Synthesis and Characterization of the Novel Rodent-Active and CNS-Penetrant P2X7 Receptor Antagonist Lu AF27139

Article abstract of DOI:10.1021/acs.jmedchem.0c02249

There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability, and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular pharmacology, s

Full text of DOI:10.1021/acs.jmedchem.0c02249

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Synthesis and Characterization of the Novel Rodent-Active and CNS-
Penetrant P2X7 Receptor Antagonist Lu AF27139
Allen T. Hopper,* Martin Juhl, Jorrit Hornberg, Lassina Badolo, John Paul Kilburn,
Annemette Thougaard, Gennady Smagin, Dekun Song, Londye Calice, Veena Menon, Elena Dale,
Hong Zhang, Manuel Cajina, Megan E. Nattini, Adarsh Gandhi, Michel Grenon, Ken Jones,
Tanzilya Khayrullina, Gamini Chandrasena, Christian Thomsen, Stevin H. Zorn, Robb Brodbeck,
Suresh Babu Poda, Roland Staal, and Thomas Möller
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ABSTRACT: There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability,
and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular
pharmacology, safety, pharmacokinetics, and functional CNS target engagement of Lu AF27139, a novel rodent-active and CNS-
penetrant P2X7 receptor antagonist. Lu AF27139 is highly selective and potent against rat, mouse, and human forms of the
receptors. The rat pharmacokinetic profile is favorable with high oral bioavailability, modest clearance (0.79 L/(h kg)), and good
CNS permeability. In vivo mouse CNS microdialysis studies of lipopolysaccharide (LPS)-primed and 2′(3′)-O-(benzoylbenzoyl)-
adenosine-5′-triphosphate (BzATP)-induced IL-1β release demonstrate functional CNS target engagement. Importantly, Lu
AF27139 was without effect in standard in vitro and in vivo toxicity studies. Based on these properties, we believe Lu AF27139 will be
a valuable tool for probing the role of the P2X7 receptor in rodent models of CNS diseases.
neuropathic pain (NPP),9 multiple sclerosis (MS), Alzheimer’s
INTRODUCTION
■
disease (AD), epilepsy, Huntington’s disease, and major
The P2X7 receptor (P2X7R) is an adenosine triphosphate
depressive disorder (MDD).10,11 While significant progress
(ATP) gated nonselective cation channel with a relatively high
has been made in the identification of human selective P2X7
km for ATP in the 100−1000 μM range in vitro.1 Existing as a
antagonists, at the start of this work, there remained an
homotrimer, P2X7Rs are widely expressed throughout the
insufficient number of CNS-permeable P2X7 receptor
CNS including the spinal cord with cellular localization
antagonist tools from which to probe rodent CNS pharmacol-
predominantly on microglia.2 Peripherally, P2X7R is expressed
ogy.12 Significant cross-species differences in molecular
on immune cells (e.g., monocytes, neutrophils, T, B, NK cells),
pharmacology and insufficient CNS permeability hampered
Schwann cells, and satellite glia. Activation of the P2X7R ion
the use of many P2X7 receptor antagonists for this purpose.13
channel by ATP leads to immediate calcium influx and a
variety of associated downstream signaling events, including
Received: December 28, 2020
Published: April 6, 2021
triggering the maturation and release of the cytokines IL-1β
and IL-18.3
Owing to its localization on microglia and importance in
regulating inflammatory cytokines, P2X7R has been suggested
to play an important role in neuroinflammation4 and has been
implicated in a variety of CNS-based diseases5−8 including
https://doi.org/10.1021/acs.jmedchem.0c02249
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Figure 1. Chemical structures of Lu AF27139 (1), Lu AF27138 (2), and P2X7R comparator compounds JNJ-54175446 (3), JNJ-54166060 (4),
JNJ analogue (5), A-804598 (6), CE224,535 (7), AZD9056 (8), GSK1482160 (9), AFC5128 (10), Actelion compound (11), A-740003 (12), A-
438079 (13), Abbott compound (14), Pfizer compound (15), JNJ-47965567 (16), JNJ-42253432 (17), and BBG (18).
Lacking were a sufficient number of structurally distinct tools
with optimized pharmacokinetic (PK) and safety profiles, to
establish the role of the P2X7R in models where chronic
dosing paradigms and near-complete target engagement would
be required to adequately test biological hypotheses. Herein,
we report the structure, synthesis, molecular pharmacology,
ADMET, and safety data for Lu AF27139 (1), a rodent-active
CNS-penetrant and highly selective P2X7R antagonist.
As part of our drug discovery research related to neuro-
inflammation, we desired to probe the role of the P2X7R in a
variety of CNS disease models (e.g., major depressive disorder,
epilepsy, multiple sclerosis, and neuropathic pain). In this vein,
we required suitable CNS-penetrant P2X7R antagonist tools
capable of giving near-complete functional inhibition of the ion
channel under a subchronic dosing paradigm. We evaluated a
variety of literature-reported compounds and found them to be
generally inadequate for this purpose, as they either lacked
sufficient rodent P2X7R potency, did not appreciably enter the
CNS, were of poor metabolic stability, or lacked appropriate
target selectivity. Therefore, part of our medicinal chemistry
work focused on identifying compounds with appropriate
characteristics to probe in preclinical rodent models of
neuroinflammation and CNS disease. To bolster this effort,
the Neurogen P2X7R patent estate was licensed by Lundbeck.
The racemic form of compound 1 was previously reported as
having an IC50 < 2 μM.14 While we synthesized and tested
numerous compounds within and near those within the
Neurogen patent estate, racemic 1 stood out as having good
potency and properties consistent with good CNS permeability
and PK, leading to the desire to separate the two enantiomers
giving 1 and 2.15
Subsequent to the conclusion of this work, several valuable
CNS-penetrant rodent-active tool compounds were de-
scribed.16 The clinical candidate JNJ-54175446 (3), with a
tetrahydro-triazolopyridine core, was reported to have near-
equal rat and human P2X7R potencies with pIC50’s of 8.8 and
8.5, respectively, and a brain-to-plasma ratio of 1.1 (Kp,uu,brain
0.46).17 A similar tetrahydro-imidazolopyridine analogue, JNJ-
54166060 (4), has rat and human P2X7R pIC50’s of 8.4 and
6.9, respectively, and a Kp,uu,brain of 0.84.13 In addition, a series
of CNS-penetrant and rat-active 1,2,4-triazolopiperidines were
described, as exemplified by 5.18 Compound 5 has rat and
human P2X7R pIC50’s of 7.10 and 9.15. Further, 5 has good
CNS permeability, as demonstrated by its activity in an ex vivo
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Table 1. P2X7 Receptor In Vitro Molecular Pharmacology, Metabolic Stability, and Permeability Data
liver microsomal
P2X7 FLIPR data
IC50 (nM)
P2X7 binding
Ki (nM)
IL-1β IC50
(nM)
stability
MDCK-1 Papp
e
a
b
c
d
(L/(h kg))
(cm/s × 10−6
)
compound
human
rat
mouse
human
rat
mouse
human
human
2.6
rat
A:B
15
ER
1
12 0.52
90 6.5
32 4.1
0.76 0.08
3.5 0.75
20 1.8
2.4 0.094
95 6.2
22 1.2
55 9.8
13 2.4
180 71
38 2.5
5.1
0.81
2
6
6.1 0.60
470 57
34 2.8
660 69
1700 340
2300 450
240 21
320 53
250 75
2900 490
300 35
8.9 0.61
3.9 0.36
60 17
15 5.7
1.5
1.4
1.2
1.2
0.3
5.3
1.6
4.3
7.2
2.6
1.0
9.3
0.6
0.7
1.5
7
27
17
0.72
8
4.4 1.1
15 1.9
5.7 1.8
130 22
510 12
1.4
9
1900 440
1500 230
260 19
300
20
5.9
25
0.2
10
11
12
13
14
15
16
17
18
72 19
32
5.1
0.7
5.5 0.81
81 5.5
5.6 1.2
130 21
68 37
7.4 1.6
53 3.0
100 28
48 13
27 9.0
290
0.83
4.0 0.20
910 160
71 6.9
59 1.9
200 42
16 2.0
4800 63
98
66
2100 390
57 15
150
1.0
4.5
46
25
3.2
140 16
40
0.77
0.6
21 1.5
5500 1300
4.7 0.82
>10 000
0.47
17
18
11
35 2.3
0.65 0.12
26
12
1.1
0.25
1.1
5800 1700
200 63
1300 65
>10 000
1000 36
700
a
P2X7R FLIPR data were determined in HEK293 cells stably transfected with either human, rat, or mouse P2X7R. The cells were preincubated
b
with antagonist and then stimulated with an EC80 concentration of BzATP. P2X7R binding was performed in membranes prepared from the same
c
cells using [3H]-A-804598 as the radio-ligand. Values are expressed as mean standard error of the mean (SEM) for n ≥ 3 experiments. IL-1β
d
inhibition data in THP-1 cells primed with lipopolysaccharide (LPS) and stimulated with BzATP. Rat and human liver microsomal intrinsic
clearance (Clint) was determined by assessing the disappearance of test compounds at six time points over a 60 min incubation period. The
compounds (1 μM) were incubated in a 1 mL reaction volume containing rat or human liver microsomes (purchased from Becton Dickinson, MA)
e
at a concentration of 0.7−1.0 mg protein/mL and fortified with NADPH. MDR1-MDCK cell permeability of the test compounds from A to B
direction or B to A direction was determined in triplicate, and the efflux ratio (ER) is the ratio of B:A/A:B permeation. Experimental details are
described in the Supporting Information.
