Preclinical characterization of substituted 6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one P2X7 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2015.12.052

The synthesis, SAR, and preclinical characterization of a series of substituted 6,7-dihydro[1,2,4]triazolo[4,3]pyrazin-8(5H)-one P2X7 receptor antagonists are described. Optimized leads from this series comprise some of the most potent human P2X7R antagonists reported to date (IC₅₀s < 1 nM). They also exhibit sufficient potency and oral bioavailability in rat to enable extensive in vivo profiling. Although many of the disclosed compounds are peripherally restricted, compound 11d is brain penetrant and upon oral administration demonstrated dose-dependent target engagement in rat hippocampus as determined by ex vivo receptor occupancy with radiotracer 5 (ED₅₀ = 0.8 mg/kg).

Full text of DOI:10.1016/j.bmcl.2015.12.052

Bioorganic & Medicinal Chemistry Letters 26 (2016) 257–261
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preclinical characterization of substituted 6,7-dihydro-[1,2,4]triazolo
[
4,3-a]pyrazin-8(5H)-one P2X7 receptor antagonists
⇑
Michael K. Ameriks , Hong Ao, Nicholas I. Carruthers, Brian Lord, Suchitra Ravula, Jason C. Rech,
Brad M. Savall, Jessica L. Wall, Qi Wang, Anindya Bhattacharya, Michael A. Letavic
Janssen Pharmaceutical Research & Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, SAR, and preclinical characterization of a series of substituted 6,7-dihydro[1,2,4]triazolo
Received 5 November 2015
Revised 12 December 2015
Accepted 15 December 2015
Available online 17 December 2015
[
4,3]pyrazin-8(5H)-one P2X7 receptor antagonists are described. Optimized leads from this series
comprise some of the most potent human P2X7R antagonists reported to date (IC50s < 1 nM). They also
exhibit sufficient potency and oral bioavailability in rat to enable extensive in vivo profiling. Although
many of the disclosed compounds are peripherally restricted, compound 11d is brain penetrant and upon
oral administration demonstrated dose-dependent target engagement in rat hippocampus as determined
by ex vivo receptor occupancy with radiotracer 5 (ED50 = 0.8 mg/kg).
Keywords:
P2X7
Ó 2015 Elsevier Ltd. All rights reserved.
6
,7-Dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8
(
5H)-one
Autoradiography
Depression
CNS
The P2X7 receptor (P2X7R) is
a
purinergic ion channel
challenges in identifying compounds capable of blocking both
1
4
expressed primarily in immune cells. In the periphery, P2X7 recep-
tors are localized on monocytes and macrophages, while in the
central nervous system (CNS) P2X7 is found predominantly in glial
cells throughout the brain.1 High concentrations of extracellular
ATP are required to activate the P2X7 ion channel and trigger
downstream signaling events, most notably secretion of the pro-
human and rat P2X7 functional activity. Recently, GlaxoSmithK-
line also disclosed a series of potent 1,2,4-triazolopiperazine antag-
1
5
onists (hP2X7 IC50s < 10 nM), exemplified by 2.
We have
subsequently demonstrated that the introduction of a methyl sub-
stituent in the 6-position of this scaffold can dramatically improve
1
5,16
potency for rat P2X7 (see compound 3).
Related 5,6-bicyclic
2
17
inflammatory cytokine IL-1 b. Since local ATP levels are frequently
heterocycles have also appeared in the literature (compounds 4
1
8
3
elevated in tumor microenvironments and at sites of inflammation
or infection, the P2X7 receptor may play a pathophysiological role
in amplifying responses to cellular stress.3 Consequently, P2X7R
antagonists have been proposed as potential therapeutics for
numerous diseases, ranging from cancer and pain5 to muscu-
and 5 ), with 5 ([ H] JNJ-54232334) representing a useful radioli-
gand for imaging ex vivo P2X7R binding in mouse and rat brain.
