animal travelled for a total of 30 min. Prior to test sessions, rats were exposed to at least three days of handling by the experimenter. Baseline sessions in which rats received a 15 min pretreatment of either 10 mg/kg (i.p.) compound 30 or vehicle before being placed in the locomotion chambers and the locomotor activity was recorded for 30 min.

Data analyses:

Data are expressed as mean ± S.E.M. For reinstatement test, differences in active lever responding between the last extinction session and reinstatement session were determined with paired t tests (within subjects comparison). The effects of compound 30 on reinstatement were analyzed by Student’s t tests (between group comparison). For locomotion data, results were expressed as locomotion distance per 5 min bins. Data were analyzed using two-way analysis of variance (ANOVA) with time as within subject factor and treatment as between group factor. P < 0.05 was considered statistically significant.

ABBREVIATIONS

ADME

absorption, distribution, metabolism and excretion

CB1

cannabinoid type-1 receptor

CPP

conditioned place preference

DCM

dichloromethane

1,2-DCE

1,2-dichloroethane

DME

1,2-dimethoxyethane

FLIPR

fluorometric imaging plate reader

GPCR

G-protein-coupled receptor

HPLC

high performance liquid chromatography

IC₅₀

half-maximum inhibitory concentration

MS

mass spectrometry

NAM

negative allosteric modulator

NMR

nuclear magnetic resonance

PAM

positive allosteric modulator

SAR

structure–activity relationship

TLC

thin-layer chromatography