Structure-activity relationships and optimization of 3,5-dichloropyridine derivatives as novel P2X7 receptor antagonists

Article abstract of DOI:10.1021/jm2012326

Screening of a library of chemical compounds showed that the dichloropyridine-based analogue 9 was a novel P2X₇ receptor antagonist. To optimize its activity, we assessed the structure-activity relationships (SAR) of 9, focusing on the hydrazide linker, the dichloropyridine skeleton, and the hydrophobic acyl (R₂) group. We found that the hydrazide linker and the 3,5-disubstituted chlorides in the pyridine skeleton were critical for P2X₇ antagonistic activity and that the presence of hydrophobic polycycloalkyl groups at the R₂ position optimized antagonistic activity. In the EtBr uptake assay in hP2X₇-expressing HEK293 cells, the optimized antagonists, 51 and 52, had IC₅₀ values of 4.9 and 13 nM, respectively. The antagonistic effects of 51 and 52 were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1β, by LPS/IFN-γ/BzATP stimulation of THP-1 cells (IC₅₀ = 1.3 and 9.2 nM, respectively). In addition, 52 strongly inhibited iNOS/COX-2 expression and NO production in THP-1 cells, further indicating that this compound blocks inflammatory signaling and suggesting that the dichloropyridine analogues may be useful in developing P2X₇ receptor targeted anti-inflammatory agents.

Full text of DOI:10.1021/jm2012326

Article
pubs.acs.org/jmc
Structure−Activity Relationships and Optimization of
3,5-Dichloropyridine Derivatives As Novel P2X7 Receptor
Antagonists
Won-Gil Lee,† So-Deok Lee,† Joong-Heui Cho,† Younghwan Jung,† Jeong-hyun Kim,† Tran T. Hien,‡
Keon-Wook Kang,‡ Hyojin Ko,§ and Yong-Chul Kim*,†,§
†School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
‡College of Pharmacy, Seoul National University, Seoul, Republic of Korea
§Graduate Program of Medical System Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712,
Republic of Korea
S
* Supporting Information
ABSTRACT: Screening of a library of chemical compounds
showed that the dichloropyridine-based analogue 9 was a novel
P2X7 receptor antagonist. To optimize its activity, we assessed the
structure−activity relationships (SAR) of 9, focusing on the
hydrazide linker, the dichloropyridine skeleton, and the hydro-
phobic acyl (R2) group. We found that the hydrazide linker and
the 3,5-disubstituted chlorides in the pyridine skeleton were
critical for P2X7 antagonistic activity and that the presence of
hydrophobic polycycloalkyl groups at the R2 position optimized
antagonistic activity. In the EtBr uptake assay in hP2X7-expressing
HEK293 cells, the optimized antagonists, 51 and 52, had IC50
values of 4.9 and 13 nM, respectively. The antagonistic effects of
51 and 52 were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1β, by LPS/IFN-γ/BzATP
stimulation of THP-1 cells (IC50 = 1.3 and 9.2 nM, respectively). In addition, 52 strongly inhibited iNOS/COX-2 expression and
NO production in THP-1 cells, further indicating that this compound blocks inflammatory signaling and suggesting that the
dichloropyridine analogues may be useful in developing P2X7 receptor targeted anti-inflammatory agents.
cathepsin S, a molecule expressed in spinal microglia that
contributes to the maintenance of persistent pain induced by
CNS or PNS injury.17
INTRODUCTION
■
The P2X7 receptor (P2X7R), a member of the ATP-gated ion
channel family, may be a target for the treatment of diseases
characterized by inflammation and chronic pain.1−4 P2X7Rs are
expressed in a variety of cell types involved in inflammation and
immune system function,5−7 including mast cells, macrophages,
and lymphocytes (T and B).8 Although its expression in
neurons is unclear,9 P2X7R is expressed in microglia and
astrocytes in the central nervous system.10,11 ATP agonism of
P2X7R has been shown to result in rapid changes in intracellular
calcium and potassium concentrations, cell maturation, and the
release of the pro-inflammatory cytokines IL-1β and IL-18 from
macrophages and microglia.12−14 In addition, prolonged
exposure of P2X7R to an agonist induces cytolytic pore forma-
tion in the plasma membrane, allowing the uptake of fluorescent
dyes and EtBr ion, a process that may lead to cell lysis and
death.15,16 Moreover, activation of P2X7R may significantly
decrease the uptake of glutamate in astrocytes via the glial-
specific glutamate/aspartate transporter (GLAST), suggesting
that antagonism of P2X7R may allow GLAST function to
recover and protect neurons from excitotoxicity induced by
traumatic injury.11 P2X7R antagonists may be utilized to treat
patients with neuropathic pain by inhibiting the release of
Recent studies with P2X7R-knockout (KO) mice clearly
indicate that P2X7R is involved in inflammation and chronic
pain.18,19 P2X7R-deficient mice showed a loss of ATP-
dependent immune cell functions, including IL-1β production
and L-selectin shedding, and significant reductions in arthritis
symptoms compared with wild type mice.18 P2X7R-KO mice
also showed reduced sensitivity to inflammation and nerve
insults,3,20 in agreement with the efficacy profile of selective
P2X7R antagonists in models of chronic inflammation and
neuropathic pain.4,21
The localization of P2X7R on immune system cells and its
pathological roles in immune-related conditions have suggested
that this receptor may be a promising target in various disease
states and conditions, including chronic inflammation,22
neurodegeneration,23,24 ischemia,25,26 and chronic pain.27,28
This has led to intensive efforts to identify and develop potent
and selective P2X7R antagonists.22,29−33 Although KN-62
Received: September 15, 2011
Published: March 8, 2012
© 2012 American Chemical Society
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Figure 1. The structures of current P2X7R antagonists.
(1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl)-4-
phenylpiperazine, 1) and its derivatives were found to act as
selective and potent P2X7R antagonists,34,35 1 is poorly soluble in
water36 and has noncompetitive and weak antagonistic activity in
rat P2X7R,37 making 1 unsuitable for use as a drug. AstraZeneca
R&D has reported that adamantane-based small molecules (2)38
and a series of cyclic imide moieties39 act as P2X7R antagonists,
and Abbott Laboratories developed N-aryl carbohydrazide (3),40
triazole (4),41 and cyanoguanidine (5)42 as novel antagonists of
P2X7R, with substantial improvements in chemical stability,
selectivity, and antagonistic activity.43 Moreover, GlaxoSmithK-
line recently described an imidazolidine carboxamide (6),44
which had low in vivo clearance and high oral bioavailability in all
species examined and appreciable efficacy in rat pain models.
Previous studies from our laboratory showed that derivatives of
pyrazolodiazepine29 and 5,6-dihydrodibenzo[a,g]quinolizinium45
could be characterized as novel P2X7R antagonists.45
In an effort to develop new P2X7R antagonists, we assessed
the structure−activity relationship (SAR) of a series of pyridinyl
hydrazide analogues of compound 9 (IC50 = 650 nM, Figure 1),
which had been identified as a new P2X7R antagonist by cell-
based screening of a representative chemical library (available at
Korean Chemical Bank). We also attempted to optimize the
biological properties of compound 9 derivatives, including their
ability to suppress IL-1β production and iNOS/COX-2
induction in BzATP-stimulated THP-1 cells.
synthesized the modified derivatives, 10, 12, and 14 and tested
their antagonistic potency at hP2X7R. The N-alkylated
compounds 10 and 11 were prepared by reductive alkylation
of 8 with phenylpropionaldehyde and formaldehyde, respec-
tively. Acylation of the N-methylated intermediate, 11, with
hydrocinnamoyl chloride yielded compound 12. An amide
derivative, 14, was synthesized by the reaction between
phenylpropionylchloride and 3,5-dichloro-4-aminopyridine for
comparison with the hydrazide linker in the lead compound 9
(Scheme 1). Hydrazinyl intermediates (15a−15e and 18) were
purchased or prepared from the corresponding halogen
substituted compounds using the same procedure used to
prepare compound 8 from 3,5-dichloro-4-iodopyridine (7). We
also made various modifications of the substitutions and the
nitrogen of the pyridine in compound 9. The pyridine analogue
16a, the 3,5-difluoropyridine analogue 16b, and the 2,6-
dichloropyridine analogue 19 were generated by nucleophilic
substitutions of 4-bromo- or 4-iodopyridine derivatives with
hydrazine followed by coupling reactions (Scheme 2). Similarly,
the phenylhydrazide derivatives, 16c−16e, instead of pyridine
skeleton of 9, were prepared similarly with 3,5-dichloro, difluoro,
and dimethyl substituents (Scheme 2). To modify the acyl group
in 9, the carbamate analogue 34 was synthesized by reacting
compound 8 with benzylchloroformate, and the urea analogue
35 was synthesized by reacting benzylamine with the activated
form of 8 after treatment with carbonyldiimidazole.
We also synthesized compounds with various aromatic,
cycloalkyl, and adamantanyl groups by the substitution of the
phenethyl group in compound 9, via coupling reactions with
the hydrazinylpyridine intermediate, 8, thus optimizing hP2X7R
antagonistic potency (Scheme 3).
