Novel Diarylurea Based Allosteric Modulators of the Cannabinoid CB1 Receptor: Evaluation of Importance of 6-Pyrrolidinylpyridinyl Substitution

Article abstract of DOI:10.1021/acs.jmedchem.7b00707

Allosteric modulators of the cannabinoid CB1 receptor have recently been reported as an alternative approach to modulate the CB1 receptor for therapeutic benefits. In this study, we report the design and synthesis of a series of diarylureas derived from PSNCBAM-1 (2). Similar to 2, these diarylureas dose-dependently inhibited CP55,940-induced intracellular calcium mobilization and [³⁵S]GTP-γ-S binding while enhancing [³H]CP55,940 binding to the CB1 receptor. Structure-activity relationship studies revealed that the pyridinyl ring of 2 could be replaced by other aromatic rings and the pyrrolidinyl ring is not required for CB1 allosteric modulation. 34 (RTICBM-74) had similar potencies as 2 in all in vitro assays but showed significantly improved metabolic stability to rat liver microsomes. More importantly, 34 was more effective than 2 in attenuating the reinstatement of extinguished cocaine-seeking behavior in rats, demonstrating the potential of this diarylurea series as promising candidates for the development of relapse treatment of cocaine addiction.

Full text of DOI:10.1021/acs.jmedchem.7b00707

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Article
Novel Diarylurea Based Allosteric Modulators of the Cannabinoid CB1
Receptor: Evaluation of Importance of 6-Pyrrolidinylpyridinyl Substitution
Thuy Nguyen, Nadezhda German, Ann M. Decker, Tifffany L Langston, Thomas F Gamage,
Charlotte E Farquhar, Jun-Xu Li, Jenny L. Wiley, Brian F Thomas, and Yanan Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00707 • Publication Date (Web): 09 Aug 2017
Downloaded from http://pubs.acs.org on August 9, 2017
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Novel Diarylurea Based Allosteric Modulators of the
Cannabinoid CB1 Receptor: Evaluation of Importance
of 6ꢀPyrrolidinylpyridinyl Substitution
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a
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a
Thuy Nguyen, Nadezhda German, Ann M. Decker, Tiffany L. Langston, Thomas F.
a
a
b
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Gamage, Charlotte E. Farquhar, Jun-Xu Li, Jenny L. Wiley, Brian F. Thomas, and Yanan
a,
Zhang *
a
Research Triangle Institute, Research Triangle Park, North Carolina 27709
b
Department of Pharmacology and Toxicology, University at Buffalo, the State University of
New York, Buffalo, NY 14214
KEYWORDS: diarylurea, CB1 receptor, cannabinoid, allosteric modulator, cocaine addiction,
structureꢀactivity relationship.
ABSTRACT: Allosteric modulators of the cannabinoid CB1 receptor have recently been
reported as an alternative approach to modulate the CB1 receptor for therapeutic benefits. In this
study, we report the design and synthesis of a series of diarylureas derived from PSNCBAMꢀ1
(2). Similar to 2, these diarylureas doseꢀdependently inhibited CP55,940ꢀinduced intracellular
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3
calcium mobilization and [ S]GTPꢀγꢀS binding while enhancing [ H]CP55,940 binding to the
CB1 receptor. Structureꢀactivity relationship studies revealed that the pyridinyl ring of 2 could be
replaced by other aromatic rings and the pyrrolidinyl ring is not required for CB1 allosteric
modulation. 34 (RTICBMꢀ74) had similar potencies as 2 in all in vitro assays but showed
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significantly improved metabolic stability to rat liver microsomes. More importantly, 34 was
more effective than 2 in attenuating the reinstatement of extinguished cocaineꢀseeking behavior
in rats, demonstrating the potential of this diarylurea series as promising candidates for the
development of relapse treatment of cocaine addiction.
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Introduction
The cannabinoid CB1 and CB2 receptors are components of the endocannabinoid system
which is involved in many important physiological processes such as appetite control,
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ꢀ3
cardiovascular regulation, learning and memory, pain regulation, and drug dependence.
Expressed abundantly in the central nervous system (CNS), the CB1 receptor has been
demonstrated to be a viable target in a number of disorders including obesity, drug addiction,
pain, inflammation, gastrointestinal diseases, multiple sclerosis, psychosis, schizophrenia, and
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, 4
osteoporosis.
The CB2 receptor is found mainly in immune cells and is responsible for
modulation of cytokine release and immune cell migration. A wide range of selective and nonꢀ
selective agonists and antagonists for CB1 and CB2 receptors have been developed to date.
9
Currently, licensed cannabinoid medications all contain tetrahydrocannabinol (∆ ꢀTHC), the
principal psychoactive constituent of the plant cannabis, or its synthetic analog (nabilone);
however, they are prescribed with many restrictions because of the adverse effects such as
marijuanaꢀlike psychoactivity and addictive tendency. The CB1 selective antagonist/inverse
agonist Rimonabant (SR141716A) was first approved for treatment of obesity, but was
subsequently withdrawn due to a risk of suicidal ideation.
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An alternate approach to target the CB1ꢀmediated signaling pathways is to develop allosteric
modulators that bind to distinct binding sites from the orthosteric site. Compared to orthosteric
ligands, allosteric modulators offer potential benefits, such as better spatial and temporal
selectivity due to their dependence on the presence of an orthosteric agonist for signaling, better
subtype selectivity due to less conserved allosteric binding sites, and improved safety profiles
due to the “ceiling” effect, i.e. their effect is saturable because of their dependence on
endogenous ligands for signaling. Recently, several allosteric modulators have been advanced to
the market as therapeutics, including cinacalcet, a positive allosteric modulator (PAM) of the
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calcium sensing receptor (CasR, a member of the GPCR C family), and maraviroc, a negative
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allosteric modulator (NAM) of the CCR5 receptor, demonstrating that allosteric modulation can
be a safe and therapeutically relevant approach to targeting GPCRs.
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Since discovery of the first CB1 modulator Org27569 (1) in 2005, several NAMs and PAMs
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have been reported. These include CB1 receptor NAMs such as PSNCBAMꢀ1 (2) , fenofibrate
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(3), and CB1 receptor PAMs ZCZ011 (4) and GAT211 (5) (Fig. 1). Compounds 1 and 2 are
two small molecule CB1 NAMs that have been the most extensively characterized. Both were
reported to enhance radioligand binding levels but decrease responses stimulated by orthosteric
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agonists in assays such as intracellular calcium mobilization, [ S]GTPꢀγꢀS binding, cAMP
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accumulation, and βꢀarrestin recruitment. Unlike 1, which displays agonist activities in some
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assays such as ERK phosphorylation,
2 showed little or no activity in the absence of an
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orthosteric agonist in many assays investigated thus far.
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Figure 1. Structures of representive small molecule CB1 allosteric modulators
Compared to the wide range of in vitro studies, the in vivo effects of these CB1 receptor
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NAMs have not been fully characterized. In animal behavioral studies, 1 had no effects on
agonistꢀinduced antinociception and catalepsy, grooming and scratching behaviors or drug
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discrimination.
However, similar to CB1 antagonists/inverse agonists, 1 has been
demonstrated to reduce food intake and to attenuate CP55,940ꢀinduced hypothermia in rats,
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albeit not in mice.
Importantly, 1 also attenuated cueꢀ and drugꢀinduced reinstatement of
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extinguished cocaineꢀ and methamphetamineꢀseeking behavior, effects that have been
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previously observed with CB1 antagonists/inverse agonists, SR141716A and AM251 (46).
On the other hand, reports on the in vivo effects of 2 have been confined to only one study, in
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which it decreased food intake and body weight. To date, 2 has not been investigated in vivo for
the potential use for the treatment of drug addiction or other centrally mediated disorders.