receptor occupancy assay in rat hippocampus with an ED50 of
0.8 mg/kg. Compound 1 adds structural diversity to the
growing list of tool P2X7R inhibitors of potential utility to
probe in preclinical rodent models of neuroinflammation and
CNS diseases.
compounds 7, 9, 10, 11, and 15, are from 100- to 1000-fold
less potent at the rodent receptor, thus limiting their usefulness
to probe rodent in vivo pharmacology. Potency shifts between
mouse and rat P2X7R were also observed, such as for 8 and 12
with 390- and 60-fold shifts in potency between rodent species,
respectively. Notable differences between the measured
binding Ki and FLIPR IC50’s for some compounds may reflect
differences in on and off rates. For example, 1 and 8 are 5- and
30-fold more potent in the rat FLIPR assay than rat binding Ki,
respectively, while 13 and 14 have rat binding Ki’s 4.5- and
4.4-fold more potent than their respective rat FLIPR IC50’s.
These compounds were also evaluated for their ability to
inhibit IL-1β release in THP-1 cells, a human cell line. With a
few exceptions, the IC50 for inhibiting IL-1β release was within
3-fold of the human P2X7 FLIPR data IC50’s. Compound 1
was 3-fold less potent at inhibiting IL-1β release in THP-1 cells
with an IC50 of 38 nM, than inhibiting Ca++ flux in the human
P2X7 FLIPR assay. A few compounds, such as 6, 8, 13, and 18,
were more potent at inhibiting IL-1β release than in the human
FLIPR assay. Despite these differences, a reasonable
correlation between the two assays was observed.
Beyond species selectivity, ADME properties associated with
low brain permeability and high intrinsic clearance have
ultimately led to insufficient exposure for hypothesis testing
with many P2X7R antagonists. Liver microsomal stability and
permeability in the MDR1-MDCK cell assay were evaluated
for representative tools (Table 1). Compounds 10, 13, 14, 16,
and 17 have particularly high rates of intrinsic clearance
measured in rat liver microsomes at 4- to 10-fold above rat
hepatic blood flow. Metabolic instability is undesirable as it
may preclude from running subchronic dosing studies and
could produce large amounts of interfering metabolites, thus
complicating interpretation of in vivo efficacy studies. The
RESULTS AND DISCUSSION
■
Compound 1,15 its enantiomer Lu AF27138 (2) and
comparator P2X7R antagonists A-804598 (6),19 CE224,535
(7),20 AZD9056 (8),21 GSK1482160 (9),22,23 AFC5128
(10),24 Actelion compound (11),25 A-740003 (12),26 A-
438079 (13),27 Abbott compound (14),28 Pfizer compound
(15),29 JNJ-47965567 (16),30 JNJ-42253432 (17),31 and BBG
(18)32 were tested in vitro against mouse, rat, and human
forms of the receptor for inhibiting 2′(3′)-O-
(benzoylbenzoyl)adenosine-5′-triphosphate (BzATP)-induced
calcium-flux using fluorescent imaging plate reader (FLIPR)-
based assays (Figure 1 and Table 1). While ATP is the
endogenous agonist for the P2X7 receptor, BzATP is a more
stable, potent, and selective agonist of the P2X7 receptor and
therefore commonly used to activate the receptor in a variety
of assays.33 Compound 1 was found to have similar P2X7R
potency against rat (IC50 2.2 nM, Ki 13 nM), mouse (IC50 22
nM, Ki 180 nM), and human (IC50 12 nM, Ki 38 nM) forms of
the receptors. Since S-enantiomer 1 was 7.5- and 40-fold more
potent against human and rat forms of the receptor,
respectively, than R-enantiomer 2, additional work focused
only on enantiomer 1. Binding experiments were performed in
cell membranes isolated from HEK293 cells stably transfected
with human, rat, or mouse P2X7R. Compound 1 was about 5-
fold less potent in the binding assays than in the functional
FLIPR assays. While many of the reference compounds have
similarly low nanomolar potency against human P2X7R,
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Figure 2. Compound 1 inhibits BzATP-induced currents in (A) HEK293 cells stably transfected with rat P2X7R and (B) rat primary microglia.
Repeated administration of 300 μM BzATP demonstrates a slowly reversible effect of a single 60 s administration of 100 nM 1.
Figure 3. Compound 1 concentration-dependently inhibits IL-1β release in rat and mouse primary cortical microglia primed with LPS and induced
with 1 mM BzATP (A) rat microglia IL-1β release with an average IC50 of 38 19 nM (n = 4). The rat IC50 of the example curve is 2.2 nM, and
(B) mouse IL-1β release with an IC50 of 26 6 nM (n = 3). The mouse IC50 for the example curve is 21.9 nM.
MDR1-MDCK cell permeability assay is a useful predictor of
CNS permeability and Pgp efflux liabilities. Compounds 7, 8,
12, and 17 have low permeability with apical to basolateral
permeation being less than 1.0 × 10−6 cm/s with varying efflux
ratios, defined as the rate going from basolateral to apical
divided by the rate moving from apical to basolateral direction.
However, compounds 1, 9, 11, 14, 15, and 16 have desired
moderate to high permeability with Pgp efflux ratios below 2.
P2X7R antagonist 6 appeared to be an excellent tool
compound candidate owing to its good rodent potency,
permeability, and metabolic stability properties. However,
possibly due to its low solubility, a formulation and dosing
paradigm to give sufficient and reproducible exposures could
not be identified, despite considerable efforts to do so
(unpublished data). During our discovery efforts, the novel
compound 1 was found to have similar P2X7R potency to rat
(IC50 2.2 nM, Ki 13 nM), mouse (IC50 22 nM, Ki 180 nM),
and human (IC50 12 nM, Ki 38 nM) forms of the receptors.
