Synthesis and structure-activity relationships of pyrazolodiazepine derivatives as human P2X7 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.09.053

Screening of library compounds has yielded pyrazolodiazepine derivatives with P2X₇ receptor antagonist activity. To explore the structure-activity relationships (SAR) of these pyrazolodiazepines as human P2X₇ receptor antagonists, de

Full text of DOI:10.1016/j.bmcl.2009.09.053

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6053–6058
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of pyrazolodiazepine derivatives
as human P2X7 receptor antagonists
Ju-Yeon Lee a, Juan Yu a, Won Je Cho b, Hyojin Ko a,c, Yong-Chul Kim a,c,
*
a Research Center for Biomolecular Nanotechnology, Department of Life Science, Gwangju Institute of Science & Technology, Gwangju, Republic of Korea
b College of Pharmacy, Chonnam National University, Gwangju, Republic of Korea
c Graduate Program of Medical System Engineering, Gwangju Institute of Science & Technology, Gwangju, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2009
Revised 9 September 2009
Accepted 12 September 2009
Available online 17 September 2009
Screening of library compounds has yielded pyrazolodiazepine derivatives with P2X7 receptor antagonist
activity. To explore the structure–activity relationships (SAR) of these pyrazolodiazepines as human P2X7
receptor antagonists, derivatives were synthesized by substitutions at positions R2 and R3 of the pyra-
zolodiazepine skeleton. Using a 20(30)-O-(4-benzoylbenzoyl)ATP (BzATP)-induced fluorescent ethidium
uptake assay, the activities of these derivatives were tested in HEK-293 cells stably expressing human
P2X7 receptors. Moreover, the effect of these derivatives was assessed by measuring their effect on
IL-1b release induced by BzATP-induced activation of differentiated THP-1 cells. A 2-phenethyl pyra-
zolodiazepine derivative with a 1-methyl-1H-3-indolyl group at position R2 had fivefold greater activity
than the derivative with a 5-isoquinolinyl at R2. Moreover, a benzyl moiety at R3 had fivefold greater
activity than a bicyclic moiety. The stereochemical effect at C-6 showed a preference for the (R)-isomer.
Among the series of active derivatives, compound 23b, with a phenethyl group at R1, a 3-methyl indole at
R2, and a benzyl at R3, exhibited activity similar to that of the positive control, KN-62, as shown by the
inhibitory effects of IL-1b release.
Keywords:
IL-1b
Privileged structure
Pyrazolodiazepine
P2X7 Receptor
Ó 2009 Elsevier Ltd. All rights reserved.
The P2X receptors are ligand-gated ion channels belonging to
the purinergic P2 receptor family. Seven subtypes of P2X1-P2X7
receptor have been identified to date, all of which are activated
by extracellular ATP (adenosine-5-triphosphate).1,2 Among these
seven subtypes, P2X7 receptor (P2X7R) is the most distinctive in
molecular structure, function and pharmacology. Receptors of this
subtype are homomeric3 and are activated by much higher ATP
concentrations (EC50 = ꢀ1 mM)4 than are the other P2X receptor
neurodegeneration, and chronic pain.9–11 Particularly, P2X7R ex-
pressed by most immune system cells has been found to play an
important role in the processing of caspase-1 and the secretion of
cytokines including IL-1b by recruiting accessory proteins such as
pannexin-1.12 In addition, the activation of P2X7R triggers several
signaling cascades, which ultimately lead to macrophage fusion,13
superoxide production in microglia,14 lymphoid cell proliferation,15
and apoptosis/necrosis.16 Thus, therapeutic interventions targeting
P2X7R have been explored as a novel approach for the prevention or
treatment of inflammatory disorders such as arthritis,17 chronic
inflammatory pain,18 neuropathic pain,19 and neurodegenerative
diseases.9
To date, several P2X7R antagonists have been described, and
their structure–activity relationships (SAR) have been studied. A
tyrosine derivative, KN-62 (1-(N,O-bis(1,5-isoquinolinesulfonyl)-
N-methyl-l-tyrosyl)-4-phenylpiperazine, compound 1, Figure 1),20
has been shown to be one of the most potent non-competitive
antagonists for human P2X7 receptors (hP2X7R), and attempts to
enhance its antagonistic activity have yielded the more potent
compounds 221 and 3.22 These KN-62 derivatives, however, are
not appropriate for therapeutic use, due to their high molecular
weight, high lipophilicity and the presence of metabolically labile
sulfonate groups. High throughput screening of drug-like small
molecules has yielded an adamantine-based derivative 4 and its
analog, AZD9056,23 the 1-benzyl-5-phenyltriazole derivative 5
subtypes (EC50 <1 lM). Molecularly, P2X7 receptors are distin-
guished by their long C terminal tails (242 residues),5 which play
a role in the formation of non-selective plasma membrane pores
during extended receptor stimulation.6 The pore function was
established by studies of the uptake of large inorganic and organic
cations, to molecular weight 900 Da, such as ethidium bromide,
propidium bromide, and 4-[(3-methyl-2-(3H)-benzoxazolylid-
ene)methyl]-1-[3-(triethylammonio)propyl]di-iodide (Yo-Pro1).7,8
P2X7R is expressed mainly in hematopoietic cells, including mast
cells, lymphocytes, erythrocytes, and macrophages, the human
monocyte cell line THP-1, epidermal Langerhans cells, fibroblasts,
and cells in the central nervous system such as microglia and Schw-
ann cells, suggesting that these receptors are involved in the path-
ophysiology of various diseases, such as chronic inflammation,
* Corresponding author. Tel.: +82 62 970 2502; fax: +82 62 970 2484.
