Design and synthesis of potent and selective P2X3 receptor antagonists derived from PPADS as potential pain modulators

Article abstract of DOI:10.1016/j.ejmech.2013.10.026

Pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate (7a, PPADS), a nonselective P2X receptor antagonist, was extensively modified to develop more stable, potent, and selective P2X₃ receptor antagonists as potential antinociceptive agents. Based on

Full text of DOI:10.1016/j.ejmech.2013.10.026

European Journal of Medicinal Chemistry 70 (2013) 811e830
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of potent and selective P2X3 receptor
antagonists derived from PPADS as potential pain modulators
Joong-Heui Cho a, Kwan-Young Jung c, Younghwan Jung a, Min Hye Kim a, Hyojin Ko a,
,b,
Chul-Seung Park a, Yong-Chul Kim a
*
a School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
b Department of Medical System Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
c Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Pyridoxalphosphate-6-azophenyl-20,40-disulfonate (7a, PPADS), a nonselective P2X receptor antagonist,
was extensively modified to develop more stable, potent, and selective P2X3 receptor antagonists as
potential antinociceptive agents. Based on the results of our previous report, all strong anionic groups in
PPADS including phosphate and sulfonate groups were changed to carboxylic acids or deleted. The un-
stable azo (eN]Ne) linkage of 7a was transformed to more stable carbon-carbon, ether or amide
linkages through the synthesis of the 5-hydroxyl-pyridine moieties with substituents at 2 position via a
Diels-Alder reaction. This resulted in the retention of antagonistic activity (IC50 ¼ 400 w 700 nM) at the
hP2X3 receptor in the two-electrode voltage clamp (TEVC) assay system on the Xenopus oocytes.
Introduction of bulky aromatic groups at the carbon linker, as in compounds 13hen, dramatically
improved the selectivity profiles of hP2X3 when compared with mP2X1 and hP2X7 receptors. Among the
substituents tested at the 2-position, the m-phenoxybenzyl group showed optimum selectivity and
potency at the hP2X3 receptor. In searching for effective substituents at the 4- and 3-positions, we found
that compound 36j, with 4-carboxaldehyde, 3-propenoic acid and 2-(m-phenoxy)benzyl groups, was the
most potent and selective hP2X3 receptor antagonist with an IC50 of 60 nM at hP2X3 and marginal
Article history:
Received 16 August 2013
Received in revised form
7 October 2013
Accepted 11 October 2013
Available online 18 October 2013
Keywords:
P2X3 receptor
Antagonists
PPADS
Structure-activity relationships
Two-electrode voltage clamp
Pain
antagonistic activities of 10
found that compound 36j potently inhibited pain signaling in the rat dorsal horn with 20
playing 65% inhibition while 20 M pregabalin, a clinically available drug, showed only 31% inhibition.
Ó 2013 Elsevier Masson SAS. All rights reserved.
m
M at mP2X1 and hP2X7. Furthermore, using an ex-vivo assay system, we
m
M 36j dis-
m
1. Introduction
P2X4 receptors [5]. Activation of P2X receptors, most of which are
nonselective cation channels permeable to Naþ, Kþ and Ca2þ, re-
sults in membrane depolarization and cell excitation [6e8].
P2X receptors are widely expressed throughout the whole body,
including in vascular smooth muscles, platelets, the nervous sys-
tem, and immune system cells [9]. Among the seven P2X receptor
subtypes, P2X3 receptors are expressed selectively and at high
levels in nociceptive primary sensory neurons in trigeminal,
nodose, and dorsal root ganglia (DRG) [10e12]. These P2X3 re-
ceptors exist as functional, homomeric ion channels, but can also
form heteromultimeric combinations with P2X2 receptors [13e15].
During inflammation and chronic constriction injuries, ATP con-
centrations and neuronal expression of P2X3 receptors were found
to be up-regulated in DRG and spinal cord [16e18]. The restricted
expression of P2X3 receptors suggests that they may be crucial in
processing pain signal.
The purine nucleotide ATP is not only a cellular energy source,
but an extracellular messenger that acts by binding to P2 nucleotide
receptors [1,2]. The P2 receptors are classified into two families:
ionotropic ligand-gated cationic channel (P2X receptors) and
metabotropic G-protein coupled receptors (P2Y receptors) [3,4].
P2X receptors are further categorized into seven subtypes P2X1 to
P2X7, with common structural features, including two trans-
membrane (TM1 and TM2) domains, a large extracellular loop
containing ATP binding sites, and intracellular N- and C-terminals.
Generally, P2X receptors form homo- or hetero-trimers to act as ion
channels and the detailed mechanism of channel opening was
elucidated by apo and open state crystal structures of zebrafish
* Corresponding author. School of Life Sciences, Gwangju Institute of Science and
Technology (GIST), Gwangju 500-712, Republic of Korea. Tel.: þ82 62 970 2502;
fax: þ82 62 970 2484.
