Synthesis, SAR, and Pharmacological Characterization of Brain Penetrant P2X7 Receptor Antagonists

Article abstract of DOI:10.1021/acsmedchemlett.5b00089

We describe the synthesis and SAR of 1,2,3-triazolopiperidines as a novel series of potent, brain penetrant P2X7 antagonists. Initial efforts yielded a series of potent human P2X7R antagonists with moderate to weak rodent potency, some CYP inhibition, poo

Full text of DOI:10.1021/acsmedchemlett.5b00089

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Letter
Synthesis, SAR and Pharmacological Characterization
of Brain Penetrant P2X7 Receptor Antagonists
Brad M. Savall, Duncan Wu, Meri De Angelis, Nicholas Iain Carruthers, Hong
Ao, Qi Wang, Brian Lord, Michael A Letavic, and Anindya Bhattacharya
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00089 • Publication Date (Web): 24 Apr 2015
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Synthesis, SAR and Pharmacological Characterization of Brain
Penetrant P2X7 Receptor Antagonists
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Brad M. Savall*, Duncan Wu, Meri De Angelis, Nicholas I. Carruthers, Hong Ao,
Qi Wang, Brian Lord, Anindya Bhattacharya, and Michael A. Letavic
Janssen Research and Development, LLC, 3210 Merryfield Row, San Diego, CA, 92121-1126, United States
KEYWORDS P2X7, neuro-inflammation, depression.
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ABSTRACT: We describe the synthesis and SAR of 1,2,3ꢀtriazolopiperidines as a novel series of potent, brain penetrant P2X7 antagoꢀ
nists. Initial efforts yielded a series of potent human P2X7R antagonists with moderate to weak rodent potency, some CYP inhibition,
poor metabolic stability and low solubility. Further work in this series, which focused on the SAR of the Nꢀlinked heterocycle, not only
increased the potency at the human P2X7R but also provided compounds with good potency at the rat P2X7R. These efforts eventually
delivered a potent rat and human P2X7R antagonist with good physicochemical properties, an excellent pharmacokinetic profile, good
partitioning into the CNS and demonstrated in vivo target engagement after oral dosing.
Introduction. Over the past few years, numerous reports
have appeared in the literature that implicate cytokines in
depression and in particular that cytokines play a role in
treatment resistant depression.1, 2 As such, several laboraꢀ
tories have reported that reduction of ILꢀ1β levels in mice
correlates to symptom improvement in a random stress
model of depression.3 Since the activation of the P2X7
receptor (P2X7R) results in the production of ILꢀ1β,4 anꢀ
tagonists of P2X7R, which are known to block ILꢀ1β reꢀ
lease, are hypothesized to be useful drugs for the treatment
of depression.5
modest affinity for the rat P2X7R. We recently disclosed
two selective brain penetrant P2X7R antagonists (5, 6)
with appreciable affinity for the rat P2X7R and subseꢀ
quently demonstrated robust P2X7R target engagement in
the CNS of the rat as measured by exꢀvivo autoradiogꢀ
raphy.15, 16
Figure 2: Brain Penetrant P2X7R Antagonists
There have been numerous reports of P2X7R antagonists
in the literature,6ꢀ10 most notably the benzamide class of
compounds from AstraꢀZeneca (1)11, 12 and a structurally
distinct benzamide from Pfizer, CEꢀ224,535 (2).13 The
Pfizer benzamide was tested in human clinical trials where
it was shown to lower peripheral levels of ILꢀ1β in a
rheumatoid arthritis trial.14 However, since CEꢀ224,535 is
not known to penetrate into the CNS, the effect on reduced
CNS levels of ILꢀ1β are not currently unknown. Herein,
we describe a series of potent, brain penetrant P2X7R anꢀ
tagonists that show robust P2X7R target engagement in
rodents.
In addition to the various P2X7R chemotypes in Figure 2,
we were aware of the 1,2,4ꢀtriazolopiperazines disclosed in
2010 (Figure 3).17 In a quest to explore other heterocyclic
cores that could serve as competent P2X7R antagonists we
decided to embark on a campaign to discover novel heterꢀ
ocycles which were potent P2X7 antagonists and this work
eventually led us to the 1,2,3ꢀtriazolopiperidines series
disclosed in this report.
