Discovery of potent and selective adamantane-based small-molecule P2X 7 receptor antagonists/interleukin-1β inhibitors

Article abstract of DOI:10.1021/jm700949w

A novel series of antagonists of the human P2X₇ receptor is described. Modification of substituents enabled identification of compounds selective for the rat P2X₇ receptor and provides useful pharmacological tools for evaluation of t

Full text of DOI:10.1021/jm700949w

5882
J. Med. Chem. 2007, 50, 5882-5885
Discovery of Potent and Selective
Adamantane-Based Small-Molecule P2X7
Receptor Antagonists/Interleukin-1â Inhibitors
Mark Furber,*,† Lilian Alcaraz,† Janice E. Bent,†
Armin Beyerbach,‡ Keith Bowers,§ Martin Braddock,§
Moya V. Caffrey,† David Cladingboel,† John Collington,‡
David K. Donald,† Malbinder Fagura,§ Frank Ince,†
Elizabeth C. Kinchin,† Celine Laurent,† Mandy Lawson,§
Timothy J. Luker,† Michael M. P. Mortimore,†
Figure 1. KN628 and an initial lead structure from high-throughput
screening and a Hit-to-Lead exercise (human P2X7 pA2 ) 8.8, rat P2X7
pA2 < 5)11.
Austen D. Pimm,† Robert J. Riley,‡ Nicola Roberts,†
Mark Robertson,§ Jill Theaker,§ Philip V. Thorne,†
Richard Weaver,‡ Peter Webborn,‡ and Paul Willis†
release of interleukin-18 (IL-18), a proinflammatory cytokine
which has also been implicated in the pathogenesis of RA.5
Additional cell types on which P2X7 receptors are located
include antigen-presenting cells, microglial cells, salivary acinar
cells, and human skin fibroblasts.6 This role of P2X7 in the
processing and release of both IL-1â and IL-18 was viewed as
an attractive approach to inhibiting levels of these cytokines
with the target of providing a treatment for rheumatoid arthritis
and other inflammatory conditions. Recently, evidence for the
role of this receptor in disease processes has been demonstrated
in mice in which the function of the P2X7 receptor has been
ablated, that is, in P2X7 receptor knock-out (ko) mice. Sequential
treatment of blood taken from wild-type mice, but not P2X7-
ko animals, with LPS and ATP yielded large amounts of cell-
free cytokine IL-1â.7 In addition, P2X7-ko mice showed a
reduced severity of arthritis in an anticollagen antibody arthritis
model.7
Few examples of P2X7 antagonists are described. 4-[(2S)-2-
[(5-Isoquinolinylsulfonyl)methylamino]-3-oxo-3-[4-phenyl-1-
piperazinyl)propyl]phenylisoquinoline sulfonic acid ester (KN-
62, IC50 51nM) (Figure 1) and derivatives have been most
studied8 but do not represent an attractive starting point for a
medicinal chemistry program. More recently, triazole9 and aryl
cyanamide10 P2X7 inhibitors have been reported. We have
recently described the discovery of a series of small-molecule
P2X7 antagonists through a high-throughput screening ap-
proach.11 The most potent compounds (e.g., 1) have low
nanomolar activity in an assay-measuring reduction of P2X7-
mediated plasma membrane pore formation in a monocytic cell
line stimulated with the synthetic ATP analogue benzoylbenzoyl
adenosine triphospate (BzATP).12 With structural simplicity and
high potency as starting point, efforts were directed toward a
number of issues with these compounds. First, the contribution
of individual structural features to receptor binding was
unknown, with only limited exploration of structure-activity
having been accomplished in the Hit-to-Lead program. Second,
the properties of these compounds were known to be far from
ideal, and the high lipophilicity, low associated solubility, and
poor in vitro metabolic stability presented challenges which
needed to be overcome. Finally, a lack of activity at the rodent
P2X7 receptor, due to poor species crossover, would make target
validation in vivo more difficult, and it was deemed necessary
to achieve this crossover within selected compounds. Each of
these three areas was targeted for investigation.
