Diphenyl purine derivatives as peripherally selective cannabinoid receptor 1 antagonists

Article abstract of DOI:10.1021/jm301181r

Cannabinoid receptor 1 (CB1) antagonists are potentially useful for the treatment of several diseases. However, clinical development of several CB1 antagonists was halted due to central nervous system (CNS)-related side effects including depression and suicidal ideation in some users. Recently, studies have indicated that selective regulation of CB1 receptors in the periphery is a viable strategy for treating several important disorders. Past efforts to develop peripherally selective antagonists of CB1 have largely targeted rimonabant, an inverse agonist of CB1. Reported here are our efforts toward developing a peripherally selective CB1 antagonist based on the otenabant scaffold. Even though otenabant penetrates the CNS, it is unique among CB1 antagonists that have been clinically tested because it has properties that are normally associated with peripherally selective compounds. Our efforts have resulted in an orally absorbed compound that is a potent and selective CB1 antagonist with limited penetration into the CNS.

Full text of DOI:10.1021/jm301181r

Article
pubs.acs.org/jmc
Diphenyl Purine Derivatives as Peripherally Selective Cannabinoid
Receptor 1 Antagonists
Alan Fulp, Katherine Bortoff, Yanan Zhang, Herbert Seltzman, James Mathews, Rodney Snyder,
Tim Fennell, and Rangan Maitra*
Discovery Sciences, Research Triangle Institute, 3040 Cornwallis Road, P.O. Box 12194, Research Triangle Park, North Carolina
27709, United States
S
* Supporting Information
ABSTRACT: Cannabinoid receptor 1 (CB1) antagonists are
potentially useful for the treatment of several diseases.
However, clinical development of several CB1 antagonists
was halted due to central nervous system (CNS)-related side
effects including depression and suicidal ideation in some
users. Recently, studies have indicated that selective regulation
of CB1 receptors in the periphery is a viable strategy for treating several important disorders. Past efforts to develop peripherally
selective antagonists of CB1 have largely targeted rimonabant, an inverse agonist of CB1. Reported here are our efforts toward
developing a peripherally selective CB1 antagonist based on the otenabant scaffold. Even though otenabant penetrates the CNS,
it is unique among CB1 antagonists that have been clinically tested because it has properties that are normally associated with
peripherally selective compounds. Our efforts have resulted in an orally absorbed compound that is a potent and selective CB1
antagonist with limited penetration into the CNS.
INTRODUCTION
■
Cannabinoid receptors belong to the endocannabinoid (EC)
system, which consists of receptors, transporters, endocanna-
binoids, and the enzymes involved in synthesis and degradation
of endocannabinoids. To date, two receptors have been
identified  CB1 and CB2.1 Both CB1 and CB2 are G
protein-coupled receptors (GPCRs) primarily activating
inhibitory G proteins (Gi/o).2 Of the two, CB1 is prominently
expressed in the central nervous system (CNS).1 However, it is
also expressed peripherally in a number of peripheral tissues.
The CB2 receptor is nominally expressed in the brain.
However, it is highly expressed in cells of the immune system.3
Figure 1. Examples of CB1 antagonists.
The CB1 receptor is also a prominent target of drugs of abuse
including (−)-Δ9-tetrahydrocannabinol (THC), the main
being able to cross the blood−brain barrier (BBB) and thus
avoiding CNS-mediated adverse effects noted with nontissue
selective compounds. A similar strategy has been successful in
the development of peripherally selective opioids.9 Several
groups are currently pursuing this strategy.10 A few peripherally
selective CB1 antagonists have been reported (Figure 2), and
their further validation and characterization are underway. It
should be noted almost all reported efforts (e.g., 3−7) at
designing peripherally restricted antagonists of CB1 to date
have focused on compounds that closely resemble 1.
Our group has been involved in the development of
peripherally selective CB1 antagonists based on amide
variations of compound 1 using two different approaches.
First, charged compounds were produced based on the
observation that such compounds cross the BBB only when
psychoactive constituent of marijuana.4
In recent years, CB1 antagonists have received attention in
the treatment of disorders that have a central nervous system
(CNS)-related craving component, including alcoholism.5
Further, CB1 is a validated drug target to treat obesity,
metabolic syndrome, liver disease, diabetes, and dyslipidemias
through both CNS (anorexic) and peripheral (metabolic)
effects.6 Despite the therapeutic promise of CB1 antagonists,
this drug class has been limited by adverse effects including
anxiety and depression, and the first clinically approved CB1
antagonist (inverse agonist) for weight loss, rimonabant (1,
SR141716A) (Figure 1), was withdrawn from Europe.7
Consequently, development or clinical trials of several CB1
antagonists, such as taranabant, otenabant (2), and ibipinabant,
were halted due to regulatory concerns generated by 1.8
An alternative strategy to target this receptor is to develop
antagonists that are peripherally restricted by virtue of not
Received: August 10, 2012
Published: October 25, 2012
© 2012 American Chemical Society
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RESULTS
■
Approaches to Ligand Design and Testing. Synthesis,
purification, and characterization of compounds are described
in the Experimental Section. Antagonism of each compound
toward CB1 was quantitatively tested using an in vitro calcium
mobilization assay for CB1 as has been previously
described,13,15 and this antagonism is expressed as an apparent
dissociation affinity constant (Ke). Compounds that demon-
strated Ke < 100 nM were further characterized using
competitive radioligand displacement of [3H]CP55940 at
CB1 and CB2 overexpressing membrane preparations as
described previously.13 Displacement data were quantified
and expressed as an equilibrium dissociation constant (Ki)
value at each receptor. Potent and selective compounds were
progressed into an in vitro model of brain penetration in
monolayers of MDCK-mdr1cells as described previously.11b,13
Certain compounds were also characterized for metabolic
stability in human hepatic microsomal fractions (S9). Finally, in
vivo testing was conducted in Sprague−Dawley (SD) rats to
measure CNS permeability of these compounds.
Compound Development Strategy and Results.
During our pursuit of peripherally selective CB1 antagonists,
8 (Figure 3) was synthesized and found to be highly potent,
Figure 2. CB1 antagonists that are reported to be selective for the
periphery.
transported by a specific transporter.11 However, the
compounds synthesized to date using this approach have low
potency as reported in our previous publication.11b The second
strategy involved generation of compounds with high
topological polar surface areas (TPSAs). Past studies indicate
that compounds with high TPSA, usually >100, have lower
permeability into the CNS.12 This strategy was more successful
and led to the identification of 7 that we have previously
reported.13 While this compound was promising, having a brain
to plasma ratio of less than 4% in a simple pharmacokinetic
experiment, oral absorption of this compound was less than
optimal.13
In this paper, we report our efforts toward the synthesis and
characterization of peripherally selective CB1 antagonists based
on 2. This compound is a highly selective CB1 receptor
antagonist developed by Pfizer that was later abandoned due to
CNS-related adverse effects during phase 3 clinical trials. This
compound is unique among CB1 antagonists that have been
clinically tested because it has properties that would be
normally associated with a peripherally selective compound,
including a TPSA of 102, three hydrogen bond donors, and
formula weight >500.14 However, 2 is CNS permeable. It was
suggested that the intramolecular H-bonding observed in the X-
ray structure between the primary amide and the ethyl amine
portion of the molecule effectively lowered the polarity of this
compound, allowing for penetration into the CNS.14 Thus
upon examination, this scaffold appeared to be more favorable
for peripheralization as synthesis of compounds based on 2 but
incapable of forming intramolecular H-bonding would be
expected to be peripherally selective. This innovative approach
to ligand design has now provided us with several promising
compounds for further refinement.
Figure 3. Design of compound 9; a hybrid of compounds 2 and 8.
selective, and to have relatively low permeability in the MDCK-
mdr1 model.13 These results along with our interest in CB1
antagonists that possess a purine core, like 2, moved us to make
purine 9. Purine 9 is a potent (Ke = 9 nM) and selective (CB2/
CB1 selectivity ratio ∼ 3077) hybrid of 2 and 8 with a TPSA of
90. Despite having a TPSA that is lower than that of 2, 9 cannot
form intramolecular hydrogen bonds, and therefore, was
predicted to be more peripherally selective than 2. This
hypothesis was tested and confirmed (Table 1) using the
MDCK-mdr1 permeability assay,13 where 9 was only 6%
Table 1. Functional Assessment of Compound 9
Ke
Ki CB1
Ki CB2
CB2/
MDCK-
CB1 (nM) [3H] (nM) [3H] CB1 Ki mdr1 A to
a
compd TPSA (nM)
CP55940
CP55940
ratio
B (%)
2
8
9
102
81
8.7
0.45
9
40
8
3.44
1.79
5504
5507
1600
3077
90
6
a
Compound’s permeability was measured from apical to basal (A to
B) sides of the membrane.
