Synthesis and evaluation of 3-amino-6-aryl-pyridazines as selective CB2 agonists for the treatment of inflammatory pain

Article abstract of DOI:10.1016/j.bmcl.2009.11.117

A series of 3-amino-6-aryl-pyridazines have been identified as CB₂ agonists with high efficacy and selectivity against the CB₁ receptor. Details of the investigation of structure-activity relationships (SAR) are disclosed, which led to the identification of pyridazine analogue 35, a compound with high potency in an in vivo model of inflammatory pain.

Full text of DOI:10.1016/j.bmcl.2009.11.117

Bioorganic & Medicinal Chemistry Letters 20 (2010) 465–468
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 3-amino-6-aryl-pyridazines as selective CB2
agonists for the treatment of inflammatory pain
*
Robert J. Gleave , Paul J. Beswick, Andrew J. Brown, Gerard M. P. Giblin, Paul Goldsmith, Carl P. Haslam,
William L. Mitchell, Neville H. Nicholson, Lee W. Page, Sadhana Patel, Susan Roomans, Brian P. Slingsby,
Martin E. Swarbrick
Pain and Neuroexcitability Discovery Performance Unit, Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Cold Harbour Lane,
Harlow, Essex CM19 5AW, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-amino-6-aryl-pyridazines have been identified as CB2 agonists with high efficacy and selec-
Received 23 October 2009
Revised 23 November 2009
Accepted 24 November 2009
Available online 27 November 2009
1
tivity against the CB receptor. Details of the investigation of structure–activity relationships (SAR) are
disclosed, which led to the identification of pyridazine analogue 35, a compound with high potency in
an in vivo model of inflammatory pain.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid
Pyridazine
Pain
Naturally occurring cannabinoids, such as D9-tetrahydrocan-
A series of amino pyridines, exemplified by compound 1 (Fig. 1),
nabinol, have been found to act as agonists at two G-protein cou-
had been discovered through optimization of a hit from high
throughput screening. The series demonstrated high potency,
1
2
14
pled receptors CB
found widely in the central nervous system (CNS) and to a lesser
1
and CB
2
and more recently at GPR55. CB
1
is
3
efficacy and selectivity and was appealing due to low molecular
weight and polar surface area. Unfortunately, high in vitro metab-
olism was a feature of the series and limited the advancement of
these compounds beyond in vitro screening.
It was decided to investigate the effect of replacing the aromatic
core on metabolic stability and a series of ring isosteres were intro-
duced (Table 1). Generally the ring substitution was well tolerated
with only small variations in agonist potency. Interestingly, the
phenyl analogue (3) had equivalent potency to the lead pyridine
(1) suggesting the absence of any role for a ring nitrogen in binding
to the receptor. Analogues 1–7 showed no significant activity at the
extent in the periphery. CB
pressed in peripheral immune tissues and activated microglia.
Recent studies have also indicated the presence of CB on neurons
2
is more localized and is highly ex-
4
–6
2
7
within the CNS. Natural product derived and synthetic cannabi-
noids have shown efficacy in animal models of inflammatory and
neuropathic pain. However despite showing potential as analge-
sics, their use as therapeutic agents has been limited due to psy-
chotropic effects such as euphoria/dysphoria, dry mouth and
8
,9
9
drowsiness. There has therefore been great interest in the possi-
bility of developing agonists devoid of these side effects.
One strategy to achieve an acceptable therapeutic index has
1
human CB receptor at concentrations up to 30 lM.
been to develop selective ligands for the CB
avoiding the behavioural effects linked to activating CB
the CNS. Several classes of selective CB ligands have demonstrated
efficacy in pre-clinical models of inflammatory pain
shown a therapeutic window with regard to CNS side-effects.
We were therefore interested in developing new structural classes
of CNS penetrant CB agonists with high receptor sub-type selec-
tivity suitable for in vivo studies.
2
receptor, therefore
Encouragingly, several of the heterocycles showed improved
metabolic stability. With the exception of triazine (7), the rat
intrinsic clearance generally decreased with lipophilicity.
1
within
2
10,11
and have
1
2,13
Cl
Cl
2
N
N
O
(
1)
*
Corresponding author.
E-mail address: robert.j.gleave@gsk.com (R.J. Gleave).
Figure 1. Pyridine lead from hit to lead chemistry.
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.117