A number of studies have evaluated the relationships between
P2X3 receptors and pain sensation. Local injection of P2X3 receptor
E-mail address: yongchul@gist.ac.kr (Y.-C. Kim).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.026

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J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
agonists, ATP or
a
,
b
-methyleneATP, into the hindpaws of rats
stability. Second, an acetyl or carboxylic acid group besides the
aldehyde group at the 4-position of 7aec was explored. Third,
the 6-azophenyl ring on the 2-position of the pyridine ring was
changed to other aromatic rings to improve selectivity at the
P2X3 receptors. The antagonistic effects of these compounds
were assessed on Xenopus oocytes expressing cloned P2X re-
ceptors using the two-electrode voltage clamp (TEVC) assay
system, and the functional activity of these compounds
was evaluated using an ex-vivo assay system measuring long-
term potentiation (LTP) as a pain signal in rat spinal dorsal
horn [38].
induced nociceptive behaviors and reduced thermal and mechan-
ical thresholds [19e21]. In animal models of inflammatory and
neuropathic pain, P2X3 receptor knock-out or downregulation of
P2X3 receptor expression by antisense oligonucleotides (ASO) or by
short interfering RNA (siRNA) significantly attenuated painful be-
haviors, including tactile allodynia and mechanical hyperalgesia
[22,23]. In addition, the P2X3 receptor antagonist TNP-ATP (20,30-O-
(2,4,6-trinitrophenyl)adenosine-50-triphosphate) and PPADS (7a,
pyridoxalphosphate-6-azophenyl-20,40-disulfonate) reduced the
excitability of DRG neurons and neuropathic pain [24]. Taken
together, the findings of these studies suggest that P2X3 receptors
play important roles in the development and maintenance of
chronic pain.
2. Results and discussion
The highly selective, non-nucleotide P2X3-P2X2/3 antagonist 1
(A-317491) was developed at Abbott Laboratories in 2002 and its
painkilling effects were validated in several animal models [25e
28]. Subsequently, the orally active P2X3-P2X2/3 antagonists, 2
(RO-4) [29,30], 3 (RO-51) [31], and 4 (RO-85) [32], at Roche and the
pyrrolo-pyrimidinone antagonist, 5 at AstraZeneca [33] were
developed (Fig. 1). The peptide antagonist, 6 (Spinorphin, IC50 ¼ 8.3
pM) [34] was reported by our group. Recently, Afferent Pharma-
ceuticals P2X3 antagonist, AF-219, entered Phase 2 clinical studies
in patients with joint pain, visceral pain, and idiopathic chronic
cough [35].
In efforts to develop new small chemical antagonists of P2X
receptors, we investigated derivatives of the nonselective P2X
receptor antagonists, 7a (PPADS), and 7b (MRS 2159) [36]. Our
preliminary structure-activity relationship (SAR) studies
showed that the aldehyde or phosphoric or sulfonic acid group
in 7a could be replaced by carboxylic acid (7c) without affecting
antagonistic potency at the mP2X1 and hP2X3 receptors [37]. To
develop novel antagonists for hP2X3 receptors, we performed
extensive modifications of the parent compounds, 7a-c. First,
the unstable azo (eN]Ne) linkages in 7aec were replaced with
stable carbon-carbon, ether, or amide linkages to increase
2.1. Chemistry
Since the parent compounds (7aec) haveunstable azo (eN]Ne)
bonds at the 2-position of the pyridine moiety, we replaced these
bonds with stable carbon-carbon or ether bonds using the Diels-
Alder reaction with bis-carbonyl based dienophiles and oxazole
based dienes (11aeo). The latter compounds were prepared from
compound 8 (L-alanine ethyl ester) using the synthesis strategy
shown in Scheme 1. Briefly, 8 was reacted with the appropriate
carboxylic acids 9aeo in the presence of general coupling reagents
to yield 10aeo, which were cyclized to their corresponding oxa-
zoles, 11aeo, using phosphorous pentoxide. The overall yield of
three steps was 92%. The 3,4-dicarboxypyridine derivatives, 12aeo,
were synthesized through Diels-Alder reactions of 11aeo with
dimethyl maleate, with subsequent hydrolysis using 20% aqueous
KOH yielding 13aeo (Scheme 1). To synthesize the 4-acetyl-pyri-
dine derivatives, the dienophile (E)-ethyl-4-oxopent-2-enoate (16)
was reacted with 11aef in Diels-Alder reactions to give 17aef, which
were hydrolyzed to the corresponding 4-acetyl-3-carboxypyridine
analogs, 18aef (Scheme 2). The acetyl group, a stronger electron-
withdrawing group, in 16 oriented the 4 position of pyridine in
18aef [39,40].
Fig. 1. The structure of P2X3 antagonists.

J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
813
Scheme 1. Synthesis of 3,4-dicarboxypyridine derivatives. Reagents and conditions: (a) EDC, TEA, DCM, RT, 2 h, 65e99%; (b) P2O5, CHCl3, reflux, 2 h, 38e99%; (c) dimethyl maleate,
neat, 5 h, 21e59%; (d) 20% KOH (aq), RT, 6 h, 27e83%.
To introduce an amide bond at the 2-position of pyridine de-
rivatives, compound 19 (4-methyl-oxazole-2-amine) was used as a
diene in a Diels-Alder reaction. This reaction yielded two com-
pounds, one with a hydrogen (20) and the other with a hydroxyl
group (21) at the 5-position of the pyridine ring. The 5-OH group in
21 was protected by benzylation to selectively acylate the 2-amino
group. The 2-amino groups of 20 and 22 were acylated using
benzoyl chloride or 3-phenoxybenzoylchloride. The protective
group of 23 was selectively removed by hydrogenolysis using Pd/C
under H2 gas to give compound 24. Lastly, the 3,4-dimethylester
groups of 24 and 26 were hydrolyzed using 20% aqueous KOH to
give 25 and 27, respectively (Scheme 3).
The 4-aldehyde-3-propenoic and -3-propionic acid derivatives,
36 and 41, respectively were synthesized via common in-
termediates, 33, which were prepared from the pyridine bis-
methylester compounds, 12 as shown in Scheme 4. The 5-OH
Scheme 2. Synthesis of 4-acetyl-3-carboxypyridine derivatives. Reagents and conditions: (a) DMF, DCM, RT, 20 h, 80%; (b) neat, 5 h, HCl (g), EtOH, 0 ꢀC, 10e51%; (c) 20% KOH (aq),
RT, 4 h, 70e90%.

