Selective cannabinoid receptor type 2 (CB2) agonists: Optimization of a series of purines leading to the identification of a clinical candidate for the treatment of osteoarthritic pain

Article abstract of DOI:10.1021/jm400305d

A focused screening strategy identified thienopyrimidine 12 as a cannabinoid receptor type 2 agonist (hCB2) with moderate selectivity over the hCB1 receptor. This initial hit suffered from poor in vitro metabolic stability and high in vivo clearance. Structure-activity relationships describe the optimization and modification to a new more polar series of purine CB2 agonists. Examples from this novel scaffold were found to be highly potent and fully efficacious agonists of the human CB2 receptor with excellent selectivity against CB1, often having no CB1 agonist activity at the highest concentration measured (>100 μM). Compound 26 is a centrally penetrant molecule which possesses good biopharmaceutical properties, is highly water-soluble, and demonstrates robust oral activity in rodent models of joint pain. In addition, the peripherally restricted molecule 22 also demonstrated significant efficacy in the same analgesic model of rodent inflammatory pain.

Full text of DOI:10.1021/jm400305d

Article
pubs.acs.org/jmc
Selective Cannabinoid Receptor Type 2 (CB2) Agonists: Optimization
of a Series of Purines Leading to the Identification of a Clinical
Candidate for the Treatment of Osteoarthritic Pain
Sean P. Hollinshead,*,† Michael W. Tidwell,†,§ John Palmer,† Rossella Guidetti,‡ Adam Sanderson,‡
Michael P. Johnson,† Mark G. Chambers,† Jennifer Oskins,† Robert Stratford,†,∥ and Peter C. Astles‡
†Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States
‡Lilly Research Center, A Division of Eli Lilly and Company, Erl Wood Manor, Sunningdale Road, Windlesham, Surrey GU20 6PH,
U.K.
ABSTRACT: A focused screening strategy identified thienopyrimidine 12 as a
cannabinoid receptor type 2 agonist (hCB2) with moderate selectivity over the
hCB1 receptor. This initial hit suffered from poor in vitro metabolic stability
and high in vivo clearance. Structure−activity relationships describe the
optimization and modification to a new more polar series of purine CB2
agonists. Examples from this novel scaffold were found to be highly potent and
fully efficacious agonists of the human CB2 receptor with excellent selectivity
against CB1, often having no CB1 agonist activity at the highest concentration
measured (>100 μM). Compound 26 is a centrally penetrant molecule which
possesses good biopharmaceutical properties, is highly water-soluble, and
demonstrates robust oral activity in rodent models of joint pain. In addition, the peripherally restricted molecule 22 also
demonstrated significant efficacy in the same analgesic model of rodent inflammatory pain.
INTRODUCTION
■
Cannabinoids belong to a class of compounds which were
originally isolated from marijuana (Cannabis sativa L.) and
partially act at two well characterized cannabinoid receptors,
CB11 and CB2,2 both of which are G-protein coupled receptors
(GPCRs). The medicinal and psychoactive properties of
cannabinoids are well documented, and marijuana has been
used for centuries as a therapeutic and a recreational drug. It
has also been used for a variety of treatments including lack of
appetite, pain, emesis, spasticity, and rheumatism.3 The primary
active and most abundant constituent of marijuana is Δ9-
tetrahydrocannabinol 1 (THC, Figure 1), which was isolated
and synthesized in 1964.4 The medical use of THC (and other
cannabinoids) has been limited by the undesirable psychotropic
side effects and potential addictive properties associated with
these compounds.5
The two human CB1 and CB2 receptors share 44% overall
homology and exhibit different pharmacologic profiles.6 Both
receptors are composed of seven transmembrane proteins and
belong to the class A rhodopsin-like GPCRs coupling to
inhibitory Gi/o proteins. Human and rat CB1 and CB2
Figure 1. Structures of 1 (Δ9-tetrahydrocannabinol, THC), 2
(AM1241), 3 (GW842166X), and 4.
receptors show 97% and 81% amino acid sequence identity
across species, repectively.7
been implicated in the analgesic activity of marijuana.
Conversely, the CB1 receptor is expressed abundantly in the
central nervous system (CNS) with the highest density in
hippocampus, cerebellum, basal ganglia, and striatum10 and
The CB2 receptor was thought to be expressed primarily in
peripheral immune cells8 such as B-cells, T-cells, monocytes,
macrophages, and in organs such as the spleen, pancreas,
thymus, lung, and tonsil. More recently, expression in the
central nervous system (CNS) has been demonstrated albeit to
a much lesser extent9 and activation of the CB2 receptor has
Received: February 27, 2013
© XXXX American Chemical Society
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Figure 2. Optimization of screening hit 12 and elaboration to purine scaffold as exemplified by 13 and 4.
a
Scheme 1. General Synthesis of Purine Analogues
a
Reagents and conditions: (i) R1-NH2, IPA, heat; (ii) 15% FeCl3 on SiO2, dioxane, heat; (iii) DDQ, DCM; (iv) amine, EtOH−THF (4:1 v/v), heat;
(v) acetone, 1 M aqueous HCl.
CB1 activation is believed to be responsible for the unwanted
side effects such as ataxia, hypothermia, and euphoria.
inherently lipophilic in nature with poor pharmacokinetic (PK)
properties, but the compounds which are the subject of more
recent disclosures have much improved properties (for example
lower log P) and also encompass a high degree of structural
diversity.22 Despite this progress, no selective CB2 agonist has
been approved for the treatment of pain. It remains unclear
from preclinical studies whether agonism of the central CB2
receptor represents a clinically useful approach.24−29
CB2 is upregulated “on demand” in the CNS and periphery
during early inflammatory events and under pathological pain
conditions8,9a,11 A recent study12 also provides the first
evidence for the localization of CB2-receptor like immunor-
eactivity in human DRG sensory neurons in vitro and,
importantly, colocalization with TRPV1 receptors in injured
nerve fibres in human osteoathritic synovium. There are several
lines of evidence to suggest CB2 receptors in tissues such as
skin and the synovial membrane surrounding the knee joint are
expressed in small and medium diameter nociceptive fibers.
The CB2 receptor is upregulated at these locations in painful or
inflammatory conditions and it has been shown that CB2
agonists block the activation of these nerve fibres,12 suggesting
this may be a useful target for the treatment of osteoarthritic
(OA) pain.
Additionally, cannabinoid CB2 agonists have been shown to
suppress nociceptive transmission in rodent models of chronic
pain with the effects being reversed by selective CB2
antagonists or absent in CB2 knockout mice.11,13−15
Importantly, CB2-selective agonists lack the centrally mediated
side effects associated with activation of CB1 receptors
including hypoactivity, hypothermia, and catalepsy.16,17 It has
been hypothesized therefore that a selective CB2 agonist could
be therapeutically useful in the treatment of pain but devoid of
the unwanted CB1-mediated CNS side effects.
Two examples of well characterized CB2 agonists include
indole 2 (AM1241), which has demonstrated efficacy in
inflammatory and neuropathic pain models,30,31 and pyrimidi-
necarboxamide 3 (GW842166X),15 which was entered into
clinical trials for the treatment of acute inflammatory pain
following third molar extraction (Figure 2); however, this drug
candidate has since been discontinued due to lack of efficacy.32
One potential reason for this failure which was offered by the
authors related to the duration of drug exposure and occupancy
at the CB2 receptor. There remains then the need for further
research into the role of CB2 agonists in the treatment of
different chronic pain conditions and the provision of CNS
penetrant molecules that are highly selective for CB2 over CB1.
The purine molecule 4 (Figure 1) represents a previously
reported example from our own internal research efforts
directed toward the discovery of such molecules.33 This
compound 4 penetrates into the brain and shows no
measurable CB1 agonist activity in vitro and demonstrates
robust oral activity in rodent models of joint pain. In this paper,
we present the SAR and optimization of this series of purine
molecules to identify candidate molecules competent to test the
utility of selective CB2 agonists in clinical studies.
