Pyridine-3-carboxamides as novel CB2 agonists for analgesia

Article abstract of DOI:10.1016/j.bmcl.2008.10.118

We describe herein the medicinal chemistry approach which led to the discovery of a novel pyridine-3-carboxamide series of CB₂ receptor agonists. The SAR of this new template was evaluated and culminated in the identification of analogue 14a wh

Full text of DOI:10.1016/j.bmcl.2008.10.118

Bioorganic & Medicinal Chemistry Letters 19 (2009) 259–263
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyridine-3-carboxamides as novel CB2 agonists for analgesia
*
William L. Mitchell , Gerard M. P. Giblin, Alan Naylor, Andrew J. Eatherton, Brian P. Slingsby,
Anthony D. Rawlings, Karamjit S. Jandu, Carl P. Haslam, Andrew J. Brown, Paul Goldsmith,
Nick M. Clayton, Alex W. Wilson, Iain P. Chessell, Richard H. Green, Andrew R. Whittington, Ian D. Wall
GlaxoSmithKline PLC, Neurosciences Centre of Excellence for Drug Discovery, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe herein the medicinal chemistry approach which led to the discovery of a novel pyridine-3-
carboxamide series of CB2 receptor agonists. The SAR of this new template was evaluated and culminated
in the identification of analogue 14a which demonstrated efficacy in an in vivo model of inflammatory
pain.
Received 17 September 2008
Revised 17 October 2008
Accepted 18 October 2008
Available online 5 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Analgesia
CB2 agonist
Inflammatory pain
Cannabinoid receptors
GW842166X
Structure–activity-relationships
Pharmacokinetics
Solubility
Weinreb reaction
Cytochrome P450, in vitro clearance
Hyperalgesia
Complete Freund’s Adjuvant
Bioavailability
4-Cyclopropyl-6-[(2,4-
dichlorophenyl)amino]-N-[(tetrahydro-2H-
pyran-4-yl)methyl]-3-pyridinecarboxamide
Cannabis sativa has been used as an analgesic as early as 2800
BC in China.1 Cannabinoids are the pharmacologically2 active com-
ponents of cannabis, and are known to mediate some of their
actions through the cannabinoid receptors3 CB1 and CB2, both of
which are G-protein coupled receptors. The CB1 receptor is found
in the central nervous system (CNS),4 as well as in the periphery,5
whereas the CB2 receptor is found mainly in the periphery,6 partic-
ularly in the immune system.7 There is also increasing recent
evidence of CB2 receptor expression in the CNS.8 Whilst stimula-
tion of the CB1 receptor mediates analgesia,9 this receptor also
mediates other undesirable CNS side-effects.10 CB2 agonists, on
the other hand, have shown efficacy in preclinical models of
inflammatory and neuropathic pain.11
novel class of selective CB2 agonists culminated in the discovery of 1
(GW842166X, Fig. 1),12 which has entered human clinical studies for
the treatment of pain. In this letter we describe our attempts to de-
O
CF3
O
O
Cl
N
N
N
N
H
O
N
H
N
Cl
1
GW842166X
N
2
A-796260
O
O
N
S
F3C
N
O
N
O
N
We have therefore sought to design selective CB2 agonists to
avoid the undesirable psychoactive effects and abuse potential asso-
ciated with agonism of the CB1 receptor. Recently our research into a
F
3
CBS-0550
4
* Corresponding author. Tel.: +44 7791634273.
E-mail address: bill.mitchell777@tiscali.co.uk (W.L. Mitchell).
Figure 1. Recent lead CB2 agonists.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.118

260
W. L. Mitchell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 259–263
CF3
sign a new series of selective CB2 agonists with improved aqueous
solubility over GW842166X (solubility at pH 7.4 is 2 g/mL).
CF3
N
CO2Me
l
a
CO2Me
R1
R1
Interest in selective CB2 agonists has been growing, and, in addi-
tion to 1, a number of other groups have reported novel selective
CB2 agonists. Abbott13 have disclosed the morpholine 2, A-
796260, whilst Taisho14 have revealed their lead compound, an
iminopyrazole 3, CBS-0550, and Boehringer Ingelheim-Evotec15
have published a novel series of selective CB2 agonists, exemplified
by the sulphonamide 4.