Our group recently reported the first structureꢀactivityꢀrelationship (SAR) study of 2 focusing
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on the 4ꢀchlorophenyl and the alkyl substitution at the 6ꢀaminopyridinyl rings (e.g. 6 and 7).
More recently, a series of analogs of 2 where the pyridinyl group was replaced by a structurally
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similar pyrimidinyl ring, which also bears an ortho nitrogen atom, were reported (e.g. 8 and 9).
In both series, the pyrrolidinyl group was either retained or replaced with substituted amino
groups, which can be considered ringꢀopened pyrrolidines. In order to expand the SAR
knowledge and investigate the importance of the 6ꢀpyrrolidinylpyridinyl group for activity as an
allosteric modulator, we designed and synthesized a series of analogs where the pyrrolidinyl
group of 2 was replaced or completely removed, and/or the pyridinyl ring was replaced with
substituted phenyl or other aromatic moieties (Fig. 2). The synthesized diarylureas were
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characterized in calcium mobilization and [ S]GTPꢀγꢀS binding assays for their potency as CB1
allosteric modulators. Select compounds were then assessed for metabolic stability in liver
microsomes. Finally, we examined the effects of 2 and one of the diarylurea analogs (34) in a rat
model of drugꢀinduced reinstatement of extinguished cocaineꢀseeking behavior.
Replacement or removal of
the pyrrolidinyl ring
Cl
O
N
N
N
N
H
H
Replacement of the pyridinyl ring
with phenyl or other aromatic groups
Figure 2. Structural modifications on compound 2 conducted in this study.
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Results and Discussion
Chemistry. Compounds 15 ꢀ 45 were synthesized as depicted in Scheme 1 following
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2
procedures described by us previously. Briefly, commercially available 3ꢀnitrophenyl boronic
acid (10) underwent Suzuki coupling with the corresponding aryl bromides under basic
conditions catalyzed by Pd(PPh ) to afford intermediates 11a–y. Reduction of the nitro group in
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11aꢀy by transfer hydrogenation with hydrazine hydrate and Raney nickel in ethanol provided 3ꢀ
substituted anilines 12aꢀy in good yields. Subsequent reaction between these anilines with 4ꢀ
chlorophenyl isocyanate afforded the final diarylureas. Alternatively, the anilines 14aꢀe could be
prepared by Suzuki coupling between 3ꢀbromoaniline (13) and corresponding aryl boronic acids,
and were then converted to the final products via reaction with 4ꢀchlorophenyl isocyanate.
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Scheme 1. Reagents and conditions: (a) corresponding aryl bromide, Pd(PPh ) , DME, aq.
3
4
o
NaHCO , reflux, 16 h (b) hydrazine hydrate, Raney Ni, ethanol, 50 C, 2 h (c) 4ꢀchlorophenyl
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isocyanate, chloroform, 50 C, 16 h (d) corresponding aryl boronic acid, Pd(PPh ) , DME, aq.
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NaHCO , reflux, 16 h.
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Biological evaluations in calcium mobilization assays. The FLIPRꢀbased calcium
mobilization assays were used as the primary screen to assess the potency of the synthesized
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diarylureas at CB1 and CB2 receptors as described previously.
In these assays, CHO cells
that overexpress the promiscuous Gα16 protein (RDꢀHGA16 cells, Molecular Devices) were
engineered to stably express the human CB1 or CB2 receptor. Compounds were evaluated for
their ability to inhibit the mobilization of intracellular calcium levels stimulated by CP55,940.
Table 1 shows IC values of the synthesized compounds against the EC concentration of
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80
CP55,940 (100 nM).
Table 1. Allosteric modulatory activities of diarylureas 2, and 15 ꢀ 45 in the CB1 calcium
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mobilization assays and [ S]GTPꢀγꢀS binding assay.
Calcium Mobilization Assay
3
5
CB1
Agonist screen
CB2
CB1 [ S]GTPꢀγꢀ
Compound
Ar
d
b
S, IC (nM)
IC50a
nM)
c
50
IC50 (nM)
(
(% CP55,940 Emax
)
2
33 ± 8
28 ± 5
> 10,000
N.D.
89 (71 – 112)
N.D.
15
231 ± 14
2 ± 0
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± 0
N.D.
N.D.
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1,320 (871 –
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33 ± 6
7 ± 13
23 ± 3
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N.D.
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2,650 (1,590 –
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44 ± 45
78 ± 38
34 ± 16
15 ± 4
N.D.
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405 (295 – 556)
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290 ± 24
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3,400 ± 700
N.D.
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3
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10 ± 7
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N.D.
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N.D.
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827 (590 –
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81 ± 10
1
,160)
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40 ± 130
N.D.
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41 ± 24
N.D.
70 ± 14
619 (497 – 772)
459 (310 – 671)
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7
4 ± 6
28
1
90 ± 45
27 ± 2
6 ± 5
> 10,000
N.D.
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9
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17 ± 6
21 ± 6
> 10,000
N.D.
452 (321 – 635)
N.D.
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16 ± 4
20 ± 3
17 ± 6
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N.D.
> 10,000
N.D.
299 (151 – 592)
464 (259 – 830)
N.D.
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38 ± 19
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2
3 ± 2
> 10,000
N.D.
153 (73 – 318)
353 (236 – 528)
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18 ± 1
9 ± 3
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91 ± 48
60 ± 39
4 ± 2
N.D.
10 ± 4
N.D.
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9
195 ± 40
1,340 ±
1 ± 0
N.D.
N.D.
559 (383 – 817)
> 10,000
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0
370
40
1,570 ±
13 ± 3
N.D.
N.D.
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1
2
134 ± 13
1 ± 1
7 ± 1
5 ± 1
N.D.
N.D.
N.D.
265 (216 – 326)
1,520 (1,120 –
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1
3
32 ± 28
34 ± 80
2
,070)
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1,890 (1,360 –
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4,100 ±
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16 ± 70
3 ± 2
0
651 (476 – 891)
N.D.
1,260 ±
160
N.D.
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a
Against EC (100 nM) of CP55,940 in stable human CB1ꢀCHOꢀRDꢀHGA16 cells. Values
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b
are the mean ± S.E.M. of at least three independent experiments in duplicate. Agonist screens
were conducted at 10 ꢁM final concentration in stable human CB1ꢀCHOꢀRDꢀHGA16 cells and
c
values are the mean ± SD of two independent experiments in duplicate. Against EC (90 nM)
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0
of CP55,940 in stable human CB2ꢀCHOꢀRDꢀHGA16 cells. IC50 values were determined for
compounds that had >50% inhibition in antagonist screens conducted at 10 ꢁM final
d
concentration. Values are the mean ± SD of two independent experiments in duplicate. Values
are expressed as mean (95% confidence interval) from at least three independent experiments in
duplicate. N.D.: Not determined.
2
2, 23
In previous studies conducted by our group and others,
the 6ꢀpyrrolidinyl group on the
pyridine ring of 2 was either retained or replaced with substituted amino groups such as
dimethylamino (6), which can be seen as the ring opened analogs of the pyrrolidinyl ring. To
explore the importance of the pyrrolidinyl substituent on the allosteric modulating activity, we
first replaced the pyrrolidinyl ring with another hydrogen bond acceptor, a methoxy group. This
alteration dampened the activity around 8ꢀfold (15, IC50 = 231 nM). The corresponding 4ꢀ
pyridinyl analog with a methoxy at the same position (17, IC = 133 nM), was slightly more
5
0
potent than its 2ꢀpyridinyl analog (15), but less potent than 2. These results suggest that a
hydrogen bond acceptor or electron donating group at this position is preferred. However,
interestingly, when the pyrrolidinyl group was completely removed, the resulting analog showed
similar potency as 2 (18, IC = 47 nM vs. 2, IC = 33 nM), implying that the pyrrolidinyl
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moiety is not required for the activity. Replacing the 2ꢀpyridinyl ring by its regioisomers, 3ꢀ or 4ꢀ
pyridinyl analogs (19 and 20), or a pyrimidinyl ring (21) resulted in slightly less potent analogs,
all of which lack the pyrrolidinyl group. Activity as an allosteric modulator was significantly
diminished with the 3ꢀmethyl substitution (16, IC = 1310 nM). Together, these results suggest
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that the pyrrolidinyl group is not required for CB1 allosteric modulator activity.