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J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
Scheme 3. Synthesis of 3,4-dicarboxypyridine derivatives with amide linker at the 2 position. Reagents and conditions: (a) dimethylmaleate, neat, 5 h, 13e17%; (b) benzoyl chloride,
DIPEA, toluene, 80 ꢀC, 2 h, 51e67%; (c) benzyl bromide, K2CO3, acetone, reflux, 2 h, 99%; (d) 3-phenoxybenzoyl chloride, DIPEA, toluene, 80 ꢀC, 2 h, 77%; (e) Pd/C, H2 (g), MeOH, RT,
0.5 h, 53e61%; (f) 20% KOH (aq), RT, 4 h, 27e35%.
groups of 12 were protected with a benzyl group and the 3,4-bis-
methylester group of 28 was reduced to a 3,4-bis-hydroxymethyl
group with LiAlH4. The protecting benzyl group was removed with
Pd/C under H2 gas from compound 29. The 4-hydroxymethyl group
and 5-phenolic alcohol of 30 were selectively protected to an ace-
tonide moiety using 2,2-dimethoxypropane and 4 equivalent of p-
toluenesulfonic acid [41]. The 3-hydroxymethyl group in 31 was
oxidized with pyridinium dichromate to yield compound 32. The
Wittig-Honor reaction of 32 with triethyl phosphonoacetate pro-
duced only the trans-isomer compound 33, which was identified by
1H NMR analysis (J value ¼ 16.4 Hz). The ethyl ester group of 33 was
hydrolyzed with 5% methanolic KOH and the acetonide group of 34
was removed with 10% formic acid followed by manganese dioxide
oxidation, yielding the 4-aldyhyde-3-propenoic acid compounds
36. In the case of the synthesis of 4-aldehyde-3-propionic acid
compounds 41, a different order of procedures for the hydrolysis
and oxidation steps was employed to achieve the best chemical
yields. The acetonide group of 33 was first deprotected with 10%
To obtain the compounds having direct substitution of a car-
boxylic acid at the 3 position, the aldehyde group of 32 was further
oxidized to carboxylic acid with OxoneÒ. The acetonide group was
removed from 43 with 10% formic acid, yielding lactone com-
pounds 44, which were formed by dehydration reactions between
the 3-carboxylic acid and 4-hydroxymethyl groups of pyridine.
Thus, an additional hydrolysis step was needed to generate com-
pounds with 4-hydroxymethyl group (45). However, oxidation re-
actions of compound 45 with manganese dioxide yielded not the
compounds with 4-aldehyde-3-carboxy pyridine compounds, but
compounds 46 as pyridine-hydroxylactone derivatives (Scheme 5).
2.2. Biology
The antagonistic activities of the synthesized compounds at the
mP2X1 and hP2X3 receptors were evaluated by two-electrode
voltage clamp (TEVC) assays on the Xenopus oocytes expressing
cloned mP2X1 and hP2X3 receptors, respectively, as described
previously [42]. hP2X7 receptor antagonistic activity was evaluated
using ethidiumþ uptake assays. Compound 1, 7b and KN-62 [43]
were used as positive control antagonists for P2X1, P2X3 and P2X7
receptors, respectively.
Compound 7a is a potent but nonselective P2X receptor antag-
onist, with potent activities at mP2X1 (IC50 ¼ 99 nM), hP2X3
(IC50 ¼ 240 nM) and hP2X7 (IC50 ¼ 1180 nM) receptors [36,43]. In
our preliminary SAR, the phosphate of 7a could be replaced by the
weak anion carboxylic acid and the number of sulfonic acid groups
formic acid to yield 38, of which a,b-unsaturated double bond was
subsequently reduced under hydrogenation conditions to give 39.
The 4-hydroxymethyl group of 39 was converted to an aldehyde
group by manganese dioxide in dichloromethane, and hydrolysis of
the ester group of 40 using 5% methanolic KOH finally afforded 4-
aldehyde-3-propionic acid derivatives, 41 (Scheme 4). Lastly, two
dicarboxylic acid analogs, 37j and 42j were synthesized from 36j
and 41j, respectively, by oxidation reactions of 4-carboxaldehyde
groups with OxoneÒ.

J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
815
Scheme 4. Synthesis of 4-aldehyde-pyridine derivatives with 3-propionic and 3-propenoic acids. Reagents and conditions: (a) benzyl bromide, K2CO3, acetone, reflux, 2 h, 85e96%;
(b) LAH, ether, 0 ꢀC, 69e88%; (c, k) Pd/C, H2 (g), MeOH, RT, 0.5 h, 82e91%; (d) 2,2-dimethoxypropane, p-TsOH, acetone, RT, 12 h, 90%; (e) PDC, DCM, RT, 12 h, 83e90%; (f) NaH, triethyl
phosphonoacetate, THF, RT, 1 h, 96%; (g, m) 5% KOH in MeOH, 53e98%; (h, j) 10% formic acid, reflux, 3 h, 33e71%; (i, l) MnO2, DCM, RT, 2 h, 66e79%; (n) OxoneÒ, DMF/ACN (1:5), RT,
2 h, 13e37%.
of 7a could be removed or replaced by carboxylic acid (7c) without
affecting antagonistic activity of 7a at mP2X1 and hP2X3 receptors
[37]. The unstable azo (eN]Ne) linkage of 7c, however, reduces its
chemical stability and bioavailability. We therefore synthesized
various 5-hydroxy-pyridine derivatives with stable carbon-carbon,
ether, or amide linkages at the 2 position using Diels-Alder re-
actions. At the 4-position, acetyl, carboxylic acid or aldehyde moi-
eties were introduced.