In support of this hypothesis, there have been a growing
number of reports from various academic and industrial groups
on the development and in vivo evaluation of selective CB2
agonists and this subject has been the subject of several
reviews.18−23 Historically these ligands have tended to be
B
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CHEMISTRY
■
The main synthetic route to obtain the desired purine
analogues in Tables 1−2 has been previously described33 and
is briefly outlined in Scheme 1.
Typically, the commercial starting material 5-amino-4,6-
dichloro-2-methyl pyrimidine 5 is reacted with an amine (R1-
NH2) to afford the monoamino substituted intermediates 6.
Subsequent reaction with aldehydes 7 is followed by cyclization
promoted by FeCl3−SiO2 according to the procedure of Dang
et al.34 This was typically followed by DDQ oxidation to ensure
complete conversion to the desired purines 8. Subsequent
reaction with amines 9 gave the desired products 10. Those
analogues containing basic amine functionality could then be
converted to the corresponding HCl salt 11 if required.
Figure 3. (A) Effect of CB2 agonist 12 (dosed po 2 h prior to testing)
and (B) blockade of the response by a CB2 selective antagonist40 (SR-
144528, dosed ip 30 min prior to dosing with 12) in the MIA model of
pain.39 Values are the mean SEM and group size N = 5−6. (**/***
p < 0.01/p < 0.001 One-way ANOVA followed by Dunnett’s test
versus vehicle).
RESULTS AND DISCUSSION
■
Our internal efforts to identify unique CB2 agonists
commenced with the screening of a 130K compound collection
(biased toward known GPCR ligands) in both human CB1 and
CB2 binding assays. We selected actives on the basis of affinity
for hCB2 (Ki < 1 μM), >10× selectivity over hCB1, a lack of
promiscuity against the panel of GPCRs, and finally CNS drug-
like properties.35 The actives from this screen were then
evaluated for agonist activity in a functional GTPγS hCB2
binding assay validated in SF9 cells or CHO cell homogenates
expressing hCB2 (Perkin-Elmer, Boston, MA) using a
scintillation proximity method.36 Selectivity over CB1 was
subsequently determined in a corresponding GTPγS hCB1
functional assay. These assays were used to drive the structure−
activity relationship (SAR) determination described in this
paper. Relative potency and selectivity across species was
determined from displacement of [3H]-CP-55,940 (a non-
selective CB1/2 agonist) from CHO cell homogenates
transiently expressing the rat or human, CB2 or CB1
receptors.37
The major routes of metabolism of 12 were established to be
N-dealkylation A, hydroxyethylation B, and aromatic oxidation
C by metabolic ID studies (Figure 4). An initial SAR focus was
to reduce the lipophilicity of the scaffold (while also improving
CB2 efficacy and selectivity against CB1) in an early attempt to
reduce hepatic microsomal metabolism (94%, 73%, and 97%
metabolized in rat, human, and dog species, respectively41)
before removing or blocking the labile sites.42
A number of more polar scaffolds were investigated as
alternatives to the thienopyrimidine, and this approach led to
the identification of the purine core as exemplified initially by
13 (see Figure 2) as an alternate structural motif. Compound
13 has a lower measured log P (3.1) when compared with 12
(log P = 3.8) and was a 5-fold more potent CB2 agonist (hCB2
EC50 = 4.6 nM) relative to 12 (see Table 1). This modification
also made 2-fold improvement in the selectivity over the CB1
receptor (CB1/2 = 206). Additionally, this compound
displayed high human and rat affinity as determined from
displacement of [3H]-CP-55,940 from CHO cell homogenates
transiently expressing human or rat CB2 receptors (h and r
CB2 Ki = 8.6 and 8.2 nM, respectively).37 Encouragingly, 13
retained excellent aqueous solubility characteristics (>100 μM
in simulated GI fluid pH 6.8) and an IC50 > 10 μM against a
panel of CYP enzymes. The Ki against the hERG ion channel
was >10 μM and displayed an improved metabolic profile (7%,
30%, and 88% metabolized in human, rat, and dog species,
respectively)41 as compared directly with 12 (see Table 1).
As outlined in our previous publication, a substituent in the
ortho position on the phenyl ring was established early in our
SAR studies to be optimal for CB2 potency.33 This group was
retained during our initial SAR studies on the purine scaffold
(Table 1). The next focus for SAR investigation was the R1
substituent on the purine nitrogen. Removal of this R1
substituent gave compound 14, which maintained CB2 potency
and a moderate selectivity over CB1. We considered the
introduction of alternative R1 substituents in a further attempt
to reduce the log P (and which would also hopefully impact
metabolism and in vivo clearance). This tactic and specific
changes in the R1 substituent was found to be a very effective
way to modulate selectivity over CB1 and also the
pharmacokinetic (PK) properties of the target molecules. For
example, the alcohol 15 maintained excellent potency and
encouragingly also increased the separation between CB2 and
CB1 agonism (CB1/CB2 = 760). The related methyl ether 16
This line of investigation led to the initial discovery of
thienopyrimidine 12 (Figure 2) which displayed high human
and rat affinity (h and r CB2 Ki = 82 and 16 nM, respectively)
and potent agonist activity (hCB2 EC50 = 24.8 nM), albeit with
modest selectivity over CB1 (104 fold).33 Historically, most
cannabinoid receptor ligands tend to be highly lipophilic and
we were encouraged that 12 contained a basic center which
conferred very good aqueous solubility (>200 μM, simulated
GI fluid pH 6.8) but did not have an affinity for the hERG ion
channel (IC50 > 10 μM, as measured in a displacement assay
using hERG channels stably expressed in HEK293 cells and
radiolabeled [3H]-dofetilide).38 Compound 12 possessed a high
oral bioavailability in rat (F = 91%), however the rate of
clearance was high (CL = 90 mL/min/kg, iv dose of 1 mg/kg).
In addition, 12 inhibited CYP2D6 activity with an IC50 = 3 μM
and also showed moderate serotonin binding most notably to
5HT1A and 1B (Kis ∼ 1 μM).
Compound 12 demonstrated in vivo efficacy in a rodent
monoiodoacetic acid (MIA) model39 of osteoarthritic (OA)
pain and significantly reduced pain compared to vehicle at a
dose of 10 mg/kg po (see Figure 3A).
This effect was reversed upon treatment of a selective CB2
antagonist SR-144528,40 confirming that this analgesic effect is
mediated by CB2 receptors (Figure 3B). Overall, compound 12
offered an attractive starting point for optimization with the
potential for improving in vitro CB2 potency, selectivity over
CB1, and in vivo clearance characteristics.
C
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Figure 4. Metabolic analysis of pooled plasma samples of 12 (10 mg/kg po, 2 h time point).
(R1 = CH2CH2OMe, R3 = Et) was a potent CB2 agonist (EC50
= 6.8 nM) and showed no measurable CB1 activity at the
highest concentration measured (CB1 EC50 > 100000 nM) and
translated into a >14700-fold selectivity over CB1. Other
substituents at R1 were also investigated including an amide
(20), lactam (21), and sulphone (22). CB2 potency was
reduced, however metabolic stability was generally improved
across species, resulting in an improvement in oral exposure.
Although accomplishing a reduction in clogP and the desired
improvement in metabolism and clearance, we found that these
R1 groups generally reduced brain penetration and in some
cases made the analogues substrates for the PgP efflux
transporter (see discussion below for compound 22).
We established that R3 = H was inactive (data not shown),
and so the R3 group was modified with the goal of stabilizing
this site to metabolism. Compound 16 and the fluoroethyl
analogue 17 possessed similar CB2 potency and excellent CB1
selectivity (CB2 EC50 = 4.5 nM, CB1 EC50 > 100000 nM,
CB1/CB2 > 22222). This change did not improve in vitro
metabolic stability, and when both compounds were dosed
orally to rats at 3 mg/kg po, AUC values were similarly low
(<50 ng·h/mL). The N−Ac analogue 18 interestingly was a
potent CB2 agonist but behaved as an inverse agonist at CB1,
illustrating a subtlety in the functional effects at this position.