Analogs of 2 reported thus far have either a pyridine or pyrimidine group, each of which has
a nitrogen atom at the ortho position. We examined the importance of the nitrogen atom and
whether the pyridinyl group can be replaced with other aromatic rings. Interestingly, substituting
the pyridinyl ring with a phenyl ring led to 22 (IC50 = 32 nM), which had nearly identical
potency to 2 (IC50 = 33 nM), suggesting that the pyridinyl group can be replaced without
compromising activity. Thus, substituents of diverse electronic and steric properties were
introduced onto the phenyl group to probe the substitution effect. The 3ꢀmethoxyphenyl (23) had
an IC of 81 nM, which was more potent than the corresponding pyridinyl analog (15, IC =
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231 nM), and only slightly less potent than 22, confirming the earlier finding that the
pyrrolidinyl group was not required for activity. Several electronꢀdonating groups such as
methyl, hydroxyl, methoxy, and isopropyloxy were incorporated in the phenyl ring at the 3
position, the same meta position as the pyrrolidinylpyridinyl group on 2. All of these analogs
(23 – 27) had lower potency than 2. The 1,3ꢀbenzodioxolꢀ5ꢀyl analog (26) was slightly weaker.
The methyl group at the 2 position (28, IC = 190 nM) had around a 3ꢀfold decrease in potency
5
0
compared to its 3ꢀposition isomer (27, IC = 74 nM).
5
0
We next investigated substitutions on the phenyl ring with a series of electronꢀwithdrawing
groups. A 3ꢀnitro group (29, IC = 27 nM) showed similar potency as 2 and the phenyl analog
5
0
22. While a weak electron withdrawing group like chloro at the 4ꢀposition (30) reduced potency
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around ~3ꢀfold compared to 2 and 22, inclusion of a chloro at the 3ꢀposition as in 3,5ꢀdichloro
(
31, IC = 23 nM) and 3,4ꢀdichloro (32, IC = 30 nM) resulted in comparable potency. The 2,6ꢀ
50 50
dichloro analog (33, IC = 338 nM) was more than 10ꢀfold weaker than its 3,5ꢀdichloro or 3,4ꢀ
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dichloro analogs (31 and 32), similar to the methyl substituent which also favored the 3ꢀposition
compared to the 2ꢀposition as described earlier. The significant drop in potency of the 2,6ꢀ
dichloro analog 33 could be due to the steric hindrance at the 2ꢀortho chloro groups that forces
the biꢀaryl system to a more nonꢀplanar spatial arrangement. The fluoro substituent, however, did
not follow the same trend as the chloro series. Both the 4ꢀfluoro analog, 3‐(4‐Chlorophenyl)‐1‐[3‐
(
4ꢀfluorophenyl)phenyl]urea (RTICBMꢀ74, 34, IC = 23 nM), and the 2,4ꢀdifluoro analog (35,
50
IC = 18 nM) displayed excellent activities. This could be attributed to the unique effect of the
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2
5, 26
highly electronegative fluorine atom which has a size as small as that of a hydrogen.
While these results suggest that electron withdrawing groups are generally preferred over
elctron donating groups for activity, the electronic property is not the only factor in determining
the potency of these compounds at the CB1 receptor. This is clearly demonstrated by the
relatively small differences in potency between the 3ꢀposition (or metaꢀ) substituted phenyl
analogs 23, 27 and 29, which have various electron densities on the phenyl group. Rather, the
substituents such as the pyrrolidinyl group in 2 at the same meta position may simply occupy a
binding pocket at this position. Given that its removal does not affect the potency, the
interactions between the pyrrolidinyl group and binding pocket of the receptor are not essential
for activity. Therefore, we briefly probed the steric requirements in the phenyl series.
Substituents of large size such as tertꢀbutyl, phenyl, and benzoyl seemed to be detrimental to the
activity. Both the 4ꢀtertꢀbutyl (36) and 3,5ꢀdiꢀtertꢀbutyl (37) substitutions showed a significant
decrease in IC values. Both 3ꢀphenyl (38) and 4ꢀphenyl (39) analogs were less potent than 2.
5
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However, 38 (IC50 = 195 nM) was significantly more potent than 39 (IC50 = 1340 nM),
consistent with the earlier observations that the metaꢀposition of the phenyl or pyridinyl ring has
better tolerance for substutition than the ortho or paraꢀpositions (27 vs. 28, 31 vs. 30). Similarly,
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4
ꢀbenzoyl analog (40) also possessed poor activity. Finally, several bicyclic and tricyclic
aromatic rings (41 – 45) were explored. In concordance with the observation that there is a
limited space at this binding pocket, the larger aromatic ring systems were not well tolerated at
2
2
this region.
All of these compounds were screened in the calcium mobilization assay for agonist activity
at the CB1 receptor; no significant agonist effects (< 30% of CP55,940 Emax) were observed for
any of the compounds. All of these compounds were also screened for agonist and antagonist
activity at the CB2 receptor to determine receptor subtype selectivity. None of the compounds
had significant CB2 agonist activity (< 10% of CP55,940 Emax). All compounds had no
significant CB2 antagonist activities (< 50% inhibition of CP55,940 EC concentration at 10
80
µM or IC values > 10 ꢁM) except compounds 21 (CB2 IC = 3.4 ꢁM ) and 44 (CB2 IC = 4.1
5
0
50
50
ꢁ
M).
Effects of diarylureas in the [ S]GTPꢀγꢀS binding assay. A number of representative
3
5
35
compounds were then further evaluated in antagonizing agonistꢀstimulated [ S]GTPꢀγꢀS binding
to CB1 receptor in mouse cerebellar membranes. The CB1 receptor agonist CP55,940 was
employed as the agonist probe. Table 1 lists the potencies (IC ’s) of these diarylureas
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35
determined against 100 nM of CB1 agonist CP55,940 in the [ S]GTPꢀγꢀS binding assay (Fig. 3).
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Figure 3. Curve fits for inhibition of CP55,940 (100 nM) stimulated [ S]GTPꢀγꢀS binding
by compounds 2, 20, and 34 used to calculate IC values in Table 1. Data are expressed as
50
35
percent of CP55,940 (100 nM) stimulated [ S]GTPꢀγꢀS binding. Symbols represent mean values
± S.E.M. from three to six independent experiments carried out in duplicate.
35
In general, the activities of the synthesized compounds in the [ S]GTPꢀγꢀS binding assay
were several fold lower than in the calcium mobilization assay but follow the same trend, with
3
5
some variations in the ranking order. In the [ S]GTPꢀγꢀS binding assay using mouse cerebellar
membranes, 2 had an IC value of 89 nM against 100 nM CP55,940. It is comparable to the
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reported values of 42ꢀ74 nM for 2 in rat cerebellar membrane and HEK293 membrane against
9, 27
50ꢀ200 nM CP55,490.
