Diarylureas Containing 5-Membered Heterocycles as CB1 Receptor Allosteric Modulators: Design, Synthesis, and Pharmacological Evaluation

Article abstract of DOI:10.1021/acschemneuro.8b00396

Allosteric modulators have attracted significant interest as an alternate strategy to modulate CB₁ receptor signaling for therapeutic benefits that may avoid the adverse effects associated with orthosteric ligands. Here we extended our previous structure-activity relationship studies on the diarylurea-based CB₁ negative allosteric modulators (NAMs) by introducing five-membered heterocycles to replace the 5-pyrrolidinylpyridinyl group in PSNCBAM-1 (1), one of the first generation CB₁ allosteric modulators. Many of these compounds had comparable potency to 1 in blocking the CB₁ agonist CP55,940 stimulated calcium mobilization and [³⁵S]GTP-γ-S binding. Similar to 1, most compounds showed positive cooperativity by increasing [³H]CP55,940 binding, consistent with the positive allosteric modulator (PAM)-antagonist mechanism. Interestingly, these compounds exhibited differences in ability to increase specific binding of [³H]CP55,940 and decrease binding of the antagonist [³H]SR141716. In saturation binding studies, only increases in [³H]CP55,940 B_max, but not K_d, were observed, suggesting that these compounds stabilize low affinity receptors into a high affinity state. Among the series, the 2-pyrrolyl analogue (13) exhibited greater potency than 1 in the [³⁵S]GTP-γ-S binding assay and significantly enhanced the maximum binding level in the [³H]CP5,5940 binding assay, indicating greater CB₁ receptor affinity and/or cooperativity.

Full text of DOI:10.1021/acschemneuro.8b00396

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Article
Diarylureas Containing 5-Membered Heterocycles as CB1 Receptor
Allosteric Modulators: Design, Synthesis, and Pharmacological Evaluation
Thuy Nguyen, Thomas F. Gamage, Ann M. Decker, Nadezhda German, Tiffany L. Langston,
Charlotte E Farquhar, Terry P. Kenakin, Jenny L. Wiley, Brian F Thomas, and Yanan Zhang
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00396 • Publication Date (Web): 06 Sep 2018
Downloaded from http://pubs.acs.org on September 7, 2018
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Diarylureas Containing 5ꢀMembered Heterocycles as CB1
Receptor Allosteric Modulators: Design, Synthesis, and
Pharmacological Evaluation
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Thuy Nguyen,1,# Thomas F. Gamage,1,# Ann M. Decker,1 Nadezhda German,1 Tiffany L.
Langston,1 Charlotte E. Farquhar,1 Terry P. Kenakin,2 Jenny L. Wiley,1 Brian F. Thomas,1
Yanan Zhang1,*
1 Research Triangle Institute, Research Triangle Park, North Carolina, United States
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Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina,
United States
# These authors contributed equally to this work.
KEYWORDS: CB1 receptor, diarylurea, allosteric modulators, fiveꢀmembered heterocycles,
structureꢀactivity relationship, PSNCBAMꢀ1.
Abstract: Allosteric modulators have attracted significant interest as an alternate strategy to
modulate CB1 receptor signaling for therapeutic benefits that may avoid the adverse effects
associated with orthosteric ligands. Here we extended our previous structureꢀactivity relationship
studies on the diarylureaꢀbased CB1 negative allosteric modulators (NAMs) by introducing fiveꢀ
membered heterocycles to replace the 5ꢀpyrrolidinylpyridinyl group in PSNCBAMꢀ1 (1), one of
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the first generation CB1 allosteric modulators. Many of these compounds had comparable
potency to 1 in blocking the CB1 agonist CP55,940 stimulated calcium mobilization and
[35S]GTPꢀγꢀS binding. Similar to 1, most compounds showed positive cooperativity by
increasing [3H]CP55,940 binding, consistent with the positive allosteric modulator (PAM)ꢀ
antagonist mechanism. Interestingly, these compounds exhibited differences in ability to increase
specific binding of [3H]CP55,940 and decrease binding of the antagonist [3H]SR141716. In
saturation binding studies, only increases in [3H]CP55,940 Bmax, but not Kd, were observed,
suggesting that these compounds stabilize low affinity receptors into a high affinity state. Among
the series, the 2ꢀpyrrolyl analogue (13) exhibited greater potency than 1 in the [35S]GTPꢀγꢀS
binding assay and significantly enhanced the maximum binding level in the [3H]CP5,5940
binding assay, indicating greater CB1 receptor affinity and/or cooperativity.
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Introduction
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The cannabinoid typeꢀ1 (CB1) receptor is one of the most abundant G proteinꢀcoupled
receptors (GPCRs) in the central nervous system (CNS) and has long been regarded as a
promising target for the treatment of many conditions such as obesity, drug addiction, pain,
inflammation, gastrointestinal diseases, multiple sclerosis, psychosis, schizophrenia, and
osteoporosis.1, 2 The CB1 receptor antagonist/inverse agonist, rimonabant (SR141716), was
previously approved in Europe for the treatment of obesity.3 It also exhibited clinical benefits in
smoking cessation,4 and it attenuated drug seeking behaviors in animal studies,5 supporting the
notion that CB1 antagonism may be useful as a treatment for drug addiction.6ꢀ8 As rimonabant
was subsequently withdrawn from the market due to its adverse effects, allosteric modulators
have attracted attention as an alternative means to target the CB1 signaling pathway for
therapeutic benefits.9ꢀ12
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PSNCBAMꢀ1 (1) was one of the first CB1 receptor allosteric modulators, identified from a
high throughput screen in a yeastꢀbased assay in 2007.13 1 shared similar in vitro
pharmacological properties to another CB1 allosteric modulator, Org27569 (2) such as enhancing
[3H]CP55,490 binding levels (positive allosteric modulation of agonist affinity) but inhibiting
agonistꢀinduced responses in functional assays (negative allosteric modulation of agonist
function).14, 15 Termed positive allosteric modulatorꢀantagonists, or PAMꢀantagonists, these
unique allosteric modulators increase the affinity of the agonist for the receptor but
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simultaneously decrease functional efficacy of the coꢀbound agonist.
As PAMꢀantagonists
‘seek and destroy’ agonistꢀactivated receptors, they offer unique advantages in contrast to
orthosteric and typical NAMs such as better reversal of ongoing persistent agonism and
favorable target coverage in vivo.16 In addition, unlike 2, 1 alone did not show agonist activities
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in several assays tested such as ERK phosphorylation.9, 14, 17 In vivo, 1 demonstrated efficacy in
reducing food intake and body weight.13 Recently, our group has demonstrated that 1 and an
analogue with greater metabolic stability, RTICBMꢀ74 (3), attenuated primeꢀinduced
reinstatement of extinguished cocaineꢀseeking behavior in rats.18 Furthermore, we have shown
that 1 attenuated tetrahydrocannabinolꢀinduced antiꢀnociception, and its analogue RTICBMꢀ28
(4) shifted the potency of THC in drug discrimination.19 The promising results from these in vivo
studies encourage further development on these diarylurea compounds for the potential use in the
treatment of CB1ꢀrelated disorders (Fig. 1).
