Discovery of APD371: Identification of a Highly Potent and Selective CB2 Agonist for the Treatment of Chronic Pain

Article abstract of DOI:10.1021/acsmedchemlett.7b00396

The discovery of a novel, selective and fully efficacious CB₂ agonist with satisfactory pharmacokinetic and pharmaceutical properties is described. Compound 6 was efficacious in a rat model of osteoarthritis pain following oral administration and, in contrast to morphine, maintained its analgesic effect throughout a 5-day subchronic treatment paradigm. These data were consistent with our hypothesis that full agonist efficacy is required for efficient internalization and recycling of the CB₂ receptor to avoid tachyphylaxis. Based on its overall favorable preclinical profile, 6 (APD371) was selected for further development for the treatment of pain.

Full text of DOI:10.1021/acsmedchemlett.7b00396

Subscriber access provided by Universitaetsbibliothek | Johann Christian Senckenberg
Letter
Discovery of APD371: Identification of a Highly Potent and
Selective CB2 Agonist for the Treatment of Chronic Pain
Sangdon Han, Lars Thoresen, Jae-Kyu Jung, Xiuwen Zhu, Jayant Thatte, Michelle Solomon, Ibragim
Gaidarov, David J Unett, Woo Hyun Yoon, Jeremy Barden, Abu Sadeque, Amin Usmani, Chuan Chen,
Graeme Semple, Andrew John Grottick, Hussein Al-Shamma, Ronald Christopher, and Robert M Jones
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00396 • Publication Date (Web): 30 Nov 2017
Downloaded from http://pubs.acs.org on December 2, 2017
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Discovery of APD371: Identification of a Highly Potent and Selective
CB2 Agonist for the Treatment of Chronic Pain
Sangdon Han¶, Lars Thoresen, JaeꢀKyu Jung, Xiuwen Zhu, Jayant Thatte, Michelle Solomon, Ibragim Gaidarov§, David J.
Unett§, Woo Hyun Yoon, Jeremy Barden, Abu Sadeque, Amin Usmani, Chuan Chen, Graeme Semple§*, Andrew J. Grotꢀ
tick§, Hussein AlꢀShamma§, Ronald Christopher and Robert M. Jones†.
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Arena Pharmaceuticals, 6154 Nancy Ridge Drive, San Diego, CA 92121, USA
KEYWORDS: cannabinoids, cannabinoid receptor type 2 (CB2), CB2 agonist, tachyphylaxis, APD371, chronic pain.
ABSTRACT: The discovery of a novel, selective and fully efficacious CB2 agonist with satisfactory pharmacokinetic and pharmaꢀ
ceutical properties is described. 6 was efficacious in a rat model of osteoarthritis pain following oral administration and, in contrast
to morphine, maintained its analgesic effect throughout a 5ꢀday subꢀchronic treatment paradigm. These data were consistent with
our hypothesis that full agonist efficacy is required for efficient internalization and recycling of the CB2 receptor to avoid tachyphyꢀ
laxis. Based on its overall favorable preclinical profile, 6 (APD371) was selected for further development for the treatment of pain
Endocannabinoids interact with a receptor system that presentꢀ
ly consists of two Class A Gꢀprotein coupled receptors
(GPCR), namely CB1 and CB2, which regulate a wide range of
pharmacological processes.1 More recently, GPR55 has been
proposed as a third member of the family, but this has yet to
be universally accepted. The ability to modulate the endocanꢀ
nabinoid system may offer opportunities to control symptoms
of pain arising from numerous clinical disorders. The CB1
receptor, which is abundantly expressed in the peripheral and
central nervous system,2 can elicit multiple actions, but the
therapeutic utility of agonists of this receptor is limited by the
psychotropic effects associated with CB1 activation in the cenꢀ
tral nervous system. The cannabinoid receptor type 2 (CB2) is
