Imidazopyridine CB2 agonists: Optimization of CB2/CB1 selectivity and implications for in vivo analgesic efficacy

Article abstract of DOI:10.1016/j.bmcl.2011.02.082

A new series of imidazopyridine CB2 agonists is described. Structural optimization improved CB2/CB1 selectivity in this series and conferred physical properties that facilitated high in vivo exposure, both centrally and peripherally. Administration of a h

Full text of DOI:10.1016/j.bmcl.2011.02.082

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2354–2358
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Imidazopyridine CB2 agonists: Optimization of CB2/CB1 selectivity
and implications for in vivo analgesic efficacy
B. Wesley Trotter a, , Kausik K. Nanda b, Christopher S. Burgey b, Craig M. Potteiger b, James Z. Deng b,
⇑
Ahren I. Green b, John C. Hartnett b, Nathan R. Kett b, Zhicai Wu b, Darrell A. Henze c, Kimberly Della Penna c,
Reshma Desai c, Michael D. Leitl c, Wei Lemaire c, Rebecca B. White d, Suzie Yeh d, Mark O. Urban c,
Stefanie A. Kane c, George D. Hartman b, Mark T. Bilodeau b
a Department of Medicinal Chemistry, Merck Research Laboratories, Boston, MA, USA
b Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA, USA
c Department of Pain Research, Merck Research Laboratories, West Point, PA, USA
d Department of Drug Metabolism & Pharmacokinetics, Merck Research Laboratories, West Point, PA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of imidazopyridine CB2 agonists is described. Structural optimization improved CB2/CB1
selectivity in this series and conferred physical properties that facilitated high in vivo exposure, both cen-
trally and peripherally. Administration of a highly selective CB2 agonist in a rat model of analgesia was
ineffective despite substantial CNS exposure, while administration of a moderately selective CB2/CB1
agonist exhibited significant analgesic effects.
Received 10 November 2010
Revised 17 February 2011
Accepted 22 February 2011
Available online 1 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
CB2 agonist
CB1 agonist
Selectivity
Pain
CFA model
The known analgesic effects of natural cannabinoids and the
identification of the CB1 and CB2 cannabinoid receptors have
engendered a large and active area of research aimed at agonists
of these receptors. Both endogenous and medicinal cannabinoids
are known to act as agonists of CB1 and CB2.1 Given the association
of CB1 agonism with the psychotropic effects of natural cannabi-
noids, selective CB2 agonism has been proposed as a potential
means of modulating pain-related behaviors without concomitant
undesirable neurological effects.2 Investigators in both academia
and industry have pursued CB2 agonists, and a substantial body
of literature has emerged detailing the design, synthesis, and
in vivo evaluation of CB2-preferring compounds with good selec-
tivity for CB2 agonism versus CB1 agonism.3
The vast majority of these studies involve molecules that retain
some measurable CB1 agonist activity.2 Given the existence of a
large CB1 receptor reserve in the central nervous system and the
demonstrated ability of weak CB1 binding to produce substantial
agonism,4 residual CB1 activity of CB2-preferring compounds
may confound the dissection of CB2 and CB1 agonism in many of
these studies. In this and the accompanying Letter,5 we present
the design and synthesis of two structurally distinct CB2 agonists
with no measurable in vitro CB1 agonist activity. Evaluation of
these highly selective compounds in vivo indicates that they do
not affect rat pain responses despite high peripheral and central
exposures. These results counter the perception that CB2 agonism
alone can produce a significant analgesic effect.
We based our initial investigations on indole A (later termed
GW405833, Fig. 1), which was discovered at Merck Frosst and
shown to bind potently to the CB2 receptor with some selectivity
over CB1.6 Given that a number of investigators have reported vari-
ants of this [6,5] heterocyclic structure to be potent CB2 agonists,7
we focused our own design efforts on closely related scaffolds. We
report here a new variant of this theme, imidazopyridine CB2 ago-
nists (Fig. 1). The imidazopyridines are potent CB2 agonists with
highly tunable CB2/CB1 selectivity and physical properties that al-
low CNS penetration. Exploration of SAR in this series has produced
both CB2-preferring agonists and agonists that appear to be com-
pletely selective for CB2 versus CB1. In vivo evaluation of these
compounds indicates a significant impact of the degree of CB2/
CB1 selectivity on analgesic effects in this series.
