Novel Phenyl-Substituted 5,6-Dihydro-[1,2,4]triazolo[4,3-a]pyrazine P2X7 Antagonists with Robust Target Engagement in Rat Brain

Article abstract of DOI:10.1021/acschemneuro.5b00303

Novel 5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine P2X7 antagonists were optimized to allow for good blood-brain barrier permeability and high P2X7 target engagement in the brain of rats. Compound 25 (huP2X7 IC₅₀ = 9 nM; rat P2X7 IC₅₀

Full text of DOI:10.1021/acschemneuro.5b00303

Letter
pubs.acs.org/chemneuro
Novel Phenyl-Substituted 5,6-Dihydro-[1,2,4]triazolo[4,3‑a]pyrazine
P2X7 Antagonists with Robust Target Engagement in Rat Brain
Christa C. Chrovian,* Akinola Soyode-Johnson, Hong Ao, Genesis M. Bacani, Nicholas I. Carruthers,
Brian Lord, Leslie Nguyen, Jason C. Rech, Qi Wang, Anindya Bhattacharya, and Michael A. Letavic
Janssen Research & Development, LLC, 3210 Merryfield Row, San Diego, California 92121-1126, United States
S
* Supporting Information
ABSTRACT: Novel 5,6-dihydro-[1,2,4]triazolo[4,3-a]-
pyrazine P2X7 antagonists were optimized to allow for good
blood-brain barrier permeability and high P2X7 target
engagement in the brain of rats. Compound 25 (huP2X7
IC50 = 9 nM; rat P2X7 IC50 = 42 nM) achieved 80% receptor
occupancy for 6 h when dosed orally at 10 mg/kg in rats as
measured by ex vivo radioligand binding autoradiography.
Structure−activity relationships within this series are described,
as well as in vitro ADME results. In vivo pharmacokinetic data
for key compounds is also included.
KEYWORDS: P2X7, SAR, brain permeability, receptor occupancy, mood disorders
that P2X7 antagonists that can cross the blood-brain barrier
2X7 is a ligand gated ion channel expressed abundantly in
PCNS glial cells. It is known to be involved in an may be the most effective at treating psychiatric conditions.16
The 5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine core has been
established in the literature as drug-like17 and is featured in a
series of P2X7 antagonists.18 We have previously published
novel compounds composed of fused triazolo-ring systems that
are potent brain penetrant P2X7 antagonists.19,20 Many of these
compounds, exemplified by 1 and 2, have excellent human and
rodent P2X7 potency. In addition, a number of compounds
from these series were found to achieve high P2X7 receptor
occupancy when measured 0.5 h after oral dosing. Our goal for
this work was to achieve high receptor occupancy over a
sustained period of time, in order to effectively interrupt the
P2X7 inflammatory pathway.
During the course of our structure−activity relationship
(SAR) studies, it was determined that the human P2X7
receptor tolerated 6-membered aliphatic carbocycle replace-
ments for the aryl and heteroaryl groups off of these cores (3).
However, reduction in ring size from cyclohexyl (3) to
cyclopropyl (4) was not tolerated. We then found that activity
could be revived with the cyclopropyl still in place, by
introducing a phenyl group on the 8-position (5).
inflammatory cascade that results in the release of inflammatory
cytokines, namely, IL-18 and IL-1β. Historical interest in this
target has focused on attenuating the release of cytokines via
P2X7 antagonism in inflammatory diseases such as rheumatoid
arthritis (RA) and pain.1−4 In the early 2000s, Pfizer2 and
AstraZeneca3 progressed P2X7 antagonists into to Phase IIb
studies for the treatment of RA. Although both compounds,
CE-224,535 and AZD9056, were effective at blocking IL-1β
release in patient plasma samples (after ex vivo stimulation with
ATP and LPS), neither compound met desired clinical end
points in RA.
More recent work has shown that P2X7 antagonists can
mitigate neuroinflammatory responses in a number of
conditions.5 In one clinical example, GSK1482160 was
administered in a Phase I trial with the intent of treating
neuropathic and inflammatory pain.6 This compound, like CE-
224,535 and AZD9056, inhibited ex vivo IL-1β release in the
study. However, due to its ability to cross the blood-brain
barrier,5−7 it would perhaps be better suited than CE-224,535
and AZD9056 to treat CNS disorders. Due to unsatisfactory
safety margins, however, GSK1482160 was not further
progressed.6
Keeping ligand efficiency (LE) in mind when embarking on a
new chemical series,21,22 efforts to further reduce molecular size
were undertaken. It was found that when the phenyl group is
introduced in the 8-position of these 5,6-dihydro-[1,2,4]-
triazolo[4,3-a]pyrazin-7(8H)-yl)methanone cores, functionality
We became interested in P2X7 modulation as a potential
treatment for psychiatric indications based on a number of
reports that have identified a neuroinflammatory tone in the
CNS of mood disorder patients.8−11 There have also been
reports of P2X7 knockout animals that exhibit a mood
stabilizing phenotype,11−14 and very recently, P2X7 antagonists
have been shown to be efficacious in preclinical chronic models
of depression.11,15,16 We presented preliminary data suggesting
Special Issue: Neuroinflammation
Received: November 20, 2015
Accepted: January 11, 2016
© XXXX American Chemical Society
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Table 1. In Vitro Antagonist Potency of 8-Phenyl-5,6-
dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanones
in a Calcium Mobilization Assay24
as small as H in the 3-position could provide excellent human
P2X7 activity (6; huP2X7 IC50 = 13 nM) as determined by a
calcium mobilization assay in 1321N1 cells expressing the
recombinant human P2X7 channel. A screen of a number of
small groups on the 3-position of the core were prepared and
tested (Table 1). Rat potency tended to improve with larger
functionality at the 3-position; for example, compare
compound 6 to both 7 and 5. When the arene of the
benzamide was removed, no activity remained up to 10 μM
(13). Chiral HPLC separation of compound 5 indicated that
P2X7 activity resides predominantly in a single enantiomer (8).
