Synthesis and structure-activity relationships of carboxylic acid derivatives of pyridoxal as P2X receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2013.01.073

Carboxylic acid derivatives of pyridoxal were developed as potent P2X ₁ and P2X₃ receptor antagonists with modifications of a lead compound, pyridoxal-5′-phosphate-6-azophenyl-2′,5′- disulfonate (5b, iso-PPADS). The designing strategies included the modifications of aldehyde, phosphate or sulfonate groups of 5b, which may be interacted with lysine residues of the receptor binding pocket, to weak anionic carboxylic acid groups. The corresponding carboxylic acid analogs of pyridoxal-5′- phosphate (1), 13 and 14, showed parallel antagonistic potencies. Also, most of 6-azophenyl derivatives (24-28) of compound 13 or 14 showed potent antagonistic activities similar to that of 5b at human P2X₃ receptors with 100 nM range of IC₅₀ values in two-electrode voltage clamp (TEVC) assay system on the Xenopus oocyte. The results indicated that aldehyde and phosphoric or sulfonic acids in 5b could be changed to a carboxylic acid without affecting antagonistic potency at mouse P2X₁ and human P2X₃ receptors.

Full text of DOI:10.1016/j.bmc.2013.01.073

Bioorganic & Medicinal Chemistry 21 (2013) 2643–2650
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of carboxylic acid
derivatives of pyridoxal as P2X receptor antagonists
Kwan-Young Jung a, , Joong-Heui Cho a, , Jung Sun Lee a, Hyo Jun Kim a, Yong-Chul Kim a,b,
⇑
a School of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea
b Department of Medical System Engineering (DMSE), Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Carboxylic acid derivatives of pyridoxal were developed as potent P2X1 and P2X3 receptor antagonists
with modifications of a lead compound, pyridoxal-50-phosphate-6-azophenyl-20,50-disulfonate (5b, iso-
PPADS). The designing strategies included the modifications of aldehyde, phosphate or sulfonate groups
of 5b, which may be interacted with lysine residues of the receptor binding pocket, to weak anionic car-
boxylic acid groups. The corresponding carboxylic acid analogs of pyridoxal-50-phosphate (1), 13 and 14,
showed parallel antagonistic potencies. Also, most of 6-azophenyl derivatives (24–28) of compound 13 or
14 showed potent antagonistic activities similar to that of 5b at human P2X3 receptors with 100 nM
range of IC50 values in two-electrode voltage clamp (TEVC) assay system on the Xenopus oocyte. The
results indicated that aldehyde and phosphoric or sulfonic acids in 5b could be changed to a carboxylic
acid without affecting antagonistic potency at mouse P2X1 and human P2X3 receptors.
Received 11 December 2012
Revised 30 January 2013
Accepted 31 January 2013
Available online 22 February 2013
Keywords:
P2X1 receptor
P2X3 receptor
Two-electrode voltage clamp
Pyridine carboxylic acids
Structure–activity relationships
Antagonists
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The gene encoding P2X1 receptor was originally cloned from rat
vas deferens,14 and its protein was shown to be distributed within
The distinct activities of extracellular adenosine 50-triphosphate
(ATP), including the transmissions of pain and inflammatory signal-
ing, are mediated through its activation of P2 receptors in various
cells and organs, including the nervous, muscular, cardiovascular,
renal and immune systems.1,2 P2 nucleotide receptors have been
classified into two families:3 P2X receptors, which are ligand-gated
cation channels; and P2Y receptors, which are G-protein coupled
receptors. Activation of neuronal P2Y receptors4 regulates both out-
wardly and inwardly rectifying K+ channels, potentiates high-volt-
age activated Ca2+ channels, and triggers InsP3-mediated release
of Ca2+ from stores in the endoplasmic reticulum.5 In contrast,
P2X receptors as ion channels play significant roles in the functions
of the peripheral and the central nervous system by impulse
generation and synaptic modulation in primary afferents.6,7 In past
decades, genes encoding seven human P2X receptor subtypes
(P2X1–7) have been cloned.8–14 Their encoded proteins are 30–50%
identical and range in length from 379–595 amino acids. P2X
receptor subunits have a similar structural topology, consisting of
two transmembrane spanning domains (TM1 and TM2) connected
by a long extracellular loop containing the ATP binding site, and
intracellular N and C termini of various lengths.15–19
the smooth muscles that line a variety of hollow organs, including
the urinary bladder, intestines, arteries, and vas deferens.20–23
These channels mediate the purinergic component of sympathetic
and parasympathetic nerve-mediated smooth muscle contraction
in a variety of tissues including the urinary bladder,24,25 vas defer-
ens,22,26 saphenous vein,27 and renal microvasculature.28 In a
mouse model lacking the gene encoding P2X1 receptor protein,
nerve-mediated vasoconstriction and contraction of the urinary
bladder and vas deferens were reduced by 50–70%.22,24,29,30
Homomeric P2X3 and heteromeric P2X2/3 receptors have been
recognized as playing a major role in mediating the primary sensory
effects of ATP.31,32 These receptors are predominantly located on
small-to-medium diameter C-fibers and Ad sensory neurons within
the dorsal root, trigeminal, and nodose sensory ganglia.33,34 Electro-
physiological studies on sensory neurons from P2X2 and P2X3 defi-
cient mice have confirmed that P2X3 and P2X2/3 receptors account
for nearly all responses to ATP in DRG sensory neurons,35,36 while
P2X2 and P2X2/3 receptors are predominant in nodose sensory
neurons.37,38 P2X3 and P2X2/3 receptors are present on both the
peripheral and central terminals of primary sensory afferents pro-
jecting to a number of somatosensory and visceral organs including
the skin, joints, bones, lungs, urinary bladder, ureter, and gastroin-
testinal tract.39 Recent studies have demonstrated that epithelial
tissues, including the bladder uroepithelium, airway epithelial cells,
and pulmonary neuroepithelial bodies, express P2X3 and P2X2/3
⇑
Corresponding author. Tel.: +82 62 715 2502; fax: +82 62 715 2484.