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permeable (A-B), while 2 was ∼40% permeable (apical to basal,
A-B) in the same assay. These positive results warranted further
SAR exploration around 9.
To further improve upon 9, the amide was changed to more
polar functionalities such as sulfonamide and urea (Figure 4,
ability to hydrogen bond in an intramolecular fashion. Two
members of structural series 13 were found to have interesting
pharmacological profiles. Urea 13a was potent (Ke = 19.2 nM),
selective (CB2/CB1 = 20000), and poorly permeable in the
MDCK-mdr1assay (A to B is <1%). Urea 13e was also found to
be potent (Ke = 62 nM) and selective (CB2/CB1 = 74) as well.
The positive result observed with compounds 10−13g led to
further exploration of the SAR around purines (Figure 5). The
Figure 4. Ligand design around compound 9, replacement of the
amide functionality.
Table 2. Functional Assessment of Analogues of Compound
9
Figure 5. Exploration of different spacers.
Ke
Ki CB1
Ki CB2
CB2/
MDCK-
CB1 (nM) [3H] (nM) [3H] CB1 Ki mdr1 A to
a
compd TPSA (nM)
CP55940
CP55940
ratio
B (%)
4-amino-4-phenylpiperidine linker of compounds 10−13g was
replaced with 4- and 3-aminopiperidine linkers to test the
importance of the juxtaposition of the terminal polar groups of
compounds 10−13g. These changes also had the benefit of
reducing the molecular weight of analogues that were
synthesized by removing the phenyl group present in 10−
13g. Structural series 14, 15, 16, and 17 were all made and
evaluated pharmacologically. The data (Table 3) for these
series show that the 4-aminopiperidine linker of structural
series 17 was superior to the linkers in structural series 14, 15,
and 16 for all direct comparisons of the R group. However, in
almost every case the 4-amino-4-phenylpiperidine linker of 10−
13g was more potent than its direct analogues in structural
series 14−17.
However, several interesting compounds were identified in
structural series 14−17. The tert-butoxycarbonyl (BOC)
analogue 14a was potent (Ke = 92 nM) and selective (CB2/
CB1 = 568). In general, compounds from structural series 15
and 16 were weakly active against CB1 in the calcium
mobilization assay and the compounds were not characterized
further. Several analogues in structural series 17 were very
interesting. The BOC analogue 17a was very potent (Ke = 11
nM) and selective (CB2/CB1 = 1142). The urea analogues
17c−f all showed good activity (Ke ranging from 49 to 149
nM). Selectivity (CB2/CB1) for ureas 17c−f ranged from 5 to
175 fold. Amides and sulfonamides 17g−k were generally active
with 3 of the 5 analogues having Ke < 10 nM. One of these
10
85
73
3.9
7.1
13947
1657
948
1959
151
153
784
24
<1
<1
<1
<1
11
15.9
2.9
11
12
101
88
6.2
13a
13b
13c
13d
13e
13f
13g
19.2
443
46
25.5
195
337
220
269
20000
4645
8946
7779
>20000
88
88
27
88
99
35
88
62
>74
114
88
171
199
a
Compound’s permeability was measured from apical to basal (A to
B) sides of the membrane.
Table 2). Carbamate 10 and amine 11 were first synthesized,
the latter of which also serves as a common synthetic
intermediate for other analogues. Interestingly, carbamate 10
was very potent (Ke = 3.9 nM), selective (CB2/CB1 = 1959),
and poorly permeable in the MDCK-mdr1 model (A to B
<1%). Similarly, amine 11 was also very potent (Ke = 15.9 nM),
selective (CB2:CB1 = 151), and poorly permeable in the
MDCK-mdr1 model (A to B <1%). With amine 11 in hand,
sulfonamide 12 and a series of ureas 13a−g were synthesized.
Sulfonamide 12 was especially interesting because it has high
potency (Ke = 2.9 nM), selectivity (CB2/CB1 = 153), poor
permeability in the MDCK-mdr1 model (A to B is <1%), and a
high TPSA (101), similar to 2 (TPSA = 102); it also lacks the
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Sulfonamide 12 was dosed orally in SD rats at 10 mg/kg,
and both plasma and brain levels were analyzed out to 8 h post
dose (Table 5). The compound showed slow absorption and
Table 3. Functional Assessment of Different Spacers and
Functional Groups
Ke CB1
compd TPSA (nM)
Ki CB1 (nM)
Ki CB2 (nM)
CB2/CB1
Ki ratio
[3H]CP55940
[3H]CP55940
Table 5. Pharmacokinetic Analyses of 12
14a
14b
14c
14d
14e
14f
85
68
92
24
14000
568
5706
1148
495
1964
71
dose
mg/kg
oral
brain/
plasma
ratio
sacrifice
time (min)
plasma conc. brain conc.
(ng/mL)
88
compd
(ng/mL)
88
12
10
30
752
38
0.05
0.08
0.10
0.11
0.10
88
60
767
58
88
3343
>20000
>5
120
240
480
1188
1653
914
122
184
89
14g
15a
15b
15c
15d
15e
16a
16b
16c
16d
16e
17a
17c
17d
17e
17f
101
85
1645
326
5546
1500
920
346
3927
8406
1153
4449
6044
11
68
88
did not reach a maximum concentration until 240 min post
dose. Sulfonamide 12 also showed diminished brain pene-
tration with the brain to plasma ratio ranging from 0.05 to 0.11.
It should be noted that unperfused brain tissues, which are
expected to contain 2−4% blood, were examined.16 Thus, it is
concluded that 12 has limited brain penetration. Furthermore,
12 demonstrated excellent oral absorption and favorable
clearance upon oral dosing in rats. Thus, this compound may
be suitable for further development.
76
101
85
68
88
76
101
85
1.5
1713
1142
88
149
133
132
49
Synthesis. Compound 9 (Scheme 1) was prepared from
commercially available piperidine 19 and the readily available
88
14
28
25
2453
153
175
5
88
88
1548
62
a
Scheme 1. Synthesis of Compound 9
17g
17h
17i
76
631
71
101
76
29
1073
37
3.2
17j
76
0.7
17k
101
0.3
1.2
5865
4888
compounds, 17k, was found to be highly selective (CB2/CB1 =
4888).
In Vitro Metabolic Stability and In Vivo Evaluation of
Brain Penetration. A small set of potent and selective
compounds were progressed into in vitro models of metabolic
stability (Table 4). Stability in human plasma and human
a
Reagents and conditions: (a) 19, triethylamine, ethanol, 55 °C, 3
days.
chloro-purine 18.14 Chloro-purine 18, piperidine 19, and
triethylamine were heated in ethanol at 55 °C for 3 days
yielding 9 in 63% yield. In a similar manner chloro-purine 18
was reacted with piperidine 20 and triethylamine in ethanol at
80 °C for 16 h to yield 10 in 84% yield (Scheme 2). The BOC
group of 10 was removed using 20% trifluoroacetic acid (TFA)
in dichloromethane to yield the primary amine 11 in 99%.
Sulfonamide 12 was prepared by reacting 11, methanesulfonyl
chloride, and triethylamine in tetrahydrofuran (THF) in 82%
yield. The ureas 13a−g were made by reacting amine 11,
triethylamine, and the appropriate isocyanate in THF in yields
ranging from 69 to 92%.
Chloro-purine 18 was converted to 14a, 15a, and 16a in
yields ranging from 86 to 95% by reaction with the appropriate
amine and triethylamine in ethanol at 80 °C for 16 h (Scheme
3). The BOC groups were removed from 14a, 15a, and 16a to
form amines 14b, 15b, and 16b using 30% TFA in
dichloromethane in yields ranging from 65 to 88%. Amines
14b, 15b, and 16b were reacted with the appropriate isocyanate
and triethylamine in THF to generate ureas 14c−f, 15c, and
16c in yields ranging from 65 to 95%. Amines 15b and 16b
were reacted with acetic anhydride in pyridine to yield amides
15d and 16d in 87% and 81% respectively. Sulfonamides 14g,
15e, and 16e were made from 14b, 15b, and 16b by reaction
Table 4. Functional Assessment of Select Compounds in for
in Vitro Metabolic Stability
in vitro metabolic
stability
Ke
CB2/
CB1
MDCK-
S9%
plasma %
CB1
mdr1 A to remaining remaining
compd TPSA (nM) CP55940
B (%)
120 min
60 min
9
90
101
88
0.45
2.9
3077
153
6
>90
>90
43
>90
>90
>90
12
13a
<1
<1
19.2
784
hepatic S9 fractions was measured to gauge potential metabolic
liabilities of these compounds. All compounds were stable in
plasma up to 1 h. The stabilities in S9 fractions for these
compounds were more variable. All compounds displayed
acceptable metabolic stabilities; however, 13a did show
significant metabolism in S9 fraction and was deemed
unsuitable for further development.