Metabolism however was not impacted by this change and the
bicyclic amide 19 prepared as a yet more stable replacement
similarly did not affect metabolic stability.
Having identified R1 as a position to impact PK and CB1
selectivity, we continued investigating additional substitutions
at this position (Table 1). The cyclopropyl compound 23 had
excellent CB2 potency (hCB2 EC50 = 4.1 nM) and very high
selectivity over CB1 (CB1 EC50 > 100000 nM, CB1/CB2 >
24390). Oral activity in the MIA pain model was demonstrated
at doses as low as 0.1 mg/kg po although the increase in
lipophilicity (clogP = 4.0) resulted in a high measured clearance
in rat and a high volume of distribution (CL = 83 mL/min/kg,
Vd = 12.4 L/kg, 1.0 mg/kg iv). We next prepared cyclic ethers
4, 24−26. The pair of 3-amino-tetrahydrofuryl enantiomers (4
and 24) were evaluated, and the (R)-isomer 4 was found to be
more potent than the corresponding (S)-isomer 24. Thus
compound 4 had a hCB2 EC50 = 8.8 nM and CB1/CB2
selectivity > 11364 with moderate metabolic turnover across
rat, human, and dog species. Relative to 23, the clearance and
volume of distribution values of 25 in rat were much improved
(CL = 30 mL/min/kg, Vd = 5.0 L/kg) and oral bioavailability
was moderate (F = 27%, dosed at 1 mg/kg). For compounds
25 and 26, excellent CB2 agonist activity (hCB2 EC50 = 11.9
and 20.1 nM, respectively) and selectivity over CB1 were
observed (both hCB1 EC50 > 100000 nM). Compound 25
demonstrated high clearance in rat (CL = 47 mL/min/kg) and
low bioavailability (<20%); however, 26 exhibited very good
PK parameters across species and the further profiling of this
compound will be discussed below.
With the R1 and R3 groups optimized in compound 26, we
returned to an investigation of the phenyl substitution R2
(compounds 27−37), and results are summarized in Table 2.
In general, the ring was insensitive to the electron donating or
withdrawing character of the substituent. Preferred substituents
in the 2-position include Cl, CF3, Me, and Et with F, OCH3,
and CN being less active. All substitutions at the 3-or 4-
positions reduced activity.
The initial screening hit 12 and optimized compounds 22
and 26 have been evaluated in vivo for efficacy in pain models
(see below for compounds 22 and 26), and confirmation of
low/no activity as CB1 agonist or antagonists in a rat native
tissue assay was desirable. Thus these three compounds were
tested for stimulation or inhibition of CP-55,940 induced
GTPγS binding in rat cerebellar homogenates43 that has
previously been characterized as a CB1-mediated activity. As
observed in the human clonal cell lines, compounds 12, 22, and
26 were all inactive in stimulating GTPγS (EC50 > 100000 nM)
or inhibiting CP-55,940 stimulated GTPγS binding (IC50
>
10000 nM). Thus these compounds appear to be highly
selective for the CB2-receptor in both human and rat.
Cross Reactivity of Target Compounds. We had
originally noted some off-target cross reactivity with our initial
screening hit 12, most notably against 5HT1A and 1B receptors
(Ki = 1.4 and 1.2 μM, respectively). However, the logical
progression of our SAR as described toward molecules having a
lower clogP also tended to provide compounds which were
largely clean against a broad panel of GPCR (including 5HT),
enzyme, and ion channel targets (data not shown).44
Pharmacokinetic Profiles in Rat and Dog. As
summarized in Table 3, compounds 26 and 22 displayed
moderate clearance (CL = 43 and 25 mL/min/kg, respectively)
and half-lives (2.4−7.4 h) in male SD rats when dosed iv and
which translated to acceptable oral bioavailability (67 and 43%,
respectively). In male beagle dogs, compound 26 had a low
clearance (18 mL/min/kg) and long half-life (7.4 h). Clearance
was moderate for 22 (35 mL/min/kg) and also had a
moderately long half-life (5 h). Both compounds had excellent
solubility as the hydrochloride salts (>100 μM in simulated GI
fluid at pH6.8) and measured IC50s at a panel of CYP enzymes
were >10 μM. However, these compounds varied in their levels
of CNS penetration, making them interesting tools to explore
analgesic efficacy. Compound 26 is not a substrate for PgP45
and is highly brain penetrant (brain/plasma = 0.9), with 17%
free fraction in rat plasma and 3% free fraction in brain. In
contrast, compound 22 is a substrate for PgP46 and brain
exposure is negligible, limited by PgP mediated efflux.
Profile of Compounds in in Vivo Animal Models.
Compound 26 was shown to be potent and efficacious in rat
D
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Table 1. Functional GTPγS and Binding Data for CB2 and CB1 Receptors and in Vitro Microsomal Stability for Compounds 4
a
and 13−26
a
b
[35S]-GTPγS and binding data results represented are the mean values SEM. Data without SEM are an n of 1. Number in parentheses is the
c
d
e
measured log P. Values and SEM are a result of n = 4 or more experiments. Values and SEM are a result of n = 2−3 experiments. ND = not
determined. Inverse agonist
f
models of knee-joint related chronic pain induced by intra-
articular monoiodoacetic acid (MIA).39 A dose related reversal
of pain is observed in this MIA pain model (see Figure 5), with
a 0.3 mg/kg po dose of 26 demonstrating an equivalent degree
of efficacy to the NSAID diclofenac, a standard of care for
osteoarthritic pain. As observed with compound 12, the efficacy
seen with a 1 mg/kg dose of 26 was completely blocked by
pretreatment with the CB2 selective antagonist SR-14452840
(data not shown).
In support of a lack of central CB1 activity oral
administration of 26 did not induce hypothermia or behavioral
impairment in a rotorod study47 in rats up to 30 mg/kg po. In
contrast, the nonselective cannabinoid agonist CP-55,940 (0.5
mg/kg ip) tested as a positive control, caused a significant
hypothermia and impaired performance on the rotorod,
E
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Table 2. Functional GTPγS and Binding Data for CB2 and CB1 Receptors and in Vitro Microsomal Stability for Compounds
a
27−37
CB2 and CB1 GTPγS assays36
CB2 binding assay37
microsomal stability41
compd
no.
hCB2 EC50
(nM)
ratio
hCB2 Ki
rCB2 Ki
R2
3-Cl
clogP
hCB2 eff (%)
hCB1 EC50 (nM)
3010 180
hCB1 eff (%) CB1/2
(nM)
(nM)
hum % rat % dog %
b
b
27
28
29
30
31
32
33
34
35
36
37
3.5
3.8
3.8
2.7
2.7
2.8
3.1
3.7
3.4
3.9
3.2
71.4 1.1
13.6 7.1
53.1 7.1
97.4 9.5
108 11.5
106 27.1
82.7 2.2
21.3 4.1
15.9 5.2
14.1 1.3
82.4 22.5
91.3 4.6
77.3 2.9
91.6 4.1
57.3 2.8
86.7 3.4
65.7 1.4
74.9 2.0
59.3 10.5
90.7 1.4
94.2 1.7
41.5 9.1
67.0 2.0
42.2
>7353
318
ND
ND
25
24
19
22
40
20
10
33
18
33
21
45
38
28
35
30
21
30
67
39
51
24
45
61
44
36
46
11
21
69
39
74
41
2-CF3
3-CF3
2-OCH3
3-OCH3
2-CN
>100000
0
62.8
40.4
b
b
16900 12000
12200 4880
22900 16800
74.0 13.7
61.1 9.4
67.8 17.9
ND
ND
b
b
125
ND
ND
b
b
212
ND
ND
b
b
>1000000
0
>943
73.1
ND
ND
b
b
2-F
6050 5080
51.5 18.7
ND
ND
2-CycPro
2-Me
>100000
0
0
0
0
>4695
>6289
>7092
>1213
56.3
104
31.8
21.8
87.1
60
>100000
>100000
>100000
2-Et
b
b
2-Me-4-
OMe
ND
ND
a
b
[35S]-GTPγS, binding data and SEM results were calculated based on at least three experiments. Data without SEM are for n = 1. ND = not
determined.