We have tentatively assigned the active isomer as the R
enantiomer (to be referred to as R*) based on data in our other
core series and pharmacophore models.19,23 Small carbocycles
and an isopropanol group were found to be tolerated (5, 8, 10,
and 12). Aromatic groups were highly active at the human and
rat receptors (7); however, these larger substituents were
expected to result in compounds with molecular weights in
excess of 500, after optimization of their physicochemical
properties. For this reason they were generally not pursued in
this series.
Compounds 5−12 were synthesized according to Scheme 1.
A coupling of the commercially available acid chloride with
Boc-protected ethylenediamine provided starting aminoamide
15 after deprotection. Compound 15 was cyclized to the
phenyl-substituted piperazinone. Piperazinone 16 was then
activated and cyclized to the 8-phenyl-5,6-dihydro-[1,2,4]-
triazolo[4,3-a]pyrazines, which were elaborated to the final
P2X7 antagonists.
The most promising compounds with potent in vitro human
and rat P2X7 IC50 values from this series underwent further
profiling for an in vitro assessment of ADME properties
(microsomal stability, cytochrome P450 (CYP) inhibition,
permeability, and plasma protein binding), solubility, and
hERG channel inhibition (Table 2). Compounds 8 and 10 both
were stable in human and rat liver microsomes and
demonstrated little or no inhibition of six CYP isoforms up
to 10 μM, no hERG channel inhibition up to 10 μM, and good
aqueous solubility. However, they were found to be substrates
for p-glycoprotein (PGP) as measured in the Caco-2 human
intestinal cell line assay, which measures the apical and
basolateral flux of the substrate across the cell monolayer.
PGP transporters are expressed in the apical membrane, and
the permeability value is reported as ratio of its secretion
(basolateral-to-apical velocity) to its absorption (apical-to-
basolateral velocity).
Compound 10 was further evaluated for its ability to cross
the blood-brain barrier in vivo. Brain levels of 10 were
measured in rat after an oral dose of 10 mg/kg at multiple time
points. The brain to plasma ratio at 30 min, 2 h, and 6 h ranged
from 0.06 to 0.07 (203 ng/mL brain at 2 h). The low brain to
plasma ratio is thought to be due to active efflux.
We next considered the possibility that the relatively basic
1,2,4-triazole may be contributing to the PGP liability of these
compounds. Consequently we investigated functional groups in
the 3-position that could influence the basicity of the triazole
ring system, such as electron withdrawing groups or groups that
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a
Scheme 1. Synthesis of 8-Phenyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanones
a
Reagents and conditions: (a) 2-bromo-2-phenylacetyl chloride, NEt3, CH2Cl2, 0 °C, 20 min (71%); (b) TFA, CH2Cl2, 16 h (99%); (c) K2CO3,
THF, 65 °C, 16 h; then di-tert-butyldicarbonate, THF, 65 °C, 5 h (79%); (d) Lawesson’s reagent, toluene, 110 °C, 3 h (23%); (e) iodomethane,
acetonitrile, rt, 16 h; (f) R-hydrazide, n-butanol, 155 °C, 3 h; then di-tert-butyldicarbonate, rt, 1 h (43−63% over 2 steps); (g) TFA, CH2Cl2, rt; (h)
2-chloro-3-(trifluoromethyl)benzoyl chloride, NEt3, CH2Cl2, 0 °C, 20 min (13%−66%).
Table 2. In Vitro ADME Data for Compounds 8 and 10
Table 3. In Vitro Antagonist Potency of and Caco-2 Data of
8R*-phenyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-
7(8H)-yl)methanones in a Calcium Mobilization Assay14
10
<0.3/<0.2
8
hu/rat microsomal
<0.3/0.21
a
stability
b
CYP inhibition
all >10 μM
all >10 μM except 2C8 = 9.6
μM
c
Caco-2 permeability
5.10
3.33
d
hERG IC50
>10 μM
>10 μM
solubility pH 2/7
>400 μM/386 μM 198 μM/67 μM
83.4/88.6 91.6/98.8
e
hu/rat plasma proteins
a
Stability in human and rat liver microsomes. Data reported as
b
extraction ratio. CYP IC50 values were obtained from human liver
microsomes for six isoforms: 1A2, 2C19, 2C8, 2C9, 2D6, and 3A4.
c
d
Papp is reported as B − A(×10−6) cm/s/A − B(×10−6) cm/s. hERG
IC50 (in μM) as measured in an [3H]-dofetolide competition binding
e
assay in HEK-293 cells expressing the hERG channel. Plasma protein
binding. Reported for human/rat as % bound.
possess steric bulk or ring strain. Several enantiopure
compounds were prepared and their activity and Caco-2 ratios
were measured. All of the compounds were highly active
huP2X7 antagonists (IC50’s < 20 nM, Table 3) and active
rP2X7 antagonists (IC50’s < 100 nM). Compounds containing
R = H, or linear or branched hydrocarbon chains generally
exhibited less desirable Caco-2 efflux ratios (20−21, 24).