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Figure 1. Structures of representative CB1 negative allosteric modulators
In previous studies, our group reported that the pyrrolidinyl ring of 1 could be replaced by
other alkylamino groups (e.g. RTICBMꢀ15, 5) or abolished completely without compromising
the CB1 activity, and the entire 5ꢀpyrrolidinylpyridinyl group could be replaced by substituted
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phenyl and pyridinyl rings.18, 20 In agreement with our results, Khurana et al recently reported
that analogues with a pyrimidinyl ring in place of the pyridinyl ring retained CB1 modulatory
activity (6 and 7, Fig. 1).21 In order to further explore the structureꢀactivity relationship (SAR)
around this scaffold and develop potent and selective CB1 NAMs as potential pharmacotherapies
for treating CNS disorders, we designed and synthesized a series of analogues in which the 5ꢀ
pyrrolidinylpyridinyl ring was replaced by other heterocyclic rings to introduce structural
diversity (Fig. 2). Specifically, we have focused our effort on investigating the effects of
substitution of various 5ꢀmembered heterocyclic rings in CB1 receptor allostery, such as furan,
thiophene, pyrrole, thiazole, and imidazole. These novel compounds were examined in calcium
mobilization, [35S]GTPꢀγꢀS binding, [3H]CP55,940 and [3H]SR141716 radioligand binding
assays to investigate their pharmacological properties as CB1 allosteric modulators.
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Figure 2. Structural modifications of the diarylurea template in this study
Results and Discussion
Chemistry. Scheme 1 depicts the synthetic routes used to prepare target compounds 8ꢀ12
and 16ꢀ18. For compounds 8ꢀ9, commercially available 3ꢀfuranylboronic acid or 3ꢀ
thienylboronic acid underwent Suzuki coupling with 3ꢀbromoaniline to provide the intermediates
20aꢀb, which reacted with 4ꢀchlorophenyl isocyanate to afford the target compounds 8 and 9. For
target compounds 10ꢀ12 and 16ꢀ18, the heterocyclic boronic acids were not commercially
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available and therefore the Suzuki coupling was carried out between the 3ꢀnitrobenzene boronic
acid and the corresponding heterocyclic bromide to give intermediates 21aꢀf. The nitro group
was then reduced to furnish the corresponding anilines 22aꢀf by Raneyꢀnickel catalyzed transfer
hydrogenation using hydrazine in ethanol. Subsequently, anilines 22aꢀf were coupled with 4ꢀ
chlorophenyl isocyanates to give the desired products 10ꢀ12 and 16ꢀ18.18, 20
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Scheme 1. Reagents and conditions (a) aryl boronic acid, Pd(PPh3)4, DME, NaHCO3, reflux,
o
16 h, 10ꢀ32% (b) 4ꢀchlorophenyl isocyanate, chloroform, 50 C, 16 h, 57ꢀ95% (c) aryl bromide,
Pd(PPh3)4, DME, NaHCO3, reflux, 16 h, 37ꢀ92% (d) hydrazine hydrate, Raney Ni, ethanol, 50
oC, 2 h, 46%ꢀquant. yield.
Similarly, Suzuki coupling between NꢀBocꢀ2ꢀpyrrole boronic acid and 1ꢀbromoꢀ3ꢀ
nitrobenzene provided the intermediate 23, which was then reduced to aniline 24 under transfer
hydrogenation conditions. Intermediate 24 was then reacted with 4ꢀchlorophenyl isocyanate to
yield the final product 15. Removal of the Boc group in 24 by refluxing in 5% w/v aqueous KOH
solution gave the aniline 25, which was then coupling with 4ꢀchlorophenyl isocyanate to afford
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the target compound 13 (Scheme 2).22 Notably, attempts to remove the Boc group of 24 under
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acidic conditions using acids such as 4N HCl in dioxane led to product degradation.
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Scheme 2. Reagents and conditions (a) 1ꢀBromoꢀ3ꢀnitrobenzne, Pd(PPh3)4, DME, NaHCO3,
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reflux, 16 h, 51% (b) hydrazine hydrate, Raney Ni, ethanol, 50 C, 2 h, 65% (c) 4ꢀchlorophenyl
isocyanate, chloroform, 50 oC, 16 h, 81ꢀ84% (d) aq. 5% KOH, reflux, 4 h, 78%.
Palladiumꢀcatalyzed direct arylation of Nꢀmethylpyrrole with 3ꢀiodoaniline gave the 3ꢀ(1ꢀ
methylꢀ1Hꢀpyrrolꢀ2ꢀyl)aniline 26,23 which was then converted to the final product 14 upon
treatment with 4ꢀchlorophenyl isocyanate (Scheme 3).
For the preparation of compound 19, initial attempts of Suzuki coupling between 2ꢀbromoꢀ
1Hꢀimidazole and 3ꢀnitrobenzene boronic acid failed to yield the desired product. Hence, the
imidazole ring was constructed by reaction between 3ꢀnitrobenzonitrile and aminoacetaldehyde
dimethyl acetal to give the intermediate 27.24 The target compound 19 was then obtained by the
reduction of the nitro group leading to the aniline 28 and the subsequent reaction with 4ꢀ
chlorophenyl isocyanate (Scheme 3).
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Scheme 3. Reagents and conditions (a) 3ꢀiodoaniline, AcOK, Pd(OAc)2, AcNMe2, sealed tube,
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150 C, 20 h, 22% (b) 4ꢀchlorophenyl isocyanate, chloroform, 50 C, 16 h, 86ꢀ98% (c) (i)
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NaOMe, MeOH, rt, 5 h (ii) (MeO)2CHCH2NH2, AcOH, 70 C, 1 h (iii) aq. 6N HCl, MeOH, 70
oC, 3 h, 61% (d) hydrazine hydrate, Raney Ni, ethanol, 50 oC, 2 h, quant. yield.
Effects of compounds 8ꢀ19 in calcium mobilization assays. All target compounds were
first evaluated for their ability to inhibit the mobilization of intracellular calcium levels
stimulated by CP55,940 in CHO cells overexpressing the promiscuous Gα16 protein (RDꢀ
HGA16 cells, Molecular Devices) and the human CB1 or CB2 receptor as previously described.25
Table 1 lists the IC50 values of the synthesized compounds against the EC80 concentration of
CP55,940 (100 nM) in the CB1 calcium mobilization assay. 1 had an IC50 value of 33 nM. When
the 5ꢀpyrrolidinylpyridinyl group was replaced with fiveꢀmembered heterocyclic rings such as
furan, thiophene, or methylthiophene, the CB1 modulatory potency was retained (8ꢀ12, IC50 = 36
ꢀ 67 nM). Replacement with pyrrole (13) or Nꢀmethylpyrrole (14) resulted in about 3ꢀfold
reduction in potencies (IC50 = 165ꢀ169 nM). The presence of the bulky Boc group in 15
significantly decreased the potency (IC50 > 3,000 nM). The three thiazolyl analogues had slightly
decreased potency compared to 1 (16ꢀ18, IC50 = 84 ꢀ 154 nM). Lastly, the potency was
dampened by around 16ꢀfold in the presence of the more polar group, imidazolyl (19, IC50 = 529
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nM). These results suggest that the CB1 receptor can accommodate a diversity of heterocylic
rings at this position.
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Table 1. Allosteric modulatory activities of compounds 8ꢀ19 in CB1 and CB2 calcium
mobilization and CB1 [35S]GTPꢀγꢀS binding assays.
CB1
CB2
Compound
Ar
[35S]GTPγS binding Antagonist screen in
Calcium assay
IC50 (nM)a
assay
calcium assay
IC50 (nM)b
(%CP55,940 Emax)
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33 ± 8
41 ± 9
115 (87, 155)
441 (339, 574)
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36 ± 1
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164 (113, 239)
573 (272, 1205)
776 (324, 1858)
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372 (182, 759)
40 (29, 55)
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169 ± 34
165 ± 14
IC50 > 10 µM e
665 (501, 883)
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3444 ± 451
3236 (1480, 7070)
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84 ± 6
94 ± 9
251 (118, 530)
331 (215, 509)
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154 ± 14
529 ± 98
1259 (562, 2818)
343 (224, 524)
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19
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Against EC80 (100 nM) of CP55,940 in stable human CB1ꢀCHOꢀRDꢀHGA16 cells. Values
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are the mean ± SEM of at least three independent experiments in duplicate. Tested in mouse
cerebellar membranes against CP55,940 (100 nM). Values are expressed as mean (95%
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confidence interval) from at least three independent experiments in duplicate. Against EC80
(100 nM) of CP55,940 in stable human CB2ꢀCHOꢀRDꢀHGA16 cells. Compounds were tested at
d
e
10 ꢁM final concentration in two independent experiments in duplicate. < 50% inhibition.