Herein, we describe the medicinal chemistry efforts that resulted
in the identification of radiotracer 5 and related 6,7-dihydro-
[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one P2X7 receptor antagonists.
Although the SAR reported for triazole cores 2–4 suggested that
an exocyclic arylamide moiety was preferred for potency, we antic-
ipated that isosteric N-benzyl lactam analogs might also be viable
P2X7R antagonists based on analogy to N-benzyl amide 1. To test
this hypothesis, 1,2,3-triazolopyridone 8 was prepared in four
4
6
7
8
loskeletal, immune, and CNS related disorders.
9
The human P2X7 receptor was first cloned in 1997, and since
then a wide variety of structurally distinct P2X7R antagonists have
1
0
been reported. At least three compounds have progressed to clin-
ical trials, primarily for the treatment of peripheral inflammatory
1
1
17
disorders. However, our longstanding interest in P2X7 relates
to the role of IL-1b in the CNS, particularly as a putative contributor
to the etiology of mood disorders.12 In this regard, pyroglutamate 1
appeared especially intriguing because it was reported to be both
brain penetrant and moderately potent at the rat P2X7 ion channel
steps from the known 1,2,3-triazolo[4,5-c]pyridine 6 (Scheme 1).
The key step in this synthetic route was a chemoselective Ru-
19
catalyzed oxidation of amine 7 to provide the desired lactam 8.
In a similar manner, 1,2,4-triazolopyrazinones 11a and 11b were
assembled in five steps from previously described triazolopyrazine
1
3
16
(
Fig. 1).
The latter attribute is notable given the historical
intermediates
9
and 10, respectively (Scheme 2).
During
this sequence, it was necessary to switch from a tert-butyl to
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.12.052
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0

2
58
M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 26 (2016) 257–261
O
R
Cl
variety of substituted benzyl bromides 17 (Scheme 4). Intermedi-
ate 16 derived from enantiopure 15, which in turn was assembled
CF3
N
O
Cl
N
N
in six steps from commercially available N-Boc-L-alaninol 14 as
N
CF3
N
O
N
H
16
previously described. Human and rat P2X7 FLIPR data for com-
pounds 11d–k are shown in Table 2. Although the 2,3-dichlorophe-
nyl analog 11e compares favorably with 11d, removing
substituents from the phenyl ring reduces potency against both
human and rat P2X7. However, comparison of 11d and 11f with
N
N
1
2
, R = H; h/r P2X7 IC50s = 2.1/92 nM
3, R = (S)-CH ; h/r P2X7 IC
h/r P2X7 IC50s = 3.2/320 nM
3
50s = 12/10 nM
1
1g reveals that monosubstitution in the meta-position of the
O
Cl
O
Cl
arene is sufficient to achieve single-digit nanomolar potency at
the human P2X7 receptor, while additional substitution in the
ortho-position can provide a synergistic improvement in rat func-
tional activity.
CF3
N
CF
3
N
N
N
N
N
N
N
3
N
T
N
Having established that the (2-chloro-3-trifluoromethyl)benzyl
group is a preferred lactam substituent, we held this moiety fixed
and optimized the 3-position of the triazole. To this end, com-
pounds 11l–t were prepared by initial activation of amide 13 as
an imidate or thioamide, followed by condensation with a variety
of aryl and heteroaryl hydrazides (Scheme 3). Due to the highly
lipophilic nature of the N-benzyl group in the 7-position, the
majority of the hydrazide inputs were selected to minimize LogP.
Gratifyingly, both five- and six-membered heteroaromatic
substituents are well tolerated by the human P2X7 receptor
(IC50s < 10 nM), but these compounds are also generally 100-fold
less potent against rat P2X7 (Table 3). Similar to the SAR trends
observed during the optimization of the 7-position, small struc-
tural changes in the 3-position dramatically affect rat P2X7 func-
tional activity (compare 11o with 11p, 11q with 11r, and 11s
with 11t). However, despite the consistent disparity in potency
between human and rat orthologs, four compounds (11d, 11m,
11q, and 11s) displayed sufficient P2X7 antagonism in the rat FLIPR
assay (IC50 < 100 nM) to warrant further evaluation.