While the 2ꢀpyridinyl (18) analog was more potent than its 4ꢀpyridinyl
35
isomer (20) in the calcium mobilization assay, the latter was more active in the [ S]GTPꢀγꢀS
binding assay (18, IC = 2,647 nM vs. 20, IC = 405 nM). Similarly, 3ꢀmethoxyꢀpyridinꢀ4ꢀyl
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50
analog (17) had comparable potency in the calcium mobilization assay but weaker activity in the
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[
S]GTPꢀγꢀS binding assay compared to the unsubtituted pyridineꢀ4ꢀyl analog (20) (17, IC =
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,320 nM vs. 20, IC = 405 nM). Within the substituted phenyl series, the 4ꢀfluorophenyl was
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the most potent analog (34, IC = 153 nM) demonstrating good potencies in both in vitro assays,
50
comparable to those of compound 2. Other phenyl analogs (22, 23, 26, 27, 29, 31, 32, 35) with
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good activities (IC values < 100 nM) in the calcium mobilization assay had potencies in the
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[ S]GTPꢀγꢀS binding assay with IC values around 300 ꢀ 800 nM. The 3ꢀbiphenyl analog (38)
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had weaker activity in the calcium mobilization assay yet displayed comparable potency in the
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[
S]GTPꢀγꢀS binding assay to other substituted phenyl analogs, whereas 4ꢀbiphenyl (39) showed
weak to no activity in both assays. Among the bicylic aromatic rings, the benzofuranꢀ5ꢀyl (41)
and quinolineꢀ3ꢀyl (44) had lower potencies in the calcium mobilization assay but exhibited
3
5
comparable potencies in the [ S]GTPꢀγꢀS binding assay to the previous phenyl analogs. On the
other hand, naphthalenꢀ2ꢀyl (42) and quinolineꢀ3ꢀyl (43) were weaker modulators in both in vitro
assays.
Characterization of allosterism. CB1 NAMs reported so far such as 2 have been
characterized as PAMꢀantagonists; i.e., increasing agonist binding but decreasing Gꢀprotein
8
9, 13, 27
functions. Consistent with previous reports,
2 doseꢀdependently decreased the Emax value
35
of [ S]GTPꢀγꢀS binding levels stimulated by CP55,940 as expected for CB1 NAMs (Fig. 4A).
Similarly, 34 showed a concentrationꢀrelated Emax reduction, confirming its allosteric mechanism
(Fig. 4B). In the calcium mobilization assay, the same effect was also observed for 34 (Fig. 4C),
2
2
similarly to 2 as previously reported by our group.
Compound 2 has been reported to demonstrate positive binding cooperativity with the CB1
7
, 9, 22
agonist CP55,940.
To determine whether 34 retains these characteristics, we conducted
3
equilibrium binding with 1 nM [ H]CP55,940 (Fig. 4D). In contrast to unlabeled CP55,940
which inhibited specific binding of the radioligand, 34 increased specific binding to 214.7 ±
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.39% (199.8 – 229.7) with an EC value of 790 nM (467 – 1,336). These results demonstrate
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positive binding cooperativity for 34 with CP55,940, consistent with the PAMꢀantagonist
mechanism.
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A
B
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D
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Figure 4. Insurmountable antagonism of CP55,940ꢀstimulated [ S]GTPꢀγꢀS binding by
diarylureas (A) compound 2 and (B) compound 34, (C) insurmountable antagonism of
CP55,940ꢀstimulated calcium mobilization by compound 34, and (D) positive binding
3
cooperativity with 1 nM [ H]CP55,940 and 34 in mouse cerebellar membranes. Data are
35
expressed as (A, B) percent stimulation of [ S]GTPꢀγꢀS binding over basal, (C) percent maximal
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CP55,940 response, or (D) percent [ H]CP55,940 bound. Symbols represent mean values ±
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+
S.E.M. from three to six independent experiments carried out in duplicate (Ca ) or triplicate
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(
[ S]GTPꢀγꢀS).
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Metabolic stability of select diarylureas. We have demonstrated that the pyrrolidinyl
moiety on the pyridine is not required for allosteric modulatory activity at the CB1 receptor.
Removal of the pyrrolidinyl moiety could result in smaller and more lipophilic molecules that
have a better chance to cross the bloodꢀbrain barrier. Moreover, the pyridine ring could be
replaced by other aromatic rings with similar size, such as substituted phenyl groups, without
losing its potency and efficacy. Moreover, the substituents of various nature including size and
electronic properties can be tolerated, thus allowing for optimization of other properties, such as
pharmacokinetics, with appropriate structural alterations. A drug must have sufficient stability to
reach its molecular targets to elicit its effects; therefore, the metabolic stability of select
compounds were evaluated in rat liver microsomes.
Table 2. Stability of select diarylureas in rat liver microsomes
a
a
Compound
Halfꢀlife (min) Clearance (µl/min/mg)
13.4 ± 4.1
113.7 ± 34.4
33.1 ± 1.0
< 4.6
2
41.9 ± 1.3
2
3
0
4
> 300
b
Values are expressed as mean ± SD from two independent experiments.
Table 2 lists the halfꢀlives (T ) and clearance (CL) of compounds 2, 20, and 34. Compound
1/2
2
was rapidly metabolized with T1/2 = 13.4 min, suggesting modest stability in first pass
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metabolism. When the pyrrolidinyl ring was absent, the metabolic stability increased by ~3ꢀfold
(20, T1/2 = 41.9 min). Finally, replacement of the pyridinyl ring with a 4ꢀfluorophenyl group
significantly improved the metabolic stability. 34 had a halfꢀlife of more than 300 min and
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clearance of less than 4.6 µl/min/mg.
Compounds 2 and 34 attenuated reinstatement of cocaineꢀseeking behavior. Blockade of
the CB1 receptor in vivo with the antagonists/inverse agonists SR141716A and 46 has been
demonstrated to reduce intake of palatable food, selfꢀadministration of several drugs of abuse,
1
8, 19, 28
and reinstatement of food and drugꢀseeking behaviors.
We previously demonstrated that
rats pretreated with 1, the first reported CB1 NAM, were less likely to seek drugs of abuse such
as cocaine or methamphetamine after a period of extinction. Therefore, we examined whether
compound 2 and one of the active diarylureas, compound 34, would achieve the same effects in
vivo.
1
50
00
Vehicle
1
50
15 mg/kg compound 2
30 mg/kg compound 2
Vehicle
0 mg/kg compound 34
1
1
100
50
0
5
0
0
Extinction Reinstatement
Extinction
Reinstatement
Figure 5. Compounds 2 (left) and 34 (right) attenuated the resumption of extinguished cocaineꢀ
seeking behavior of active lever responding as compared with saline treatment in rats.
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As shown in Fig. 5, a cocaine prime significantly reinstated the extinguished active lever
response (t test: t[7] = 16.29, p < 0.0001). Pretreatment with 2 attenuated cocaineꢀinduced
reinstatement of cocaineꢀseeking behavior (one way ANOVA: F[2, 22] = 5.174, p < 0.05). Post
hoc analysis revealed that at the dose of 30 mg/kg, 2 significantly reduced cocaineꢀinduced
reinstatement behavior. Excitingly, 34 at 10 mg/kg produced the same degree of attenuation as
that of 30 mg/kg of 2 (t test: t[20] = 1.84, p < 0.05). While 2 and 34 had equivalent in vitro IC50
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values in both calcium mobilization and [ S]GTPꢀγꢀS binding assays, 34 was effective at a
lower dose than 2 in attenuating cocaineꢀseeking behavior. The difference in in vivo potency is
likely due to the improved metabolic stability of 34 producing higher concentrations at sites of
action within the CNS.