Biology. BzATP-induced pore formation in hP2X7-express-
ing HEK293 cells is the primary assay used to test the
antagonistic effects of the pyridine-4-hydrazide derivatives at
hP2X7R, with activity measured as ethidium bromide (EtBr)
uptake.46 1 was used as the positive control. The SAR at the
RESULTS AND DISCUSSION
■
Chemistry. The synthetic routes used to prepare compound
9 and its derivatives are shown in three schemes (Schemes 1, 2,
and 3). Compound 9 was synthesized from the conventional
coupling reaction of hydrocinnamic acid and 3,5-dichloro-4-
hydrazinylpyridine (8), which had been synthesized by the
nucleophilic aromatic substitution of 3,5-dichloro-4-iodopyr-
idine (7) by hydrazine (Scheme 1). To explore the SAR of
the phenylpropanehydrazide moiety of compound 9, we
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a
Scheme 1
a
Reagents and conditions: (a) hydrazine hydrate, EtOH, 90 °C, 16 h, 78%; (b) EDC, DIPEA, hydrocinnamic acid, CH2Cl2, RT, 1 h, 88%;
(c) 3-phenylpropionaldehyde, NaBH3CN, 1% AcOH in DCE, RT, 2.5 h, 99%; (d) formaldehyde, NaBH3CN, 1% AcOH in DCE, RT, 2.5 h, 80%.
a
a
Scheme 2
Scheme 3
a
Reagents and conditions: (a) benzyl chloroformate, CH2Cl2, RT, 2 h,
71%; (b) (i) carbonyl diimidazole, TEA, CH2Cl2, RT, 3.5 h, (ii) benzyl
amine, TEA, RT, 1 h, 49%; (c) EDC, DIPEA, acid compound, CH2Cl2
or DMF, RT, 1−4 h, 66−94%. (Each structure of R group can be
referred from Table 3.)
a
Reagents and conditions: (a) hydrazine hydrate, n-BuOH, 90−100 °C,
18 h, 50−60%; (b) EDC, DIPEA, hydrocinnamic acid, CH2Cl2, RT, 1 h,
32−94%.
analogues 10, 12, and 14, in which 9 had been modified with
hydrazine, N′-methyl hydrazide, and amide linkers, respectively.
We found that all three modified analogues were inactive in
hP2X7R antagonism, suggesting that the intact hydrazide linker
hydrazide linker in the structure of lead compound 9 (IC50
650 nM) was analyzed by evaluating the activity of the
=
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is essential and important for the antagonism of compound 9 at
hP2X7R (Table 1) and that this hydrazide moiety may
activity, indicating that dichlorides and their positions in the
pyridine ring are important in the modulation of P2X7R
activity. We also found that the 3,5-difluoride derivatives 16b
and 16d, with relatively smaller halogen isosteres, were totally
inactive in the ethidium uptake assay.
Because the antagonistic activity of the 3,5-dimethylbenzene
derivative 16e was comparable to that of compound 9, certain
sized substituents at the 3 and 5 positions of pyridine, such as
chloro and methyl groups, may have steric effects on the
branched hydrazide moiety, resulting in a conformation
favorable to the efficient antagonism of P2X7R. Although
none of the modified compounds described above had
antagonistic potency greater than that of the lead compound
9, the initial SAR studies of the linker and skeleton confirmed
the importance of the hydrazide linker and positions of the
chlorides in the pyridine ring.
Table 1. P2X7R Antagonistic Activities of 3,5-
Dichloropyridine Derivatives with Modifications of
Hydrazide Linker of the Lead Compound 9
a
compd
X
IC50 (nM)
We attempted to optimize the P2X7R antagonism of 9 by
modifying its phenethyl group (Tables 3 and 4). We therefore
synthesized compounds 20−23, containing fluoride, chloride,
methyl, and methoxy groups, respectively, at the para position,
to test the activities of compounds with different substituents.
We found that the antagonistic activity of these four derivatives
ranged from 0.9 to 1.5 μM (Table 3). When analyzing the
effects of substitutions at 2 positions, we found that the
antagonistic activity of the 2,4-dichloro compound 25 was signifi-
cantly weaker than that of the 4-chloro compound, 24,
suggesting that the presence of multiple substituents on the
phenyl group did not favor antagonism. We also found that the
incorporation of hydrophilic groups, such as phenol (26) and
pyridine (27) moieties, resulted in a loss of antagonistic potency.
These findings indicate that compact and hydrophobic groups
are the preferred pharmacophores at this position.
We also attempted to optimize the spatial distance between
the phenyl ring and the 3,5-dichloropyridine-4-hydrazide in
compound 9 by testing compounds 28−33, with carbon chains
ranging from 0 to 6 in length. We found that the optimum
number of carbons was 2, corresponding to the structure of the
lead compound 9. Interestingly, compound 33 with a six-carbon
chain showed antagonistic potency with an IC50 value of
950 nM, suggesting that more lipophilic groups may be
pharmacophores at that position, in addition to the optimized
phenethyl group. To test the effects of a hydrogen bond
acceptor and donor in place of the α-carbon, we assayed the
activities of the carbamate analogue, 34, and the urea analogue,
35, respectively. Compared with compound 9, compound 34
showed a nearly 2-fold increase in antagonistic potency at
hP2X7R with an IC50 value of 370 nM, whereas 35 did not have
an antagonistic effect. These results suggest that the presence
of an additional hydrogen bond acceptor at the position of the
α-carbon may play a role in the interaction with hP2X7R.
We also synthesized several conformationally constrained
derivatives with restricted free rotation of the phenethyl moiety,
containing unsaturated carbon chains and bicyclic ring moieties.
In general, constrained analogues with extended forms of
aromatic groups, such as the trans-cinnamoyl (36), ( )-tetra-
hydronaphthalene (37), dihydro-1H-indene (38), and 2-indole
(40) derivatives, were inactive or weak P2X7R antagonists.
However, the ( )-benzocyclobutyl derivative 39, possessing a
highly compact structure with a bent form of conformation,
showed a 2-fold increase in potency, with an IC50 value of
320 nM.
1
280 40
9
H
650 170
b
10
12
14
<20%
b
CH3
<20%
b
<20%
a
b
Number of determinations ≥3. Percent inhibition at concentration
10 μM and number of determination ≥3.
participate in crucial interactions such as hydrogen bonding
with P2X7R.
We also tested the activity of compound 9 derivatives in
which the nitrogen of the pyridine ring and dichloride groups
had been modified (Table 2). We found that compound 16c,
Table 2. P2X7R Antagonistic Activities of Derivatives with
Modifications of Dichloropyridine Skeleton of the Lead
Compound 9
a
compd
X
Y
IC50 (nM)
9
N
N
N
C
C
C
Cl
650 170
b
16a
16b
16c
16d
16e
19
H
<20%
b
F
<20%
Cl
F
1400 200
<20%
CH3
900 90
b
<20%
a
b
Number of determinations ≥3. Percent inhibition at concentration
10 μM and number of determination ≥3.
the benzene derivative of 9, had a 2-fold lower antagonistic
activity than 9, with an IC50 value of 1.4 μM. In contrast, the
antagonistic effect of the 3,5-dimethylbenzene derivative 16e
(IC50 = 900 nM) was only slightly lower than that of 9,
demonstrating that the benzene ring is tolerated at the receptor.
In the analysis of the effect of the 3,5-dichloro substituents of 9,
both the unsubstituted (16a) and 2,6-dichloro (19) pyridine
derivatives of 9 showed significantly reduced antagonistic
When we analyzed the effects of π−π interaction using derivatives
with bulkier naphthalene groups (41−43), we found that the
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Table 3. P2X7R Antagonistic Activities of
3,5-Dichloropyridine Derivatives with Modifications of
Phenylpropanehydrazide Moiety of the Lead Compound 9
Table 4. P2X7R Antagonistic Activities of
3,5-Dichloropyridine Derivatives with Cycloalkyl and
Hydrophobic Rings
a
b
Number of determinations ≥3. Percent inhibition at concentration
10 μM and number of determination ≥3.
aromatic group of the ligand and the receptor may not be
essential or a bulky R1 group may attenuate binding to P2X7R.
On the basis of our findings with compound 33, we tested
another approach to optimize the antagonistic potency of these
compounds by using highly lipophilic groups such as cycloalkyl
and adamantanyl at the R1 position (Table 4). Cycloalkyl
derivatives with 5-, 6-, and 7-membered rings (compounds 44−
48) at the R1 position displayed good antagonistic profile when
compared with analogues containing aromatic groups. Several
cycloalkyl derivatives showed potent antagonistic activity
comparable to or better than those of compounds 1 and 9,
with IC50 values ranging from 130 to 380 nM. We found that the
cyclohexyl derivative 45 (IC50 = 130 nM) was the most potent
antagonist among this series of compounds, with a greater than
2-fold increase in antagonistic activity compared with the positive
control, compound 1. Testing of the chain length of this class of
compounds (compounds 45, 47, and 48) yielded results similar
a
b
Number of determinations ≥3. Percent inhibition at concentration
10 μM and number of determination ≥3.
antagonistic effect of the 3-(naphthalen-1-yl)propanehydrazide
derivative 43 was 2-fold lower, whereas the 2-(naphthalen-1-
yl)acetohydrazide derivatives 41 and 42 with shorter carbon
chains were inactive. Thus π−π interactions between the
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to those of the analogues described in Table 3, with a two-carbon
chain being the optimal spacer length between the hydrazide and
ring moieties for antagonism. To investigate the effects of more
hydrophobic moieties at the R1 position, we synthesized and
tested several adamantane, noradamantane, and norbornane
analogues (compounds 49−56). Among this series of poly-
cycloalkyl derivatives, the adamantane-based molecules 49−52
were more potent antagonists at hP2X7R than the other
compounds 53−56. Although the potency of the adamantany-
lacetohydrazide derivative 49 was not significantly higher than
that of compound 9, the adamantanylhydrazide derivative 50,
which was one carbon shorter, showed enhanced antagonistic
potency, with an IC50 value of 77 nM. Further optimization of
this series yielded the 3-bromoadamantanyl (51) and 3,5-
dimethyladamantanyl (52) derivatives, which had IC50 values
of 4.9 and 13 nM, respectively. However, other stereoscopic
derivatives except for 55 displayed very weak antagonistic activity
(53−56).