NH2
Cl
N
7
N
H
6
b
c, H2NCH2R3
CF3
O
Following the identification of 1 as a clinical candidate, we began
to look for a CB2 agonist back-up candidate with improved aqueous
solubility over 1. As part of this programme, we investigated replac-
ing the central pyrimidine core with a pyridine core, since the basic
pKa of pyridines (5.2) is higher16 than pyrimidines (1.3), and molec-
ular overlays suggested good alignment17 of key features of the pyr-
idine 5 with 1. In the current research presented here, we describe
the structure–activity-relationships (SAR), the pharmacokinetics,
and efficacy in a pain model of these novel pyridine CB2 agonists.
R2
N
R1
H
N
H
N
8a-j
Scheme 1. Reagents and conditions: (a) Neat, 120 °C; (b) KOH, H2O, reflux; (c) EDC,
HOBT or HATU, PS-carbodiimide, HOBT or PS-diisopropylethylamine, 1-butyl-3-
methylimidazolium hexafluorophosphate.
Table 1
R2
O
CB2 Agonist potencies18 and efficacies for compounds (8a–j).
NHR3
CF3
O
5
R1
N
R2
N
N
R1
H
H
8a-j
N
N
H
Initially we chose to prepare the 4-(trifluoromethyl)pyridines
8a–j, which retained the 4-(trifluoromethyl)-substituent, so that
the direct effects of changing the pyrimidine core to a pyridine core
could be compared with our earlier SAR evaluation which led to
the discovery of 1. Condensation18 of the anilines 6 with the chlo-
ropyridine 7, followed by hydrolysis and amide formation gave the
final products 8a–j (Scheme 1).
All but one (8j) of the analogues 8a–j were found to be CB2 ago-
nists with an efficacy not significantly different from the reference
CB2 agonist HU210 (Table 1—the assay for CB2 was run using a
yeast CB2 reporter assay in yeast cells, Saccharomyces cerevisiae,
expressing the human CB2 receptor18). Initially the amide nitrogen
a
Compound
R1
R2
pEC50
SDb
E %c
8a
8b
8c
8d
8e
8f
8g
8h
8i
2,4-di-Cl
3-Cl
3-F
4-Cl,2-dMe
2-MeO,5-Cl
2-MeO,5-Cl
2,4-di-Cl
3-Cl
4-Tetrahydropyranyl
4-Tetrahydropyranyl
4-Tetrahydropyranyl
4-Tetrahydropyranyl
4-Tetrahydropyranyl
Cyclobutyl
Cyclopentyl
Cyclohexyl
4-Fluorophenyl
4-H2NCO-Phenyl
7.3
6.6
6.0
6.6
6.4
6.7
7.0
7.5
7.1
5.8
0.3
0.3
0.1
0.1
0.2
0.05
0.2
0.3
0.05
0.1
100
95
90
94
89
87
86
84
84
10
3-Br
3-Cl
8j
a
CB2 assay data is the mean of at least two determinations, see Ref. 18 for the
method, HU210 had pEC50 8.6 SD 0.2 and was used as a reference.
substituent was kept fixed as
a (4-tetrahydropyranyl)methyl
b
Standard deviation.
group, 8a–e, whilst the aniline substituent R1 was varied. This re-
vealed that a 2,4-dichloro-substituent, as in 8a, was optimal for
CB2 potency. However, variation of the amide N-substituent was
well tolerated and other cycloalkyl groups, 8f–h, gave CB2 agonists
of comparable potency and efficacy to the tetrahydropyranyl
group. Indeed, the cyclohexyl group improved CB2 potency, (8h
cf 8b). A 4-fluorobenzyl group 8i was similarly tolerated on the
amide nitrogen, but the 4-carboxamidobenzyl group in 8j was not.
All of the analogues showed selectivity over CB1 with a pEC50 of
less than 5.5 and/or an efficacy of less than 10% at CB1.
c
Efficacy (E) is expressed as a percentage relative to the efficacy of HU210 (an
unselective CB1/CB2 agonist. All analogues show little or no activity at the human
CB1 receptor, that is, CB1 pEC50 < 5.5 and/or E < 10% (data not shown).