Table 1 summarizes the antagonistic effects of these 3,4-
dicarboxypyridine (13aeg) and 4-acetyl-3-carboxypyridine de-
rivatives (18aef) towards mP2X1, hP2X3, and hP2X7 receptors.
Although these analogs were less potent than their parent com-
pounds (7aec), and their selectivity for hP2X3 receptor was not
improved, we could establish structure-activity relationships of
the series of new analogs. We first investigated the effects of the
carbon chain at the 2-position of the pyridine moiety. Two carbon
linkages, by changing 2-benzyl group (13a) to 2-phenethyl group
(13b), greatly increased the antagonistic activities, showing IC50
values of 645 nM at mP2X1 and 797 nM at hP2X3 receptors. The
conformational lock of two carbon linkage by employing a trans
form of double bond (13c) did not improve the antagonistic ac-
45.5% and 34.5%, respectively, and the similar activity profile was
observed when the compounds were tested at mP2X1 receptors.
Replacement of the p-methoxy of 13d with carboxylic acid (13g) did
not affect the antagonistic activity at the hP2X3 receptor, but
enhanced the activity at the mP2X1 receptor, showing increased
IC50 value of 267 nM (13g) from 446 nM (13d). This result suggested
that the polarity at the p-position of the phenyl group may shift
selectivity profiles toward mP2X1 rather than toward hP2X3
receptors.
In the case of the 4-acetylcarboxypyridine analogs, 18aef, the
antagonistic potencies markedly decreased at both of mP2X1 and
hP2X3 receptors than 3,4-dicarboxypyridine analogs, 13aeg. This
result may be due to the lack of affinity of the 4-acetyl group on the
pyridine moiety, compared with 4-carboxylic acid group, for the
interaction with lysine residues in the ATP binding site of the P2X
receptors [8,44]. All compounds in this series showed negligible
antagonistic activity for hP2X7 receptor.
To increase potency and selectivity for hP2X3 receptor, we
exploited our findings on the effect of polarity of the substituents at
the phenyl group connected to the 2-position of pyridine moiety on
the selectivity profiling between mP2X1 and hP2X3 receptors.
Therefore, we introduced non-polar bulky aromatic groups, such as
naphthyl (compound 13h), diphenyl (compound 13i), 3-
pheonoxyphenyl (compound 13jel), and 4-pheonoxyphenyl
(compound 13men) groups at the 2-position of the pyridine
moiety. Strikingly, all the compounds in Table 2,13hen showed less
tivities, both at mP2X1 (IC50
(IC50 ¼ 920 nM) receptors.
¼
768 nM) and at hP2X3
We next investigated the effects of the methoxy position on the
phenyl ring substituted at the carbon linkage, finding that the p-
position (13d) was best rather than the m- (13e) or o- (13f) position.
For example, compound 13d had an IC50 value of 460 nM at hP2X3
than 50% inhibition in 10
mM concentrations at mP2X1 receptors,
receptors, whereas 10 mM 13e and 13f inhibited the receptors only
while the compounds antagonized hP2X3 receptors with 400 nMe

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J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
Scheme 5. Synthesis of pyridine-hydroxylactone derivatives. Reagents and conditions: (a) OxoneÒ, DMF/ACN (1:5), RT, 2 h, 59e94%; (b) 10% formic acid, reflux, 3 h, 70e92%; (c) 5%
KOH in MeOH, RT, 4 h, 48e53%; (d) MnO2, THF/H2O (3:7), RT, 2 h, 21e26%.
3
m
M range of IC50 values. The antagonistic potency of these com-
pounds at hP2X3 receptors was increased in the order of 3-
phenoxyphenyl diphenyl naphthyl (13j 13i 13h)
In an attempt to optimize the hP2X3 antagonism of 13j, the po-
sition of phenoxy substituents and carbon-chain length between the
pyridine and phenoxyphenyl rings were modified (Table 2). Among
the derivatives with phenoxy substituents at various positions of the
2-benzyl moiety, we found that the m-phenoxyphenyl compound
13j was 2- and 6-fold more active than the p-phenoxyphenyl com-
pound 13m (IC50 ¼ 1050 nM) and the o-phenoxyphenyl compound
13n (IC50 ¼ 2930 nM), respectively. In analyzing the effect of carbon-
chain length (n) of the phenoxyphenyl moiety, we found that com-
pound 13j with n ¼ 1 was slightly more potent than compound 13k
>
>
>
>
(Table 2). The biological activity of the most potent analog in this
series, 13j (IC50 ¼ 474 nM) was parallel to that of the 4-
methoxyphenyl derivative, 13d (IC50 ¼ 460 nM), of which antago-
nistic potency at mP2X1 receptor was also similar (IC50 ¼ 446 nM).
However, as expected, 13j displayed dramatically increased selec-
tivity for hP2X3 versus mP2X1 and hP2X7 receptors, at which 10 mM
13j showed only 42% antagonism and no activity, respectively.