Synthesis of imidazopyridines relied on elaboration of appropri-
ate aminomethyl pyridines (Scheme 1). Condensation of 2-amino-
methyl pyridine with ethyl oxalyl chloride provided ester 1.
⇑
Corresponding author.
E-mail address: bwesley_trotter@merck.com (B.W. Trotter).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.082

B. W. Trotter et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2354–2358
2355
O
O
Cl
Cl
MeO
N
O
OMe
NHR
a,b
c,d
O
A / GW405833
NH2
N
N
N
N
N
40%
Me
N
Human CB2 Ki 10 nM
Human CB1 Ki 2000 nM
8
9
10
MeO
e
83%
O
O
O
O
OMe
OMe
O
OMe
g
f
N
N
N
N
N
N
R1
NH
80%
100%
O
O
O
R1
R1
11
13
12
H
R2N
N
N
h
63%
N
N
N
N
N
R2
R2
R2
O
c,d
NHR
N
O
OMe
Figure 1. Imidazopyridine analogs of A.
N
N
N
14
15
Formylation with POCl3/DMF then gave imidazopyridine aldehyde
2. Reductive amination was employed to afford 3, which could be
transformed into ketone derivatives such as 6a via hydrolysis,
Weinreb amide formation, and Grignard addition. Carboxylic acid
4 also underwent routine amide coupling reactions to provide
amides of type 7.
Br
R2N
O
O
OMe
NHR
60%
c,d
i
N
N
N
N
17
16
R
R
Isomeric imidazopyridines were prepared using aza-phenylgly-
cine ester 8 (Scheme 2).8 In order to prepare alkyl-substituted ana-
logs 10, nitrogen acylation followed by cyclization was employed
to provide ester 9. Ester hydrolysis and amide coupling afforded
amides 10. Alternatively, treatment of 8 with DMF dimethylacetal
provided imidazopyridine 11, which was a versatile intermediate
for preparation of multiple 1-substituted variants. Formylation of
11 proceeded in high yield to afford 12, which could be reductively
aminated to provide esters such as 13. Standard hydrolysis and
coupling reactions provided CB2 agonists of type 14. Bromination
of 11 provided 15, which underwent palladium-catalyzed coupling
reactions with amines9 to give 16. The ester hydrolysis and cou-
pling sequence then provided amides 17.
Scheme 2. Reagents and conditions: (a) cyclopropylcarbonyl chloride, sodium
bicarbonate, dichloromethane, rt; (b) POCl3, DMF; (c) NaOH, MeOH/water, rt; (d) R-
NH2, EDC, iPr2NEt, HOAt, DMF, rt; (e) DMF dimethyl acetal, toluene, reflux; (f) POCl3,
DMF, 115 °C; (g) morpholine, sodium triacetoxy-borohydride, dichloromethane, rt;
(h) Br2, acetic acid; (i) morpholine, Cs2CO3, XantPhos, Pd2dba3, 100 °C.
O
O
OEt
OEt
a, b
c
N
N
N
N
NH2
18
N
N
N
39%
N
88%
Br
HCl
19
20
A facile synthesis of related imidazopyrazine analogs was also
developed to access compounds of type 22 (Scheme 3). Application
F3C
O
O
OEt
N
d
90%
NH
e,f
N
N
N
N
N
O
N
O
OEt
22
OEt
65%
N
a
b
N
21
N
N
N
N
NH2
N
O
97%
65%
O
2
1
O
H
Scheme 3. Reagents and conditions: (a) ethyl oxalyl chloride, triethylamine,
THF,0 °C to rt; (b) POCl3, DMF, 100 °C; (c) NBS, triethylamine, acetonitrile, 0 °C to
rt; (d) morpholine, Cs2CO3, XantPhos, Pd2dba3, 100 °C; (e) NaOH, MeOH/water, rt;
(f) R-trifluoromethyl(amino)methyl-2-pyridine, EDC, iPr2NEt, HOAt, DMF, rt.
O
N
O
N
OH
OEt
N
N
86%
N
g
c
d
N
100%
of the acylation/cyclization sequence to aminomethylpyrazine 18
provided the imidazopyrazine scaffold 19, which underwent
NBS-mediated bromination and palladium-catalyzed amination
to provide 21. Ester hydrolysis and amide coupling provided ago-
nists 22.