Gratifyingly, the cyclobutyl, trifluoromethyl, and difluoromethyl
functionalities showed good B → A/A → B ratios (<2) in the
Caco-2 cell line and were poised for further investigation.
Compounds 25 and 26 contain a −CF3 and a −CF2H group,
respectively, and are thought to be less susceptible to active
transport due to the electron-withdrawing nature of the
fluoromethyl groups.
Compounds 20−24 were synthesized according to Scheme 1
followed by chiral HPLC separation of enantiomers. Com-
pounds 25−26 were synthesized by condensing the desired
fluorinated anhydride onto the requisite hydrazine to form the
unsaturated fused ring system (Scheme 2). The triazolopyr-
azine core could then be reduced using Pd/C, and elaborated
to the final P2X7 antagonists.
a
Papp is reported as B − A(×10−6) cm/s/A − B(×10−6) cm/s.
methyl and 3-cyclopropyl examples (32 and 33, respectively).
However, in both of these cases, the rat potency was reduced
compared to their desfluoro- parent compounds 8 and 10.
Pyridines and pyrazoles (34−36) were found to be more than
10-fold less potent than their phenyl parent compounds in
human and >100-fold in rat. A benzyl group in the 8-position
was tolerated at the human receptor but not the rat (37).
Compounds 23, 25, and 26 were profiled in vivo. The
cyclobutyl compound 23, despite good in vitro potency, was
found to have low levels of P2X7 receptor occupancy as
determined by an ex vivo radioligand binding autoradiography
experiment in rat (Figure 1). The animal groups were dosed
orally at 10 mg/kg and P2X7 receptor occupancy was
determined at 0.5, 2, and 6 h. The brain and plasma levels
revealed low brain levels of 76 ng/mL of 23 at 0.5 h, and a
brain/plasma ratio of 0.05, likely contributing to the low
observed level of receptor occupancy (Table 5). In vitro ADME
Next, the structure−activity relationship of 8-phenyl replace-
ments on 5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-
methanones was investigated (Table 4). A para-fluorophenyl
group was found to have human P2X7 potency for both the 3-
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Scheme 2. Synthesis of 3-Substituted −CF3 and −CF2H
a
Derivatives
Figure 1. Ex vivo receptor occupancies of 23, 25, and 26 at 10 mg/kg
dosed orally in rat.
a
Reagents and conditions: (a) H2NNH2−H2O, 120 °C, 1 h (89%);
rat Ki values or rat brain protein binding (Table 6). The values
measured in these experiments are comparable between 25 and
26. Both compounds also both showed good volumes of
distribution and bioavailabilities (Table 5).
Examining the in vitro ADME and safety data (Table 6), 25
and 26 showed no CYP inhibition in six isoforms and no hERG
channel inhibition. In an in vitro selectivity panel of 55 targets
(CEREP ExpresSProfile), compound 25 showed little or no
measurable inhibition of 45 receptors, 5 ion channels, and 3
transporters at 1 μM. It is of note that plasma free fraction for
25 was found to be about five times higher in human than in
rat, and a similar variation was observed for 23.
(b) di- or trifluoroacetic anhydride, rt, 2 h; concentrate then add
polyphosphoric acid, 140 °C, 16 h (57%−70%); (c) Pd/C, H2, rt, 16 h
(44−92%); (d) 2-chloro-3-(trifluoromethyl)benzoyl chloride; chiral
HPLC (19−23% yield of desired enantiomer).
Table 4. In Vitro Antagonist Potency of Phenyl Replacement
at the 8-Position of 5,6-Dihydro-[1,2,4]triazolo[4,3-
a]pyrazin-7(8H)-yl)methanones
Compound 25 was investigated next in a dose response ex
vivo radioligand binding autoradiography experiment (Figure
2). It was dosed orally as a suspension in 0.5% HPMC to rats at
seven concentrations. Drug levels were measured after 2 h, and
they were found to be dose-dependent in both the brain and
plasma. The highest levels of ex vivo receptor occupancy
measured was 90% at 10 mg/kg. The effective dose for 50%
occupancy was 4.3 mg/kg, with a corresponding plasma EC50 of
748 ng/mL (total plasma concentration). Total brain levels at
this dose were calculated to be 408 ng/mL. It is not known the
degree of P2X7 blockade that will be efficacious in neuro-
inflammatory and psychiatric indications. Nonetheless, it is
highly desirable to dose high enough in proof of concept
studies to be able to achieve full receptor inhibition in a clinical
setting. Based on plasma concentrations measured in the dose
response receptor occupancy study, total plasma concentrations
for 90% receptor occupancy would be approximately 2000 ng/
mL in rat.