E-mail address: yongchul@gist.ac.kr (Y.-C. Kim).
These authors equally contributed to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.073

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K.-Y. Jung et al. / Bioorg. Med. Chem. 21 (2013) 2643–2650
Figure 1. Structures of P2X3 receptor antagonists (1–4) and pyridoxal-5-phosphate class P2X receptor antagonists (5a–5c).
Scheme 1. Synthesis of the pyridoxic-b5-carboxylic acid. Reagents and conditions: (a) 2,2-dimethoxypropane, p-TsOH, acetone, 65.7%; (b) pyridinium dichromate, CH2Cl2,
67.5%; (c) triethylphosphoacetate, NaH, THF, 81.3%; (d) 10% formic acid in water, reflux, 3 h, 97.8%; (e) Pd/C, H2, MeOH, 89.2%; (f) MnO2, CH2Cl2, 71.1%; (g) 5% KOH, MeOH,
53.9%; (h) OXONE, 0.2 M DMF/CH3CN, 47.7%.
channels, which may modulate certain mechanosensory and
chemosensory responses.40,41 The therapeutic effects of blocking
P2X receptors have been studied using potent and selective antag-
onists, such as TNP-ATP (1) at P2X1, P2X3, and P2X2/3 receptors,42 A-
317491 (2) at P2X3 and P2X2/3 receptors,43 Spinorphin (3) at P2X3
receptors,44 and RO-85 (4) at P2X3, and P2X2/3 receptors (Fig. 1).45
These studies showed that peripheral and spinal P2X3 and P2X2/3
receptors are involved in transmitting persistent, chronic inflam-
matory and neuropathic pain signals. Another series of P2 receptor
antagonists, pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid
(PPADS), and its derivatives (5a–c), were reported to be potent
antagonists at P2X1,3 receptors with IC50 values in the nanomolar
range and selectivity over P2Y1 receptors.46,47
evaluated using a two-electrode voltage clamp (TEVC) assay sys-
tem on Xenopus oocytes expressing cloned mP2X1 and hP2X3
receptors.
2. Results and discussion
2.1. Chemistry
A series of carboxylic acid derivatives of pyridoxal and pyridoxic
acid derivatives of 5b was synthesized using the routes shown in
Schemes 1–3. Pyridoxine 6 was treated with 2,2-dimethoxypro-
pane in the presence of p-toluenesulfonic acid in acetone, followed
by the oxidation of the 3-hydroxyl group with pyridinium dichro-
mate in dichloromethane to yield the 5-aldehyde derivative 8. A
Wittig–Horner reaction of 8 with triethylphosphoacetate produced
compound 9 and subsequent deprotection of the acetonide with
Herein, we report the development of novel antagonists for
mP2X1 and hP2X3 receptors by modification of the aldehyde, phos-
phate or sulfonate group of 5b, which may interact with lysine res-
idues in the ATP binding pocket of the receptors, to a weak anionic
carboxylic acid group. The antagonistic effect of these analogs was
10% formic acid yielded compound 10. After reduction of the
a,b-
unsaturated double bond of compound 10 by catalytic hydrogena-

K.-Y. Jung et al. / Bioorg. Med. Chem. 21 (2013) 2643–2650
2645
Scheme 2. Synthesis of the pyridoxic-b5-acrylic acid. Reagents and conditions: (a) 5% KOH in MeOH, rt, 3 h, 74.5%; (b) 10% formic acid in water, reflux, 3 h, 60.5%; (c) MnO2,
0.3 M THF/Water, 52.4%; (d) OXONE, 0.2 M DMF/ACN, 71.3%.
Scheme 3. Synthesis of the 5-((benzyl(2-carboxyetyl)amino)methyl pyridoxic acid. Reagents and conditions: (a) benzylamine, NaBH3CN, 1% AcOH in DCM, 50.7%; (b) ethyl
acrylate, EtOH, reflux, 24 h, 34.6%; (c) 10% formic acid in water, reflux, 3 h; (d) 5% KOH in MeOH, rt, 3 h, 46.9%; (e) MnO2, 0.3 M THF/Water, 34.5%; (f) OXONE, 0.2 M DMF/ACN,
42.6%.
tion, the 4-hydroxymethyl group was selectively converted to an
aldehyde using manganese dioxide. The ester group of 12 was
hydrolyzed to produce the corresponding acid 13, which was, in
turn, converted to the 4-carboxylic acid analog, 14 by oxidation
with OXONE (Scheme 1).
produce compound 21. Using oxidation reactions similar to those
in Scheme 1 yielded the, 4-aldehyde analog, 22 and the 4-carbox-
ylic acid analog 23. Compounds 13 and 14 were transformed to
compounds 24–29 by diazo coupling reactions with various aniline
derivatives substituted with sulfonic or carboxylic acids
(Scheme 4). However, the diazo coupling reactions with 17, 18,
22 and 23 failed to yield the 6-substituted derivatives.