The aqueous solubility19 of the 4-(trifluoromethyl)pyridines 8a-
j was investigated, but it was not improved compared to 1: for
example, 8a has a solubility of <1 lg/ml at pH 7.4. We therefore
sought to improve the solubility by synthesizing18 the 4-(isopro-
pyl)pyridines 12a–o (R2 = iPr, Scheme 2, Table 2), as the pKa of
4-(isopropyl)pyridine20 is 6.02 compared to the pKa of 2.63 for
4-(trifluoromethyl)pyridine.21
R2
COCl
CONHR3
CONHR3
a H2NR3
b
R1
11
Cl
N
Cl
N
9
Cl
N
R2
NH2
10
c
CONHR3
R1
N
N
H
R1
e
12a-o
NH2
CO2H
H2NR3
CO2H
CO2H
(11)
c
d
R1
N
N
Cl
N
Cl
N
H
13
i
Scheme 2. Reagents and conditions: (a) Et3N, 0 °C; (b) R2MgBr, 0 °C ? rt, then DDQ.; (c) microwave 150–180 °C or microwave, MsOH, 180 °C; (d) PrMgBr, 0 °C ? rt, then
Mn(OAc)3 À60 °C ? rt; microwave, 120 °C; (e) EDC, HOBT.

W. L. Mitchell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 259–263
261
Table 2
The in vitro data (Table 2) showed that these 4-isopropylpyri-
dines 12a–o were potent CB2 agonists with an efficacy not signifi-
cantly different from the reference CB2 agonist HU210. Exploration
of the aniline substituent R1 to investigate the electronic require-
ments showed that the optimal 3-substituent for CB2 potency
was a halogen, either chlorine or bromine 12a,b. However, di-sub-
stitution was tolerated, 12f–h, and yielded analogues of similar po-
tency to 12a. Modification of the amide N-substituent showed that
the cyclobutyl ring increased the CB2 potency, 12j cf 12d, and that
a number of other rings, but not all (e.g., 12l), were tolerated 12i–
k. The isobutyl group gave a potent CB2 agonist, 12m, with a supra-
maximal response. Other N-alkyl substituents were less potent
12n,o. All analogues in this series were selective over CB1, with
CB1 pEC50 6 5.5 and/or an efficacy of <7%.
CB2 agonist potencies18 and efficacies for compounds 12a–o
O
NHR3
R1
N
N
H
(12a-o)
.
R1
R3a
pEC50
SDb
E %b
b
Compound
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
3-Cl
3-Br
(4-THP)methyl–
(4-THP)methyl–
(4-THP)methyl–
(4-THP)methyl–
(4-THP)methyl–
(4-THP)methyl–
(4-THP)methyl–
(4-THP)methyl–
Cyclopentylmethyl–
Cyclobutylmethyl–
4-Fluorobenzyl–
4-THP–
6.9
7.2
6.4
6.3
6.4
7.3
7.1
7.2
7.4
7.3
7.0
6.3
7.4
6.6
6.4
0.15
0.1
0.1
0.2
0.1
0.04
0.15
0.1
0.15
0.2
101
105
82
88
100
80
75
98
105
87
89
3-MeO–
3-Me–
3-NC–
2,4-di-Cl–
3-Cl, 4-NC–
2-F,3-CF3–
3-Cl
3-Me
3-Cl
3-Cl
3-Cl
However, the solubility of the 4-(isopropyl)pyridine series, de-
spite a small improvement, was still unacceptably low, for exam-
ple, 12f has a solubility of 5 lg/mL at pH 7.4. In addition,
preliminary in vitro rat clearance22 for many of the more potent
analogues, where measured, was also unacceptably high (e.g., for
12b,f,i,m, rat CLi >5 mL mÀ1 gÀ1 liver), and so further optimization
was undertaken.
0.1
0.2
0.1
0.4
85
121
97
iPrCH2–
Our attention turned to investigate the SAR of the 4-substituent,
that is, R2, (Table 3). Analogues with an R2 cycloalkyl group 14a,b
were prepared23 by the routes previously used (Scheme 2),
however, the introduction of a 4-(2-hydroxyprop-2-yl)-group or
a 4-(tert-butyl) group required new syntheses (Scheme 3). Ortho-
lithiation of the acid 13, followed by treatment with acetone gave
the lactone 15. Displacement of the chlorine with an aniline gave
the precursor 16. Reaction23 of 16 with an amine using the
conditions described by Weinreb,24 effected ring opening to give
the 4-(2-hydroxyprop-2-yl)-analogue 14c. The isopropyl interme-
diate 17, (prepared by the routes shown in Scheme 2) was alkyl-
ated25a to give the tert-butylpyridine 18, and this converted by
the usual methods into the final tert-butyl analogue 14d.