Several compounds showed excellent in vitro properties,
including compounds 17a−k. Because of limited resources only
sulfonamide 12 was advanced into an in vivo experiment to
evaluate its brain penetration in SD rats upon oral dosing.
Future studies are planned with additional compounds.
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a
Scheme 2. Synthesis of Analogues of Compound 9
Scheme 3. Synthesis of Compounds with Different Spacers
and Functional Groups
a
a
Reagents and conditions: (a) 20, triethylamine, ethanol, 80 °C, 16 h;
(b) 20% TFA in dichloromethane; (c) methanesulfonyl chloride,
triethylamine, THF; (d) appropriate isocyanate, triethylamine, THF.
with methanesulfonyl chloride and triethylamine in THF in
yields ranging from 51 to 91%.
Chloro-purine 18 and piperidine 21 were heated in ethanol
at 80 °C for 16 h yielding 17a in 99% yield (Scheme 4). The
remaining chemistry, to generate 17b−k, was performed in a
similar manner to reactions discussed above.
a
Reagents and conditions: (a) triethylamine, ethanol, 80 °C, 16 h; (b)
30% TFA in dichloromethane; (c) appropriate isocyanate, triethyl-
amine, THF, rt; (d) acetic anhydride, pyridine; (e) methanesulfonyl
chloride, triethylamine, THF.
DISCUSSION
■
CB1 receptor antagonism is a validated strategy to treat several
important disorders including diabetes, obesity, metabolic
syndromes, liver diseases, and dyslipidemias. However, non-
tissue selective antagonists of this receptor are not clinically
useful due to adverse CNS effects. CB1 receptors are also
expressed in peripheral organs and targeting of these peripheral
receptors has produced promising data in experimental models
of important diseases. Thus, several groups are trying to
develop peripherally selective antagonists of CB1 for
therapeutic use. While some progress has been made in this
direction, currently disclosed CB1 antagonists that are
peripherally selective need further improvement and validation
prior to clinical use. Furthermore, most of these compounds are
closely related to 1. In this report, we disclose our efforts to
develop peripherally selective antagonists of CB1 based on 2,
which we believe is a promising scaffold for optimization. This
is because 2 has several chemical properties that would indicate
poor brain penetration. However, intramolecular hydrogen
bonding may effectively lower the polarity of 2 and this has
been suggested by Griffith and co-workers.14 Thus, we
hypothesized that optimized design and synthesis of com-
pounds based on 2 that do not have the ability to form
intramolecular hydrogen bonds could lead to CB1 antagonists
with limited brain penetration.
Several potent and selective CB1 antagonists have been
reported in this paper including nine compounds that have Ke <
100 nM and selectivity >100-fold versus CB2. Potency at CB1
has been obtained with a variety of functional groups on the
terminus of the aminopiperidine in structures 10−17k.
Carbamates, amines, sulfonamides, ureas, and amides have all
been well tolerated at this position. The 4-amino-4-phenyl-
piperidine linker present in compounds 10−13g was the most
potent linker noted in this study. 4-Aminopiperidine derivatives
were more potent that 3-aminopiperidine analogues. However,
of the 1−3 aminopiperidine compounds in structural series 15
and 16, the R enantiomers (15) were generally more potent at
CB1 (four out of five examples) than the S enantiomers (16) in
head to head comparisons. Of the 4-aminopiperidine linker
compounds, lacking a phenyl group on the piperidine ring, in
structural series 14 and 17, the analogues where the piperidine
nitrogen was bonded to the purine core (structural series 17)
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significant reduction in brain penetration in comparison to 2 in
SD rats, which has ∼33% brain exposure at 2−3 h postdose.17
In conclusion, through our studies, we have discovered novel
analogues of 2 with vastly reduced brain penetration. Of the
compounds reported, 12 had excellent potency, selectivity, and
oral absorption in rats. This compound has low levels of brain
penetration as well. This compound will be evaluated in proof
of concept studies for diseases such as obesity, liver fibroses,
and diabetes in the future. Furthermore, sulfonamide 12 will
also serve as a starting point for refinement to further lower the
brain penetration of future compounds, if necessary. However,
any such endeavor must be dictated by the proposed use, and
an acceptable balance between therapeutic benefits and
undesirable effects must be achieved. For example, antiobesity
drugs should have a very favorable profile as far as adverse
effects are concerned. By contrast, in other diseases such as liver
disease or diabetes, a more permissive profile of adverse effects
could be tolerated.
Scheme 4. Synthesis of Compounds with Different Spacers
and Functional Groups Continued
a
EXPERIMENTAL SECTION
■
Compound Synthesis and Characterization. Chemistry
General. Purity and characterization of compounds were established
by a combination of HPLC, TLC, and NMR analytical techniques
1
described below. H and 13CNMR spectra were recorded on a Bruker
Avance DPX-300 (300 MHz) spectrometer and were determined in
CHCl3-d or MeOH-d4 with tetramethylsilane (TMS) (0.00 ppm) or
solvent peaks as the internal reference unless otherwise noted.
Chemical shifts are reported in ppm relative to the solvent 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 I2 detection.
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 × 150 mm, 5 μm column with
gradient elution using the mobile phases (A) H2O containing 0.05%
CF3COOH and (B) methanol. A flow rate of 1.0 mL/min was used.
All compounds were isolated as white or off-white solids.
a
Reagents and conditions: (a) 21, ethanol, 80 °C, 16 h; (b) 30% TFA
in dichloromethane; (c) appropriate isocyanate, triethylamine, THF;
(d) appropriate anhydride, triethylamine, THF; (e) appropriate
sulfonyl chloride, triethylamine, THF.
were favored over the isomers where the 4-amino group was
bonded to the core (structural series 14). Compounds with
branched terminal R groups were found to be more selective
for CB1 over CB2 than compounds with less sterically
demanding R groups. This trend was especially prevalent in
ureas 13a−d and 17c−f. In both of these series, 13a−d and
17c−f, the compounds with the most branched R group have
the highest selectivity ratio for CB2/CB1. This selectivity is
achieved not by gains in CB1 binding affinity, but by lowering
the binding affinity for CB2. While 2 is an inverse agonist at
CB1, it is currently not known if the compounds described in
this publication act as inverse agonist or neutral antagonists at
the present time. Those studies will be performed in the future.
The removal of the intramolecular hydrogen bond found in 2
allowed us to synthesize compounds with significantly lower
permeability in the MDCK-mdr1 assay. This can be
demonstrated by comparing 2 and 12. While 2 and 12 have
almost identical TPSAs (102 vs 101), 12 which lacks the ability
to hydrogen bond in an intramolecular fashion has vastly
reduced permeability in the MDCK-mdr1 assay. Thus,
confirming Griffith and co-worker’s hypothesis.14
Compounds 9, 12, and 13a were identified for further
evaluation. All of these compounds showed reasonable stability
to human plasma. Both 9 and 12 had good stability in human
S9 fraction with over 90% of compound remaining after 120
min of incubation. The stability of 13a to S9 fraction was
acceptable, but it did show significant metabolism in the assay.
Compound 12 was advanced into in vivo models to assess its
brain penetration. In a rat PK assay, 12 showed brain to plasma
ratios ranging from 0.05 to 0.11, indicating limited brain
penetration. Generally, compounds that have <10% brain
penetration are considered peripherally selective. This is also a
1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-phe-
nylpiperidine-4-carboxamide (9). To a solution of 6-chloro-8-(2-
chlorophenyl)-9-(4-chlorophenyl)-9H-purine (9) (23.1 mg, 0.062
mmol, 1 equiv) in 2 mL of ethanol was added 4-carbamoyl-4-
phenylpiperidin-1-ium trifluoroacetate (19) (25 mg, 0.123 mmol, 2
equiv) and triethylamine (0.03 mL, 0.19 mmol, 3 equiv). The reaction
was heated to 55 °C for 3 days. The reaction was concentrated in
vacuo. The crude material was purified by silica gel column
chromatography using 0−100% ethyl acetate/hexanes to yield 21 mg
1
(63%) of desired product. H NMR (300 MHz, methanol-d4) δ ppm
2.02−2.21 (m, 2 H) 2.62 (d, J = 13.37 Hz, 2 H) 3.82 (br. s., 2 H) 4.98
(d, J = 15.54 Hz, 2 H) 7.07−7.67 (m, 13 H) 8.02−8.29 (m, 1 H), [M
+ H]+ 543.6.
tert-Butyl N-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-
6-yl]-4-phenylpiperidin-4-yl}carbamate (10). To a solution of 18 (50
mg, 0.133 mmol, 1 equiv) in 2 mL of ethanol was added tert-butyl N-
(4-phenylpiperidin-4-yl)carbamate (20) (44 mg, 0.16 mmol, 1.2
equiv) and triethylamine (0.03 mL, 0.20 mmol, 1.5 equiv). The
reaction was heated to 80 °C for 16 h. The reaction was concentrated
in vacuo. The crude material was purified by silica gel column
chromatography using 0−100% ethyl acetate/hexanes to yield 69 mg
(84%) of desired product. 1H NMR (300 MHz, chloroform-d) δ ppm
1.31−1.49 (m, 9 H) 2.09−2.28 (m, 2 H) 2.43 (br. s., 2 H) 3.62 (br. s.,
2 H) 4.96 (s, 1 H) 5.30 (br. s., 2 H) 7.03 - 7.61 (m, 13 H) 8.40 (s, 1
H), [M + H]+ 615.4.