N
N
4
5
h/r P2X7 IC50s = 2.3/1900 nM
[ H] JNJ-54232334
Figure 1. Aryl and N-benzyl amide based P2X7 antagonists.
silicon-based carbamate protecting group due to the poor solubil-
ity of 9 in the benzylic oxidation step.
Lactams 8, 11a, and 11b were evaluated for their ability to block
Bz-ATP induced Ca flux in 1321N1 glial cells expressing human
or rat P2X7 (Table 1). Although 1,2,3-triazolopyridone 8 is inac-
tive in both cell constructs (IC50s > 10 M), the analogous 1,2,4-
triazole isomer 11a is a sub-micromolar antagonist of the human
P2X7 receptor (IC50 = 440 nM). Furthermore, the introduction of a
methyl group in the 6-position of this scaffold provided >100-fold
improvement in potency for both human and rat P2X7 (compare
2
+
2
0
l
2
1
1
1a and 11b). Racemate 11b was separated into individual anti-
podes 11c and 11d using chiral HPLC, and subsequent profiling
revealed that only the (À) enantiomer 11d is a potent P2X7R
antagonist. The absolute configuration of 11d was established by
asymmetric synthesis, starting from commercially available (S)-
All four lead compounds were predicted to be promising CNS
candidates based on their high MPO (multiparameter optimiza-
tion) scores (Table 4). This algorithm utilizes six calculated physic-
1
,2-diaminopropane 12 (Scheme 3). It is worth noting that this is
ochemical properties (cLogP, cLogD, MW, TPSA, HBD, and pK ) to
a
2
3
the same absolute stereochemistry previously determined for
related 6-substituted 1,2,4-triazolopyrazines containing an exo-
cyclic amide (see compound 3).16 However, there appear to be
important speciation differences in the SAR between the two ser-
rate compounds on a scale of zero to six. In general, experimental
ADME data support these favorable in silico evaluations, as all four
leads exhibit minimal turnover in human and rat liver microsomes,
along with low plasma protein binding and encouraging aqueous
solubility across a physiologically relevant pH range. However, in
the Caco-2 cell permeability assay, only pyrazine 11d shows com-
parable rates of transport in both directions (B–A/A–B = 1.1),
whereas the remaining three compounds are actively effluxed
(B–A/A–B > 10).
ies:
a methyl substituent in the (6S)-position dramatically
improves potency at the rat P2X7R for both chemotypes, but a sim-
ilar beneficial effect for the human ortholog is only observed
within the lactam series (compare compounds 2 vs 3 and 11a vs
2
2
1
1d). As a consequence, even though compound 11d is only
moderately potent against rat P2X7, it is one of the most potent
human P2X7 receptor antagonists reported to date.
In order to explore the SAR around the 7-position of 11d,
analogs 11e–k were prepared by alkylating lactam 16 with a
O
N
N
NBoc
R
NTeoc
R
c
N
a,b
NTeoc
R
N
N
N
N
N
N
O
2
N
N
N
N
N
9, R = H
10, R = (+)-CH3
N
a,b
NH *HCl
N
N
N
3 steps
ref 17
N
N
N
N
N
N
Cl
6
d
N
N
N
N
Cl
e
CF3
Br
O
Cl
O
Cl
O
Cl
CF
3
N
N
d
N
N
NH
CF3
CF3
N
N
N
N
c
N
7
N
N
N
N
N
R
R
N
N
N
11a,b
N
N
N
N
8
N
N
Scheme 2. Synthesis of compounds 11a and 11b. Reagents and conditions: (a)
1.25 M HCl/EtOH, DCM, rt (61%); (b) TeocSuc, iPr NEt, DMF, rt (57–89%); (c) cat
RuO , NaIO , MeCN/CHCl /H O, rt (29–44%); (d) TFA, DCM, rt (71–95%); (e) Cs CO
DMF, rt (63–79%).