3-Cl
3-Cl
tBuCH2–
Et2CHCH2–
0.25
86
a
THP, tetrahydropyranyl.
See footnotes in Table 1 for definition of these terms. All analogues show little
b
or no activity at the human CB1 receptor, that is, CB1 pEC50 < 5.5 and/or E 6 7% (data
not shown).
The 4-(isopropyl)pyridines 12a–o (R2 = iPr) were prepared18 by
addition of a Grignard reagent to the pyridinecarboxamide 9, fol-
lowed by reaction of 10 with an aniline 11. Alternatively, addition
of isopropylmagnesium chloride to the nicotinic acid 13, followed
by reaction with an aniline 11, and standard amide formation also
gave the desired 4-(isopropyl)pyridines 12a–o (R2 = iPr).
Table 3
CB2 potencies, in vitro and in vivo pharmacokinetics, and aqueous solubility
R2
N
O
N
H
O
R1
N
H
12f and 14a-o
.
Compound
R1
R2
CB2
SDa
rCLib
hCLib
CYP450 IC50
(
l
M)
AUC
Solubility
a
pEC50
E%a
1A2
2C9
2C19
2D6
3A4
(0 À t)/dose (min kg/L)c
l
g/mL at pH 7.4
12f
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
3-Cl
3-Br
3-CF3
3-OCF3
3-NC
3-MeO
3-Me
3-F
2,5-dichloro
3,4-dichloro
3,5-dichloro
iPr
7.3
7.1
6.8
5.9
7.9
6.8
6.7
6.7
6.6
6.1
5.7
5.8
5.6
6.4
6.8
7.2
0.04
0.2
0.1
0.05
0.1
0.1
0.1
0.3
0.1
0.2
0.3
0.2
0.1
0.1
0.1
0.4
80
90
61
79
100
82
93
76
87
78
65
76
87
92
70
57
7.6
4.3
nd
nd
2.3
nd
1.6
nd
3.5
nd
nd
nd
nd
12.0
nd
nd
4.0
1.7
nd
nd
<0.5
nd
<0.5
nd
1.0
nd
nd
nd
60
nd
nd
nd
22
nd
23
nd
NT
nd
nd
nd
nd
53
nd
nd
40
43
nd
nd
17
nd
24
nd
16
nd
nd
nd
nd
27
nd
nd
43
82e
nd
nd
28
nd
29
nd
65
nd
nd
nd
nd
49
nd
nd
28
55
nd
nd
>100
nd
16
nd
24
nd
nd
nd
nd
42
nd
nd
58
>100
nd
nd
64
nd
15
nd
nd
16
nd
5
5
7
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
14o
Cyclopropyl
Cyclopentyl
–C(OH)Me2
tBu
<1
150
23
91
45
19
<1
4
168
26
125
12
6
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
nd
>100e
nd
nd
3
57d
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
>100
nd
nd
2.4
nd
nd
nd
7
a
See footnotes in Table 1 for definition of these terms. All analogues show little or no activity at the human CB1 receptor, that is, CB1 pEC50 < 5.5 and/or E 6 5% (data not
shown).
b
c
In vitro clearance, mL minÀ1 gÀ1 liver; r, rat; h, human; see Ref. 22 for procedures.
Administered to rats as a suspension in aqueous 1% methylcellulose.
Interference in the assay. nd, not determined.
d

262
W. L. Mitchell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 259–263
OH
O
O
O
CO2H
Cl
Cl
O
O
c
a
b
N
H
O
Cl
N
N
N
Cl
N
N
H
N
H
Cl
Cl
13
14c
15
16
O
O
O
Cl
b
d
N
H
N
N
H
H
O
O
O
Cl
N
Cl
N
N
N
H
Cl
17
18
14d
Scheme 3. Reagents and conditions: (a) LiTMP, À55 °C, then Me2CO, À71 °C ? rt; (b) 2,4-dichloroaniline, MsOH, microwave, 180 °C; (c) (4-THP)methylamine (5 equiv),
Me3Al (5 equiv), CH2Cl2, 40 °C; (d) n-BuLi (2 equiv) À65 °C, MeI.