mainly expressed on the cells of the immune system.3 Precliniꢀ
cal studies have consistently demonstrated that activation of
CB2 receptors reverses pain responses in a wide range of acute
and chronic pain models.4ꢀ9 A lack of selectivity inherent in
some of the pharmacological tools used in these studies howꢀ
ever, has plagued the field and doubts remain as to the true
potential of selective CB2 agonists for pain management. The
precise mechanism of action underlying antinociceptive efꢀ
fects mediated by CB2 receptor activation remains elusive too,
despite several proposed hypotheses.10ꢀ13 Importantly though,
this mode of intervention could avoid issues such as tolerance
and addiction as well as some of the severe side effects associꢀ
ated with current pain therapies. The pharmaceutical industry
has made considerable efforts to identify potent and selective
CB2 agonists for the treatment of pain,14 and several compaꢀ
nies have developed CB2 selective molecules for evaluation in
human trials, so far without success.15
nists that demonstrated robust acute efficacy in several pain
models in rodents, we failed with this first iteration to identify
a molecule that produced a sustained response upon repeated
dosing. This potential for in vivo desensitization of the CB2
receptor has not previously been critically examined. Whereas
it is often assumed that agonists of GPCRs may induce tachyꢀ
phylaxis, it is known that agonistꢀinduced endocytosis reguꢀ
lates many GPCRs by a specific membrane trafficking pathꢀ
way.17 Typically, the rapid recycling of agonistꢀinduced interꢀ
nalized receptors back to the plasma membrane can facilitate
the maintenance of the cellular response to the agonist during
long term agonist exposure. In the case of the µꢀopioid recepꢀ
tor for example, activation of the receptor with morphine, a
partial µꢀopioid receptor agonist, results in desensitization of
the response due to a weak (or partial) internalization reꢀ
sponse. The resultant lack of receptor recycling results in the
receptor being essentially removed from the cell surface and
thus unavailable to the agonist ligand.18 In contrast, activation
with the full agonists etorphin or DAMGO allows for efficient
receptor recycling and no desensitization is observed.
Figure 1. SAR approach to the discovery of 6
OH
R1
OH
OH
R2
HN
HN
N
HN
N
O
O
O
H
H
*
H
N
N
N
N
*
H
1-6
H
R
F
H
N
N
O
We recently disclosed a tricyclic 3ꢀ5ꢀ5ꢀfused pyrazole 3ꢀ
carboxamide series (e.g. 1) as a novel CB2 receptor chemoꢀ
type.16 Despite the discovery of potent and selective CB2 agoꢀ
F
1
6 (APD371)
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1
2
Table 1. In vitro EC50 data and Liver Microsomal stability
βꢀArrestin, EC50 (nM)a
3
4
5
6
7
8
9
LM Stabilityb
(h, r, m)
Cmpd
R
R1 / R2
Stereoꢀ
chemistry
hCB2 (Emax
)
hCB1
rCB2 (Emax
)
rCB1
1
2
3
4
5
6
(R,R)ꢀ
(R,R)ꢀ
(R,R)ꢀ
(S,S)ꢀ
(S,S)ꢀ
(S,S)ꢀ
CH3 / CH3
CH3 / CH3
64 (83)
ꢀ
>10,000
ꢀ
70 (46)
320 (85)
21 (78)
ꢀ
>10,000
>10,000
>10,000
ꢀ
>60, >60, 13
>60, >60, >60
>60, 38, 9
ꢀ
2,4ꢀdifluoroꢀPh
2ꢀpyrazinyl
2ꢀpyrazinyl
2ꢀpyrazinyl
(S)ꢀtꢀbutyl / H
(R)ꢀtꢀbutyl / H
(S)ꢀtꢀbutyl / H
(S)ꢀtꢀbutyl / H
26 (104)
560 (78)
59 (107)
6.2 (106)
>10,000
>10,000
>10,000
>10,000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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55
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59
60
2ꢀpyrazinyl
53 (95)
7.6 (100)
>10,000
>10,000
>60, >60, 24
>60, >60, >60
4ꢀoxoꢀ2ꢀpyrazinyl
R, R1 and R2 refer to substituents in Fig. 1 for the general structure above the arrow. a EC50 values are the mean of three or more
independent experiments with logSD < 0.4; Intrinsic activity (Emax) was determined relative to CPꢀ55,940 (100). halfꢀlife (t1/2,
min).