Scheme 1. Synthesis of compound 8. Reagents and conditions: (a) HCO
60 °C (36%); (b) 6 N HCl, rt (94%); (c) 2-chloro-3-(trifluoromethyl)benzyl bromide,
Et N, DMF, rt (82%); (d) cat RuO , NaIO , MeCN/CHCl /H O, rt (43%).
2
H, Et
3
N,
2
1
2
4
3
2
2
3
,
3
2
4
3
2

M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 26 (2016) 257–261
259
Table 1
N
NBoc
NHBoc
In vitro potency of lactams 8, 11a–d
6 steps
ref. 16
N
a, b
N
HO
O
Cl
O
Cl
14
15
N
N
N
N
CF
3
N
CF
3
N
N
N
N
N
N
R
R
O
O
R
N
8
N
N
Br
N
N
1
1a-d
NH
N
N
N
R'
R'
N
17
N
a
a
Compound
R
hP2X7 IC50 (nM)
rP2X7 IC50 (nM)
c
16
11e-k
N
N
N
N
8
H
H
(±) CH
(+) CH
>10,000 (n = 1)
440 (n = 1)
3.4 ± 0.1
4710 ± 3930
0.7 ± 0.3
>10,000 (n = 1)
8670 (n = 1)
36 ± 16
11a
11b
11c
11d
3
Scheme 4. Synthesis of compounds 11e–k. Reagents and conditions: (a) cat RuO
NaIO , MeCN/CHCl /H O, rt (48%); (b) TFA, DCM, rt (64%); (c) Cs CO , DMF, rt (26–
76%).
2
,
3
>10,000
79 ± 39
4
3
2
2
3
(À) CH
3
a
IC50s determined using a FLIPR Ca2+ flux assay. Values represent the mean of at
least three experiments run in triplicate, unless otherwise noted. h denotes human,
and r is rat.
Table 2
In vitro potency of compounds 11d–k
The Caco-2 assay is generally used as an in vitro screen to pre-
dict intestinal absorption, but in the case of compounds 11d, 11m,
1q, and 11s, rat PK experiments revealed a much stronger associ-
R
O
N
N
N
R'
1
N
ation between Caco-2 efflux ratios and brain penetration (Table 5).
Whereas all four leads reach micromolar plasma levels following a
single 10 mg/kg oral dose, only 11d partitions effectively into the
brain ([brain]/[plasma] = 0.7). The degree of brain penetration also
correlates well with receptor occupancy in the hippocampus,
which was assessed by ex vivo autoradiography using the compet-
(S)
N
N
Compound
R
R’
hP2X7 IC50a
nM)
rP2X7 IC50a
(nM)
(
3
24
itive P2X7 receptor antagonist [ H] A-804598. In particular, 11d
achieves significant central target engagement, whereas 11q and
Cl
CF
3
1
1d
1e
H
H
0.7 ± 0.3
79 ± 39
1
1s are both peripherally restricted and show little to no receptor
occupancy in the brain. However, all of the compounds depicted in
Table 5 are orally bioavailable in rat (%F > 80) and possess suitable
PK profiles to support further in vivo evaluation. Given their struc-
tural similarity and differing tissue distribution in rat, 11d and 11s
could potentially serve as complementary tool compounds for
selectively probing the central and systemic effects of P2X7 antag-
onism in various preclinical disease models.