Compound showed > 50% inhibition and was tested for potency.
None of the compounds showed significant CB2 antagonist activity (< 50% inhibition of
CP55,940 EC80 concentration at 10 ꢁM or IC50 values >10 ꢁM) (Table 1). All compounds were
additionally tested for agonist activity at both CB1 and CB2 receptors. No significant CB1 agonist
activity (< 30% of CP55,940 Emax) or CB2 agonist activity (< 30% of CP55,940 Emax) at
concentrations of 10 ꢀM was observed (Supporting Information).
Effects of diarylureas in [35S]GTPꢀγꢀS binding assay. All compounds were then evaluated
as NAMs at the CB1 receptor in the [35S]GTPꢀγꢀS assay using the CB1 agonist CP55,940 (Table
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1). While 3ꢀfuranyl (8) showed similar potency in the calcium assay to 1, it was ~4 fold less
potent in the GTPꢀγꢀS assay. The 3ꢀthiophenyl analogue (9, IC50 = 164 nM) had comparable
potency to 1 (IC50 = 115 nM), and the 2ꢀthiophenyl analogue (10) was slightly weaker (IC50 =
573 nM). Introduction of a methyl group resulted in a 5ꢀfold reduction in inhibitory potency in
the 3ꢀthiophenyl analogue (11), whereas the potency slightly increased in the 2ꢀthiophenyl
analogue ( 12 vs. 10). The lower potencies of both Nꢀmethylpyrrolyl (14) and NꢀBoc pyrrolyl
(15) analogues were consistent with the results from the calcium assay. Similarly, the three
thiazolyl analogues (16ꢀ18) shared the same trend in activities in both assays.
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In general, the compounds were more potent in the calcium assay than in GTPꢀγꢀS. However,
the pyrrolyl analogue (13, IC50 = 40 nM) appeared to be more potent than 1 in the GTPꢀγꢀS
binding assay even though it was weaker in the calcium assay. Similarly, the imidazolyl
analogue (19) demonstrated better activity in the GTPꢀγꢀS binding assay than the calcium assay.
This difference in the potency ranking order between the two assays could result from the
differences in affinity for human versus mouse CB1 receptor and/or G protein subtype coupling
involved in the response (e.g. overexpression of Gα16 in the calcium assay).
Effects of diarylureas on [3H]CP55,940 radioligand binding. Effects of representative
allosteric modulators on equilibrium binding of 1 nM [3H]CP55,940 and 1 nM [3H]SR141716
are depicted in Figure 3. The curveꢀfit values of all target compounds are listed in Table 2.
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Figure 3. (A) Effects of representative diarylureas on the equilibrium binding levels of 1 nM
of [3H]CP55,940, and (B) Effects of representative diarylureas on the equilibrium binding
levels of 1 nM of [3H]SR141716.
Table 2. Allosteric modulatory activities of diarylureas 1 and 3ꢀ14 in the CB1 [3H]CP55,940 and
[3H]SR141716 binding assays a
[3H]
[3H]CP55,940
pEC50 ± S.E.
[3H]SR141716 %[3H]SR141716
pIC50 ± S.E.
Compound
Ar
CP55,940 Curveꢀ
fit top ± S.E.
Binding a,b ± S.E.
5.69 ± 0.08
5.13 ± 0.18
256.0 ± 5.59
4.91 ± 0.11
20.42 ± 3.05
1
8
174.1 ± 8.86
4.76 ± 0.14
34.87 ± 2.43
5.34 ± 0.01
5.24 ± 0.14
5.41 ± 0.08
4.88 ± 0.18
264.6 ± 9.81
199.8 ± 8.78
226.0 ± 5.82
233.3 ± 20.53
4.95 ± 0.15
4.54 ± 0.21
4.08 ± 0.83
4.64 ± 0.74
20.44 ± 2.13
45.54 ± 0.15
73.19 ± 5.77
86.90 ± 5.87
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5.86 ± 0.09
458.3 ± 16.25
164.5 ± 8.61
4.85 ± 0.18
4.75 ± 0.20
26.60 ± 5.68
41.41 ± 3.26
13
14
5.25 ± 0.19
4.83 ± 0.27
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15.5 ± 24.12
5.07 ± 0.11
4.59 ± 0.17
17.20 ± 4.12
15
4.97 ± 0.17
5.19 ± 0.17
224.2 ± 15.85
181.2 ± 8.15
44.34 ± 1.42
50.58 ± 2.04
16
17
5.53 ± 0.11
4.78 ± 0.17
5.29 ± 0.27
4.99 ± 0.12
134.2 ± 4.47
355.7 ± 27.39
37.68 ± 0.76
34.83 ± 8.94
18
19
3.91 ± 0.79
a Compounds tested in presence of 1 nM [3H]CP55,940 or 1 nM [3H]SR141716. percent specific
b
[3H]SR141716 binding at 32 µM of test compound.
Similar to previously reported CB1 allosteric modulators such as 1, most analogues increased
specific binding of [3H]CP55,940, demonstrating positive cooperativity with the agonist
radioligand. At the highest tested concentration of 10 µM, the 3ꢀthiophenyl analogue (9) had
relatively similar maximal increases in specific [3H]CP55,940 binding as compared to 1, whereas
the 2ꢀthiophenyl analogue (10) only exhibited approximately half the increase in specific
binding. The 3ꢀfuranyl analogue (8) exhibited a similar increase in specific [3H]CP55,940
binding as compared to the 2ꢀthiophenyl analogue (10), suggesting that both the position and
nature of heteroatom are important for imparting cooperativity. Introduction of a methyl at the 5
position of the 3ꢀthiophenyl (11) or 2ꢀthiophenyl analogue (12) resulted in a slight lower increase
in [3H]CP55,940 binding level compared to 9. Within the pyrrolyl series, the unsubstituted 2ꢀ
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pyrrolyl analogue (13, maximum binding level: 458%) increased the [3H]CP55,90 binding level
more than 2ꢀfold compared to 1 (maximum binding level: 256%), whereas Nꢀmethylpyrrolꢀ2ꢀyl
(14) only slightly enhanced the level of radioligand binding. Interestingly, the NꢀBocꢀ2ꢀpyrrolyl
(15) significantly decreased the binding of the radioligand.
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The trend of cooperativity in agonist binding positively correlates with their potency in the
GTPꢀγꢀS binding assay (r(13) = 0.65, p<0.05). For instance, 13 was the most potent compound
of the series in the GTPꢀγꢀS assay (IC50 = 40 nM), and it also increased the maximal
[3H]CP55,940 binding to the largest extent (458%). In contrast, consistent with its low activity in
both the calcium and GTPꢀγꢀS assays, 15 displayed no increase in [3H]CP55,940 binding but
instead decreased the maximal level.