1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-phe-
nylpiperidin-4-amine (11). A solution of 10 (2.65 g, 4.3 mmol) was
stirred in dichloromethane (32 mL) and trifluoroacetic acid (8 mL) for
1.5 h. The reaction was concentrated in vacuo. The crude product was
dissolved in ethyl acetate and washed with 3.8 M NaOH. The aqueous
10027
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Article
layer was extracted twice with ethyl acetate. The combined organic
layer was washed with brine and dried with MgSO4 to yield 2.21 g
(99%) of pure desired product. 1H NMR (300 MHz, chloroform-d) δ
ppm 1.87 (d, J = 13.56 Hz, 2 H) 2.15−2.39 (m, 2 H) 3.82−4.11 (m, 2
H) 5.10 (br. s., 2 H) 7.11−7.62 (m, 13 H) 8.40 (s, 1 H), [M + H]+
515.8.
tert-Butyl 4-{[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-
yl]amino}piperidine-1-carboxylate (14a). To a solution of 18 (151
mg, 0.403 mmol, 1 equiv) in 4 mL of ethanol was added tert-butyl 4-
aminopiperidine-1-carboxylate (96 mg, 0.483 mmol, 1.2 equiv) and
triethylamine (0.08 mL, 0.6 mmol, 1.5 equiv). The reaction was heated
to 80 °C for 16 h. The reaction was concentrated in vacuo. The crude
material was purified by silica gel column chromatography using 0−
100% ethyl acetate/hexanes to yield 203 mg (94%) of desired product.
1H NMR (300 MHz, chloroform-d) δ ppm 1.38−1.55 (m, 11 H) 2.14
(d, J = 12.34 Hz, 2 H) 2.98 (t, J = 12.10 Hz, 2 H) 4.00 - 4.21 (m, 2 H)
4.28−4.52 (m, 1 H) 5.83 (d, J = 7.63 Hz, 1 H) 7.17−7.27 (m, 2 H)
7.31−7.44 (m, 5 H) 7.50 (d, J = 6.88 Hz, 1 H) 8.44 (s, 1 H), [M +
H]+ 539.4.
8-(2-Chlorophenyl)-9-(4-chlorophenyl)-N-(piperidin-4-yl)-9H-
purin-6-amine (14b). A solution of 14a (180 mg, 0.33 mmol) was
stirred in dichloromethane (9 mL) and trifluoroacetic acid (4 mL) for
16 h. The reaction was concentrated in vacuo. The crude product was
dissolved in ethyl acetate and washed with saturated NaHCO3. The
aqueous layer was extracted twice with ethyl acetate. The combined
organic layer was washed with brine and dried with MgSO4. The crude
material was purified by silica gel column chromatography using 0−
100% CMA 80 (80% chloroform, 18% methanol, and 2% ammonium
hydroxide)/ethyl acetate to yield 129 mg (88%) of desired product.
1H NMR (300 MHz, chloroform-d) δ ppm 1.45 (qd, J = 11.66, 3.81
N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-
phenylpiperidin-4-yl}methanesulfonamide (12). To a solution of 11
(8.4 mg, 0.016 mmol, 1 equiv) in 2 mL of THF was added
methanesulfonyl chloride (0.01 mL) and triethylamine (0.02 mL). The
mixture was stirred for 16 h. The reaction was concentrated in vacuo.
The crude material was purified by silica gel column chromatography
using 0−100% ethyl acetate/hexanes to yield 8 mg (82%) of desired
1
product. H NMR (300 MHz, chloroform-d) δ ppm 2.22 (s, 3 H)
2.30−2.44 (m, 2 H) 2.47−2.66 (m, 2 H) 4.20 (br. s., 2 H) 4.75 (s, 2
H) 7.08−7.64 (m, 13 H) 8.38 (s, 1 H), [M + H]+ 593.3.
General Procedure for Making Ureas from 1-[8-(2-Chlorophenyl)-
9-(4-chlorophenyl)-9H-purin-6-yl]-4-phenylpiperidin-4-amine (11).
To a solution of 11 (19 mg, 0.036 mmol, 1 equiv) in 2 mL of THF
was added triethylamine (0.015 mL, 0.108 mmol, 3 equiv) and the
appropriate isocyanate (1.5 equiv). The reaction was stirred for 16 h.
The reaction was concentrated in vacuo. The crude material was
purified by silica gel column chromatography using 0−100% ethyl
acetate/hexanes to yield pure compound.
3-tert-Butyl-1-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-
Hz, 2 H) 2.10 (d, J = 11.87 Hz, 2 H) 2.64−2.88 (m, 2 H) 3.10 (d, J =
12.62 Hz, 2 H) 4.27 (br. s., 1 H) 5.80 (d, J = 6.31 Hz, 1 H) 7.01−7.55
(m, 8 H) 8.37 (s, 1 H), [M + H]+ 439.6.
purin-6-yl]-4-phenylpiperidin-4-yl}urea (13a). Reaction proceeded in
1
81% yield. H NMR (300 MHz, chloroform-d) δ ppm 1.12 (s, 9 H)
2.13−2.27 (m, 2 H) 2.27−2.43 (m, 2 H) 3.67 (br. s., 2 H) 3.96 (br. s.,
1 H) 4.78 (br. s., 1 H) 5.10−5.68 (m, 2 H) 7.07−7.63 (m, 13 H) 8.40
(s, 1 H), [M + H]+ 614.7.
General Procedure for Making Ureas from 8-(2-chlorophenyl)-9-
(4-chlorophenyl)-N-(piperidin-4-yl)-9H-purin-6-amine 14b. To a
solution of 14b (18.4 mg, 0.042 mmol, 1 equiv) in 2 mL of THF
was added triethylamine (0.02 mL, 0.126 mmol, 3 equiv) and the
appropriate isocyanate (1.5 equiv). The reaction is stirred for 16 h.
The reaction was concentrated in vacuo. The crude material was
purified by silica gel column chromatography using 0−100% CMA 80/
ethyl acetate to yield pure compound.
4-{[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]amino}-
N-ethylpiperidine-1-carboxamide (14c). Reaction proceeded in 86%
yield. 1H NMR (300 MHz, chloroform-d) δ ppm 1.09 (t, J = 7.21 Hz,
3 H) 1.34−1.60 (m, 2 H) 2.11 (d, J = 12.34 Hz, 2 H) 2.83−3.06 (m, 2
H) 3.13−3.32 (m, 2 H) 3.92 (d, J = 13.37 Hz, 2 H) 4.40 (d, J = 4.90
Hz, 2 H) 5.80 (d, J = 7.82 Hz, 1 H) 7.04−7.51 (m, 8 H) 8.37 (s, 1 H),
[M + H]+ 510.4.
4-{[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]amino}-
N-(propan-2-yl)piperidine-1-carboxamide (14d). Reaction pro-
ceeded in 91% yield. 1H NMR (300 MHz, chloroform-d) δ ppm
1.18 (d, J = 6.50 Hz, 6 H) 1.46−1.68 (m, 2 H) 2.19 (d, J = 10.55 Hz, 2
H) 3.03 (t, J = 11.59 Hz, 2 H) 3.89−4.10 (m, 3 H) 4.29 (d, J = 7.16
Hz, 1 H) 4.42 (br. s., 1 H) 5.88 (d, J = 7.82 Hz, 1 H) 7.13−7.64 (m, 8
H) 8.45 (s, 1 H), [M + H]+ 524.7.
1-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-
phenylpiperidin-4-yl}-3-ethylurea (13b). Reaction proceeded in 81%
yield. 1H NMR (300 MHz, chloroform-d) δ ppm 0.85−1.01 (m, 3 H)
2.11−2.29 (m, 2 H) 2.30−2.47 (m, 2 H) 2.96−3.20 (m, 2 H) 3.64 (br.
s., 2 H) 4.10−4.24 (m, 1 H) 5.00 (s, 1 H) 5.32 (d, J = 14.41 Hz, 2 H)
7.10−7.60 (m, 13 H) 8.40 (s, 1 H), [M + H]+ 586.8.