Cl
Cl
1
1.0 ± 0.1
103 ± 11
CH
3
CF
3
1
1
1
1f
H
H
H
3.4 ± 1.5
3.6 ± 1.4
216 ± 28
506 ± 209
4140 ± 990
>10,000
CF
3
To guide further development of 11d for CNS indications,
in vitro and ex vivo P2X7R occupancy dose–responses were
obtained in rat brain hippocampal tissue sections. In these studies,
1g
1h
Cl
3
pyrazinone 5 was used in place of [ H] A-804598 as the radiotracer
1
8
due to its reduced non-specific binding in rat brain tissue. Com-
pound 5 is a tritiated derivative of pyrimidine 11m, which was
selected for radiolabeling based on improved physicochemical
properties (i.e., free fraction and aqueous solubility) and rat
potency compared to 11d. In rat hippocampal tissue sections,
Cl
11i
H
72 ± 21
2384 ± 879
Cl
1
1d completely displaced 5 with an IC50 = 28 ± 12 nm (Fig. 2).
1
1
1j
H
6960 ± 2300
65 ± 20
>10,000
Cl
Cl
CF3
1k
(±) CH
3
2690 ± 317
NH *2HCl
NH2
CF3
2
a
b
HN
H
2
N
BocHN
Cl
BocHN
O
a
IC50s determined using a FLIPR Ca2+ flux assay. Values represent the mean of at
1
2
least three experiments run in triplicate, unless otherwise noted. h denotes human,
and r is rat.
O
Cl
O
CF3
N
CF3
c,d
N
e,f
N
N
HN
N
Similarly, following oral administration, 11d exhibited dose-
dependent ex vivo P2X7 receptor occupancy in rat brain, reaching
full occupancy at 10 mg/kg (Fig. 3). The calculated ED50 (0.8 mg/kg)
corresponds to plasma and brain EC50s of 62 ng/mL and 66 ng/mL,
respectively. However, accounting for rat brain and plasma protein
binding (95.4%/90.4%), the unbound brain-to-plasma partition
ratio (Kp u,u) at this dose is 0.51, which suggests that brain uptake
of 11d may be limited by efflux mechanisms.
R
13
1
1d and 11l-t
Scheme 3. Synthesis of compounds 11d and 11l–t. Reagents and conditions: (a)
Boc O, NaOH, H O/MeOH, 0 °C?rt (42%); (b) 2-chloro-3-trifluoromethylbenzalde-
hyde, Na(OAc) BH, DCE, rt (85%); (c) methyl chlorooxoacetate, Et N, CH Cl , 0 °C?rt
97%); (d) 4 N HCl/dioxane, rt, then Et N, CH Cl , rt (100%); (e) Et , DCM, rt, or
Lawesson’s reagent, THF, 55 °C (67–99%); (f) RCONHNH , 1-butanol, 130 °C (27–
0%).
2
2
3
3
2
2
+
À
(
3
2
2
3
O BF
4
2
9

2
60
M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 26 (2016) 257–261
Table 3
Table 5
In vitro potency of compounds 11d and 11l–t
In vivo profile of lead compounds
Compound
Rat PK parametersa
Rat BBBb
B/P @ P2X7 R.O.
O
Cl
CF3
Vss (L/kg) Cl (mL/
T1/2 (h) %F
N
N
c
d
min/kg)
T
max
@ Tmax
N
N
1
1
1
1d
1m
1q
1.5
3.3
5.0
4.2
19
11
21
13
1.0
4.8
2.8
3.9
125
672/945 78
80 116/2788 41
(S)
R
82
81
43/1064
41/1311
0
3
a
a
Compound
R
hP2X7 IC50 (nM)
rP2X7 IC50 (nM)
11s
a
Compounds dosed as solutions in 20% HP-b-CD at 1 mg/kg (iv) and 5 mg/kg
po).
N
0.7 ± 0.3
79 ± 39
>10,000
(
1
1d
1l
b
Compounds dosed at 10 mpk, po in Sprague Dawley rats (n = 2 or 3).
Brain (B) and plasma (P) concentrations listed in ng/mL.
N
c
d
P2X7 receptor occupancy in rat hippocampus as measured by ex vivo autora-
N
3
24
1
3.8 ± 1.5
diography using [ H] A-804598 as the radiotracer.
