Diarylureas as allosteric modulators of the cannabinoid CB1 receptor: Structure-activity relationship studies on 1-(4-chlorophenyl)-3-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]phenyl}urea (PSNCBAM-1)

Article abstract of DOI:10.1021/jm501042u

The recent discovery of allosteric modulators of the CB1 receptor including PSNCBAM-1 (4) has generated significant interest in CB1 receptor allosteric modulation. Here in the first SAR study on 4, we have designed and synthesized a series of analogs focusing on modifications at two positions. Pharmacological evaluation in calcium mobilization and binding assays revealed the importance of alkyl substitution at the 2-aminopyridine moiety and electron deficient aromatic groups at the 4-chlorophenyl position for activity at the CB1 receptor, resulting in several analogs with comparable potency to 4. These compounds increased the specific binding of [³H]CP55,940, in agreement with previous reports. Importantly, 4 and two analogs dose-dependently reduced the E_maxof the agonist curve in the CB1 calcium mobilization assays, confirming their negative allosteric modulator characteristics. Given the side effects associated with CB1 receptor orthosteric antagonists, negative allosteric modulators provide an alternative approach to modulate the pharmacologically important CB1 receptor.

Full text of DOI:10.1021/jm501042u

Article
pubs.acs.org/jmc
Open Access on 08/27/2015
Diarylureas as Allosteric Modulators of the Cannabinoid CB1
Receptor: Structure−Activity Relationship Studies on 1‑(4-
Chlorophenyl)-3-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]phenyl}urea
(PSNCBAM-1)
Nadezhda German, Ann M. Decker, Brian P. Gilmour, Elaine A. Gay, Jenny L. Wiley, Brian F. Thomas,
and Yanan Zhang*
Research Triangle Institute, Research Triangle Park, North Carolina 27709, United States
S
* Supporting Information
ABSTRACT: The recent discovery of allosteric modulators of
the CB1 receptor including PSNCBAM-1 (4) has generated
significant interest in CB1 receptor allosteric modulation. Here
in the first SAR study on 4, we have designed and synthesized
a series of analogs focusing on modifications at two positions.
Pharmacological evaluation in calcium mobilization and
binding assays revealed the importance of alkyl substitution
at the 2-aminopyridine moiety and electron deficient aromatic
groups at the 4-chlorophenyl position for activity at the CB1
receptor, resulting in several analogs with comparable potency
to 4. These compounds increased the specific binding of
[3H]CP55,940, in agreement with previous reports. Impor-
tantly, 4 and two analogs dose-dependently reduced the Emax of the agonist curve in the CB1 calcium mobilization assays,
confirming their negative allosteric modulator characteristics. Given the side effects associated with CB1 receptor orthosteric
antagonists, negative allosteric modulators provide an alternative approach to modulate the pharmacologically important CB1
receptor.
Compared to traditional orthosteric drugs, allosteric modu-
lators may offer several unique advantages. First, allosteric
modulators may exhibit greater subtype selectivity because of
the higher sequence divergence at extracellular allosteric
binding sites,17 in contrast to the often conserved orthosteric
domains for certain GPCR subtypes. Second, allosteric
modulators may have tissue selectivity, exerting effects only
where endogenous ligands are present. Finally, the effect of
allosteric modulators is saturable because of their dependence
on endogenous ligands for signaling.18,19
Several allosteric ligands for the CB1 receptor, including both
small molecules and peptides, have been recently reported.20−22
Among them, two series, the Org compounds (Org27569,
-27759, and -29647; 1−3) and PSNCBAM-1 (4) (Figure 1),
have been more widely studied.23−28 Interestingly, the
pharmacological profile of these allosteric modulators is
complex. While these compounds all enhanced the specific
binding of the CB1 agonist [3H]CP55,940, they behaved as
negative allosteric modulators, or allosteric antagonists, in a
number of functional assays including reporter gene, GTP-γ-S,
and mouse vas deferens assays.23,24,29,30 Subsequently, using a
site-directed fluorescence labeling (SDFL) approach, Fay et al.
INTRODUCTION
■
Cannabinoid CB1 and CB2 receptors are G-protein-coupled
receptors (GPCRs) and are key components of the
endocannabinoid system.1−3 The CB1 receptor is one of the
most highly expressed GPCRs in the central nervous system
(CNS) and plays a role in retrograde neuronal signaling and
attenuation of neurotransmitter release. The CB2 receptor is
primarily located in immune cells, regulating cell differentiation
and migration. The CB1 receptor in particular is involved in
many disorders, such as obesity, mental illness, pain, multiple
sclerosis, smoking, and drug addiction, and has therefore been
considered a promising target for the treatment of these
pathological conditions.4−6 A plethora of selective and
nonselective ligands for the CB1 receptor have been developed,
some of which are used extensively as research tools.7−10
However, use of CB1 agonists has been associated with
marijuana-like psychoactivity and clinical trials have raised
concerns that CB1 antagonists could promote a state of
depression and anxiety.11,12
An alternative strategy for regulating GPCRs has recently
emerged which involves the allosteric binding site, one that is
topographically distinct from the orthosteric site. Allosteric
modulators are ligands that bind to these sites to alter the
receptor signaling properties of the orthosteric ligand, changing
ligand affinity, functional efficacy, and functional potency.13−16
Received: July 10, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Structures of reported allosteric modulators of the CB1 receptor.
a
suggested that 1 binding to the CB1 receptor produced a
unique agonist-bound conformation, one that does not lead to
protein binding and/or activation, therefore behaving as a
negative modulator in functional assays.31 Compound 4 caused
noncompetitive antagonism in [35S]GTP-γ-S binding stud-
ies.24,29,30 In electrophysiological studies, 4 pretreatment
revealed agonist-dependent functional antagonism, abolishing
CP55,940-induced reductions in miniature inhibitory post-
synaptic currents (mIPSCs) frequency.29 When tested in vivo,
4 reduced feeding and body weight in rats in an acute feeding
study, confirming its allosteric antagonist character.24 These
findings indicate that the negative modulation of the CB1
receptor may provide an alternative strategy to regulate the
endocannabinoid system and hence may represent a valid
approach for medication development for the treatment of CB1
receptor mediated diseases.26
More recently, structure−activity relationship (SAR) studies
have been reported by several groups on the Org com-
pounds.28,32−34 To the best of our knowledge, no such study
has been published on 4. Here, we report our initial SAR
studies on compound 4 and the pharmacological evaluation of
its analogs in calcium mobilization and radioligand binding
assays. We have focused on two main structural areas: the 2-
pyrrolidinyl position on the pyridine and the 4-chlorophenyl
group (Figure 2).
Scheme 1. Synthesis of Intermediates 6a−n
a
Reagents and conditions: (a) acetic anhydride, DCM, rt, 16 h (for
6a) or corresponding aldehyde, Na(OAc)3BH, acetic acid, 1,2-DCE;
(b) corresponding amine, reflux, 15 min to 3 h.
presence of Pd(PPh3)4 afforded compounds 8a−n.39,40
Reduction of intermediates 8a−n with hydrazine hydrate and
Raney nickel in ethanol at 50 °C provided intermediates 9a−n,
followed by final coupling of 9a−n with the corresponding
phenyl isocyanates in chloroform to give the final ureas 4, 10−
21, and 25.36 Compounds 22−24 were obtained through
methylation of the corresponding monoalkyl derivatives 12−
14, respectively, using reductive amination with formaldehyde.
Compounds 26−45 were synthesized from intermediates 8h
using the same two-step procedure in the synthesis of 4.
Pharmacological Evaluations. FLIPR-based CB1 and
CB2 calcium mobilization assays were developed in our
laboratories to characterize the target compounds. In these
assays, CHO cells that have been engineered to overexpress the
promiscuous G protein, Gα16 (Molecular Devices), were
further engineered to also stably express the CB1 and CB2
receptors, respectively. Therefore, activation of the CB1 or CB2
receptors, which are primarily coupled to Gαi/o proteins, is now
coupled to the mobilization of internal calcium through the
Gα16 protein. These assays have shown strong correlation with
other CB1 and CB2 assays such as radioligand binding assays
and are routinely used in our laboratories.41−43 These assays
were also used previously by our group to characterize the CB1
receptor modulator RTI-371.20
Figure 2. Proposed structural modifications on 4.
RESULTS AND DISCUSSION
■
Chemistry. Compounds 4 and 10−45 were synthesized
following procedures depicted in Scheme 1 and Scheme 2. 2,6-
Disubstituted pyridines 6a−g were prepared by reacting 2-
amino-6-bromopyridine (5) with either acetic anhydride (6a)
or the corresponding aldehydes (6b−g) in the presence of
sodium triacetoxyborohydride using 1,2-dichloroethane as a
solvent.35,36 The synthesis of intermediates 6h−n consisted of
displacement of one of the bromides of 2,6-dibromopyridine
(7) by refluxing in the appropriate amine.37,38
In the CB1 calcium assay, 4 dose-dependently reduced the
Emax values of CP55,940, as expected for negative allosteric
modulators (Figure 3). 1 displayed a similar dose-dependent
reduction in Emax (unpublished results). These results confirm
that the CB1 calcium assay used here is a suitable system for
evaluating allosteric modulators. The potencies of the
Suzuki coupling reactions of intermediates 6a−n with 3-
nitrophenylboronic acid under standard conditions in the
B
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2. Synthesis of Compounds 4 and 10−45
a
Reagents and conditions: (a) 3-nitrophenylboronic acid, Pd(PPh3)4, DME, NaHCO3, reflux, 12 h; (b) hydrazine hydrate, Raney Ni, 50 °C, ethanol,
2 h; (c) corresponding isocyanate, chloroform, rt or 55 °C, 16 h; (d) formaldehyde (37% aq), Na(OAc)3BH, acetic acid, 1,2-DCE.
increasing the size of the alkyl groups to propyl and butyl
resulted in further reduced potency (13, 14), the smaller
dimethyl analog 11 showed comparable potency to 4 (27.4 nM
vs 32.5 nM). These results suggested that a ring system is not
required for activity at the CB1 receptor in this series. In the N-
monoalkyl series (15−21), potency first increases and then
decreases with elongation of the alkyl substituent, except for the
propyl analog 17 (IC50 = 311 nM). The N-butyl analog 18
showed the highest potency among the series (93 nM). The
cyclopentyl analog (21) showed similar potency to that of
pentyl (19). Comparison of the monoalkyl and dialkyl series
suggests that the presence of the N-hydrogen is tolerated for
Figure 3. Allosteric modulation of 4 in CB1 Ca2+ assay.
activity. Moreover, the determining factor for CB1 activity
appears to be the size of the N-alkyl substituent(s), as the
basicity of the nitrogen remained little changed (e.g., pKa = 3.83
(4) vs 3.55 (12) vs 3.83 (16), as calculated in ACD Labs). For
instance, the N-butyl group (18) is similar in size to the N-
diethyl (12) and they had similar potency at the CB1 receptor.
synthesized compounds at the CB1 receptor were obtained by
calculating IC50 values for attenuation of the effects of the EC80
concentration of the CB1 agonist CP55,940 (Table 1). In our
assays, the EC80 of CP55,940 averaged 100 nM. Under these
To further investigate the size requirement at this position, a set
conditions, 4 had IC50 = 32.5 nM, consistent with its potency in
other assays.24,29
of mixed alkyl analogs was examined (22−24). In these series,
one of the alkyl groups is a methyl and the second substituent is
an ethyl (22), propyl (23), or butyl (24). As expected, the
smaller ethyl group showed higher potency (IC50 = 53.7 nM)
than the propyl and butyl derivatives. Taken together, these
data suggest the binding pocket prefers substitution on the
nitrogen but has a limited space for size. While an NH is
tolerated with another alkyl substituent of the appropriate size,
the optimal substitution pattern includes a methyl group and an
alkyl substituent with one to four carbons in length.