Table 1
Antagonistic effects of 3,4-dicarboxypyridine and 4-acetyl-3-carboxy pyridine derivatives at mP2X1, hP2X3 and hP2X7 receptors.
compounds
X
R1
mP2Xa1
hP2Xa3
hP2Xd7
1e
e
35 ꢁ 4 nM
e
e
7bf
30 ꢁ 13 nM
e
KN-62g
13a
13b
13c
13d
13e
13f
13g
18a
18b
18c
18d
18e
18f
e
e
158 ꢁ 18 nM
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
eOH
eOH
eOH
eOH
eOH
eOH
eOH
eCH3
eCH3
eCH3
eCH3
eCH3
eCH3
eCH2C6H5
eCH2CH2C6H5
eCH]CHC6H5
eCH2CH2C6H4-p-OCH3
eCH2CH2C6H4-m-OCH3
eCH2CH2C6H4-o-OCH3
eCH2CH2C6H4-p-COOH
eCH2C6H5
eCH2CH2C6H5
eCH]CHC6H5
eCH2CH2C6H4-p-OCH3
eCH2CH2C6H4-m-OCH3
eCH2CH2C6H4-o-OCH3
54.2 ꢁ 10.6%b
645 ꢁ 23 nMc
768 ꢁ 12 nMc
446 ꢁ 32 nMc
52.4 ꢁ 6.2%b
49.8 ꢁ 5.4%b
267 ꢁ 9 nMc
44.2 ꢁ 4.0%b
47.9 ꢁ 8.1%b
52.4 ꢁ 6.2%b
52.0 ꢁ 7.1%b
29.9 ꢁ 5.7%b
11.4 ꢁ 3.3%b
31.4 ꢁ 6.2%b
797 ꢁ 99 nMc
920 ꢁ 155 nMc
460 ꢁ 69 nMc
45.5 ꢁ 6.4%b
34.5 ꢁ 4.3%b
458 ꢁ 35 nMc
18.3 ꢁ 6.4%b
37.3 ꢁ 10.0%b
27.4 ꢁ 5.5%b
42.2 ꢁ 7.5%b
15.8 ꢁ 9.0%b
6.3 ꢁ 2.3%b
aThe ion current was induced by 2
m
M ATP at the recombinant P2X receptors expressed in Xenopus oocytes, and the percentage of inhibition of the ion current by 10
m
Mb or IC50
mM BzATP at the
valuesc of compounds was measured for mP2X1 and hP2X3 receptors, respectively (mean ꢁ SEM, n ꢂ 3). dThe accumulation of ethidiumþ was induced by 6
hP2X7 receptors expressed in HEK293 cells and % inhibition of the accumulation by 10
m
M compounds was measured (mean ꢁ SEM, n ꢂ 3). eReported IC50 value is 97 nM at
hP2X3 receptors [25]. Reported IC50 value is 43 nM at rP2X1 receptors [36]. gReported IC50 value is 210 nM at hP2X7 receptors [43].
f

J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
817
Table 2
antagonistic activity (Table 3). As a result, compound 27, which has
no hydroxyl group, was inactive in hP2X3 receptor antagonism,
suggesting that an intact hydroxyl group is required for a key
interaction, such as hydrogen bonding, with the binding site of
hP2X3 receptor.
Antagonistic effects of 3,4-dicarboxypyridine derivatives with bulky aromatic
groups at 2-position of pyridine moiety for mP2X1, hP2X3 and hP2X7 receptors.
Our previous study showed that an aldehyde group at the 4-
position of the pyridine moiety in 7c resulted in enhanced antag-
onistic activity at the hP2X3 receptor [37]. Therefore, we employed
an aldehyde group at the 4-position (Table 4). Also, modifications
were made at the 3-position with propionic acid or propenoic acid
to match the corresponding spacer for the phosphate in the parent
compound 7a. Generally, propenoic acid showed dramatically
increased antagonistic activity for hP2X3 receptor than propionic
acid analogs (36h vs 41h, 36j vs 41j). The effect of 2-position sub-
stituents was similar to the results shown in Table 2, in which the 3-
phenoxyphenyl group showed greater activity than did naphthyl
groups (36j vs 36h, 41j vs 41h). To assess the effects of aldehyde at
the 4-position of pyridine, the aldehyde groups of 36j and 41j were
further oxidized to prepare 37j and 42j, respectively. As expected,
the 4-carboxaldehyde derivatives, 36j and 41j had 2- and 6-fold
greater antagonistic activity than the corresponding carboxylic
acid derivatives, 37j and 42j, respectively. To investigate the effects
of the spacer between the pyridine ring and the 3-position of car-
boxylic acid, we attempted to synthesize 4-aldehyde-3-
carboxypyridine derivatives which had no carbon-chain. Howev-
er, the oxidation of compound 45 to aldehyde resulted in the
generation of pyridine-hydroxylactone analogs, 46 via intra-
molecular cyclization (Scheme 5). The antagonistic potency of
these two compounds, 46h and 46j, was significantly reduced
(IC50 ¼ 856 and 652 nM), respectively.
compounds
n
R2
mP2Xa1
hP2Xa3
hP2Xd7
13h
13i
13j
13k
13l
13m
13n
1
1
1
2
0
1
1
eC10H7
eCH(C6H5)2
48.7 ꢁ 3.1%b 1250 ꢁ 41 nM
Inactive
Inactive
Inactive
Inactive
42.0 ꢁ 6.6%b 824 ꢁ 67 nM
eC6H4-m-O-C6H5 41.8 ꢁ 8.9%b 474 ꢁ 23 nM
eC6H4-m-O-C6H5 38.3 ꢁ 2.5%b 587 ꢁ 42 nM
eC6H4-m-O-C6H5 49.7 ꢁ 4.1%b 3300 ꢁ 640 nM Inactive
eC6H4-p-O-C6H5 40.0 ꢁ 2.6%b 1050 ꢁ 196 nM Inactive
eC6H4-o-O-C6H5 49.6 ꢁ 3.5%b 2930 ꢁ 525 nM Inactive
aThe ion current was induced by 2
expressed in Xenopus oocytes, and the percentage of inhibition of the ion current by
mM ATP at the recombinant P2X receptors
10 m
Mb or IC50 valuesc of compounds was measured for mP2X1 and hP2X3 receptors,
respectively (mean ꢁ SEM, n ꢂ 3). dThe accumulation of ethidiumþ was induced by
6 mM BzATP at the hP2X7 receptors expressed in HEK293 cells and % inhibition of the
accumulation by 10
mM compounds was measured (mean ꢁ SEM, n ꢂ 3).
with n ¼ 2, whereas the potency of compound 13l with n ¼ 0 was
significantly reduced. These results suggest that antagonistic activity
for hP2X3 receptors can be optimized by placing the phenoxy group
at the m-position of the phenyl group with a carbon chain length of 1,
linking to the 2 position of pyridine moiety.