Modification of the R2 substituent had a significant effect on the
CB2 potency of these analogues (Table 3). A cyclopropyl group in
14a maintained the same level of CB2 potency as an isopropyl
O
Cl
N
14a
H
group in 12f, and the tert-butyl group in 14d increased CB2 po-
tency. However, the cyclopentyl group in 14b reduced CB2 efficacy
and the (2-hydroxyprop-2-yl) group in 14c reduced CB2 potency.
Following preliminary in vitro pharmacokinetic analysis, we no-
ticed that the rat in vitro clearance22 for the cyclopropylpyridine
14a was significantly lower than that of the isopropylpyridine
12f. A final round of optimization of this 4-(cyclopropyl)pyridine
sub-series was therefore undertaken prior to evaluation in vivo
to ensure the best chance of a compound with high oral bioavail-
ability and also activity in vivo.
Further 4-(cyclopropyl)pyridine analogues 14e–o (Table 3)
were prepared25b as shown in Scheme 2. Amongst the substituents
investigated at the 3-position of the aniline, the halogen containing
substituents appeared to give the more potent analogues 14e–h
but 2,4- and 3,5-dichloro-substitution, gave the most potent CB2
agonists 14a,o.
O
N
N
H
100
90
80
70
60
50
40
30
20
10
0
Cl
The solubility data for this series revealed that two of the oxy-
gen-containing analogues, 14c and 14j, as expected, had the great-
est aqueous solubility at pH 7.4. Some of the less lipophilic
analogues, for example, 14l, also had improved solubility. However
these three analogues also had weaker CB2 potency and were thus
not progressed any further.
0.01
0.1
1
Dose (mg/kg p.o.)
The in vitro clearance and inhibition of cytochrome P450 for 4-
(cyclopropyl)pyridines with CB2 pEC50 P 6.4 and E > 85% was
further assessed to prioritise them for in vivo pharmacokinetic
analysis. Gratifyingly, a number of these, 14a,d,f,h, appeared to
have low in vitro clearance in both rat and human microsomes,
combined with no significant P450 interactions.
Figure 2. CFA model of inflammatory pain.
and shows full reversal of hyperalgesia at a dose of 0.3 mg/kg po
as determined by the weight bearing protocol (Fig. 2).
In conclusion, a novel series of potent, selective pyridine-3-car-
boxamide CB2 agonists has been discovered. One of these, 14a, has
good oral bioavailability, low clearance, and potent oral activity in
the CFA model of inflammatory pain. Whilst some pyridine ana-
logues have improved aqueous solubility, the lower solubility of
14a is not a barrier to good oral bioavailability and oral activity
in the CFA model of inflammatory pain.
These four analogues were next evaluated in a pharmacokinetic
study in conscious rats, to assess their exposure in the blood up to
6 h after an oral dose of 3 mg/kg. Two of these, 14a,d, had good oral
exposure [as expressed by dose normalised area under the curve,
(AUC(0 À t)/dose)] despite having low aqueous solubility.
Since 14a showed no activity at CB1 at concentrations up to
30 l
M (cf 14d26), further pharmacokinetic studies were under-
References and notes
taken. In conscious rats, following an oral dose of 3 mg/kg of 14a,
the bioavailability was 39%, whilst an iv dose of 1 mg/kg of 14a
produced a clearance of 14 mL/min/kg, a volume of distribution
of 2.4 L/kg, and a half life of 3.1 h.
14a has high in vitro CB2 potency and efficacy, with excellent
selectivity over CB1, and good in vitro and in vivo pharmacokinetic
properties. Despite its low aqueous solubility, it was selected for
further evaluation in an in vivo model to give encouragement for
further research around this template in the future. In the estab-
lished Complete Freund’s Adjuvant (CFA) model of inflammatory
pain27, 14a has high potency with an oral ED50 of 0.07 mg/kg po,
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ˇ
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18. Procedures for the syntheses of compounds and details of the yeast CB2
reporter assay used to assess their potency at the CB2 receptor, are given in:
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19. The solubility was determined in a high-throughput solubility assay, using the
principle of precipitative solubility. A 10 mM DMSO stock solution of the
analyte was diluted down in phosphate-buffered saline at pH 7.4. The resulting
solution was equilibrated at room temperature for 1 h and filtered to remove
any undissolved material. The solubility of the analyte in the filtrate was
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