Among the thiazolyl analogues (16ꢀ18), the 4ꢀthiazolyl analogue (16) increased the
radioligand binding level nearly as much as 1. However, the positive effect was diminished in
the case of the 5ꢀthiazolyl (17), and markedly reduced in case of the 2ꢀthiazolyl (18). The
correlation between [3H]CP55,940 binding maximum level and potency in the GTPꢀγꢀS binding
assays seen in the pyrrole series was also observed in the thiazole series. On the other hand, the
imidazolyl analogue (19, maximum binding level 355%) demonstrated stronger binding
cooperativity with [3H]CP55,940 yet was less potent in the GTPꢀγꢀS binding assay compared to
1, which may reflect a lower affinity (e.g. pEC50 value appeared right shifted in comparison)
despite greater cooperativity.
Effects of diarylureas on [3H]SR141716 radioligand binding. As opposed to the
[3H]CP55,940 binding studies where most compounds increase radioligand binding, most of the
analogues reduced [3H]SR141716 binding in a similar fashion to previously reported CB1
26
allosteric modulators 1 and 2.13,
These results are consistent with the PAMꢀantagonist
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mechanism.16 Cannabinoid agonists exhibit two distinct affinities for the CB1 receptor,27 which
reflect two receptor states, one coupled to the cognate G protein (active) and the other uncoupled
(inactive). Some reports suggest that allosteric regulation by G proteins does not affect the
9
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affinity of the CB1 antagonist/inverse agonist SR141716 for the receptor, i.e. it binds with equal
29
affinity to either receptor state,28,
and that SR141716 may sequester G proteins in a
nonsignaling complex; however, Bouaboula et al. reported an increase in SR141716 affinity for
the CB1 receptor when it is uncoupled from G protein by coincubation with GTPꢀγꢀS,30 an effect
that has been reported for other inverse agonists of G protein coupled receptors.31 Therefore, an
increase in affinity of the modulator for the agonist highꢀaffinity receptor state (as reflected by
the ability of the modulator to increase binding of [3H]CP55,940) would also result in an increase
in the ability of the modulator to reduce SR141716 binding. In other words, if SR141716 has a
higher affinity for receptors inactive/uncoupled from G protein, then structural changes that
increase the affinity of the modulator for the agonist highꢀaffinity conformation will result in a
concomitant increase in the modulator’s ability to reduce [3H]SR141716 binding and vice versa,
since the distribution of receptor states would be shifted towards those with which SR141716 has
higher affinity.
Interestingly, the 5ꢀmethylthiophenyl analogues (11 and 12) only exhibited a slight reduction
in [3H]SR141716 binding that was significantly less than observed in the rest of the series.
However, as discussed above, both 11 and 12 displayed a similar ability relative to 1 in
increasing [3H]CP55,940 binding. It has suggested that the binding pocket for the structurally
similar CB1 allosteric modulator Org27569 overlaps with that of SR141716.32 Thus, it is possible
that the binding site for these allosteric modulators also partially overlaps with that of SR141716,
with the methyl group interfering with the ability of the modulator to occupy SR141716’s
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binding site. Alternatively, it could be that 11 and 12 have lower affinity for receptors uncoupled
from their cognate G protein, for which SR141716 may have higher affinity.
7
8
9
Notably, calculated pIC50 values from [35S]GTPꢀγꢀS binding studies were positively
correlated with pEC50 values from [3H]CP55,940 equilibrium binding (r(13) = 0.65, p<0.05)
(Table 2) but not pIC50 values from displacement binding of [3H]SR141716 (r(13) = 0.06,
p=0.82). This suggests that affinity for the agonistꢀbound conformation, but not the antagonist,
predicts the allosteric modulator’s inhibitory potency, which is consistent with positive
cooperativity between the modulator and the probe agonist (PAMꢀantagonist).19
10
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Effects of diarylureas on [3H]CP55,940 saturation binding. Saturation binding
experiments were conducted in the absence and presence (10 µM) of a subset of synthesized
modulators to determine their effects on [3H]CP55,940’s Bmax and Kd values. Under the vehicle
condition, [3H]CP55,940 exhibited a Bmax of 1.56 ± 0.09 pmol/mg, which was increased by coꢀ
incubation with each allosteric modulator (Table 3) as previously reported for 1.14 Compounds
11 and 17 exhibited comparable Bmax and Kd values to 1. Intriguingly, 13, which exhibited a
larger increase in specific [3H]CP55,940 binding than PSNCBAMꢀ1 (Table 2), also produced a
larger Bmax in saturation experiments (Table 3). Additionally, 8, which exhibited a smaller
increase in [3H]CP55,940 binding as compared to 1 (Table 2), also resulted in a lower Bmax for
[3H]CP55,940 in saturation binding (Table 3). In contrast, Kd values for [3H]CP55,940 were not
significantly affected by the presence of the allosteric modulators. Considering the low range of
concentrations for [3H]CP55,940, it is likely the Bmax values observed in these saturation binding
plots reflect the population of high affinity receptors that are coupled to the G proteins, as
CP55,940 exhibits a Ki in the 100 nM range for the uncoupled low affinity receptor.33 Thus, the
increase in Bmax following coꢀincubation with allosteric modulators reflects an increase in
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affinity of the nonꢀcoupled receptors resulting in a Bmax shift rather than a Kd shift. These results
combined with the elevated Bmax values suggest that these compounds are not modulating highꢀ
affinity binding but rather altering the equilibrium of lowꢀ and highꢀaffinity receptors. The
enhanced Bmax values and unchanged Kd values of these diarylureas correspond to the same trend
observed for Org27569 previously.14 Therefore, differences in maximal increases observed in
specific [3H]CP55,940 binding [Table 2; Figure 3A] between allosteric modulators likely reflect
the proportion of low affinity receptors shifted into a high affinity state.
10
11
12
13
14
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17
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Table 3. Allosteric effects of diarylureas on [3H]CP55,940 saturation binding at 10 µM of test
compound
[3H]CP55,940 Bmax Bmax (pmol/mg)
[3H]CP55,940
Kd (nM) ± S.E.
Compound
Kd (nM) 95% CI
1.31 to 2.52
1.27 to 2.60
1.98 to 3.71
1.98 to 3.29
1.20 to 1.63
2.02 to 4.17
(pmol/mg) ± S.E.
95% CI
Vehicle
1.56 ± 0.09
1.38 to 1.74
1.92 ± 0.30
1.93 ± 0.33
2.85 ± 0.41
2.63 ± 0.31
1.41 ± 0.10
3.10 ± 0.51
4.53 ± 0.29
3.22 ± 0.19
3.64 ± 0.17
5.87 ± 0.15
3.57 ± 0.25
3.95 to 5.11
2.82 to 3.62
3.28 to 4.00
5.55 to 6.19
3.05 to 4.10
1
8
11
13
17
Conclusions
In our previous study, we demonstrated that the 5ꢀpyrrolidinylpyridinyl of 1 could be
replaced by substituted phenyl and pyridinyl groups and modifications at this position led to CB1
allosteric modulators with better metabolic stability and greater potency in vivo.18 This study
further elucidates the structural requirements at the place of 5ꢀpyrrolidinylpyridinyl group of 1.
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We showed that various fiveꢀmembered heterocyclic rings could be accommodated at this
position, resulting in the generation of interesting pharmacological activities at the CB1 receptor.
Replacement by furanyl, thiophenyl, or 5ꢀmethylthiophenyl groups (8ꢀ12) maintained similar
allosteric modulating potency in the calcium mobilization assay against human CB1 receptor.