We also investigated the effects of other linkages, including
ether and amide bonds, at the 2-positon of the pyridine moiety
(Table 3). Compound 13o with an ether linkage showed very weak
The subtype selectivity profiles of the series of 4-aldehyde de-
rivatives in Table 4 for hP2X3 receptor vs mP2X1 and hP2X7 re-
ceptors were also significantly improved. For example, the most
potent antagonists, 36h and 36j with IC50 values of 145 and 60 nM
at hP2X3 receptor, respectively, showed only 15w48% inhibition
antagonism for hP2X3 receptors even at 10
mM concentrations.
Compound 25a with amide linkage had similar antagonistic ac-
tivity (IC50 ¼ 723 nM) compared with compound 13b having
carbon-carbon linker. However, the combination of m-phenox-
yphenyl group and amide linkage (25b) showed reduced antago-
nism at hP2X3 receptors, compared with compound 13k. Since
compounds 20 and 21 were simultaneously obtained in the Diels-
Alder reaction with compound 19 in Scheme 3, we could evaluate
the importance of 5-hydroxyl group in the pyridine ring for the
towards mP2X1 and hP2X7 receptors at concentrations of 10 mM.
Furthermore, the heterogeneously expressed rP2X2/3 receptors,
which are more physiologically relevant, were also effectively
inhibited by compound 36j with parallel potency at hP2X3 receptor
(data shown in Figure S1 of Supporting information). These results
indicate that our designing strategies to improve potency and
selectivity were successfully employed.
Table 3
Antagonistic effects of 3,4-dicarboxypyridine derivatives with various modifications of pyridine moiety for hP2X3 and hP2X7 receptors.
Compounds
R1
R2
R3
hP2Xa3
hP2Xd7
13b
13o
25a
27
13k
25b
eCH2C6H5
eO-CH2C6H5
797 ꢁ 99 nMc
23.1 ꢁ 2.9%b
723 ꢁ 100 nMc
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
eC6H5
eC6H5
eOH
eH
eCH2C6H4-m-O-C6H5
587 ꢁ 42 nMc
eC6H4-m-O-C6H5
eOH
928 ꢁ 82 nMc
aThe ion current was induced by 2
m
M ATP at the recombinant P2X receptors expressed in Xenopus oocytes, and the percentage of inhibition of the ion current by 10
m
Mb or IC50
valuesc compounds was measured for hP2X3 receptors, respectively (mean ꢁ SEM, n ꢂ 3). dThe accumulation of ethidiumþ was induced by 6
mM BzATP at the hP2X7 receptors
expressed in HEK293 cells and % inhibition of the accumulation by 10
m
M compounds was measured (mean ꢁ SEM, n ꢂ 3).

818
J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
Table 4
Antagonistic effects of 4-aldehyde-3-carboxypyridine derivatives at hP2X3 and
hP2X7 receptors.
Compounds X
R
mP2Xa1
hP2Xa3
hP2Xd7
36h
41h
36j
41j
37j
42j
46h
46j
eH eC10H7
eH eC10H7
22.0 ꢁ 8.0%b 145 ꢁ 41 nMc 48.4 ꢁ 3.8%
e
636 ꢁ 86 nMc 43.4 ꢁ 5.3%
eH eC6H4-m-O-C6H5 18.3 ꢁ 5.8%b 60 ꢁ 25 nMc 15.1 ꢁ 5.0%
eH eC6H4-m-O-C6H5 23.0 ꢁ 5.3%b 366 ꢁ 63 nMc 10.2 ꢁ 6.2%
eOH eC6H4-m-O-C6H5 33.7 ꢁ 3.8%b 377 ꢁ 29 nMc Inactive
eOH eC6H4-m-O-C6H5 31.7 ꢁ 5.9%b 638 ꢁ 73 nMc Inactive
eC10H7
e
856 ꢁ 76 nMc 14.8 ꢁ 6.1%
eC6H4-m-O-C6H5 45.0 ꢁ 7.0%b 652 ꢁ 60 nMc 27.1 ꢁ 10.6%
Fig. 2. Inhibition effects of pain signals (Long-Term Potentiation) in rat dorsal horn.
The optical signal is monitored in the spinal dorsal horn stained with the voltage
sensitive dye (Di-4-ANEPPS). The optical signal is analyzed and calculated as % inhi-
bition of LTP by the compound treatment. Data represents means ꢁ SD with 4 different
samples (significant versus Pregabalin/Compound, *p < 0.05, **p < 0.01).
aThe ion current was induced by 2
m
M ATP at the recombinant P2X receptors
expressed in Xenopus oocytes, and the percentage of inhibition of the ion current by
10 m
Mb or IC50 valuesc of compounds was measured for mP2X1 and hP2X3 receptors,
respectively (mean ꢁ SEM, n ꢂ 3). dThe accumulation of ethidiumþ was induced by
6 mM BzATP at the hP2X7 receptors expressed in HEK293 cells and % inhibition of the
accumulation by 10
m
M compounds was measured (mean ꢁ SEM, n ꢂ 3).