Figure 1: Previously Reported P2X7 Antagonists
Figure 3: 1,2,4ꢀTriazolopiperazine P2X7R Antagonists
Results and Discussion. Glaxo SmithKline recently disꢀ
closed a series of brain penetrant P2X7R antagonists (3,
4)6, 7 that had good affinity for the human P2X7R but only
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Scheme 2: Synthetic Route to 1,2,3ꢀTriazolopiperidines
Chemistry. Our initial synthesis began with commercially
available 1Hꢀ[1,2,3]triazolo[4,5ꢀc]pyridine (9) (Scheme 1).
Although arylation of the 1Hꢀ[1,2,3]ꢀtriazoloꢀ[4,5ꢀc]ꢀ
pyridine had the potential to give several regioisomers, we
anticipated that the electronic effect of the pyridyl nitrogen
would favor arylation to the 1ꢀposition. In practice, the use
of Buchwald’s copper mediated arylation18, 19 furnished the
desired arylated products in a 5:1 ratio favoring the N-1
aryl regioisomer albeit in low yield and as an inseparable
mixture.20 Subsequent hydrogenation of the Nꢀ1, Nꢀ2 mixꢀ
ture (Hꢀcube, Pt2O, 90 bar, MeOH) did furnish a small
amount the 1,2,3ꢀtriazolopiperidine however this method
suffered from incomplete / irreproducible conversion, even
after conducting the reaction in continuous flow mode.
Regardless, the regioisomers were separated at the 1,2,3ꢀ
triazolopiperidine stage and then coupled with the correꢀ
sponding benzoic acids to furnish the desired product(s)
(Scheme 1). As the first analog made (12a) exhibited an
hP2X7R IC50 = 2.7 nM, our interest in this series grew.
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Scheme 1: First Generation Synthesis of 1,2,3ꢀ
Triazolopiperidines: Triazole Arylation Route
Alternatively, we were aware that an acyl pyridinium21, 22
species (18) could enable the synthesis of the final prodꢀ
ucts directly. As such, the acyl pyridinium species (18)
was formed by the addition of an acid chloride to the triaꢀ
zolopyridine 16 in THF, however, this species (18) was
unstable and readily decomposed in the presence of protic
solvents such as methanol. Treatment of the intermediate
acyl pyridinium (18) with sodium or lithium borohydride
did not result in reduction to the desired product. However,
use of the Hantzsch ester23 cleanly provided the partially
reduced derivatives (19); which were subsequently reduced
with hydrogen over Pd/C in ethanol to provide final prodꢀ
ucts (12). In some cases the pendant heterocycles were
cleaved and a transfer hydrogenation with ammonium
formate and Pd/C was more productive.
The low yield and irreproducibility of the hydrogenation
reaction coupled with difficulties encountered with the
purification of the regioisomers prompted us to look at
alternative methods to more efficiently prepare these comꢀ
pounds. The regiochemical issue was addressed by using
4ꢀchloroꢀ3ꢀnitropyridine as the starting material as shown
in Scheme 2. An amino heterocycle displacement of chloꢀ
roꢀnitroꢀpyridine (13), was followed by reduction which
furnished the diamino pyridine (15), which was subseꢀ
quently converted to the 1,2,3ꢀtriazolopyridine core (16)
after treatment with tꢀbutyl nitrite. Reduction as in Scheme
1 provided 17, which was coupled to the desired carboxꢀ
ylic acid or acid chloride to provide compounds 12.
The first entry into this series of antagonists was comꢀ
pound 12a (Table 1), which had a potent hP2X7 FLIPR
IC50 of 2.7 nM, but was significantly less potent at the rat
P2X7R with a IC50 = 1900 nM. In addition, compound
12a was rapidly metabolized inꢀvitro and was a CYP 2C19
inhibitor with an IC50 of 0.1 ꢀM. Compound 12a was not
cross reactive in a commercial panel of 50 receptor, ion
channel and transporter assays (EurofinsꢀCEREP,
http://www.eurofins.com/) at a screening concentration of
1.0 ꢀM. Since compound 12 had a clogP of ~4.0, we reaꢀ
soned that reducing lipophilicity (lower cLogP) could adꢀ
dress both the metabolism issues and the CYP profile.