Departments of Medicinal Chemistry, DiscoVery Bioscience, and
Physical and Metabolic Science, AstraZeneca R&D Charnwood,
Bakewell Road, Loughborough, Leics LE11 5RH, U.K.
ReceiVed August 1, 2007
Abstract: A novel series of antagonists of the human P2X7 re-
ceptor is described. Modification of substituents enabled identifi-
cation of compounds selective for the rat P2X7 receptor and provides
useful pharmacological tools for evaluation of the role of P2X7 in
disease.
The cytokines interleukin-1â (IL-1â) and tumor necrosis
factor R (TNF-R) are produced in response to inflammation,
tissue injury, and microbial infection.1 Overexpression of these
cytokines has been implicated in the pathogenesis of a number
of chronic inflammatory disorders, most notably, rheumatoid
arthritis (RA). RA is a chronic disease characterized by joint
inflammation accompanied by concurrent joint erosion and
destruction, and extensive evidence from both in vitro and in
vivo studies indicates that IL-1â, a proinflammatory cytokine,
is involved in mechanisms leading to the progressive joint
destruction observed in RA. Anakinra (Kineret), a recombinant
human IL-1â receptor antagonist (IL-1Ra), is the first marketed
treatment for RA that neutralizes IL-1â activity.2 Anakinra is
indicated as a monotherapeutic agent or can be used in
combination with other disease-modifying antirheumatic drugs
(DMARDs), such as methotrexate, and has shown significant
improvement in patients with active RA, including a reduction
in the rate of radiological disease progression.2 While providing
significant patient benefits, the possibility of providing a small-
molecule IL-1â inhibitor that can be delivered by the oral route
should provide the stimulus for further developments in this
area; likely advantages would encompass patient compliance
and health economics.
The P2X7 receptor is a ligand-gated ion channel present on
a variety of cell types involved in the inflammatory and immune
process, specifically macrophages, mast cells, T lymphocytes,
and B lymphocytes.3 Activation of the P2X7 receptor by
extracellular adenosine triphosphate (ATP) leads to the process-
ing and release of the proinflammatory cytokine IL-1â
from monocytes and macrophages and to the degranulation of
mast cells and the shedding of surface molecules L-selectin and
CD23 from lymphocytes.4 The P2X7 receptor also regulates the
The central adamantylmethylbenzamide unit was readily
constructed by a simple amide coupling of adamantylmethy-
lamine with an appropriate acid chloride. Compounds with a
directly linked amine substituent 2 and 3 were prepared from
the corresponding bromo compound either by a palladium-
* To whom correspondence should be addressed. Tel (+44) 01509
644880. Fax. (+44) 01509 645520. E-mail: mark1.furber @astrazeneca.
com.
† Department of Medicinal Chemistry.
‡ Department of Physical and Metabolic Science.
§ Department of Discovery Bioscience.
10.1021/jm700949w CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/27/2007

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 5883
Table 2. Effect of Aromatic Substituent Regiochemistry (2, 3, 4, and 6
Positions) on P2X7 Activity
Scheme 1. General Method for the Synthesis of
Adamantylmethylbenzamidesa
human
P2X7 pA2
rat
12
12
compound
X
amine
P2X7 pIC50
13
14
15
16
O
2-(4-piperidinyl)
3-(1-piperazinyl)
4-(1-piperazinyl)
6-(4-piperidinyl)
<5
6.7
6.9
<5
<5
<5
6.1
<5
CH2
CH2
O
Table 3. Replacement of Phenyl with Heterocycles
a Reagents: (a) (COCl)2/CH2Cl2/cat. DMF; (b) adamantylmethylamine/
iPr2NEt/CH2Cl2; (c) mono-N-BOC-diamine/Pd(OAc)2/ BINAP/Cs2CO3/
toluene; (d) HCl/MeOH; (e) MeLi /THF/-70 °C then nBuLi/-70 °C; (f)
N-BOC-4-piperidone; (g) cHCl; (h) H2/PtO2; (i) KMnO4 then (a), (b); (j)
BBr3/CH2Cl2; (k) N-BOC-aminoalcohol; (l) mono-N-BOC-diamine; (m)
P(OMe)3/toluene; (n) LDA 2.3 equiv/THF/-70 °C.