1-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-
phenylpiperidin-4-yl}-3-(propan-2-yl)urea (13c). Reaction proceeded
1
in 79% yield. H NMR (300 MHz, chloroform-d) δ ppm 0.94 (d, J =
6.50 Hz, 6 H) 2.13−2.29 (m, 2 H) 2.29−2.42 (m, 2 H) 3.51−3.76 (m,
2 H) 3.95 (d, J = 7.82 Hz, 1 H) 4.93 (s, 1 H) 5.32 (d, J = 13.56 Hz, 2
H) 7.07−7.63 (m, 13 H) 8.40 (s, 1 H), [M + H]+ 600.7.
1-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-
phenylpiperidin-4-yl}-3-propylurea (13d). Reaction proceeded in
88% yield. 1H NMR (300 MHz, chloroform-d) δ ppm 0.64−0.77
(m, 3 H) 1.19−1.37 (m, 2 H) 2.10−2.27 (m, 2 H) 2.29−2.45 (m, 2
H) 2.87−3.08 (m, 2 H) 3.63 (br. s., 2 H) 4.28 (br. s., 1 H) 5.11 (s, 1
H) 5.36 (br. s., 2 H) 7.10−7.60 (m, 13 H) 8.40 (s, 1 H), [M + H]+
600.5.
3-Butyl-1-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-
yl]-4-phenylpiperidin-4-yl}urea (13e). Reaction proceeded in 82%
yield. 1H NMR (300 MHz, chloroform-d) δ ppm 0.74−0.86 (m, 3 H)
1.11 (dq, J = 14.79, 7.22 Hz, 2 H) 1.20−1.31 (m, 2 H) 2.11 - 2.27 (m,
2 H) 2.29−2.41 (m, 2 H) 3.05 (q, J = 6.59 Hz, 2 H) 3.63 (br. s., 2 H)
4.22 (t, J = 5.13 Hz, 1 H) 5.07 (s, 1 H) 5.34 (br. s., 2 H) 7.12−7.56
(m, 13 H) 8.40 (s, 1 H), [M + H]+ 614.7.
4-{[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]amino}-
N-propylpiperidine-1-carboxamide (14e). Reaction proceeded in
95% yield. 1H NMR (300 MHz, chloroform-d) δ ppm 0.86−1.01
(m, 3 H) 1.44−1.67 (m, 4 H) 2.19 (d, J = 10.64 Hz, 2 H) 3.05 (t, J =
11.73 Hz, 2 H) 3.22 (q, J = 6.59 Hz, 2 H) 3.92−4.08 (m, 2 H) 4.43
(br. s., 1 H) 4.54 (t, J = 5.04 Hz, 1 H) 5.88 (d, J = 7.72 Hz, 1 H) 7.09−
7.58 (m, 8 H) 8.45 (s, 1 H), [M + H]+ 524.8.
Ethyl 2-[({1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-
N-Butyl-4-{[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-
yl]-4-phenylpiperidin-4-yl}carbamoyl)amino]acetate (13f). Reaction
yl]amino}piperidine-1-carboxamide (14f). Reaction proceeded in
1
1
proceeded in 92% yield. H NMR (300 MHz, chloroform-d) δ ppm
91% yield. H NMR (300 MHz, chloroform-d) δ ppm 0.79−0.92 (m,
1.18−1.28 (m, 3 H) 2.11−2.29 (m, 2 H) 2.33−2.52 (m, 2 H) 3.64 (br.
s., 2 H) 3.82−3.92 (m, 2 H) 4.09−4.24 (m, 2 H) 5.13 (br. s., 1 H)
5.23−5.52 (m, 2 H) 5.60 (s, 1 H) 7.05−7.59 (m, 13 H) 8.39 (s, 1 H),
[M + H]+ 644.4.
3 H) 1.29 (dq, J = 14.75, 7.20 Hz, 2 H) 1.36−1.58 (m, 4 H) 2.11 (d, J
= 10.64 Hz, 2 H) 2.96 (t, J = 11.73 Hz, 2 H) 3.17 (q, J = 6.75 Hz, 2 H)
3.81−4.01 (m, 2 H) 4.22−4.51 (m, 2 H) 5.79 (d, J = 7.54 Hz, 1 H)
7.01−7.53 (m, 8 H) 8.37 (s, 1 H), [M + H]+ 538.4.
3-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-
8-(2-Chlorophenyl)-9-(4-chlorophenyl)-N-(1-methanesulfonylpi-
peridin-4-yl)-9H-purin-6-amine (14g). To a solution of 14b (18.4 mg,
0.042 mmol, 1 equiv) in 2 mL of THF was added methanesulfonyl
chloride (0.005 mL, 0.063 mmol, 1.5 equiv) and triethylamine (0.02
mL, 0.126 mmol, 3 equiv). The mixture was stirred for 16 h. The
reaction was concentrated in vacuo. The crude material was purified by
silica gel column chromatography using 0−100% ethyl acetate/
phenylpiperidin-4-yl}-1-cyclohexylurea (13g). Reaction proceeded in
1
69% yield. H NMR (300 MHz, chloroform-d) δ ppm 0.77−1.13 (m,
3 H) 1.15−1.36 (m, 3 H) 1.36−1.54 (m, 3 H) 1.56−1.76 (m, 1 H)
2.12−2.28 (m, 2 H) 2.29−2.42 (m, 2 H) 3.34−3.55 (m, 1 H) 3.65 (br.
s., 2 H) 3.96−4.20 (m, 1 H) 4.96 (s, 1 H) 5.34 (br. s., 2 H) 7.06−7.61
(m, 13 H) 8.40 (s, 1 H), [M + H]+ 640.5.
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1
hexanes to yield 20 mg (91%) of desired product. H NMR (300
MHz, chloroform-d) δ ppm 1.58−1.76 (m, 2 H) 2.21 (d, J = 10.64 Hz,
2 H) 2.76 (s, 3 H) 2.90 (t, J = 10.93 Hz, 2 H) 3.77 (d, J = 12.15 Hz, 2
H) 4.33 (br. s., 1 H) 5.84 (br. s., 1 H) 7.04−7.51 (m, 8 H) 8.36 (s, 1
H), [M − H]− 515.7.
was heated to 80 °C for 16 h. The reaction was concentrated in vacuo.
The crude material was purified by silica gel column chromatography
using 0−100% ethyl acetate/hexanes to yield 137 mg (95%) of desired
product. 1H NMR (300 MHz, chloroform-d) δ ppm 1.43 (br. s., 9 H)
1.60−1.76 (m, 2 H) 1.77−1.93 (m, 1 H) 2.08−2.22 (m, 1 H) 3.30 (br.
s., 2 H) 3.60 (br. s., 1 H) 3.98 (br. s., 1 H) 4.42 (br. s., 1 H) 5.96 (d, J
= 7.54 Hz, 1 H) 7.14−7.68 (m, 8 H) 8.48 (s, 1 H), [M + H]+ 539.4.
8-(2-Chlorophenyl)-9-(4-chlorophenyl)-N-[(3S)-piperidin-3-yl]-
9H-purin-6-amine (16b). A solution of 16a (120 mg, 0.22 mmol) was
stirred in dichloromethane (7 mL) and trifluoroacetic acid (3 mL) for
16 h. The reaction was concentrated in vacuo. The crude product was
dissolved in ethyl acetate and washed with saturated NaHCO3. The
aqueous layer was extracted twice with ethyl acetate. The combined
organic layer was washed with brine and dried with MgSO4. The crude
material was purified by silica gel column chromatography using 0−
100% CMA 80/ethyl acetate to yield 64 mg (65%) of desired product.
1H NMR (300 MHz, chloroform-d) δ ppm 1.55−1.86 (m, 4 H) 2.01−
tert-Butyl (3R)-3-{[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-
purin-6-yl]amino}piperidine-1-carboxylate (15a). To a solution of
18 (100 mg, 0.267 mmol, 1 equiv) in 3 mL of ethanol was added tert-
butyl (3R)-3-aminopiperidine-1-carboxylate (64 mg, 0.32 mmol, 1.2
equiv) and triethylamine (0.06 mL, 0.4 mmol, 1.5 equiv). The reaction
was heated to 80 °C for 16 h. The reaction was concentrated in vacuo.
The crude material was purified by silica gel column chromatography
using 0−100% ethyl acetate/hexanes to yield 124 mg (86%) of desired
product. 1H NMR (300 MHz, chloroform-d) δ ppm 1.33 (br. s., 9 H)
1.49−1.67 (m, 2 H) 1.74 (d, J = 6.97 Hz, 1 H) 2.00 (d, J = 2.83 Hz, 1
H) 3.20 (br. s., 2 H) 3.50 (br. s., 1 H) 3.88 (br. s., 1 H) 4.32 (br. s., 1
H) 5.87 (d, J = 7.44 Hz, 1 H) 7.02−7.54 (m, 8 H) 8.38 (s, 1 H), [M +
H]+ 539.3.