Conclusions
The endocannabinoid system, particularly the CB1 recpeotor, has shown considerable
potential as a therapeutic target in a number of neuropsychiatric conditions such as mood
29
disorders and drug addiction. Although CB1 antagonists/inverse agonists have shown potential
therapeutic effects in these conditions, their activity also produces untoward effects limiting their
development as drug candidates. The present study describes a series of novel diarylureas
derived from 2, a CB1 NAM without known agonist activities, as an alternate approach to
manipulate this important signaling pathway and to potentially circumvent adverse side effects of
orthosteric ligands.
Optimization of 2 resulted in a series of diarylureas with comparable or even greater potency
3
5
as determined in calcium mobilization and [ S]GTPꢀγꢀS binding assays. SAR studies of this
series of diarylureas indicated that the pyrrolidinyl ring of 2 was not necessary for the allosteric
modulation of the CB1 receptor and that the pyridinyl ring of 2 could be replaced by other
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aromatic rings. Introducing a phenyl ring in place of the pyridinyl ring led to 34 with similar in
vitro potency but with significantly improved microsomal stability. We have demonstrated for
the first time that these diarylureas were effective in attenuating reinstatement of cocaine taking
after a period of extinction. In this model, the optimized analog 34 appeared more effective than
the lead compound 2, which may be partially ascribed to its improved metabolic stability.
Although the in vivo effects of these diarylureas in attenuating cocaine reinstatement could not
be exclusively attributed to the CB1 signaling pathway at present, the involvement of the CB1
receptor is a plausible mode of action given that similar effects were observed for the CB1
0
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21
antagonist/inverse agonists SR141716A and 46, and the structurally distinct CB1 NAM 1,
which has been reported to possess high selectivities against more than 40 GPCRs, including
1
7
many that have been indicated in drug addiction.
Currently, there are no FDA approved medications for the treatment of cocaine addiction, the
30
relapse rate of which remains as high as 40 to 60%. With the in vivo efficacy to attenuate
reinstatement of cocaine craving, these CB1 allosteric modulators may represent promising
candidates for the development of therapeutics for relapse prevention of cocaine addiction and
abuse, an urgent need that is currently unfilled.
EXPERIMENTAL SECTION
Chemistry. All solvents and chemicals were reagent grade. Unless otherwise mentioned, all
reagents and solvents were purchased from commercial vendors and used as received. Flash
column chromatography was carried out on a Teledyne ISCO CombiFlash Rf system using
prepacked columns. Solvents used include hexane, ethyl acetate (EtOAc), dichloromethane,
methanol, and chloroform/methanol/ammonium hydroxide (80:18:2) (CMAꢀ80). Purity and
characterization of compounds were established by a combination of HPLC, TLC, mass
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spectrometry, and NMR analyses. Melting point was recorded by the MelꢀTemp II instrument
1
13
(
Laboratory Devices Inc. , U.S.). H and C NMR spectra were recorded on a Bruker Avance
DPXꢀ300 (300 MHz) spectrometer and were determined in chloroformꢀd, DMSOꢀd6, or
methanolꢀd4 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
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(ESI). All test 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).
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 NaHCO solution (3 eq) followed by
3
Pd(Ph ) (0.075 eq). The reaction mixture was refluxed overnight under nitrogen atmosphere.
3
4
The reaction mixture was diluted with ethyl acetate, washed with a saturated NaHCO solution
3
and brine. The combined organic layers were dried over anhydrous MgSO and filtered. The
4
filtrate was concentrated in vacuo and the residue was purified by column chromatography
(
SiO , ethyl acetate/hexanes) to give the desired product.
2
2
‐Methoxy‐6‐(3‐nitrophenyl)pyridine
(11a)
was
prepared
from
2‐bromo‐6‐
methoxypyridine (0.12 ml, 1.00 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol)
1
following the general procedure A as white solid (0.12 g, 50%). H NMR (300 MHz, CDCl ) δ
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2
2
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3
3
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.89 (t, J = 1.98 Hz, 1H), 8.01 (dd, J = 0.57, 7.35 Hz, 1H), 7.64 ꢀ 7.74 (m, 2H), 7.41 (d, J = 7.35
+
Hz, 1H), 6.72 ꢀ 6.80 (m, 2H), 4.04 (s, 3H). MS (ESI) m/z for C H N O [M+H] : calcd: 231.1;
12 10
2
3
found: 231.4.
‐Methyl‐2‐(3‐nitrophenyl)pyridine (11b) was prepared from 2‐bromo‐4‐methylpyridine
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
(0.11 ml, 1.00 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol) following the general
1
procedure A as white solid (0.16 g, 73%). H NMR (300 MHz, CDCl ) δ 8.84 (t, J = 1.98 Hz,
3
1
7
2
H), 8.58 (d, J = 4.90 Hz, 1H), 8.34 ꢀ 8.39 (m, 1H), 8.23 ꢀ 8.27 (m, 1H), 7.60 ꢀ 7.67 (m, 2H),
+
.15 (dd, J = 0.66, 4.99 Hz, 1H), 2.46 (s, 3H). MS (ESI) m/z for C H N O [M+H] : calcd:
1
2
10
2
2
14.1; found: 214.4.
2‐Methoxy‐4‐(3‐nitrophenyl)pyridine (11c) was prepared from 2ꢀmethoxy‐5‐
bromopyridine (0.21 ml, 1.60 mmol) and 3ꢀnitrophenylboronic acid (0.29 g, 1.76 mmol)
1
following the general procedure A as white solid (0.34 g, 85%). H NMR (300 MHz, CDCl ) δ
3
8.36 ꢀ 8.46 (m, 2H), 8.17 ꢀ 8.25 (m, 1H), 7.79 ꢀ 7.88 (m, 2H), 7.58 ꢀ 7.66 (m, 1H), 6.88 (d, J =
+
8.48 Hz, 1H), 4.00 (s, 3H). MS (ESI) m/z for C H N O [M+H] : calcd: 231.1; found: 231.5.
1
2
10
2
3
2‐(3‐Nitrophenyl)pyridine (11d) was prepared from 2ꢀbromopyridine (1.00 g, 6.33 mmol)
and 3ꢀnitrophenylboronic acid (1.16 g, 6.96 mmol) following the general procedure A as orange
1
solid (0.85 g, 67%). H NMR (300 MHz, CDCl ) δ 8.86 (t, J = 1.88 Hz, 1H), 8.75 (td, J = 1.20,
3
4
.94 Hz, 1H), 8.37 (td, J = 1.39, 7.77 Hz, 1H), 8.27 (ddd, J = 0.94, 2.21, 8.15 Hz, 1H), 7.85 (d, J
=
1.70 Hz, 2H), 7.66 (t, J = 8.01 Hz, 10H), 7.62 ꢀ 7.70 (m, 1H), 7.34 (ddd, J = 2.45, 4.76, 6.17
+
Hz, 1H). MS (ESI) m/z for C H N O [M+H] : calcd: 201.1; found: 201.4.
1
1
8
2
2
4
‐(3‐Nitrophenyl)pyridine (11e) was prepared from 4ꢀbromopyridine hydrochloride (0.19 g,
1
.0 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.1 mmol) following the general procedure A
1
as brown solid (0.13 g, 67%). H NMR (300 MHz, CDCl ) δ 8.73 ꢀ 8.78 (m, 1H), 7.62 ꢀ 7.74 (m,
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+
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H), 7.51 ꢀ 7.59 (m, 2H), 7.43 ꢀ 7.50 (m, 2H). MS (ESI) m/z for C H N O [M+H] : calcd:
11 8 2 2
2
01.1; found: 201.4.