b
group also played a crucial role in engaging the receptor as
demonstrated by the weaker effect of the (R)ꢀtert butyl anaꢀ
logue 4. Finally, an additional modest decrease in lipophilicity
by incorporation of a pyrazine Nꢀoxide into the scaffold proꢀ
vided the highly potent and fully efficacious CB2 agonist 6
(cLogP = ꢀ 0.65). To focus further on this promising scaffold,
an additional series of 3ꢀcarboxamide analogues (10ꢀ21) were
prepared using starting from commercially available and optiꢀ
The intrinsic efficacy (Emax) of 1 is significantly attenuated in
both human and rat CB2 receptor assays relative to the nonꢀ
selective cannabinoid receptor full agonist CPꢀ55,940 (Table
1). In addition, 1 failed to induce the same level of internalizaꢀ
tion of rat CB2 receptors compared to CPꢀ55,940, as measured
by time resolved flow cytometry (~68% relative to CPꢀ55,940,
Figure S6). We hypothesized that this inefficient internalizaꢀ
tion by 1 may be responsible for the tachyphylaxis observed in
cally pure (S)ꢀ2ꢀ(butꢀ3ꢀenꢀ1ꢀyl)oxirane and utilizing the synꢀ
thetic sequence illustrated in Scheme 1. The significant reducꢀ
tion in the lipophilicity of the core now allowed for the use of
vivo by limiting the receptor endocytic recycling pathway in a
similar way to the morphineꢀꢀꢀopioid receptor interaction.18
Furthermore, we wanted to investigate whether internalization
more lipophilic amines in the exploration of this series while
efficiency could be improved by increasing agonist efficacy
still maintaining a good balance of physical properties.
which could then reduce or eliminate the tachyphylaxis effect.
(1S,5R)ꢀBicyclo[3.1.0]hexanꢀ2ꢀone (7) was prepared through a
catalytic intramolecular cyclopropanation of the oxirane, folꢀ
We therefore focused on identifying a highly selective and
lowed by a TEMPO/bleach oxidation.20 A facile oneꢀpot twoꢀ
fully efficacious CB2 agonist to test our hypothesis. With this
step synthesis involving acylation of ketone 7 with diethyl
goal in mind we further explored aryl group substitution in our
pyrazole N-1, 3ꢀcarboxamide series, as well as stereoisomers
oxalate followed by condensation with 2ꢀhydrazinylpyrazine
yielded the tricyclic pyrazole core 8.21 The resultant ethyl ester
of the tricyclic core (Figure 1) using our previously described
synthetic approaches.16 An additional objective was to imꢀ
was hydrolyzed with LiOH to an acid intermediate which was
then selectively oxidized with mCPBA to form the Nꢀoxide
acid 9. We found that hydrolysis of the ester followed by oxiꢀ
dation, provided better control over the regioselectivity of the
prove the drug like properties of our initial series, that was
rather lipophilic and had only marginal solubility, and to idenꢀ
tify compounds that maintained full intrinsic efficacy regardꢀ
less of species. To assess potency and efficacy, we employed a
oxidation step than using the reverse sequence. 9 was then
DiscoverX PathHunterTM βꢀarrestin assay, which was reported
converted to the target compounds via a HATUꢀmediated amꢀ
ide coupling. Screening data for selected examples prepared in
to exhibit a much more robust signal for CB2 ligands than a
this manner are shown in Table 2.
homogenous timeꢀresolved fluorescence (HTRF) cyclase asꢀ
say.19 Indeed, the βꢀarrestin assay proved to more discerning
Scheme 1:
General synthesis of tricyclo pyrazoleꢀ3ꢀ
in identifying small differences in intrinsic efficacy of our
novel series than the cAMP assay, although EC50 values in this
platform were generally five to tenꢀfold rightꢀshifted. In this
assay (Table 1), 1 appeared even more partial relative to the
full agonist CPꢀ55,940 (rCB2 Emax = 46) than in the cAMP
platform (rCB2 Emax = 80). Incorporation of a pyrazine to give
2, both significantly decreased the lipophilicity of the moleꢀ
cule (cLogP for 1 = 2.1; for 2 = ꢀ 0.36) and increased the inꢀ
trinsic efficacy for rat CB2 albeit with a loss of potency. Reꢀ
placing the gemꢀdimethyl feature with a sterically bulky
group, (S)ꢀtertꢀbutyl (3), resulted in dramatically improved
CB2 potency without any meaningful change in efficacy relaꢀ
tive to 2. Although the intrinsic efficacy for the human recepꢀ
tor was already at a maximum level, the (S,S) analogue 5 furꢀ
ther improved intrinsic efficacy at the rat CB2 receptor, while
retaining good potency. The stereochemistry of the tert-butyl
carboxamides
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Reagents (a) Lithium 2,2,6,6ꢀtetramethylpiperidine, MTBE,
(b) TEMPO (2.5 mol %), NaOCl, 67% for a and b (c) tꢀBuOK,
steps (e) LiOH, 100 % (f) MCPBA, 81 % (g) HATU,
NH2CR3R4R5, 67% for 6.