And compound 1 with high permeability in the MDR1-MDCK
assay without efflux and suitable metabolic stability became a
compound of interest for additional profiling.
To evaluate the effects of 1 on the P2X7R ion channel, it
was profiled in several additional P2X7R functional cell-based
assays. In electrophysiology experiments, 1 inhibited 100 μM
BzATP-induced current in HEK293 cells stably transfected
with the rat P2X7R (Figure 2A) in a dose response manner
with an IC50 of 66 nM. Similarly, 1 inhibited 300 μM BzATP-
induced current in primary rat microglia (Figure 2B) with 80%
inhibition occurring at a 100 nM dose. This experimental
paradigm reveals a slow off-rate of the compound, as it took
17.6 min to regain 50% of the normal response to repeated
application of 300 μM BzATP. This can be observed in Figure
2B, where the first two repeat applications of 300 μM BzATP
are administered after repeated 80 s of washout with a slow
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Table 2. Exposure of Lu AF27139 (1) in Rat after Oral Administration
a
a
a
dose
Cu, plasma (nM)
Cu, brain (nM)
Cu, spinal cord (nM)
(mg/kg, po)
(1 h)
(2 h)
(1 h)
(2 h)
(1 h)
(2 h)
1
3
10
30
100
22.4 4.2
71.2 14
381 97
414 100
1460 240
22.8 10
91.4 20
164 63
556 67
497 140
5.4 2.6
42.2 4.6
89.6 16
272 53
6.4 2.0
5.20 0.80
33.0 3.8
106 19
283 100
705 87
10.0 2.0
38.2 9.6
96.8 22
293 130
647 110
30.4 9.4
94.2 53
436 61
743 110
1120 180
a
Free plasma, brain, and spinal cord concentrations of Lu AF27139 in rat were determined by the formula (Ct*fu), where Ct is the total tissue
(plasma, brain, or spinal cord) drug concentration and fu is the fraction unbound in these tissues as determined by ex vivo equilibrium dialysis.
Values are expressed as mean SEM for n = 3 animals. fu, plasma = 0.02 0.00, fu, spinal cord = 0.07 0.03, and fu, brain = 0.09 0.03. Values are
expressed as mean SEM for n ≥ 3 experiments.
rate of current recovery. The difference in potency for 1
between the rat FLIPR assay (IC50 2.3 nM) and the
electrophysiology experiments may be due to the different
levels of BzATP used. The rat FLIPR assay was run by
stimulating cells with an EC80 of BzATP (c.a. ∼ 4 μM), which
is 25- to 75-fold below that used in the electrophysiology
experiments. Data from the electrophysiology assays along with
that from the binding assay (rat Ki 13 nM) confirm that 1
antagonizes P2X7R through direct receptor interaction. The
finding that 1 is effective in primary rat microglia confirms the
presence of functional P2X7R on microglia.
Beyond its ion channel function, P2X7R activation has long
been known to regulate IL-1β.3 IL-1β is carefully regulated in
vivo, and its production requires two steps: a priming step via
activation of the TLR4 receptor to generate pro-IL-1β and
subsequent activation of the P2X7R with ATP or the agonist
BzATP, thereby triggering inflammasome assembly and
activation of caspase 1, which cleaves pro-IL-1β to give mature
active IL-1β.34 Consistent with being a P2X7R antagonist, 1
inhibits LPS-primed and BzATP-induced IL-1β release from
THP-1 cells with an IC50 of 38 2.5 nM. Similarly, P2X7R
antagonist 1 concentration-dependently blocked IL-1β release
in rat and mouse primary cortical microglia primed with LPS
and induced with 1 mM BzATP with IC50’s of 38 19 nM in
rat and 26 6 nM in mice (Figure 3).
The TPSA is 80, c log P is 2.2 (determined by Daylight
software version 2.2), and the measured log D is 3.3. Intrinsic
clearances for 1 in rat and human liver microsomes are 5.1 and
2.6 L/(h kg) (Table 1), and in rat and human hepatocytes are
4.8 and 0.85 L/(kg h), respectively. Compound 1 does not
inhibit CYP3A4, 1A2, 2C9, 2C19, 2D6, 2B6, or 2C8 at up to
25 μM, nor is there a time-dependent inhibition against these
cyps at a similar concentration based on a high-throughput
assay developed for a parallel measure of both reversible and
time-dependent inhibition. However, compound 1 does have
mild to moderate CYP3A4 induction potential at 10 μM, as
measured in human hepatocytes with a relative increase of
26.9% in CYP3A4 mRNA. This is comparable to levels
obtained with the clinically relevant CYP3A inducer, rifampin,
at a concentration of 10 μM.
The rat pharmacokinetics of plasma concentration−time
profile of 1 following intravenous (iv) (1 mg/kg) and per os
(po) (2 mg/kg) administration was characterized by a
noncompartment model. The modeling parameter estimates
show a low plasma clearance (Cl) of 0.79 L/(h kg) and a
moderate volume of distribution at a steady state (Vss) of 0.95
L/kg, resulting in a modest terminal half-life (T1/2) of 2.7 h.
Following oral administration, 1 was rapidly absorbed (Tmax
=
0.58 h) with a high Cmax (2.7 μM) and high absolute oral
bioavailability (F% ≥ 100).
The cross-reactivity of 1 was evaluated at a 10 μM
concentration against a combination of 84 binding, cellular,
and nuclear receptor functional and enzyme assays at CEREP
and in the KINOMEscan screen against 468 kinases at
DiscoveRx. In the CEREP screen, a minor signal from the A2A
binding assay (34% inhibition of agonist binding) was followed
up, but no agonistic or antagonistic activity could be detected.
In the KINOMEscan, five kinases were inhibited by 65−90%
relative to controls in the initial screen, but these hits were not
confirmed upon retesting, suggesting that the initial findings
were false-positives. Compound 1 was also clean when tested
(n = 4) against the available P2X and P2Y receptors at Axxam.
Testing was performed in both agonist and antagonist modes
in an eight-point dose response starting at a top dose of 10 μM
against hP2RX1, hP2RX2, hP2RX3, hP2RX2/3, hP2RX4, rat
P2RX4, hP2RY1, hP2RY2, hP2RY6, hP2RY11, hP2RY12, and
hP2RY13. In summary, 1 was without significant effect against
the targets tested. The likelihood of off-target activity
contributing to the pharmacology of 1 is low based on its
selectivity profile.
Compound 1 is considered to have desirable physical−
chemical and DMPK properties supportive of good oral
bioavailability and CNS permeability. Two issues however,
high liver microsomal clearance and mild to moderate CYP3A4
induction potential, were identified. Concerns related to the
rate of rat liver microsomal clearance (1.5-fold rat liver blood
flow, LBF) are tempered owing to the observed low rate of in
vivo plasma clearance of 0.79 L/(h kg) (0.25-fold rat LBF) and
high oral bioavailability in rat.
Exposure assessment was performed in rat (Table 2), and
drug concentrations were measured in plasma, brain, and
spinal cord at 1 and 2 h after oral administration of 1 at 1, 3,
10, 30, and 100 mg/kg. Exposure was dose-proportional,
achieving greater than 700 nM free drug levels in the CNS 1 h
and 2 h after a 100 mg/kg, po dose. Based on unbound drug
concentrations (Cu), the brain-to-plasma ratio varied from
about 25% at 1 mg/kg to near 75% at 30 and 100 mg/kg doses.