Table 3. Pharmacokinetic (PK) Properties for Compounds 26 and 22
compd
species
CL (mL/min/kg)
Vd (L/kg)
T1/2 po (h)
AUC (ng·h/mL)
Cmax (ng/mL)
bioavailability (%)
a
26
rat PK
43
18
25
35
6
2.4
7.4
1.4
5
265
1,940
312
60
352
76
67
76
43
60
b
dog PK
5.5
1.9
a
22
rat PK
b
dog PK
9.2
286
62
a
b
Rat (Male Sprague−Dawley) 1 mg/kg iv, 1 mg/kg po. Dog (Beagle) 1 mg/kg iv, 3 mg/kg po.
We were interested in testing the peripherally restricted CB2
agonist 22 in the in vivo MIA model of rat joint pain and were
surprised to find that a 0.3 mg/kg po dose of 22 demonstrated
an equivalent degree of efficacy (see Figure 6). Similarly, 22
also did not induce hypothermia48 in rats up to 30 mg/kg po
(data not shown).
While this data may suggest that CNS penetration is not
required for efficacy of a CB2 agonist in the MIA model of
osteoarthritic pain, whether this would similarly be true in any
other preclinical models of pain or spinal pain transmission will
require further study and relevance to any human pain
condition remains unknown at this time.
CONCLUSIONS
■
Figure 5. Dose dependent effect of 26 (dosed by oral gavage 1 h prior
to testing) in the MIA model.39 Compound 26 at 0.1, 0.3, and 1 mg/
kg significantly reduced weight bearing deficits compared to vehicle as
did diclofenac at 5 mg/kg. The 1 mg/kg dose of 26 was significantly
different from diclofenac whereas the 0.1 and 0.3 mg/kg doses of 26
were not. Values are presented as mean SEM, N = 5. (*/***p <
0.05/0.001 vs vehicle, One-Way ANOVA followed by Tukey HSD.
From the initial starting thienopyrimidine 12, we have
discovered and optimized a novel series of purines as orally
bioavailable, selective CB2 agonists. The results have
demonstrated that the selectivity against CB1 could be
modulated by a judicious choice of R1 substituent, and several
highly selective CB2 agonists were prepared (many having no
measurable in vitro CB1 agonist activity up to 100 μmol).
Finally, the in vitro and in vivo clearance characteristics of this
series of purines were optimized, and compound 26 emerged as
a leading molecule demonstrating good CNS penetration and
thereby confirming the high selectivity for CB2 in vivo (data
not shown).48
F
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Wheatgerm Agglutinin SPA bead (GE Healthcare, Piscataway, NJ) was
added, and the plates were sealed, vortexed, and incubated for an
additional hour. Plates were then centrifuged at 700g for 10 min and
amount of bound [35S] using a Wallac MicroBeta TriLux scintillation
counter (PerkinElmer, Boston, MA). Similarly, hCB2-Sf9 membranes
(Perkin-Elmer, Boston, MA) and GDP (1 μM final, Sigma, St Louis,
MO) were added to assay buffer above and homogenized with a
polytron. Diluted compounds and membranes were added to the assay
plate and preincubated for 15 min at room temperature. This was
followed by addition of [35S]-GTPγ (500 pM final) for an additional
35 min. Subsequently, a mixture containing Nonidet P40 detergent
(0.2% final, Roche, Indianapolis, IN), anti-Gi antibody (final dilution
of 1:362; Covance, Princeton, NJ), and 1.25 mg antirabbit antibody
scintillation proximity assay beads (GE Healthcare, Piscataway, NJ)
was added. The plates were then sealed, vortexed, and incubated for an
additional 2 h before centrifugation and determination of [35S] bound
as above. Percent agonist effect was determined by normalizing the
agonist/inverse agonist dose response data to the response seen with
the CB1/2 agonist (methanandamide). Analysis of the resulting
sigmoidal shaped curve using a four-parameter logistic reduced fit with
Activity Base and XLFit3 (IDBS, Emeryville, CA) resulted in the
reported EC50 and percent relative efficacy. Ability to stimulate [35S]-
GTPγ binding or inhibit CP-55,940 stimulated [35S]-GTPγ binding in
rat cerebellar homogenates was determined using the methods of
Griffin, et al.43
Figure 6. Dose dependent effect of CB2 agonist 22 (dosed by oral
gavage 1 h prior to testing) in the MIA model.39 Both doses of
compound 22 significantly reduced weight bearing deficits and the 1
mg/kg doses was significantly more efficacious than 5 mg/kg
diclofenac. Values are the mean SEM, N = 6 (*** p < 0.001 vs
vehicle, One-Way ANOVA followed by Tukey−Kramer HSD).
Monoiodoacetate (MIA) Model.39 For all studies, male Lewis
rats of approximately 8 weeks of age at the time of MIA injection were
used to measure pain in the MIA model. The rats were housed in
groups of 2 or 3 per cage and maintained in a constant temperature
and on a 12 h light/12 h dark cycle. Animals had free access to food
and water at all times except during data collection.
potent oral activity in a preclinical model of joint pain. This
compound showed no central CB1 agonist effects as measured
in preclinical models and demonstrated an overall safety profile,
allowing evaluation in clinical studies of OA pain. The results of
these studies will be reported in subsequent communications
from these laboratories.
In the MIA model, the right knees of each rat were injected with 0.3
mg of MIA in 50 μL of saline and the left knees with 50 μL of saline.
Pain was measured at varying times after MIA injection (not normally
before 10 day post MIA injection) using incapacitance testing. This
measured the difference in hind paw weight bearing between the MIA
and saline injected knees, and each measurement was the average of
three separate measurements each measured over 1 s.
For studies with CB2 agonists, rats were randomized into dose
groups (n = 5 or 6) and then dosed once with the compound under
investigation. Dosing was staggered by 15 min for each rat and at a
predetermined time postdose (usually corresponding to the Tmax),
pain measured using incapacitance testing. Studies were routinely run
with fourgroups, vehicle (1% carboxy methyl cellulose in water plus
0.25% polysorbate 80), and three compound groups which can be
either single compounds at a single dose or the same compound at
three doses. Results were reported as the difference in weight bearing
between saline and MIA injected knees and statistical comparisons
were made between vehicle treated and compound treated animals to
assess the effect of compounds on knee pain in the model.
Chemistry: General Methods. All reagents and anhydrous
solvents were obtained from commercial sources and used without
further purification unless noted otherwise. 1H NMR spectra were
recorded on a Bruker 400 MHz spectrometer. 1H NMR chemical
shifts are reported in ppm with the solvent resonance as the internal
standard (CDCl3 7.26 ppm, methanol-d4 3.31 ppm, DMSO-d6 2.49
ppm).
Optical rotations were measured with a 341 polarimeter (Perkin-
Elmer) at 20 °C and at 589 nm (sodium lamp).
Compounds were analyzed for purity by HPLC and HPLC-MS and
purities of synthesized compounds were all found to be >95% by
methods 1 and/or 2, as specified below.
EXPERIMENTAL SECTION
■
Radioligand Binding Assays. Displacement of [3H]-CP-55,940
from human and rat CB2 receptors. The methods of Felder et al. were
utilized with minor modifications.37 Specifically, membrane homoge-
nates from cells stably or transiently expressing the human or rat CB2
receptor were washed by centrifugation and diluted into a 50 mM Tris
HCl (pH 7.4), 5 mM MgCl2, 2.5 mM EDTA, and 0.1% BSA 20 buffer.
Specific binding of [3H]-CP-55,940 was defined with 1 μM CP-55,940.
The ability of compounds to displace specific [3H]-CP-55,940 binding
was tested over a range of concentrations in the Tris, MgCl2, EDTA,
and BSA buffer in the presence of 1% dimethyl sulfoxide by incubating
at room temperature for 90 min in a volume of 300 μL. Unifiltered 96-
well microplates pretreated with 0.5% polyvinylpyrrolidone, 0.1% 25
polysorbate 20 in water were washed three times with cold Tris buffer.