‐(3‐Nitrophenyl)pyrimidine (11f) was prepared from 5ꢀbromopyrimidine (0.16 g, 1.0
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.1 mmol) following the general procedure A as
1
light yellow solid (0.10 g, 52%). H NMR (300 MHz, CDCl ) δ 9.29 ꢀ 9.32 (m, 1H), 9.03 (s, 2H),
3
8.48 (t, J = 1.98 Hz, 1H), 8.33 ꢀ 8.38 (m, 1H), 7.94 (qd, J = 0.93, 7.75 Hz, 1H), 7.71 ꢀ 7.79 (m,
+
1
H). MS (ESI) m/z for C H N O [M+H] : calcd: 202.1; found: 202.4.
10
7
3
2
1
‐(3‐Methoxyphenyl)‐3‐nitrobenzene (11g) was prepared from 2ꢀbromoꢀ3ꢀmethoxybenzene
(0.30 g, 1.6 mmol) and 3ꢀnitrophenylboronic acid (0.29 g, 1.8 mmol) following the general
1
procedure A as white solid (0.24 g, 65%). H NMR (300 MHz, CDCl ) δ 8.33 ꢀ 8.40 (m, 1H),
3
8
.13 (td, J = 1.06, 8.24 Hz, 1H), 7.80 ꢀ 7.88 (m, 1H), 7.54 (t, J = 8.01 Hz, 1H), 7.29 ꢀ 7.41 (m,
1H), 7.13 (dd, J = 0.85, 7.63 Hz, 1H), 7.04 ꢀ 7.09 (m, 1H), 6.86 ꢀ 6.94 (m, 1H), 3.82 (s, 3H).
‐(3‐Hydroxyphenyl)‐3‐nitrobenzene (11h) was prepared from 2ꢀbromoꢀ3ꢀhydroxybenzene
1
(0.30 g, 1.7 mmol) and 3ꢀnitrophenylboronic acid (0.32 g, 1.9 mmol) following the general
1
procedure A as white solid (0.24 g, 64%). H NMR (300 MHz, CDCl ) δ 8.44 (d, J = 1.70 Hz,
3
1
H), 8.16 ꢀ 8.25 (m, 1H), 7.85 ꢀ 7.95 (m, 1H), 7.56 ꢀ 7.65 (m, 1H), 7.33 ꢀ 7.40 (m, 1H), 7.20 (br.
s., 1H), 7.13 (br. s., 1H), 6.88 ꢀ 7.00 (m, 1H), 5.42 (br. s., 1H).
‐(3‐Nitrophenyl)‐3‐(propan‐2‐yloxy)benzene (11i) was prepared from 1‐bromo‐3‐
1
(propan‐2‐yloxy)benzene (0.30 g, 1.7 mmol) and 3ꢀnitrophenylboronic acid (0.28 g, 1.7 mmol)
1
following the general procedure A as yellow liquid (0.30 g, 84%). H NMR (300 MHz, CDCl ) δ
3
8
.44 (t, J = 1.98 Hz, 1H), 8.15 ꢀ 8.24 (m, 1H), 7.90 (td, J = 1.25, 7.86 Hz, 1H), 7.54 ꢀ 7.65 (m,
1H), 7.33 ꢀ 7.44 (m, 1H), 7.10 ꢀ 7.20 (m, 2H), 6.95 (dd, J = 1.88, 8.10 Hz, 1H), 4.64 (spt, J =
.06 Hz, 1H), 1.34 ꢀ 1.43 (m, 6H).
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‐(4‐Chlorophenyl)‐3‐nitrobenzene (11j) was prepared from 1‐iodo‐4‐chlorobenzene (0.30
g, 1.25 mmol) and 3ꢀnitrophenylboronic acid (0.23 g, 1.38 mmol) following the general
1
procedure A as white solid (0.24 g, 82%). H NMR (300 MHz, CDCl ) δ 8.37 ꢀ 8.43 (m, 1H),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
.21 (ddd, J = 1.04, 2.26, 8.19 Hz, 1H), 7.88 (ddd, J = 1.04, 1.74, 7.77 Hz, 1H), 7.62 (t, J = 8.01
Hz, 1H), 7.51 ꢀ 7.58 (m, 2H), 7.41 ꢀ 7.50 (m, 2H).
,3‐Dichloro‐4‐(3‐nitrophenyl)benzene (11k) was prepared from 1‐bromo‐3,5‐
1
dichlorobenzene (0.30 g, 1.32 mmol) and 3ꢀnitrophenylboronic acid (0.24 g, 1.46 mmol)
1
following the general procedure A as white solid (0.29 g, 82%). H NMR (300 MHz, CDCl ) δ
3
8
.41 (t, J = 1.79 Hz, 1H), 8.27 (dd, J = 1.22, 8.19 Hz, 1H), 7.87 (d, J = 7.72 Hz, 1H), 7.62 ꢀ 7.70
m, 1H), 7.50 (d, J = 1.70 Hz, 2H), 7.43 (t, J = 1.70 Hz, 1H).
,2ꢀDichloro‐4‐(3‐nitrophenyl)benzene (11l) was prepared
(
1
from 1‐iodo‐3,4‐
dichlorobenzene (0.30 g, 1.10 mmol) and 3ꢀnitrophenylboronic acid (0.20 g, 1.21 mmol)
1
following the general procedure A as white solid (0.19 g, 65%). H NMR (300 MHz, CDCl ) δ
3
8.40 (t, J = 1.98 Hz, 1H), 8.21 ꢀ 8.27 (m, 1H), 7.87 (td, J = 1.25, 7.68 Hz, 1H), 7.71 (d, J = 2.07
Hz, 1H), 7.64 (t, J = 8.01 Hz, 1H), 7.55 ꢀ 7.59 (m, 1H), 7.46 (dd, J = 2.17, 8.38 Hz, 1H).
1
,3‐Dichloro‐2‐(3‐nitrophenyl)benzene (11m) was prepared from 2‐bromo‐1,3‐
dichlorobenzene (0.30 g, 1.32 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol)
1
following the general procedure A as yellow solid (0.23 g, 65%). H NMR (300 MHz, CDCl ) δ
3
8.26 ꢀ 8.34 (m, 1H), 8.18 (d, J = 1.70 Hz, 1H), 7.59 ꢀ 7.68 (m, 2H), 7.41 ꢀ 7.49 (m, 2H), 7.29 ꢀ
7
.35 (m, 1H).
1
‐ (4‐ Fluorophenyl)‐ 3‐ nitrobenzene (11n) was prepared from 1‐bromo‐4‐fluorobenzene
(0.30 g, 1.71 mmol) and 3ꢀnitrophenylboronic acid (0.31 g, 1.88 mmol) following the general
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procedure A as white solid (0.35 g, 93%). H NMR (300 MHz, CDCl ) δ 8.39 (s, 1H), 8.19 (d, J
3
= 7.54 Hz, 1H), 7.86 (d, J = 7.72 Hz, 1H), 7.57 ꢀ 7.62 (m, 2H), 7.18 (t, J = 8.57 Hz, 2H).
1
‐ (2,4‐ Difluorophenyl)‐ 3‐ nitrobenzene (11o) was prepared from 1‐bromo‐2,4‐
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
difluorobenzene (0.19 g, 1.00 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol)
1
following the general procedure A as white solid (0.17 g, 71%). H NMR (300 MHz, CDCl ) δ
3
8
.37 (s, 1H), 8.23 (td, J = 1.08, 8.19 Hz, 1H), 7.85 (dd, J = 1.13, 7.72 Hz, 1H), 7.59 ꢀ 7.67 (m,
1H), 7.46 (dt, J = 6.31, 8.62 Hz, 1H), 6.92 ꢀ 7.06 (m, 2H).