It remains unclear if the observed differences in B/P ratios
across doses are due to efflux mechanisms only apparent at low
concentrations. Drug concentrations did not vary between 1
and 2 h post-dose nor did they vary between brain and spinal
cord tissue. Importantly, the Cu,brain and Cu,spinal cord values were
reasonably dose-proportional. Unbound concentrations of 1
are presented for better comparisons of drug exposure across
Compound 1 was further characterized in a variety of
physical−chemical and ADME assays. Thermodynamic sol-
ubility is 16 μg/mL (32 μM), measured using the shake flask
high-performance liquid chromatography (HPLC) method.
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Figure 4. Oral administration of 1 inhibits icv administered LPS-primed and icv BzATP-stimulated IL-1β production in (A) rat and (B) mouse
cortex. LPS was administered at time 1 h, compound 1 at time 0, and BzATP at time 1 h. Microdialysis samples were collected every 60 min and
IL-1β in the microdialysate samples analyzed using a V-PLEX assay from MesoScale Discovery (Rockville, MD), n = 6 or 7 per time-point. The
lower limit of detection was 2 pg/mL for IL-1β. Graphical and statistical analyses performed using GraphPad Prism software (version 5.01). Area
under the curve (AUC) was calculated from baseline for each animal between time 0 and 5 h. Analysis of variance (ANOVA) with Dunnett’s
multiple comparison test was used to determine the significance between treatment groups.
Scheme 1. Synthesis of Lu AF27139 (1); Reagents and Conditions: (a) (i) Morpholine, Tetrahydrofuran (THF); (ii) 2M HCl
in Ether; (b) Zn, HCl, EtoH, 87% Crude Yield, Two Steps; (c) N-Acetyl-L-leucine, MeCN/H2O (30:1), 30% Yield, 97% ee; (d)
20, 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane-2,4,6-trioxide (T3P), Triethanolamine (TEA), THF, Room Temperature
(RT), 3 h, 62% Yield
matrices and in line with the free-drug hypothesis where only
the unbound concentration of drug is available to bind to the
target. Since nonspecific binding of compounds varies in
different tissues, ratios such as the brain-to-plasma ratio of drug
should be based on the unbound concentrations.35 Fraction
unbound (fu) of 1 was determined ex vivo for rat plasma, brain,
and spinal cord tissues utilizing an equilibrium dialysis method
(see the Supporting Information). The fu’s in plasma, brain,
and spinal cord for 1 are 0.02, 0.09, and 0.07, respectively.
In vivo CNS functional activity was evaluated by measuring
IL-1β in the frontal cortex (FC) of freely moving rats and mice
using microdialysis (Figure 4). Orally administered 1 reduced
intracerebroventricular (icv) administered LPS-primed and
BzATP-triggered IL-1β release in the frontal cortex of rats and
mice. In contrast to other reports,30 we did not observe any IL-
1β release without LPS priming (data not shown). This is most
likely due to the use of low endotoxin human serum albumin
(HSA) in the microdialysis perfusion medium. Other
investigators report the use of bovine serum albumin (BSA),
which is frequently contaminated with endotoxin.36 Com-
pound 1 was dosed orally 1 h prior to icv BzATP
administration and produced minimal effective doses of 3
mg/kg and 10 mg/kg in rats and mice, respectively.
As part of a strategy to identify and mitigate potential safety
issues as early as possible during the drug discovery process, 1
was characterized in an exploratory toxicology screening
battery.37 In high content screening and oxidative stress assays,
which identify roughly 50% of idiosyncratic hepatotoxic
drugs,38 1 did not test positive for any of the parameters,
including mitochondrial membrane potential, cell count, cell
membrane integrity, lysosomal activity, and ROS formation
when tested in a dose response manner up to 100 μM. There
were no signs of genotoxicity in the miniaturized Ames,39 in
vitro micronucleus,40 and GreenScreen41 assays. At 30 μM, 1
showed 44% inhibition of the hERG ion channel. Other
cardiac ion channels tested (i.e., hNav1.5, hCav1.2, and
hKCNQ1/min K) were not inhibited at 10 μM.
Compound 1 was well tolerated up to 150 mg/kg, po in a 7-
day exploratory toxicology study in rats, as well as up to 100
mg/kg, po bid in a 14-day exploratory toxicology study in
mice. No macroscopic pathology, histological lesions, or
adverse hematological changes were observed up to the
indicated doses. In male rats, there was a dose-proportional
increase in exposure (AUC0−8 h) between 3 mg/kg (day 7, Cmax
4640 nM, Tmax 1 h, AUC0−8 h 7160 nM*h) and 150 mg/kg
(day 7, Cmax 39 600 nM, Tmax 4 h, AUC0−8 h 210 000 nM*h).
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(ee) as determined by chiral HPLC or supercritical fluid
chromatography (SFC). The comparator P2X7R antagonists A-
804598 (6),19 CE224,535 (7),20 AZD9056 (8),21 GSK1482160
(9),22,23 AFC5128 (10),24 Actelion compound (11),25 A-740003
(12),26 A-438079 (13),27 Abbott compound (14),28 Pfizer compound
(15),29 JNJ-47965567 (16),30 JNJ-42253432 (17),31 and BBG (18)32
were either synthesized as previously described or were readily
available from commercial vendors. The 1H NMR and liquid
chromatography−mass spectrometry (LC/MS) data for all com-
pounds were consistent with the structure and literature reports. All
compounds were greater than 97% pure based on 1H NMR and LC/
MS data.
The exposure also increased in a dose-proportional manner
between 3 and 30 mg/kg in female rats, but at 150 mg/kg, the
exposure did not increase compared with that at 30 mg/kg.
Exposures decreased in both sexes after repeated dosing at 30
mg/kg, as the AUC0−8 h values after 7-day dosing were 55 and
74% of that obtained after a single dose in male and female
rats, respectively. In female mice, repeated dosing at 100 mg/
kg (day 14, Cmax 61 640 nM, Tmax 1 h, AUC0−8 h 131 000
nM*h) also reduced the exposure to 33% of that obtained after
a single 100 mg/kg dose (day 1, Cmax 140 000 nM, Tmax 1 h,
AUC0−8 h 399 000 nM*h). Additional exposure data can be
found in Table S1a,b in the Supporting Information.
General Methods. Analytical LC−MS and HPLC data were
obtained using one of the methods described below.
The reduced exposure after repeated dosing suggests that 1
is enhancing its own rate of clearance, possibly through CYP
induction of the rat CYP3A4 orthologue as was observed with
human hepatocytes. Induction of metabolism might also
explain why the exposure in female rats did not increase at
the highest dose. The impact of repeat dosing on exposure
should be considered if planning to run repeat dosing
experiments. Overall, 1 is considered to have a good safety
and tolerability profile supportive of subchronic dosing studies.
To synthesize 1 in sufficient amounts to broadly support its
use as a tool, development of scalable routes toward the two
coupling partners, thiazole carboxylic acid 23 and diamine 22,
was developed. The optimized synthesis of 1 (Scheme 1) was
initiated with a 1,4-conjugated addition of morpholine to
nitroolefin 19, and the corresponding adduct precipitated
directly by the addition of 2 M hydrochloric acid. The resulting
white solid was isolated via filtration of the reaction mixture
affording hydrochloride salt 20. Attempts to dry hydrochloride
20 in a vacuum oven at 40 °C resulted in the elimination of
morpholine affording back nitroolefin 19; therefore, hydro-
chloride salt 20 was used directly in the next step. Reduction of
the nitro moiety by exposure to zinc in an aqueous ethanolic
solution provided racemic diamine 21 in 87% yield. After
extensive screening, an enantioselective crystallization reso-
lution method of diamine 21 using N-acetyl-L-leucine in a
mixture of acetonitrile and water was discovered to afford the
S-enantiomer 22 in 30% yield and >97% ee. Diamine 22 was
subsequently coupled with corresponding carboxylic acid 23 to
afford 1 in 16% overall yield, 99% UV purity, and >97% ee.