Excess nitric oxide (NO) produced by iNOS is an important
mediator of acute and chronic inflammation.48 COX-2, which is
induced by LPS or a mixture of several cytokines, is also
involved in the pathology of chronic inflammation.49 To test
whether derivative 52 suppresses the expression of inflamma-
tory proteins, we tested its effect on BzATP-stimulated iNOS
and COX-2 expression in THP-1 cells. Western blot analyses
showed that exposure of THP-1 cells to LPS/IFN-γ slightly
increased cellular levels of iNOS and COX-2 proteins and
that BzATP treatment potentiated the expression of both
(Figure. 2A). However, compound 52, at a concentration of
Among the optimized derivatives, compound 52 was further
evaluated for its selectivity at the mP2X1 and hP2X3 receptors.
Two-electrode voltage clamping (TEVC) experiments were
carried out on Xenopus oocytes with the overexpression of
mP2X1 or hP2X3 receptors by microinjection of corresponding
capped ribonucleic acids (cRNA). As a result, 10 μM 52
showed only 10−20% reduction of the 2 μM ATP-induced ion
current of P2X1 and P2X3 receptors, suggesting high selectivity
of the antagonist for P2X7 receptor vs P2X1 and P2X3
receptors. The detailed data was described in the Supporting
Information (Figure S1).
IL-1β is a major mediator of inflammatory signaling in
activated immune system cells in chronic inflammatory diseases
and is produced in THP-1 cells by LPS and IFN-γ-
stimulation.47 Because the activation of P2X7R is an important
upstream event for IL-1β processing and release following LPS/
IFN-γ stimulation of THP-1 cells, we tested whether the novel
3,5-dichloropyridine-4-hydrazide analogues with potent antag-
onistic activity in the EtBr assay could inhibit the release of
IL-1β by LPS/IFN-γ/BzATP stimulated THP-1 cells, as
determined by ELISA (Table 5). We found that selected
Figure 2. Inhibition of iNOS and COX-2 expression by 52. Effects of
52 on (A) protein expression of iNOS/COX-2 and (B) NO
production. For the differentiation into macrophage phenotype,
THP-1 cells were exposed to 25 ng/mL LPS and 10 ng/mL IFNγ
for 3 h and the differentiated cells were incubated with 52 for 30 min
and then incubated with 1 mM BzATP for an additional 24 h. Total
cell lysates were subjected to immunoblottings for the detection of
iNOS and COX-2. Culture media were used for the detection of
medium nitrite levels. Data represents means SD with 4 different
samples (significant versus LPS/IFN-γ-treated group, *p < 0.05; signi-
ficant versus LPS/IFN-γ + BzATP-treated group, #p < 0.05).
Table 5. Inhibitory Activities of Selected P2X7R Antagonists
against BzATP-Induced IL-1β Release in Differentiated
THP-1 Cells
a
a
IC50 (nM)
IC50 (nM)
compd EtBr assay
compd
EtBr assay
IL-1β
IL-1β
1
280 40
650 170
370 130
320 10
130 60
120 20
46
50
51
52
200 40
77 28
4.9 0.5
42 13
130 20
1.3 0.4
9.2 2.4
9
320 120
b
34
39
45
20%
b
13%
13
1
1 μM, almost completely reduced iNOS and COX-2 protein
expression in response to P2X7R activation (Figure 2A),
showing that 52 efficiently suppresses the de novo synthesis
of the inflammatory proteins, iNOS and COX-2. The previous
literature reported that the activation of P2X7 receptor
enhances interferon-γ (IFNγ)-induced type II nitric oxide
synthase activity in BV-2 microglial cells and the IFNγ-induced
NO production was suppressed by nonselective P2X
antagonists including suramine and oxidized ATP (oxATP).50
Another report suggested that the action of P2X7R receptor was
related with the regulation of downstream transcription factor,
FosB/AP-1, in multiple cell types including the role of
nucleotide-induced COX-2 expression in murine astrocytes.49
56 11
a
b
Number of determinations ≥3. Percent inhibition at concentration
10 μM and number of determination ≥3.
derivatives with IC50 values in submicromolar ranges in the EtBr
assay showed generally parallel order of potency of inhibition in
the assay of IL-1β production, except for 34 and 39. Especially,
the adamantane-based derivatives (50−52) significantly in-
hibited the release of IL-1β, with IC50 values in the range of
1.3−130 nM, while the range of IC50 values in the EtBr assay
was 4.9−77 nM. Thus, the potent antagonists we synthesized
were confirmed as having functional activity in modulating
inflammatory signals mediated by the activation of P2X7R.
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40:1) to give hydrazine intermediate 8 (500 mg, 78%). 1H NMR
(CDCl3, 300 MHz): δ 8.24 (2H, s), 6.10 (1H, s), 4.16 (2H, s). ESI
[M − H] = 177.0.
Therefore, we suggested that compound 52 may attenuate the
production of iNOS/COX-2 in activated THP-1 cells through
P2X7R receptor antagonism and subsequent regulation of
the downstream signaling pathway. To assess whether the
inhibition of iNOS expression by compound 52 reduces NO
production, we further evaluated nitrite concentrations in the
culture media. As a result, the accumulation of nitrite in the
culture media by addition of BzATP to THP-1 cells primed
with LPS/IFN-γ significantly attenuated by 1 μM compound
52 (Figure 2B), confirming that compound 52 efficiently
suppresses the NO production mediated by P2X7R activation
and LPS induction in macrophages.
These results suggest that the novel P2X7R antagonists
developed in this study may be useful as therapeutic agents for
inflammatory disorders related with the upregulation of iNOS/
COX-2 such as NO-mediated inflammatory bowel disease51
(IBD) and others.52,53
General Procedure for the Preparation of Hydrazide Compounds
Using Coupling Reaction. Intermediate 8 (1 equiv) was dissolved in
anhydrous dichloromethane, followed by addition of acidic compound
(1.5 equiv), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydro-
chloride (EDC, 1.5 equiv), and diisopropylethylamine (1.5 equiv).
The reaction mixture was stirred for 30−90 min at room temperature.
The solvent was removed by rotary evaporator. The residue was taken
up in saturated aqueous NaHCO3 solution and extracted with ethyl
acetate (2×). The combined organic later was dried over sodium
sulfate, filtered, and evaporated. The residue was purified by silica gel
column chromatography to give tested compounds. (9, 14, 16a, 16c−
16e, 19−34, 36−56).
N′-(3,5-Dichloropyridin-4-yl)-3-phenylpropanehydrazide (9). Fol-
lowing the general procedure using 8 and hydrocinnamic acid, 9 was
obtained as a white solid (30.8 mg, 88%); mp 149−151 °C. 1H NMR
(CDCl3, 300 MHz): δ 8.28 (2H, s), 7.61 (1H, d, J = 3.3 Hz, N−H),
7.31−6.83 (5H, m), 6.82 (1H, d, J = 3.9 Hz, N−H), 2.97 (2H, t, J =
7.7 Hz), 2.55 (2H, t, J = 7.5 Hz). 13C NMR (CDCl3, 300 MHz): δ
172.9, 154.1, 143.0, 137.1, 136.5, 134.9, 124.6, 120.9, 37.3, 31.2. ESI
[M + H] = 311.9.
3,5-Dichloro-4-(2-(3-phenylpropyl)hydrazinyl)pyridine (10). 3,5-
Dichloro-4-hydrazinylpyridine (8, 40.0 mg, 0.2 mmol) and
3-phenylpropionaldehyde (40.0 μL, 0.3 mmol) were dissolved in
0.1% acetic acid of anhydrous 1,2-dichloroethane (2.0 mL). The
reaction mixture was stirred at room temperature for 30 min. Sodium
cyanoborohydride (18.0 mg, 0.3 mmol) was added in the mixture and
stirred at room temperature for 2 h. Brine (10.0 mL) was added to
quench the reaction, and the mixture was transferred to a separatory
funnel and then extracted with dichloromethane (2×). The combined
organic layer was dried over sodium sulfate, filtered, and evaporated.
The residue was purified by silica gel column chromatography to give
10 as a sticky oil (66.1 mg, 99%). 1H NMR (CDCl3, 400 MHz): δ 8.22
(2H, s), 7.28−7.14 (5H, m), 6.09 (1H, s, N−H), 4.31 (1H, d, J = 4.8
Hz, N−H), 2.83−2.78 (2H, m), 2.66 (2H, t, J = 7.8 Hz), 1.81−1.74
(2H, m). 13C NMR (CDCl3, 400 MHz): δ 154.5, 152.6, 147.3, 132.9,
132.8, 130.2, 122.6, 48.8, 29.0, 24.3. ESI [M + H] = 297.3.
N′-(3,5-Dichloropyridin-4-yl)-N-methyl-3-phenylpropanehydra-
zide (12). 3,5-Dichloro-4-hydrazinylpyridine (8, 50.0 mg, 0.3 mmol)
and formaldehyde (14.0 μL, 0.4 mmol) were dissolved in 1% acetic acid
of anhydrous 1,2-dichloroethane (2.0 mL). The reaction mixture stirred
at room temperature for 30 min. Sodium cyanoborohydride (27.0 mg,
0.4 mmol) was added in the mixture and stirred at room temperature
for 2 h. Brine (10.0 mL) was added to quench the reaction, and the
mixture was transferred to a separatory funnel and then extracted with
dichloromethane (2×). The combined organic layer was dried over
sodium sulfate, filtered, and evaporated. The residue was purified by
silica gel column chromatography to give 11 as a white solid (28.4 mg,
52%). Following the general procedure using the 11 and hydro-
cinnamic acid, 12 was obtained as a white solid (39.2 mg, 80%); mp
113−115 °C. 1H NMR (CDCl3, 400 MHz): δ 8.28 (2H, s), 7.31−7.18
(5H, m), 6.49 (1H, s, N−H), 3.06 (3H, s), 2.98 (2H, t, J = 7.4 Hz),
2.67 (2H, t, J = 7.8 Hz). 13C NMR (CDCl3, 400 MHz): δ 194.0, 153.8,
151.5, 149.7, 135.6, 135.5, 133.0, 123.3, 20.1, 18.9, 14.0. ESI
[M + H] = 325.8.