Notably, modification to 2ꢀpyrrolyl group (13) significantly enhanced the potency in the GTPꢀγꢀ
S assay, doubled the [3H]CP55,940 binding level in the competitive binding assay and markedly
increased Bmax in the [3H]CP55,940 saturation binding assay. These results indicate that 13 had
greater affinity and/or cooperativity at the CB1 receptor. The fact that Kd values in the
[3H]CP55,940 saturation binding assay remained unchanged suggested that the these compounds
modulated the CB1 receptor by stabilizing agonist lowꢀaffinity receptors into an agonist highꢀ
affinity state. In the competitive [3H]SR141716 binding assay, while most synthesized
diarylureas retained the ability to reduce SR141716 binding to CB1 receptor as expected for
PAMꢀantagonists, the 5ꢀmethylthiophenyl analogues (11 and 12) only slightly reduced
[3H]SR141716 binding, possibly due to the partially overlapping binding pockets.32
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A number of CB1 NAMs have been reported and their in vitro pharmacological properties
have been thoroughly characterized to date, most of which appear to be PAMꢀantagonists. Since
PAMꢀantagonists and agonists increase the other’s binding affinity to the receptor, this class of
NAMs “seek out and destroy” agonistꢀbound receptor complexes, therefore providing the unique
benefit of reversing ongoing persistent agonism and resulting in favorable in vivo target
coverage.16 Despite these, the translation of in vitro to in vivo effects has not been well
established with these CB1 NAMs. For instance, the subtle attenuation of THCꢀinduced antiꢀ
nociception by 1 and the small reduction in THC’s potency by 4 in drug discrimination assays
were not seen with their structurallyꢀrelated analogue 5.19 This may be attributed to
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pharmacokinetic profiles of these allosteric modulators or slower association kinetics commonly
observed of allosteric modulators compared to the orthosteric ligands,34 leading to less
pronounced in vivo effects. Indeed, we have demonstrated that 3, a more metabolically stable
analogue of 1, produced a more pronounced effect in attenuating reinstatement of cocaine selfꢀ
administration.18 By replacing the 5ꢀpyrrolidinylpyridinyl group of 1 with a diversity of fiveꢀ
membered heterocycles, this work illustrated that structural modification at this position may
allow further fineꢀtuning of pharmacological properties to achieve desired therapeutic effects.
Continued SAR studies and optimization effort by improving potency and pharmacokinetic
properties and/or modifying receptor binding kinetics may lead to CB1 allosteric modulators as
improved in vivo probes and potential clinical therapeutics.
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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 hexanes, ethyl acetate (EtOAc), dichloromethane, and
methanol. Purity and characterization of compounds were established by a combination of
HPLC, TLC, mass spectrometry, and NMR analyses. 1H and 13C NMR spectra were recorded on
a Bruker Avance DPXꢀ300 (300 MHz) spectrometer and were determined in CDCl3, DMSOꢀd6,
or CD3OD 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.
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Nominal mass spectra were obtained using a Waters Alliance HT/Micromass ZQ system (ESI).
High resolution mass spectra were obtained using Agilent 1290 Infinity UHPLCꢀ6230 TOF
system (ESI). All final compounds were greater than 95% pure as determined by HPLC on an
Agilent 1100 system using an Agilent Zorbax SBꢀPhenyl, 2.1 mm × 150 mm, 5 ꢁm column
using a 15 minute gradient elution of 5ꢀ95% 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).
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General procedure A. To a mixture of aryl bromide or aryl iodide (1 eq), boronic acid (1.1
eq) in dimethoxyethane (0.1 M) was added 1M aqueous NaHCO3 solution (3 eq) followed by
Pd(Ph3)4 (0.075 eq). The reaction mixture was refluxed overnight under nitrogen atmosphere.
The reaction mixture was diluted with ethyl acetate, washed with a saturated NaHCO3 solution
and brine. The combined organic layers were dried over anhydrous MgSO4 and filtered. The
filtrate was concentrated in vacuo and the residue was purified by column chromatography
(SiO2, ethyl acetate/hexanes) to give the desired product.
3‐(Furan‐3‐yl)aniline (20a) was prepared from 3‐bromoaniline (0.19 ml, 1.74 mmol) and
(furan‐3‐yl)boronic acid (0.21 g, 1.91 mmol) following the general procedure A as white solid
(0.09 g, 32%). 1H NMR (300 MHz, CDCl3) δ 7.66 ꢀ 7.71 (m, 1H), 7.45 (t, J = 1.70 Hz, 1H), 7.12
ꢀ 7.20 (m, 1H), 6.90 (td, J = 1.30, 7.58 Hz, 1H), 6.81 (t, J = 1.88 Hz, 1H), 6.66 (d, J = 1.13 Hz,
1H), 6.60 (ddd, J = 0.94, 2.45, 7.91 Hz, 1H), 3.69 (br. s., 2H). MS (ESI) m/z for C10H9NO
[M+H]+: calcd: 160.1; found: 160.1.
3‐(Thiophen‐3‐yl)aniline (20b) was prepared from 3‐bromoaniline (0.19 ml, 1.74 mmol)
and (thiophen‐3‐yl)boronic acid (0.25 g, 1.91 mmol) following the general procedure A as white
solid (0.03 g, 10%). 1H NMR (300 MHz, CDCl3) δ 7.38 ꢀ 7.42 (m, 1H), 7.32 ꢀ 7.37 (m, 2H), 7.15
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ꢀ 7.22 (m, 1H), 7.00 (td, J = 1.25, 7.68 Hz, 1H), 6.92 (t, J = 1.88 Hz, 1H), 6.63 (ddd, J = 0.94,
2.26, 7.91 Hz, 1H), 3.71 (br. s., 2H). MS (ESI) m/z for C10H9NS [M+H]+: calcd: 176.1; found:
176.3.
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2‐(3‐Nitrophenyl)thiophene (21a) was prepared from 2‐bromothiophene (0.10 ml, 1.00
mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol) following the general procedure A as
1
yellow solid (0.18 g, 92%). H NMR (300 MHz, CDCl3) δ 8.42 (t, J = 1.88 Hz, 1H), 8.06 ꢀ 8.13
(m, 1H), 7.89 (d, J = 7.72 Hz, 1H), 7.53 (t, J = 8.01 Hz, 1H), 7.42 (dd, J = 0.85, 3.67 Hz, 1H),
7.37 (dd, J = 0.75, 5.09 Hz, 1H), 7.12 (dd, J = 3.77, 5.09 Hz, 1H).
2‐Methyl‐4‐(3‐nitrophenyl)thiophene
(21b)
was
prepared
from
4‐bromo‐2‐
methylthiophene (0.10 ml, 1.00 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol)
1
following the general procedure A as yellow solid (0.09 g, 42%). H NMR (300 MHz, CDCl3)
δ 8.37 (t, J = 1.88 Hz, 1H), 8.09 (td, J = 1.06, 8.24 Hz, 1H), 7.85 (d, J = 7.72 Hz, 1H), 7.52 (t, J
= 8.01 Hz, 1H), 7.32 (d, J = 1.51 Hz, 1H), 7.08 (s, 1H), 2.54 (s, 1H).
2‐Methyl‐5‐(3‐nitrophenyl)thiophene (21c) was prepared from 4‐bromo‐2‐methylthiophene
(0.10 ml, 1.00 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol) following the general
1
procedure A as yellow solid (0.09 g, 42%). H NMR (300 MHz, CDCl3) δ 8.36 (t, J = 1.98 Hz,
1H), 8.05 (ddd, J = 0.94, 2.12, 8.24 Hz, 1H), 7.79 ꢀ 7.84 (m, 1H), 7.50 (t, J = 8.01 Hz, 1H), 7.22
(d, J = 3.58 Hz, 1H), 6.75 ꢀ 6.79 (m, 1H), 2.50 ꢀ 2.56 (m, 3H).