While a synthesis for intermediate thiazole carboxylic acid
20 was previously reported,14 an alternative inexpensive,
robust, and scalable procedure was developed to support
large-scale production of 1 (see Supporting Information
Scheme S1 and the associated synthetic procedures).
Method A: System controller, Shimadzu CBM-20A; UV detector,
Shimadzu SPD-M20A. Column: C-18 4.6 × 30 mm, 3.5 μm
symmetry; column temperature: 60 °C. Mobile phase gradient:
solvent A: H2O with 0.05% v/v trifluoroacetic acid (TFA) and solvent
B: Methanol with 0.05% TFA. Flow: 3.0 mL/min
Method B: A Waters Acquity UPLC-MS was used. Column:
Acquity UPLC BEH C18 1.7 μm; 2.1 × 50 mm; column temperature:
60 °C; solvent system: A = water/TFA (99.95:0.05) and solvent B =
acetonitrile/water/TFA (94.965:5:0.035); Method: Linear gradient
elution with A:B = 90:10 to 0:100 in 1.0 min and with a flow rate of
1.2 mL/min.
Method C: HPLC column: Merck LiChroCart 250-4, LiChroSorb
RP-8, 5 μm, 250 ×4.6 mm Mobile phase: 20 mM triethylamine/
phosphate buffer in water/acetonitrile 50/50, pH = 3.0. Flow: 1.0
mL/min. Temp.: 30 °C.
Method D: Chiral HPLC column: Chiralpak AD-H, 5 my, 250 ×
4.6 mm eluent: heptane:ethanol:diethylamine = 90:10:0.1, flow 1.0
mL/min. Temp: 30 °C
1H NMR spectra were recorded at 500 or 600 MHz on Bruker
Avance instruments. Tetramethylsilane (TMS) was used as internal
reference standard. Chemical shift values are expressed in ppm. The
following abbreviations are used for multiplicity of NMR signals: s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet in
accordance with the ACS Style Guide.42
Synthesis of Lu AF27139. The synthesis of racemic ( )-N-(2-
(4-chlorophenyl)-2-morpholinoethyl)-2-(pyrimidin-2-yl)-4-
(trifluoromethyl)thiazole-5-carboxamide has previously been de-
scribed.14 Herein is a convenient enantiomeric synthesis protocol
for the preparation of (S)-N-(2-(4-chlorophenyl)-2-morpholinoeth-
yl)-2-(pyrimidin-2-yl)-4-(trifluoromethyl)thiazole-5-carboxamide (1).
1-Chloro-4-((E)-2-nitro-vinyl)-benzene (19). 4-Chlorobenzalde-
hyde (200.0 g, 1423 mmol) was dissolved in acetic acid (600.0 mL,
10 550 mmol) and nitromethane (308.2 mL, 5691 mmol) was added,
then acetic anhydride (26.85 mL, 284.6 mmol) and ammonium
acetate (120.6 g, 1565 mmol) was added. The reaction was heated at
reflux for 2 h, then volatiles were removed by evaporation and water
was added (400 mL, 20 000 mmol) followed by ethyl acetate (400
mL, 4000 mmol). The solids were filtered and dried to yield the title
1
compound 19 as a brown solid (178.8 g). H NMR (CDCl3 500
CONCLUSIONS
■
MHz): δ ppm 6.88 (d, 1H), 6.48 (d, 1H), 6.47-6.32 (m, 4H). HPLC,
tR (min, Method A) = 1.48.
In conclusion, herein, we provide molecular pharmacology,
ADME/PK, in vivo CNS functional activity, and safety data for
1, which support its utility as a brain-penetrant chemical probe
to interrogate the role of the P2X7R in preclinical models of
neuroinflammation and CNS diseases. Importantly, a robust
synthetic route for this novel small molecule is also described,
thus enabling its availability. We believe 1 will be a valuable
addition to the existing tools for evaluating the role of the
P2X7 receptor in rodent models of CNS diseases.
4-[1-(4-Chlorophenyl)-2-nitro-ethyl]-morpholine Hydrochloride
Salt (20). 1-Chloro-4-((E)-2-nitro-vinyl)-benzene 19 (178.8 g,
973.9 mmol) was dissolved in tetrahydrofuran (383.9 mL, 4733
mmol). Morpholine (84.93 mL, 973.9 mmol) was added, and the
mixture was stirred for 30 min at RT. Diethyl ether (1329 mL, 12660
mmol) was added followed by 2.0 M HCl in diethyl ether (584.3
mL). Internal temperature increased to 36 °C, and the slurry was
stirred for 15 min before being cooled on an ice bath with stirring for
15 min. The solids were filtered and washed with diethyl ether. The
resulting title compound 20 was used without further purification in
the next step.
EXPERIMENTAL SECTION
■
2-(4-Chlorophenyl)-2-morpholin-4-yl-ethylamine (21). 4-[1-(4-
Chloro-phenyl)-2-nitro-ethyl]-morpholine, hydrochloride salt 20
(59.0 g, 192 mmol), was suspended in ethanol (680 mL, 12 000
mmol), then 37% aqueous HCl solution (200 mL) was added
Materials and Methods. Drugs. The synthesis of Lu AF27139,
(S)-N-(2-(4-chlorophenyl)-2-morpholinoethyl)-2-(pyrimidin-2-yl)-4-
(trifluoromethyl)thiazole-5-carboxamide 1, and enantiomer 2 is
described below. The final products have >97% enantiomeric excess
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followed by the addition of zinc (42.8 g, 654 mmol) in four portions.
The mixture was stirred for 50 min, during which the mixture became
a clear almost colorless solution. The volatiles were removed by
evaporation, and the aqueous mixture was quenched by pouring into
32% aqueous ammonia solution (400 mL). The organics were
extracted with iPrOAc (2 × 100 mL). The combined organic phases
were dried (sodium sulfate), filtered, and evaporated to dryness in
vacuo using MeCN (3 × 100 mL) to co-evaporate, affording the title
compound 21 (45.1 g). 1H NMR (CDCl3 600 MHz): δ ppm 7.31 (d,
2H), 7.20 (d, 2H), 3.71−3.65 (m, 4H), 3.26 (t, 1H), 3.09−3.04 (m,
1H), 2.96−2.91 (m, 1H), 2.43−2.37 (m, 4H).
with heptane (2 × 50 mL), and dried under vacuum to constant
weight to yield the title compound (1) (40 g). H NMR (DMSO-d6
1
600 MHz): δ ppm 9.02 (d, 2H), 8.94 (t, 1H), 7.71 (t, 1H), 7.44 (d,
2H), 7.33 (d, 2H), 3.92−3.86 (m, 1H), 3.67−3.64 (m, 1H), 3.60−
3.48 (m, 5H), 2.40−2.30 (m, 4H). LCMS (MH+): m/z = 498.3, tR
(min, Method B) = 0.49. HPLC tR (min, Method C) = 5.16. [α]D20
=
−34.89 (c = 1% in MeOH). Elemental Analysis (CHN) and KF H2O:
Found C 50.67%, H 3.90%, N 13.76%, KF H2O < 0.1%. Expected C
50.66%, H 3.85%, N 14.07%.