Compounds 17 and 18, the
a,b-unsaturated derivatives of 13
and 14, respectively, were synthesized from compound 9 as shown
in Scheme 2. Compound 23 was synthesized using a 6-step proce-
dure (Scheme 3). The aldehyde group of compound 8 was reacted
with benzylamine under conditions of reductive amination in the
presence of NaBH3CN to produce 19. A Michael type 1,4-addition
reaction of the secondary amine of compound 19 with ethyl acry-
late yielded compound 20, followed by deprotection reactions with
10% formic acid for the acetonide and 5% KOH for the ethyl ester to
2.2. Biological evaluation
The antagonistic activity of the synthesized compounds at the
mP2X1 and hP2X3 receptors was evaluated by performing
two-electrode voltage clamp (TEVC) assays on Xenopus oocytes
expressing cloned mP2X1 and hP2X3 receptors.48 These
Scheme 4. General synthesis of pyridoxine-6-azophenyl-b5-carboxylic acid derivatives by azo coupling reactions.

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K.-Y. Jung et al. / Bioorg. Med. Chem. 21 (2013) 2643–2650
Table 1
Structures and antagonistic effects of pyridoxal carboxylic acid derivatives at mP2X1 and hP2X3 receptors
Compound
Position
% Inhibition
a
a
20
30
40
50
X
Y
mP2X1
hP2X3
1b
13.2 3.1
19.5 5.1
14.8 1.7
7.4 1.9
Inactive
Inactive
20.1 4.2
22.3 8.1
15.2 7.7
9.3 2.3
Inactive
Inactive
13b
H
OH
H
OH
H
OH
CH2CH2
CH2CH2
CH@CH
CH@CH
14b
17b
18b
22b
23b
Inactive
Inactive
5bc (iso-PPADS)
SO3H
SO3H
SO3H
H
H
H
H
H
H
H
H
H
H
H
H
H
SO3H
SO3H
CO2H
CO2H
SO3H
SO3H
SO3H
H
H
H
62.4 3.1
58.6 4.5
60.2 4.9
41.8 9.8
57.6 8.8
48.5 10.5
51.7 13.4
48.3 5.6
40.1 8.4
45.7 10.8
46.5 11.2
37.6 6.9
56.3 8.8
25.7 6.9
24c
25c
26c
27c
28c
29c
H
OH
H
OH
H
OH
H
H
a
The ion current was induced by 2
lM ATP at the recombinant P2X receptors expressed in Xenopus oocytes, and the percentage inhibition of ion current by 10 lM and 100
nMof each compound was measured at mP2X1 and hP2X3 receptors (mean SEM, n = 4).
b
At concentrations of 10
At concentrations of 100 nM.
l
M.
c
compounds were also evaluated for selectivity at hP2X7 receptors
using an ethidium bromide (EtBr) uptake assay.
dent antagonisms. Compound 24 showed dose-dependent antago-
nism against ATP-induced ion currents at mP2X1 receptors, with a
complete reversal of antagonistic effects after wash-out (Fig. 2A).
The full dose-response curves of 5b, 24 and 28 at the mP2X1 and
hP2X3 receptors are depicted in Figures 2B and C, respectively.
The antagonistic potency of 24 and 28 at P2X1 and P2X3 receptors
were maintained compared with 5b, showing IC50 values in the
range of 100 nM. However, the IC50 ratios for P2X1 receptors rela-
tive to P2X3 receptors were 0.3 for 5b, 0.7 for 24 and 1.6 for 28.
Therefore, modifications of the pyridoxal-50-phosphate and 6-phe-
nylsulfonic acid moieties of 5b by incorporation of carboxylic acids
may shift selectivity profiles toward greater antagonisms toward
P2X3 receptors than toward P2X1 receptors.
Since pyridoxal-50-phosphate, 1 has been reported to antago-
nize P2X1 receptors with an IC50 value of roughly 10 lM (refer-
ence), the inhibitory activities of compound 1 and its carboxylic
acid analogs at both receptors were investigated at concentrations
of 10 lM. Compounds 13 and 14 showed similar antagonistic
potencies as pyridoxal-50-phosphate, 1 at mP2X1 and hP2X3 recep-
tors, whereas compounds 17, 18, 22, and 23 displayed weak or no
activity (Table 1). The results obtained with compounds 22 and 23
suggest that there is no additional hydrophobic binding pocket
around the 5-position carboxylic acid. Also, the comparison of
compounds 13 and 17 indicated that a flexible propionic acid
group may be associated with greater antagonistic activity than a
more rigid propenic acid group. Therefore, 13 and 14 were selected
as pyridine carboxylic acid analogs and subjected to diazo coupling
reactions to prepare compounds 24–29, 6-substituted derivatives
with aniline-2,3-disulfonic acid, sulfanilic acid, and 4-aminoben-
zoic acid groups.
In the investigation of selectivity profiles of compounds 24–29
at P2X7 receptor using ethidium bromide (EtBr) uptake assays in
hP2X7-expressing HEK293 cells, all the derivatives showed less
than 50% antagonistic activity at a concentration of 10 lM (Supple-
mentary Fig. S1). Thus, the selectivity of new pyridine carboxylic
acid derivatives for P2X1 and P2X3 versus P2X7 receptors was
greatly improved, since the IC50 values of PPADS analogs, such as
PPNDS, MRS 2159 have been reported to be in the range of 0.6–
Most of these compounds showed antagonistic activities gener-
ally similar to that of the positive control, 5b (iso-PPADS) with
100 nM concentrations showing 50–60% inhibition at P2X1 recep-
tors, indicating that pyridoxal-50-phosphate could be replaced with
pyridine carboxylic acid analogs. We found that the 6-substituted
pyridine-4-carboxylic acid analogs 25, 27 and 29 were more potent
antagonists at mP2X1 than at hP2X3 receptors. In contrast, com-
pounds 26 and 28, with aldehyde groups at the 4-position, dis-
played greater antagonistic activities at hP2X3 receptors than
compounds 27 and 29 (Table 1). Therefore, substitutions at both
the 4-carboxaldehyde and 4-acidic groups may enhance antago-
nism at P2X3 relative to P2X1 receptors.