The SAR studies focused on two main structural areas of
compound 4: the 2-pyrrolidinyl position on the pyridine and
the 4-chlorophenyl group. Specifically, the modifications on the
pyrrolidinyl portion of the molecule were designed in order to
understand the spatial requirements, as well as the necessity of
the ring’s presence in this region. The first analog examined was
the piperidyl analog 10. This ring expanded analog showed
slightly decreased potency (∼3-fold) relative to 4. Next, a series
of ring opened analogs were investigated. These efforts resulted
in a series of compounds bearing various dialkyl and monoalkyl
substituents on the nitrogen atom (Table 1). The diethylamino
analog 12, the ring opened analog of compound 4, showed ∼4-
fold lower potency (125 nM vs 32.5 nM). Interestingly, while
Compound 25, the last compound of the series, was used to
investigate the effect of an electron withdrawing group.
Inclusion of an acetamide group (as in compound 25)
decreased CB1 antagonistic potency by roughly 8-fold when
C
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
withdrawing groups at the 4-position provided good potency,
with the fluoro (27, IC50 = 32 nM) and the cyano analogs (29,
IC50 = 33 nM) being the most active of the series, showing
potency similar to 4. Introduction of electron-donating moieties
(31−33) led to reduced potency, although the reduction was
only modest (5- to 6-fold). The greatest loss of potency was
observed for dimethylamino analog 33 which had an IC50 of
1640 nM. Together, these results suggest that functional groups
with electron withdrawing properties are favored at the 4-
position for activity at the CB1 receptor.
Table 1. Compounds 4 and 10−25 and Inhibition of
CP55,940 Activity at CB1 and CB2 Receptors
We next investigated whether substitutions at other positions
are tolerated. The importance of 4-phenyl substitution was
immediately confirmed by moving the chloro group to position
3, where compound 40 showed a roughly 7-fold drop in
activity. In addition, 41, bearing chloro groups at the 3 and 4
positions, showed higher potency (IC50 = 161 nM) than 42
(IC50 = 716 nM) with its 3,5-dichloro substitutions. These data
highlighted the importance of 4-position substitution for CB1
modulatory activity. Interestingly, none of the compounds with
substitutions at the 2-position (37−39) showed activity when
tested at concentrations up to 10 000 nM. While electronic
properties may contribute to the loss of activity, the presence of
the 2-methyl group most likely forces the structure into a
nonplanar conformation which may not be favored for
interaction with the binding site.
To probe the effect of substitution patterns on activity, we
synthesized a series of disubstituted and trisubstituted analogs,
bearing either electron withdrawing or donating functionalities.
The disubstituted analogs had substitutions at the 3,4-, 3,5- and
2,6-positions with groups such as chloro, methyl, or methoxy.
Among these, the 3,4-disubstitution pattern showed the highest
potency (e.g., 41 vs 42), but potencies were still lower than
those of the corresponding 4-substituted analogs (41 vs 4).
Within the same substitution pattern, electron withdrawing
groups showed higher potency than the electron donating
groups (42 vs 36 and 41 vs 34), where the dimethyl analog 36
showed ∼2-fold decreased activity compared to the dichloro
analog 42. Clearly, these data confirmed that electronic
characteristics of substituents play an important role in CB1
receptor binding and activation. The trisubstituted compound
35 showed little activity.
Finally, other aromatic systems were investigated. For
instance, replacing the phenyl group with the electron-deficient
pyridine moiety (43) led to a slight decline in activity,
suggesting the possibility of introducing heterocyclic structures
in this part of the molecule. Next, larger naphthyl groups (44,
45) were used to probe possible spatial constraint of the
binding pocket. The 2-naphthyl analog (45) showed a
significant decline in activity, and the 1-naphthyl analog (44)
had an almost total loss of activity. These results suggest that
rigid bulky structures are not well tolerated at this position.
Moreover, these observations are in agreement with the earlier
results in the 2-substituted analogs (37−39), as the 1-naphthyl
analog is most likely to adopt a more nonplanar conformation
than the 2-naphthyl because of steric repulsion.
a
Against EC80 (100 nM) of CP55,940. Values are the mean SEM of
b
at least three independent experiments in duplicate. Agonist screen at
10 000 nM. Values are the mean SEM of at least two independent
experiments in duplicate. Activity was less than 15% of CP55,940 Emax
at maximum concentration tested (10 000 nM). Against EC80 (100
nM) of CP55,940. Values are the mean
independent experiments in duplicate. Less than 35% inhibition of
CP55,940 EC80 (100 nM) at concentration of 10 000 nM.
c
d
SEM of at least two
e
compared to the parent compound 4. Compound 25 also had
slightly reduced potency in comparison to 22 with an ethyl
group that is similar in size, suggesting that electron donating
alkyl groups are preferred at this position.
All compounds were also tested for their antagonist activity
at the CB2 receptor, and the percent inhibition of CP55,940
activity at this receptor is reported (Table 1). Most of the
compounds showed little or no antagonism. For those
compounds that exhibited over 35% inhibition, IC50 values
against the EC80 concentration (100 nM) of CP55,940 at the
CB2 receptor were obtained. None had an IC50 lower than 10
000 nM (Table 1). All compounds were also screened for
agonist activity at both CB1 and CB2 receptors. At the CB1
receptor, none of the compounds showed activity greater than
35% of the CB1 agonist CP55,940 Emax when tested at 10 000
nM. Similarly, no compounds displayed agonist activity greater
than 15% of the CP55,940 Emax at the maximum concentration
tested (10 000 nM) at the CB2 receptor.
The next set of compounds was designed to examine the
effects of substitution pattern on the 4-chlorophenyl portion of
the parent compound 4 (Table 2). First, the phenyl analog
(26), bearing no substituents, showed a 5-fold decrease in
activity, suggesting that substitution at the 4-position is
preferred for specific interactions with the binding pocket. To
identify effects caused by electronic properties of the 4-
substituent, we tested a series of compounds (27−33) bearing
groups with a range of electronic characteristics. Electron
Again, all the compounds were screened at the CB2 receptor
for antagonist activity, most of which showed minimal
inhibition (Table 2). IC50 values were obtained for compounds
that showed over 35% inhibition at 10 000 nM, and none
showed IC50 values less than 10 000 nM. All compounds were
also screened for agonist activity at both the CB1 and CB2
receptors. No significant agonist activity was observed for any
D
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. Compounds 26−45 and Their Activity at the CB1 and CB2 Receptors
a
b
Against EC80 (100 nM) of CP55,940. Values are the mean SEM of at least three independent experiments in duplicate. Agonist screen at 10 000
c
nM. Values are the mean SEM of at least two independent experiments in duplicate. Activity was less than 15% of CP55,940 Emax at maximum
concentration tested (10 000 nM). Against EC80 (100 nM) of CP55,940. Values are the mean SEM of at least two independent experiments in
duplicate. Less than 35% inhibition of CP55,940 EC80 (100 nM) at concentration of 10 000 nM.
d
e
of the compounds at either receptor (<30% and 15% of
CP55,940 Emax at the CB1 and CB2 receptors, respectively).
Two compounds (11 and 29) were further evaluated for
their allosteric modulation as compared to 4. As shown in
Figure 4, both compounds were able to dose-dependently
lower the top of the agonist curve of CP55,940, confirming
their allosteric characteristics.
A total of seven compounds that showed good potency in the
calcium assay were also evaluated at the CB1 receptor in
competitive radioligand binding assays against [3H]CP55,940
in hCB1 cell membranes. The binding experiments on two of
the compounds, 4 and 29, are shown in Figure 5. As expected,
SR141716 dose-dependently displaced the radioligand [3H]-
CP55,940. Similar to previously described CB1 receptor
allosteric modulators, all the compounds caused significant
and concentration-dependent enhancement in the binding of
[3H]CP55,940 (Table 3). Horswill et al. previously reported
that compound 4 dose-dependently increased [3H]CP55,940
binding by 58% with an EC50 of 14.4 nM.24 In our hands,
compound 4 had an EC50 of 167 nM, with an increase of Emax
of ∼74% (Emax = 174%, Table 3). Compounds 11 and 27 both
had similar potencies to 4 in the calcium assay. Interestingly, 11
showed a 4-fold decrease in potency, whereas 27 had almost
identical EC50 values in the binding assay. Compounds 18 and
E
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
modulation.23,24 Results of this first SAR study of 4 revealed
that a series of analogs of 4 showed a similar complex
pharmacological profile, enhancing [3H]CP55,940 binding
while antagonizing activity of the agonist CP55,940 in the
calcium assays. Our initial SAR studies showed that a cyclic
system at the 2-pyrrolidylpyridine position was not required for
activity. Moreover, tertiary amine substitution was more
favorable than secondary and the optimal pattern for
substitution was obtained when one of the alkyl groups was
methyl and the other was one to four carbons in length. The
most active compound of the series was the dimethyl analog 11,
which showed almost identical potency to 4 in the calcium
assay and 4-fold deceased potency in the binding enhancement
of [3H]CP55940. At the 4-chlorophenyl position, our data
suggest that the 4-position on the phenyl group tolerates
structural modifications but favors electron withdrawing
functionalities. Compound 29 with a 4-cyano group showed
the highest potency in both CB1 calcium mobilization and
radioligand binding assays.
4 has been shown to possess hypophagic activity in vivo, but
its therapeutic potential has yet to be fully explored. Our study
further supports the earlier finding that 4 acts as a
noncompetitive, allosteric antagonist of the CB1 receptor.29,30
Importantly, 4 has shown no adverse or toxic effects after
administration in vivo,24 as might be expected with allosteric
modulators because of their dependence on endogenous
ligands for signaling. Given the recent adverse effects observed
with orthosteric antagonists, these allosteric antagonists
represent a promising alternative to modulate CB1 function
and may serve as much needed tools to identify alternative
treatment strategies with reduced CNS side effects involving
the CB1 receptor system.