4‐(3‐Nitrophenyl)‐1,3‐thiazole (21d) was prepared from 4‐bromo‐1,3‐thiazole (0.09 ml,
1.00 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol) following the general procedure
1
A as yellow solid (0.12 g, 58%). H NMR (300 MHz, CDCl3) δ 8.93 (d, J = 1.88 Hz, 1H), 8.78
(t, J = 1.98 Hz, 1H), 8.26 ꢀ 8.31 (m, 1H), 8.17 ꢀ 8.22 (m, 1H), 7.72 (d, J = 2.07 Hz, 1H), 7.62 (t, J
= 8.01 Hz, 1H).
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5‐(3‐Nitrophenyl)‐1,3‐thiazole (21e) was prepared from 5‐bromo‐1,3‐thiazole (0.09 ml, 1.00
mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol) following the general procedure A as
yellow solid (0.10 g, 46%). 1H NMR (300 MHz, CDCl3) δ 8.86 (s, 1H), 8.43 (t, J = 1.88 Hz, 1H),
8.17 ꢀ 8.24 (m, 2H), 7.88 ꢀ 7.94 (m, 1H), 7.58 ꢀ 7.66 (m, 1H).
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2‐(3‐Nitrophenyl)‐1,3‐thiazole (21f) was prepared from 4‐bromo‐1,3‐thiazole (0.09 ml, 1.00
mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol) following the general procedure A as
yellow solid (0.08 g, 37%). 1H NMR (300 MHz, CDCl3) δ 8.80 (t, J = 1.88 Hz, 1H), 8.24 ꢀ 8.32
(m, 1H), 7.87 ꢀ 7.96 (m, 2H), 7.43 ꢀ 7.47 (m, 2H).
tert‐Butyl 2‐(3‐nitrophenyl)‐1H‐pyrrole‐1‐carboxylate (23) was prepared from 1ꢀbromoꢀ3ꢀ
nitrobenzene (0.20 g, 1 mmol) and NꢀBocꢀ2ꢀpyrroleboronic acid (0.23 g, 1.1 mmol) following
the general procedure A as yellow solid (0.15 g, 51%). 1H NMR (300 MHz, CDCl3) δ 8.12 ꢀ 8.28
(m, 2H), 7.69 (d, J = 6.22 Hz, 1H), 7.47 ꢀ 7.58 (m, 1H), 7.36 ꢀ 7.44 (m, 1H), 6.22 ꢀ 6.35 (m, 2H),
1.40 (s, 9H).
2‐(3‐Nitrophenyl)‐1H‐imidazole (27). To a solution of 3ꢀnitrobenzonitrile (0.50 g, 3.37
mmol) in anhydrous methanol (17 ml) was added sodium methoxide (0.18 g, 3.37 mmol). The
reaction mixture was stirred at room temperature for 5 h. Acid acetic (0.39 ml, 6.82 mmol) and
aminoacetaldehyde dimethyl acetal (0.37 ml, 3.37 mmol) were then added and the reaction
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mixture was heated at 70 C with stirring for 1 h. After cooling to room temperature, methanol
(2.25 ml) and 6N aqueous HCl (1.7 ml) solution were added to the reaction mixture. The reaction
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temperature was subsequently raised to 70 C for 1 h. After cooling to room temperature, the
solvent was removed under reduced pressure. Saturated aqueous potassium carbonate was added
slowly until pH 8ꢀ10. The desired product precipitated and was collected by filtration as white
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solid (0.39 g, 61%). H NMR (300 MHz, CD3OD) δ 7.21 ꢀ 7.27 (m, 1H), 6.66 ꢀ 6.77 (m, 2H),
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General procedure B. To a solution of nitrobenzene derivative (1 eq) in ethanol (0.1 M) was
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added hydrazine hydrate (15 eq). The reaction was stirred at 50 C for 15 min and an excess of
Raney nickel slurry in water (1.2 eq) was added slowly. After 1 h, the bubbling ceased, the
mixture was cooled to room temperature and filtered through Celite. The filtrate was condensed
under reduced pressured and the residue was either used for the next step without purification or
purified by column chromatography (SiO2, ethyl acetate/hexanes) to afford the desired product.
3‐(Thiophen‐2‐yl)aniline (22a) was prepared from 21a (0.18 g, 0.89 mmol) following the
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general procedure B as yellow solid (0.10 g, 64%). H NMR (300 MHz, CDCl3) δ 7.20 ꢀ 7.29
(m, 2H), 7.10 ꢀ 7.18 (m, 1H), 6.98 ꢀ 7.08 (m, 2H), 6.92 (s, 1H), 6.59 (dd, J = 2.07, 7.91 Hz, 1H),
3.69 (br. s., 2H). MS (ESI) m/z for C12H9Cl2N [M+H]+: calcd: 238.0; found: 238.1.
3‐(5‐Methylthiophen‐3‐yl)aniline (22b) was prepared from 21b (0.09 g, 0.42 mmol)
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following the general procedure B as white solid (0.08 g, quant.). H NMR (300 MHz, CDCl3) δ
7.12 ꢀ 7.17 (m, 2H), 6.93 ꢀ 7.02 (m, 2H), 6.87 (d, J = 1.88 Hz, 1H), 6.59 (dd, J = 1.51, 7.91 Hz,
1H), 3.68 (br. s., 2H), 2.50 (s, 3H). MS (ESI) m/z for C11H11NS [M+H]+: calcd: 190.1; found:
190.2.
3‐(5‐Methylthiophen‐2‐yl)aniline (22c) was prepared from 21c (0.09 g, 0.42 mmol)
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following the general procedure B as colorless liquid (0.08 g, quant.). H NMR (300 MHz,
CDCl3) δ 7.09 ꢀ 7.16 (m, 1H), 7.05 (d, J = 3.39 Hz, 1H), 6.93 ꢀ 6.99 (m, 1H), 6.86 (t, J = 1.88
Hz, 1H), 6.67 ꢀ 6.72 (m, 1H), 6.56 (ddd, J = 0.94, 2.26, 7.91 Hz, 1H), 3.65 (br. s., 2H), 2.48 (s,
2H). MS (ESI) m/z for C11H11NS [M+H]+: calcd: 190.1; found: 190.3.
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3‐(1,3‐Thiazol‐4‐yl)aniline (22d) was prepared from 21d (0.12 g, 0.58 mmol) following the
general procedure B as colorless liquid (0.06 g, 61%). 1H NMR (300 MHz, CDCl3) δ 8.86 (d, J =
1.88 Hz, 1H), 7.48 (d, J = 1.88 Hz, 1H), 7.26 ꢀ 7.35 (m, 2H), 6.69 (dd, J = 1.22, 2.35 Hz, 1H),
6.23 (dd, J = 2.26, 7.91 Hz, 1H), 3.71 (br. s., 2H). MS (ESI) m/z for C9H8N2S [M+H]+: calcd:
177.1; found: 177.5.
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3‐(1,3‐Thiazol‐5‐yl)aniline (22e) was prepared from 21e (0.09 g, 0.46 mmol) following the
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general procedure B as colorless liquid (0.06 g, 73%). H NMR (300 MHz, CDCl3) δ 8.72 (s,
1H), 8.03 (s, 1H), 7.13 ꢀ 7.23 (m, 1H), 6.97 (dd, J = 0.94, 7.72 Hz, 1H), 6.88 (d, J = 3.77 Hz,
1H), 6.66 (td, J = 1.06, 8.05 Hz, 1H), 3.80 (br. s., 2H). MS (ESI) m/z for C9H8N2S [M+H]+:
calcd: 177.1; found: 177.4.