E-mail address: yongchul@gist.ac.kr (Y.-C. Kim).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.053

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J.-Y. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6053–6058
Figure 1. P2X7 receptor antagonists.
and the terpene-derived acyl hydrazide 6. Compound 4 showed
improved solubility and appreciable stability to metabolism,24
whereas compounds 5 and 6 effectively attenuated allodynia in a
rat model of neuropathic pain.25,26 Compound 7, an iminium qua-
ternary protoberberine alkaloid (QPA) was reported as a new
hP2X7R antagonist by our research group.27
Recently, we demonstrated that pyrazolodiazepine skeleton
could be a potential privileged structure by identifying the biolog-
ical activity of various analogs at different class of target proteins,
including melanocortin-4 receptor, b-secretase and hP2X7R.28 As
an extension of this work, we report here the full SAR analysis
and optimization of the antagonistic profile of various pyra-
zolodiazepine derivatives at the hP2X7R. We tested the ability
of these derivatives to inhibit fluorescent ethidium ion uptake
into HEK293 cells stably expressing hP2X7R and to block IL-1b re-
lease by differentiated THP-1 cells after receptor activation by
20(30)-O-(4-benzoylbenzoyl)-ATP (BzATP),
a
selective P2X7R
agonist.
Scheme 1. General synthetic scheme for the preparation of 6,2-substituted tetrahydropyrazolo[4,3-e][1,4]diazepin-8(2H)-ones. Reagents and conditions: (a) CH3OH, acetyl
chloride, 24 h, 95%; (b) phenethyl bromide or 1-Boc 4-(2-(methylsulfonyloxy)ethyl)piperidine, NaH, DMF, 12 h, 65–84%; (c) (i) 1 m NaOH MeOH, 1 h, 98–99%; (ii) (1)
L-
Ser(tBu)-methyl ester, (2) L-Phe-methyl ester, EDC, HOBt, TEA (triethylamine), DCM, 8 h, 78%; (d) Pd/C, H2, MeOH, 4 h, 98%; (e) DIBAL-H, toluene, 3 h, 60–70%; (f) NaBH(OAc)3,
1% AcOH, DCM, 55–60%.

J.-Y. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6053–6058
6055
Scheme 2. Synthesis of 2-piperidinylethyl pyrazolodiazepine derivatives of P2X7 antagonist. Reagents and condition: (a) cyclopropane carbonyl chloride, TEA (triethylamine),
DCM, 80%; (b) 20% TFA, anisole, DCM, 97%; (c) R3–CHO, NaBH(OAc)3, DCM, 67%.
Although the initial compounds were weakly active (e.g., 8,
The piperidine moiety of the pyrazolodiazepine-8-one ana-
logue, 17a, was further derivatized, as shown in Scheme 2. The
N-4 position of the diazepine ring was first substituted with cyclo-
propane carbonyl chloride to yield the tertiary amide, 18, and the
Boc group of 18 was deprotected with 20% TFA using anisole as a
scavenger to yield 19 for the introduction of bicyclic aryl group
at the piperidine moiety. The desired substituted piperidine-based
pyrazolodiazepine-8-one analogs, 20a–c, were obtained by react-
ing 19 with 5-quinoline and 5-isoquinoline carboxaldehydes, both
of which are building blocks for P2X7R antagonists, and 3-methyl-
1H-indole carboxaldehyde.