‐(4‐tertꢀButylphenyl)‐3‐nitrobenzene (11p) was prepared from 1‐bromo‐4‐tertꢀ
1
butylbenzene (0.30 g, 1.41 mmol) and 3ꢀnitrophenylboronic acid (0.26 g, 1.55 mmol) following
1
the general procedure A as colorless liquid (0.15 g, 42%). H NMR (300 MHz, CDCl ) δ 8.43 (t,
3
J = 1.88 Hz, 1H), 8.12 ꢀ 8.18 (m, 1H), 7.87 ꢀ 7.92 (m, 1H), 7.48 ꢀ 7.60 (m, 5H), 1.37 (s, 9H).
1,3‐Di‐tert‐butyl‐5‐(3‐nitrophenyl)benzene (11q) was prepared from 1‐bromo‐3,5‐di‐tert‐
butylbenzene (0.30 g, 1.1 mmol) and 3ꢀnitrophenylboronic acid (0.20 g, 1.2 mmol) following
1
the general procedure A as colorless liquid (0.22 g, 63%). H NMR (300 MHz, CDCl ) δ 8.41 ꢀ
3
8
.44 (m, 1H), 8.19 (td, J = 1.08, 8.19 Hz, 1H), 7.91 (dd, J = 0.47, 7.82 Hz, 1H), 7.59 ꢀ 7.63 (m,
H), 7.52 (t, J = 1.51 Hz, 1H), 7.42 (d, J = 1.70 Hz, 2H), 1.40 (s, 21H).
1
1‐(3‐Nitrophenyl)‐3‐phenylbenzene (11r) was prepared from 1‐bromo‐3‐phenylbenzene
(0.17 ml, 1.0 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.1 mmol) following the general
1
procedure A as colorless liquid (0.27 g, 98%). H NMR (300 MHz, CDCl ) δ 8.52 (t, J = 1.98
3
Hz, 1H), 8.21 ꢀ 8.26 (m, 1H), 7.98 (td, J = 1.27, 7.82 Hz, 1H), 7.81 ꢀ 7.84 (m, 1H), 7.63 ꢀ 7.69
(m, 4H), 7.59 ꢀ 7.63 (m, 2H), 7.45 ꢀ 7.50 (m, 2H), 7.39 ꢀ 7.43 (m, 1H).
1‐(4‐Phenylphenyl)‐3‐nitrobenzene (11s) was prepared from 1‐bromo‐4‐phenylbenzene
0.30 g, 1.29 mmol) and 3ꢀnitrophenylboronic acid (0.24 g, 1.42 mmol) following the general
(
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procedure A as white solid (0.21 g, 58%). H NMR (300 MHz, CDCl ) δ 8.50 (t, J = 1.98 Hz,
3
1H), 8.20 (ddd, J = 0.75, 2.12, 8.24 Hz, 1H), 7.95 (td, J = 1.18, 7.82 Hz, 1H), 7.71 (d, J = 0.94
Hz, 4H), 7.62 ꢀ 7.66 (m, 3H), 7.44 ꢀ 7.51 (m, 2H), 7.37 ꢀ 7.42 (m, 1H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
4
5
6
7
8
9
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[
4‐(3‐Nitrophenyl)phenyl](phenyl)methanone
(11t)
was
prepared
from
(4‐
iodophenyl)(phenyl)methanone (0.31 g, 1.00 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10
1
mmol) following the general procedure A as brown solid (0.18 g, 60%). H NMR (300 MHz,
CDCl ) δ 8.49 ꢀ 8.55 (m, 1H), 8.28 (dd, J = 1.79, 7.63 Hz, 1H), 7.91 ꢀ 8.01 (m, 3H), 7.81 ꢀ 7.87
3
(
m, 2H), 7.73 ꢀ 7.78 (m, 2H), 7.60 ꢀ 7.71 (m, 2H), 7.49 ꢀ 7.56 (m, 2H).
5
‐(3‐Nitrophenyl)‐1‐benzofuran (11u) was prepared from 5‐bromo‐1‐benzofuran (0.13 ml,
1
.00 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol) following the general procedure
1
A as white solid (0.20 g, 84%). H NMR (300 MHz, CDCl ) δ 8.47 (t, J = 1.98 Hz, 1H), 8.18
3
(ddd, J = 1.04, 2.21, 8.24 Hz, 1H), 7.90 ꢀ 7.96 (m, 1H), 7.81 ꢀ 7.85 (m, 1H), 7.68 ꢀ 7.71 (m, 1H),
7
.58 ꢀ 7.64 (m, 2H), 7.51 ꢀ 7.56 (m, 1H), 6.85 (dd, J = 0.85, 2.17 Hz, 1H).
2
‐(3‐Nitrophenyl)naphthalene (11v) was prepared from 2‐bromonaphthalene (0.41 g, 2.00
mmol) and 3ꢀnitrophenylboronic acid (0.37 g, 2.20 mmol) following the general procedure A as
1
white solid (0.37 g, 74%). H NMR (300 MHz, CDCl ) δ 8.57 (t, J = 1.98 Hz, 1H), 8.22 (ddd, J
3
= 0.94, 2.17, 8.19 Hz, 1H), 8.08 (d, J = 1.51 Hz, 1H), 8.01 ꢀ 8.06 (m, 1H), 7.96 (d, J = 8.48 Hz,
1
H), 7.86 ꢀ 7.93 (m, 2H), 7.74 (dd, J = 1.88, 8.48 Hz, 1H), 7.64 (t, J = 8.01 Hz, 1H), 7.52 ꢀ 7.57
m, 2H).
‐(3‐Nitrophenyl)quinoline (11w) was prepared from 2‐bromoquinoline (0.18 g, 0.84
(
2
mmol) and 3ꢀnitrophenylboronic acid (0.15 g, 0.93 mmol) following the general procedure A as
1
white solid (0.02 g, 11%). H NMR (300 MHz, CDCl ) δ 9.05 (t, J = 1.88 Hz, 1H), 8.53 ꢀ 8.58
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(
m, 1H), 8.28 ꢀ 8.34 (m, 2H), 8.20 (d, J = 8.48 Hz, 1H), 7.94 (d, J = 8.67 Hz, 1H), 7.88 (d, J =
8
.10 Hz, 1H), 7.75 ꢀ 7.82 (m, 1H), 7.70 (t, J = 8.01 Hz, 1H), 7.55 ꢀ 7.63 (m, 1H).
3
‐(3‐Nitrophenyl)quinoline (11x) was prepared from 3‐bromoquinoline (0.14 ml, 1.00
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol) following the general procedure A as
1
white solid (0.08 g, 33%). H NMR (300 MHz, CDCl ) δ 9.19 (d, J = 2.26 Hz, 1H), 8.55 ꢀ 8.60
3
(m, 1H), 8.38 (d, J = 2.26 Hz, 1H), 8.29 (dd, J = 1.41, 8.19 Hz, 1H), 8.17 (d, J = 8.48 Hz, 1H),
8.05 (d, J = 7.91 Hz, 1H), 7.93 (d, J = 8.10 Hz, 1H), 7.79 (dt, J = 1.32, 7.72 Hz, 1H), 7.72 (t, J =
8.01 Hz, 1H), 7.60 ꢀ 7.67 (m, 1H).
2
‐(3‐Nitrophenyl)‐9H‐fluorene (11y) was prepared from 2‐bromo‐9H‐fluorene (0.25 ml,
1.00 mmol) and 3ꢀnitrophenylboronic acid (0.18 g, 1.10 mmol) following the general procedure
1
A as yellow solid (0.07 g, 26%). H NMR (300 MHz, CDCl ) δ 8.51 (t, J = 1.98 Hz, 1H), 8.19
3
(ddd, J = 0.94, 2.26, 8.10 Hz, 1H), 7.97 (qd, J = 0.94, 7.72 Hz, 1H), 7.84 ꢀ 7.91 (m, 2H), 7.81 (d,
J = 4.71 Hz, 2H), 7.62 ꢀ 7.67 (m, 1H), 7.56 ꢀ 7.60 (m, 1H), 7.40 (d, J = 6.97 Hz, 1H), 7.36 (dd, J
=
1.32, 7.35 Hz, 1H), 3.99 (s, 2H).