In conclusion, 8-phenyl substituted 5,6-dihydro-[1,2,4]-
triazolo[4,3-a]pyrazin-7(8H)-yl)methanones are potent P2X7
antagonists. The substituent at the 3-position is an important
determinant of both P2X7 rat potency and efflux propensity.
We additionally found that fluorinated methyl groups
significantly improved brain penetration compared to other
small alkyl groups. Compounds 25 and 26 both showed good
receptor occupancy at 10 mg/kg as determined by ex vivo
autoradiography experiments in rat. Compound 25 had good
brain/plasma levels and a linear dose-dependent response in
the rat ex vivo receptor occupancy assay. From these studies,
we calculated a high total plasma EC90 of approximately 2000
ng/mL in rat. Nonetheless, further study of 26 as a P2X7
antagonist is warranted.
assays also revealed 23 to have moderate stability in liver
microsomes and high plasma protein binding in rat (Table 5).
Compounds 25 and 26 both showed good brain penetration,
achieving brain/plasma ratios of 0.5 (Table 6). Maximum P2X7
occupancies of 82% and 58% were measured at 10 mg/kg for
25 and 26, respectively, in the ex vivo radioligand binding
autoradiography experiment (Figure 1). The lower measured
receptor occupancy for 26 vs 25 cannot be explained by
comparing their brain levels or iv clearances (Table 5), in vitro
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Table 5. Average Brain and Plasma Levels in Rats after a 10 mg/kg oral dose of 23, 25, or 26, and in Vivo Pharmacokinetic Data
a
for 23, 25, and 26 in Rats (5 mg/kg Oral and 1 mk/kg iv Doses)
brain/plasma concn (ng/mL) 10 mg/kg
rat PK
iv Cl
(mL/min/kg)
Vss
(L/kg)
compd
0.5 h
2.0 h
6.0 h
B/P @ 2 h
iv T1/2 (h)
F
23
25
26
76 3/1288 137
846 317/1461 341
927 318/1502 384
39 11/848 71
26 16/745 144
1162 55/3034 170
792 158/1549 147
0.05
0.5
ND
1.1
3.0
ND
20.5
5.9
ND
2.4
1.7
ND
78%
82%
881 401/1618 537
990 313/2047 438
0.5
a
Brain (B) and plasma (P) concentrations listed in ng/mL.
aminoethyl)carbamate (10 g, 59.29 mmol) in 40 mL of CH2Cl2 was
cooled to −78 °C. Triethylamine (16.48 mL, 118.59 mmol) and 2-
bromo-2-phenylacetyl chloride (13.85 g, 59.29 mmol) were sub-
sequently added. The reaction was stirred for 20 min, warmed to 0 °C,
and stirred for 1 h. The reaction was quenched with water and
extracted three times with CH2Cl2. The combined organic layers were
washed with brine, dried with MgSO4, and concentrated. The resulting
residue was purified via silica gel chromatography (0−50% ethyl
acetate/hexanes) to provide the desired product (15.09 g, 71%) as a
white solid. 1H NMR (500 MHz, CDCl3) δ 7.51−7.28 (m, 5H), 5.43−
5.31 (m, 1H), 4.89 (s, 2H), 3.58−3.16 (m, 4H), 1.56−1.32 (m, 9H).
MS (ESI) mass calcd. C15H21BrN2O3, 357.2; m/z found, 358.2 [M +
H]+.
N-(2-Aminoethyl)-2-bromo-2-phenylacetamide (15). To a sol-
ution of tert-butyl (2-(2-bromo-2-phenylacetamido)ethyl)carbamate
(7.8 g, 21.75 mmol) in 30 mL of CH2Cl2 was added trifluoroacetic
acid (16.6 mL, 217.49 mmol). The reaction was allowed to stir at
room temperature overnight then concentrated and washed with satd.
NaHCO3 and extracted three times with CH2Cl2. The combined
organic layers were dried using MgSO4, filtered, and concentrated to
provide the desired product (10.54 g, 99%). MS (ESI) mass calcd.
C10H13BrN2O, 257.2; m/z found, 258.2 [M + H]+.
tert-Butyl 3-oxo-2-phenylpiperazine-1-carboxylate (16). To a
solution of 15 (19.28 g, 43.74 mmol) in 430 mL of THF was added
anhydrous K2CO3 (60.46 g, 437.46 mmol). The reaction was refluxed
at 65 °C overnight. Di-tert-butyldicarbonate (19.28 g, 87.49 mmol)
was then added, and the reaction was refluxed at 65 °C for an
additional 5 h and then cooled to room temperature, diluted with ethyl
acetate, and washed with water. The organic layer was partitioned,
dried with MgSO4, filtered, concentrated, and purified via silica gel
chromatography (0−30% ethyl acetate/hexanes) to provide the
desired product (9.54 g, 79%) as a white solid. 1H NMR (500
MHz, CDCl3) δ 7.42−7.27 (m, 5H), 6.02−5.71 (m, 1H), 4.15−4.06
(m, 1H), 3.80−3.69 (m, 1H), 3.69−3.61 (m, 1H), 3.42−3.30 (m, 1H),
1.51 (s, 9H). MS (ESI) mass calcd. C15H20N2O3, 276.3; m/z found,
277.2 [M + H]+.