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Table 1: SAR for N1ꢀAryl 1,2,3ꢀTriazolopiperidines
(IC50 = 3.8 nM) and provided a slight improvement in the
CYP 2C19 inhibition (IC50 = 0.3 ꢀM) though the rat affiniꢀ
ty and metabolic stability were not improved. The addition
of a second nitrogen, as in the pyrimidine (12c), also mainꢀ
tained affinity for the human receptor (IC50 = 2.3 nM),
showed a further improvement in the CYP 2C19 inhibition
(IC50 = 3.2 ꢀM), and a modest improvement in the human
and rat microsomal stability. The pyrimidine compound
(12d) was especially gratifying as it was potent at both the
human (IC50 = 4.2 nM) and rat (IC50 = 6.8 nM) P2X7R in
#
hP2X7
rP2X7
HLM/
CYP 2C19
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Ar
cLogP
IC50
IC50
RLM
ER c
(IC50) ꢀ
M d
(nM)a
(nM)b
addition to further improving the CYP inhibition (IC50
=
12a
8.5 ꢀM) and the microsomal stability. This was one of the
first compounds in this series that showed substantial affinꢀ
ity for the rat P2X7R. The 4ꢀfluoropyridine (12e) also had
0.94 /
0.92
2.7
3.8
2.3
4.2
2.1
1900
1400
1900
6.8
0.10
0.30
3.2
4.0
significantly improved potency at the rat receptor (IC50
=
30 nM), relative to the pyridyl parent (12b, IC50 = 1400
nM), and reduced CYP inhibition (IC50 = 1.3 ꢀM), but was
still rapidly metabolized. The 5ꢀfluoro pyrimidine (12f)
had good human potency (IC50 = 24 nM), improved CYP
inhibition (IC50 = >10 ꢀM), and microsomal stability when
compared to the parent pyrimidine (12d) though the rat
affinity (IC50 = 3000 nM) was reduced. Finally, the pyraꢀ
zole (12g) had good human affinity (IC50 = 39 nM), reꢀ
duced rat affinity (IC50 = 209 nM), moderate microsomal
stability and no CYP inhibition (IC50 = >10 ꢀM).
12b
12c
12d
12e
0.90 /
>0.92
3.2
2.3
2.3
3.3
0.80 /
0.86
After optimizing the N-1ꢀaryl portion of the molecule, we
prepared a limited number of benzamides while keeping
the aryl group as a pyrimidine (Table 2). The 2ꢀchloroꢀ3ꢀ
trifluoromethyl (12d) had an IC50 = 4.2 nM. Replacing the
chloro (12c) with a fluoro (12h) provided a compound
with reduced potency at the human (IC50 = 22 nM) and rat
(IC50 = 258 nM) receptor, though human microsomal staꢀ
bility and CYP 2C19 liability were slightly improved. The
2ꢀmethyl (12h) analog was slightly more potent at the huꢀ
man receptor (IC50 = 2.2 nM), though significantly less
potent at the rat receptor (349 nM). Human and rat microꢀ
somal stability and CYP 2C19 inhibition were also imꢀ
proved. The 2,3ꢀdichloro derivative (12j) was potent at the
human receptor (IC50 = 2.0 nM), less potent at rat receptor
(IC50 = 53 nM), had improved microsomal stability, but
slightly worse in the CYP 2C19 assay. Finally, both of the
2,3ꢀbis halo derivatives (12k, l) were less active.