Stereochemistry Determination of (S)-2-(4-Chlorophenyl)-2-
morpholinoethylamine (22). The absolute stereochemistry of chiral
amine 22 was determined by XRPD analysis of the corresponding
Mosher’s amide,43 which confirmed the S-stereochemistry. Stereo-
chemistry assignment of (2R)-N-[(2S)-2-(4-chlorophenyl)-2-mor-
pholino-ethyl]-3,3,3-trifluoro-2-methoxy-2-phenyl-propanamide was
(S)-2-(4-Chlorophenyl)-2-morpholin-4-yl-ethylamine-(S)-2-ace-
tylamino-4-methyl-pentanoate. To a solution of 2-(4-chlorophen-
yl)-2-morpholinoethylamine 21 (106.7 g, 0.443 mol) dissolved in
acetonitrile (3005 mL) was added (S)-2-acetamido-4-methylpenta-
noic acid (N-acetyl-L-leucine 76.80 g, 0.443 mol), and the mixture was
heated to reflux and water (105 mL) was added, giving a clear
solution. The heating was stopped, and the solution was allowed to
cool slowly to room temperature. At ca. 45 °C, crystallization started
and continued over 4 h while cooling slowly to 20 °C. The product
was isolated by filtration and washed on the filter with acetonitrile (2
× 100 mL) and dried in vacuo. The solid was resuspended in 240 mL
of acetonitrile:water (v/v 96.5:3.5). The slurry was then heated slowly
to reflux for 1 h, heating was removed, and stirring was continued
while slowly cooling to room temperature (RT). The slurry was
stirred for 16 h at RT. The precipitated compound was isolated by
filtration, washed on the filter with 2 × 20 mL of acetonitrile:water
(v/v 96.5/3.5) and 1 × 30 mL of acetonitrile, and dried. The product
was then dried to constant weight in vacuo at 40 °C to yield the title
+
−
determined from a combination of R-factor tests [R1 = 0.0414, R1
= 0.0561], Flack parameter [x+ = −0.02(2), x− = 1.01(2)], and
Bayesian statistics on Bijvoet differences, p3(ok), p3(twin), and
p3(wrong) 1.000, 0.000, and 0.000, respectively. Calculation based on
6994 Bijvoet pairs. XRPD was measured on a sample of the batch and
gave the same pattern as the one used for the structure determination.
This means that the crystal selected for the structure determination is
representative of the batch.
P2X7 Inhibition Measured by FLIPR Assays. Human
(NM_002562.5), mouse (NM_002562.5), and rat
(NM_002562.5) P2X7 receptors were cloned into a pCDNA3.1
vector and stably expressed in HEK 293s cells (passage number <25,
ATCC Manassas, VA). The stable cells (15 000/well) were plated in
384-well poly-D-lysine (PDL)-coated black FLIPR plates (Greiner Bio
One, Monroe, NC) using 1.5% fetal bovine serum (FBS) (Atlanta
Biologicals, Lawrenceville, GA), in Dulbecco’s modified Eagle’s
medium (DMEM) (GibcoThermo Fischer Scientific, Waltham
MA) 24 h before the assay. The plates were washed 2× with the assay
buffer. For the human P2X7R assay, sucrose buffer was used to
improve the signal-to-noise ratio (sucrose assay buffer, pH 7.4, 5 mM
potassium chloride, 9.6 mM NaH2PO4·2H2O, 25 mM 2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (HEPES), 0.5
mM CaCl2, 5 mM glucose, 280 mM sucrose, and 1.0 mM probenecid
(Sigma, St. Louis, MO)), whereas for rat and mouse P2X7R, regular
FLIPR buffer was used [Hank’s balanced salt solution buffer (HBSS),
pH 7.4 1× buffer supplemented with 20 mM HEPES plus 2.5 mM
probenecid (Sigma) and 0.05% bovine serum albumin (Sigma)]. After
washing, the plates were loaded with 30 μL of Fluo-4 NW in Fluo-4
NW Calcium Assay Kit buffer (Molecular Probes, Eugene, OR) and
were incubated at 37 °C for 30 min in a humidified chamber (5%
CO2/95% air) and for 30 min at RT. Mobilization of intracellular
Ca+2 in response to the various P2X7 antagonists was measured
online using the FLIPR Tetra reader. In the assay, baseline
fluorescence was measured for 15 s, and then 15 μL of compounds
(40 μM) was added and fluorescence was monitored for 3 min for
agonist activity. Subsequently, 15 μL of BzATP (hP2X7 1 × 8 μM,
rP2X7 1 × 15 μM) was added, and after a 30 min incubation at room
temperature, the fluorescence was read for 3 min for IC50
determination. Data were analyzed using Lundbeck’s LSP curve-fit
software, which is similar to Prism nonlinear regression curve-fit
analysis.
1
compound as a white solid (55.1 g). H NMR (CDCl3 600 MHz): δ
ppm 7.68 (d, 1H), 7.44 (d, 2H), 7.26 (d, 2H), 4.08−4.03 (m, 1H),
3.71−3.68 (m, 1H), 3.58−3.52 (m, 4H), 3.38−3.32 (m, 1H), 2.88−
2.83 (m, 1H), 2.40−2.35 (m, 2H), 2.22−2.15 (m, 2H), 1.81 (s, 3H),
1.62−1.55 (m, 1H), 1.50−1.45 (m, 1H), 1.41−1.36 (m, 1H), 0.85 (d,
3H), 0.81 (d, 3H). HPLC tR (min, Method C) = 2.53.
(S)-2-(4-Chlorophenyl)-2-morpholinoethylamine (22). To a sol-
ution of 2-(4-chlorophenyl)-2-morpholinoethylamine (S)-2-acetami-
do-4-methylpentanoate (55.1 g) in iPrOAc/H2O (400 mL) was
added 24% aqueous ammonia. The phases were separated, and the
water phase was extracted once more with iPrOAc (50 mL) and the
combined organic phases were washed with water (2 ×50 mL), dried
over MgSO4, and the solvent was removed in vacuo. NaCl (ca. 20 g)
was dissolved in the water phase and then extracted with iPrOAc (3 ×
100 mL). The combined organic phases were washed with brine (2 ×
50 mL), dried over MgSO4, evaporated, and dried to constant weight
to yield the title compound 22 (31.3 g). HPLC tR (min, Method C) =
2.53, Chiral HPLC tR (min, Method D) = 14.96
(S)-N-(2-(4-Chlorophenyl)-2-morpholinoethyl)-2-(pyrimidin-2-
yl)-4-(trifluoromethyl)thiazole-5-carboxamide (1). (S)-2-(4-Chlor-
ophenyl)-2-morpholinoethylamine 22 (31.3 g, 130 mmol) was
dissolved in dry THF (700 mL) at RT. 2-(Pyrimidin-2-yl)-4-
(trifluoromethyl)thiazole-5-carboxylic acid 23 (39.4 g, 143 mmol,
preparation described in supplemental materials) was added followed
by triethylamine (54.4 mL, 390 mmol). 2,4,6-Tripropyl-1,3,5,2,4,6-
trioxatriphosphinane-2,4,6-trioxide (T3P) (122 mL, 50% solution in
ethyl acetate, dissolved in THF 100 mL) was added slowly and
resulted quickly in a clear solution. The reaction was stirred at RT for
3 h. To the reaction was added iPrOAc (500 mL), and the organic
phase was washed with 0.5 M NaOH (200 mL) and 0.1 M NaOH
(200 mL), followed by washing with water (2 × 200 mL). The
product was then extracted with 1 M aqueous H2SO4 (4 × 200 mL),
and the combined water phases were washed with iPrOAc (2 × 200
mL) and was again made basic with 24% aqueous ammonia (strong
exotherm, cooled with ice) and extracted with iPrOAc (2 × 400 mL).