1
diethyl oxalate, (d) 2ꢀhydrazinylpyrazine, EtOH, 73% two
2
3
4
Table 2. 3ꢀCarboxamide SAR
5
6
7
8
βꢀarrestin, EC50, nMa
hCB1 (Emax rCB2 (Emax
LM stabilityb
(h, r)
Cmpd
R3 / R4
(S)ꢀtꢀbutyl / H
R5
hCB2 (Emax
6.2 (106)
46 (103)
105 (96)
17.2 (119)
280 (98)
5.6 (112)
1.2 (107)
3.8 (98)
)
)
)
rCB1 (Emax
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
840 (107)
2700 (74)
>10,000
>10,000
>10,000
>10,000
)
6
ꢀCH2OH
ꢀCH2OH
ꢀCH2OH
ꢀCH2OH
ꢀCH2OH
ꢀCH2OH
ꢀCH2F
ꢀCH2F
ꢀCF3
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1100 (105)
6100 (96)
>10,000
>10,000
>10,000
>10,000
7.6 (100)
55 (88)
>60, >60
>60, >60
>60, >60
>60, >60
>60, >60
>60, >60
>60, >60
>60, >60
>60, >60
>60, >60
>60, >60
ꢀ
10
11
12
13
14
15
16
17
18
19
20
(S)ꢀisopropyl / H
(S)ꢀphenyl / H
(S)ꢀCF3 / H
9
60 (72)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
35 (107)
530 (85)
6.8 (106)
1.5 (97)
6.4 (87)
6.3 (98)
1.6 (106)
14.5 (103)
290 (81)
(R)ꢀCF3 / H
ꢀCyclopentylꢀc
(S)ꢀtꢀbutyl / H
(S)ꢀisopropyl / H
CH3 / CH3
ꢀCyclobutylꢀc
ꢀCyclopropylꢀc
ꢀCyclopropylꢀc
2.3 (108)
1.2 (115)
5.9 (108)
91 (102)
ꢀCF3
ꢀCF3
ꢀCH3
a EC50 values are the mean of three or more independent experiments with logSD < 0.4; Intrinsic activity (Emax) was determined
relative to CPꢀ55,940 (100). b halfꢀlife (t1/2, min).c R3 and R4 are connected to form a carbocycle.
I
n general, the carboxamides maintained excellent stability in
efficacy were maintained in all species assessed, and that 6
was highly selective for CB2 over CB1 in both binding and
functional assays. Furthermore, 6 induced efficient receptor
internalization (~106% relative to the CB1/2 agonist CP55,940,
Figure S6) in CHO cells expressing HAꢀtagged rat CB2 sugꢀ
gesting that, according to our hypothesis, 6 would be able to
drive agonistꢀinduced receptor recycling.
both human and rat liver microsomes (LM), but CB2 potency
and efficacy were somewhat sensitive to variations in R3, R4,
and R5. For example, replacing the tertꢀbutyl with less stericalꢀ
ly hindered groups, including fluoroꢀalkyl groups (e.g. 10, 12-
13) or phenyl (11) led to a decrease in CB2 potency. Although
the stereochemistry of a single substituent had a significant
impact on CB2 potency (e.g. 12 vs. 13 or 4 vs. 5), achiral anaꢀ
logues such as cyclopentane (14) were well tolerated, affordꢀ
ing an excellent potency and efficacy profile. However, further
characterization of 14 later showed it did not have a signifiꢀ
cantly improved pharmacokinetic profile in rodents compared
to 6 (Table S4). Isosteric replacement of –OH with –F (e.g.
15ꢀ16) resulted in improved potency for CB2, but reduced
selectivity against CB1. On the other hand, incorporating the
lipophilic CF3 group into either the gemꢀdimethyl array (17) or
small ring systems (e.g. 18ꢀ19) allowed us to achieve an outꢀ
standing balance of potency and selectivity. 17 showed favorꢀ
able rat PK albeit with a somewhat elongated Tmax, (Table
S5) and was selected for evaluation in several in vivo rodent
pain models. 17 displayed good efficacy in these models after
oral administration.