Figure 4. Allosteric modulation of compounds 11 (top) and 29
(bottom) in CB1 Ca2+ assay.
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 chromatog-
raphy 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 character-
ization 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 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 with gradient
elution using the mobile phases (A) H2O containing 0.1% CF3COOH
and (B) MeCN, with a flow rate of 1.0 mL/min.
Figure 5. Competitive binding studies on compounds 4 and 29.
Table 3. Radioligand Binding Studies at the CB1 Receptor
against [3H]CP55,940
a
a
compd
EC50 (nM)
Emax (% specific binding)
4
167 37
b
174
132
184
8
6
3
10
11
18
27
29
43
663 92
1520 630
207 62
161 10
172
172
196
3
3
5
55.2 2.8
408 127
a
Values are the mean
SEM of at least three independent
b
experiments in duplicate. EC50 could not be obtained because of
the limited Emax enhancement (∼32%).
43 both had lower potency than compound 4 in binding
enhancement, in line with the calcium results. Finally, the 4-
cyano analog 29 had the highest potency among the series
(EC50 = 55.2 nM) in increasing [3H]CP55,940 binding, with a
3-fold increase in potency compared to 4.
1-(4-Chlorophenyl)-3-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (4). Hydrazine hydrate (3.2 mL, 66 mmol) was added to
a suspension of 8h (1 g, 4.4 mmol) in ethanol (50 mL). The mixture
was stirred at 50 °C for 15 min, and a clear solution was obtained. An
excess of Raney nickel (∼1 g) was added portionwise. After the
bubbling ceased, the mixture was cooled to room temperature and
filtered. The filtrate was condensed to afford intermediate 9h (0.6 g),
which was used in the next step without further purification.
CONCLUSIONS
■
A number of recently discovered allosteric modulators of the
CB1 receptor display opposing pharmacological properties,
with both positive (in radiolabeled binding studies against
[3H]CP55,940) and negative (in several functional assays)
F
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4-Chlorophenyl isocyanate (19 mg, 0.12 mmol) was added to a
stirred solution of 9h (30 mg, 0.12 mmol) in anhydrous chloroform (5
mL). The reaction mixture was stirred overnight at room temperature.
The formed precipitate was filtered and thoroughly washed with
dichloromethane. Final product 4 was obtained as an off-white solid
1H), 6.23 (d, J = 8.29 Hz, 1H), 3.43 (t, J = 6.59 Hz, 4H), 1.99 (dd, J =
3.39, 9.80 Hz, 4H).
6-Bromo-N-methylpyridin-2-amine (6i). 6i was prepared using
1
the procedure for compound 6h in 63% yield. H NMR (300 MHz,
chloroform-d) δ 7.23−7.31 (m, 1H), 6.73 (d, J = 7.54 Hz, 1H), 6.29
(d, J = 8.10 Hz, 1H), 4.73 (br s, 1H), 2.90 (d, J = 5.09 Hz, 3H).
6-Bromo-N,N-dimethylpyridin-2-amine (6j). 6j was prepared
1
(20 mg, 45%). H NMR (300 MHz, DMSO-d6) δ 8.82 (d, J = 11.87
Hz, 2H), 8.08 (s, 1H), 7.43−7.71 (m, 5H), 7.23−7.40 (m, 3H), 7.06
(d, J = 7.35 Hz, 1H), 6.42 (d, J = 8.29 Hz, 1H), 3.48 (br s, 4H), 1.97
(br s, 4H).
1
using the procedure for compound 6h in 87% yield. H NMR (300
MHz, chloroform-d) δ 7.20−7.28 (m, 1H), 6.66 (d, J = 7.54 Hz, 1H),
6.37 (d, J = 8.48 Hz, 1H), 3.06 (s, 6H).
N-(6-Bromopyridin-2-yl)acetamide (6a). Acetic anhydride was
added to a solution of the commercially available 2-amino-6-
bromopyridine (5, 0.3 g, 1.73 mmol) in DCM (7 mL), and the
reaction mixture was stirred at room temperature for 16 h. Upon
completion reaction mixture was diluted with DCM, washed with
saturated sodium carbonate, dried over MgSO4, and concentrated
under reduced pressure. Final product was obtained as a white solid
(0.36 g, 97%). 1H NMR (300 MHz, chloroform-d) δ 8.15 (d, J = 8.10
Hz, 1H), 7.56 (t, J = 7.91 Hz, 1H), 7.21 (d, J = 7.72 Hz, 1H), 2.20 (s,
3H).
6-Bromo-N-propylpyridin-2-amine (6b). A solution of propanal
(0.16 mL, 2.2 mmol) and 2-amino-6-bromopyridine (5, 0.30 g, 1.7
mmol) in 1,2-DCE (10 mL) was treated with Na(OAc)3BH (0.92 g,
4.3 mmol), and the reaction mixture was stirred at room temperature
for 24 h. After completion the reaction was quenched with aqueous
NaOH (1 M) and then extracted with DCM (3 × 10 mL). Combined
organic layers were washed with water (2 × 15 mL), brine (15 mL),
dried over MgSO4, and concentrated under reduced pressure. Crude
product was obtained as a clear oil (0.41 g, 92%) and used in the next
step without purification. 1H NMR (300 MHz, chloroform-d) δ 7.15−
7.35 (m, 1H), 6.63−6.78 (m, 1H), 6.27 (d, J = 8.29 Hz, 1H), 4.71 (br
s, 1H), 3.09−3.24 (m, 2H), 1.63 (sxt, J = 7.27 Hz, 2H), 0.98 (t, J =
7.44 Hz, 3H).
6-Bromo-N-ethylpyridin-2-amine (6k). 6k was prepared using
1
the procedure for compound 6h in 26% yield. H NMR (300 MHz,
chloroform-d) δ 7.24 (t, J = 7.82 Hz, 1H), 6.69 (d, J = 7.44 Hz, 1H),
6.27 (d, J = 8.19 Hz, 1H), 4.74 (br s, 1H), 3.16−3.33 (m, 2H), 1.22 (t,
J = 7.16 Hz, 3H).
6-Bromo-N,N-diethylpyridin-2-amine (6l). 6l was prepared
1
using the procedure for compound 6h in 41% yield. H NMR (300
MHz, chloroform-d) δ 6.98−7.63 (m, 1H), 6.61 (d, J = 7.35 Hz, 1H),
6.33 (d, J = 8.48 Hz, 1H), 3.48 (q, J = 7.03 Hz, 4H), 1.16 (t, J = 6.97
Hz, 6H).
2-Bromo-6-(piperidin-1-yl)pyridine (6m). 6m was prepared
1
using the procedure for compound 6h in 63% yield. H NMR (300
MHz, chloroform-d) δ 7.19−7.25 (m, 1H), 6.67 (d, J = 7.35 Hz, 1H),
6.50 (d, J = 8.48 Hz, 1H), 3.51 (br s, 4H), 1.64 (br s, 6H).
6-Bromo-N-cyclopentylpyridin-2-amine (6n). 6n was prepared
1
using the procedure for compound 6h in 52% yield. H NMR (300
MHz, chloroform-d) δ 8.85 (s, 1H), 8.30 (d, J = 7.72 Hz, 1H), 8.21 (d,
J = 8.10 Hz, 1H), 7.48−7.64 (m, 2H), 7.08 (d, J = 7.54 Hz, 1H), 6.43
(d, J = 8.29 Hz, 1H), 4.71 (br s, 1H), 3.96−4.18 (m, 1H), 2.10 (dd, J =
5.65, 12.06 Hz, 2H), 1.63−1.88 (m, 4H), 1.53−1.55 (m, 2H).
N-[6-(3-Nitrophenyl)pyridin-2-yl]acetamide (8a). 8a was pre-
1
pared using the procedure for compound 8h in 80% yield. H NMR
(300 MHz, chloroform-d) δ 8.88 (s, 1H), 8.19−8.32 (m, 3H), 8.05 (br
s, 1H), 7.83 (t, J = 8.01 Hz, 1H), 7.60−7.68 (m, 1H), 7.55 (d, J = 7.72
Hz, 1H), 2.28 (s, 3H).
N-Propyl-6-(3-nitrophenyl)pyridine-2-amine (8b). 8b was
prepared using the procedure for compound 8h in 81% yield. 1H
NMR (300 MHz, chloroform-d) δ 8.90 (s, 1H), 8.36 (d, J = 7.72 Hz,
1H), 8.19 (dd, J = 1.13, 8.10 Hz, 1H), 7.53 (td, J = 8.03, 19.92 Hz,
2H), 7.04 (d, J = 7.35 Hz, 1H), 6.51 (d, J = 8.67 Hz, 1H), 3.38−3.56
(m, 2H), 1.59−1.79 (m, 2H), 0.99 (t, J = 7.44 Hz, 3H).
6-Bromo-N,N-dipropylpyridin-2-amine (6c). 6c was prepared
1
using the procedure for compound 6b in 46% yield. H NMR (300
MHz, chloroform-d) δ 7.10−7.23 (m, 1H), 6.51−6.67 (m, 1H), 6.30
(d, J = 8.48 Hz, 1H), 3.31−3.41 (m, 4H), 1.51−1.66 (m, 4H), 0.88−
0.96 (m, 6H).
6-Bromo-N-butylpyridin-2-amine (6d). 6d was prepared using
1
the procedure for compound 6b in 41% yield. H NMR (300 MHz,
chloroform-d) δ 7.24 (t, J = 7.91 Hz, 1H), 6.70 (d, J = 7.35 Hz, 1H),
6.27 (d, J = 8.10 Hz, 1H), 4.70 (br s, 1H), 3.20 (q, J = 6.78 Hz, 2H),
1.51−1.64 (m, 2H), 1.33−1.46 (m, 2H), 0.94 (t, J = 7.35 Hz, 3H).
6-Bromo-N,N-dibutylpyridin-2-amine (6e). 6e was prepared
N,N-Dipropyl-6-(3-nitrophenyl)pyridine-2-amine (8c). 8c was
prepared using the procedure for compound 8h in 80% yield. 1H
NMR (300 MHz, chloroform-d) δ 8.92 (s, 1H), 8.34 (d, J = 7.72 Hz,
1H), 8.19 (dd, J = 1.13, 8.10 Hz, 1H), 7.55 (td, J = 8.03, 19.92 Hz,
2H), 7.04 (d, J = 7.35 Hz, 1H), 6.48 (d, J = 8.67 Hz, 1H), 3.43−3.55
(m, 4H), 1.61−1.78 (m, 4H), 0.99 (t, J = 7.44 Hz, 6H).