Table 6. In Vitro ADME and Selectivity Data for 25, 26, and
23
a
25
26
23
b
rat Ki
9.1 4.0 nM
11.9 6.9 nM
6.6 3.1 nM
4.1 2.3 nM
5.2 1.8 nM
b
hu Ki
10.2 8.0 nM
(n = 2)
hu/rat
<0.30/<0.20
<0.30/0.20
0.72/0.50
microsomal
stability
CYP inhibition
>10 μM
>10 μM
>10 μM
permeability
(Caco-2)
0.75
0.78
1.2
hERG IC50
>10 μM
>10 μM
>10 μM
solubility pH 2/7 22 μM/18 μM
38 μM/122 μM 100 μM/12 μM
hu/rat plasma
proteins
92.0/98.6
90.7/91.1
96.9/99.4
rat brain proteins 96.4
% bound
95.9
96.9
a
See Table 2 for assay descriptions for microsomal stability, CYP
b
inhibition, permeability, hERG, and plasma protein binding assays. Ki
values are reported as the mean of three experiments in triplicate,
unless otherwise stated. hu is human, and r is rat. Ki sd is reported.
tert-Butyl 2-phenyl-3-thioxopiperazine-1-carboxylate (17). To a
mixture of Lawesson’s reagent (4.15 g, 9.95 mmol) in 125 mL of
toluene was added 16 (2.5 g, 9.05 mmol) in toluene (approx. 10 mL).
The mixture was heated at 110 °C for 3 h in a sealed tube. The
reaction was worked up with 10% NaOH and extracted three times
with ethyl acetate. The combined organic layers were dried with
MgSO4, filtered, concentrated, and purified via silica gel chromatog-
raphy (0−50% ethyl acetate/hexanes) to provide the desired product
1
(614 mg, 23%) as a crystalline orange solid. H NMR (500 MHz,
CDCl3) δ 9.67 (s, 1H), 7.51−7.41 (m, 2H), 7.37−7.27 (m, 3H), 6.13
(s, 1H), 4.08−3.77 (m, 1H), 3.53−3.38 (m, 1H), 3.38−3.26 (m, 2H),
1.50 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 199.5, 153.5, 138.7,
128.6, 128.1, 127.5, 81.4, 65.5, 43.1, 36.1, 28.4. MS (ESI) mass calcd.
C15H20N2SO2, 292.1; m/z found, 293.2 [M + H]+.
tert-Butyl 3-(methylthio)-2-phenyl-5,6-dihydropyrazine-1(2H)-
carboxylate (18). To a stirred solution of 17 (390 mg, 1.33 mmol))
in 3 mL of acetonitrile was added iodomethane (227 mg, 1.60 mmol).
The reaction was stirred at room temperature overnight and then
concentrated to provide the desired product (407 mg, 99%). 1H NMR
(500 MHz, CDCl3) δ 7.51−7.41 (m, 3H), 7.40−7.31 (m, 2H), 6.17 (s,
1H), 4.25−4.08 (m, 2H), 4.06−3.90 (m, 1H), 3.50−3.37 (m, 1H),
Figure 2. (a) Dose response analysis of the P2X7 antagonist 25 at 1 h
in the ex vivo ligand binding autoradiography assay in rat. Receptor
occupancy given the average % value of three animals per dose. (b)
Corresponding brain and plasma concentrations for the P2X7
antagonist 25 at 1 h in rat in the dose response ex vivo ligand
binding autoradiography assay. Concentrations shown are the average
of n = 3 data points per dose.
METHODS
■
Chemistry. Synthesis of 8 and 10. tert-Butyl (2-(2-bromo-2-
phenylacetamido)ethyl)carbamate. A solution of tert-butyl N-(2-
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3.08 (s, 3H), 1.48 (s, 9H). MS (ESI) mass calcd. C16H22N2SO2, 306.1;
m/z found, 307.2 [M + H]+.
(ESI) mass calcd. C14H16N4, 240.31: m/z found, 241.2 [M + H]+. The
enantiomers of compound 5 were separated via chiral SFC (stationary
phase: CHIRALPAK AD-H 5 μm 250 × 20 mm; mobile phase: 70%
CO2, 30% iPrOH) yielding the desired product as a mixture of several
tert-Butyl 3-methyl-8-phenyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]-
pyrazine-7(8H)-carboxylate (19a). To a round-bottom flask was
added 18 (606 mg, 1.978 mmol), acetic hydrazide (1.48 g, 19.76
mmol) followed by 10 mL of n-butanol. The reaction was heated to
155 °C and stirred for 3 h then cooled to room temperature and di-
tert-butyl dicarbonate (436 mg, 1.978 mmol) was added. The reaction
was subsequently stirred for 1 h at room temperature and then
isolated, concentrated, and purified via silica gel chromatography (0−
10% 2 M NH3-MeOH/CH2Cl2) to produce the desired product (390
mg, 63%). 1H NMR (400 MHz, CDCl3) δ 7.40−7.20 (m, 5H), 6.67 (s,
1H), 4.45 (s, 1H), 3.98−3.77 (m, 2H), 3.32−3.16 (m, 1H), 2.44 (s,
3H), 1.54−1.48 (m, 9H). MS (ESI) mass calcd. C17H22N4O2, 314.2;
m/z found, 315.0 [M + H]+.