4.6 l
M in various assay systems.49
3. Conclusion
We have described the successful modification of non-selective
P2 receptor antagonists PPADS and iso-PPADS, by the replacement
of strong anionic moieties such as phosphoric or sulfonic acids
with weaker carboxylic acids. Overall, modification of the moiety
at position 4 or 5 in the pyridine skeleton to a carboxylic acid group
antagonized mP2X1 and hP2X3 receptors without loss of activity. In
particular, a non-phosphate and non-sulfonate analog, 28, showed
similar antagonistic potencies as its parent compound, 5b, with
Among the series of derivatives, compounds 24 and 28 were
chosen as representative analogs for the evaluation of dose-depen-

K.-Y. Jung et al. / Bioorg. Med. Chem. 21 (2013) 2643–2650
2647
TCI (Tokyo). Aniline-2,5-disulfonic acid was obtained from K&K
Laboratories, Inc. (Hollywood, CA). All other reagents and solvents
were of analytical grade and purchased from Merck (Darmstadt,
Germany). Solutions of agonists and antagonists were prepared
daily from stock solutions (10 mM, stored frozen) made up in
extracellular bathing solution. Analytical TLC was carried out on
precoated silica glass plates (Merck silica gel 60, F254) and visual-
ized with UV light or stained with sulfonic acid stain. Flash column
chromatography was performed with silica (Merck, 70–230 mesh).
Proton nuclear magnetic resonance spectroscopy was performed
on a JEOL JNM-LA 300WB spectrometer, and spectra were taken
in DMSO-d6. Unless otherwise noted, chemical shifts are expressed
as ppm downfield from internal tetramethylsilane, or relative ppm
from DMSO (2.5 ppm). Data are reported as follows: chemical shift,
multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; b, broad;
app., apparent), coupling constants, and integration. Mass spec-
troscopy was carried out on MALDI-TOF and ESI instruments. The
determination of purity was performed on a Shimadzu SCL-10A
VP HPLC system using a Shimadzu Shim-pack C18 analytical col-
umn (250 mm ꢀ 4.6 mm, 5
lm, 100 Å) in linear gradient solvent
systems. One solvent system (A) was 0.1 M triethylammonium
acetate buffer/CH3CN = 95:5 to 40:60 for 30 min with flow rate
1 mL/min. The other (B) was 5 mM tetrabutylammonium phos-
phate buffer/CH3CN = 80:20 to 40:60 in 30 min with flow rate
1 mL/min. The cRNA of hP2X3 receptor was obtained by reverse
transcription of the cDNA of hP2X3 receptor, which was generously
provided by Dr. W. Stuhmer and Dr. F. Soto of the Max-Plank Insti-
tute. The EST clones containing full-length cDNAs of mP2X1 (clone
ID: 4189541) and hP2X7 receptor (clone ID: 5286944) were pur-
chased (Invitrogen, CA, U.S.A.), and their sequences were confirmed
by DNA sequencing.
4.1.1. 2,2,8-Trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-
yl)methanol (7)
To a mixture of 20 g (0.1 mol) of pyridoxine and 244 mL
(1.9 mol) of 2,2-dimethoxypropane was added 90 g (0.4 mol) of
para-toluenesulfonic acid in flame-dried 1 L three-neck flask. This
mixture was stirred for 30 h at room temperature and neutralized
with NaHCO3 and concentrated in evaporation. After extraction
with dichloromethane, the combined organic layer was washed
with brine, dried over MgSO4, filtered, concentrated, and purified
by column chromatography with dichloromethane/metha-
nol = 20:1. Yield 65.7% 1H NMR (CDCl3) d 1.52 (6H, s, 2 ꢀ CH3),
2.34 (3H, s, CH3), 4.52 (2H, s, CH2), 4.91 (2H, s, CH2), 7.78 (1H, s,
H-6). MS (ESI): m/z = 210.2 (M++1).
Figure 2. Inhibition of mP2X1 and hP2X3 receptor-mediated currents by com-
pounds 24 and 28. Inward currents were elicited by 2 M ATP in the control and in
l
the presence of two concentrations of compound 24 in Xenopus oocytes expressing
mP2X1 receptor subtypes (A). Concentration–inhibition curves of compounds 24
(j), 28 (d), and iso-PPADS 5b (N) in cells expressing (B) mP2X1 and (C) hP2X3
4.1.2. 2,2,8-Trimethyl-4H-[1,3]dioxino[4,5-c]pyridine-5-
carbaldehyde (8)
receptors. The continuous line for ATP was fit to the data using the equation I = Imax
/
(1 + IC50/L)nH, where I is the actual current for a ligand concentration (L), nH is the
Hill coefficient, and Imax is the maximal current. In B, the IC50 values and Hill
coefficients were: (j) 70.5 2.7 nM, and ꢁ1.1 0.4, respectively, for compound 24;
(d) 130 14 nM, and ꢁ1.2 0.5, respectively, for compound 28; (N) 30.7 5.1 nM,
and ꢁ1.1 0.1, respectively, for compound 5b. In C, the IC50 values and Hill
coefficients were: (j) 106 18 nM, and ꢁ1.2 0.3, respectively, for compound 24;
(d) 79.8 1.2 nM, and ꢁ1.1 0.02, respectively, for compound 28; (N)
96.2 21.0 nM, and ꢁ1.1 0.3, respectively, for compound 5b.