The combined organic phases were washed with water (1 × 300 mL)
and brine (2 × 200 mL), dried over MgSO4, and the solvent was
partly removed in vacuo (volume, ca. 400 mL) and heptane (600 mL)
was added. The mixture was stirred on a rotary evaporator for 16 h
while slowly cooling to room temperature. The reaction was cooled
on ice for 30 min, and the product was isolated by filtration, washed
P2X7R Binding Assays. In vitro binding was performed in
membranes prepared from HEK293 cells stably transfected with
mouse, rat, or human P2X7 using [3H]-A-804598 as the radio-ligand.
Binding was performed in assay buffer (50 mM Tris-HCL, 0.1% BSA,
pH 7.4) at 4 °C for 1 h in a total volume of 250 μL using either 10 μg
of rat P2X7R, 40 μg of human P2X7R, or 60 μg of mouse P2X7R
membranes per well. The membranes were prepared using 25 mM
Tris-HCl, pH 7.4, 250 mM sucrose, 2.5 mM ethylenediaminetetra-
acetic acid (EDTA), 2 μg/mL aprotinin, 0.5 μg/mL leupeptin, and
200 nM phenylmethanesulfonyl fluoride (PMSF). Nonspecific
binding was defined using 10 μM cold A-804598. Kd was determined
by concentration-dependent saturation binding using 10 concen-
trations of [3H]-A-804598 (0.5−50 nM).
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THP-1 Cell IL-1β Assay. THP-1 cells (passage number <15,
ATCC, Manassas, VA) were maintained in growth media
(RPMI1640, 10% FBS (Atlanta Biologicals), 100 units/mL penicillin
and 100 μg/mL streptomycin (P/S), and 0.05 mM β-mercaptoetha-
nol) (all except FBS from Gibco)) and differentiated with 10 ng/mL
of interferon γ (IFN-γ) (R&D Systems, Minneapolis, MN) over 48 h
at 37 °C and 5% CO2. The differentiated cells were removed from the
T150 flasks and resuspended in CTL test media (Cellular Technology
Limited, OH) containing 1% Glutamax. Cells were primed by adding
lipopolysaccharide (LPS) (Sigma) to a final concentration of 100 ng/
mL, and then plated into PDL-coated 96-well plates at a density of
10k cells/well. The plates were covered with Breathe-Easier
microplate sealing tape and incubated at 37 °C and 5% CO2 for 3.5
h. Test compounds diluted in CTL test media were added to the cells
and incubated for 20 min at 37 °C and 5% CO2. BzATP was then
added to the cells at a final concentration of 1 mM and incubated for
an additional 30 min at 37 °C and 5% CO2. The supernatant from the
wells was transferred into a 384-well polypropylene plate. The IL-1β
levels in the supernatants were measured using human AlphaLISA IL-
1β immunoassay (PerkinElmer) and an Envision microplate reader
(PerkinElmer, Waltham, MA).
and GraphPad Prism 6 software (GraphPad Software, Inc) for further
analysis.
Electrophysiology Recordings from Rat Primary Microglia.
Primary rat microglia were seeded at a density of 300 000/35 mm dish
for 24 h. Whole-cell patch-clamp recordings were performed using an
EPC9 patch-clamp amplifier and PatchMaster software (HEKA
Instruments, Inc., Holliston, MA). The cell’s membrane potential
was held at −60 mV. External solution contained 140 mM NaCl, 4
mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM HEPES, and 10 mM
glucose at pH 7.4. The patch solution contained 140 mM CsCl2, 11
mM EGTA, 2 mM MgCl2, and 10 mM HEPES, pH 7.2. Compound 1
was applied by gravity via a capillary tube placed in close proximity to
the recording dish. P2X7R currents were elicited by application of 300
μM BzATP for 10 s.
IL-1β Measurements in Mouse and Rat Microglia. The IL-1β
assay was conducted 1 day after rat microglia (100 000 cells/well/48
well plate) or mouse microglia (50 000 cells/well/96-well plate) were
plated on PDL-coated plates (BD Falcon). The cells were primed
with 3 EU/mL LPS (Control Standard Endotoxin, Associates of Cape
Cod) for 3.5 h. Vehicle or 1 was added and incubated for an
additional 30 min. BzATP was then added to the cells at a final
concentration of 1 mM and incubated for an additional 30 min at 37
°C and 5% CO2. All treatments were added to wells sequentially
without any media being removed or changed. Post incubation, the
supernatant was collected and analyzed for IL-1β as per the
manufacturer’s protocol (mouse: N45ZA-1, rat: N45IA-1; MesoScale
Discovery, Rockville, MD). IL-1β levels were normalized to total
protein as determined using the BCA protein assay kit (Thermo
Scientific, Rockford, IL). IC50 values were generated using GraphPad
Prism 6 software (GraphPad Software, Inc., San Diego, CA). The data
were expressed as mean standard deviation (SD).
Mouse and Rat Microdialysis. Male Sprague−Dawley rats
(Crl:SD, Charles River) (280−350 g) were implanted with a guide
cannulas (BrainLink, Groningen, The Netherlands) into the frontal
cortex (FC), and an additional cannula for intracerebroventricular
(icv) injections was implanted into the lateral cerebral ventricle. Male
C57BL (C57BL/6NCrl, Charles River, Kingston, NY) mice (18−25
g) were implanted with guide cannulas (BrainLink, Groningen, The
Netherlands) into the frontal cortex (FC) and an additional cannula
for intracerebroventricular (icv) injections into the lateral cerebral
ventricle (N = 6−7). Both implantations were completed in one
surgery. Seven days after surgery, microdialysis probes (2 mm
polyethylene membrane), 3000 kDa molecular weight cutoff
(BrainLink, Groningen, The Netherlands), were inserted via the
guide cannula into the FC. The animals were then placed in plastic
observation cages and connected to a two-channel swivel. The input
of the dialysis probe was connected to a syringe pump, which
delivered artificial cerebrospinal fluid (CSF, perfusion fluid CNS, M
Dialysis AB, Stockholm, Sweden) with 0.15% human serum albumin
to the probe at a rate of 0.7 μL/min overnight. The output from the
swivel was attached to a peristaltic pump (EICOM USA, San Diego,
CA) and a refrigerated fraction collector using a “push−pull”
microdialysis method. After overnight perfusion, samples were
collected every 60 min. LPS (rat: 3 μg/5 μL; mouse: 0.1 μg/3 μL)
was injected icv 1 h prior to oral administration of Lu AF27139 or
vehicle (20% 2-hydroxypropyl-β-cyclodextrin). BzATP (rat: 50 μg/5
μL; mouse: 5 μg/3 μL) was given icv 1 h after administration of 1 or
vehicle. During microdialysis sampling, the conscious, unrestrained
animals were housed in the animal containment system with free
access to food and water. Microdialysis samples were kept at −80 °C
until IL-1β analyses. IL-1β in the microdialysate samples was analyzed
using custom and standard V-PLEX assays from MesoScale Discovery
(Rockville, MD). The lower limit of detection was 2 pg/mL for IL-1β.
Graphical and statistical analysis was done using GraphPad Prism
software (version 5.01). An area under the curve (AUC) value was
calculated from the baseline for each animal from time 0 to 5 h. A
one-way analysis of variance (ANOVA) with Dunnett’s multiple
comparison test was used to determine significance between
treatment groups.
Rat Primary Microglia Cultures. Female Sprague−Dawley rats
(Crl:SD, Charles River, Kingston, NY; E14 timed pregnant at
delivery) were singly housed at 20−22 °C on a 12 h dark/light cycle
with food and water ad libitum. Primary microglia cultures were
prepared from postnatal day 3 (P3).