1
rotamers. H NMR (400 MHz, CDCl3) δ 7.86−7.71 (m, 1H), 7.60−
7.29 (m, 7H), 6.15−5.99; 5.20−5.02 (series of m, 1H), 4.25−3.30
(series of m, 4H), 1.78−1.68 (m, 1H), 1.28−1.16 (m, 2H), 1.16−0.99
(m, 2H). For the major rotamer: 13C NMR (151 MHz, DMSO) δ
165.5, 154.6, 147.8, 137.2, 137.0, 132.1, 128.7, 128.6, 128.5, 127.1,
125.3, 123.4, 121.6, 120.0, 50.8, 41.4, 6.6, 6.1, 4.4. MS (ESI) mass
calcd. C22H18ClF3N4O+H, 447.1194; m/z found, 447.1190 [M + H]+.
The enantiomeric ratio was determined to be 100% by chiral SFC
analysis (Chiralpak AD, 30% iPrOH + (0.3% iPrNH2), 35 °C, 3 mL/
min), tR (major) = 4.08 min, tR (minor) = 4.89 min.
Synthesis of 25 and 26. 2-Chloro-3-phenylpyrazine (27). To a
solution of 2,3-dichloropyrazine (1.50 g, 10.07 mmol) and phenyl-
boronic acid (1.23 g, 10.07 mmol) in 35 mL of DME was added
Na2CO3 (1.07 g, 10.07 mmol) in 15 mL of water. N2 gas was bubbled
through the reaction mixture for 15 min then the flask was equipped
with a condenser and purged with N2 for another 15 in before adding
tetrakis(triphenylphosphine)palladium (582 mg, 0.503 mmol). The
reaction was heated to reflux and allowed to stir overnight. The
reaction was cooled to rt, diluted with 80 mL of water, and extracted
three times with CH2Cl2. The combined organic extracts were dried
with MgSO4, filtered, concentrated, and purified via silica gel
chromatography (0−30% ethyl acetate/hexanes) to provide the
desired product (1.39 g, 72%) as a white solid. MS (ESI) mass
calcd. C10H7ClN2, 190.0; m/z found, 191.0 [M + H]+.
2-Hydrazinyl-3-phenylpyrazine (28). A neat suspension of 27
(1.39 g, 7.23 mmol) in hydrazine monohydrate (3.6 mL, 72.78 mmol)
was placed in microwave vial and irradiated at 120 °C for 1 h. The
resulting reaction mixture was cooled down to rt, diluted with 30 mL
water, and extracted three times with 30 mL of CH2Cl2. The combined
organic extracts were dried using MgSO4 and concentrated under
reduced pressure to provide the desired product (1.21 g, 89%). MS
(ESI) mass calcd. C10H10N4, 186.1; m/z found, 187.2 [M + H]+.
3-(Difluoromethyl)-8-phenyl-[1,2,4]triazolo[4,3-a]pyrazine (29).
A neat residue of 28 (665 mg, 3.57 mmol) was cooled to 0 °C and
difluoroacetic anhydride (4.44 mL, 35.7 mmol) was added dropwise.
The reaction was allowed to stir at room temperature for 2 h then
concentrated. The residue was suspended in 4 mL of polyphosphoric
acid to form a gelatinous mixture, which was heated to 140 °C and
stirred overnight. The reaction was neutralized to pH 7 with NaOH
pellets and ice water. The resulting aqueous solution was extracted
three times with ethyl acetate. The combined organic extracts were
dried with MgSO4, concentrated, and purified via silica gel
chromatography (0−50% ethyl acetate/hexanes) to provide the
desired product (500 mg, 57%). 1H NMR (500 MHz, CDCl3) δ
8.84−8.77 (m, 2H), 8.20 (d, J = 4.6 Hz, 1H), 8.12 (d, J = 4.6 Hz, 1H),
7.60−7.53 (m, 3H), 7.45−7.22 (m, 1H). MS (ESI) mass calcd.
C12H8F2N4, 246.1; m/z found, 247.1 [M + H]+.
3-(Difluoromethyl)-8-phenyl-5,6,7,8-tetrahydro-[1,2,4]triazolo-
[4,3-a]pyrazine (30a). To a round-bottom flask containing a solution
of 29 (500 mg, 2.03 mmol) in 5 mL ethanol was added 10% palladium
on carbon (wet Degussa powder, 108 mg, 0.102 mmol). The reaction
vessel was purged with N2 gas, fitted with a hydrogen balloon (1 atm),
and stirred at rt overnight. The reaction mixture was then filtered
through a pad of Celite, and the filtrate was concentrated under
reduced pressure to provide the desired product (470 mg, 92%). MS
(ESI) mass calcd. C12H12F2N4, 250.1; m/z found, 251.1 [M + H]+.