As a preliminary animal experiment for the anti-pain signaling
activities of new P2X3 receptor antagonists, an ex-vivo assay system
was employed in the rat dorsal horn, which is involved in neural
pain mechanisms. Compounds 13j, 36j, 41j, and 46j having 3-
phenoxyphenyl group at 2-position of pyridine were selected
along with pregabalin as a positive control for the ex-vivo tests to
investigate the relevance between the results of in-vitro and ex-vivo
assay systems. The latter may be affected by the polarity of sub-
stituents at 3 and 4 position due to the different permeability in rat
dorsal horn. As the results, the profile of hP2X3 receptor antago-
nism of compounds in the TEVC assay system was similar to their
efficacy in the ex-vivo assay system. Moreover, most of the antag-
4. Experimental section
4.1. Chemistry
All reagents and solvents were purchased from commercial
suppliers and used without further purification. Proton nuclear
magnetic resonance spectroscopy was performed on a JEOL JNM-LA
300WB spectrometer at 300 MHz or JEOL JNM-ECX 400P spec-
trometer at 400 MHz, and spectra were taken in CDCl3 or 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,
integration, multiplicity (s, singlet; d, doublet; t, triplet; m, multi-
plet; br, broad; app., apparent), and coupling constants. Mass
spectroscopy was carried out on MALDI-TOF and Electrospray
ionization (ESI) instruments.
onists showed greater efficacy than pregabalin at 20 mM concen-
trations (Fig. 2). Compound 36j, the most potent and selective
of the hP2X3 receptor antagonists, inhibited the pain signal
65%, compared with 31% for pregabalin. These findings show
that the optimized P2X3 receptor antagonists, derived from
the parent PPADS analogs (7aec) have sufficient potential to be
antinociceptive agents with in-vivo efficacy.
The purity of all final compounds was determined by HPLC (at
least 95% purity unless otherwise noted). The determination of
purity was performed on
system using Shimadzu Shim-pack C18 analytical column
(250 mm ꢃ 4.6 mm, 5
a Shimadzu SCL-10A VP HPLC
a
ꢀ
3. Conclusions
mm, 100 A) in two solvent systems. The
solvent systems were H2O: CH3CN ¼ 70:30 or 0.1% TFA (Trifluoro-
acetic acid) in H2O: CH3CN ¼ 65:35 to 5:95 for 30 min with flow
rate 1 mL/min.
In this study, we successfully optimized a nonselective P2X re-
ceptor antagonist, 7a (PPADS), generating potent and selective
hP2X3 receptor antagonists, by chemical modifications and SAR
studies. These included the replacement of the unstable azo (eN]
Ne) linker in 7aec with stable carbon-carbon linkers, and sub-
stitutions with 4-carboxaldehyde, 3-propenoic acid and bulky hy-
drophobic aromatic groups at the 2-positon. SAR analysis suggested
that the 5-hydroxyl group of pyridine was essential for the antag-
onism and that bulky aromatic groups at the 2-position were very
important for selectivity toward hP2X3 receptors. The optimized
compounds, including the most potent and selective hP2X3 re-
ceptor antagonist, 36j, with an IC50 value of 60 nM, also signifi-
cantly inhibited pain signaling in the rat dorsal horn, suggesting
that the carboxypyridine derivatives developed in this study may
have the potential to contribute to the development of pain mod-
ulators targeting P2X3 receptors.
Compound 1 and KN-62 were purchased from Sigma-Aldrich.
Compound 7b was prepared according to literature procedures
[36].
4.1.1. General procedure for the preparation of compounds (10aeo)
To a suspension of various carboxylic acid 9aeo (1.0 equiv) and
L-alanine ethyl ester (8, 1.0 equiv) in dichloromethane was treated
with EDC (2.0 equiv). Triethylamine (1.0 equiv) was added drop
wise to the mixture for a period of 30 min. After this mixture was
stirred for 12 h at room temperature, it was neutralized with
saturated aqueous sodium bicarbonate and extracted with
dichloromethane twice. The combined organic layer was dried with
anhydrous Na2SO4. After Na2SO4 was filtered, solvent was removed
in vacuum. The residue was purified by silica gel column

J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
819
chromatography with n-hexanes/ethyl acetate ¼ 1:1 to afford 10ae
o as sticky oil.
10g (5.8 g). Yield 59%; 1H NMR (CDCl3, 300 MHz)
d
1.25 (3H, t,
J ¼ 7.2 Hz, CH3), 1.33 (3H, d, J ¼ 6.9 Hz, CH3), 2.48 (2H, t, J ¼ 5.1 Hz,
CH2), 2.89 (2H, t, J ¼ 5.1 Hz, CH2), 3.78 (3H, s, COOCH3), 4.15 (2H, q,
J ¼ 7.2 Hz, CH2), 4.49e4.52 (1H, m, CH), 5.95 (1H, d, J ¼ 5.4 Hz, NH),
6.81 (2H, d, J ¼ 7.2 Hz, phenyl), 7.10 (2H, d, J ¼ 7.2 Hz, phenyl); MS
(ESI): m/z ¼ 307.5 [M þ H].
4.1.1.1. Ethyl 2-(2-phenylacetamido)propanoate (10a). Following the
general procedure for the synthesis of (10aeo), coupling reaction of
8 (20 g, 0.13 mol) and phenyl acetic acid (20 g, 0.15 mol) afforded 10a
(27 g). Yield 96%; 1H NMR (CDCl3, 300 MHz)
d
1.22 (3H, t, J ¼ 7.2 Hz,
CH3), 1.33 (3H, d, J ¼ 7.2 Hz, CH3), 3.59 (2H, s, CH2), 4.13 (2H, q,
J ¼ 7.2 Hz, CH2), 4.54 (1H, q, J ¼ 7.2 Hz, CH), 6.12 (1H, d, J ¼ 6.3 Hz,
NH), 7.30e35 (5H, m, phenyl); MS (ESI): m/z ¼ 236.1 [M þ H].