CONCLUSIONS
■
Conventional screening for P2X7R antagonists using the
ethidium bromide uptake assay led to the identification of the
3,5-dichloropyridine analogue, 9, as the lead compound. To
optimize P2X7R antagonistic potency, we tested the effects of
modifications at the hydrazide, dichloropyridine, and phenyl-
ethyl groups of 9. SAR analysis suggested that (1) the hydrazide
linker was essential, (2) dichloro-substitution at the 3- and
5-positions of the pyridine was favorable for the antagonism of
P2X7R activity, (3) two-carbon chain between the phenyl ring
and 3,5-dichloropyridine-4-hydrazide was the optimal spatial
distance, (4) a hydrogen bond acceptor at the α-carbon (34)
enhanced P2X7R antagonism, and (5) derivatives (50−52) with
compact hydrophobic adamantane groups instead of phenyl
group showed optimum antagonism. The potent antagonists
also showed parallel activities for the inhibition of IL-1β release
from differentiated THP-1 cells. Especially, derivative 52 was
highly selective for hP2X7 receptor vs mP2X1 and hP2X3
receptors and significantly suppressed the expression of
inflammatory proteins such as COX-2 and iNOS in LPS/IFN-
γ/BzATP-stimulated THP-1 cells. These P2X7R functional assays
indicate that 3,5-dichloropyridine-based P2X7R antagonists may
hold promise in the treatment of inflammatory disorders.
EXPERIMENTAL SECTION
Chemistry. Reagents and solvents were purchased from
commercial suppliers and used without further purification. H and
■
1
13C NMR spectra were determined with a JEOL JNM-LA 300WB
spectrometer at 300 MHz or JEOL JNM-ECX 400P spectrometer
at 400 MHz, and spectra were taken in CDCl3 or DMSO-d6. Unless
otherwise noted, chemical shifts are expressed as δ units (ppm)
downfield from internal tetramethylsilane or relative ppm from DMSO
(2.5 ppm) and coupling constants (J) are in Hertz (Hz). Mass
spectroscopy was performed on ESI. Melting points were determined
in open capillary tubes using a Stuart melting point apparatus SMP10.
The purity of compounds was determined by elemental analysis,
carried out with an elemental analyzer (Flash EA 1112series/CE
Instruments). All compounds tested in the biological assay showed
more than 95% purity unless noted.
N-(3,5-Dichloropyridin-4-yl)-3-phenylpropanamide (14). Follow-
ing the general procedure using the 4-amino-3,5-dichloropyridine (13)
and hydrocinnamic acid, 14 was obtained as a light-yellow gel
(21.6 mg, 93%). 1H NMR (CDCl3, 400 MHz): δ 8.63 (2H, S), 7.30−
7.17 (5H, m), 2.98 (2H, t, J = 7.6 Hz), 2.85 (2H, t, J = 7.4 Hz). 13C
NMR (CDCl3, 400 MHz): δ 172.5, 156.3, 148.9, 142.2, 140.0, 132.1,
128.5, 126.3, 39.3, 30.2. ESI [M + H] = 296.0.
3-Phenyl-N′-(pyridin-4-yl)propanehydrazide (16a). Following the
general procedure using the 4-hydrazinypyridine (15a, purchased from
Sigma Aldrich) and hydrocinnamic acid, 16a was obtained as a white
solid (19.8 mg, 33%); mp 186−188 °C. 1H NMR (CDCl3, 400 MHz): δ
7.94 (2H, d, J = 5.7 Hz), 7.66 (1H, s, N−H), 7.41−7.15 (5H, m), 6.83
(2H, d, J = 5.7 Hz), 6.67 (1H, s, N−H), 2.95 (2H, t, J = 7.7 Hz), 2.49
Preparation of 3,5-Dichloro-4-hydrazinylpyridine (8). 3,5-Di-
chlroro 4-iodopyridine (7, 1.0 g, 3.6 mmol) and hydrazine hydrate
(540 μL, 18.0 mmol) were dissolved in ethanol (20 mL). The reaction
mixture was refluxed for 16 h. The reaction solution was dried by rotary
evaporator. The residue was taken up in saturated aqueous NaHCO3
solution and extracted with ethyl acetate (2×). The combined extracts
were dried over sodium sulfate, filtered, and evaporated. The residue
was purified by silica gel column chromatography (CH3Cl:methanol =
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(2H, t, J = 7.8 Hz). 13C NMR (CDCl3, 400 MHz): δ 176.2, 156.1, 151.9,
142.6, 133.5, 130.0, 125.4, 119.8, 36.2, 28.2. ESI [M + H] = 242.1.
N′-(3,5-Difluoropyridin-4-yl)-3-phenylpropanehydrazide (16b).
4-Chloro-3,5-difluoropyridine (300 mg, 2.0 mmol) and hydrazine
hydrate (380 μL, 8.0 mmol) were dissolved in ethanol (20 mL).
The reaction mixture was refluxed for 14 h. The reaction solution was
dried by rotary evaporator. The residue was taken up in saturated
aqueous NaHCO3 solution and extracted with ethyl acetate (2×). The
combined organic layer was dried over sodium sulfate, filtered, and
evaporated. The residue was purified by silica gel column chromatog-
raphy (CH3Cl:methanol = 50:1) to give hydrazine intermediate 15b as a
pink solid (143 mg, 49%). 1H NMR (CDCl3, 300 MHz): δ 8.14 (2H, s),
6.39 (1H, s), 4.21 (2H, s). ESI [M − H] = 144.2. Next, following the
general procedure using the hydrazine intermediate (15b) and
hydrocinnamic acid, 16b was obtained as a sticky oil (26.8 mg, 51%).
1H NMR (CDCl3, 400 MHz): δ 8.15 (2H, s), 7.36 (1H, s, N−H),
7.30−7.16 (5H, m), 6.39 (1H, s, N−H), 2.97 (2H, t, J = 7.6 Hz), 2.53
(2H, t, J = 7.4 Hz). 13C NMR (CDCl3, 300 MHz): δ 180.5, 165.3 (d, J =
1062.4 Hz), 143.0, 135.2, 133.4, 128.2, 124.6, 113.7, 29.8, 25.7. ESI
[M + H] = 278.0.
7.4 Hz), 2.50 (2H, t, J = 7.6 Hz). 13C NMR (CDCl3, 400 MHz): δ
178.2, 168.5, 152.5, 151.3, 139.3, 132.5, 121.9, 116.9, 29.3, 23.7. ESI
[M + H] = 328.9.
3-(4-Chlorophenyl)-N′-(3,5-dichloropyridin-4-yl)-
propanehydrazide (21). Following the general procedure using the 8
and 4-chloroophenylpropionic acid, 21 was obtained as a white solid
1
(28.0 mg, 86%); mp 172−174 °C. H NMR (CDCl3, 400 MHz): δ
8.29 (2H, s), 7.46 (1H, d, J = 4.0 Hz, N−H), 7.22 (2H, d, J = 8.4 Hz),
7.08 (2H, d, J = 8.8 Hz), 6.80 (1H, d, J = 4.0 Hz, N−H), 2.93 (2H, t,
J = 7.4 Hz), 2.50 (2H, t, J = 7.4 Hz). 13C NMR (CDCl3, 400 MHz): δ
171.5, 148.0, 146.9, 138.6, 132.1, 129.6, 128.5, 119.9, 34.7, 30.1. ESI
[M + H] = 346.9.
N′-(3,5-Dichloropyridin-4-yl)-3-p-tolylpropanehydrazide (22).
Following the general procedure using the 8 and 4-toluylpropionic
acid, 22 was obtained as a white solid (22.6 mg, 90%); mp 185−
187 °C. 1H NMR (CDCl3, 400 MHz): δ 8.28 (2H, s), 7.51 (1H, d, J =
3.6 Hz, N−H), 7.10−7.04 (4H, m), 6.82 (1H, d, J = 4.0 Hz, N−H),
2.92 (2H, t, J = 7.8 Hz), 2.51 (2H, t, J = 7.6 Hz), 2.31 (3H, s). 13C
NMR (CDCl3, 400 MHz): δ 176.6, 152.3, 148.3, 146.2, 138.1, 129.3,
128.1, 120.5, 35.5, 30.4, 21.0. ESI [M + H] = 326.0.
N′-(3,5-Dichloropyridin-4-yl)-3-(4-methoxyphenyl)-
propanehydrazide (23). Following the general procedure using the 8
and 4-methoxyphenylpropionic acid, 23 was obtained as a white solid
N′-(2,6-Dichlorophenyl)-3-phenylpropanehydrazide (16c). Fol-
lowing the general procedure using the (2,6-dichlorophenyl)hydrazine
(15c, purchased from Sigma Aldrich) and hydrocinnamic acid, 16c
1
1
(27.9 mg, 90%); mp 148−150 °C. H NMR (CDCl3, 400 MHz): δ
was obtained as a transparent oil (25.1 mg, 83%). H NMR (CDCl3,
8.28 (2H, s), 7.52 (1H, d, J = 4.0 Hz, N−H), 7.07 (2H, d, J = 6.4 Hz),
6.80 (2H, d, J = 6.4 Hz, N−H), 2.90 (2H, t, J = 7.6 Hz), 2.50 (2H, t,
J = 7.6 Hz). 13C NMR (CDCl3, 400 MHz): δ 171.7, 158.5, 148.6,
147.1, 132.4, 129.5, 120.7, 114.3, 55.6, 35.9, 30.3. ESI [M + H] = 342.0.