The reaction mixture was then transferred to the filter plate
immediately before terminating the incubation by rapid filtration,
and three 200 μL washes with cold Tris buffer. After the filter plates
were dried, microscint 20 (Perkin-Elmer, Boston, MA) was added to
each well, the plate sealed, and counted for determination of
disintegrations per minute. The displacement curves were graphed
and the resulting Ki values determined utilizing Graphpad Prism.
CB1 and CB2 in Vitro Functional Assays. Exemplified
compounds are tested for agonist activity against cloned human
CB1 and CB2 receptors using a SPA (scintillation proximity assay)
based [35S]-GTPγ binding assay.36 All assay components were
prepared in assay buffer made up of 20 mM HEPES (pH 7.4), 100
mM NaCl, 5 mM MgCl2, and 0.125% bovine serum albumin. [35S]-
GTPγ binding was measured in a 96-well format using a whole
membrane capture technique for the CB1 assay and a modified
antibody capture technique previously described for the CB2 assay.36
Human CB1-CHO membranes (Applied Cell Sciences, Rockville,
MD), GDP (1 μM final, Sigma, St Louis, MO), and saponin (10 μg/
mL final, Sigma, St Louis, MO) were added to assay buffer and
homogenized with a Brinkman KINEMATICA AG homogenizer.
Diluted compounds, [35S]-GTPγ (500 pM final, PerkinElmer Life
Sciences, Boston, MA), and membranes were added to the assay plate
and incubated for 30 min at room temperature. Then 1 mg/well
HPLC-MS Method 1. Agilent 1100 HPLC, Phenomenex Gemini-
NX 2 mm × 50 mm × 3 μm C18 110A column; solvent A, 95/5
water/acetonitrile with 10 mM ammonium bicarbonate pH = 10;
solvent B, acetonitrile; gradient 5−100% B in 3 min with 0.75 min
hold of 100% B. Flow rate 1.0 mL/min. Atmospheric pressure
ionization-electrospray (API-ES) mass spectra data were acquired with
an Agilent Technologies MSD single quadrupole coupled to an
HP1100 LC system. DAD 200−400 nM scan.
G
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HPLC-MS Method 2. Agilent 1100 HPLC, Phenomenex Gemini-
NX 2 mm × 50 mm × 3 μm C18 110A column; solvent A, water with
0.1% formic acid; solvent B, acetonitrile with 0.1% formic acid;
gradient 5−100% B in 3 min with 0.75 min hold of 100% B. Flow rate
1.0 mL/min. Atmospheric pressure ionization-electrospray (API-ES)
mass spectra data were acquired with an Agilent Technologies MSD
single quadrupole coupled to an HP1100 LC system. DAD 200−400
nM scan.
Chiral HPLC Method 1. HPLC, Chiralpak AD-H 4.6 mm × 150
mm eluting with 20% methanol (containing 0.2% iso-propyl amine) in
CO2. Flow rate 5 mL/min. λ = 225 nM.
Chiral HPLC Method 2. HPLC, Chiralpak AS-H 4.6 mm × 150 mm
eluting with 0.2% N,N-dimethylethylamine in methanol. Flow rate 1.0
mL/min. λ = 225 nM.
10 (R1 = Me, ortho-Cl, R3 = Et) as a white solid (0.5 g, 78%). HPLC
= 100% at 2.1 min by HPLC-MS method 1, mass spectrum (m/z):
371 (M + 1). H NMR (400 MHz, CD3OD) 7.52−7.60 (m, 3H),
1
7.44−7.48 (m, 1H), 4.22−4.32 (br, 4H), 3.54 (s, 3H), 2.55 (t, 4H),
2.51 (s, 3H), 2.44 (q, J = 7.35 Hz, 2H), 1.10 (t, J = 7.25 Hz, 3H).
Hydrochloride Salt Formation, General Procedure D: 8-(2-
Chlorophenyl)-6-(4-ethylpiperazin-1-yl)-2,9-dimethyl-purine
Hydrochloride (13). As an example, 8-(2-chlorophenyl)-6-(4-ethyl-
piperazin-1-yl)-2,9-dimethyl-purine 10 (R1 = Me, ortho-Cl, R3 = Et)
(118 mg) was dissolved in a 1:1 mixture of acetone−acetonitrile (6
mL), and a 1 M solution of HCl in ether (1.1 equiv, 0.4 mL) was
added. The reaction mixture was stirred at room temperature for 1 h,
and the solvent was removed. The residue was dissolved in water (1
mL) and lyophilized overnight to afford the title compound 13 (0.13
g, 100%) as a white solid. HPLC = 100% at 2.16 min by HPLC-MS
8-(2-Chlorophenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-9-
[(3R)-tetrahydrofuran-3-yl]purine Hydrochloride (4). The title
compound was prepared by the general methods A−D (see below).
HPLC = 100% at 2.30 min by HPLC-MS method 1, mass spectrum
(m/z): 413 (M + 1). Chiral HPLC = 96% at 2.68 min by chiral HPLC
1
method 1, mass spectrum (m/z): 371 (M + 1). H NMR (400 MHz,
CD3OD) 7.47−7.62 (m, 4H), 5.60−5.68 (br m, 2H), 3.64−3.70 (br
m, 2H), 3.58 (s, 3H), 3.42−3.56 (br m, 2H), 3.22 (q, J = 7.2 Hz, 2H),
3.12−3.19 (br m, 2H), 2.58 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H).
8-(2-Chlorophenyl)-6-(4-ethylpiperazin-1-yl)-2-methyl-9H-
purine Hydrochloride (14). The title compound was prepared by
the general methods A−D. HPLC = 100% at 1.86 min by HPLC-MS
1
method 2. H NMR (400 MHz, CD3OD) 7.48−7.64 (m, 4H), 5.54−
5.64 (2H, br m), 4.70−4.77 (m, 1H), 4.30 (q, J = 7.6 Hz, 1H), 4.14−
4.24 (m, 1H), 3.95−4.04 (br, 1H), 3.86 (q, J = 7.4 Hz, 1H), 3.53−3.73
(br m, 4H), 3.19−3.31 (br m, 2H), 2.93 (s, 3H), 2.62 (s, 3H), 2.52−
1
method 1, mass spectrum (m/z): 357 (M + 1). H NMR (400 MHz,
CD3OD) 7.77 (dd, J = 1.9, 7.6 Hz, 1H), 7.43−7.58 (m, 3H), 5.62−
5.78 (br s, 2H), 3.62−3.78 (br s, 2H), 3.50−3.62 (br s, 2H), 3.24 (q, J
= 7.2 Hz, 2H), 3.15−3.26 (br s, 2H), 2.54 (s, 3H), 1.37 (t, J = 7.3 Hz,
3H).
2.63 (br m, 1H), 2.27−2.39 (br m, 1H). Specific rotation = [α]20
12.8 (c 10, MeOH).
+
D
SNAr, General Procedure A: 6-Chloro-N4,2-dimethyl-pyr-
imidine-4,5-diamine (6, R1 = Me). As an example, 40%
monomethylamine in water (1 equiv, 11.2 mmol) was added to a
solution of 2-amino-4,6-dichloro-2-methylpyrimidine 5 (1 equiv, 11.2
mmol) and diisopropylethylamine (1 equiv, 11.2 mmol) in isopropyl
alcohol (36 mL). The mixture was heated in a sealed tube at 150 °C
for 4 h. The reaction mixture was cooled and directly adsorbed onto
silica and purified by flash chromatography on silica gel eluting with
30−50% acetone in hexanes to afford the title compound 6 (R1 = Me)
(1.5 g, 77%). 1H NMR (400 MHz, CDCl3) 4.85 (br s, 1H), 3.18 (br s,
2H), 3.01 (d, J = 5.3 Hz, 3H), 2.42 (s, 3H).