1
using the procedure for compound 6b in 43% yield. H NMR (300
MHz, chloroform-d) δ ppm 7.13−7.24 (m, 1 H), 6.60 (d, J = 7.35 Hz,
1 H), 6.30 (d, J = 8.38 Hz, 1 H), 3.34−3.46 (m, 4 H), 1.47−1.63 (m, 4
H), 1.27−1.43 (m, 4 H), 0.90−1.02 (m, 6 H).
N-Butyl-6-(3-nitrophenyl)pyridine-2-amine (8d). 8d was pre-
1
6-Bromo-N-pentylpyridin-2-amine (6f). 6f was prepared using
pared using the procedure for compound 8h in 68% yield. H NMR
1
the procedure for compound 6b in 38% yield. H NMR (300 MHz,
(300 MHz, chloroform-d) δ 8.85 (s, 1H), 8.36 (d, J = 7.91 Hz, 1H),
8.21 (dd, J = 1.32, 8.10 Hz, 1H), 7.58 (q, J = 7.54 Hz, 2H), 7.10 (d, J =
7.35 Hz, 1H), 6.68 (d, J = 8.48 Hz, 1H), 3.18 (t, J = 6.97 Hz, 1H), 1.53
(m, 2H), 1.24−1.41 (m, 2H), 0.81−0.95 (m, 3H).
N,N-Dibutyl-6-(3-nitrophenyl)pyridine-2-amine (8e). 8e was
prepared using the procedure for compound 8h in 73% yield. 1H
NMR (300 MHz, chloroform-d) δ 8.84 (br s, 1H), 8.25 (d, J = 7.54
Hz, 1H), 8.11 (d, J = 7.25 Hz, 1H), 7.32−7.58 (m, 2H), 6.95 (d, J =
7.16 Hz, 1H), 6.39 (d, J = 8.38 Hz, 1H), 3.45 (br s, 4H), 1.57 (br s,
4H), 1.34 (d, J = 6.78 Hz, 4H), 0.92 (t, J = 7.06 Hz, 6H).
chloroform-d) δ 8.85 (t, J = 1.74 Hz, 1H), 8.31 (d, J = 7.82 Hz, 1H),
8.20 (dd, J = 1.32, 8.19 Hz, 1H), 7.56 (td, J = 7.89, 15.68 Hz, 2H),
7.07 (d, J = 9.70 Hz, 1H), 6.39 (d, J = 9.70 Hz, 1H), 3.35 (q, J = 6.81
Hz, 2H), 1.54−1.75 (m, 2H), 1.21−1.40 (m, 4H), 0.83−1.00 (m, 3H).
6-Bromo-N-hexylpyridin-2-amine (6g). 6g was prepared using
1
the procedure for compound 6b in 41% yield. H NMR (300 MHz,
chloroform-d) δ 8.85 (s, 1H), 8.31 (d, J = 7.72 Hz, 1H), 8.09−8.26
(m, 1H), 7.43−7.69 (m, 2H), 7.08 (d, J = 7.44 Hz, 1H), 6.41 (d, J =
8.19 Hz, 1H), 4.65 (br s, 1H), 3.35 (q, J = 6.59 Hz, 2H), 1.56−1.75
(m, 2H), 1.26−1.49 (m, 6H), 0.91 (t, J = 6.59 Hz, 3H).
N-Pentyl-6-(3-nitrophenyl)pyridine-2-amine (8f). 8f was pre-
1
2-Bromo-6-(pyrrolidin-1-yl)pyridine (6h). A solution of 2,6-
dibromopyridine (7, 5g, 21 mmol) in pyrrolidine (30 mL) was heated
to reflux for 10 min until no starting material was detected by TLC.
Pyrrolidine was removed under reduced pressure and the residue was
dissolved in a 30% solution of ethyl acetate in dichloromethane. The
organic portion was washed with NaOH (1 M, 20 mL), dried over
MgSO4, and concentrated under reduced pressure. The crude product
was purified by chromatography on silica (0−15% EtOAc in hexane)
pared using the procedure for compound 8h in 79% yield. H NMR
(300 MHz, chloroform-d) δ 8.85 (t, J = 1.74 Hz, 1H), 8.31 (d, J = 7.82
Hz, 1H), 8.20 (dd, J = 1.32, 8.19 Hz, 1H), 7.56 (td, J = 7.89, 15.68 Hz,
2H), 7.07 (d, J = 9.70 Hz, 1H), 6.39 (d, J = 9.70 Hz, 1H), 3.35 (q, J =
6.81 Hz, 2H), 1.54−1.75 (m, 2H), 1.21−1.40 (m, 4H), 0.83−1.00 (m,
3H).
N-Hexyl-6-(3-nitrophenyl)pyridine-2-amine (8g). 8g was
prepared using the procedure for compound 8h in 82% yield. 1H
NMR (300 MHz, chloroform-d) δ 8.85 (s, 1H), 8.31 (d, J = 7.72 Hz,
1H), 8.09−8.26 (m, 1H), 7.43−7.69 (m, 2H), 7.08 (d, J = 7.44 Hz,
1
to give the desired product as a white solid (4.0 g, 83%). H NMR
(300 MHz, chloroform-d) δ 7.19−7.25 (m, 1H), 6.64 (d, J = 7.16 Hz,
G
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
1H), 6.41 (d, J = 8.19 Hz, 1H), 4.65 (br s, 1H), 3.35 (q, J = 6.59 Hz,
2H), 1.56−1.75 (m, 2H), 1.26−1.49 (m, 6H), 0.91 (t, J = 6.59 Hz,
3H).
compound 4 in 43% yield. H NMR (300 MHz, methanol-d4) δ 8.11
(s, 1H), 7.68 (d, J = 7.72 Hz, 1H), 7.37−7.53 (m, 4H), 7.17−7.36 (m,
4H), 7.00 (d, J = 7.35 Hz, 1H), 6.47 (d, J = 8.48 Hz, 1H), 3.43−3.58
(m, 4H), 1.58−1.78 (m, 4H), 0.97 (t, J = 7.35 Hz, 6H).
2-(3-Nitrophenyl)-6-(pyrrolidine-1-yl)pyridine (8h). Nitrogen
was bubbled through a mixture of 3-nitrophenylboronic acid (0.81 g,
4.84 mmol), 6h (1.00 g, 4.4 mmol), and NaHCO3 (1.10 g, 13.20
mmol) in DME (60 mL) and water (25 mL) for 15 min. Pd(Ph3)4
(0.38 g, 0.33 mmol) was added, and reaction mixture was refluxed
overnight under nitrogen atmosphere. Reaction solvent was removed
under reduced pressure and the resulting residue was purified by
chromatography on silica (0−10% EtOAc in hexane) to give the
3-(4-Chlorophenyl)-1-{3-[6-(dibutylamino)pyridin-2-yl]-
phenyl}urea (14). 14 was prepared from 9e using the procedure for
compound 4 in 54% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.87 (s,
1H), 8.77 (s, 1H), 8.18 (s, 1H), 7.62 (d, J = 7.54 Hz, 1H), 7.51 (d, J =
8.90 Hz, 3H), 7.28−7.43 (m, 4H), 7.01 (d, J = 7.39 Hz, 1H), 6.53 (d, J
= 8.52 Hz, 1H), 3.51 (t, J = 7.32 Hz, 4H), 1.47−1.66 (m, 4H), 1.21−
1.43 (m, 4H), 0.93 (t, J = 7.32 Hz, 6H).
3-(4-Chlorophenyl)-1-{3-[6-(methylamino)pyridin-2-yl]-
phenyl}urea (15). 15 was prepared from 9i using the procedure for
compound 4 in 53% in yield. 1H NMR (300 MHz, DMSO-d6) δ 8.83
(s, 1H), 8.80 (s, 1H), 7.61 (d, J = 7.72 Hz, 1H), 7.47−7.55 (m, 4H),
7.30−7.38 (m, 3H), 6.99 (d, J = 7.35 Hz, 1H), 6.42 (d, J = 8.19 Hz,
1H), 2.87 (d, J = 4.71 Hz, 3H).
3-(4-Chlorophenyl)-1-{3-[6-(ethylamino)pyridin-2-yl]-
phenyl}urea (16). 16 was prepared from 9k using the procedure for
compound 4 in 47% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.10 (s,
1H), 7.76 (s, 3H), 7.63−7.70 (m, 3H), 7.59 (br s, 3H), 7.36 (d, J =
15.82 Hz, 1H), 7.08 (s, 1H), 6.42 (d, J = 8.29 Hz, 1H), 3.41−3.57 (m,
2H), 1.87−2.10 (m, 3H).
3-(4-Chlorophenyl)-1-{3-[6-(propylamino)pyridin-2-yl]-
phenyl}urea (17). 17 was prepared from 9b using the procedure for
compound 4 in 49% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.81 (d,
J = 16.77 Hz, 2H), 8.05 (s, 1H), 7.60 (d, J = 7.72 Hz, 1H), 7.39−7.55
(m, 4H), 7.27−7.38 (m, 3H), 6.96 (d, J = 7.35 Hz, 1H), 6.56 (t, J =
5.46 Hz, 1H), 6.43 (d, J = 8.29 Hz, 1H), 1.50−1.69 (m, 2H), 0.96 (t, J
= 7.35 Hz, 3H).
3-(4-Chlorophenyl)-1-{3-[6-(butylamino)pyridin-2-yl]-
phenyl}urea (18). 18 was prepared from 9d using the procedure for
compound 4 in 49% yield. 1H NMR (300 MHz, chloroform-d) δ 7.40
(t, J = 7.82 Hz, 4H), 7.05−7.20 (m, 2H), 6.91 (d, J = 7.35 Hz, 1H),
6.62 (dd, J = 1.51, 6.97 Hz, 1H), 6.24 (d, J = 8.29 Hz, 1H), 3.13−3.30
(m, 2H), 1.46−1.66 (m, 2H), 1.27−1.45 (m, 2H), 0.89 (t, J = 7.35 Hz,
3H).
3-(4-Chlorophenyl)-1-{3-[6-(pentylamino)pyridin-2-yl]-
phenyl}urea (19). 19 was prepared from 9f using the procedure for
compound 4 in 57% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.84 (s,
1H), 8.78 (s, 1H), 8.07 (s, 1H), 7.60 (d, J = 7.72 Hz, 1H), 7.38−7.54
(m, 5H), 7.27−7.37 (m, 3H), 6.96 (d, J = 7.35 Hz, 1H), 6.42 (d, J =
8.29 Hz, 1H), 1.51−1.64 (m, 2H), 1.18−1.44 (m, 6H), 0.86 (t, J =
6.50 Hz, 3H).
1
desired product (1.0 g, 84%). H NMR (300 MHz, chloroform-d) δ
7.27−7.46 (m, 3H), 7.06−7.20 (m, 1H), 6.88 (d, J = 7.54 Hz, 1H),
6.61 (d, J = 7.72 Hz, 1H), 6.21 (d, J = 8.29 Hz, 1H), 3.48 (br s, 4H),
1.79−2.04 (m, 4H).