These routes allowed facile variation of substituents on both
carbon positions of the imidazo ring of the core. Optimization of
compounds focused on functional CB2 agonist efficacy and CB2/
CB1 selectivity.10 We also targeted agonists with high plasma free
fraction; for compounds attaining similar plasma exposures, this
strategy was expected to produce compounds capable of achieving
significant free concentrations in the brain.11 As shown in Table 1,
both ketone (6) and amide (7) derivatives of the ‘Type I’ imidazo-
pyridine scaffold were potent CB2 agonists.
3
O
O
4
Cl
Cl
O
N
O
N
Me
O
N
NHR
N
e
78%
OMe
f
N
N
N
N
N
85%
N
6a
7
5
O
O
O
Scheme 1. Reagents and conditions: (a) ethyl oxalyl chloride, triethylamine, THF,
rt; (b) POCl3, DMF, reflux; (c) morpholine, sodium triacetoxyborohydride, 1,2-
dichloroethane, rt; (d) NaOH, MeOH/water, rt; (e) EDC, iPr2NEt, DMF, MeONHMe–
HCl, rt; (f) (2,3-dichlorophenyl)magnesium iodide, THF, 0 °C to rt; (g) EDC, iPr2NEt,
DMF, HOAt, RNH2, rt.

2356
B. W. Trotter et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2354–2358
Table 1
Table 2
Type I imidazopyridine CB2 agonists
Type II imidazopyridine CB2 agonists
O
R1
O
R1
N
N
N
N
R2
hCB2
cAMP
IC50 nM
hCB1
cAMP
IC50 nM
Plasma
free
R2
fractionb
hCB2
cAMP
hCB1 cAMP Plasma
Entry R1
R2
a
a
(Emax
)
(Emax)
IC50 nMa
free
IC50 nMa (Emax
)
fractionb
OH
Entry R1
Cl
R2
(Emax
)
F
F
N
14a
3 (88%)
68 (99%) ND
Cl
F
NH
N
N
N
N
O
O
6a
6b
6c
5 (94%)
2565 (66%) 24%
2229 (97%) 3%
90 (101%) ND
10a
10b
7 (99%)
>17000 3%
>17000 5%
F3C
Cl
NH
NH
NH
0.7 (98%)
0.3 (110%)
Me
Me
Me
21 (97%)
Cl
F
F
F3C
N
N
17a
17b
21 (97%) 705 (55%) 10%
N
CF3
CF3
F
F
O
N
7a
1.7 (98%)
347 (100%) ND
OH
NH
33 (99%)
>17000 16%
O
N
NH
S
O
O
N
7b
44 (80%) 12700 (54%) 11%
a
NH
See Ref. 10.
See Ref. 10.
b
Measured via ultrafiltration or equilibrium dialysis following incubation with
a
rat plasma.
b
Measured via ultrafiltration or equilibrium dialysis following incubation with
rat plasma.
Table 3
Imidazopyrazine CB2 agonists
O
R1
Moderate CB2/CB1 selectivity (ca. 500-fold) was generally ob-
N
N
served. A survey of aminomethyl substituents identified moder-
ately basic amines including morpholine and 4-difluoropiperidine
that conferred good CB2 functional efficacy. Amines containing po-
lar functionality (6a, 7b) exhibited high plasma free fraction, with
morpholine analogs exhibiting more potent CB2 agonism.
Investigation of the ‘Type II’ scaffold (Table 2) revealed that, in
contrast to aminomethyl-substituted agonists (14a), analogs bear-
ing cyclopropyl substitution (10a,b) or a directly attached morpho-
line substituent (17a,b) displayed improved CB2/CB1 selectivity.
Variation of the amide substituent was also found to modulate effi-
cacy of CB2 agonism as well as selectivity over CB1. Again, incorpo-
ration of polarity was pursued in order to reduce plasma protein
binding. Hydroxymethyl-containing amide 17b was a potent CB2
agonist that displayed no CB1 agonism in vitro.