Initial SAR-studies around compound 8 were aimed at investigat-
ing the effect on P2X7R antagonism of the substituents, known
heterocyclic pharmacophore of P2X7R antagonists, in the piperidine
N-1 and the stereochemistry of C-5 (R to S). As shown in Table 1,
compound 18, an enantiomer of 8, where N-4 substitution of the dia-
zepine ring was fixed with cyclopropane carbonyl and the stereo-
chemistry of C-6 was changed to the S-configuration, showed a
IC50 = 4.31 lM, 9 = 18.6 lM), screening of library compounds
showed that the combinations of Boc-piperidine 4-ethyl at R1
and benzyl at R2 and phenethyl at R1 and tert-butoxymethyl at
R
2 were preferred pharmacophores for antagonistic activity. Using
the structural information obtained from compounds 8 and 9, we
established a strategy for further design of derivatives of the pyra-
zolodiazepine skeleton. We assessed the effect of stereochemistry
at the C-6 position and the structure–activity relationships of
substituted groups at the N-1 position of the piperidine of com-
pound 8. Boc was changed to a bicyclic aryl group, which is present
as a pharmacophore in the structures of other hP2X7R antagonists.
We also evaluated the importance of the thiophene carbonyl and
tert-butoxymethyl groups by replacing more flexible amine moiety
instead of tertiary amide at the N-4 position and the methyl bicy-
clic ester at C-6 of the diazepine structure in compound 9.
The pyrazolodiazepine-8-one skeleton was synthesized by a
standard procedure (Scheme 1).28 The N-2 position of the pyrazole
ring of methyl 4-nitro-1H-pyrazole-3-carboxylate 11 was alkylated
with 1-Boc 4-(2-(methylsulfonyloxy)ethyl)piperidine or phenethyl
bromide since library screening showed that these moieties at the
R1 position contributed to their antagonistic activity (compounds 8
and 9). The resulting carboxylic acid group from hydrolysis of ester
threefold decrease in antagonistic activity, with an IC50 of 13 lM.
Thus, the R-configuration of the diazepine skeleton was more benefi-
cial than the S-configuration. Removal of the Boc group of the piperi-
dine moiety, 19, dramatically decreased the activity, showing only
13% inhibition at 10 lM. Loss of activity was also observed for all
12 was coupled with
L
-Ser or
L
-Phe esters. After reduction of nitro
the analogs of 20, suggesting that the N-1 position of piperidine is
not an appropriate site for functionalization with heterocyclic phar-
macophores suchasisoquinoline andindolegroups. Thus, these alter-
ations did not result in any increase in antagonistic activity at P2X7R.
group of 13 to amine, ester 14 was subjected to reductive intramo-
lecular cyclization and subsequent reduction of imine 16 afforded
the tetrahydro-1,4-pyrazolodiazepin-8-one skeleton 17.
Table 1
Activities of synthesized compounds 18–20c and KN-62 on the ethidium accumulation in hP2X7-expressing HEK293 cellsa
R3
% Inhibition (10
lM)
IC50 (lM)
b
Compds
KN62 (positive control)
(R)-8
18
85
70
61
13
24
27
39
2
3
2
6
3
4
7
0.11 0.02
4.31 0.50
13.0 2.4
Boc
Boc
H
19
20a
20b
20c
5-Methylquinoline
5-Methylisoquinoline
3-Methyl-1-methyl-1H-indole
a
Data are expressed as means SD. All experiments were repeated three times.
IC50 = 50% inhibitory concentration, representing the mean from dose–response curves.
b

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J.-Y. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6053–6058
as shown in Scheme 3. The tert-butyl group of compound 17b
was removed by treatment with 50% trifluoroacetic acid in dichlo-
romethane to yield the free hydroxyl analog, 21, which was subse-
quently functionalized with 2-naphthalene carbonyl 22a, benzoyl
22b, 1-methyl-1H-3-indole carbonyl 22c, and 5-isoquinoline car-
bonyl 22d under standard coupling conditions using EDAC. Finally,
a series of compounds 23a–u was synthesized by alkylation with
various substituted aryl halides using DBU as base, or by reductive
alkylation with various substituted aryl aldehydes using standard
reducing agents.