2-(4-Chlorophenyl)-N′-(3,5-dichloropyridin-4-yl)acetohydrazide
(24). Following the general procedure using the 8 and 4-chlorophenyl-
acetic acid, 24 was obtained as a white solid (30.3 mg, 86%); mp 178−
300 MHz): δ 7.57 (1H, d, J = 4.8 Hz, N−H), 7.29−7.14 (7H, m), 6.91
(1H, t, J = 8.1 Hz), 6.80 (1H, d, J = 5.1 Hz, N−H), 2.95 (2H, t, J = 7.7
Hz), 2.49 (2H, t, J = 7.8 Hz). 13C NMR (CDCl3, 300 MHz): δ 180.0,
147.0, 146.3, 133.8, 133.5, 133.1, 131.0, 130.6, 128.5, 32.1, 27.1. ESI
[M + H] = 311.0.
N′-(2,6-Difluorophenyl)-3-phenylpropanehydrazide (16d). Fol-
lowing the general procedure using the (2,6-fluorophenyl)hydarzine
(15d, purchased from Apolloscientific) and hydrocinnamic acid, 16d
was obtained as a light-pink solid (30.7 mg, 94%); mp 119−121 °C.
1H NMR (CDCl3, 400 MHz): δ 7.59 (1H, d, J = 5.2 Hz, N−H), 7.29−
7.16 (5H, m), 6.91−6.81 (3H, m), 6.41−6.39 (1H, m), 2.96 (2H, t, J =
7.8 Hz), 2.48 (2H, t, J = 7.8 Hz). 13C NMR (CDCl3, 400 MHz): δ
180.5, 161.3, 146.4, 133.4, 133.2, 131.1, 126.6, 115.1, 111.7, 32.3, 27.2.
ESI [M + H] = 278.0. Purity: 92%.
N′-(2,6-Dimethylphenyl)-3-phenylpropanehydrazide (16e). Fol-
lowing the general procedure using the (2,6-dimethylphenyl)hydarzine
(15e, purchased from Apolloscientific) and hydrocinnamic acid, 16e
was obtained as a gel (31.9 mg, 93%); mp 78−80 °C. 1H NMR
(CDCl3, 400 MHz): δ 7.26−7.11 (6H, m), 6.96 (2H, d, J = 7.2 Hz),
6.90−6.86 (1H, m), 6.09 (1H, d, J = 3.6 Hz, N−H), 2.93 (2H, d, J =
7.6 Hz), 2.42 (2H, d, J = 7.6 Hz), 2.31 (3H, s). 13C NMR (CDCl3,
400 MHz): δ 178.0, 149.8, 146.2, 133.6, 133.2, 132.9, 130.8, 127.7,
32.3, 27.1, 13.0. ESI [M + H] = 268.9.
1
180 °C. H NMR (CDCl3, 400 MHz): δ 8.29 (2H, s), 7.63 (1H, bs),
7.33−7.24 (4H, m), 6.79 (1H, s, N−H), 3.59 (2H, s). 13C NMR
(CDCl3, 400 MHz): δ 175.8, 154.3, 146.1, 139.6, 135.3, 132.8, 126.1,
120.7, 36.9. ESI [M + H] = 332.0.
2-(2,4-Dichlorophenyl)-N′-(3,5-dichloropyridin-4-yl)-
acetohydrazide (25). Following the general procedure using the 8
and 2,4-dichlorophenylacetic acid, 25 was obtained as a white solid
1
(20.3 mg, 77%); mp 191−193 °C. H NMR (CDCl3, 400 MHz): δ
8.29 (2H, s), 7.51 (1H, d, J = 4.0 Hz, N−H), 7.37 (1H, d, J = 1.6 Hz),
7.09 (2H, d, J = 1.6 Hz), 6.79 (1H, d, J = 4.0 Hz, N−H), 3.03(2H, d,
J = 7.4 Hz), 2.53 (2H, d, J = 7.4 Hz). 13C NMR (CDCl3, 400 MHz): δ
177.6, 152.6, 151.3, 139.6, 137.6, 136.1, 133.8, 132.1, 129.8, 122.2,
37.5. ESI [M + H] = 366.0.
N′-(3,5-Dichloropyridin-4-yl)-3-(4-hydroxyphenyl)-
propanehydrazide (26). Following the general procedure using the 8
and 4-hydroxyphenylpropionic acid, 26 was obtained as a light-yellow
solid (29.6 mg, 93%); mp 154−156 °C. 1H NMR (CDCl3, 400 MHz,
δ ppm, J in Hz): δ 10.12 (1H, s, O−H), 9.14 (1H, s, N−H), 8.20
(2H, s), 8.14 (1H, s, N−H), 6.95 (2H, d, J = 8.4 Hz), 6.62 (2H, d, J =
8.0 Hz), 2.67 (2H, t, J = 7.8 Hz), 2.35 (2H, t, J = 7.8 Hz). 13C NMR
(CDCl3, 400 MHz): δ 177.8, 161.1, 153.0, 151.6, 134.4, 132.3, 119.2,
117.0, 29.3, 23.8. ESI [M + H] = 328.0.
N′-(2,6-Dichloropyridin-4-yl)-3-phenylpropanehydrazide (19).
2,6-Dichloro-4-iodopyridine (17, 300 mg, 1.1 mmol) and hydrazine
hydrate (175 μL, 5.5 mmol) were dissolved in ethanol (20 mL). The
reaction mixture was refluxed for 16 h. The reaction solution was dried
by rotary evaporator. The residue was taken up in saturated aqueous
NaHCO3 solution and extracted with ethyl acetate (2×). The combined
organic layer was dried over sodium sulfate, filtered, and evaporated.
The residue was purified by silica gel column chromatography
(CH3Cl:methanol = 45:1) to give hydrazine intermediate 18 as
N′-(3,5-Dichloropyridin-4-yl)-2-(pyridin-4-yl)acetohydrazide (27).
Following the general procedure using the 8 and 3-(pyridin-4-
yl)propanoic acid, 27 was obtained as a white solid (27.7 mg, 92%);
1
1
mp 220−222 °C. H NMR (CDCl3, 400 MHz): δ 8.59 (2H, d, J =
a white solid (186 mg, 57%). H NMR (CDCl3, 300 MHz): δ 6.85
5.6 Hz), 8.30 (2H, s), 7.68 (1H, s, N−H), 7.23 (2H, d, J = 6.0 Hz),
6.82 (1H, d, J = 3.2 Hz, N−H), 3.59 (2H, s). 13C NMR (CDCl3,
400 MHz): δ 177.3, 155.8, 154.5, 153.2, 149.6, 129.2, 123.6, 36.0. ESI
[M + H] = 299.0.
(2H, s), 6.50 (1H, s), 4.78 (2H, s). ESI [M − H] = 177.2. Next,
following the general procedure using the hydrazine intermediate 18 and
hydrocinnamic acid, 19 was obtained as a white solid (20.6 mg, 71%);
mp 154−156 °C. 1H NMR (CDCl3, 300 MHz): δ 7.36−7.21 (5H, m),
6.99 (1H, s, N−H), 6.45 (2H, s), 6.34 (1H, s, N−H). 13C NMR
(CDCl3, 300 MHz): δ 177.9, 152.8, 151.2, 143.8, 131.3, 130.9, 129.0,
122.3, 35.7, 29.5. ESI [M + H] = 312.0.
N′-(3,5-Dichloropyridin-4-yl)benzohydrazide (28). Following the
general procedure using the 8 and benzoic acid, 28 was obtained as
1
a white solid (25.4 mg, 90%); mp 160−162 °C. H NMR (CDCl3,
400 MHz): δ 9.00 (1H, s, N−H), 8.27 (2H, S), 7.80 (2H, d, J = 7.2
Hz), 7.54 (1H, t, J = 7.6 Hz), 7.44 (2H, t, J = 7.6 Hz), 7.07 (1H, s,
N−H). 13C NMR (CDCl3, 400 MHz): δ 171.4, 148.2, 147.1, 132.5,
131.3, 128.8, 127.3, 120.4. ESI [M + H] = 283.9. Purity: 92%.
N′-(3,5-Dichloropyridin-4-yl)-2-phenylacetohydrazide (29). Fol-
lowing the general procedure using the 8 and phenylacetic acid, 29 was
N′-(3, 5-Dichloropyridin-4-yl)-3-(4-fluorophenyl)-
propanehydrazide (20). Following the general procedure using the 8
and 4-fluorophenylpropionic acid, 20 was obtained as a white solid
(18.4 mg, 84%); mp 182−184 °C. H NMR (CDCl3, 400 MHz): δ
8.28 (2H, s), 7.58 (1H, d, J = 3.6 Hz, N−H), 7.12−7.09 (2H, m),
6.96−6.92 (2H, m), 6.80 (1H, d, J = 4.0 Hz, N−H), 2.93 (2H, t, J =
1
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obtained as a white solid (21.2 mg, 78%); mp 216−218 °C. 1H NMR
(CDCl3, 400 MHz): δ 8.24 (2H, s), 7.96 (1H, s, N−H), 7.38−7.27
(5H, m), 6.80 (1H, s, N−H), 3.59 (2H, s). 13C NMR (CDCl3, 400
MHz): δ 167.9, 146.3, 145.3, 132.2, 128.4, 127.9, 126.6, 119.3, 43.1.
ESI [M + H] = 298.0.