Microglia were cultured as described.44 Briefly, the animals were
euthanized by CO2 anesthesia followed by decapitation. The brains
were removed, and cortices were dissected out and chopped in ice-
cold HBSS (Gibco). The tissue was mechanically triturated by pipette
to obtain a single-cell suspension. Cortical suspension from one rat or
two mice were plated into a single T150 flask (BD Falcon, Corning,
NY) in DMEM-GlutaMax (Gibco) containing 4.5 g/L of D-glucose
and supplemented with 10% low endotoxin (0.06 EU/mL) FBS
(Atlanta Biologicals) and 1% P/S (Gibco).
Cultures were grown in a humidified incubator at 37 °C under 5%
CO2 for 10−14 days, during which time microglia were harvested by
tapping the flasks and collecting microglia floating in the medium.
Microglia were pelleted by centrifugation at 1100 rpm (276g) on a
Beckman Coulter (Brea, CA) Allegra 6 series with a GH-3.8 swing
bucket rotor for 5 min, resuspended in DMEM/10%FBS/P/S
medium, and plated at an assay-specific density in poly-D-lysine
(PDL)-coated plates (BD Falcon). Purity was assessed by labeling
with the microglial maker CD11b, which routinely identified >95% of
cells as microglia.
Electrophysiology. Compound Preparation. A 10 mM stock
solution of Lu AF27139 in dimethyl sulfoxide (DMSO) was prepared
immediately before use and diluted with extracellular solution buffer
to the desired concentrations. Glass vial inserts were filled with 350−
400 μL of compound solution and placed into the glass insert base
plate for use in the QPatch assay, or in a capillary tube for recording
in rat primary microglia.
QPatch DRC in HEK293 Cells Expressing the Rat P2X7R. HEK293
cells stably expressing rat P2X7 were grown in T75 tissue culture
flasks to 70−80% confluence. On the day of the experiment, the cells
were washed with D-PBS, lifted with 2 mL of Detachin, and
centrifuged (250g, 2 min). The supernatant was removed, and the
cells were washed and resuspended in extracellular solution to achieve
a final cell density of ∼3 × 106 cells/mL.
QPatch Electrophysiology. Whole-cell patch-clamp experiments
were carried out on a QPatch-16 automated electrophysiology
platform (Sophion Biosciences, Paramus, NJ). Following establish-
ment of the whole-cell configuration, the cells were held at −60 mV.
The extracellular solution contained 140 mM NaCl, 4 mM KCl, 0.1
mM MgCl2, 0.3 mM CaCl2, 10 mM HEPES, and 10 mM glucose at
pH 7.4. The intracellular solution contained 140 mM CsCl2, 11 mM
EGTA, 2 mM MgCl2, and 10 mM HEPES at pH 7.2. P2X7 currents
were elicited with 100 μM BzATP (Sigma). Currents were analyzed
using the Sophion QPatch software and exported to Microsoft Excel
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Ethical Statement. All animal procedures were conducted in
accordance with the Guide for Care and Use of Laboratory Animals45
and with the approval of the Institutional Animal Care and Use
Committees at Lundbeck Research USA.
Experimental Animals. Unless noted otherwise, all in vivo
experiments were conducted with male Sprague−Dawley (Crl:SD,
Charles River) rats weighing 150−200 g. The animals were subjected
to an acclimation period of 7 days prior to using for experiments and
were maintained under standard husbandry conditions (22 °C, 35−
50% relative humidity, 12 h light/dark cycle), with unrestricted access
to standard rat chow and drinking water.
Hong Zhang − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Manuel Cajina − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Megan E. Nattini − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Adarsh Gandhi − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Michel Grenon − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Ken Jones − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Tanzilya Khayrullina − Neuroinflammation Disease Biology
Unit Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Gamini Chandrasena − Neuroinflammation Disease Biology
Unit Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Formulation and Dosing Compound 1. Unless mentioned
otherwise, for in vivo experiments, 1 was formulated as a solution or
fine suspension in 20% hydroxypropyl-β-cyclodextrin and adminis-
tered via oral gavage at a volume of 5 or 10 mL/kg.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02249.
■
sı
Scheme S1; synthesis of intermediate 20 and associated
detailed synthetic methods; liver microsomal stability
assay; hepatocyte stability assay; MDCK-MDR1 perme-
ability assay; compound 1 exposure and fraction
unbound in plasma, brain, and spinal cord; LC−MS/
MS method for analysis of compound 1 from exposure
studies; Rat PK methods; in vivo exploratory toxicology
study methods; and Tables S1a and S1b with rat and
mouse toxicokinetic exposure data (PDF)
Christian Thomsen − Neuroinflammation Disease Biology
Unit Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Stevin H. Zorn − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Structure smiles (CSV)
Robb Brodbeck − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Suresh Babu Poda − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Roland Staal − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Thomas Möller − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
AUTHOR INFORMATION
Corresponding Author
Allen T. Hopper − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States; orcid.org/0000-0001-8797-7882;
Email: athopper@aol.com
■
Authors
Martin Juhl − Process Research Lundbeck A/S, 2500 Valby,
Denmark
Jorrit Hornberg − Toxicology Research Lundbeck A/S, 2500
Valby, Denmark
Lassina Badolo − Chemistry and DMPK Lundbeck A/S, 2500
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02249
Valby, Denmark
John Paul Kilburn − Chemistry and DMPK Lundbeck A/S,
2500 Valby, Denmark
Annemette Thougaard − Toxicology Research Lundbeck A/S,
2500 Valby, Denmark
Author Contributions
A.T.H., M.J., M.G., and P.K. supported Lu AF27139
identification, synthesis, and project leadership. G.S. and D.S.
supported microdialysis studies. J.H., A.T., M.C., and G.C.
supported safety evaluation and toxicokinetics. L.B., M.C.,
A.G., M.E.N., and G.C. supported ADME/PK. R.S., L.D.,
V.M., E.D., H.Z., C.T., S.H.Z., K.J., T.K., R.M.B., S.B.P., and
T.M. supported in vitro molecular pharmacological character-
ization. These authors contributed equally.
Funding
Gennady Smagin − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Dekun Song − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Londye Calice − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Veena Menon − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
This research was completely funded by Lundbeck Research
USA or H. Lundbeck A/S.
Elena Dale − Neuroinflammation Disease Biology Unit
Lundbeck Research USA, Paramus, New Jersey 07652,
United States
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors kindly acknowledge and thank the Lundbeck
organization for their complete support, including financial
support, for this project. They also acknowledge Dario Dollar
and Benny Bang-Anderson for their input, challenge, and
feedback in support of this work.
ABBREVIATIONS
■
AD, Alzheimer’s disease; ATP, adenosine triphosphate;
BzATP, 2′(3′)-O-(benzoylbenzoyl)adenosine-5′-triphosphate;
Clint, intrinsic clearance; CSF, cerebrospinal fluid; Cu,
concentration unbound; DMEM, Dulbecco’s modified Eagle’s
medium; D-PBS, Dulbecco’s phosphate-buffered saline; FBS,
fetal bovine serum; FC, frontal cortex; FLIPR, fluorescent
imaging plate reader; fu, fraction unbound; HBSS, Hank’s
balanced salt solution buffer; HEPES, 2-[4-(2-hydroxyethyl)-
piperazin-1-yl]ethanesulfonic acid buffer; ICV, intracerebro-
ventricular; LBF, liver blood flow; LPS, lipopolysaccharide;
MDD, major depressive disorder; MS, multiple sclerosis; NPP,
neuropathic pain; PMSF, phenylmethanesulfonyl fluoride;
P2X7R, P2X purinoceptor 7; P/S, penicillin−streptomycin
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