3‐(1,3‐Thiazol‐2‐yl)aniline (22f) was prepared from 21f (0.08 g, 0.37 mmol) following the
general procedure B as colorless liquid (0.03 g, 46%). 1H NMR (300 MHz, CDCl3) δ 7.84 (d, J =
3.39 Hz, 1H), 7.29 ꢀ 7.35 (m, 3H), 7.22 (t, J = 8.01 Hz, 1H), 6.72 ꢀ 6.77 (m, 1H), 3.80 (br. s.,
2H). MS (ESI) m/z for C9H8N2S [M+H]+: calcd: 177.1; found: 177.3.
tert‐Butyl 2‐(3‐aminophenyl)‐1H‐pyrrole‐1‐carboxylate (24) was prepared from 23 (0.147
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g, 0.51 mmol) following the general procedure B as white solid (0.09 g, 65%). H NMR (300
MHz, CDCl3) δ 7.21 ꢀ 7.37 (m, 1H), 7.06 ꢀ 7.18 (m, 1H), 6.70 ꢀ 6.78 (m, 1H), 6.57 ꢀ 6.68 (m,
2H), 6.18 (d, J = 10.74 Hz, 2H), 3.63 (br. s., 2H), 1.37 (s, 9H). MS (ESI) m/z for C15H18N2O2
[M+H]+: calcd: 259.1; found: 259.5.
3‐(1H‐Pyrrol‐2‐yl)aniline (25). A solution of 24 (0.06 g, 0.23 mmol) in 5% aqueous
potassium hydroxide (23 ml) was refluxed for 4 h. After cooling to room temperature, the
mixture was poured into water and extracted with dichloromethane. The organic phase was dried
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with anhydrous magnesium sulfate, filtered, concentrated in vacuo to the desired product as
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white solid (0.03 g, 78%). H NMR (300 MHz, CDCl3) δ 8.41 (br. s., 1H), 7.05 ꢀ 7.22 (m, 1H),
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6.73 ꢀ 6.94 (m, 3H), 6.40 ꢀ 6.60 (m, 2H), 6.28 (s, 1H), 3.67 (br. s., 2H). MS (ESI) m/z for
C10H10N2 [M+H]+: calcd: 159.1; found: 159.2.
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3‐(1‐Methyl‐1H‐pyrrol‐2‐yl)aniline (26). To a solution of 3ꢀiodoaniline (0.24 ml, 2 mmol)
in N,Nꢀdimethylacetamide (8 ml) in a sealed tube was added Nꢀmethylpyrrole (0.36 ml, 8
mmol), potassium acetate (0.39 g, 8 mmol), and palladium (II) acetate (0.005 g, 0.02 mmol). The
reaction mixture was stirred at 150 oC for 20 h. The reaction mixture was then diluted with ethyl
acetate, washed three times with water and once with brine. The organic layer was dried with
anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue
was purified by column chromatography (SiO2, ethyl acetate/ hexanes) to give the desired
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product was yellow liquid (0.08 g, 22%). H NMR (300 MHz, CDCl3) δ 7.17 (t, J = 7.82 Hz,
1H), 6.76 ꢀ 6.81 (m, 1H), 6.69 (td, J = 2.10, 8.24 Hz, 2H), 6.61 (ddd, J = 0.75, 2.31, 8.05 Hz,
1H), 6.16 ꢀ 6.21 (m, 2H), 3.68 (br. s., 2H), 3.64 (s, 3H). MS (ESI) m/z for C11H12N2 [M+H]+:
calcd: 173.1; found: 173.5.
3‐(1H‐Imidazol‐2‐yl)aniline (28) was prepared from 27 (0.30 g, 1.58 mmol) following the
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general procedure B as white solid (0.25 g, quant.). H NMR (300 MHz, CDCl3) δ 7.05 ꢀ 7.23
(m, 5H), 6.69 (d, J = 8.10 Hz, 1H), 3.74 (br. s., 2H). MS (ESI) m/z for C9H9N3 [M+H]+: calcd:
160.1; found: 160.2.
General procedure C. To a solution of aryl amine (1 eq) in anhydrous chloroform (0.04 M)
was added 4ꢀchlorophenyl isocyanate (1eq) at room temperature. The reaction mixture was then
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heated at 60 C for 16 h. The precipitated product was filtered and thoroughly washed with
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3‐(4‐Chlorophenyl)‐1‐[3‐(furan‐3‐yl)phenyl]urea (8) was prepared from 20a (0.03 g, 0.16
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mmol) following the general procedure C as white solid (0.05 g, 95%). H NMR (300 MHz,
DMSOꢀd6) δ 8.86 (s, 1H), 8.73 (s, 1H), 8.12 (s, 1H), 7.75 (t, J = 1.70 Hz, 1H), 7.65 (s, 1H), 7.47
ꢀ 7.52 (m, 2H), 7.29 ꢀ 7.36 (m, 4H), 7.20 ꢀ 7.28 (m, 1H), 6.88 (d, J = 0.94 Hz, 1H). HRMS (ESI)
m/z for C17H13ClN2O2 [M+H]+: calcd: 313.0738; found: 313.0744.
3‐(4‐Chlorophenyl)‐1‐[3‐(thiophen‐3‐yl)phenyl]urea (9) was prepared from 20b (0.03 g,
0.15 mmol) following the general procedure C as white solid (0.03 g, 63%). 1H NMR (300 MHz,
DMSOꢀd6) δ 8.85 (s, 1H), 8.76 (s, 1H), 7.75 ꢀ 7.79 (m, 2H), 7.63 (dd, J = 2.92, 4.99 Hz, 1H),
7.45 ꢀ 7.51 (m, 3H), 7.30 ꢀ 7.36 (m, 5H). HRMS (ESI) m/z for C17H13ClN2OS [M+H]+: calcd:
329.0510; found: 329.0504.
3‐(4‐Chlorophenyl)‐1‐[3‐(thiophen‐2‐yl)phenyl]urea (10) was prepared from 22a (0.10 g,
0.67 mmol) following the general procedure C as white solid (0.13 g, 57%). 1H NMR (300 MHz,
DMSOꢀd6) δ 8.84 (s, 2H), 7.82 (s, 1H), 7.55 (d, J = 5.09 Hz, 1H), 7.50 (d, J = 8.85 Hz, 2H), 7.44
ꢀ 7.47 (m, 1H), 7.28 ꢀ 7.36 (m, 5H), 7.14 (dd, J = 3.67, 4.99 Hz, 1H). HRMS (ESI) m/z for
C17H13ClN2OS [M+H]+: calcd: 329.0510; found: 329.0508.
3‐(4‐Chlorophenyl)‐1‐[3‐(5‐methylthiophen‐3‐yl)phenyl]urea (11) was prepared from 22b
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(0.03 g, 0.17 mmol) following the general procedure C as white solid (0.04 g, 70%). H NMR
(300 MHz, CD3OD) δ 7.73 (br. s., 1H), 7.61 (d, J = 7.54 Hz, 1H), 7.39 ꢀ 7.48 (m, 4H), 7.32 ꢀ
7.38 (m, 2H), 7.24 ꢀ 7.31 (m, 4H), 2.51 (s, 3H). HRMS (ESI) m/z for C18H15ClN2OS [M+H]+:
calcd: 343.0666; found: 343.0666.
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3‐(4‐Chlorophenyl)‐1‐[3‐(5‐methylthiophen‐2‐yl)phenyl]urea (12) was prepared from 22c
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(0.04 g, 0.19 mmol) following the general procedure C as white solid (0.05 g, 77%). H NMR
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(300 MHz, CD3OD) δ 7.71 (s, 1H), 7.44 (d, J = 8.85 Hz, 2H), 7.21 ꢀ 7.32 (m, 5H), 7.16 (d, J =
3.58 Hz, 1H), 6.74 (d, J = 2.64 Hz, 1H), 2.49 (s, 3H). HRMS (ESI) m/z for C18H15ClN2OS
[M+H]+: calcd: 343.0666; found: 343.0665.