N
R2
hCB2
hCB1
Plasma
cAMP IC50 cAMP IC50 free
nMa (Emax nMa (Emax fractionb
Entry R1
R2
)
)
F
N
N
22a
92 (96%) >17000
36 (99%) >17000
3%
F
N
F
NH
Me
Me
Me
Me
22b
6%
NH
Imidazopyrazines incorporating directly attached amino sub-
stituents were also found to display generally high CB2/CB1 selec-
tivity (Table 3). As observed for the des–aza scaffolds, CB2 efficacy
could be tuned by variation of both the amide and amino function-
ality, and plasma free fraction was impacted by incorporation of
polar atoms (22a,b vs 22c,d). The impact of nitrogen incorporation
in the core is illustrated by comparison of 22c and 17a (Table 2);
while CB2 efficacy is reduced somewhat, both selectivity and free
fraction are significantly improved upon migration to the imidazo-
pyrazine core.
F
F
F3C
NH
N
22c
22d
139 (94%) >17000
69 (98%) >17000
18%
25%
N
O
F3C
NH
N
N
O
These efforts provided us with a collection of imidazopyridine
CB2 agonists with a range of selectivity profiles and physicochem-
ical attributes. Compounds 6a and 17b (Fig. 2) were identified as
a
See Ref. 10.
b
Measured via ultrafiltration or equilibrium dialysis following incubation with
rat plasma.

B. W. Trotter et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2354–2358
2357
Cl
reversals in paw withdrawal threshold at 100 mpk (Fig. 3). Plasma,
brain, and CSF levels at the 100 mpk dose (60 min timepoint) were
4500, 5400, and 110 nM, respectively.
In contrast, the more selective agonist 17b elicited no change in
paw withdrawal threshold when dosed at 100 mpk (Fig. 4). Plasma,
brain, and CSF levels (120 min timepoint) were measured at 4300,
1300, and 300 nM, respectively, all well above the in vitro CB2 IC50
values.
Cl
N
hCB2 cAMP IC50 5 nM (94% activity)
O
hCB1 cAMP IC50 2565 nM (66% activity)
N
Rat CB2 cAMP IC50 11 nM (87% activity)
Rat CB1 cAMP IC50 1068 nM (104% activity)
N
O
6a
Rat Plasma Protein Binding 76%
Our results indicate that two compounds with a high degree of
structural similarity, differing mainly with respect to their CB2/CB1
selectivity profiles, exhibit markedly different efficacy profiles in
the rat CFA model. By obtaining plasma, brain, and CSF exposure
data, we have demonstrated that both compounds are present in
both the systemic circulation and the CNS in levels exceeding their
CB2 IC50 values. The analgesic effect observed for 6a combined
with the lack of effect seen with the more selective agonist 17b
suggests that CB1 agonism may play a role in the efficacy of 6a.
In fact, almost all selective CB2 agonists that have been evaluated
in rodent pain models do exhibit measurable CB1 agonism.13,14 The
in vitro profile of 6a is similar to many agonists reported in the lit-
erature. It is selective against CB1 (rat CB2/CB1 ꢀ 100-fold) but
nonetheless exhibits significant efficacy as a CB1 agonist.15
Relevant to these observations is the demonstration by Gifford
et al. that a large CB1 receptor reserve exists in the CNS. These
investigators demonstrated that major downstream effects of
CB1 agonism can be observed at <1% occupancy and that 95% inhi-
bition of these effects occurs at <8% occupancy of the CB1 receptor.
Thus even a compound with a relatively high EC50 for CB1 agonism
may significantly affect CB1 signaling.4
OH
hCB2 cAMP IC50 33 nM (99% activity)
hCB1 cAMP IC50 >17000 nM
O
NH
Rat CB2 cAMP IC50 58 nM (87% activity)
Rat CB1 cAMP IC50 >17000 nM
N
N
N
Rat Plasma Protein Binding 84%
17b
O
Figure 2. CB2 agonists for in vivo dosing.
appropriate tools to elucidate the impact of CB2/CB1 selectivity on
in vivo analgesic effects. Both compounds exhibited potent CB2
agonism in vitro as well as high rat plasma free fraction. Thus it
was anticipated that both compounds would effect CB2-mediated
pharmacology both in the CNS and the periphery when dosed to
rats. The key difference between the two compounds concerned
CB2/CB1 selectivity. 6a retained moderate but significant CB1
agonism in vitro, while 17b exhibited no in vitro agonism against
either the human or rat CB1 receptor (Fig. 2).