SAR for the R2 position was initially assessed for a small group
of compounds (22a-23c), as shown in Table 2. In general, the het-
erocyclic group-containing compounds 22c, 22d, 23b and 23c (1-
methyl-1H-3-indolyl, and 5-isoquinolinyl, >68% inhibition at
10
lM) had greater antagonistic activity than the simple aromatic
ring-containing compounds 22a, 22b, and 23a (naphthyl and phe-
nyl, <46% inhibition at 10 lM). Therefore, subsequent SAR study
Scheme 3. Synthesis of 2-phenethyl pyrazolodiazepine derivatives of P2X7 antag-
onist. Reagents and conditions: (a) 50% TFA, anisole, DCM, 95%; (b) substituted
carboxylic acid, EDC, DMAP, DCM, 75%; (c) substituted aryl halide, DBU, DMSO,
heating, 57% or aryl aldehyde, NaBH(OAc)3, DCM, 45%.
assays utilized a combination of substitutions at the R3 position,
together with 1-methyl-1H-3-indolyl, and 5-isoquinolinyl substi-
tutions at the R2 position. In the case of 5-isoquinolinyl analogs
(22d, 23c–i), despite the appreciable activity (IC50 = 0.79 lM) of
22d, where there is no substitution at R3 position, neither substitu-
tion of a benzyl (23c–g) or 1-methyl indole (23h) moiety enhanced
activity. However, a p-OH-m-CH3-benzyl moiety at the R3 position
In a second attempt to optimize activity, the N-2 position of the
pyrazole ring was fixed with a phenethyl group, as in compound 9,
and the SAR of the remaining two diversity points, the N-4 position
and the OH group originating from the serine side chain of the dia-
zepine ring, was assessed. The phenethyl-based pyrazolodiaze-
pine-8-one derivatives were synthesized by three-step reactions,
(23i) had comparable activity (IC50 = 0.74 lM) as 22d. In contrast,
for the 1-methyl-1H-3-indolyl analog, 23b, introduction of a ben-
zyl group at the R3 position dramatically increased the inhibitory
Table 2
Activities of synthesized compounds 22a–23u and KN-62 on ethidium accumulation in hP2X7-expressing HEK293 cells and on IL-1b release by LPS/IFNc-differentiated human
THP-1 cellsa
c
Compd
R2
R3b
% Inhibition (10
HEK293 cells29
92
46 13
l
M)
THP-1 cells30
103
IC50 on HEK293 cells (
lM)
KN62 (positive control)
3
5
0.11 0.02
22a
22b
22c
22d
23a
23b
23c
23d
23e
23f
23g
23h
23i
23j
23k
23l
23m
23n
23o
23p
23q
23r
23s
23t
23u
1Nph
phenyl
3MIn
5-IQ
1Nph
3MIn
5-IQ
5-IQ
5-IQ
5-IQ
5-IQ
H
H
H
H
33
43
71
44
94
68
75
6
3
5
0
4
9
2
44 26
—
105
42 37
65 24
0.79 0.19
Bn
Bn
Bn
p-F-Bn
9
0.31 0.08
1.45 0.18
1.28 0.23
p-Br-Bn
38 14
69 19
42 32
p-CH3-Bn
m-CH3-Bn
1-methyl indole
p-OH-m-CH3-Bn
p-F-Bn
48 12
51 17
1.18 0.28
0.74 0.05
5-IQ
5-IQ
33
84
39
2
4
9
3MIn
3MIn
3MIn
3MIn
3MIn
3MIn
3MIn
3MIn
3MIn
3MIn
3MIn
(S)-3MIn
p-Cl-Bn
p-Br-Bn
94 10
55 10
50 22
38 27
0.25 0.20
0.85 0.36
0.52 0.04
88
87
29
65
9
5
3
9
p-CH3-Bn
p-OCH3-Bn
p-NO2-Bn
m-CH3-Bn
m-F-Bn
p-OH-m-CH3-Bn
1-Methyl indole
1-Methyl naphthyl
p-OH-m-CH3-Bn
41 16
1.42 0.29
46 11
45 14
88
68
4
1
37 18
65 15
0.18 0.04
1.07 0.24
48 10
30
5
a
b
c
Data are expressed as means SDs. All experiments were repeated at least 2–3 times.