2-[8-(2-Chlorophenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-
purin-9-yl]ethanol Hydrochloride (15). The title compound was
prepared by the general methods A−D. HPLC = 100% at 1.66 min by
1
HPLC-MS method 1, mass spectrum (m/z): 387 (M + 1). H NMR
(400 MHz, CD3OD) 7.57−7.62 (m, 3H), 7.47−7.54 (m, 1H), 5.61−
5.74 (br, 2H), 4.20 (t, J = 5.42 Hz, 2H), 3.67 (t, J = 5.35 Hz, 2H),
3.55−3.68 (m, 4H), 3.21−3.33 (m, 2H), 2.93 (s, 3H), 2.65 (s, 3H).
8-(2-Chlorophenyl)-6-(4-ethylpiperazin-1-yl)-9-(2-methox-
yethyl)-2-methyl-purine Hydrochloride (16). The title compound
was prepared by the general methods A−D. HPLC = 100% at 2.12
min by HPLC-MS method 1, mass spectrum (m/z): 415 (M + 1). 1H
NMR (400 MHz, DMSO-d6) 7.56−7.65 (m, 3H), 7.48−7.52 (m, 1H),
5.25−5.50 (br, 2H), 4.11 (t, J = 5.5 Hz, 2H), 3.46−3.60 (br, 4H), 3.44
(t, J = 5.5 Hz, 2H), 2.96−3.13 (br, 4H), 2.94 (s, 3H), 2.47 (s, 3H), (br
t, 3H).
Purine Formation, General Procedure B: 6-Chloro-8-(2-
chlorophenyl)-2,9-dimethyl-purine (8, R1 = Me, R2 = ortho-
Cl). As an example, 6-chloro-N4,2-dimethyl-pyrimidine-4,5-diamine 6
(R1 = Me) (1 equiv, 40.0 mmol), 2-chloro benzaldehyde 7 (R2 =
ortho-Cl) (2 equiv, 80.0 mmol), and 15% FeCl3 on SiO2 (21g)34 were
combined in dioxane (90 mL) and heated to 100 °C for 16 h. The
mixture was cooled and filtered through silica. The filtrates were
evaporated and the residue dissolved in dichloromethane (50 mL).
The mixture was cooled in an ice-bath, and 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (1.0 equiv, 40 mmol) was added. The cooling bath
was removed, and the reaction mixture was then stirred at room
temperature for 2 h. The reaction mixture was diluted with
dichloromethane and sequentially washed with 1N aqueous sodium
hydroxide solution, water, and brine. The organic layer was dried over
anhydrous sodium sulfate and filtered and concentrated to afford a
residue which was purified on silica gel eluting with 15% ethyl acetate
in hexanes to provide the title compound 8 (R1 = Me, R2 = ortho-Cl)
(7.0 g, 59%) as a white solid. HPLC = 100% at 2.07 min by HPLC-MS
8-(2-Chlorophenyl)-6-[4-(2-fluoroethyl)piperazin-1-yl]-9-(2-
methoxyethyl)-2-methyl-purine Hydrochloride (17). The title
compound was prepared by the general methods A−D. HPLC = 100%
at 2.07 min by HPLC-MS method 1, mass spectrum (m/z): 433 (M +
1
1). H NMR (400 MHz, CDCl3) 7.39−7.53 (m, 4H), 5.54−6.14 (br,
2H), 5.09−5.14 (m, 1H), 4.97−5.02 (m, 1H), 4.37−4.68 (m, 2H),
4.01−4.31 (m, 2H), 3.65−3.82 (m, 2H), 3.59 (br t, J = 4.70 Hz, 2H),
3.38−3.46 (m, 1H), 3.31−3.38 (m, 1H), 3.07−3.24 (m, 2H), 3.06 (s,
3H), 2.86 (s, 3H).
1-[4-[8-(2-Chlorophenyl)-9-(2-methoxyethyl)-2-methyl-
purin-6-yl]piperazin-1-yl]ethanone Hydrochloride (18). The
title compound was prepared by the same general methods A−D.
HPLC = 100% at 1.82 min by HPLC-MS method 1, mass spectrum
1
1
method 2, mass spectrum (m/z): 293 (M + 1). H NMR (400 MHz,
(m/z): 429 (M + 1). H NMR (400 MHz, CD3OD) 7.51−7.67 (m,
CDCl3) 7.50−7.60 (m, 3H), 7.40−7.42 (m, 1H), 3.68 (s, 3H), 2.80 (s,
3H).
4H), 4.40−4.80 (br, 4H), 4.34 (t, J = 5.05 Hz, 2H), 3.78−3.84 (m,
4H), 3.51 (t, J = 5.05 Hz, 2H), 3.12 (s, 3H), 2.74 (s, 3H), 2.16 (s, 3H).
Isomer 2,2-[8-(2-Chlorophenyl)-9-(2-methoxyethyl)-2-meth-
yl-purin-6-yl]-1,3,4,7,8,8a-hexahydropyrrolo[1,2-a]pyrazin-6-
one Hydrochloride (19). The racemic title compound (as the free
base) was prepared by the general methods A−C. This material (694
mg) was separated into enantiomers by supercritical fluid chromatog-
raphy (SFC) utilizing a Berger Multigram Supercritical Fluid
chromatograph with a Chiralpak AD-H column (2.1 cm × 25 cm ×
5 μm). The components were eluted with 20% methanol (containing
0.2% DMEA) in CO2 at 70 mL/min monitoring at λ = 225 nM.
Fractions containing each separated enantiomer were collected and
evaporated to yield isomer 1 (230 mg) and isomer 2 (220 mg). Data
for isomer 2: chiral HPLC = 98% at 1.95 min by chiral HPLC method
1. Isomer 2 (220 mg) was directly converted into the corresponding
SNAr, General Procedure C: 8-(2-Chlorophenyl)-6-(4-ethyl-
piperazin-1-yl)-2,9-dimethyl-purine (10, R1 = Me, R2 = ortho-
Cl, R3 = Et). As an example, 6-chloro-8-(2-chlorophenyl)-2,9-
dimethyl-purine 8 (R1 = Me, R2 = ortho-Cl) (1 equiv, 1.7 mmol),
N-ethyl piperazine 9 (R3 = Et) (1.1 equiv, 1.8 mmol), and
triethylamine (1.1 equiv, 1.8 mmol) were combined together in
ethanol (5 mL) and heated at 90 °C for 16 h. The reaction mixture
was cooled and concentrated under reduced pressure. The residue was
dissolved in dichloromethane and sequentially washed with saturated
sodium bicarbonate solution, water, and brine. The organics were
dried over anhydrous sodium sulfate and filtered and concentrate to
provide a residue which was purified on silica gel column using 2%
methanol in dichloromethane as eluent to afford the title compound
H
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Journal of Medicinal Chemistry
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hydrochloride salt using general procedure D to provide the title
compound 19 (240 mg) as a white solid. HPLC = 100% at 3.15 min
by HPLC-MS method 1, mass spectrum (m/z): 441 (M + 1). Chiral
4H), 3.20 (t, J = 11.4 Hz, 2H), 3.00−3.11 (m, 2H), 2.72 (s, 3H), 2.48
(s, 3H), 1.59−1.76 (m, 2H).
8-(3-Chlorophenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-9-
tetrahydropyran-4-yl-purine Hydrochloride (27). The title
compound was prepared by the general methods A−D. HPLC =
100% at 2.31 min by HPLC-MS method 1, mass spectrum (m/z): 427
(M + 1). 1H NMR (400 MHz, CD3OD) 7.69 (s, 1H), 7.54−7.64 (m,
3H), 5.52−5.65 (br, 2H), 4.47−4.56 (m, 1H), 4.02 (dd, J = 11.78, 4.52
Hz, 2H), 3.61−3.78 (m, 4H), 3.41 (t, J = 11.61 Hz, 2H), 3.27−3.36
(m, 2H), 2.94 (s, 3H), 2.86 (qd, J = 12.35, 4.60 Hz, 2H), 2.68 (s, 3H),
1.78−1.84 (m, 2H).