To a stirred solution of 5 g (23.9 mmol) of 7 in 50 mL of anhy-
drous dichloromethane was added pyridinium dichromate with
vigorous stirring. After overnight reaction, the mixture was filtered
through celatum bed with methanol. The filtrate was evaporated
under vacuum and purified by column chromagotraphy with n-
hexanes/ethyl acetate = 3:1. Yield 67.5% 1H NMR (CDCl3) d 1.57
(6H, s, 2 ꢀ CH3), 2.51 (3H, s, CH3), 5.18 (2H, s, CH3), 8.48 (1H, s,
H-6), 10.05 (1H, s, CHO). MS (ESI): m/z = 208.0 (M++1).
slightly improved selectivity for P2X3 receptors. Moreover, all new
pyridine carboxylic acid derivatives were highly selective for
mP2X1 and hP2X3 receptors when compared with hP2X7 receptors.
4.1.3. Ethyl 3-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-
yl)acrylate (9)
To a suspension of 500 mg (16.9 mmol) of NaH in anhydrous
tetrahydrofuran was added dropwise 3 mL (16.8 mmol) of triethyl-
phosphoacetate. The mixture color was changed to clear yellow
solution with H2 gas release. After 5 min stirring, 3.2 g (15.3 mmol)
of 8 in 40 mL tetrahydrofuran was added and the reaction solution
was stirred for 1 h. The mixture extracted with chloroform and
brine. The organic residue was combined and dried over MgSO4, fil-
4. Experimental section
4.1. Chemistry
ATP (disodium salt), Pyridoxine, and the reagents for diazo cou-
pling reactions were purchased from Aldrich (St. Louis, MO) and

2648
K.-Y. Jung et al. / Bioorg. Med. Chem. 21 (2013) 2643–2650
tered, concentrated, and purified by column chromatography with
n-hexanes/ethyl acetate = 3:1. Yield 81.3% 1H NMR (CDCl3) d 1.33
(3H, s, CH3), 1.57 (6H, s, 2 ꢀ CH3), 2.44 (3H, s, CH3), 4.28 (2H, q,
J = 8.8 Hz, CH2), 4.93 (2H, s, CH2), 6.37 (1H, d, J = 16.6 Hz, CH),
7.54 (1H, d, J = 16.6 Hz, CH), 8.26 (1H, s, H-6). MS (ESI): m/
z = 278.3 (M++1).
The reaction solvent was evaporated in vacuum and purified by
ion-exchange column chromatography suing Amberlite CG-50.
Yield 74.5% 1H NMR (D2O) d 1.57 (6H, s, 2 ꢀ CH3), 2.38 (3H, s,
CH3), 5.08 (2H, s, CH2), 6.43 (1H, d, J = 16.6 Hz, CH), 7.20 (1H, d,
J = 16.6 Hz), 8.17 (1H, s, H-6). MS (ESI): m/z = 250.2 (M++1).
4.1.10. 3-(5-Hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-
yl)acrylic acid (16)
4.1.4. Ethyl 3-(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-
3-yl)acrylate (10)
To a solution of aqueous 4 N HCl in methanol was added 2 g
(8.0 mmol) of 14 and stirred for 2 days. Reaction was monitored
by analytical HPLC system and purified by ion-exchange column
chromatography suing Amberlite CG-50. Yield 60.5% 1H NMR
A solution of 3 g (10.8 mmol) of 9 in 20 mL solution of 10% for-
mic acid was refluxed for 2 h. After cooling at room temperature,
the mixture was extracted with chloroform and saturated NaHCO3.
The organic layer was combined and dried over MgSO4, filtered,
concentrated, and purified by column chromatography with
dichloromethane/methanol = 10:1. Yield 97.8% 1H NMR (CDCl3) d
1.35 (3H, s, CH3), 2.48 (3H, s, CH3), 4.28 (2H, q, J = 8.8 Hz, CH2),
5.12 (2H, s, CH2), 6.30 (1H, d, J = 16.6 Hz, CH), 7.67 (1H, d,
J = 16.6 Hz, CH), 8.14 (1H, s, H-6). MS (ESI): m/z = 238.1 (M++1).
(D2O)
d 2.45 (3H, s, CH3), 4.87 (2H, s, CH2), 6.43 (1H, d,
J = 16.6 Hz, CH), 7.26 (1H, d, J = 16.6 Hz, CH), 7.98 (1H, s, H-6). MS
(ESI): m/z = 210.1 (M++1).
4.1.11. 3-(4-Formyl-5-hydroxy-6-methylpyridin-3-yl)acrylic
acid (17)
4.1.5. Ethyl 3-(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-
3-yl)propanoate (11)
Following the compound 13 procedure. Yield 52.4% 1H NMR
(DMSO-d6) d 2.39 (3H, s, CH3), 6.95 (1H, d, CH, J = 16.1 Hz), 7.58
(1H, d, CH, J = 16.1 Hz), 8.34 (1H, s, H-6). MS (ESI): m/z = 208.2
(M++1).
The solution of 2 g (8.4 mmol) of 10 in dichloromethane was
added 0.5 g of Pd/C and stirred for 4 h under H2 atmosphere. The
mixture was filtered through celatum bed. The filtrate was evapo-
rated in vacuum and purified by column chromatography with
chloroform/methanol = 10:1. Yield 89.2% 1H NMR (CDCl3) d 1.23
(3H, t, J = 7.1 Hz, CH3), 2.40 (3H, s, CH3), 2.50 (2H, t, J = 7.6 Hz,
CH2), 2.80 (2H, t, J = 7.6 Hz, CH2), 4.10 (2H, q, J = 7.1 Hz, CH2),
4.99 (2H, s, CH2), 7.73 (1H, s, H-6). MS (ESI): m/z = 240.1 (M++1).