2.14 (m, 1 H) 2.66−2.85 (m, 2 H) 2.87−3.04 (m, 1 H) 3.32 (dd, J =
11.77, 2.35 Hz, 1 H) 4.39 (br. s., 1 H) 6.20 (d, J = 5.84 Hz, 1 H)
7.12−7.66 (m, 8 H) 8.47 (s, 1 H), [M + H]+ 439.6.
8-(2-Chlorophenyl)-9-(4-chlorophenyl)-N-[(3R)-piperidin-3-yl]-
9H-purin-6-amine (15b). A solution of tert-butyl 15a (109 mg, 0.202
mmol) was stirred in dichloromethane (7 mL) and trifluoroacetic acid
(3 mL) for 16 h. The reaction was concentrated in vacuo. The crude
product was dissolved in ethyl acetate and washed with saturated
NaHCO3. The aqueous layer was extracted twice with ethyl acetate.
The combined organic layer was washed with brine and dried with
MgSO4. The crude material was purified by silica gel column
chromatography using 0−100% CMA 80/ethyl acetate to yield 70
(3S)-3-{[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
amino}-N-ethylpiperidine-1-carboxamide (16c). To a solution of
16b (16 mg, 0.036 mmol, 1 equiv) in 2 mL of THF was added
triethylamine (0.015 mL, 0.109 mmol, 3 equiv) and ethyl isocyanate
(0.004 mL, 0.055 mmol, 1.5 equiv). The reaction is stirred for 16 h.
The reaction was concentrated in vacuo. The crude material was
purified by silica gel column chromatography using 0−100% ethyl
1
1
mg (79%) of desired product. H NMR (300 MHz, chloroform-d) δ
acetate/hexanes to yield 16 mg (86%) of desired product. H NMR
ppm 1.43−1.82 (m, 4 H) 1.91−2.03 (m, 1 H) 2.67 (dt, J = 11.68, 7.16
Hz, 2 H) 2.76−2.94 (m, 1 H) 3.21 (dd, J = 11.82, 2.87 Hz, 1 H) 4.28
(br. s., 1 H) 6.11 (d, J = 6.12 Hz, 1 H) 6.98−7.57 (m, 8 H) 8.36 (s, 1
H), [M + H]+ 439.6.
(300 MHz, chloroform-d) δ ppm 1.06−1.34 (m, 3 H) 1.53−1.95 (m, 4
H) 2.12−2.29 (m, 1 H) 3.07 (br. s., 2 H) 3.32 (br. s., 1 H) 3.72−4.41
(m, 3 H) 5.06 (br. s., 1 H) 6.02 (d, J = 6.12 Hz, 1 H) 7.08 - 7.66 (m, 8
H) 8.45 (br. s., 1 H), [M + H]+ 510.3.
(3R)-3-{[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
amino}-N-ethylpiperidine-1-carboxamide (15c). To a solution of
15b (20 mg, 0.046 mmol, 1 equiv) in 2 mL of THF was added
triethylamine (0.02 mL, 0.137 mmol, 3 equiv) and ethyl isocyanate
(0.005 mL, 0.068 mmol, 1.5 equiv). The reaction is stirred for 16 h.
The reaction was concentrated in vacuo. The crude material was
purified by silica gel column chromatography using 0−100% ethyl
1-[(3S)-3-{[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
amino}piperidin-1-yl]ethan-1-one (16d). A solution of 16b (15.7 mg,
0.036 mmol, 1 equiv) in 1 mL of acetic anhydride and 1 mL of
pyridine was stirred for 16 h. The reaction was concentrated in vacuo.
The crude material was purified by silica gel column chromatography
using 0−100% ethyl acetate/hexanes to yield 14 mg (81%) of desired
1
product. H NMR (300 MHz, chloroform-d) δ ppm 1.48−1.91 (m, 3
1
acetate/hexanes to yield 15 mg (65%) of desired product. H NMR
H) 2.07 (s, 4 H) 2.90−3.39 (m, 2 H) 3.96−4.57 (m, 3 H) 5.83 (d, J =
6.50 Hz, 1 H) 7.04−7.56 (m, 8 H) 8.37 (s, 1 H), [M + H]+ 481.3.
8-(2-Chlorophenyl)-9-(4-chlorophenyl)-N-[(3S)-1-methanesulfo-
nylpiperidin-3-yl]-9H-purin-6-amine (16e). To a solution of 16b
(15.3 mg, 0.035 mmol, 1 equiv) in 2 mL of THF was added
methanesulfonyl chloride (0.005 mL, 0.07 mmol, 2 equiv) and
triethylamine (0.015 mL, 0.11 mmol, 3 equiv). The mixture was stirred
for 16 h. The reaction was concentrated in vacuo. The crude material
was purified by silica gel column chromatography using 0−100% ethyl
(300 MHz, chloroform-d) δ ppm 1.03−1.34 (m, 3 H) 1.56−1.96 (m, 4
H) 2.18 (br. s., 1 H) 3.07 (br. s., 2 H) 3.32 (br. s., 1 H) 3.73−4.42 (m,
3 H) 5.05 (br. s., 1 H) 6.02 (d, J = 5.84 Hz, 1 H) 7.09−7.63 (m, 8 H)
8.45 (br. s., 1 H), [M + H]+ 510.3.
1-[(3R)-3-{[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
amino}piperidin-1-yl]ethan-1-one (15d). A solution of 15b (20 mg,
0.046 mmol, 1 equiv) in 1 mL of acetic anhydride and 1 mL of
pyridine was stirred for 16 h. The reaction was concentrated in vacuo.
The crude material was purified by silica gel column chromatography
using 0−100% ethyl acetate/hexanes to yield 19 mg (87%) of desired
1
acetate/hexanes to yield 16 mg (89%) of desired product. H NMR
(300 MHz, chloroform-d) δ ppm 1.63−2.02 (m, 4 H) 2.77 (s, 3 H)
2.98−3.19 (m, 2 H) 3.31 (br. s., 1 H) 3.71 (d, J = 10.55 Hz, 1 H) 4.56
(br. s., 1 H) 6.01 (d, J = 7.54 Hz, 1 H) 6.97−7.56 (m, 8 H) 8.37 (s, 1
H), [M + H]+ 517.4.
1
product. H NMR (300 MHz, chloroform-d) δ ppm 1.53−1.92 (m, 3
H) 2.07 (br. s., 4 H) 2.84−3.37 (m, 2 H) 3.86−4.59 (m, 3 H) 5.88 (br.
s., 1 H) 7.04−7.60 (m, 8 H) 8.37 (br. s., 1 H), [M + H]+ 481.3.
8-(2-Chlorophenyl)-9-(4-chlorophenyl)-N-[(3R)-1-methanesulfo-
nylpiperidin-3-yl]-9H-purin-6-amine (15e). To a solution of 15b (20
mg, 0.046 mmol, 1eq.) in 2 mL of dichloromethane was added
methanesulfonyl chloride (0.007 mL, 0.091 mmol, 2 equiv) and
triethylamine (0.02 mL, 0.137 mmol, 3 equiv). The mixture was stirred
for 16 h. The reaction was concentrated in vacuo. The crude material
was purified by silica gel column chromatography using 0−100% ethyl
tert-Butyl N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-
6-yl]piperidin-4-yl}carbamate (17a). To a solution of 18 (309 mg,
0.824 mmol, 1 equiv) in 10 mL of ethanol was added tert-butyl N-
(piperidin-4-yl)carbamate (21) (329 mg, 1.65 mmol, 2 equiv). The
reaction was heated to 80 °C for 16 h. The reaction was concentrated
in vacuo. The crude material was purified by silica gel column
chromatography using 0−100% ethyl acetate/hexanes to yield 442 mg
(99%) of desired product. 1H NMR (300 MHz, chloroform-d) δ ppm
1.40−1.53 (m, 11 H) 2.12 (d, J = 11.40 Hz, 2 H) 3.32 (t, J = 11.63 Hz,
2 H) 3.80 (br. s., 1 H) 4.41−4.68 (m, 1 H) 5.40 (br. s., 2 H) 7.16−
7.24 (m, 2 H) 7.26−7.43 (m, 5 H) 7.51 (d, J = 6.59 Hz, 1 H) 8.38 (s, 1
H), [M + H]+ 539.2.