N-Methyl-6-(3-nitrophenyl)pyridine-2-amine (8i). 8i was
prepared using the procedure for compound 8h in 79% yield. 1H
NMR (300 MHz, chloroform-d) δ 8.85 (s, 1H), 8.32 (d, J = 7.91 Hz,
1H), 8.21 (dd, J = 0.94, 7.16 Hz, 1H), 7.57 (q, J = 7.79 Hz, 2H), 7.10
(d, J = 7.54 Hz, 1H), 6.44 (d, J = 8.29 Hz, 1H), 4.69 (br s, 1H), 3.01
(d, J = 4.90 Hz, 3H).
N,N-Dimethyl-6-(3-nitrophenyl)pyridine-2-amine (8j). 8j was
prepared using the procedure for compound 8h in 85% yield. 1H
NMR (300 MHz, chloroform-d) δ 8.90 (s, 1H), 8.38 (d, J = 7.72 Hz,
1H), 8.21 (d, J = 8.29 Hz, 1H), 7.47−7.70 (m, 2H), 7.09 (d, J = 7.35
Hz, 1H), 6.56 (d, J = 8.48 Hz, 1H), 3.19 (s, 6H).
N-Ethyl-6-(3-nitrophenyl)pyridine-2-amine (8k). 8k was pre-
pared in using the procedure for compound 8h in 81% yield. 1H NMR
(300 MHz, chloroform-d) δ 8.85 (t, J = 1.84 Hz, 1H), 8.31 (d, J = 7.82
Hz, 1H), 8.20 (dd, J = 1.32, 8.19 Hz, 1H), 7.56 (td, J = 7.94, 13.99 Hz,
2H), 7.09 (d, J = 7.44 Hz, 1H), 6.41 (d, J = 8.29 Hz, 1H), 4.61 (br s,
1H), 3.34−3.46 (m, 2H), 1.31 (t, J = 7.16 Hz, 3H).
N,N-Diethyl-6-(3-nitrophenyl)pyridine-2-amine (8l). 8l was
prepared using the procedure for compound 8h in 73% yield. 1H
NMR (300 MHz, chloroform-d) δ 8.88 (s, 1H), 8.38 (s, 1H), 8.20 (d,
J = 7.91 Hz, 1H), 7.47−7.63 (m, 2H), 7.04 (d, J = 7.35 Hz, 1H), 6.50
(d, J = 8.48 Hz, 1H), 3.61 (q, J = 6.97 Hz, 4H), 1.25 (t, J = 7.06 Hz,
6H).
2-(3-Nitrophenyl)-6-(piperidin-1-yl)pyridine (8m). 8m was
prepared using the procedure for compound 8h in 67% yield. 1H
NMR (300 MHz, chloroform-d) δ 8.86 (s, 1H), 8.36 (d, J = 7.91 Hz,
1H), 8.21 (dd, J = 1.32, 8.10 Hz, 1H), 7.58 (q, J = 7.54 Hz, 3H), 7.10
(d, J = 7.35 Hz, 1H), 6.68 (d, J = 8.48 Hz, 1H), 3.65 (br s, 4H), 1.69
(s, 6H).
3-(4-Chlorophenyl)-1-{3-[6-(hexylamino)pyridin-2-yl]-
phenyl}urea (20). 20 was prepared from 9g using the procedure for
compound 4 in 40% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.84 (s,
1H), 8.78 (s, 1H), 7.60 (d, J = 7.72 Hz, 1H), 7.40 (s, 4H), 7.28−7.37
(m, 3H), 6.96 (d, J = 7.35 Hz, 1H), 6.53 (t, J = 5.32 Hz, 1H), 6.42 (d, J
= 8.29 Hz, 1H), 1.48−1.66 (m, 2H), 1.17−1.45 (m, 8H), 0.86 (t, J =
6.45 Hz, 3H).
3-(4-Chlorophenyl)-1-{3-[6-(cyclopentylamino)pyridin-2-yl]-
phenyl}urea (21). 21 was prepared from 9n using the procedure for
compound 4 in 56% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.74 (s,
1H), 8.46 (s, 1H), 7.83 (s, 1H), 7.68 (t, J = 8.10 Hz, 1H), 7.61 (br s,
1H), 7.43 (d, J = 7.91 Hz, 1H), 7.04−7.35 (m, 6H), 6.86 (d, J = 6.78
Hz, 1H), 6.59 (d, J = 9.04 Hz, 1H), 3.85−3.98 (m, 1H), 1.97−2.17
(m, 2H), 1.78−1.93 (m, 2H), 1.60−1.77 (m, 4H).
N-Cyclopentyl-6-(3-nitrophenyl)pyridine-2-amine (8n). 8n
was prepared using the procedure for compound 8h in 65% yield.
1H NMR (300 MHz, chloroform-d) δ 8.85 (s, 1H), 8.30 (d, J = 7.72
Hz, 1H), 8.21 (d, J = 8.10 Hz, 1H), 7.48−7.64 (m, 2H), 7.08 (d, J =
7.54 Hz, 1H), 6.43 (d, J = 8.29 Hz, 1H), 4.71 (br s, 1H), 3.96−4.18
(m, 1H), 2.10 (dd, J = 5.65, 12.06 Hz, 2H), 1.63−1.88 (m, 4H), 1.53−
1.55 (m, 2H).
3-(4-Chlorophenyl)-1-{3-[6-(piperidin-1-yl)pyridin-2-yl]-
phenyl}urea (10). 10 was prepared from 9m using the procedure for
compound 4 in 42% yield. 1H NMR (300 MHz, DMSO-d6) δ 9.03 (d,
J = 9.80 Hz, 2H), 8.03 (s, 1H), 7.44−7.68 (m, 4H), 7.23−7.40 (m,
2H), 7.09 (d, J = 7.54 Hz, 1H), 6.78 (d, J = 8.48 Hz, 1H), 3.61 (m,
4H), 1.61 (m, 6H).
3-(4-Chlorophenyl)-1-{3-[6-(dimethylamino)pyridin-2-yl]-
3-(4-Chlorophenyl)-1-(3-{6-[ethyl(methyl)amino]pyridin-2-
yl}phenyl)urea (22). A solution of 37% aqueous formaldehyde
solution (0.01 mL, 0.11 mmol) and 12 (0.03 g, 0.082 mmol) in 1,2-
DCE (3 mL) was treated with Na(OAc)3BH (0.063 g, 0.3 mmol), and
the reaction mixture was stirred at room temperature for 24 h. After
completion the reaction was quenched with aqueous NaOH (1 M)
and then extracted with DCM (3 × 20 mL). Combined organic layers
were washed with water (2 × 15 mL), brine (15 mL), dried over
MgSO4, and concentrated under reduced pressure. The resulting slurry
phenyl}urea (11). 11 was prepared from 9j using the procedure for
1
compound 4 in 49% yield. H NMR (300 MHz, DMSO-d6) δ 9.11−
9.52 (m, 2H), 8.11 (s, 1H), 7.42−7.69 (m, 5H), 7.28−7.40 (m, 3H),
7.08 (d, J = 7.35 Hz, 1H), 6.62 (d, J = 8.48 Hz, 1H), 3.11 (s, 6H).
3-(4-Chlorophenyl)-1-{3-[6-(diethylamino)pyridin-2-yl]-
phenyl}urea (12). 12 was prepared from 9l using the procedure for
1
compound 4 in 46% yield. H NMR (300 MHz, methanol-d4) δ 8.06
(t, J = 1.79 Hz, 1H), 7.69 (d, J = 7.72 Hz, 1H), 7.41−7.55 (m, 4H),
7.23−7.38 (m, 3H), 7.02 (d, J = 7.35 Hz, 1H), 6.52 (d, J = 8.48 Hz,
1H), 3.63 (q, J = 7.16 Hz, 4H), 1.23 (t, J = 6.97 Hz, 6H).
3-(4-Chlorophenyl)-1-{3-[6-(dipropylamino)pyridin-2-yl]-
phenyl}urea (13). 13 was prepared from 9c using the procedure for
1
was purified on silica to give 22 (14 mg, 43%). H NMR (300 MHz,
DMSO-d6) δ 8.86 (s, 1H), 8.81 (s, 1H), 8.04−8.13 (m, 1H), 7.44−
7.68 (m, 5H), 7.28−7.39 (m, 3H), 7.05 (d, J = 7.25 Hz, 1H), 6.59 (d, J
H
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
= 8.48 Hz, 1H), 3.65 (q, J = 7.19 Hz, 2H), 3.00−3.08 (m, 3H), 1.12 (t,
J = 7.02 Hz, 3H).
Hz, 2H), 6.41 (d, J = 8.29 Hz, 1H), 3.48 (br s, 4H), 2.84 (s, 6H), 1.98
(br s, 4H).
3-(4-Chlorophenyl)-1-(3-{6-[propyl(methyl)amino]pyridin-2-
yl}phenyl)urea (23). 23 was prepared from 13 using the procedure
for compound 22 in 41% yield. 1H NMR (300 MHz, chloroform-d) δ
7.91 (s, 1H), 7.69 (d, J = 6.88 Hz, 1H), 7.37−7.49 (m, 1H), 7.27 (d, J
= 6.03 Hz, 4H), 7.04−7.15 (m, 5H), 6.88 (d, J = 7.35 Hz, 1H), 6.42
(d, J = 8.48 Hz, 1H), 3.49 (t, J = 7.30 Hz, 2H), 3.08 (s, 3H), 1.54−
1.70 (m, 2H), 0.89 (t, J = 7.35 Hz, 3H).
3-(2H-1,3-Benzodioxol-5-yl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-
2-yl]phenyl}urea (34). 34 was prepared using the procedure for
compound 4 in 52% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.75 (s,
1H), 8.64 (s, 1H), 8.06 (s, 1H), 7.48−7.67 (m, 3H), 7.26−7.40 (m,
1H), 7.22 (s, 1H), 7.05 (d, J = 7.35 Hz, 1H), 6.69−6.89 (m, 2H), 6.41
(d, J = 8.29 Hz, 1H), 5.97 (s, 2H), 3.48 (br s, 4H), 1.98 (br s, 4H).