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3‐(4‐Chlorophenyl)‐1‐[3‐(1H‐pyrrol‐2‐yl)phenyl]urea (13) was prepared from 25 (0.03 g,
0.18 mmol) following the general procedure C as white solid (0.05 g, 81%). 1H NMR (300 MHz,
DMSOꢀd6) δ 11.26 (br. s., 1H), 8.87 (br. s., 1H), 8.67 (br. s., 1H), 7.67 (s, 1H), 7.47 ꢀ 7.56 (m,
2H), 7.30 ꢀ 7.39 (m, J = 5.70 Hz, 2H), 7.22 ꢀ 7.29 (m, 3H), 6.84 (s, 1H), 6.44 (s, 1H), 6.12 (s,
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1H). C NMR (75 MHz, DMSOꢀd6) δ 152.5, 139.8, 138.7, 133.6, 131.1, 129.0, 128.6, 125.3,
119.7, 119.3, 117.5, 115.9, 113.7, 109.0, 105.5. HRMS (ESI) m/z for C17H14ClN3O [M+H]+:
calcd: 312.0898; found: 312.0894.
3‐(4‐Chlorophenyl)‐1‐[3‐(1‐methyl‐1H‐pyrrol‐2‐yl)phenyl]urea (14) was prepared from
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26 (0.08 g, 0.45 mmol) following the general procedure C as white solid (0.14 g, 98%). H NMR
(300 MHz, DMSOꢀd6) δ 8.86 (s, 1H), 8.77 (s, 1H), 7.56 (s, 1H), 7.50 (d, J = 8.85 Hz, 2H), 7.30 ꢀ
7.36 (m, 4H), 7.03 ꢀ 7.08 (m, 1H), 6.83 (t, J = 2.17 Hz, 1H), 6.15 (dd, J = 1.88, 3.58 Hz, 1H),
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6.04 ꢀ 6.08 (m, 1H), 3.66 (s, 3H). C NMR (75 MHz, DMSOꢀd6) δ 152.5, 139.6, 138.6, 133.4,
128.8, 128.6, 125.4, 124.3, 121.6, 119.8, 117.8, 116.6, 108.3, 107.3, 34.9. HRMS (ESI) m/z for
C18H16ClN3O [M+H]+: calcd: 326.1055; found: 326.1049.
tert‐Butyl
2‐[3‐({1‐[(4‐chlorophenyl)amino]ethenyl}amino)phenyl]‐1H‐pyrrole‐1‐
carboxylate (15) was prepared from 24 (0.03 g, 0.10 mmol) following the general procedure C
as white solid (0.04 g, 84%). 1H NMR (300 MHz, DMSOꢀd6) δ 8.83 (s, 1H), 8.75 (s, 1H), 7.46 ꢀ
7.51 (m, 2H), 7.31 ꢀ 7.39 (m, 3H), 7.23 ꢀ 7.30 (m, 1H), 6.94 (d, J = 7.54 Hz, 1H), 6.26 ꢀ 6.30 (m,
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1H), 6.22 ꢀ 6.26 (m, 1H), 1.31 (s, 9H). C NMR (75 MHz, DMSOꢀd6) δ 152.4, 148.8, 138.9,
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138.6, 134.4, 134.2, 128.6, 128.0, 125.3, 122.5, 119.7, 118.6, 117.0, 114.1, 110.7, 83.5, 27.1.
HRMS (ESI) m/z for C22H22ClN3O3 [M+H]+: calcd: 412.1422; found: 412.1423.
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3‐(4‐Chlorophenyl)‐1‐[3‐(1,3‐thiazol‐4‐yl)phenyl]urea (16) was prepared from 22d (0.03
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g, 0.19 mmol) following the general procedure C as white solid (0.05 g, 74%). H NMR (300
MHz, DMSOꢀd6) δ 9.20 (d, J = 1.88 Hz, 1H), 8.85 (s, 2H), 8.12 ꢀ 8.15 (m, 1H), 8.11 (d, J = 1.88
Hz, 1H), 7.59 (d, J = 7.35 Hz, 1H), 7.48 ꢀ 7.53 (m, 2H), 7.31 ꢀ 7.45 (m, 5H). HRMS (ESI) m/z
for C16H12ClN3OS [M+H]+: calcd: 330.0462; found: 330.0460.
3‐(4‐Chlorophenyl)‐1‐[3‐(1,3‐thiazol‐5‐yl)phenyl]urea (17) was prepared from 21e (0.03 g,
0.16 mmol) following the general procedure C as white solid (0.04 g, 66%). 1H NMR (300 MHz,
DMSOꢀd6) δ 9.09 (s, 1H), 8.88 (s, 2H), 8.26 (s, 1H), 7.83 (s, 1H), 7.50 (d, J = 8.85 Hz, 2H), 7.30
ꢀ 7.41 (m, 5H). HRMS (ESI) m/z for C16H12ClN3OS [M+H]+: calcd: 330.0462; found: 330.0454.
3‐(4‐Chlorophenyl)‐1‐[3‐(1,3‐thiazol‐2‐yl)phenyl]urea (18) was prepared from 22f (0.03 g,
0.16 mmol) following the general procedure C as white solid (0.04 g, 72%). 1H NMR (300 MHz,
DMSOꢀd6) δ 8.96 (s, 1H), 8.87 (s, 1H), 8.22 (s, 1H), 7.93 (d, J = 3.20 Hz, 1H), 7.80 (d, J = 3.01
Hz, 1H), 7.48 ꢀ 7.59 (m, 3H), 7.40 ꢀ 7.47 (m, 2H), 7.30 ꢀ 7.39 (m, 2H). HRMS (ESI) m/z for
C16H12ClN3OS [M+H]+: calcd: 330.0462; found: 330.0464.
3‐(4‐Chlorophenyl)‐1‐[3‐(1H‐imidazol‐2‐yl)phenyl]urea (19) was prepared from 28 (0.13
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g, 0.82 mmol) following the general procedure C as white solid (0.22 g, 86%). H NMR (300
MHz, DMSOꢀd6) δ 12.52 (br. s., 1H), 8.84 (d, J = 11.30 Hz, 2H), 8.07 (br. s., 1H), 7.52 (br. s.,
3H), 7.44 (br. s., 1H), 7.34 (d, J = 7.16 Hz, 3H), 7.03 ꢀ 7.20 (m, 2H). 13C NMR (75 MHz,
DMSOꢀd6) δ 152.4, 145.5, 139.8, 138.7, 131.4, 129.1, 128.6, 125.4, 119.7, 118.6, 118.0, 115.1.
HRMS (ESI) m/z for C16H13ClN4O [M+H]+: calcd: 313.0851; found: 313.0846.
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Calcium Mobilization Assay. CHOꢀRDꢀHGA16 cells (Molecular Devices, CA) stably
expressing the human CB1 receptor were plated into 96ꢀwell blackꢀwalled assay plates at 25,000
cells/well in 100 ꢁL of Ham’s F12 (supplemented with 10% fetal bovine serum, 100 units of
penicillin/streptomycin, and 100 ꢁg/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 ꢁL of prewarmed (37 °C) assay buffer. The cells were
incubated for 45 min at 37 °C, 5% CO2 in 200 ꢁL of the diluted Calcium 5 dye solution. For
antagonist assays to determine IC50 values, the EC80 concentration of CP55,940 was prepared at
10x the desired final concentration in 0.25% BSA/0.5% DMSO/0.5% EtOH/assay buffer,
aliquoted into 96ꢀwell 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 ꢁL 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). Calciumꢀmediated changes in fluorescence were monitored every 1 s over a 90 s
time period, with the Tetra adding 25 ꢁL 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. 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
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