4.1.12. 5-(2-Carboxyvinyl)-3-hydroxy-2-methylisonicotinic acid
(18)
Following the compound 14 procedure. Yield 71.3% 1H NMR
(DMSO-d6) d 2.31 (3H, s, CH3), 6.12(1H, d, CH, J = 16.8 Hz), 7.89
(1H, d, CH, J = 16.8 Hz), 8.76 (1H, s, H-6). MS (ESI): m/z = 224.2
(M++1).
4.1.6. Ethyl 3-(4-formyl-5-hydroxy-6-methylpyridin-3-
yl)propanoate (12)
To a solution of 1 g (4.2 mmol) of 11 in 50 mL of anhydrous
dichloromethane was added activated manganese dioxide with vig-
orous stirring for 2 h at room temperature under N2 atmosphere. The
mixture was filtered through celatum bed and filtrated was evapo-
rated in vacuum and purified by column chromatography with n-
hexanes/ethyl acetate = 4:1. Yield 71.1% 1H NMR (CDCl3) d 1.24
(3H, t, J = 7.1 Hz, CH3), 2.51 (3H, s, CH3), 2.69 (2H, t, J = 7.6 Hz, CH2),
3.25 (2H, t, J = 7.6 Hz, CH2), 4.12 (2H, q, J = 7.1 Hz, CH2), 8.02 (1H, s,
H-6), 10.46 (1H, s, CHO). MS (ESI): m/z = 238.1 (M++1).
4.1.13. N-Benzyl-1-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-
c]pyridin-5-yl) methanamine (19)
To a solution of 200 mg (1.0 mmol) of 8 in 1.0% acetic acid in
dichloromethane was added 126 lL (1.2 mmol) of benzylamine
and stirred for 30 min. 90 mg (1.4 mmol) of NaBH3CN was added
reaction mixture and stirred for 4 h. The reaction mixture was ex-
tracted with chloroform and saturated NaHCO3, dried over MgSO4,
filtered, concentrated, and purified by column chromatography
with dichloromethane/methanol = 30:1.Yield 50.7% 1H NMR
(CDCl3) d 1.54 (6H, s, 2 ꢀ CH3), 2.39 (3H, s, CH3), 3.64 (2H, s,
CH2), 3.78 (2H, s, CH2), 4.93 (2H, s, CH2), 7.25–7.40 (5H, m, phenyl),
7.92 (1H, s, H-6). MS (ESI): m/z = 299.4 (M++1).
4.1.7. 3-(4-Formyl-5-hydroxy-6-methylpyridin-3-yl)propanoic
acid (13)
To a solution of 5% KOH in methanol was added 330 mg
(1.4 mmol) of 12 with vigorous stirring for 1 h. The reaction solvent
was removed by evaporation and product was purified by ion-ex-
change column chromatography using Amberlite CG-50 resin.
Yield 53.9% 1H NMR (D2O) d 2.47 (3H, s, CH3), 2.55 (2H, t,
J = 7.5 Hz, CH2), 3.15 (2H, t, J = 7.5 Hz, CH2), 7.58 (1H, s, H-6),
10.44 (1H, s, CHO). MS (ESI): m/z = 210.2 (M++1).
4.1.14. Ethyl 3-(benzyl((2,2,8-trimethyl-4H-[1,3]dioxino[4,5-
c]pyridin-5-yl)methyl) amino)propanoate (20)
To a solution of 50 mg (0.2 mmol) of 19 in ethanol was added
37 lL (0.3 mmol) of ethyl acrylate and stirred for overnight at
room temperature. The mixture was concentrated in vacuum and
purified by column chromatography with chloroform/metha-
nol = 50:1. Yield 34.6% 1H NMR (CDCl3) d 1.14 (3H, t, J = 7.2 Hz,
CH3), 1.45 (6H, s, 2 ꢀ CH3), 2.31 (3H, s, CH3), 2.39 (2H, t,
J = 7.2 Hz, CH2), 2.68 (2H, t, J = 7.2 Hz, CH2), 3.36 (2H, s, CH2),
3.46 (2H, s, CH2), 4.01 (2H, q, J = 7.2 Hz, CH2), 4.69 (2H, s, CH2),,
7.17ꢂ7.26 (5H, m, phenyl), 7.84 (1H, s, H-6). MS (ESI): m/
z = 399.5 (M++1).
4.1.8. 5-(2-Carboxyethyl)-3-hydroxy-2-methylisonicotinic acid
(14)
To a solution of 177 mg (0.8 mmol) in 0.2 M DMF in acetonitrile
was added 520 mg (0.8 mmol) of oxone with vigorous stirring for
3 h. The reactant color was changed from yellow to colorless. Prod-
uct was purified by ion-exchange column chromatography using
Amberlite CG-50 resin and lyophilized. Yield 47.7% 1H NMR
(D2O) d 2.34 (3H, s, CH3), 2.50 (2H, t, J = 7.5 Hz, CH2), 2.74 (2H, t,
J = 7.5 Hz, CH2), 7.53 (1H, s, H-6). MS (ESI): m/z = 226.3 (M++1).