0.61 /
0.82
8.5
0.85 /
0.89
30
1.3
12f
0.54 /
0.65
24
39
3000
209
>10
>10
2.5
2.9
12g
0.70 /
0.82
ahuman FLIPR pIC50 measured in a Ca2+ flux assay, brat FLIPR pIC50
measured in a Ca2+ flux assay, all data are the result of at least three
assays run in triplicate with the mean value reported; The standard
deviation is all cases was less than two fold. cHLM and RLM refer to
human and rat liver microsomal preparations with data reported as the
extraction ratio (ER), CYP2C19 data was obtained from human liver
microsomes. Details of all assay conditions are provided in the sup-
plemental information.
d
Indeed, alternative heterocycles proved to be a fruitful exꢀ
pedition (Table 1). Changing from a phenyl (12a) to a
pyridyl (12b) maintained affinity at the human receptor
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Table 2: SAR for Benzamide of 1,2,3ꢀTriazolopiperidines
Figure 4: Rat PK Data for compound 12c, 12d, and 12f.
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5
Compound 12c
Rat PK
Vehicle: 20% HPꢀbꢀCD
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9
10000
1000
100
10
IV 1.0 mg/kg
PO 5.0 mg/kg
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#
CYP
2C19
hP2X7
rP2X7
HLM/
Ar
cLogP
IC50
IC50
RLM
ER c
(IC50) ꢀM
(nM)a
(nM)b
d
1
12d
12h
12i
0.1
0
2
4
6
8
0.61 /
0.82
Time (hr)
4.2
22
6.8
258
349
53
8.5
>10
>10
6.3
2.3
1.9
2.3
2.2
Compound 12d
Rat PK
Vehicle: 20% HPꢀbꢀCD
0.50 /
0.85
10000
1000
100
10
IV 1 mg/kg
PO 5 mg/kg
0.47 /
0.72
2.2
2.0
1
12j
12k
0.1
0.49 /
0.82
0
1
2
3
Time (hr)
Compound 12f
Rat PK
Vehicle: 20% HPbCD
387
255
4300
1300
N/D
N/D
N/D
N/D
2.3
1.7
10000
1000
100
10
IV 1 mg/kg
PO 5 mg/kg
12l
a-d See Table 1
1
Since we had now generated potent P2X7 compounds in
this series, we were interested to see how these compounds
behaved inꢀvivo, and in particular if the compounds would
distribute efficiently into the CNS. Although we were
especially interested in compound (12d), as it was active at
both the human and the rat P2X7R, we obtained additional
inꢀvitro ADME characterization of three of the more promꢀ
ising compounds (12c, 12d, and 12f). The profiles of
those three compounds are shown in Table 3 and 4. In
general all three compounds have reasonable physical
properties, including solubility, permeability and protein
binding to warrant inꢀvivo testing.
0
4
8
12
16
20
24
Time (hr)
The rat pharmacokinetic data for all three compounds is
shown in Figure 4. The pyrazine (12c) had bioavailability
of 33% and reached a Cmax of 1.0 ꢀM. The halfꢀlife of
0.7 h is consistent with the relatively high microsomal exꢀ
traction ratio (0.82) as clearance of 12c was near the rat
hepatic blood flow. Disappointingly, compound 12d
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which had the best rat P2X7R affinity (4.2 nM) had the
lowest oral exposure of all three compounds with a bioaviꢀ
Acknowledgements.
The authors would like to thank Professor David MacMilꢀ
lan for useful discussions toward optimizing the synthetic
strategy, and especially the use of the Hantzsch ester reꢀ
duction of the acyl pyridinium species.
lability of 1%. Compound 12f proved to have the best PK
with measured bioavailability greater than 100%, and a
halfꢀlife of 2.3 hrs, which is slightly longer than predicted
by the rat extraction ratio (0.65). Although we do not
know the reason for the low bioavailability of compound
12d, it is interesting to note that 12d has the lowest solubilꢀ
ity of the three compounds.
Supporting Information Available: Supporting information
for “Synthesis, SAR and Pharmacological Characterization
of Brain Penetrant P2X7 Receptor Antagonists” including
synthesis, characterization and assay conditions is availaꢀ
ble free of charge via the Internet at http://pubs.acs.org.
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Figure 5: Rat P2X7 Receptor Occupancy Data for 12f.