Table 1. Human and Rat P2X7 Activities for Amine-Substituted
Adamantylmethylaminebenzamides
this benzylic bromide with lithium tetrakis(N-dihydropyridine)-
aluminate13 followed by reduction of the resulting pyridine ring
afforded the racemic 3-piperidinyl compound 9. Analogous
chemistry was used to prepare compounds described in Tables
2 and 3.
humana
rata
SAR from the Hit-to-Lead exercise,11 combined with the
additional complexity and lack of a demonstrable advantage
from functionalizing the adamantane ring itself, led us to explore
substitution on the aromatic ring as the favored approach to
introduce groups which could improve the physical properties
of the compounds. In particular, we were interested in incor-
poration of polar and charged groups into this region of the
molecule if this could be achieved without adversely affecting
potency. Initial attempts to introduce a carboxylic acid func-
tionality were not promising, and direct replacement of the
O-methyl group in the 5 position of the aryl ring of compound
1 with a carboxylate group gave a large drop in potency (pA2
6.2). Incorporation of amine substituents, however, was more
beneficial (Table 1).
compound
X
amine
P2X7 pA2
P2X7 pIC50
2
3
4
5
6
7
8
9
bond
bond
O
O
O
NH
NH
CH2
CH2
CH2
CH2
1-piperazinyl
4-piperidinyl
4-piperidinyl
3(R)-piperidinyl
3(S)-piperidinyl
3-(()-piperidinyl
4-piperidinyl
3-(()-piperidinyl
4-piperidinyl
1-piperazinyl
(1S,4S)-2,5-diaza-
bicyclo[2.2.1]-
heptane
7.5
7.8
7.8
8.2
8.0
8.3
7.6
8.2
7.8
8.0
8.3
5.9
6.5
6.2
6.5
7.0
7.5
6.3
7.5
7.2
6.4
6.6
10
11
12
a This screen relies on entry of the fluorescent compound ethidium
bromide through P2X7 receptor-activated pores. The increase in fluorescence
of the intracellular DNA-bound ethidium bromide from cells is a measure
of P2X7 receptor activation, and this can be used to quantify the effect of
test compounds on the P2X7 receptor.12
While a drop in potency is seen compared to 1, most of these
cyclic amine substituted compounds retain a good level of
activity and, importantly, have improved physical properties
(e.g., solubility) and are appreciably more stable to metabolism.
The most promising example from this series was judged to be
compound 11. The compound is stable in in vitro hepatocyte
and microsomal preparations and displays good pharmacoki-
netics in vivo.14 Further studies have also shown this compound
to be selective for the P2X7 receptor after screening widely
against other receptor types, including related P2X receptors.
An X-ray structure (Figure 2) shows the amide carbonyl to be
twisted 44° out of the plane of the phenyl group by the ortho-
catalyzed coupling with the appropriate amine or, in the case
of compound 3, by a lithiation approach followed by addition
to N-BOC 4-piperidone, acid-catalyzed dehydration, and selec-
tive hydrogenation of the resulting olefin. The same arylbromide
was employed in palladium-catalyzed couplings to afford the
NH-linked analogues 7 and 8. Oxygen-linked compounds 4-6
were prepared using standard Mitsonobu chemistry and the
methylene-linked analogues 9-12 by displacement of the
corresponding benzylic bromide (Scheme 1). Displacement of

5884 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Letters
Figure 2. The molecular conformation of compound 11 dihydrochlo-
ride monohydrate. The two chloride ions and the water molecule are
excluded for clarity. The angle between the carboxamide plane [Od
C-NH-C] and the aromatic Cl-phenyl plane is 44.4°.