4
66
R. J. Gleave et al. / Bioorg. Med. Chem. Lett. 20 (2010) 465–468
Table 1
X
a
X
N
SAR of aryl core (compounds 1–7)
R1
N
N
N
b
X
N
R2
Cl
X = Halogen
b
Cl
N
O
Aryl
CB
2
pEC50
Rat CLi
(mL/min/g)
Aqueous
Solubility
c log D
@pH7.4
a
b
c
(Efficacy)
(lg/mL)
Ar
a
Ar
R1
N
1
2
3
4
5
6
7
7.1 (88%)
6.7 (67%)
7.4 (86%)
7.1 (76%)
7.4 (89%)
7.3 (92%)
7.0 (78%)
9.5
—
—
—
—
6
3.2
3.4
4.0
2.1
3.0
2.3
0.6
N
N
N
X
N
N
N
R2
1
2
N
3
Scheme 1. Reagents and conditions: (a) R NHR , MeCN, NEt , microwave, 160 °C,
3
0 min–5 h, 2–89%; (b) (i) ArB(OH)
2 3 4 2 3
, Pd(PPh ) , 2 N Na CO , DME, 90 °C or (ii)
ArB(OH) , KF, Pd dba , [tBu PH]BF , 1,4-dioxane, 100 °C, 10–68%.
2
2
3
3
4
20
2.8
—
decrease in stability which was not affected by the introduction
of fluorine substituents (9). Pyrrolidine was also a successful
replacement for potency and this could be increased further
through the introduction of cis-2,5-methyl substituents (12, 13).
2
agonism and a small
amount of CB partial agonism although a significant selectivity
window remained. Secondary amines, as exemplified by com-
pounds 14 and 15, maintained potency only with the most lipo-
philic group.
N
N
N
These analogues showed very high CB
1
1
N
N
2.8
4.7
<1
—
N
From this initial investigation the observed SAR seemed to cor-
relate well with the pyridine template previously investigated.14
N
N
N
Table 2
a
SAR of amine substituent (compounds 4, 8–15)
Human CB
variability is monitored by the use of a cannabinoid agonist HU210. Efficacy at CB
CB is expressed as a percentage relative to the efficacy of HU210. See Ref. 15 for
assay method.
1 2
/CB assay data is the mean of at least two determinations. Assay
1
/
2
Cl
b
CLi = microsomal clearance, see Ref. 16 for procedure.
Calculated log D acquired using ACD v8.0 (ACDlabs).
N
Cl
c
X
N
X
CB
2
pEC50
CB
(Efficacy)
1
pEC50
Rat CLi
a
a
(mL/min/g)b
(
Efficacy)
Pyridazine (4) and pyrimidine (6) demonstrated particularly
encouraging profiles. Of concern was the low solubility, however
pyridazine (4) showed measurable aqueous solubility and it was
found this could be improved through salt formation (HCl salt,
N
4
8
7.1 (76%)
8.2 (86%)
<4.5
2.8
35
O
N
<4.5
<4.5
aqueous solubility = 120 lg/mL). This promising data led us to ini-
tiate further SAR studies around the pyridazine template. The aim
of this investigation was to increase the potency of the lead pyrid-
azine (4), whilst maintaining or reducing the level of in vitro met-
abolic turnover. Improvements in solubility would also be
beneficial in achieving oral bioavailability. It was hoped that this
investigation would lead to analogues with suitable properties to
profile in vivo.
N
N
9
F
7.8 (83%)
29
F
O
1
1
0
1
S
O
6.2 (89%)
7.6 (56%)
<4.5
<4.5
—
—
N
Pyridazine analogues were synthesized from the commercially
available 3,6-dichloro and 3,6-dibromo pyridazines according to
Scheme 1. Synthesis proceeded via a palladium catalysed cross
coupling combined with an aromatic halide displacement. In cases
where the yield from the Suzuki–Miyaura coupling was low, 3-
chloro-6-iodopyridazine could be prepared via an aromatic Finkel-
stein reaction from 3,6-dichloropyridazine. Halide displacement
was carried out at high temperature in a microwave reactor. Reac-
tion times were significantly longer for sterically hindered amines.
The order of the two reactions was dependant upon the desired
point of variation, however carrying out the amine displacement
first avoided the separation of mono and bis-arylated pyridazines.
Initial work concentrated on the amine substituent and a range
of tertiary and secondary amines were introduced (Table 2).
Increasing the lipophilicity, for example, piperidine (8), led to an
1
1
1
2
3
4
N
N
8.9 (83%)
9.2 (93%)
6.7 (69%)
5.6 (57%)
5.2 (21%)
<4.5
9.7
8.7
—
1
7
N
H
O
1
5
<4.5
<4.5
—
N
H
a/b
See footnotes to Table 1 for details.
2
increase in CB activity. This however was accompanied by a