4.1.15. 3-(Benzyl((5-hydroxy-4-(hydroxymethyl)-6-
methylpyridin-3-yl)methyl) amino) propanoic acid (21)
Following the compound 13 procedure. Yield 46.9% 1H NMR
(D2O) d 2.42(3H, s, CH3), 2.53 (2H, t, J = 7 .2 Hz, CH2), 3.02 (2H, t,
J = 7.2 Hz, CH2), 3.95 (2H, s, CH2), 4.62 (2H, s, CH2), 7.35–7.45
(5H, m, phenyl), 7.62 (1H, s, H-6). MS (ESI): m/z = 331.4 (M++1).
4.1.9. 3-(2,2,8-Trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-
yl)acrylic acid (15)
To a solution of 50 mL of aqueous 5% KOH in 100 mL of metha-
nol was added 3 g (12.1 mmol) of 9 with vigorous stirring for 3 h.

K.-Y. Jung et al. / Bioorg. Med. Chem. 21 (2013) 2643–2650
2649
4.1.16. 3-(Benzyl((4-formyl-5-hydroxy-6-methylpyridin-3-
yl)methyl) amino) propanoic acid (22)
4.1.22. 3-(2-Carboxyethyl)-5-hydroxy-6-methyl-2-((4-
sulfophenyl)diazenyl) isonicotinic acid (27)
Following the compound 12 procedure. Yield 34.5% 1H NMR
(CDCl3) d 2.47 (3H, s, CH3), 2.51 (2H, t, J = 6.9 Hz, CH2), 2.78 (2H,
t, J = 6.9 Hz, CH2), 3.61 (2H, s, CH2), 3.76 (2H, s, CH2), 7.24–7.33
(5H, m, phenyl), 8.01 (1H, s, H-6), 10.21 (1H, s, CHO). MS (ESI):
m/z = 329.4 (M++1).
Following the general procedure for the synthesis of azo group
containing compounds. Yield 28.3% 1H NMR (D2O) d 2.65 (3H, s,
CH3), 2.73 (2H, t, J = 7.2 Hz, CH2), 3.68 (2H, t, J = 7.2 Hz, CH2), 7.79
(2H, d, J = 7.2 Hz, phenyl), 7.98 (2H, d, J = 7.2 Hz, phenyl). 13C
NMR (DMSO-d6, 400 MHz): d 19.2, 23.9, 35.9, 121.6, 122.4, 127.3,
129.3, 131.5, 147.4, 148.4, 156.7, 168.3, 173.9, 175.3. MS (MALDI-
TOF): m/z = 410.3 (M++1).
4.1.17. 5-((Benzyl(2-carboxyethyl)amino)methyl)-3-hydroxy-2-
methylisonicotinic acid (23)
4.1.23. 4-((3-(2-Carboxyethyl)-4-formyl-5-hydroxy-6-
methylpyridin-2-yl) diazenyl) benzoic acid (28)
Following the compound 14 procedure. Yield 42.6% 1H NMR
(D2O) d 2.58 (3H, s, CH3), 2.55 (2H, t, J = 6.9 Hz, CH2), 2.82 (2H, t,
J = 6.9 Hz, CH2), 3.71 (2H, s, CH2), 3.82 (2H, s, CH2), 7.24–7.33
(5H, m, phenyl), 8.01 (1H, s, H-6). MS (ESI): m/z = 345.4 (M++1).
Following the general procedure for the synthesis of azo group
containing compounds. Yield 32.5% 1H NMR (D2O) d 2.53 (3H, S,
CH3), 2.62 (2H, t, J = 6.9 Hz, CH2), 3.75 (2H, t, J = 6.9 Hz, CH2), 7.94
(2H, d, J = 8.1 Hz, phenyl), 8.06 (2H, d, J = 8.1 Hz, phenyl), 10.52
(1H, s, CHO).). 13C NMR (DMSO-d6, 400 MHz): d 18.3, 21.1, 32.9,
128.1, 128.6, 130.6, 131.9, 132.1, 149.6, 156.7, 168.4, 170.4,
176.9, 178.3, 199.2. MS (MALDI-TOF): m/z = 358.3 (M++1).
4.1.18. General procedure for the synthesis of azo group
containing compounds (24–29)
8 mg (0.1 mmol) of NaNO2 was added to a stirred solution of an
aniline analogue (0.1 mmol) and 13 mg (0.1 mmol) of Na2CO3 in
4.1.24. 3-(2-Carboxyethyl)-2-((4-carboxyphenyl)diazenyl)-5-
hydroxy-6-methy lisonicotinic acid (29)
1 mL of H2O at 0 °C. 60 lL (0.3 mmol) of 6 N HCl was added drop-
wise, and the mixture was stirred for 5–10 min at 0 °C. A solution
of carboxylic acid analogues of pyridoxal- or pyridoxic acid
(0.1 mmol) in 1 mL of H2O was added at once, and the pH of the
mixture was adjusted to 10–11 by the addition of 1 N NaOH
Following the general procedure for the synthesis of azo group
containing compounds. Yield 24.5% 1H NMR (D2O) d 2.43 (3H, s,
CH3), 2.58 (2H, t, J = 7.5 Hz, CH2), 3.52 (2H, t, J = 7.5 Hz, CH2), 7.63
(2H, d, J = 8.1 Hz, phenyl), 7.98 (2H, d, J = 8.1 Hz, phenyl). 13C
NMR (DMSO-d6, 400 MHz): d 19.6, 22.4, 36.4, 120.7, 122.5, 130.9,
132.1, 139.3, 147.8, 153.9, 165.2, 167.7, 174.7, 175.5. MS (MALDI-
TOF): m/z = 374.3 (M++1).