(2-Chloro-3-(trifluoromethyl)phenyl)(3-(difluoromethyl)-8-phe-
nyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone
(26). To a solution of 30 (150 mg, 0.60 mmol) in 5 mL of CH2Cl2 was
added triethylamine (0.25 mL, 1.8 mmol). The reaction was stirred for
5 min at room temperature and then cooled to 0 °C. 2-Chloro-3-
(trifluoromethyl)benzoyl chloride (291 mg, 1.20 mmol) was added
and the reaction was stirred at 0 °C for 20 min. The reaction was
quenched with water and warmed to room temperature then extracted
three times with CH2Cl2. The combined organic layers were dried
using MgSO4, concentrated, and purified via basic HPLC to provide
(2-Chloro-3-(trifluoromethyl)phenyl)(3-methyl-8-phenyl-5,6-di-
hydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone (14). To a
solution of 19a (390 mg, 1.24 mmol) in 5 mL of CH2Cl2 was added
trifluoroacetic acid (0.390 mL, 5.10 mmol). The reaction was allowed
to stir at room temperature overnight and then concentrated, washed
with conc. NaHCO3, and extracted three times with CH2Cl2. The
combined organic layers were dried using MgSO4, filtered, and
concentrated to give 3-methyl-8-phenyl-5,6,7,8-tetrahydro-[1,2,4]-
triazolo[4,3-a]pyrazine. 1H NMR (400 MHz, CDCl3) δ 7.42−7.37
(m, 2H), 7.36−7.26 (m, 3H), 5.22 (s, 1H), 3.92−3.80 (m, 2H), 3.36−
3.27 (m, 1H), 3.24−3.16 (m, 1H), 2.39 (s, 3H), 2.36 (2, 1H). 13C
NMR (101 MHz, CDCl3) δ 151.4, 150.0, 139.3, 128.6, 128.13, 128.08,
57.0, 42.9, 40.6, 10.2. MS (ESI) mass calcd. C12H14N4, 214.27; m/z
found, 215.0 [M + H]+. The crude compound was dissolved in 5 mL
of CH2Cl2, and triethylamine was added (0.234 mL, 1.68 mmol). The
reaction was stirred for 5 min at room temperature and then cooled to
0 °C. 2-Chloro-3-(trifluoromethyl)benzoyl chloride (272 mg, 1.120
mmol) was added, and the reaction was stirred at 0 °C for 20 min. The
reaction was quenched with water, warmed to room temperature, and
extracted three times with CH2Cl2. The combined organic layers were
dried using MgSO4, concentrated, and purified via basic HPLC
(Agilent prep system, Waters XBridge C18 5 μm 50 × 150 mm
column, 5−95% MeCN/20 mM NH4OH over 22 min at 80 mL/min)
1
to provide the racemic product (157 mg, 30%). H NMR (500 MHz,
CDCl3) δ 7.87−7.70 (m, 1H), 7.60−7.29 (m, 7H), 6.21−5.93; 5.21−
5.01 (series of m, 1H), 4.16−3.30 (series of m, 4H), 2.51−2.45 (m,
3H). MS (ESI): mass calcd. for C20H16ClF3N4O+H, 421.1037; m/z
found, 421.1055 [M + H]+.
(2-Chloro-3-(trifluoromethyl)phenyl)(3-methyl-8-phenyl-5,6-di-
hydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone (10). Com-
pound 14 (222 mg, 0.528 mmol) was separated via chiral SFC
(stationary phase: CHIRALPAK AD-H 5 μm 250 × 20 mm, mobile
phase: 70% CO2, 30% iPrOH), yielding the desired product (98 mg,
44%) as a mixture of several rotamers. 1H NMR (500 MHz, CDCl3) δ
7.87−7.70 (m, 1H), 7.60−7.29 (m, 7H), 6.21−5.93; 5.21−5.01 (series
of m, 1H), 4.16−3.30 (series of m, 4H), 2.51−2.45 (m, 3H). For the
major rotamer: 13C NMR (151 MHz, DMSO) δ 165.6, 150.1, 147.9,
137.3, 137.0, 132.0, 128.7, 128.63, 128.60, 128.1, 127.1, 125.3, 123.5,
121.7, 119.9, 50.9, 41.4, 9.6. MS (ESI): mass calcd. for
C20H16ClF3N4O+H, 421.1037; m/z found, 421.1051 [M + H]+. The
enantiomeric ratio was determined to be 100% by chiral SFC analysis
(Chiralpak AD, 30% iPrOH + (0.3% iPrNH2), 35 °C, 3 mL/min),
20
tR(major) = 3.45 min, tR (minor) = 5.32 min. [α]D = −82.8 (c =
0.344, MeOH).
(2-Chloro-3-(trifluoromethyl)phenyl)(3-cyclopropyl-8-phenyl-5,6-
dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone (8). The
desired product was prepared in an analogous manner to compound
14 (using cyclopropanecarbohydrazide instead of acetic hydrazide to
form 19). The intermediate used in the final coupling step, 3-
cyclopropyl-8-phenyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine,
was characterized: 1H NMR (400 MHz, CDCl3) δ 7.41−7.35 (m, 2H),
7.34−7.23 (m, 3H), 5.21 (s, 1H), 4.02−3.90 (m, 2H), 3.32−3.23 (m,
1H), 3.22−3.12 (m, 1H), 2.32 (s, 1H), 1.75−1.63 (m, 1H), 1.13−1.06
(m, 2H), 1.04−0.94 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 154.9,
151.2, 139.4, 128.5, 128.1, 128.0, 56.7, 42.9, 40.3, 6.6, 6.4, 5.0. MS
F
DOI: 10.1021/acschemneuro.5b00303
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ACS Chemical Neuroscience
Letter
the racemate (132 mg, 48%). The racemate was separated via chiral
SFC (stationary phase: CHIRALPAK AD-H 5um 250 × 20 mm;
mobile phase: 70% CO2, 30% iPrOH) yielding the desired product as
AUTHOR INFORMATION
Corresponding Author
*E-mail: cchrovia@its.jnj.com.