1-{3-[6-(Pyrrolidin-1-yl)pyridin-2-yl]phenyl}-3-(3,4,5-
trimethoxyphenyl)urea (35). 35 was prepared using the procedure
3-(4-Chlorophenyl)-1-(3-{6-[butyl(methyl)amino]pyridin-2-
1
yl}phenyl)urea (24). 24 was prepared from 14 using the procedure
for compound 4 in 46% yield. H NMR (300 MHz, DMSO-d6) δ
1
for compound 22 in 61% yield. H NMR (300 MHz, DMSO-d6) δ
8.45−8.78 (m, 2H), 7.90−8.14 (m, 1H), 7.44−7.70 (m, 3H), 7.25−
7.40 (m, 1H), 6.91−7.17 (m, 1H), 6.81 (s, 2H), 6.31−6.54 (m, 1H),
3.76 (s, 6H), 3.62 (s, 3H), 3.42−3.53 (m, 4H), 1.91−2.03 (m, 4H).
3-(3,5-Dimethylphenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (36). 36 was prepared using the procedure for
8.25 (s, 1H), 7.95 (t, J = 8.01 Hz, 1H), 7.60 (d, J = 7.72 Hz, 1H),
7.42−7.54 (m, 4H), 7.29 (dd, J = 5.56, 8.57 Hz, 5H), 4.01 (t, J = 7.25
Hz, 2H), 3.26 (br s, 3H), 1.64 (t, J = 6.97 Hz, 2H), 1.27−1.45 (m,
2H), 0.92 (t, J = 7.25 Hz, 3H).
1
compound 4 in 50% yield. H NMR (300 MHz, DMSO-d6) δ 8.80
N-[6-(3-{[(4-Chlorophenyl)carbamoyl]amino}phenyl)pyridin-
2-yl]acetamide (25). 25 was prepared from 9a using the procedure
(s, 1H), 8.60 (s, 1H), 8.06 (s, 1H), 7.46−7.73 (m, 3H), 7.22−7.42 (m,
1H), 6.97−7.16 (m, 3H), 6.62 (s, 1H), 6.42 (d, J = 8.48 Hz, 1H), 3.48
(br s, 4H), 2.24 (s, 6H), 1.98 (br s, 4H).
1
for compound 4 in 47% yield. H NMR (300 MHz, DMSO-d6) δ
10.47 (s, 1H), 8.90 (d, J = 15.64 Hz, 2H), 8.15 (s, 1H), 8.03 (d, J =
8.10 Hz, 1H), 7.85 (t, J = 7.91 Hz, 1H), 7.65 (d, J = 7.54 Hz, 1H),
7.29−7.58 (m, 6H), 2.14 (s, 3H).
3-(2,6-Dimethylphenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (37). 37 was prepared using the procedure for
1
compound 4 in 60% yield. H NMR (300 MHz, DMSO-d6) δ 8.95
3-Phenyl-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]phenyl}urea
(26). 26 was prepared using the procedure for compound 4 in 49%
(br s, 1H), 8.10 (s, 1H), 7.86 (s, 1H), 7.56 (q, J = 7.03 Hz, 2H), 7.22−
7.37 (m, 1H), 6.91−7.14 (m, 3H), 6.40 (d, J = 8.29 Hz, 1H), 3.47 (br
s, 4H), 2.23 (s, 6H), 1.97 (br s, 4H).
1
yield. H NMR (300 MHz, DMSO-d6) δ 8.75 (s, 1H), 8.68 (s, 1H),
8.07 (br s, 1H), 7.51−7.69 (m, 3H), 7.46 (d, J = 7.91 Hz, 2H), 7.21−
7.39 (m, 3H), 7.06 (d, J = 7.35 Hz, 1H), 6.97 (t, J = 7.11 Hz, 1H), 6.42
(d, J = 8.29 Hz, 1H), 3.48 (br s, 4H), 1.97 (br s, 4H).
3-(2-Ethylphenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (38). 38 was prepared using the procedure for
1
compound 4 in 41% yield. H NMR (300 MHz, DMSO-d6) δ 9.10
3-(4-Fluorophenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (27). 27 was prepared using the procedure for
(s, 1H), 8.07 (s, 1H), 7.93 (s, 1H), 7.77 (d, J = 7.82 Hz, 1H), 7.49−
7.66 (m, 3H), 7.30−7.39 (m, 1H), 7.11−7.24 (m, 2H), 6.92−7.08 (m,
2H), 6.42 (d, J = 8.29 Hz, 1H), 3.43−3.61 (m, 4H),2.62 (q, J = 7.47
Hz, 2H), 1.96 (t, J = 6.31 Hz, 4H), 1.18 (t, J = 7.49 Hz, 3H).
3-[2-(Propan-2-yl)phenyl]-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-
yl]phenyl}urea (39). 39 was prepared using the procedure for
1
compound 4 in 61% yield. H NMR (300 MHz, DMSO-d6) δ 8.81
(d, J = 9.14 Hz, 2H), 7.96−8.14 (m, 1H), 7.41−7.73 (m, 5H), 7.25−
7.41 (m, 1H), 7.13 (s, 3H), 6.30−6.52 (m, 1H), 3.48 (br s, 4H), 1.83−
2.10 (m, 4H).
3-(4-Bromophenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
1
phenyl}urea (28). 28 was prepared using the procedure for
compound 4 in 49% yield. H NMR (300 MHz, DMSO-d6) δ 8.04−
1
compound 4 in 53% yield. H NMR (300 MHz, DMSO-d6) δ 8.82
8.15 (m, 1H), 7.91−8.00 (m, 1H), 7.50−7.70 (m, 4H), 7.24−7.40 (m,
2H), 7.01−7.20 (m, 3H), 6.33−6.48 (m, 1H), 3.43−3.61 (m, 4H),
3.09−3.21 (m, 1H), 1.88−2.06 (m, 4H), 1.12−1.28 (m, 6H).
3-(3-Chlorophenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (40). 40 was prepared using the procedure for
compound 4 in 64% yield. 1H NMR (300 MHz, DMSO-d6) δ 9.05
(s, 1H), 8.98 (s, 1H), 8.18 (s, 1H), 7.83 (s, 1H), 7.75 (d, J = 7.72 Hz,
1H), 7.62−7.71 (m, 2H), 7.34−7.51 (m, 3H), 7.07−7.22 (m, 2H),
6.53 (d, J = 8.29 Hz, 1H), 3.58 (br s, 4H), 2.08 (br s, 4H).
(d, J = 12.06 Hz, 2H), 8.07 (s, 1H), 7.51−7.70 (m, 3H), 7.45 (s, 4H),
7.28−7.39 (m, 1H), 7.06 (d, J = 7.35 Hz, 1H), 6.42 (d, J = 8.29 Hz,
1H), 3.48 (br s, 4H), 1.98 (br s, 4H).
3-(4-Cyanophenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (29). 29 was prepared using the procedure for
1
compound 4 in 49% yield. H NMR (300 MHz, DMSO-d6) δ 9.31
(br s, 1H), 9.04 (br s, 1H), 8.10 (s, 1H), 7.68−7.81 (m, 2H), 7.62−
7.69 (m, 3H), 7.57 (t, J = 7.72 Hz, 2H), 7.31−7.42 (m, 1H), 7.06 (d, J
= 7.35 Hz, 1H), 6.42 (d, J = 8.29 Hz, 1H), 3.40−3.55 (m, 4H), 1.97 (t,
J = 6.31 Hz, 4H).
3-(3,4-Dichlorophenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (41). 41 was prepared using the procedure for
1
compound 4 in 43% yield. H NMR (300 MHz, DMSO-d6) δ 9.02
3-(4-Nitrophenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (30). 30 was prepared using the procedure for
(s, 1H), 8.90 (s, 1H), 8.08 (s, 1H), 7.90 (d, J = 2.26 Hz, 1H), 7.66 (d, J
= 7.72 Hz, 1H), 7.45−7.59 (m, 3H), 7.27−7.42 (m, 2H), 7.06 (d, J =
7.35 Hz, 1H), 6.42 (d, J = 8.29 Hz, 1H), 3.48 (br s, 4H), 1.97 (br s,
4H).
1
compound 4 in 45% yield. H NMR (300 MHz, DMSO-d6) δ 9.55
(br s, 1H), 9.10 (br s, 1H), 8.22 (s, 2H), 8.12 (s, 1H), 7.71 (d, J = 9.42
Hz, 3H), 7.49−7.62 (m, 2H), 7.30−7.43 (m, 1H), 7.07 (d, J = 7.35
Hz, 1H), 6.42 (d, J = 8.48 Hz, 1H), 3.48 (br s, 4H), 1.98 (br s, 4H).
3-(4-Methylphenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (31). 31 was prepared using the procedure for
3-(3,5-Dichlorophenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (42). 42 was prepared using the procedure for
1
compound 4 in 52% yield. H NMR (300 MHz, DMSO-d6) δ 9.08
1
compound 4 in 63% yield. H NMR (300 MHz, DMSO-d6) δ 8.78
(s, 1H), 8.97 (s, 1H), 8.08 (s, 1H), 7.66 (d, J = 7.72 Hz, 1H), 7.48−
7.61 (m, 3H), 7.26−7.41 (m, 1H), 7.06 (d, J = 7.35 Hz, 1H), 6.42 (d, J
= 8.48 Hz, 1H), 3.47 (br s, 4H), 1.97 (br s, 4H).
(s, 1H), 8.64 (s, 1H), 8.06 (s, 1H), 7.46−7.74 (m, 3H), 7.20−7.41 (m,
3H), 6.90−7.15 (m, 3H), 6.42 (d, J = 8.29 Hz, 1H), 3.48 (br s, 4H),
2.25 (s, 3H), 1.98 (br s, 4H).
3-(6-Chloropyridin-3-yl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-
yl]phenyl}urea (43). 43 was prepared using the procedure for
compound 4 in 62% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.99 (d,
J = 19.03 Hz, 2H), 8.48 (d, J = 2.45 Hz, 1H), 8.09 (s, 1H), 8.01 (dd, J
= 2.64, 8.67 Hz, 1H), 7.66 (d, J = 7.72 Hz, 1H), 7.50−7.61 (m, 2H),
7.44 (d, J = 8.67 Hz, 1H), 7.31−7.40 (m, 1H), 7.06 (d, J = 7.54 Hz,
1H), 6.42 (d, J = 8.48 Hz, 1H), 3.48 (br s, 4H), 1.98 (br s, 4H).