1
HPLC method 1 = 99% at 2.10 min. H NMR (400 MHz, CD3OD)
7.58−7.64 (m, 3H), 7.49−7.54 (m, 1H), 5.45−5.80 (br, 2H), 4.32 (t, J
= 5.0 Hz, 2H), 4.13 (dd, J = 2.8, 12.9 Hz, 1H), 3.80−3.89 (m, 1H),
3.48 (t, J = 5.0 Hz, 2H), 3.24−3.27 (m, 1H), 3.10 (s, 3H), 3.02−3.16
(m, 2H), 2.72 (s, 3H), 2.40−2.46 (m, 2H), 2.25−2.33 (m, 1H), 1.72−
1.82 (m, 1H). Specific rotation = [α]20 − 19.4 (c 10, EtOH).
D
N-[2-[8-(2-Chlorophenyl)-2-methyl-6-(4-methylpiperazin-1-
yl)purin-9-yl]ethyl]acetamide Hydrochloride (20). The title
compound was prepared by the same general methods A−D. HPLC
= 100% at 1.59 min by HPLC-MS method 1, mass spectrum (m/z):
428 (M + 1). 1H NMR (CD3OD) 7.58−7.63 (m, 3H), 7.49−7.54 (m,
1H), 5.59−5.69 (br, 2H), 4.20 (t, 2H, J = 5.5 Hz), 3.54−3.68 (m, 4H),
3.39 (t, 2H, J = 5.65 Hz), 3.20−3.27 (m, 2H), 2.93 (s, 3H), 2.64 (s,
3H), 1.67 (s, 3H).
2-Methyl-6-(4-methylpiperazin-1-yl)-9-tetrahydropyran-4-
yl-8-[2-(trifluoromethyl)phenyl]purine Hydrochloride (28). The
title compound was prepared by the general methods A−D. HPLC =
100% at 2.20 min by HPLC-MS method 1, mass spectrum (m/z): 461
1
(M + 1). H NMR (400 MHz, CD3OD) 7.92−7.97 (m, 1H), 7.80−
7.85 (m, 2H), 7.60−7.65 (m, 1H), 5.48−5.66 (m, 2H), 4.10 (td, J =
12.25, 4.10 Hz, 1H), 3.96 (dd, J = 11.80, 4.35 Hz, 2H), 3.55−3.76 (m,
4H), 3.29 (t, J = 12.3 Hz, 2H), 2.93 (s, 3H), 2.70−2.96 (m, 4H), 2.68
(s, 3H), 1.55−1.8 (m, 2H).
1-[2-[8-(2-Chlorophenyl)-2-methyl-6-(4-methylpiperazin-1-
yl)purin-9-yl]ethyl]pyrrolidin-2-one Hydrochloride (21). The
title compound was prepared by the same general methods A−D.
HPLC = 100% at 0.935 min by HPLC-MS method 1, mass spectrum
2-Methyl-6-(4-methylpiperazin-1-yl)-9-tetrahydropyran-4-
yl-8-[3-(trifluoromethyl)phenyl]purine Hydrochloride (29). The
title compound was prepared by the general methods A−D. HPLC =
100% at 2.38 min by HPLC-MS method 1, mass spectrum (m/z): 461
(M + 1). 1H NMR (400 MHz, CD3OD) 7.95 (s, 1H), 7.87−7.91 (m,
2H), 7.76−7.80 (m, 1H), 5.55−5.67 (m, 2H), 4.42−4.51 (m, 1H),
4.02 (dd, J = 11.66, 4.42 Hz, 2H), 3.51−3.69 (m, 4H), 3.39 (t, J = 12.1
Hz, 2H), 3.19−3.29 (m, 2H), 2.94 (qd, J = 12.50, 4.70 Hz, 2H), 2.93
(s, 3H), 2.62 (s, 3H), 1.76−1.83 (m, 2H).
1
(m/z): 454 (M + 1). H NMR (400 MHz, CD3OD) 7.61−7.69 (m,
3H), 7.53−7.58 (m, 1H), 5.55−5.71 (m, 2H), 4.34 (t, J = 5.4 Hz, 2H),
3.60−3.78 (m, 4H), 3.48 (t, J = 5.40 Hz, 2H), 3.29−3.38 (m, 2H),
3.16 (t, J = 7.10 Hz, 2H), 2.95 (s, 3H), 2.72 (s, 3H), 2.09 (t, J = 8.20
Hz, 2H), 1.76−1.86 (m, 2H).
8-(2-Chlorophenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-9-
(2-methylsulfonylethyl)purine Hydrochloride (22). The title
compound was prepared by the same general methods A−D. HPLC
= 100% at 1.86 min by HPLC-MS method 1, mass spectrum (m/z):
449 (M + 1). High resolution mass spectrum: calcd 448.1448, found
8-(2-Methoxyphenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-
9-tetrahydropyran-4-yl-purine Hydrochloride (30). The title
compound was prepared by the general methods A−D. HPLC = 100%
at 2.03 min by HPLC-MS method 1, mass spectrum (m/z): 423 (M +
1
448.1460. H NMR (400 MHz, CD3OD) 7.58−7.65 (m, 3H), 7.49−
1
7.53 (m, 1H), 5.56−5.67 (br m, 2H), 4.57 (t, J = 6.6 Hz, 2H), 3.56−
3.68 (br m, 4H), 3.55 (t, J = 6.5 Hz, 2H), 3.21−3.30 (br m, 2H), 2.93
(s, 3H), 2.82 (s, 3H), 2.65 (s, 3H).
1). H NMR (400 MHz, CD3OD) 7.55−7.60 (m, 1H), 7.42 (dd, J =
7.50, 1.75 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 7.11 (td, J = 7.5, 0.9 Hz,
1H), 5.46−5.62 (br, 2H), 4.08−4.17 (m, 1H), 3.95−4.03 (m, 2H),
3.80 (s, 3H), 3.45−3.67 (m, 4H), 3.28−3.38 (m, 2H), 3.14−3.28 (m,
2H), 2.92 (s, 3H), 2.76−2.93 (br, 2H), 2.60 (s, 3H), 1.55−1.84 (br,
2H).
8-(2-Chlorophenyl)-9-cyclopropyl-2-methyl-6-(4-methylpi-
perazin-1-yl)purine Hydrochloride (23). The title compound was
prepared by the same general methods A−D. HPLC = 100% at 2.23
min by HPLC-MS method 1, mass spectrum (m/z): 383 (M + 1). 1H
NMR (400 MHz, CDCl3) 7.38−7.52 (m, 4H), 5.59−5.80 (br, 2H),
3.94−4.10 (br, 2H), 3.42−3.62 (br, 3H), 2.90−3.07 (br, 2H), 2.81 (s,
3H), 2.77 (s, 3H), 0.97−1.04 (br, 2H), 0.67−0.72 (br, 2H).
8-(2-Chlorophenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-9-
[(3S)-tetrahydrofuran-3-yl]purine Hydrochloride (24). The title
compound was prepared by the same general methods A−D. HPLC =
100% at 2.24 min by HPLC-MS method 1, mass spectrum (m/z): 413
(M + 1). Chiral HPLC = 99% at 3.71 min by chiral HPLC method 2.
1H NMR (400 MHz, CD3OD) 7.49−7.64 (m, 4H), 5.59 (br d, J =
8-(3-Methoxyphenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-
9-tetrahydropyran-4-yl-purine Hydrochloride (31). The title
compound was prepared by the general methods A−D. HPLC = 100%
at 2.11 min by HPLC-MS method 1, mass spectrum (m/z): 423 (M +
1
1). H NMR (400 MHz, CD3OD) 7.49 (t, J = 7.35 Hz, 1H), 7.15−
7.19 (m, 3H), 5.49−5.63 (br, 2H), 4.53−4.63 (m, 1H), 4.02 (dd, J =
12.10, 4.61 Hz, 2H), 3.84 (s, 3H), 3.64−3.85 (m, 4H), 3.29−3.43 (m,
4H), 2.95 (s, 3H), 2.82 (qd, J = 12.55, 4.55 Hz, 2H), 2.72 (s, 3H),
1.78−1.84 (m, 2H).