Author Contributions
C.C. and M.L. conceived and oversaw the project series. A.S.-J.
and C.C. conceived, synthesized and characterized final
compounds. J.R. and G.B. conceived and synthesized
intermediates. B.L. conducted receptor occupancy experiments.
L.N. analyzed PK and receptor occupancy experiments. Q.W.
and H.A. conducted P2X7 assays. M.L., A.B., and N.C. oversaw
the project. C.C. drafted the manuscript. C.C. and A.S.-J.
revised the manuscript.
■
1
a mixture of several rotamers (63 mg, 23% overall yield). H NMR
(400 MHz, CDCl3) δ 7.87−7.77 (m, 1H), 7.61−7.32 (m, 7H), 7.14−
6.84 (m, 1H), 6.29−6.08; 5.17−5.10 (series of m, 1H), 4.43−3.27
(series of m, 4H). For the major rotamer: 13C NMR (126 MHz,
DMSO) δ 165.6, 150.7, 146.5, 137.2, 136.5, 132.2, 128.9, 128.7, 128.4,
127.2, 125.9, 123.7, 121.5, 119.3, 110.7, 108.8, 106.9, 50.9, 42.9. MS
(ESI) mass calcd. C20H14ClF5N4O + H, 457.0849; m/z found,
457.0832 [M + H]+. The enantiomeric ratio was determined to be
100% by chiral SFC analysis (Chiralpak AD, 30% iPrOH+(0.3%
iPrNH2), 35 °C, 3 mL/min), tR (major) = 2.08 min, tR (minor) = 3.41
20
min. [α]D = −87.8 (c = 0.319, MeOH).
Notes
3-(Trifluoromethyl)-8-phenyl-5,6,7,8-tetrahydro-[1,2,4]triazolo-
[4,3-a]pyrazine (30b). The desired product was prepared in an
analogous manner to 30a (using trifluoracetic anhydride instead of
The authors declare no competing financial interest.
1
ACKNOWLEDGMENTS
difluoroacetic anhydride to form 29). H NMR (500 MHz, CDCl3) δ
■
7.44−7.31 (m, 5H), 5.34 (s, 1H), 4.20−4.15 (m, 2H), 3.44−3.37 (m,
1H), 3.32−3.25 (m, 1H), 2.16 (br s, 1H). 13C NMR (126 MHz,
CDCl3) δ 154.2, 144.1, 143.8, 143.5, 143.2, 138.2, 128.8, 128.6, 128.0,
57.1, 44.7, 44.7, 40.4.
The authors would like to thank David Speybrouck for enabling
chiral separations, Heather McAllister for HRMS data, and
Warren Wade for optical rotation data.
(2-Chloro-3-(trifluoromethyl)phenyl)(8-phenyl-3-(trifluorometh-
yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone
(25). The desired product was prepared in an analogous manner to 26.
The racemate was separated via chiral SFC (stationary phase:
CHIRALPAK AD-H 5 μm 250 × 20 mm; mobile phase: 80% CO2,
20% iPrOH) yielding the desired product as a mixture of several
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rotamers. H NMR (400 MHz, CDCl3) δ 7.86−7.77 (m, 1H), 7.60−
7.32 (m, 7H), 6.26−6.08; 5.23−5.09 (m, 1H), 4.40−3.36 (m, 4H). For
the major rotamer: 13C NMR (151 MHz, DMSO) δ 165.5, 151.6,
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475.0755; m/z found, 475.0762 [M + H]+. The enantiomeric purity
was determined to be 100% by chiral SFC analysis (Chiralpak AD,
20% iPrOH + (0.3% iPrNH2), 35 °C, 3 mL/min), tR (major) = 2.76
25
min, tR (minor) = 3.91 min. [α]D = −84.1 (c = 0.607, MeOH).
Pharmacological Assays. Primary Pharmacological Assays.
Lipopolysaccharide (LPS)-primed, Bz-ATP induced IL-1β release
from human peripheral blood mononuclear cells (PBMC) was used as
the primary screen to test for P2X7 antagonism and was conducted as
previously described.25,26
P2X7 ex Vivo Radioligand Binding Autoradiography. Animal
work was done in accordance with the Guide Care for and Use of
Laboratory Animals adopted by the United States National Institutes
of Health. Animals were allowed to acclimate for 7 days after receipt.
They were group housed in accordance with institutional standards,
received food and water ad libitum, and were maintained on a 12 h
light/dark cycle. Male Sprague Dawley rats approximately 300−400 g
in body weight were used. For time course studies, two animals per
time point over three time points were used. For dose response
studies, three animals per dose over 7−10 doses were tested. Animals
were euthanized with carbon dioxide, and plasma and tissue removed.
Tissue sections were prepared as previously described.17
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschemneur-
o.5b00303.
Analytical data and synthetic routes for compounds 5−7,
9, 11, 12, 20−24, and 32−37 and experimental protocols
for pharmacological assays, ADME and hERG assays
(PDF)
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DOI: 10.1021/acschemneuro.5b00303
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ACS Chemical Neuroscience
Letter
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