R. J. Gleave et al. / Bioorg. Med. Chem. Lett. 20 (2010) 465–468
467
Table 3
Table 4
SAR of aryl substituent (compounds 16–30)
SAR of aryl substituent (compounds 31–36)
R
N
N
N
N
N
N
O
Aryl
CB
2
pEC50
CB
(Efficacy)
1
pEC50
Rat CLi
R
CB
2
pEC50 (Efficacy)a
CB
1
pEC50 (Efficacy)a
a
a
(mL/min/g)b
(
Efficacy)
4
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
2,3-Di-Cl
2-Cl
2-Me
7.1 (76%)
5.8 (77%)
5.1 (38%)
5.9 (78%)
4.9 (37%)
5.9 (55%)
6.0 (15%)
5.7 (71%)
6.8 (75%)
6.4 (39%)
7.3 (78%)
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
<4.5
H
N
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
31
6.6 (61%)
6.8 (70%)
6.7 (75%)
<4.5
—
—
—
2-CF
2-OMe
2-OCF
3-CF
2,3-Di-Me
3
3
O
3
3
2
3
<4.5
<4.5
3
2,3-Di-CF
2,5-Di-Cl
3
2,3,5-Tri-Cl
2,4-Di-Cl
2-Cl-4-Me
2-Cl-4-OMe
2-OBn
N
N
<4.5
<4.5
6.2 (45%)
<4.5
3
3
3
4
5
6
7.1 (82%)
8.0 (85%)
7.4 (93%)
<5.0
6.0
3.7
5.1
5.4 (11%)
<4.5
3-OBn
a
See footnotes to Table 1 for details.
5.1 (71%)
4.7 (52%)
N
Although high levels of potency had been obtained, this set of pyr-
idazine analogues indicated that metabolic stability remained a
key challenge and it looked difficult to replace the morpholine
group and retain a low turnover in vitro. It was decided to look
at whether the potency could be improved through modification
of the aryl substituent and a number of analogues were prepared
with the morpholine kept constant (Table 3).
N
a/b
See footnotes to Table 1 for details.
(
as a di-hydrochloride salt) in order to see how the in vitro data
translated in vivo (Table 5).
Generally changes to the aryl substitution pattern were not well
tolerated. A comparison of the mono substituted analogues, for
The in vivo DMPK profile was characterized by a moderate-high
blood clearance, a volume of distribution which indicated a good
potential to distribute into the tissues and oral bioavailability of
ca. 26%. This data enabled us to select 35 as a tool for further inves-
tigation of the in vivo profile of this class of compounds. The com-
pound was initially evaluated in the rat Freund’s Complete
Adjuvant (FCA) model of inflammatory pain using a weight bearing
protocol, and produced a significant reversal of the FCA induced
hypersensitivity at all doses tested (0.1–1 mg/kg po) (Fig. 2). Tissue
samples from this study at 1 mg/kg (1 h post dose) showed a mean
3 3
example, 18 (2-CF ) and 21 (3-CF ), indicated that 2-substitution
was important for maintaining efficacy. As observed in the pyri-
dine template, 2,4-disubstituted analogues (26–28) lost all po-
tency, possibly indicating restricted space within the binding site.
Overall, only 2,3-di-trifluoromethyl (23) and 2,4,5-trichloro (25)
maintain equivalent agonism to the original 2,3-dichloro. As 2,3-
substitution seemed optimal we decided to look at bi-aryl substit-
uents. For heterocyclic bi-aryls, it was decided to synthesise ana-
logues using the potent cis-2,5-dimethlpyrrolidine group,
therefore balancing the reduced aryl lipophilicity and hopefully
obtaining high potency (Table 4).
1
8
blood concentration of 0.3
lM and a brain concentration of 0.5 lM
indicating a brain:blood ratio of approximately 1.7:1.
2
Encouragingly, CB agonism was observed across a range of het-
erocycles in particular quinolines and iso-quinolines (33–36).
The 4-substituted isoquinoline (35) showed a promising combi-
nation of agonist activity, in vitro metabolic stability and high
aqueous solubility (di-hydrochloride salt, >1 mg/mL). The com-
pound was therefore progressed into a rat in vivo DMPK study
Table 5
Rat DMPK parameters for analogue 35
Dose route
Parameter
Mean (n = 3)
Intravenousa
Clb (mL/min/kg)
57 ± 5
V
SS (L/kg)
(h)
1.7 ± 0.3
0.3 ± 0.1
T
½
Oralb
C
T
(lM)
0.340 ± 0.181
0.9 (0.5–1.0)
26 ± 13
max
max (h)
F (%)
a
Compound dissolved in 0.9% (w/v) aqueous saline. Rats received an infusion of
3
5 at a target dose of 1 mg free base/kg.
Compound formulated in 1% (w/v) aqueous methylcellulose. Single oral dose
b
Figure 2. Dose–response effect of compound 35 and Celebrex (10 mg/kg po) in the
FCA model of inflammatory pain.
via gastric gavage to achieve a target dose level of 3 mg/kg of free base.