N′-(3,5-Dichloropyridin-4-yl)-4-phenylbutanehydrazide (30). Fol-
lowing the general procedure using the 8 and phenylbutanoic acid, 30
was obtained as a white solid (29.8 mg, 91%); mp 117−119 °C.
1H NMR (CDCl3, 400 MHz): δ 8.30 (2H, s), 7.51 (1H, d, J = 4.0 Hz,
N−H), 7.30−7.15 (5H, m), 6.81 (1H, d, J = 4.4 Hz, N−H), 2.65
(2H, t, J = 7.4 Hz), 2.22 (2H, t, J = 7.4 Hz), 2.02−1.95 (2H, m). 13C
NMR (CDCl3, 400 MHz): δ 178.6, 152.9, 151.3, 144.8, 131.2, 131.0,
128.6, 122.2, 29.2, 26.6, 19.6. ESI [M + H] = 325.9.
N′-(3,5-Dichloropyridin-4-yl)-5-phenylpentanehydrazide (31).
Following the general procedure using the 8 and phenylpentanoic
acid, 31 was obtained as a white solid (28.2 mg, 81%); mp 118−
120 °C. 1H NMR (CDCl3, 400 MHz): δ 8.30 (2H, s), 7.49 (1H, d, J =
4.0 Hz, N−H), 7.29−7.14 (5H, m), 6.83 (1H, d, J = 3.6 Hz, N−H),
2.61 (2H, t, J = 7.2 Hz), 2.23 (2H, t, J = 7.2 Hz), 1.69−1.62 (4H, m).
13C NMR (CDCl3, 400 MHz): δ 193.7, 164.6, 162.8, 156.6, 141.0,
140.0, 136.9, 130.1, 25.8, 23.1, 20.1, 12.3. ESI [M − H] = 337.7.
N′-(3,5-Dichloropyridin-4-yl)-6-phenylhexanehydrazide (32). Fol-
lowing the general procedure using the 8 and phenylhexanoic acid, 32
was obtained as a white solid (27.6 mg, 71%); mp 128−130 °C.
1H NMR (CDCl3, 400 MHz): δ 8.29 (2H, s), 7.71 (1H, d, J = 2.4 Hz,
N−H), 7.28−7.25 (2H, m), 7.19−7.14 (3H, m), 6.83 (1H, d, J =
3.2 Hz, N−H), 2.58 (2H, t, J = 7.6 Hz), 2.21 (2H, t, J = 7.4 Hz), 1.68−
1.61 (4H, m), 1.38−1.24 (2H, m). 13C NMR (CDCl3, 400 MHz): δ
180.4, 154.3, 152.8, 147.8, 132.5, 129.6, 129.5, 123.7, 31.4, 28.9, 26.4,
23.9, 19.5. ESI [M + H] = 353.3.
(CDCl3, 400 MHz): δ 8.33 (2H, s), 7.71 (1H, d, J = 16.0 Hz), 7.68
(1H, d, J = 4.0 Hz, N−H), 7.53−7.38 (5H, m), 7.01 (1H, d, J =
4.0 Hz, N−H), 6.42 (1H, d, J = 15.6 Hz). 13C NMR (CDCl3,
400 MHz): δ 172.4, 152.2, 151.6, 147.2, 137.5, 133.0, 131.5, 130.5,
121.6, 118.2. ESI [M + H] = 309.0. Purity: 94%.
( )-N′-(3,5-Dichloropyridin-4-yl)-1,2,3,4-tetrahydronaphthalene-
2-carbohydrazide (37). Following the general procedure using the
8 and ( )-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid, 37 was
obtained as a white solid (25.1 mg, 79%); mp 219−211 °C. 1H NMR
(CDCl3, 400 MHz): δ 9.15 (2H, s), 8.48 (1H, d, J = 4.8 Hz, N−H),
7.67−7.56 (4H, m), 7.36 (1H, d, J = 4.8 Hz, N−H), 3.05−2.84
(4H, m), 2.64−2.61 (1H, m), 2.14−1.87 (2H, m). 13C NMR (CDCl3,
400 MHz): δ 181.2, 152.9, 151.4, 138.6, 137.5, 131.8, 131.7, 128.7,
128.5, 122.5, 34.3, 25.7, 21.9, 19.6. ESI [M + H] = 337.9.
N′-(3,5-Dichloropyridin-4-yl)-2,3-dihydro-1H-indene-2-carbohy-
drazide (38). Following the general procedure using the 8 and Indane-
2-carboxylic acid, 38 was obtained as a white solid (19.8 mg, 70%);
1
mp 237−239 °C. H NMR (DMSO-d6, 400 MHz): δ 8.31 (2H, s),
7.68 (1H, d, J = 3.2 Hz, N−H), 7.22−7.16 (4H, m), 6.86 (1H, d, J =
4.0 Hz, N−H), 3.26−3.18 (5H, m). 13C NMR (DMSO-d6, 400 MHz):
δ 181.5, 152.5, 151.6, 145.1, 129.2, 126.5, 121.7, 38.0, 30.6. ESI [M +
H] = 323.9. Purity: 92%.
( )-N′-(3,5-Dichloropyridin-4-yl)-1,2-dihydrocyclobutabenzene-
1-carbohydrazide (39). Following the general procedure using the 8
and ( )-benzocyclobutanoic acid, 39 was obtained as a white solid
1
(18.3 mg, 67%); mp 237−239 °C. H NMR (CDCl3, 400 MHz): δ
8.31 (2H, s), 8.01 (1H, d, J = 4.0 Hz, N−H), 7.32−7.30 (2H, m), 7.22
(1H, t, J = 4.0 Hz), 7.17 (1H, d, J = 4.0 Hz), 6.85 (1H, d, J = 4.0 Hz,
N−H). 4.30−4.28 (1H, m), 3.60 (1H, dd, J = 14.0, 6.4 Hz), 3.397
(1H, dd, J = 14.2, 2.4 Hz). 13C NMR (CDCl3, 400 MHz): δ 178.6,
166.5, 155.9, 154.2, 141.0, 136.4, 135.7, 129.4, 129.4, 122.5, 40.0, 32.0.
ESI [M + H] = 309.3. Purity: 94%.
N′-(3,5-Dichloropyridin-4-yl)-7-phenylheptanehydrazide (33).
Following the general procedure using the 8 and phenylheptanoic
acid, 33 was obtained as a white solid (31.6 mg, 81%); mp 131−
N′-(3,5-Dichloropyridin-4-yl)-1H-indole-2-carbohydrazide (40).
Following the general procedure using the 8 and 2-indolic acid, 40
was obtained as a brown solid (29.3 mg, 94%); mp 273−275 °C.
1H NMR (DMSO-d6, 400 MHz): δ 11.65 (1H, s, N−Hindole), 10.77
(1H, s), 8.52 (1H, s, N−H), 8.24 (2H, s), 7.62 (1H, d, J = 8.4 Hz),
7.40 (1H, d, J = 8.4 Hz), 7.22 (1H, s, N−H), 7.17(1H, t, J = 7.8 Hz),
7.03 (1H, t, J = 7.6 Hz). 13C NMR (DMSO-d6, 400 MHz): δ 166.8,
153.2, 151.6, 140.6, 132.2, 130.0, 126.5, 124.3, 122.3, 119.0, 114.1,
104.5. ESI [M + H] = 322.6. Purity: 94%.
1
133 °C. H NMR (CDCl3, 400 MHz): δ 8.29 (2H, s), 7.77 (1H, s,
N−H), 7.28−7.24 (2H, m), 7.18−7.14 (3H, m), 6.83 (1H, d, J =
2.8 Hz, N−H), 2.57 (2H, t, J = 7.6 Hz), 2.20 (2H, t, J = 7.6 Hz), 1.64−
1.57 (4H, m), 1.33−1.31 (4H, m). 13C NMR (CDCl3, 400 MHz): δ
181.0, 154.8, 153.4, 148.6, 133.1, 132.6, 130.1, 124.2, 32.1, 29.5, 26.8,
24.7, 24.3, 20.2. ESI [M + H] = 367.4.
Benzyl-2-(3,5-dichloropyridin-4-yl)hydrazinecarboxylate (34).
Following the general procedure using the 8 and benzyl chloroformate,
N′-(3,5-Dichloropyridin-4-yl)-2-(naphthalen-1-yl)acetohydrazide
(41). Following the general procedure using the 8 and naphthalen-1-
acetic acid, 41 was obtained as a white solid (28.4 mg, 71%); mp 221−
1
34 was obtained as a yellow oil (26.6 mg, 71%). H NMR (CDCl3,
400 MHz): δ 8.29 (2H, s), 7.71 (1H, s, N−H), 7.36−7.21 (5H, m),
6.80 (1H, s, N−H), 3.58 (2H, s). 13C NMR (CDCl3, 400 MHz): δ
158.7, 155.0, 150.4, 139.4, 133.4, 133.1, 131.5, 121.9, 69.1. ESI [M +
H] = 312.9. Purity: 88%.
1
223 °C. H NMR (CDCl3, 400 MHz): δ 8.47 (1H, s, N−H), 8.11
(2H, s), 7.91−7.76 (3H, m), 7.49−7.37 (4H, m), 6.84 (1H, s, N−H),
3.99 (2H, s). 13C NMR (CDCl3, 400 MHz): δ 179.3, 154.5, 153.5,
139.2, 137.1, 133.6, 133.4, 133.3, 133.1, 130.7, 130.1, 128.0, 123.9,
112.9, 38.1. ESI [M + H] = 347.5.
N-Benzyl-2-(3,5-dichloropyridin-4-yl)hydrazinecarboxamide (35).