6 had a favorable overall ADME profile (see Table S1 for
parameters). It was stable in mouse, rat, dog, monkey, and
human liver microsomes, and showed moderate plasma proꢀ
tein binding (PPB; ~10 to 20 % unbound fraction) in all five
species examined. In vivo pharmacokinetic studies in multiple
preclinical species showed that 6 had good plasma exposure,
moderate systemic clearance, low steadyꢀstate volume of disꢀ
tribution across species, and good oral bioavailability in roꢀ
dents and dogs. 6 had a relatively short halfꢀlife in most speꢀ
cies, something that we regarded as a potential advantage for
pain treatments if it were to translate into humans. The outlyꢀ
ing species in terms of oral bioavailability, was the cynomolꢀ
gus monkey. Additional studies to address this species differꢀ
ence suggested that cytosolic aldehyde oxidases (AO), which
are highly expressed in monkeys,22 were likely responsible for
the in vivo biotransformation of the Nꢀoxide to the reduced
form 5. These data (not shown) were consistent with the high
concentrations of 5 observed in the plasma of monkeys
(AUClast ratio 5:6 = 4.89). In contrast, the AUClast ratio beꢀ
tween 5 and 6 in rats and dogs were only 0.17 and 0.12 respecꢀ
tively, after a dose of 10 mg/kg PO, reflecting the much lower
expression of cytosolic aldehyde oxidases in these species. We
were concerned that the generation of an active metabolite,
even at low levels, may result in undesirable in vivo activity,
particularly if it lacked the optimized CB2 efficacy, selectivity
and partitioning profile of the parent. However, although it
was a less potent agonist at the CB2 receptor, 5 maintained full
agonist efficacy, along with high selectivity against CB1 and in
a broader panel (Table S12). In addition, 5 was largely periphꢀ
erally restricted in abbreviated CNS studies in the rat (b/p ratio
However, 17 had relatively high CNS penetration (b/p ratio =
0.83 following a 3 mg/kg, PO dose, Table S6). Despite this
profile 17 was dosed up to 100 mg/kg in rats without inducing
any change on body temperature and locomotor activity which
are known to be characteristic of CB1 activation. This apparent
lack of CB1 engagement in vivo is consistent with the in vitro
data that shows no measurable activity at the rat CB1 receptor.
However, as a further mitigation strategy with respect to poꢀ
tential central CB1 activity in the clinic, we focused our further
efforts on 6, which was equally selective compared to 17 and
in CNS partitioning studies had significantly lower brain exꢀ
posure in the rat. (b/p ratio = 0.04 at 3 mg/kg, PO, Table S3).
A more comprehensive in vitro profile of 6 (see Table S10)
showed that single digit nanomolar potency and full intrinsic
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~ 0.1, Table S7). Thus, with a pharmacokinetic and metabolic
profile suitable for in vivo testing, the acute analgesic effect of
6 was evaluated in the monosodium iodoacetate (MIA) model
of osteoarthritis (OA) pain in the
rat.23
1
2
3
4
5
6
7
8
9
Figure 2. (a) Doseꢀresponse in the MIAꢀinduced model of
OA pain, (b) Blocking experiments with a CB2 antagonist or a
CB1 antagonist. Doses: 6 (3 mg/kg, IP), AR401733 (10 mg/kg,
PO) Rimonabant (Rim, 10 mg/kg, PO).
Having achieved a good in vivo analgesic effect in our preclinꢀ
ical models, the potential candidate was further profiled. An
Xꢀray crystal structure showed the absolute stereochemistry
and the position of the Nꢀoxide function to be consistent with
the original assignments from 1HꢀNmr (Figure S4). Solid form
characterization revealed 6 to have excellent physicochemical
properties, including high aqueous solubility (~2 mg/mL @
pH 2 to 7), a high melting point (164 °C), and excellent stabilꢀ
ity. 6 showed no significant inhibition of major CYP P450
enzymes (i.e., 1A2, 2C9, 2C19, 2D6, 3A4; IC50 >50 ꢀM) indiꢀ
cating a low risk for drugꢀdrug interactions. 6 had no effect on
the hERG channel in either [3H]ꢀAstemizole binding or patch
clamp assays (IC50 > 30 ꢀM) and showed no cytotoxic potenꢀ
tial in an Essential Cell Function (ECF) panel measuring celluꢀ
lar parameters in live HepG2 cells up to 1000 ꢀM. It was negꢀ
ative for mutagenic activity in the Ames bacterial reverse muꢀ
tation assay in the presence and absence of rat liver S9 fracꢀ
tion. Additionally, both 6 and its predominant metabolite 5,
showed only weak or no offꢀtarget activity (at 10 ꢀM) in in
vitro binding assays that cover a broad range of receptors, ion
channels, and transporters (Eurofins/CEREP panel, Tables
S11, S12).