3-(Naphthalen-1-yl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (44). 44 was prepared using the procedure for
3-(4-Methoxyphenyl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (32). 32 was prepared using the procedure for
1
compound 4 in 49% yield. H NMR (300 MHz, DMSO-d6) δ 8.79
(s, 1H), 8.60 (s, 1H), 8.16 (s, 1H), 7.57−7.79 (m, 3H), 7.34−7.53 (m,
3H), 7.16 (d, J = 7.35 Hz, 1H), 6.98 (d, J = 8.85 Hz, 2H), 6.52 (d, J =
8.29 Hz, 1H), 3.83 (s, 3H), 3.58 (br s, 4H), 2.08 (br s, 4H).
3-[4-(Dimethylamino)phenyl]-1-{3-[6-(pyrrolidin-1-yl)-
pyridin-2-yl]phenyl}urea (33). 33 was prepared using the
procedure for compound 4 in 56% yield. 1H NMR (300 MHz,
DMSO-d6) δ 8.60 (s, 1H), 8.30 (s, 1H), 8.05 (s, 1H), 7.45−7.70 (m,
3H), 7.17−7.38 (m, 3H), 7.05 (d, J = 7.35 Hz, 1H), 6.71 (d, J = 8.67
1
compound 4 in 36% yield. H NMR (300 MHz, DMF) δ 9.22 (s,
1H), 8.84 (s, 1H), 8.09−8.23 (m, 2H), 8.04 (d, J = 7.44 Hz, 1H), 7.94
(d, J = 7.54 Hz, 1H), 7.44−7.71 (m, 5H), 7.29−7.42 (m, 1H), 7.08 (d,
I
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
J = 7.35 Hz, 1H), 6.42 (d, J = 8.29 Hz, 1H), 3.43−3.56 (m, 4H), 1.83−
2.07 (m, 4H).
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 dilutions and incubated for 15 min at 37 °C. After the
pretreatment incubation period, the plate was read with a FlexStation
II (Molecular Devices). Calcium-mediated changes in fluorescence
were monitored every 1.52 s over a 60 s time period, with the
FlexStation II adding 25 μL of the CP55,940 EC80 concentration at the
19 s time point (excitation at 485 nm, detection at 525 nm). Peak
kinetic reduction (SoftMax, Molecular Devices) relative fluorescence
units (RFUs) were generated. Test compound RFUs were compared
to the CP55,940 EC80 RFUs to generate % inhibition values. 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 FlexStation II added single concentration dilutions of
the test compounds prepared at 10× 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 Emax RFUs to generate % Emax
values. For IC50 assays, the above procedure was followed except that
cells were pretreated with serial dilutions of the test compounds
(prepared at 10× the desired final concentration in 2.25% BSA/4.5%
DMSO/4.5% EtOH/assay buffer) and the FlexStation II added the
EC80 concentration of CP55,940 (prepared at 10× the desired
concentration in 0.25% BSA/0.5% DMSO/0.5% EtOH/assay buffer).
Peak kinetic reduction (SoftMax, Molecular Devices) relative
fluorescence units were plotted against log compound concentration
(nM). Data were fit to a three-parameter logistic curve to generate
IC50 values (GraphPad Prism 6.0, GraphPad Software, Inc., San Diego,
CA).
3-(Naphthalen-2-yl)-1-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]-
phenyl}urea (45). 45 was prepared using the procedure for
1
compound 4 in 34% yield. H NMR (300 MHz, DMSO-d6) δ 8.96
(s, 1H), 8.90 (s, 1H), 8.12 (d, J = 4.05 Hz, 2H), 7.75−7.92 (m, 4H),
7.42−7.71 (m, 5H), 7.37 (t, J = 7.86 Hz, 2H), 7.07 (d, J = 7.44 Hz,
1H), 6.43 (d, J = 8.29 Hz, 1H), 3.43−3.61 (m, 4H), 1.98 (t, J = 6.40
Hz, 4H).
CB1 Calcium Mobilization Assay. RD-HGA16 cells (Molecular
Devices) stably expressing the human CB1 receptor were used. The
day before the assay, cells were plated into 96-well black-walled assay
plates at 25 000 cells/well in Ham’s F12 supplemented with 10% fetal
bovine serum, 100 units of penicillin, 100 units of streptomycin, and
100 μg/mL Normocin. The cells were incubated overnight at 37 °C,
5% CO2. Prior to the assay, Calcium 5 dye (Molecular Devices) was
reconstituted according to the manufacturer’s instructions. The
reconstituted dye was diluted 1:40 in prewarmed (37 °C) assay buffer
(1× 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.
For antagonist (IC50) assays, the EC80 concentration of CP55,940 was
prepared at 10× the desired final concentration in 0.25% BSA/0.5%
DMSO/0.5% EtOH/assay buffer, aliquoted into 96-well polypropy-
lene plates, and warmed to 37 °C. Serial dilutions of the test
compounds were prepared at 10× 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). 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 10
s time point (excitation at 470−495 nm, detection at 515−575 nm).
Maximum kinetic reduction (ScreenWorks, Molecular Devices)
relative fluorescence units (RFU) were plotted against log compound
concentration. Data were fit to a three-parameter logistic curve to
generate IC50 values (GraphPad Prism 6.0, GraphPad Software, Inc.,
San Diego, CA). For the modulation experiments, the above
procedure was followed except that cells were pretreated with a single
concentration of test compound (prepared at 10× the desired
concentration in 2.25% BSA/4.5% DMSO/4.5% EtOH/assay buffer)
and the Tetra added serial dilutions of CP55,940 (prepared at 10× the
desired concentration in 0.25% BSA/0.5% DMSO/0.5% EtOH/assay
buffer). 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 10× 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 Emax RFUs to generate % Emax
values.
[3H]CP55,940 Competitive Binding Assay. Binding assays were
preformed to determine the effect of test compounds on the binding
of [3H]CP55,940 to the human CB1 receptor. Assays were conducted
with 0.62 nM [3H]CP55,944, varying concentrations of unlabeled test
compounds, and membranes from HEK293 cells expressing human
CB1 receptors (PerkinElmer) in a final volume of 500 mL of assay
buffer (50 mM TRIZMA HCl, 5 mM MgCl2, 1 mM EDTA, 0.5% BSA,
1% DMSO, pH 7.4). Specific binding was defined as the difference
between [3H]CP55,940 binding in the absence and presence of 10 μM
nonradiolabeled CP55,940. Eight concentrations of each test
compound were run in duplicate. Binding was initiated by the
addition of CB1 membranes (8 mg protein). Assay tubes were
incubated at 30 °C in a shaking water bath for 1 h. The binding assay
was terminated by vacuum filtration onto a 96-well Unifilter GF/B
glass-fiber filter plate using a cell harvester (Brandel), followed by four
washes with ice-cold wash buffer (50 mM TRIZMA HCl, 5 mM
MgCl2, 1 mM EDTA, 0.1% BSA, pH 7.4). The filter plate was
presoaked in 0.1% PEI for half an hour and rinsed with cold wash
buffer just before filtration of the assay tubes. The filter plate was
allowed to dry, and 35 mL of MicroScint 20 (PerkinElmer) was added
to each well. Radioactivity was measured using a Top Count NXT
(Packard). The exact concentration of [3H]CP55,940 used in each
assay was determined (0.644−0.804 nM) using a TriCarb 2200CA
liquid scintillation analyzer. Binding data were expressed as a
percentage of specific [3H]CP55,940 binding.
CB2 Calcium Mobilization Assay. CHO-RD-HGA16 (Molecular
Devices) cells stably expressing the human CB2 receptor were used.
The day before the assay, cells were plated into 96-well black-walled
assay plates at 30 000 cells/well in Ham’s F12 supplemented with 10%
fetal bovine serum, 100 units of penicillin and streptomycin, and 100
μg/mL Normocin. The cells were incubated overnight at 37 °C, 5%
CO2. Prior to the assay, Calcium 5 dye (Molecular Devices) was
reconstituted according to the manufacturer’s instructions. The
reconstituted dye was diluted 1:40 in prewarmed (37 °C) assay buffer
(1× 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.
For antagonist screens, the EC80 concentration of CP55,940 was
prepared at 10× the desired final concentration in 0.25% BSA/0.5%
DMSO/0.5% EtOH/assay buffer, aliquoted into 96-well polypropy-
lene plates, and warmed to 37 °C. Single concentration dilutions of the
test compounds were prepared at 10× the desired final concentration
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC analysis results of target compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: yzhang@rti.org. Phone: 919-541-1235. Fax: 919-541-
6499.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript
J
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
functional selectivity on new drug discovery. Pharmacol. Rev. 2010,
62, 265−304.
(15) Christopoulos, A.; Kenakin, T. G protein-coupled receptor
allosterism and complexing. Pharmacol. Rev. 2002, 54, 323−374.
(16) Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Allosteric
modulators of GPCRs: a novel approach for the treatment of CNS
disorders. Nat. Rev. Drug Discovery 2009, 8, 41−54.
(17) Shore, D. M.; Baillie, G. L.; Hurst, D. H.; Navas, F., III;
Seltzman, H. H.; Marcu, J. P.; Abood, M. E.; Ross, R. A.; Reggio, P. H.
Allosteric modulation of a cannabinoid G protein-coupled receptor:
binding site elucidation and relationship to G protein signaling. J. Biol.
Chem. 2014, 289, 5828−5845.
(18) Christopoulos, A. Allosteric binding sites on cell-surface
receptors: novel targets for drug discovery. Nat. Rev. Drug Discovery
2002, 1, 198−210.
(19) Bridges, T. M.; Lindsley, C. W. G-protein-coupled receptors:
from classical modes of modulation to allosteric mechanisms. ACS
Chem. Biol. 2008, 3, 530−541.
(20) Navarro, H. A.; Howard, J. L.; Pollard, G. T.; Carroll, F. I.
Positive allosteric modulation of the human cannabinoid (CB)
receptor by RTI-371, a selective inhibitor of the dopamine transporter.
Br. J. Pharmacol. 2009, 156, 1178−1184.
(21) Pamplona, F. A.; Ferreira, J.; Menezes de Lima, O., Jr.; Duarte,
F. S.; Bento, A. F.; Forner, S.; Villarinho, J. G.; Bellocchio, L.; Wotjak,
C. T.; Lerner, R.; Monory, K.; Lutz, B.; Canetti, C.; Matias, I.; Calixto,
J. B.; Marsicano, G.; Guimaraes, M. Z.; Takahashi, R. N. Anti-
inflammatory lipoxin A4 is an endogenous allosteric enhancer of CB1
cannabinoid receptor. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 21134−
21139.
(22) Bauer, M.; Chicca, A.; Tamborrini, M.; Eisen, D.; Lerner, R.;
Lutz, B.; Poetz, O.; Pluschke, G.; Gertsch, J. Identification and
quantification of a new family of peptide endocannabinoids (Pepcans)
showing negative allosteric modulation at CB1 receptors. J. Biol. Chem.
2012, 287, 36944−36967.