1
acetate/hexanes to yield 12 mg (51%) of desired product. H NMR
(300 MHz, chloroform-d) δ ppm 1.81−2.18 (m, 4 H) 2.77−2.94 (m, 3
H) 3.07−3.29 (m, 2 H) 3.42 (br. s., 1 H) 3.82 (d, J = 9.89 Hz, 1 H)
4.68 (br. s., 1 H) 6.13 (br. s., 1 H) 7.12−7.65 (m, 8 H) 8.48 (s, 1 H),
[M + H]+ 517.8.
tert-Butyl (3S)-3-{[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-
purin-6-yl]amino}piperidine-1-carboxylate (16a). To a solution of
18 (100 mg, 0.267 mmol, 1 equiv) in 3 mL of ethanol was added tert-
butyl (3S)-3-aminopiperidine-1-carboxylate (64 mg, 0.32 mmol, 1.2
equiv) and triethylamine (0.06 mL, 0.4 mmol, 1.5 equiv). The reaction
1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
piperidin-4-amine (17b). A solution of 17a (380 mg, 0.705 mmol)
was stirred in dichloromethane (7 mL) and trifluoroacetic acid (3 mL)
for 16 h. The reaction was concentrated in vacuo. The crude product
was dissolved in ethyl acetate and washed with saturated NaHCO3.
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The aqueous layer was extracted twice with ethyl acetate. The
combined organic layer was washed with brine and dried with MgSO4.
The crude material was purified by silica gel column chromatography
using 0−100% CMA 80/ethyl acetate to yield 294 mg (95%) of the
desired compound. Compound was determined to be 95% pure by 1H
General Procedure for Making Sulfonamides from 1-[8-(2-
Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-amine
(17b). To a solution of 17b (21 mg, 0.048 mmol, 1 equiv) in 2 mL of
THF was added triethylamine (0.02 mL, 0.143 mmol, 3 equiv) and the
appropriate sulfonyl chloride (2 equiv). The reaction is stirred for 16
h. The reaction was concentrated in vacuo. The crude material was
purified by silica gel column chromatography using 0−100% ethyl
acetate/hexane to yield pure compound.
1
NMR and was carried forward into further chemistry. H NMR (300
MHz, chloroform-d) δ ppm 1.31−1.54 (m, 2 H) 1.92−2.08 (m, 2 H)
2.92−3.12 (m, 1 H) 3.27 (t, J = 11.87 Hz, 2 H) 5.42 (br. s., 2 H)
7.07−7.61 (m, 8 H) 8.38 (s, 1 H), [M + H]+ 439.4.
General Procedure for Making Ureas from 1-[8-(2-Chlorophenyl)-
9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-amine (17b). To a
solution of 17b (20 mg, 0.046 mmol, 1 equiv) in 2 mL of THF was
added triethylamine (0.02 mL, 0.136 mmol, 3 equiv) and the
appropriate isocyanate (1.5 equiv). The reaction was stirred for 16 h.
The reaction was concentrated in vacuo. The crude material was
purified by silica gel column chromatography using 0−100% ethyl
acetate/hexane to yield pure compound.
N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
piperidin-4-yl}methanesulfonamide (17h). Reaction proceeded in
1
32% yield. H NMR (300 MHz, chloroform-d) δ ppm 1.55−1.67 (m,
2 H) 2.18 (d, J = 12.53 Hz, 2 H) 3.03 (s, 3 H) 3.36 (t, J = 12.29 Hz, 2
H) 3.59−3.80 (m, 1 H) 4.31 (d, J = 7.44 Hz, 1 H) 5.43 (d, J = 9.89
Hz, 2 H) 7.13−7.44 (m, 7 H) 7.51 (d, J = 6.78 Hz, 1 H) 8.39 (s, 1 H),
[M + H]+ 517.6.
N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
piperidin-4-yl}benzenesulfonamide (17k). Reaction proceeded in
1
1-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
piperidin-4-yl}-3-ethylurea (17c). Reaction proceeded in 56% yield.
1H NMR (300 MHz, chloroform-d) δ ppm 1.13 (t, J = 7.21 Hz, 3 H)
53% yield. H NMR (300 MHz, chloroform-d) δ ppm 1.43−1.59 (m,
2 H) 1.94 (d, J = 10.46 Hz, 2 H) 3.30 (t, J = 11.96 Hz, 2 H) 3.41−3.66
(m, 1 H) 4.62 (d, J = 7.54 Hz, 1 H) 5.26 (d, J = 10.08 Hz, 2 H) 7.07−
7.69 (m, 10 H) 7.92 (d, J = 7.35 Hz, 2 H) 8.35 (s, 1 H), [M + H]+
579.4.
1.33−1.57 (m, 2 H) 2.04−2.20 (m, 2 H) 3.14−3.25 (m, 2 H) 3.32 (t, J
= 12.06 Hz, 2 H) 3.86−4.05 (m, 1 H) 4.23−4.45 (m, 2 H) 5.39 (br. s.,
2 H) 7.03−7.59 (m, 8 H) 8.37 (s, 1 H), [M + H]+ 510.2.
Calcium Mobilization and Radioligand Displacement As-
says. Each compound was pharmacologically characterized using a
functional fluorescent CB1 activated Gαq16-coupled intracellular
calcium mobilization assay in CHO-K1 cells, as has been described
in our previous publications and apparent affinity (Ke) values were
determined.13 Briefly, CHO-K1 cells were engineered to coexpress
human CB1 and Gαq16. Activation of CB1 by an agonist then leads to
generation of inositol phospahatase 3 (IP3) and activation of IP3
receptors, which leads to mobilization of intracellular calcium. Calcium
flux was monitored in a 96-well format using the fluorescent dye
Calcein-4 a.m. in an automated platereader (Flexstation, Molecular
Devices). The antagonism of a test compound was measured by its
ability to shift the concentration response curve of the synthetic CB1
agonist CP55940 rightwards using the equation:
Ke = [Ligand]/[DR-1] where DR is the EC50 ratio of CP55940 in
the presence or absence of a test agent. Standard errors were between
5 and 25% of mean in most cases and have been left out for clarity.
Further characterization of select compounds was performed using
radioligand displacement of [3H]SR141716 and equilibrium dissoci-
ation constant (Ki) values were determined as described previously.13
Selectivity of these compounds at CB1 versus CB2 was also
determined by obtaining Ki values at either receptor using displace-
ment of [3H]CP55940 in membranes of CHO-K1 cells overexpressing
either receptor. Data reported are average values from 3 to 6
measurements. The standard errors for most measurements were
between 5 and 25% of mean and have been left out from the tables and
figures for clarity.
MDCK-mdr1 Permeability Assays. MDCK-mdr1 cells perme-
ability assays were preformed as previously described.11b,13 MDCK-
mdr1 cells obtained from Netherlands Cancer Institute were grown on
Transwell type filters (Corning) for 4 days to confluence in DMEM/
F12 media containing 10% fetal bovine serum and antibiotics as has
been described previously.13 Compounds were added to the apical side
at a concentration of 10 μM in a transport buffer comprising of 1×
Hank’s balanced salt solution, 25 mM D-glucose and buffered with
HEPES to pH 7.4. Samples were incubated for 1 h at 37 °C and
carefully collected from both the apical and basal side of the filters.
Compounds selected for MDCK-mdr1 cell assays were infused on an
Applied Biosystems API-4000 mass spectrometer to optimize for
analysis using multiple reaction monitoring (MRM). Flow injection
analysis was also conducted to optimize for mass spectrometer
parameters. Samples from the apical and basolateral side of the MDCK
cell assay were dried under nitrogen on a Turbovap LV. The
chromatography was conducted with an Agilent 1100 binary pump
with a flow rate of 0.5 mL/min. Mobile phase solvents were A, 0.1%
formic acid in water, and B, 0.1% formic acid in methanol. The initial
solvent conditions were 10% B for 1 min, then a gradient was used by
1-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
piperidin-4-yl}-3-(propan-2-yl)urea (17d). Reaction proceeded in
1
84% yield. H NMR (300 MHz, chloroform-d) δ ppm 1.14 (d, J =
6.50 Hz, 6 H) 1.35−1.55 (m, 2 H) 2.12 (d, J = 10.46 Hz, 2 H) 3.32 (t,
J = 12.10 Hz, 2 H) 3.84 (dd, J = 13.66, 6.69 Hz, 1 H) 3.89−4.04 (m, 1
H) 4.12−4.36 (m, 2 H) 5.39 (br. s., 2 H) 7.19 (d, J = 8.67 Hz, 2 H)
7.28−7.43 (m, 5 H) 7.51 (d, J = 6.69 Hz, 1 H) 8.37 (s, 1 H), [M +
H]+ 524.6.
1-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
piperidin-4-yl}-3-propylurea (17e). Reaction proceeded in 71% yield.