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In this model, knee cartilage damaged by MIA injection into
the joint precipitates osteoarthritis and an associated hyperalꢀ
gesia that can be measured by von Frey hair stimulation.
Eighteen days postꢀMIA injection, MIA treated rats received
an oral dose of 6 (0.3 ꢀ 30 mg/kg). Von Frey paw withdrawal
thresholds were measured 1 h postꢀdose to assess the analgesic
effect in MIAꢀinduced late phase hypersensitivity. 6 signifiꢀ
cantly increased paw withdrawal thresholds at doses ≥ 3
mg/kg PO (ED50 = 2.3 mg/kg; Figure 2a), an effect that was
not statistically different from that observed with 3 mg/kg IP
of morphine (data not shown). In a separate experiment, a
single dose of 6 (10 mg/kg, PO) inhibited paw withdrawal
threshold for up to 4 hours after administration. Based on the
PK data for this dose, there appeared to be a good correlation
between serum drug concentrations and pharmacological reꢀ
sponse over this time period. Seperately, the analgesic effects
of 6 were shown to be highly likely mediated via activity at
CB2 receptors, since the effects were blocked by a novel CB2
inverse agonist AR401733, a much more selective CB2 inverse
agonist in the rat than the literature antagonists AMꢀ630 and
SR144528 (Figure S1), but not by the selective CB1 antagonist
rimonabant (Figure 2b). Being from the same scaffold as 6, it
is possible that AR401733 has equal and opposite functional
activity via another target. However, we regard this possibility
as remote as the analgesic effect of 1, with the same tricyclic
core as both AR401733 and 6, was reported to be blocked by
AMꢀ630,16 which while less selective for CB2 over CB1 has
significant structural differences.
In vivo, 6 did not induce any change in core body temperature
(CBT) up to a dose of 100 mg/kg PO whereas, CPꢀ55940
showed a clear decrease in CBT at 0.3mg/kg (Figure S7). Theꢀ
se data are consistent with an absence of measurable functionꢀ
al activity at the CNS expressed CB1 receptor (EC50 > 30 ꢀM)
and/or the peripheral restriction observed in vivo (b/p ratio =
0.04). A doseꢀrange finding 14ꢀday toxicity study was conꢀ
ducted in male and female SpragueꢀDawley rats using 0, 30,
100, and 300 mg/kg (PO) dose groups. 6 was well tolerated at
all doses, and only at the highest dose of 300 mg/kg was tranꢀ
sient lethargy observed on the first day of dosing, which reꢀ
solved with subsequent doses. No treatment related changes in
hematology and histopathological abnormalities were obꢀ
served.
Figure 3. 5ꢀDay subꢀchronic study: 6 (3 mg/kg, BID, IP),
morphine (10 mg/kg, BID, IP)
With encouraging acute in vivo data, we investigated the effect
of subꢀchronic treatment with 6 in the OA pain model (Figure
3). Because of its short halfꢀlife and moderate Cmax after PO
administration 6 was dosed IP twice daily for 5 days. This
route gave greater acute exposure (~ 2ꢀfold higher Cmax and
5ꢀfold higher AUC after dose correction ꢀ Table S2) and was
calculated to have the best chance of maintaining receptor
coverage over the dosing period. Reversal of MIAꢀinduced
hypersensitivity was assessed 1 h postꢀdose following the
morning dose on days 1, 3, and 5. An effect comparable to the
acute drug response was retained over the duration of the
study at each time point tested, suggesting that dosing with 6
does not induce tachyphylaxis in this model at least under this
dosing paradigm. In contrast, animals treated with morphine
using the same dosing regimen exhibited a significant decrease
in drug response after 3 or more days of dosing.
In summary, we have discovered a selective, and fully efficaꢀ
cious CB2 agonist with an overall satisfactory pharmacokinetic
profile, good pharmaceutical properties and that met our preꢀ
clinical efficacy and safety criteria. 6 was as efficacious as
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ACS Medicinal Chemistry Letters
protein expression and pain behavior in
inflammation. Neuroscience 2003, 119, 747ꢀ757.
a rat model of
morphine in a rat model of OA pain following oral administraꢀ
tion and unlike morphine, maintained its analgesic effect
throughout a subꢀchronic treatment paradigm without tachyꢀ
phylaxis. These data are consistent with our developing hyꢀ
pothesis that a full agonist is required for efficient internalizaꢀ
tion and recycling of the CB2 receptor which we believe may
be a viable approach to avoiding tachyphylaxis. Based on its
favorable profile, 6 (APD371) was selected for clinical develꢀ
opment for the treatment of pain. The outcome of further studꢀ
ies will be disclosed in due course.