PO BBB Penetration of the P2X7 Antagonist 12f
at 10 mg/kg in the Rat, n=2 per time point
10000
1000
100
100
90
80
70
60
50
40
30
20
10
0
10
0
2
4
6
8
Time (hr)
Plasma
Brain
Occupancy
Compound 12f was also tested in a rat exꢀvivo autoradiogꢀ
raphy (ARG) experiment to assess the brain P2X7 receptor
occupancy (Figure 5). When 12f was dosed at 10 mg/Kg
PO in the rat it achieved a maximum occupancy of 45% at
30 minutes. The occupancy decreased over time in conꢀ
junction with decreasing concentrations of 12f in the brain
and plasma, which were measured in the same experiment.
Therefore, compound 12f demonstrated proof of concept
that the 1,2,3ꢀtriazolopiperidines are a new class of brain
penetrant P2X7R antagonists that are capable of occupying
the receptor when dosed orally.
Conclusions.
We describe the synthesis and SAR of 1,2,3ꢀ
triazolopiperidines as a new series of potent and brain penꢀ
etrant P2X7 antagonists. The prototype N-1ꢀphenyl triaꢀ
zole (12a) was potent at the human P2X7R, but had poor
affinity for the rat P2X7R, poor microsomal stability, was
a strong inhibitor of CYP 2C19, and had a high cLogP. By
replacing the phenyl group with various heterocycles we
discovered compound 12f, a potent human and rat P2X7R
ligand with sufficiently good physicochemical properties
to enable oral dosing in rats and provide high levels of the
compound in the plasma and the brain, and demonstrated
target engagement as measured by the rat ARG. Additionꢀ
al studies of this novel class of P2X7R antagonists will be
the subject of future publications.
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Table 3: In Vitro Pharmacology for Compounds 12c, 12d, and 12f.
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6
7
8
Human
rat
mouse
FLIPR IC50
human Ki
rat Ki
hERGd
IC50 (ꢀM)
>10
human
e
b
c
a
FLIPR IC50
WB IC50
FLIPR IC50
12c
12d
12f
2.3 nM
4.2 nM
24 nM
15 nM
4.0 nM
588 nM
23
16
32
5.0
2.8
20
316 nM
79 nM
1.9 ꢀM
6.8 nM
3.0 ꢀM
9
>10
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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251 nM
ahuman FLIPR pIC50 measured in a Ca2+ flux assay, brat FLIPR pIC50 measured in a Ca2+ flux assay, cmouse FLIPR pIC50 measured in a Ca2+ flux as-
say, dhERH IC50 determined in a tritiated dofetilide binding assay, eHuman blood was primed with lipopolysaccharide (LPS; 30 ng/ml) followed by addi-
tion of the test antagonist or vehicle with the final P2X7 stimulus of Bz-ATP. Test compounds were added and incubated for an additional 30 minutes.
The P2X7 agonist, Bz-ATP (1 mM) was finally added and incubated for 1.5 hours at 37°C. Post incubation, the plates were centrifuged (low-speed spin)
and the supernatant collected for IL-1F ELISA analysis as per manufacturer’s protocol (human IL-1F: Thermo Scientific; catalogue # EH2IL1B5;). All
data are the result of at least three assays run in triplicate with the mean value reported.
Table 4: Physical Properties and In Vitro DMPK Parameters for Compounds 12c, 12d, and 12f.
MW
clog P
2.3
human
ERa
rat
mouse
ERa
human/rat
ppbb
Caco-2
brain pbd
Solubility pH2/pH7e
ERa
A to B/B to Ac
12c
408.
7
0.80
0.61
0.54
0.86
0.82
0.65
0.84
0.89
0.87
89.6 / 91.6
84.3 / 88.6
83.4 / 83.9
81.2 / 38.4
71.4 / 45.4
75.5 / 55.4
94.0
304 / 235
48 / 50
49 / 202
c
12d 408.
8
2.3
90.9
12f
426.
8
2.5
92.5
ahuman, rat or mouse extraction ratio as measured in a microsomal preparation, human or rat protein binding reported as % bound, Papp reported in
b
units of cm/sec x 10-6, drat brain protein binding reported as % bound, eReported in ꢀM.
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