3‐(Pyridin‐3‐yl)aniline (14a) was prepared from 3‐bromoaniline (0.11 ml, 1.00 mmol) and
3ꢀpyridinylboronic acid (0.18 g, 1.10 mmol) following the general procedure A as white solid
1
(
0.10 g, 59%). H NMR (300 MHz, CDCl ) δ 8.81 (d, J = 1.70 Hz, 1H), 8.56 (dd, J = 1.41, 4.80
3
Hz, 1H), 7.77 ꢀ 7.85 (m, 1H), 7.31 (dd, J = 4.71, 7.91 Hz, 1H), 7.24 (t, J = 7.72 Hz, 1H), 6.91 ꢀ
6.98 (m, 1H), 6.83 ꢀ 6.88 (m, 1H), 6.71 (td, J = 1.11, 7.96 Hz, 1H), 3.83 (br. s., 2H). MS (ESI)
+
m/z for C H N [M+H] : calcd: 171.1; found: 171.0.
1
1
10
2
3
‐(2H‐1,3‐Benzodioxol‐5‐yl)aniline (14b) was prepared from 3‐bromoaniline (0.19 ml, 1.74
mmol) and (2H‐1,3‐benzodioxol‐5‐yl)boronic acid (0.32 g, 2.08 mmol) following the general
1
procedure A as yellow liquid (0.08 g, 60%). H NMR (300 MHz, CDCl ) δ 6.95 ꢀ 7.04 (m, 2H),
3
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.81 ꢀ 6.90 (m, 4H), 6.61 ꢀ 6.66 (m, 1H), 6.55 ꢀ 6.61 (m, 2H), 6.41 (d, J = 2.64 Hz, 1H), 6.24 (dd,
+
J = 2.54, 8.38 Hz, 1H), 5.89 (s, 2H), 3.73 (br. s., 2H). MS (ESI) m/z for C H NO [M+H] :
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calcd: 213.1; found: 213.2.
‐(3‐Methylphenyl)aniline (14c) was prepared from 3‐bromoaniline (0.11 ml, 1.00 mmol)
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and 3ꢀmethylphenylboronic acid (0.16 g, 1.10 mmol) following the general procedure A as
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yellow liquid (0.12 g, 66%). H NMR (300 MHz, CDCl ) δ 7.29 ꢀ 7.39 (m, 3H), 7.12 ꢀ 7.22 (m,
3
2
H), 6.96 ꢀ 7.00 (m, 1H), 6.90 (t, J = 1.98 Hz, 1H), 6.67 (ddd, J = 0.94, 2.26, 7.91 Hz, 1H), 3.73
br. s., 2H), 2.41 (s, 3H).
‐(2‐Methylphenyl)aniline (14d) was prepared from 3‐bromoaniline (0.11 ml, 1.00 mmol)
(
3
and 2ꢀmnethylphenylboronic acid (0.15 g, 1.10 mmol) following the general procedure A as
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yellow liquid (0.15 g, 82%). H NMR (300 MHz, CDCl ) δ 7.20 ꢀ 7.29 (m, 5H), 6.61 ꢀ 6.74 (m,
3
+
3H), 3.69 (br. s., 2H), 2.28 (s, 3H). MS (ESI) m/z for C H N [M+H] : calcd: 184.1; found:
13
13
1
84.2.
3
‐(3‐Nitrophenyl)aniline (14e) was prepared from 3‐bromoaniline (0.19 ml, 1.74 mmol) and
3ꢀnitrophenylboronic acid (0.32 g, 1.91 mmol) following the general procedure A as white solid
1
(0.08 g, 21%). H NMR (300 MHz, CDCl ) δ 8.37 ꢀ 8.45 (m, 1H), 8.17 (ddd, J = 0.94, 2.26, 8.10
3
Hz, 1H), 7.83 ꢀ 7.92 (m, 1H), 7.57 (t, J = 7.91 Hz, 1H), 7.21 ꢀ 7.28 (m, 1H), 6.96 ꢀ 7.03 (m, 1H),
.92 (t, J = 1.88 Hz, 1H), 6.72 ꢀ 6.78 (m, 1H), 3.82 (br. s., 2H). MS (ESI) m/z for C H N O
2
6
1
2
10
2
+
[
M+H] : calcd: 215.1; found: 215.0.
General procedure B. To a solution of nitrobenzene derivative (1 eq) in ethanol (0.1 M) was
o
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
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under reduced pressured and the residue was either used for the next step without purification or
purified by column chromatography (SiO , ethyl acetate/hexanes) to afford the desired product.
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‐(6‐Methoxypyridin‐2‐yl)aniline (12a) was prepared from 11a (0.23 g, 1.00 mmol)
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following the general procedure B as colorless liquid (0.20 g, quant. yield). H NMR (300 MHz,
CDCl ) δ 7.54 (dd, J = 7.54, 8.10 Hz, 1H), 7.36 ꢀ 7.41 (m, 2H), 7.17 ꢀ 7.27 (m, 2H), 6.62 ꢀ 6.70
3
+
(
m, 2H), 4.00 (s, 3H), 3.65 ꢀ 3.81 (m, 2H). MS (ESI) m/z for C H N O [M+H] : calcd: 201.1;
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found: 201.1.
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‐(4‐Methylpyridin‐2‐yl)aniline (12b) was prepared from 11b (0.16 g, 0.73 mmol)
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following the general procedure B as yellow solid (0.10 g, 76%). H NMR (300 MHz, CDCl ) δ
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.52 (d, J = 3.96 Hz, 1H), 7.51 (s, 1H), 7.27 (d, J = 5.09 Hz, 3H), 7.04 (d, J = 3.58 Hz, 1H), 6.65
+
(
br. s., 1H), 3.63 (br. s, 2H), 2.39 (s, 3H). MS (ESI) m/z for C H N [M+H] : calcd: 184.1;
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2
12
2
found: 184.4.
3
‐(2‐Methoxypyridin‐4‐yl)aniline (12c) was prepared from 11c (0.30 g, 1.30 mmol)
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following the general procedure B as colorless liquid (0.17 g, 65%). H NMR (300 MHz, CDCl )
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δ 8.35 (d, J = 2.07 Hz, 1H), 7.71 ꢀ 7.78 (m, 1H), 7.18 ꢀ 7.28 (m, 1H), 6.91 (td, J = 1.25, 7.68 Hz,
1
H), 6.76 ꢀ 6.85 (m, 2H), 6.67 (ddd, J = 0.75, 2.26, 7.91 Hz, 1H), 3.97 (s, 3H), 3.34 (br. s., 2H).
+
MS (ESI) m/z for C H N O [M+H] : calcd: 201.1; found: 201.2.
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2
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‐(Pyridin‐2‐yl)aniline (12d) was prepared from 11d (0.85 g, 4.24 mmol) following the
general procedure B and the crude product was used for the next step without purification.
3‐(Pyridin‐4‐yl)aniline (12e) was prepared from 11e (0.13 g, 0.65 mmol) following the
1
general procedure B as white solid (0.07 g, 65%). H NMR (300 MHz, CDCl ) δ 8.61 ꢀ 8.66 (m,
3
1H), 7.63 ꢀ 7.72 (m, 3H), 7.52 ꢀ 7.56 (m, 1H), 7.45 ꢀ 7.50 (m, 3H). MS (ESI) m/z for C H N
2
1
1
10
+
[
M+H] : calcd: 171.1; found: 171.0.
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