(23) Price, M. R.; Baillie, G. L.; Thomas, A.; Stevenson, L. A.; Easson,
M.; Goodwin, R.; McLean, A.; McIntosh, L.; Goodwin, G.; Walker, G.;
Westwood, P.; Marrs, J.; Thomson, F.; Cowley, P.; Christopoulos, A.;
Pertwee, R. G.; Ross, R. A. Allosteric modulation of the cannabinoid
CB1 receptor. Mol. Pharmacol. 2005, 68, 1484−1495.
(24) Horswill, J. G.; Bali, U.; Shaaban, S.; Keily, J. F.; Jeevaratnam, P.;
Babbs, A. J.; Reynet, C.; Wong Kai In, P. PSNCBAM-1, a novel
allosteric antagonist at cannabinoid CB1 receptors with hypophagic
effects in rats. Br. J. Pharmacol. 2007, 152, 805−814.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Tiffany Langston and Allyson Smith for technical
assistance and David Perrey for the critical reading of the
manuscript. This work was supported by National Institute on
Drug Abuse, National Institutes of Health, U.S. (Grant
DA032837 to Y.Z. and Grant DA003672 to J.L.W.).
ABBREVIATIONS USED
■
CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2;
GPCR, G-protein-coupled receptor; SAR, structure−activity
relationship; IC50, half-maximum inhibitory concentration;
DCM, dichloromethane; 1,2-DCE, 1,2-dichloroethane; DME,
1,2-dimethoxyethane; TLC, thin-layer chromatography; MS,
mass spectrometry; HPLC, high performance liquid chroma-
tography; FLIPR, fluorometric imaging plate reader
REFERENCES
■
(1) Pertwee, R. G. The therapeutic potential of drugs that target
cannabinoid receptors or modulate the tissue levels or actions of
endocannabinoids. AAPS J. 2005, 7, E625−E654.
(2) Howlett, A. C.; Breivogel, C. S.; Childers, S. R.; Deadwyler, S. A.;
Hampson, R. E.; Porrino, L. J. Cannabinoid physiology and
pharmacology: 30 years of progress. Neuropharmacology 2004, 47
(Suppl. 1), 345−358.
(3) Pertwee, R. G. Cannabinoid pharmacology: the first 66 years. Br.
J. Pharmacol. 2006, 147 (Suppl. 1), S163−S171.
(4) Jagerovic, N.; Hernandez-Folgado, L.; Alkorta, I.; Goya, P.;
Martin, M. I.; Dannert, M. T.; Alsasua, A.; Frigola, J.; Cuberes, M. R.;
Dordal, A.; Holenz, J. Structural−activity relationship study on C-4
carbon atom of the CB1 antagonist SR141716: synthesis and
pharmacological evaluation of 1,2,4-triazole-3-carboxamides. Eur. J.
Med. Chem. 2006, 41, 114−120.
(5) Mackie, K. Cannabinoid receptors as therapeutic targets. Annu.
Rev. Pharmacol. Toxicol. 2006, 46, 101−122.
(6) Pacher, P.; Batkai, S.; Kunos, G. The endocannabinoid system as
an emerging target of pharmacotherapy. Pharmacol. Rev. 2006, 58,
389−462.
(25) Ross, R. A. Allosterism and cannabinoid CB(1) receptors: the
shape of things to come. Trends Pharmacol. Sci. 2007, 28, 567−572.
(26) Ross, R. A. Tuning the endocannabinoid system: allosteric
modulators of the CB1 receptor. Br. J. Pharmacol. 2007, 152, 565−566.
(27) Ahn, K. H.; Mahmoud, M. M.; Kendall, D. A. Allosteric
modulator ORG27569 induces CB1 cannabinoid receptor high affinity
agonist binding state, receptor internalization, and Gi protein-
independent ERK1/2 kinase activation. J. Biol. Chem. 2012, 287,
12070−12082.
(7) Di Marzo, V. Targeting the endocannabinoid system: to enhance
or reduce? Nat. Rev. Drug Discovery 2008, 7, 438−455.
(8) Makriyannis, A. 2012 Division of Medicinal Chemistry Award
Address. Trekking the cannabinoid road: a personal perspective. J.
Med. Chem. 2014, 57, 3891−3911.
(9) Muccioli, G. G.; Lambert, D. M. Current knowledge on the
antagonists and inverse agonists of cannabinoid receptors. Curr. Med.
Chem. 2005, 12, 1361−1394.
(10) Jagerovic, N.; Fernandez-Fernandez, C.; Goya, P. CB1
cannabinoid antagonists: structure−activity relationships and potential
therapeutic applications. Curr. Top. Med. Chem. 2008, 8, 205−230.
(11) Moreira, F. A.; Grieb, M.; Lutz, B. Central side-effects of
therapies based on CB1 cannabinoid receptor agonists and
antagonists: focus on anxiety and depression. Best Pract. Res., Clin.
Endocrinol. Metab. 2009, 23, 133−144.
(12) Pertwee, R. G. Targeting the endocannabinoid system with
cannabinoid receptor agonists: pharmacological strategies and
therapeutic possibilities. Philos. Trans. R. Soc. London, Ser. B 2012,
367, 3353−3363.
(28) Ahn, K. H.; Mahmoud, M. M.; Samala, S.; Lu, D.; Kendall, D. A.
Profiling two indole-2-carboxamides for allosteric modulation of the
CB1 receptor. J. Neurochem. 2013, 124, 584−589.
(29) Wang, X.; Horswill, J. G.; Whalley, B. J.; Stephens, G. J. Effects
of the allosteric antagonist 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-
ylpyridin-2-yl)phenyl]urea (PSNCBAM-1) on CB1 receptor modu-
lation in the cerebellum. Mol. Pharmacol. 2011, 79, 758−767.
(30) Baillie, G. L.; Horswill, J. G.; Anavi-Goffer, S.; Reggio, P. H.;
Bolognini, D.; Abood, M. E.; McAllister, S.; Strange, P. G.; Stephens,
G. J.; Pertwee, R. G.; Ross, R. A. CB(1) receptor allosteric modulators
display both agonist and signaling pathway specificity. Mol. Pharmacol.
2013, 83, 322−338.
(31) Fay, J. F.; Farrens, D. L. A key agonist-induced conformational
change in the cannabinoid receptor CB1 is blocked by the allosteric
ligand Org 27569. J. Biol. Chem. 2012, 287, 33873−33882.
(32) Piscitelli, F.; Ligresti, A.; La Regina, G.; Coluccia, A.; Morera, L.;
Allara, M.; Novellino, E.; Di Marzo, V.; Silvestri, R. Indole-2-
(13) Melancon, B. J.; Hopkins, C. R.; Wood, M. R.; Emmitte, K. A.;
Niswender, C. M.; Christopoulos, A.; Conn, P. J.; Lindsley, C. W.
Allosteric modulation of seven transmembrane spanning receptors:
theory, practice, and opportunities for central nervous system drug
discovery. J. Med. Chem. 2012, 55, 1445−1464.
(14) Kenakin, T.; Miller, L. J. Seven transmembrane receptors as
shapeshifting proteins: the impact of allosteric modulation and
K
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
carboxamides as allosteric modulators of the cannabinoid CB(1)
receptor. J. Med. Chem. 2012, 55, 5627−5631.
(33) Mahmoud, M. M.; Ali, H. I.; Ahn, K. H.; Damaraju, A.; Samala,
S.; Pulipati, V. K.; Kolluru, S.; Kendall, D. A.; Lu, D. Structure−activity
relationship study of indole-2-carboxamides identifies a potent
allosteric modulator for the cannabinoid receptor 1 (CB1). J. Med.
Chem. 2013, 56, 7965−7975.
(34) Khurana, L.; Ali, H. I.; Olszewska, T.; Ahn, K. H.; Damaraju, A.;
Kendall, D. A.; Lu, D. Optimization of chemical functionalities of
indole-2-carboxamides to improve allosteric parameters for the
cannabinoid receptor 1 (CB1). J. Med. Chem. 2014, 57, 3040−3052.
(35) Stuart, D. R.; Bertrand-Laperle, M.; Burgess, K. M.; Fagnou, K.
Indole synthesis via rhodium catalyzed oxidative coupling of
acetanilides and internal alkynes. J. Am. Chem. Soc. 2008, 130,
16474−16475.
(36) Perrey, D. A.; German, N. A.; Gilmour, B. P.; Li, J. X.; Harris, D.
L.; Thomas, B. F.; Zhang, Y. Substituted tetrahydroisoquinolines as
selective antagonists for the orexin 1 receptor. J. Med. Chem. 2013, 56,
6901−6916.
(37) Sammakia, T.; Hurley, T. B. Studies on the mechanism of action
of 2-formyl-4-pyrrolidinopyridine: isolation and characterization of a
reactive intermediate. J. Org. Chem. 1999, 64, 4652−4664.
(38) Tsukamoto, I.; Koshio, H.; Kuramochi, T.; Saitoh, C.; Yanai-
Inamura, H.; Kitada-Nozawa, C.; Yamamoto, E.; Yatsu, T.; Shimada,
Y.; Sakamoto, S.; Tsukamoto, S. Synthesis and structure−activity
relationships of amide derivatives of (4,4-difluoro-1,2,3,4-tetrahydro-
5H-1-benzazepin-5-ylidene)acetic acid as selective arginine vasopressin
V2 receptor agonists. Bioorg. Med. Chem. 2009, 17, 3130−3141.
(39) Handy, S. T.; Zhang, Y.; Bregman, H. A modular synthesis of
the lamellarins: total synthesis of lamellarin G trimethyl ether. J. Org.
Chem. 2004, 69, 2362−2366.
(40) Handy, S. T.; Zhang, Y. N. Approaches to the synthesis of the
lamellarins and related natural products. A review. Org. Prep. Proced.
Int. 2005, 37, 411−445.
(41) Zhang, Y.; Gilliam, A.; Maitra, R.; Damaj, M. I.; Tajuba, J. M.;
Seltzman, H. H.; Thomas, B. F. Synthesis and biological evaluation of
bivalent ligands for the cannabinoid 1 receptor. J. Med. Chem. 2010,
53, 7048−7060.
(42) Fulp, A.; Bortoff, K.; Zhang, Y.; Seltzman, H.; Mathews, J.;
Snyder, R.; Fennell, T.; Maitra, R. Diphenyl purine derivatives as
peripherally selective cannabinoid receptor 1 antagonists. J. Med.
Chem. 2012, 55, 10022−10032.
(43) Fulp, A.; Bortoff, K.; Seltzman, H.; Zhang, Y.; Mathews, J.;
Snyder, R.; Fennell, T.; Maitra, R. Design and synthesis of cannabinoid
receptor 1 antagonists for peripheral selectivity. J. Med. Chem. 2012,
55, 2820−2834.
L
dx.doi.org/10.1021/jm501042u | J. Med. Chem. XXXX, XXX, XXX−XXX