N
1
1m
1n
N
N
0.5 ± 0.06
0.5 ± 0.08
32 ± 7
1
N
350 ± 34
N
1
1
1o
1p
0.7 ± 0.2
380 ± 220
F
F
0.2 ± 0.02
3610 ± 2280
Figure 2. Competition of [3H]
5 binding by 11d in membranes from rat
hippocampus. Data are expressed as mean ± SD from n = 3 replicates per data point.
1
1q
1r
1.9 ± 1.2
4.3 ± 1.7
88 ± 74
N
1
6670 ± 2110
N
Cl
1
1s
1t
N
0.6 ± 0.4
4.2 ± 1.5
7.1 ± 3.6
N
H
1
N
960 ± 720
N
a
IC50s determined using a FLIPR Ca2+ flux assay. Values represent the mean of at
least three experiments run in triplicate, unless otherwise noted. h denotes human,
r is rat, and m is mouse.
Figure 3. Ex vivo P2X7 receptor occupancy with compound 11d in rat brain: dose
dependency following po administration (n = 3 per dose ± SEM). P2X7 occupancy
Table 4
In vitro ADME/DMPK data for lead compounds
was measured 30 min after drug administration using compound
radiotracer.
5 as the
Compound
CNS
MPO
HLM/RLM
stability
%PPBb
(h/r)
Solubilityc
(pH 2/pH 7)
Caco-2d
(A–B/B–A)
a
1
1
1
1
1d
1m
1q
1s
5.6
5.6
5.5
5.5
0.4/0.4
<0.3/<0.2
0.3/0.3
87/90
79/83
78/83
92/90
89/56
176/201
>400/375
41/95
47/50
4.4/64
6.2/73
5.4/65
exhibit any cross-reactivity in a Eurofins Cerep panel of ion-chan-
nels and GPCRs (0/50 > 50% inhibition at 1 M), and demonstrates
minimal potential for drug–drug interactions (2C19 IC50 = 7.2 M,
all other CYPs > 10 M). However, despite this promising preclini-
cal profile, further development of 11d was ultimately suspended
in favor of antagonists from related series due to their superior
potency in the rat ex vivo P2X7R occupancy assay.
l
<0.3/0.2
l
l
a
Stability in human and rat liver microsomes at 1
tion ratios.
lM. Data reported as extrac-
b
Plasma protein binding for human/rat reported as % bound at 1
Aqueous equilibrium solubility at pH 2 and pH 7 in
Data shown are Papp A–B/B–A(Â10 ) cm/s.
lM.
c
l
M.
d
À6
In summary, we have described the synthesis, SAR, and preclin-
ical characterization of a series of 6,7-dihydro[1,2,4]triazolo[4,3]
pyrazin-8(5H)-one P2X7 receptor antagonists. The introduction of
a methyl group in the 6-position dramatically improved potency
In addition to being a very potent human P2X7R antagonist, 11d
appears extremely selective. The compound lacks affinity for the
hERG channel (IC50 > 10 M, dofetilide binding assay), does not
2
+
l
in both human and rat FLIPR Ca flux assays. Many of the disclosed

M. K. Ameriks et al. / Bioorg. Med. Chem. Lett. 26 (2016) 257–261
261
compounds have outstanding physicochemical properties, which
helped guide the identification of 5 as a novel radioligand for
imaging ex vivo P2X7R binding in rat brain. Optimized leads from
this series are orally bioavailable in rat and possess sufficient PK
profiles to enable further in vivo profiling. In particular, 11d is a
potent, selective, and brain-penetrant P2X7R antagonist that
exhibits dose-dependent target engagement in rat hippocampus
following oral administration.
12. (a) Sperlagh, B.; Illes, P. Trends Pharmacol. Sci. 2014, 35, 537; (b) Koo, J. W.;
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G.; Medhurst, S. J.; Michel, A. D.; Moses, A. P.; Page, L.; Patel, S.; Roman, S. A.;
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Jiang, L.-H. Bioorg. Med. Chem. Lett. 2015, 25, 3164; (b) Michel, A. D.; Clay, W. C.;
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