Analgesic effects of the two compounds were evaluated in a rat
CFA hyperalgesia model.12 Both 6a and 17b were dosed either or-
ally or subcutaneously to rats in order to maximize peripheral and
CNS exposure. Plasma, brain, and CSF11 levels were measured for
each compound in order to verify that adequate exposure for CB2
activity was achieved. Moderately selective agonist 6a exhibited
dose-dependent effects in the CFA model, giving naproxen-like
An important caveat to this work (and to published CB2 agonist
studies to date) is the absence of a direct demonstration of CB2 and
CB1 agonism in vivo by our compounds. However, we have demon-
strated that the compounds achieve high exposure in vivo, both
peripherally and centrally, with a significant fraction of unbound
compound available. In the accompanying paper, study of a struc-
turally dissimilar series of CB2 agonists provides further evidence
that CB2 agonism alone may be insufficient for analgesic activity.5
450
Vehicle
20 mpk Naproxen
10 mpk Cmpd 6a
30 mpk Cmpd 6a
100 mpk Cmpd 6a
400
100
75
50
25
0
350
300
250
200
150
100
50
67%
57%
*
*
*
*
*
*
*
25%
18%
-6%
0
BL
CFA
30 min
60 min
60 min post-dose
Figure 3. Agonist 6a dosed in the rat CFA model.
100
450
400
350
300
250
200
150
100
50
Vehicle
20 mg/kg Naproxen
100 mg/kg Cmpd 17b
75
50
*
*
25
0
0
120 min post-administration
BL
CFA
60 min 120 min
Figure 4. Agonist 17b dosed in the rat CFA model.

2358
B. W. Trotter et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2354–2358
temperature for 15 min. Then, forskolin at EC70 was added and incubated at
Acknowledgments
room temperature for an additional 30 min. Detection of cAMP was performed
using Cisbio’s HRTF cAMP dynamic 2 kit following the manufacturer’s protocol
for cAMP detection. After adding d2-cAMP and anti-cAMP-cryptate to all the
wells, there was a final incubation at room temperature for 1 h. Plates were
then read on an EnVision plate reader (Perkin Elmer). CV values calculated for a
positive control compound (>250 replicates) were 59% and 70%, respectively,
for CB1 and CB2 IP values.
We would like to thank Joan S. Murphy and Dr. Charles W. Ross
III for HRMS analysis. We would also like to thank Janine N. Brouil-
lette and Dr. Steven M. Pitzenberger for NMR support.
11. Liu, X. R.; Smith, B. J.; Chen, C.; Callegari, E.; Becker, S. L.; Chen, X.; Cianfrogna,
J.; Doran, A. C.; Doran, S. D.; Gibbs, J. P.; Hosea, N.; Liu, J. H.; Nelson, F. R.; Szewc,
M. A.; Van Deusen, J. Drug Metab. Dispos. 2006, 34, 1443.
12. Nagakura, Y.; Okada, M.; Kohara, A.; Kiso, T.; Toya, T.; Iwai, A.; Wanibuchi, F.;
Yamaguchi, T. J. Pharmacol. Exp. Ther. 2003, 306, 490.
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10. Compounds were added to a Greiner black 384 well low volume assay plate.
1000 CHO-K1 cells (expressing human or rat CB1 or CB2) were added to each
well of the assay plate containing compound, then incubated at room
13. GSK CB2 agonist clinical candidate GW-842166X is unique in that it is reported
to produce analgesia in preclinical models despite exceedingly weak CB1
agonist activity. However, investigators at Abbott (Ref. 14) did observe
measurable rat CB1 agonist activity for this compound. Giblin, G. M. P.;
O’Shaughnessy, C. T.; Naylor, A.; Mitchell, W. L.; Eatherton, A. J.; Slingsby, B. P.;
Rawlings, D. A.; Goldsmith, P.; Brown, A. J.; Haslam, C. P.; Clayton, N. M.;
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15. Consistent with residual CB1 agonism, 6a exhibited slight but significant
impairment of motor coordination when dosed at 100 mpk po in a rat rotarod
model. Rotarod experiments were performed as described in: Boyce, S.; Wyatt,
A.; Webb, J. K.; O’Donnell, R.; Mason, G.; Rigby, M.; Sirinathsinghji, D.; Hill, R.
G.; Rupniak, N. M. J. Neuropharmacol. 1999, 38, 611.