3,5-Dichloro-4-hydrazinylpyridine (8, 40.0 mg, 0.2 mmol) was
dissolved in dichloromethane (20 mL), and then carbonyl diimidazole
(36.4 mg, 0.4 mmol) and triethylamine (40.0 μL, 0.4 mmol) were
added. The reaction mixture was stirred at room temperature for 1 h.
The white precipitate was filtered, washed with dichloromethane, and
dried under reduced pressure to give N′-(3,5-dichloropyridin-4-yl)-1H-
imidazole-1-carbohydrazide (46.5 mg, 76%) The compound was
dissolved in dimethylformaimide (2.0 mL), and then benzyl amine
(36.5 μL, 0.3 mmol) and triethylamine (35.0 μL, 0.4 mmol) were
added. The reaction mixture was stirred at room temperature for 2 h.
Then the dimethylformamide was removed by rotary evaporator. The
residue was taken up in water and extracted with ethyl acetate (2×).
The combined organic layer was dried over sodium sulfate, filtered,
and evaporated. The residue was purified by silica gel column
chromatography (CH3Cl:methanol = 20:1) to give 35 as a white solid
N′-(3,5-Dichloropyridin-4-yl)-2-(naphthalen-2-yl)acetohydrazide
(42). Following the general procedure using the 8 and naphthalen-2-
acetic acid, 42 was obtained as a white solid (31.6 mg, 81%); mp 240−
1
242 °C. H NMR (CDCl3, 400 MHz): δ 10.47 (1H, s, N−H), 8.24
(2H, s), 7.86−7.81 (4H, m), 7.47−7.40 (2H + N − H, m), 3.64
(2H, s). 13C NMR (CDCl3, 400 MHz): δ 176.1, 153.1, 151.5, 136.5,
136.4, 135.2, 131.0, 130.8, 130.6, 130.5, 130.4, 129.1, 128.5, 119.3, 35.6.
ESI [M + H] = 347.6. Purity: 90%.
N′-(3,5-Dichloropyridin-4-yl)-3-(naphthalen-1-yl)propanehydrazide
(43). Following the general procedure using the 8 and naphthalen-1-
propionic acid, 43 was obtained as a white solid (36.3 mg, 90%); mp
1
206−208 °C. H NMR (CDCl3, 400 MHz): δ 8.39 (1H, s, N−H),
8.21 (2H, s), 7.99−7.96 (1H, m), 7.85−7.83 (1H, m), 7.71 (1H, d, J =
8.4 Hz), 7.48−7.45 (2H, m), 7.34 (1H, t, J = 7.6 Hz), 7.27 (1H, d, J =
6.8 Hz), 6.82 (1H, s, N−H), 3.39 (2H, t, J = 7.8 Hz), 2.62 (2H, t, J =
7.6 Hz). 13C NMR (CDCl3, 400 MHz): δ 178.4, 152.6, 139.6, 137.1,
134.4, 131.6, 129.9, 128.6, 128.3, 128.1, 128.0, 126.9, 125.4, 122.0,
28.6, 23.4. ESI [M + H] = 361.1.
1
(34.5 mg, 65%); mp 215−217 °C. H NMR (CDCl3, 400 MHz): δ
8.18 (2H, s), 7.39 (1H, s, N−H), 7.28−7.18 (5H, m), 6.97 (1H, s,
N−H), 6.10 (1H, t, J = 5.0 Hz, N−H), 4.37 (2H, d, J = 4.8 Hz).
13C NMR (CDCl3, 400 MHz): δ 166.2, 154.7, 152.9, 144.2, 133.2,
132.1, 132.0, 123.4, 40.8. ESI [M + H] = 312.9.
3-Cyclopentyl-N′-(3,5-dichloropyridin-4-yl)propanehydrazide
(44). Following the general procedure using the 8 and cyclo-
pentylpropionic acid, 44 was obtained as a white solid (23.0 mg,
93%); mp 181−183 °C. 1H NMR (CDCl3, 400 MHz): δ 8.29 (2H, s),
N′-(3,5-Dichloropyridin-4-yl)cinnamohydrazide (36). Following
the general procedure using the 8 and trans-cinnamoic acid, 36 was
obtained as a white solid (23.7 mg, 78%); mp 184−186 °C. 1H NMR
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7.69 (1H, d, J = 3.6 Hz, N−H), 6.83 (1H, d, J = 3.6 Hz, N−H),
2.23 (2H, t, J = 7.6 Hz), 1.76−1.48 (11H, m). 13C NMR (CDCl3,
400 MHz): δ 181.1, 154.8, 153.4, 124.2, 36.1, 28.9, 28.4, 27.0, 20.4.
ESI [M + H] = 303.6.
boxylic acid, 53 was obtained as a white solid (24.0 mg, 71%); mp
198−200 °C. 1H NMR (CDCl3, 400 MHz): δ 8.30 (2H, s), 7.74 (1H,
d, J = 4.0 Hz, N−H), 6.85 (1H, d, J = 3.6 Hz, N−H), 2.70 (1H, t,
J = 6.6 Hz), 2.33 (2H, s), 2.01−1.99 (2H, m), 1.48−1.66 (4H, m),
1.64−1.59 (4H, m). 13C NMR (CDCl3, 400 MHz): δ 185.1, 154.6,
153.6, 124.4, 51.5, 44.2, 43.8, 41.1, 40.5, 33.7, 33.6, 30.6. ESI [M + H]
= 328.0. Purity: 92%.
3-Cyclohexyl-N′-(3,5-dichloropyridin-4-yl)propanehydrazide (45).
Following the general procedure using the 8 and cyclohexylpropionic
acid, 45 was obtained as a white solid (23.7 mg, 90%); mp 198−
200 °C. 1H NMR (CDCl3, 400 MHz): δ 8.30 (2H, s), 7.55 (1H, d, J =
3.6 Hz, N−H), 6.84 (1H, d, J = 3.6 Hz, N−H), 2.23 (2H, t, J =
7.8 Hz), 1.68−1.62 (5H, m), 1.52 (2H, q, J = 7.6 Hz), 1.23−1.12 (4H,
m), 0.90−0.81 (2H, m). 13C NMR (CDCl3, 400 MHz): δ 172.5, 148.3,
147.0, 122.8, 37.1, 32.9, 32.3, 31.0, 26.4, 26.1. ESI [M + H] = 318.0.
2-Cyclohexyl-N′-(3,5-dichloropyridin-4-yl)acetohydrazide (46).
Following the general procedure using the 8 and cycloheptylacetic
acid, 46 was obtained as a white solid (18.1 mg, 78%); mp 193−
N′-(3,5-Dichloropyridin-4-yl)bicyclo[2.2.1]heptane-2-carbohydra-
zide (54). Following the general procedure using the 8 and
2-norbornylcarboxylic acid, 54 was obtained as a white solid (26.1 mg,
1
84%); mp 180−182 °C. H NMR (CDCl3, 400 MHz): δ 8.31 (2H, s),
7.60 (1H, d, J = 3.6 Hz, N−H), 6.84 (1H, d, J = 4.0 Hz, N−H), 2.68−
2.64 (1H, m), 2.48−2.26 (3H, m), 1.65−1.27 (7H, m). 13C NMR
(CDCl3, 400 MHz): δ 182.5, 154.8, 153.6, 124.5, 41.9, 37.7, 37.3, 33.3,
27.1, 24.8, 19.6. ESI [M + H] = 301.7.
1
195 °C. H NMR (CDCl3, 400 MHz): δ 8.30 (2H, s), 7.60 (1H, s,
2-(Bicyclo[2.2.1]heptan-2-yl)-N′-(3,5-dichloropyridin-4-yl)-
acetohydrazide (55). Following the general procedure using the 8 and
2-norbornylacetic acid, 55 was obtained as a white solid (29.9 mg,
80%); mp 173−175 °C. 1H NMR (CDCl3, 400 MHz): δ 8.30 (2H, s),
7.55 (1H, d, J = 3.6 Hz, N−H), 6.86 (1H, d, J = 4.0 Hz, N−H), 2.13
(2H, d, J = 7.6 Hz), 2.04−1.99 (1H, m), 1.72−1.40 (8H, m), 1.24−
1.09 (2H, m). 13C NMR (CDCl3, 400 MHz): δ 179.5, 154.8, 149.6,
127.4, 52.6, 48.6, 39.4, 28.7, 27.4, 19.5, 18.4. ESI [M + H] = 315.7.
N′-(3,5-Dichloropyridin-4-yl)bicyclo[2.2.1]hept-5-ene-2-carbohy-
drazide (56). Following the general procedure using the 8 and
5-norbornenyl-2-carboxylic acid, 56 was obtained as a white solid
N−H), 6.85 (1H, d, J = 2.8 Hz, N−H), 2.08 (2H, d, J = 6.8 Hz),
1.83−1.61 (5H, m), 1.28−1.00 (4H, m), 0.98−0.88 (2H, m).
13C NMR (CDCl3, 400 MHz): δ 180.4, 155.0, 153.6, 124.6, 38.4,
31.2, 29.3, 21.6, 21.4. ESI [M + H] = 303.8.
2-Cycloheptyl-N′-(3,5-dichloropyridin-4-yl)acetohydrazide (47).
Following the general procedure using the 8 and cyclohexylacetic
acid, 47 was obtained as a white solid (18.2 mg, 79%); mp 181−
183 °C. 1H NMR (CDCl3, 400 MHz): δ 8.29 (2H, s), 7.59 (1H, d, J =
3.6 Hz, N−H), 6.85 (1H, d, J = 3.6 Hz, N−H), 2.21−1.89 (6H, m),
1.54−1.47 (3H, m), 1.28−1.05 (6H, m). 13C NMR (CDCl3, 400
MHz): δ 189.6, 160.6, 158.9, 125.6, 26.5, 25.9, 23.0, 21.0, 19.1. ESI
[M + H] = 317.5.