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7. Quartilho, A.; Meta, H. P.; Ibrahim, M. M.; Vanderah, T. W.;
Porreca, F.; Makriyannis, A.; Malan, T. P. Inhibition of
inflammatory hyperalgesia by activation of peripheral CB2
cannabinoid receptors. Anesthesiology 2003, 99, 955ꢀ960.
8. Yao, B. B.; Hsieh, G. C.; Frost, J. M.; Fan, Y.; Garrison, T. R.;
Daza, A. V.; Grayson, G. K.; Zhu, C. Z.; Pai, M.; Chandran, P.;
Salyers, A. K.; Wensink, E. J.; Honore, P.; Sullivan, J. P.; Dart,
M. J.; Meyer, M. D. In vitro and In vivo chracterization of Aꢀ
796260: a selective cannabinoid CB2 receptor agonist exhibiting
analgesic activity in rodent pain models. Br. J. Pharmacol. 2008,
153, 390ꢀ401.
9. Guidetti, R.; Astles, P. C.; Sanderson, A. J.; Hollinshead, S. P.;
Johnson, M. P.; Chambers, M. G. The SAR development of
substituted purine derivatives as selective CB2 agonists for the
treatment of chronic pain. Bioorg. Med. Chem. Lett., 2014, 24,
5572ꢀ5575.
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Supporting Information
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The Supporting Information is available in pdf format free of
charge on the ACS Publications website which includes: chemisꢀ
try methods, full characterization data for APD371, in vitro
screening and in vivo pharmacology methods, additional functionꢀ
al and pharmacokinetic data for compounds discussed in the text.
10. Ibrahim, M. M.; Porreca, F.; Lai, J.; Albrecht, P. J.; Rice, F. L.;
Khodorova, A.; Davar, G.; Makriyannis, A.; Vanderah, T. W.;
Mata, H. P.; Malan, T. P. Jr. CB2 cannabinoid receptor activation
produces antinociception by stimulating peripheral release of
endogenous opioids. Proc. Natl. Acad. Sci. USA. 2005, 102, 3093ꢀ
3098.
AUTHOR INFORMATION
Corresponding Author
11. Anand, U.; Otto, W. R.; SanchezꢀHerrera, D.; Facer, P.; Yiangou,
Y.; Korchev, Y.; Birch, R.; Benham, C.; Bountra, C.; Chessell, I.
P.; Anand, P. Cannabinoid receptor CB2 localization and agonistꢀ
mediated inhibition of capsaicin responses in human sensory
neurons. Pain, 2008, 138, 667ꢀ680.
* gsemple@beacondiscovery.com
Present Addresses
¶ Crinetics Pharmaceuticals, 6197, Cornerstone Ct, San Diego,
CA 92121
§ Beacon Discovery, 6118 Nancy Ridge Dr. San Diego CA 92121
† Blackthorn Therapeutics, 329 Oyster Point Blvd, 3rd Floor,
South San Francisco, CA 94080
12. Wilkinson, J. D.; Kendall, D. A.; Ralevic, V. ꢁ9ꢀ
Tetrahydrocannabinol inhibits electrically evoked CGRP release
and capsaicinꢀsensitive sensory neurogenic vasodilatation in the
rat mesenteric arterial bed. Br. J. Pharmacol. 2007, 152, 709ꢀ716.
13. Nagarkatti, P.; Pandey, R.; Rieder S. A.; Hegde, V.; Nagarkatti,
M. Cannabinoids as novel antiꢀinflammatory drugs. Future Med.
Chem. 2009, 1, 1333ꢀ1349.
ABBREVIATIONS
14. (a) Han, S.; Thatte, J.; Buzard, D. J.; Jones, R. M. Therapeutic
utility of cannabinoid receptor type 2 (CB2) selective agonists. J.
Med. Chem. 2013, 56, 8224ꢀ8256. (b) Morales, P.; Hernandezꢀ
Folgado, L.; Goya, P.; Jagerovic, N.; Cannabinoid receptor 2
(CB2) agonists and antagonists: a patent update. Expert Opin.