(ꢂ300
lL). The yellow color changed to red (sometimes gradually),
indicating the reaction had occurred. After stirring the mixture for
30 min at 0 °C and 30 min at 25 °C, it was then purified by ion-ex-
change column chromatography using Amberlite CG-50 resin (H+
form, weakly acidic) with the elution of water (flow rate 0.5 mL/
min). The red fraction showing a single peak in HPLC was collected
and lyophilized to give the desired compound with more than 95%
purity.
4.2. Biology
4.2.1. Antagonistic activity at recombinant mP2X1 and hP2X3
receptors
Xenopus oocytes were harvested and prepared as previously de-
4.1.19. 3-(2-((2,5-Disulfophenyl)diazenyl)-4-formyl-5-hydroxy-
6-methylpyridin-3-yl) propanoic acid (24)
scribed.50 Defolliculated oocytes were injected cytosolically with
mP2X1 and hP2X3 receptor cRNA (40 nL, 1 lg/mL), respectively,
Following the general procedure for the synthesis of azo group
containing compounds. Yield 28.5% 1H NMR (D2O) d 2.39 (3H, s,
CH3), 2.65 (2H, t, J = 6.9 Hz, CH2), 3.54 (2H, t, J = 6.9 Hz, CH2), 8.34
(1H, d, J = 8.1 Hz, phenyl), 8.62 (1H, d, J = 8.1 Hz, phenyl), 9.06
(1H, s, phenyl), 10.20 (1H, s, CHO). 13C NMR (DMSO-d6,
400 MHz): d 19.5, 21.3, 35.2, 126.6, 127.5, 129.9, 131.8, 138.0,
150.0, 150.9, 151.9, 173.7, 170.4, 179.5, 198.1. MS (MALDI-TOF):
m/z = 474.4 (M++1).
incubated for 24 h at 18 °C in Barth’s solution and kept for up to
12 days at 4 °C until used in electrophysiological experiments.
ATP-activated membrane currents (Vh = ꢁ70 mV) were recorded
from cRNA-injected oocytes using the two-electrode voltage-
clamp technique (Axoclamp 2B amplifier). Voltage recording (1–
2 M
(5 M
X
X
tip resistance) and current-recording microelectrodes
tip resistance) were filled with 3.0 M KCl. Oocytes were held
in an electrophysiological chamber and superfused with Ringer’s
solution (5 mL/min, at 18 °C) containing (mM) NaCl, 110; KCl,
2.5; HEPES, 5; BaCl2, 1.8, adjusted to pH 7.5. ATP was superfused
over the oocytes for 60–120 s then washed out for a period of
20 min. For inhibition curves, data were normalized to the current
evoked by ATP, at pH 7.5. Test substances were added for 20 min
prior to ATP exposure; all peptides were tested for reversibility
of their effects. The concentration required to inhibit the ATP re-
sponse by 50% (IC50) was taken from Hill plots constructed using
the formula: log(I/Imax ꢁ I), where I is the current evoked by ATP
in the presence of an antagonist. Data are presented as mean SEM
(n = 4) for the data from different batches of oocytes.
4.1.20. 3-(2-Carboxyethyl)-2-((2,5-disulfophenyl)diazenyl)-5-
hydroxy-6-methylisonicotinic acid (25)
Following the general procedure for the synthesis of azo group
containing compounds. Yield 32.4% 1H NMR (D2O) d 2.74 (3H, s,
CH3), 2.85 (2H, t, J = 8.7 Hz, CH2), 3.84 (2H, t, J = 8.7 Hz, CH2), 8.03
(1H, d, J = 8.1 Hz, phenyl), 8.13 (1H, s, phenyl), 8.22 (1H, d,
J = 8.1 Hz, phenyl). 13C NMR (DMSO-d6, 400 MHz): d 19.0, 21.5,
34.5, 125.5, 126.8, 128.0, 129.7, 131.4, 149.7, 150.7, 159.2, 160.2,
167.4, 173.5, 174.2. MS (MALDI-TOF): m/z = 489.4 (M++1).
4.1.21. 3-(4-Formyl-5-hydroxy-6-methyl-2-((4-
4.2.2. Ethidium bromide (EtBr) accumulation assay at hP2X7
receptors
sulfophenyl)diazenyl)pyridin-3-yl) propanoic acid (26)
Following the general procedure for the synthesis of azo group
containing compounds. Yield 26.2% 1H NMR (D2O) d 2.43 (3H, s,
CH3), 2.65 (2H, t, J = 6.9 Hz, CH2), 3.50 (2H, t, J = 6.9 Hz, CH2), 7.67
(2H, d, J = 8.4 Hz, phenyl), 7.82 (2H, d, J = 8.4 Hz, phenyl), 10.29
(1H, s, CHO). 13C NMR (DMSO-d6, 400 MHz): d 19.5, 21.6, 35.9,
124.8, 127.3, 128.8, 130.2, 131.5, 148.1, 149.8, 156.0, 167.2,
174.0, 196.6. MS (MALDI-TOF): m/z = 394.3 (M++1).
hP2X7-expressing HEK293 cells, a human embryonic kidney
cells, were grown in DMEM supplemented with 10% fetal bovine
serum as monolayer culture at 37 °C in a humidified atmosphere
of 5% CO2. Cells were harvested with treatment of Trypsin/EDTA
solution, collected by centrifugation (200g for 5 min). The cells
were resuspended at 2.5 ꢀ 106 cells/mL in assay buffers, consisting
of (in mM) HEPES 10, N-methyl-D-glutamine 5, KCl 5.6, D-glucose

2650
K.-Y. Jung et al. / Bioorg. Med. Chem. 21 (2013) 2643–2650
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.073.
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