4
68
R. J. Gleave et al. / Bioorg. Med. Chem. Lett. 20 (2010) 465–468
6. Stella, N. Glia 2004, 48, 267.
Compound 35 had shown the ability to penetrate the CNS and
had measurable CB agonism, therefore an in vivo study was
undertaken to look into the effect of 35 on rat core temperature
after dosing, an indicator for CB
mediated side effects.19 Com-
pound 35 showed no significant hypothermia at doses up to and
including 300 mg/kg po (Cmax blood 6.6 M and brain ca 11 M)
7.
Van Sickle, M. D.; Duncan, M.; Kingsley, P. J.; Mouihate, A.; Urbani, P.; Mackie,
K.; Stella, N.; Makriyannis, A.; Piomelli, D.; Davison, J. S.; Marnett, L. J.; Di
Marzo, V.; Pittman, Q. J.; Patel, K. D.; Sharkey, K. A. Science 2005, 310, 329.
1
8. Fox, A.; Bevan, S. Expert Opin. Investig. Drugs 2005, 14, 695.
Burns, T. L.; Ineck, J. R. Ann. Pharmacother. 2006, 40, 251.
1
9.
1
0. Jhaveri, M. D.; Sagar, D. R.; Elmes, S. J. R.; Kendall, D. A.; Chapman, V. Mol.
l
l
Neurobiol. 2007, 36, 26.
and therefore demonstrated the potential to achieve a high thera-
peutic index with this class of compound.
11. Cheng, Y.; Hitchcock, S. A. Expert Opin. Investig. Drugs 2007, 16, 951.
1
2. 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.; Wilson, A. W.; Chessell, I. P.; Wittington, A. R.; Green, R. J. Med.
Chem. 2007, 50, 2597.
In summary, replacement of the pyridine core of the initial lead
(
1) with a pyridazine led to the discovery of a number of analogues
with increased in vitro metabolic stability. Introduction of the cis-
,5-dimethylpyrrolidine moiety was found to greatly increase the
CB activity. Combining this group with a number of bicyclic het-
13. Malan, T. P.; Ibrahim, M. M.; Deng, H.; Liu, Q.; Mata, H. P.; Vanderah, T.;
Porreca, F.; Makriyannis, A. Pain 2001, 93, 239.
2
1
4. Gleave, R. J.; Beswick, P. J.; Brown, A. J.; Giblin, G. M. P.; Haslam, C. P.;
Livermore, D.; Moses, A.; Nicholson, N. H.; Page, L. W.; Slingsby, B.; Swarbrick,
M. E. Bioorg. Med. Chem. Lett. 2009, 19, 6578.
2
erocycles led to the identification of compound 35, which showed
high potency in a model of inflammatory pain.
1
5. Details of the yeast CB-2 reporter assay used to assess potency are given in:
Eartherton, A. J.; Giblin, G. M. P.; Mitchell, W. L.; Naylor, A.; Rawlings, D. A.
World Patent 74,939, 2005.
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