Ther. Pat. 2016, 26, 843ꢀ56.
CB2, Cannabinoid Receptorꢀ2; CB1, Cannabinoid Receptorꢀ1;
cLogP, calculated LogP; hERG, human EtherꢀàꢀgoꢀgoꢀRelated
Gene; CYP, Cytochrome P450; CHO cell, Chinese Hamster Ovaꢀ
ry cell; CNS, Central Nervous System; HAꢀtag, Hemagglutinin
fragment tag; PO, per oral; IP, intraꢀperitoneal; SC, subꢀ
cutaneous;
TEMPO,
2,2,6,6ꢀtetramethylpiperidineꢀ1ꢀoxyl;
15. Gilron, I.; Dickenson, A.H. Emerging drugs for neuropathic pain.
Expert Opin. Emerg. Drugs, 2014 , 19, 329ꢀ41.
mCPBA, metaꢀchloro perbenzoic acid; PK, pharmacokinetics;
ADME, Adsorption, Distribution, Metabolism and Excretion;
MIA, monoiodoacetate; MTBE, methyl tertꢀbutyl ether; OA, osꢀ
teoarthritis; HATU, 1ꢀ[Bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀ
triazolo[4,5ꢀb]pyridinium 3ꢀoxide hexafluorophosphate; PPB,
Plasma protein binding; AUC, area under the curve. DAMGO, Dꢀ
Ala2, NꢀMePhe4, Glyꢀol]ꢀenkephalin.
16. Han, S.; Thoresen, L.; Zhu, X.; Narayanan, S.; Jung, JꢀK.; Strahꢀ
Pleynet, S.; Decaire, M.; Choi, K.; Xing, Y.; Yue, D.; Semple, G.;
Thatte, J.; Solomon, M.; Fu, L.; Whelan, K.; AlꢀShamma, H.;
Gatlin, G.; Chen, R.; Dang, H.; Pride, C.; Gaidarov, I.; Unett, D.
J.; Behan, D. P.; Sadeque, A.; Usmani, K. A.; Chen, C.; Edwards,
J.; Morgan, M.; Jones, R. M. Discovery of 1a,2,5,5aꢀtetrahydroꢀ
1Hꢀ2,3ꢀdiazaꢀcyclopropa[a]pentalenꢀ4ꢀcarboxamides as potent
and selective CB2 receptor agonists. Bioorg. Med. Chem. Lett.
2015, 25, 322ꢀ326.
17. Ferguson, S. S. G. Evolving concepts in G proteinꢀcoupled
receptor endocyctosis: The role in receptor desensitization and
signaling. Pharmacol. Rev. 2001, 53, 1ꢀ24.
18. Cox, B. M. Agonists at µꢀopioid receptors spin the wheels to keep
the action going. Mol. Pharmacol. 2005, 67, 12ꢀ14.
19. Mcguinness, D.; Malikzay, A.; Visconti, R.; Lin, K.; Bayne, M.;
Monsma, F.; Lunn, C. A. Characterizing Cannabinoid CB2
receptor ligands using DiscoveRx PathHunterTM βꢀArrestin assay.
J. Biomol. Screen, 2009, 14(1), 49ꢀ58.
20. Alorati, A. D.; Bio, M. M.; Brands, K. M. J.; Cleator, E.; Davis,
A. J.; Wilson, R. D.; Wise, C. S., A practical and scalable syntheꢀ
sis of 1R,5Sꢀbicyclo[3.1.0]hexanꢀ2ꢀone: The development of a
catalytic lithium 2,2,6,6ꢀtetramethylpiperidide (LTMP) mediated
intramolecular cyclopropanation of (R)ꢀ1,2ꢀepoxyhexꢀ5ꢀene. Org.
Process. Res. Dev., 2007, 11, 637ꢀ641.
21. Boatman, P. D.; Schrader, T. O.; Kasem, M.; Johnson, B. R.;
Skinner, P. J.; Jung, JꢀK.; Xu, J.; Cherrier, M. C.; Webb, P. J.;
Semple, G.; Sage, C. R.; Knudsen, J.; Chen, R.; Tassart, A. K.;
CarballoꢀJane, E.; Richman, J. G. Potent tricyclic pyrazole teꢀ
trazole agonists of the nicotinic acid receptor (GPR109a). Bioorg.
Med. Chem. Lett. 2010, 20, 2797ꢀ2800.
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