1H NMR (300 MHz, chloroform-d) δ ppm 0.83−0.98 (m, 3 H) 1.37−
1.59 (m, 4 H) 2.13 (d, J = 10.27 Hz, 2 H) 3.12 (q, J = 6.66 Hz, 2 H)
3.32 (t, J = 12.01 Hz, 2 H) 3.83−4.07 (m, 1 H) 4.20−4.47 (m, 2 H)
5.40 (br. s., 2 H) 7.13−7.24 (m, 2 H) 7.28−7.44 (m, 5 H) 7.51 (d, J =
6.69 Hz, 1 H) 8.37 (s, 1 H), [M + H]+ 524.1.
3-Butyl-1-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-
yl]piperidin-4-yl}urea (17f). Reaction proceeded in 69% yield. 1H
NMR (300 MHz, chloroform-d) δ ppm 0.84−0.99 (m, 3 H) 1.28−
1.55 (m, 6 H) 2.04−2.20 (m, 2 H) 3.15 (q, J = 6.75 Hz, 2 H) 3.32 (t, J
= 12.15 Hz, 2 H) 3.83−4.08 (m, 1 H) 4.18−4.39 (m, 2 H) 5.40 (br. s.,
2 H) 7.08−7.43 (m, 7 H) 7.51 (d, J = 6.69 Hz, 1 H) 8.37 (s, 1 H), [M
+ H]+ 538.4.
General Procedure for Making Amides from 1-[8-(2-Chlorophen-
yl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-amine (17b). To a
solution of 17b (21 mg, 0.048 mmol, 1 equiv) in 2 mL of THF was
added triethylamine (0.02 mL, 0.143 mmol, 3 equiv) and the
appropriate anhydride (2 equiv). The reaction is stirred for 16 h. The
reaction was concentrated in vacuo. The crude material was purified by
silica gel column chromatography using 0−100% ethyl acetate/hexane
to yield pure compound.
N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
piperidin-4-yl}acetamide (17g). Reaction proceeded in 65% yield. 1H
NMR (300 MHz, chloroform-d) δ ppm 1.42−1.56 (m, 2 H) 1.99 (s, 3
H) 2.12 (d, J = 10.08 Hz, 2 H) 3.30 (t, J = 12.24 Hz, 2 H) 4.09−4.25
(m, 1 H) 5.41 (d, J = 8.01 Hz, 3 H) 7.13 - 7.43 (m, 7 H) 7.51 (d, J =
6.78 Hz, 1 H) 8.38 (s, 1 H), [M + H]+ 481.4.
N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
piperidin-4-yl}-2,2,2-trifluoroacetamide (17i). Reaction proceeded in
1
39% yield. H NMR (300 MHz, chloroform-d) δ ppm 1.62 (qd, J =
11.99, 3.86 Hz, 2 H) 2.18 (d, J = 10.83 Hz, 2 H) 3.31 (t, J = 12.43 Hz,
2 H) 4.11−4.29 (m, 1 H) 5.53 (br. s., 2 H) 6.19 (d, J = 7.06 Hz, 1 H)
7.01−7.57 (m, 8 H) 8.40 (s, 1 H), [M + H]+ 535.4.
N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-
piperidin-4-yl}benzamide (17j). Reaction proceeded in 19% yield. 1H
NMR (300 MHz, chloroform-d) δ ppm 1.82−1.97 (m, 2 H) 2.77 (d, J
= 12.34 Hz, 2 H) 3.08 (br. s., 2 H) 3.46−3.62 (m, 1 H) 3.66−3.80 (m,
1 H) 4.01−4.13 (m, 1 H) 5.26−5.46 (m, 1 H) 7.12−7.59 (m, 13 H)
8.38 (s, 1 H), [M + H]+ 543.6.
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increasing to 95% B over 5 min, then returning to initial conditions.
Data reported are average values from 2 to 3 measurements.
In Vitro Stability Testing. Stability of compounds to plasma and
S9 fraction was preformed as previously described.13 In vitro testing for
metabolic stability was conducted in pooled samples of mixed gender
human plasma from BioChemed Services, Winchester, VA and human
mixed gender pooled hepatic S9 fraction supplied by Xenotech, LLC,
Lenexa, KS. Identity of the donors was unknown.
For the hepatic S9 metabolism studies, all samples were tested at 10
μM final concentration in a 1 mL volume containing 1 mg/mL S9.
Samples were incubated in a buffer containing 50 mM potassium
phosphate, pH 7.4 with 3 mM MgCl2 and a NADPH regeration
system comprising of NADP (1 mM), glucose-6-phosphate (5 mM)
and glucose-6-phosphate dehydrogenase (1 unit/mL). Triplicate
samples were incubated for 0, 15, 30, 60, and 120 min. Reactions
were terminated by addition of 3 vol of acetonitrile and processed as
described for the MDCK-mdr1 assays, but standard curves were
prepared in blank matrix for each compound for quantitative
assessment.
AUTHOR INFORMATION
Corresponding Author
*Tel: 919-541-6795. Fax: 919-541-8868. E-mail: rmaitra@rti.
■
org.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Ann Gilliam for performing
the binding assays, and Ms. Sherry Black and Ms. Purvi Patel
for help with metabolic stability studies. We express our
gratitude to the NIDA drug supply program for providing
radiolabeled probes and to Dr. Brian Thomas for supplying the
CB1 cells. This research was funded by research grants
1R21AA019740-01 and 1R03AA017514-01 to R.M. from
NIAAA.
ABBREVIATIONS USED
■
The plasma stability studies were conducted at 37 °C in a volume of
1 mL plasma per sample. All compounds were tested at 10 μM final
concentration at 0, 30, and 60 min after a 5 min preincubation.
Reactions were terminated by addition of acetonitrile and analyzed as
described above.
CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2;
CMA 80, 80% chloroform, 18% methanol, and 2% ammonium
hydroxide; CNS, central nervous system; BBB, blood−brain
barrier; TPSA, topological polar surface area; ECS, endocanna-
binoid system; CBR, cannabinoid receptors; Ke, apparent
affinity constant, MDCK-mdr1, Madin-Darby canine kidney
cells transfected with the human MDR1 gene; A, apical; B,
basal; BOP, benzotriazole-1-yl-oxytris(dimethylamino)-
phosphonium hexafluorophosphate; CHO-K1, Chinese ham-
ster ovary cells; IP3, inositol phospahatase 3; MRM, multiple
reaction monitoring; LOQ, below limit of quantitation; NA,
not applicable
Pharmacokinetic Evaluation in Vivo. Male Sprague−Dawley
rats aged 8 weeks at time of dosing were acquired from Charles River
Laboratories and were dosed orally. Three to four animals were used
per time point. Dose was formulated in food-grade corn oil at 10 mg/
kg. Plasma, liver, and brain were taken from all rats at 0.5, 1, 2, 4, and 8
h postdose. Samples were prepared and analyzed as follows: Plasma
(50 μL) was mixed with 10 μL of 10 μL of acetonitrile, and 300 μL of
acetonitrile, vortexed, and centrifuged at 9000g for 5 min. Super-
natants were transferred to autosampler vials with low-volume inserts
and injected without dilution. Brains were homogenized with 50:50
ethanol/water (3:1, v/v) using a Potter Elvehjem type homogenizer.
Homogenate (50 μL) was mixed with 10 μL of acetonitrile, and 300
μL of acetonitrile, vortexed and centrifuged at 9000g for 5 min.
Supernatant was transferred to autosampler vials with low-volume
inserts and injected without dilution. Standards were prepared as
above for each compound in blank plasma, blank liver homogenate,
and blank brain homogenate. Standards used were within 15% of
nominal; except for 20% at LOQ. Compounds for LC-MS/MS
analyses were supplied at 1 mg/mL in methanol. The stock solutions
were further diluted to ∼100 ng/mL. The 100 ng/mL solutions were
used to optimize the mass spectrometer for MRM transitions and mass
spectrometer parameters. Infusion and flow injection optimization
were also performed. LC-MS/MS was conducted using an Applied
Biosystems API 4000 coupled with an Agilent 1100 HPLC system.
Chromatography was performed with a Phenomenex Luna C18 (50 ×
2 mm, 5 μm) column. Mobile phases were 0.1% formic acid and 10
mM ammonium formate in water (A), and 0.1% formice acid and 10
mM ammonium formate in methanol. Initial conditions were 10% B
and held for one minute, followed by a linear gradient to 90% B over 5
min. 90% B was held for 2 min before returning to initial conditions.
Compound 12 was analyzed with multiple reaction monitoring in the
positive mode with a transition of 595.28 → 370.0. The following
parameters were used, DP = 116, CE = 45, CXP = 36, CAD = 4, CUR
= 10, GS1 = 40, GS2 = 60, IS = 4000, and TEM = 650.
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S
* Supporting Information
HPLC and melting point data of target compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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