Figure 4. BzATP-induced IL-18 release from isolated human
monocytesseffect of compounds 11 and 17 (n ) 3)15.
chlorine atom, a common feature within this series of com-
pounds and one associated with high activity at the P2X7
receptor.
It is apparent from this small series of compounds that
introduction of an amine substituent had introduced activity at
the rat P2X7 receptor which was not present in the starting
compound 1. Although this activity varied considerably between
the compounds and compound 11 had relatively weak activity,
at least two members of the series (7 and 9) had activity
approaching IC50 ) 30 nM. Further investigation led us to move
the amine substituent into the other available positions on the
aromatic ring in compound 1 and to examine a greater variety
of amine groups. The results of the first exercise can be
illustrated with just four examples (Table 2). Depending on the
chemistry used to construct the compounds, the linking group
X was either oxygen or a methylene unit. Attachment of the
amine group through the 2 (replacing the Cl group) or 6
positions led to inactive compounds. Attachment to the 3 or 4
position retained a degree of activity, but this was substantially
reduced compared to that of the 5 position.
Having established the 5 position as optimal for attaching a
charged group, we investigated replacement of the phenyl ring
itself with a limited selection of heterocyclic groups (Table 3).
While this work did not provide an advance in terms of
properties, it did reveal the furan ring compound 17 as an
inactive structural analogue which could be used as a standard
for in vitro and in vivo testing. In an assay-measuring release
of IL-1â from monocytes, compound 11 showed complete
inhibition of cytokine release (pIC50 7.1 ( 0.1), whereas
compound 17 showed no activity in this assay (Figure 3).
Compounds 11 and 17 displayed a similar pattern of activity in
an IL-18 release assay (Figure 4), compound 11 giving complete
inhibition of cytokine release (pIC50 7.4 ( 0.1).
Figure 5. Compounds used for in vivo evaluation.
good exposure when dosed orally. Although promising activity
at the rat P2X7 receptor was observed in some of the compounds
illustrated in Table 1, the more metabolically stable members,
4, 11, and 12, were those with lower activity. Extensive
screening of analogues led to the discovery of compound 21,
which has the desired properties of high activity at the rat P2X7
receptor (pA2 7.6) combined with good in vitro and in vivo
stability but poor oral bioavailability. Further modification of
this compound yielded compound 22, which combined rat P2X7
activity (pA2 7.8), low rat plasma protein binding (66%), and
high exposure when dosed orally, and compound 23, with very
similar properties. Interestingly, both compounds were selective
for the rat versus the human P2X7 receptor (pIC50 <6.0) and
were highly selective when screened against a wide variety of
receptor and enzyme systems (MDS Pharma),16 including
P2X1-5 receptors.17P2X7 antagonists, through their ability to
inhibit release of IL-1â and IL-18, should have utility in the
treatment of a range of inflammatory diseases where these
cytokines play a role. The discovery of highly selective and
metabolically stable small-molecule antagonists of the human
P2X7 receptor such as compound 11, alongside the discovery
of close structural analogues possessing activity at the rat P2X7
receptor (21-23), will enable an evaluation of P2X7 antagonists
in both disease models and in human disease.
Target validation studies required a compound with activity
at the rat P2X7 receptor combined with the ability to achieve
1
Supporting Information Available: Experimental details, H
NMR chemical shifts, HPLC and MS data are provided, including
X-ray crystallographic data for compound 11. This material is
available free of charge via the Internet at http://pubs.acs.org.
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V
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1A, IC50 7 µM, and nonselective sigma receptor activity, IC50 4
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(17) Inactive when tested at 10 µM against human P2X1, P2X3, P2X4,
and P2X5 and rat P2X2 and P2X2/3 (heteromeric) receptors.
JM700949W