4-Cyclohexyl-N′-(3,5-dichloropyridin-4-yl)butanehydrazide (48).
Following the general procedure using the 8 and cyclohexylbutanoic
acid, 48 was obtained as a white solid (17.9 mg, 77%); mp 159−
161 °C. 1H NMR (CDCl3, 400 MHz): δ 8.29 (2H, s), 7.58 (1H, d, J =
3.2 Hz, N−H), 6.83 (1H, d, J = 4.0 Hz, N−H), 2.19 (2H, t, J =
7.6 Hz), 1.81−1.55 (9H, m), 1.32−1.07 (4H, m), 0.98−0.78 (2H, m).
13C NMR (CDCl3, 400 MHz): δ 179.1, 152.9, 151.5, 122.4, 31.9, 31.4,
28.1, 27.3, 20.1, 19.8, 15.5. ESI [M + H] = 312.9.
N′-(3,5-Dichloropyridin-4-yl)-2-adamantylacetohydrazide (49).
Following the general procedure using the 8 and 2-adamantanylacetic
acid, 49 was obtained as a white solid (30.7 mg, 90%); mp 195−
197 °C. 1H NMR (CDCl3, 400 MHz): δ 8.29 (2H, s), 7.52 (1H, d, J =
4.0 Hz, N−H), 6.90 (1H, d, J = 4.0 Hz, N−H), 1.90−1.86 (5H, m),
1.69−1.59 (12H, m). 13C NMR (CDCl3, 400 MHz): δ 176.8, 152.8,
151.5, 122.5, 43.6, 37.3, 30.9, 27.0, 22.1. ESI [M + H] = 355.9.
N′-(3,5-Dichloropyridin-4-yl)-2-adamantyhydrazide (50). Follow-
ing the general procedure using the 8 and 2-adamantanylcarboxylic
acid, 50 was obtained as a white solid (27.6 mg, 90%); mp 207−
209 °C. 1H NMR (CDCl3, 400 MHz): δ 8.30 (2H, s), 7.82 (1H, d, J =
4.0 Hz, N−H), 6.83 (1H, d, J = 4.0 Hz, N−H), 2.05−2.01 (3H, m),
1.93−1.88 (6H, m), 1.77−1.68 (6H, m). 13C NMR (CDCl3,
400 MHz): δ 183.5, 152.8, 151.9, 122.6, 35.2, 33.4, 30.8, 30.6, 21.4.
ESI [M + H] = 341.9.
1
(20.1 mg, 69%); mp 186−188 °C. H NMR (CDCl3, 400 MHz): δ
8.29 (2H, s), 7.57 (1H, d, J = 4.0 Hz, N−H), 6.80 (1H, d, J = 4.0 Hz,
N−H), 6.22 (1H, q, J = 3.0 Hz), 5.93 (1H, q, J = 3.0 Hz), 3.18 (1H, s),
2.94−2.88 (2H, m), 1.98−1.91 (1H, m), 1.58−1.24 (3H, m). 13C
NMR (CDCl3, 400 MHz): δ 181.9, 154.5, 153.3, 143.4, 136.5, 124.2,
49.5, 47.2, 43.0, 39.2, 24.8. ESI [M + H] = 299.7.
Biological Methods. Measurement of Ethidium Bromide
Accumulation in hP2X7-Expressing HEK 293 Cells. hP2X7-exp-
ressing HEK 293 cells were resuspended at 2.5 × 106 cells/mL
in assay buffer composed of 10 mM HEPES, 5 mM N-methyl-
D
-glutamine, 5.6 mM KCl, 10 mM D-glucose, and 0.5 mM
CaCl2 (pH 7.4) supplemented with either 280 mM sucrose or
140 mM NaCl, and an 80 μL aliquot was added to each well of
a 96-well plates. To each was added test compounds or 1-[N,O-
bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpi-
perazine (1) as the positive control, followed by the hP2X7R
agonist benzoylbenzoyl ATP (BzATP). The plates were
incubated at 37 °C for 2 h, and the cellular accumulation of
ethidium ion was determined by measuring fluorescence with a
Bio-Tek FL600 fluorescent plate reader (excitation wavelength
530 nm; emission wavelength 590 nm). The IC50 value of each
compound was measured and compared with that of 9.
Enzyme-Linked Immunosorbent Assay (ELISA) of Human IL-1β in
Differentiated THP-1 Cells. Human monocytic leukemia THP-1 cells
were plated at 2 × 105 cells/well of 96-well plates. To differentiate
THP-1 cells into macrophages, the cells were incubated with 25 ng/mL
LPS and 10 ng/mL IFN-γ for 3 h. The differentiated cells were
incubated at 37 °C with KN-62 (1) or test compounds for 30 min,
followed by 1 mM BzATP for an additional 30 min. The culture
supernatants were collected by centrifugation at ∼250g for 5 min and
stored as aliquots at −70 °C. IL-1β concentrations were measured by
ELISA, using antihuman IL-1β antibody as the capture antibody and a
biotinylated antihuman IL-1β antibody for detection (eBioscience).
The concentration of IL-1β in each sample was compared with that of
a standard curve of recombinant human IL-1β, with the basal level of
IL-1β subtracted from all measurements.
N′-(3,5-Dichloropyridin-4-yl)-3-bromoadamantylhydrazide (51).
Following the general procedure using the 8 and 3-bromoadamantyl-
carboxylic acid, 51 was obtained as a white solid (28.3 mg, 79%); mp
1
214−216 °C. H NMR (CDCl3, 400 MHz): δ 8.31 (2H, s), 7.78
(1H, d, J = 4.0 Hz, N−H), 6.79 (1H, d, J = 4.0 Hz, N−H), 2.46 (2H,
s), 2.36−2.24 (6H, m), 1.73−1.55 (6H, m). 13C NMR (CDCl3,
400 MHz): δ 183.7, 155.7, 154.3, 125.4, 61.9, 47.9, 46.0, 34.3, 31.2,
28.2. ESI [M + H] = 419.9.
N′-(3,5-Dichloropyridin-4-yl)-3,5-dimethyladamantylhydrazide
(52). Following the general procedure using the 8 and 3,5-
dimethyladamantylcarboxylic acid, 52 was obtained as a white solid
1
(31.6 mg 93%); mp 162−164 °C. H NMR (CDCl3, 400 MHz): δ
Measurement of iNOS and COX-2 Expression by Western
Blotting. Treated cells were collected, washed with cold phosphate-
buffered saline (PBS), lysed on ice for 30 min in 100 μL lysis buffer
[120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP40 (Nonidet P-40)],
and centrifuged at ∼10000g for 30 min at 4 °C. The supernatants were
withdrawn and their protein concentrations were determined using
a BCA protein assay kit (Pierce, Rockford, IL). Aliquots of the lysates,
containing 30 μg of protein, were boiled for 5 min and electrophoresed
8.29 (2H, s), 7.82 (1H, d, J = 4.0 Hz, N−H), 6.79 (1H, d, J = 4.4 Hz,
N−H), 2.14−2.13 (1H, m), 1.71 (2H, d, J = 2.8 Hz), 1.51 (4H, q, J =
12.6 Hz), 1.51 (4H, q, J = 10.6 Hz), 1.36−1.15 (2H, m), 0.85 (6H, s).
13C NMR (CDCl3, 400 MHz): δ 183.3, 152.8, 151.8, 122.7, 46.1, 40.1,
37.5, 37.2, 32.0, 24.8, 24.1, 22.7. ESI [M + H] = 369.8.
N′-(3,5-Dichloropyridin-4-yl)-1-noradamantylhydrazide (53).
Following the general procedure using the 8 and 1-noradamantylcar-
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on 10% SDS-polyacrylamide gels. The proteins were transferred onto
nitrocellulose membranes, which were incubated with primary
antibodies against iNOS or COX-2 or mouse monoclonal anti-β-actin
antibodies, followed by incubation with secondary antimouse or
antirabbit antibodies. Protein bands were detected using an enhanced
chemiluminescence Western blotting detection kit (Pierce Biotechnol-
ogy, Rockford, IL).
Measurement of Nitrite Production in Differentiated THP-1 Cells.
THP-1 (2 × 105 cells/mL) prepared as above were cultured in 48-well
plates for 24 h, and nitrite concentrations in the culture media were
measured by incubating each with Griess reagent (1% sulfanilamide,
0.1% N-1-naphthylenediamine dihydrochloride, and 2.5% phosphoric
acid) for 10 min and measuring absorbance at 540 nm.
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(21) Taylor, S. R. J.; Gonzalez-Begne, M.; Sojka, D. K.; Richardson, J.
C.; Sheardown, S. A.; Harrison, S. M.; Pusey, C. D.; Tam, F. W. K.;
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Purinergic Signalling 2009, 5 (1), 63−73.
ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical data of element analysis of final compounds for
purity. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +82-62-715-2502. Fax: +82-62-715-2484. E-mail:
yongchul@gist.ac.kr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by a Basic Science Research
Program of the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(grant no. 2010-0013872) and by “Studies on the Neural
Specific Functions and Regulation of Orphan GPCRs”,
Gwangju Institute of Science and Technology (GIST), Korea.
ABBREVIATIONS USED
■
ATP, adenosine 5′-triphosphate; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; BCA, bicinchoninic acid; ELISA,
enzyme-linked immunosorbent assay; SDS, sodium dodecyl
sulfate; IC50, half-maximal inhibitory concentration; IL,
interleukin; LPS, lipopolysaccharide; IFN, interferon; COX,
cyclooxygenase; NOS, nitric oxide synthase; AP-1, activator
protein 1; ESI, electrospray ionization; NMR, nuclear magnetic
resonance
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