Abbreviations: 1Nph, 1-naphthyl; 3Min, 1-methyl-1H-3-indolyl; 5-IQ, 5-isoquinolinyl.
IC50 = 50% inhibitory concentration, representing the mean from dose–response curves of at least three experiments.

J.-Y. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6053–6058
6057
activity from 43% to 94% at 10
l
M, showing an IC50 value of
and (3) the size and position of substituents on the benzyl moiety
at the R3 position had an important effect on the biological activity
of 1-methyl-1H-3-indolyl pyrazolodiazepine derivatives. In partic-
ular, compound 23b, which contains a phenethyl group at R1, a 1-
methyl-1H-3-indolyl group at R2 and a benzyl group at R3, pre-
sented a combination of potent antagonism, for both IL-1b release
and EtBr uptake. These results indicate that modification of the
pyrazolodiazepin-8-one skeleton may be useful in designing com-
pounds for therapeutic intervention in P2X7 receptor related
diseases.
0.31 lM. Therefore, the effect of positional substitutions of benzyl
group at R3 position was assessed by comparing compounds 23j–r
with 23b.
The antagonistic activity of p-F and m-F substituted compounds
23j and 23q was significantly reduced. In the case of methyl substi-
tutions, the compound containing a p-CH3 benzyl group (23m) had
an IC50 of 0.52 lM, whereas the m-CH3 substituted compound
(23p) showed reduced activity. Interestingly, the p-methoxy ana-
log, 23n, showed a significant decrease in activity, with only 29%
inhibition at 10 lM. The activity of the p-nitro substituted com-
pound (23o) was somewhat decreased. Unlike the compound with
fluoro substitution, compounds with p-Cl (23k) and p-Br-benzyl
(23l) groups recovered activity. In particular, the p-Cl-substituted
analog, 23k, showed slightly higher activity than 23b with an
IC50 of 0.25 lM. Notably, the para and meta disubstituted benzyl
moiety (23r) was the most potent antagonist among the series of
Acknowledgements
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (Grant No.
2009-0074289) and by a grant from the institute of Medical Sys-
tem Engineering (iMSE) in the GIST, Korea.
pyrazolodiazepine-8-ones we tested. In the EtBr-uptake inhibition
assay, this compound had an IC50 of 0.18 lM, and there was an
apparent enantiomeric preference for the (R)-isomer of the 1-
methyl-1H-3-indolyl group when compared with the correspond-
ing (S)-isomer, 23u.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.09.053.
As a second assay for functional antagonism, compounds dis-
playing >60% inhibition at 10
say were also investigated for their ability to inhibit IL-1b release
from 1 mM BzATP-activated LPS/IFN -differentiated human THP-
lM in the EtBr uptake inhibition as-
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Figure 2. Concentration-dependent inhibition of BzATP-stimulated IL-1b release in
LPS/IFN
c-differentiated human THP-1 cells by compound 23b and KN-62. Data
points represent means SD of values obtained (n = 3).

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J.-Y. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6053–6058
28. Lee, J.-Y.; Kim, Y.-C. ChemMedChem 2009, 4, 733.
were incubated at 37 °C for 120 min, and cellular accumulation of ethidium+
was determined by measuring fluorescence with a fluorescent plate reader
(excitation filter of 530/20 and emission filter of 590/20).
29. All experiments were performed using adherent HEK293 cells stably
transfected with cDNA encoding the human P2X7 receptor. Synthesized
pyrazolodiazepin-8-one derivatives were added to each well of 96-well plate
(black, clear bottom). hP2X7-Expressing HEK293 cells were then re-suspended
at 2.5 ꢁ 106 cells/mL in HEPES-buffered salt solution that comprised (in mM):
ethidium bromide 0.1, ethylene diamine tetraacetic acid (EDTA) 1, glucose 5,
HEPES 20, and potassium chloride 140 (pH 7.4). The cell suspension was
treated to the wells of 96-well plate followed by addition of BzATP. The plates
30. IL-1b release was measured in differentiated THP-1 cells primed for 3 h with
25 ng/mL LPS and 10 ng/ml IFN
for 30 min. Synthesized pyrazolodiazepin-8-one derivatives at 10
c
, and then was stimulated with 1 mM BzATP
M were
l
treated for 30 min prior to BzATP. Supernatants were collected by
centrifugation at 1000 rpm for 5 min and assayed for the presence of mature
human IL-1b using an ELISA kit.