2-[2-Methyl-6-(4-methylpiperazin-1-yl)-9-tetrahydropyran-
4-yl-purin-8-yl]benzonitrile Hydrochloride (32). The title com-
pound was prepared by the general methods A−D. HPLC = 100% at
1.90 min by HPLC-MS method 1, mass spectrum (m/z): 418 (M + 1).
1H NMR (400 MHz, CD3OD) 7.96−7.98 (m, 1H), 7.89 (td, J = 7.56,
13.6 Hz, 2H), 4.69−4.76 (m, 1H), 4.30 (q, J = 7.6 Hz, 1H), 4.15−4.22
(br, 1H), 3.95−4.05 (br, 1H), 3.86 (q, J = 7.4 Hz, 1H), 3.50−3.66 (br
m, 4H), 3.18−3.27 (br m, 2H), 2.93 (s, 3H), 2.60 (s, 3H), 2.52−2.63
(br m, 1H), 2.27−2.39 (br m, 1H). Specific rotation = [α]20 − 13.6
D
1.5 Hz, 1H), 7.71−7.79 (m, 2H), 5.56−5.70 (br, 2H), 4.32 (tt, J =
12.05, 4.19 Hz, 1H), 3.98 (dd, J = 11.7, 4.50 Hz, 2H), 3.49−3.66 (m,
4H), 3.37 (td, 12.1, 1.65 Hz, 2H), 3.15−3.27 (m, 2H), 2.91 (s, 3H),
2.87 (qd, J = 12.35, 4.45 Hz, 2H), 2.60 (s, 3H), 1.77−1.84 (m, 2H).
8-(2-Fluorophenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-9-
tetrahydropyran-4-yl-purine Hydrochloride (33). The title
compound was prepared by the general methods A−D. HPLC =
100% at 2.09 min by HPLC-MS method 1, mass spectrum (m/z): 411
(c 10, MeOH).
8-(2-Chlorophenyl)-6-(4-ethylpiperazin-1-yl)-2-methyl-9-
(tetrahydropyran-4-ylmethyl)purine Hydrochloride (25). The
title compound was prepared by the general methods A−D. HPLC =
100% at 2.17 min by HPLC-MS method 1, mass spectrum (m/z): 455
(M + 1). 1H NMR (400 MHz, CDCl3) 7.44−7.57 (m, 4H), 5.53−5.78
(m, 2H), 3.91−4.06 (m, 4H), 3.78−3.88 (m, 2H), 3.60 (d, J = 11.5
Hz, 2H), 3.22 (t, J = 11.5 Hz, 2H), 3.05−3.14 (m, 2H), 2.79−2.92 (m,
2H), 2.61 (s, 3H), 2.00−2.13 (m, 1H), 1.52 (t, J = 7.34 Hz, 3H),
1.25−1.35 (m, 2H), 1.04−1.16 (m, 2H).
8-(2-Chlorophenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-9-
tetrahydropyran-4-yl-purine Hydrochloride (26). The title
compound was prepared by the general methods A−D. HPLC =
100% at 2.31 min by HPLC-MS method 1, mass spectrum (m/z): 427
(M + 1). High resolution mass spectrum: calcd 426.1935, found
426.1947. 1H NMR (400 MHz, DMSO-d6) 7.56−7.67 (m, 3H), 7.48−
7.52 (m, 1H), 5.20−5.50 (br, 2H), 3.83−4.04 (m, 5H), 3.39−3.50 (m,
1
(M + 1). H NMR (400 MHz, CD3OD) 7.55−7.66 (m, 2H), 7.29−
7.40 (m, 2H), 7.54−7.64 (m, 2H), 4.22 (td, J = 12.05, 4.02 Hz, 1H),
3.99 (dd, J = 11.78 Hz, 2H), 3.43−3.63 (m, 4H), 3.36 (td, J = 12.10,
1.72 Hz, 2H), 3.14−3.24 (m, 2H), 2.91 (s, 3H), 2.88 (qd, J = 12.40,
4.50 Hz, 2H), 2.57 (s, 3H), 1.67−1.73 (m, 2H).
8-(2-Cyclopropylphenyl)-2-methyl-6-(4-methylpiperazin-1-
yl)-9-tetrahydropyran-4-yl-purine Hydrochloride (34). The title
compound was prepared by the general methods A−D. HPLC = 100%
at 2.26 min by HPLC-MS method 1, mass spectrum (m/z): 433 (M +
1). 1H NMR (400 MHz, CD3OD) 7.45−7.50 (m, 1H), 7.26−7.32 (m,
I
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2H), 7.02 (d, J = 8.01 Hz, 1H), 5.50−5.61 (br, 2H), 4.18 (tt, J = 12.20,
4.10 Hz, 1H), 3.90−4.03 (m, 2H), 3.47−3.67 (m, 4H), 3.17−3.34 (m,
4H), 2.83−3.07 (m, 2H), 2.92 (s, 3H), 2.60 (s, 3H), 1.61−1.81 (br,
2H), 1.52−1.60 (m, 1H), 0.59−0.89 (m, 4H).
2-Methyl-6-(4-methylpiperazin-1-yl)-8-(o-tolyl)-9-tetrahy-
dropyran-4-yl-purine Hydrochloride (35). The title compound
was prepared by the general methods A−D. HPLC = 100% at 2.13
min by HPLC-MS method 1, mass spectrum (m/z): 407 (M + 1). 1H
NMR (400 MHz, CD3OD) 7.44−7.49 (m, 1H), 7.38−7.41 (m, 1H),
7.30−7.36 (m, 2H), 5.45−6.62 (br, 2H), 4.13 (tt, J = 12.05, 4.20 Hz,
1H), 3.95 (dd, J = 11.60, 4.25 Hz, 2H), 3.34−3.62 (m, 4H), 3.29 (td, J
= 12.10, 1.41 Hz, 2H), 3.11−3.25 (m, 2H), 2.91 (s, 3H), 2.76−2.89
(m, 2H), 2.55 (s, 3H), 2.17 (s, 3H), 1.65−1.69 (m, 2H).
NT, not tested; OA, osteoarthritis; PgP, P-glycoprotein 1; po,
per os (by mouth); SAR, structure−activity relationship; SEM,
standard error of the mean; THC, tetrahydrocannabinol; THP,
tetrahydropyran; T1/2, half-life; TRPV1, transient receptor
potential cation channel subfamily V member 1
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 1-317-277-9146. Fax: +1-317-276-6545. E-mail:
hollinshead_sean_p@lilly.com.
Present Addresses
§M.W.T.: Southwest Research Institute, 6220 Culebra Road,
San Antonio, Texas 78238−5166, United States.
∥R.S.: Xavier University of Louisiana, College of Pharmacy,
New Orleans, Louisiana 70125, United States.
Br. J. Pharmacol. 2008, 153, 240−251. (b) Fernan
́
dez-Ruiz, J.; Romero,
, M. Cannabinoid
Notes
J.; Velasco, G.; Tolon, R. M.; Ramos, J. A.; Guzman
́
́
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Arunendra Pathak and Somesh Sharma and their
chemistry team at Jubilant Chemsys. We also acknowledge the
support of Wendy Gough, Sherri Andis, and Laura Martin (Eli
Lilly and Company) for providing in vitro characterization of
the molecules at CB1 and CB2. We also thank Jeffrey
Richardson (DCSG) for scale up of key compounds and to
1
Robert Boyer for assistance with H NMR assignments.
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ABBREVIATIONS USED
■
ANOVA, analysis of variance; CB1, cannabinoid receptor 1;
CB2, cannabinoid receptor 2; CHO, Chinese hamster ovary;
CL, clearance; CNS, central nervous system; DDQ, 2,3-
dichloro-5,6-dicyanobenzoquinone; DRG, dorsal root ganglia;
GDP, guanosine diphosphate; GI, gastrointestinal; GPCR, G-
protein coupled receptor; GTPγS, guanosine 5′-O-[γ-thio]-
̀
triphosphate; hERG, human ether-a-go-go-related gene; HSD,
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