4.1.1.8. Ethyl 2-(2-(naphthalen-2-yl)acetamido)propanoate (10h).
Following the general procedure for the synthesis of 10aeo,
coupling reaction of 8 (3.0 g, 19 mmol) and 2-naphthylacetic acid
(4.0 g, 21 mmol) afforded 10h (4.1 g). Yield 74%; 1H NMR (CDCl3,
4.1.1.2. Ethyl
2-(3-phenylpropanamido)propanoate
(10b).
400 MHz)
d
1.20 (3H, t, J ¼ 7.2 Hz, CH3), 1.31 (3H, d, J ¼ 7.2 Hz, CH3),
Following the general procedure for the synthesis of 10aeo,
coupling reaction of 8 (7.5 g, 48 mmol) and 3-phenylpropionic acid
(11 g, 73 mmol) afforded 10b (11 g). Yield 92%; 1H NMR (CDCl3,
3.74 (2H, s, CH2), 4.12 (2H, q, J ¼ 7.2 Hz, CH2), 4.55e4.62 (1H, m, CH),
6.01 (1H, d, J ¼ 6.8 Hz, NH), 7.38e7.50 (3H, m, naphthyl), 7.73e7.85
(4H, m, naphthyl); MS (ESI): m/z ¼ 285.8 [M þ H].
300 MHz)
d
1.24 (3H, t, J ¼ 7.2 Hz, CH3), 1.32 (3H, d, J ¼ 7.2 Hz, CH3),
2.48 (2H, t, J ¼ 7.2 Hz, CH2), 2.94 (2H, t, J ¼ 7.2 Hz, CH2), 4.14 (2H, q,
J ¼ 7.2 Hz, CH2), 4.53e4.58 (1H, m, CH), 6.12 (1H, d, J ¼ 6.6 Hz, NH),
7.18e28 (5H, m, phenyl); MS (MALDI-TOF): m/z ¼ 248.4 [M þ H].
4.1.1.9. Ethyl
2-(3,3-diphenylpropanamido)propanoate
(10i).
Following the general procedure for the synthesis of 10aeo,
coupling reaction of 8 (3.0 g, 19 mmol) and 3,3-diphenylpropanoic
acid (4.8 g, 21 mmol) afforded 10i (4.1 g). Yield 65%; 1H NMR (CDCl3,
4.1.1.3. Ethyl 2-cinnamamidopropanoate (10c). Following the gen-
eral procedure for the synthesis of 10aeo, coupling reaction of 8
(5.0 g, 32 mmol) and cinnamic acid (5.6 g, 38 mmol) afforded 10c
400 MHz)
d
1.12 (3H, d, J ¼ 7.2 Hz, CH3), 1.22 (3H, t, J ¼ 7.2 Hz, CH3),
2.67e2.99 (2H, m, CH2), 4.12 (2H, q, J ¼ 7.2 Hz, CH2), 4.42e4.48 (1H,
m, CH), 4.55e4.59 (1H, m, CH), 5.80 (1H, d, J ¼ 7.2 Hz, NH), 7.15e
7.29 (10H, m, phenyl); MS (ESI): m/z ¼ 325.7 [M þ H].
(7.6 g). Yield 95%; 1H NMR (CDCl3, 300 MHz)
d
1.30 (3H, t, J ¼ 7.2 Hz,
CH3), 1.49 (3H, d, J ¼ 6.9 Hz, CH3), 4.22 (2H, q, J ¼ 7.2 Hz, CH2), 4.70e
4.75 (1H, m, CH), 6.27 (1H, d, J ¼ 6.0 Hz, NH), 6.43 (1H, d, J ¼ 15.6 Hz,
]CH), 7.35e7.39 (3H, m, phenyl), 7.48e7.52 (2H, m, phenyl), 7.63
(1H, d, J ¼ 15.6 Hz, ]CH); MS (ESI): m/z ¼ 248.2 [M þ H].
4.1.1.10. Ethyl 2-(2-(3-phenoxyphenyl)acetamido)propanoate (10j).
Following the general procedure for the synthesis of 10aeo,
coupling reaction of 8 (3.0 g, 19 mmol) and 3-phenoxyphenylacetic
acid (5.0 g, 21 mol) afforded 10j (7.3 g). Yield 95%; 1H NMR (CDCl3,
4.1.1.4. 2-[3-(4-Methoxy-phenyl)-propionylamino]-propionic acid ethyl
ester (10d). Following the general procedure for the synthesis of
400 MHz)
d
1.26 (3H, t, J ¼ 7.2 Hz, CH3) 1.34 (3H, d, J ¼ 6.8 Hz, CH3),
3.56 (2H, s, CH2), 4.17 (2H, q, J ¼ 7.2 Hz, CH2), 4.56 (1H, q, J ¼ 6.8 Hz,
CH), 6.04 (1H, d, J ¼ 4.8 Hz, NH), 6.94 (2H, s, phenyl), 7.03 (2H, d,
J ¼ 7.6 Hz, phenyl), 7.12 (1H, t, J ¼ 7.6 Hz, phenyl), 7.25e7.35 (4H, m,
phenyl); MS (ESI): m/z ¼ 328.3 [M þ H].
10aeo, coupling reaction of
methoxyphenyl)propionic acid (6.8 g, 37 mmol) afforded 10d
(7.5 g). Yield 81%; 1H NMR (CDCl3, 300 MHz)
8 (5.0 g, 33 mmol) and 3-(4-
d
1.25 (3H, t, J ¼ 7.2 Hz,
CH3), 1.33 (3H, d, J ¼ 6.9 Hz, CH3), 2.48 (2H, t, J ¼ 5.1 Hz, CH2), 2.89
(2H, t, J ¼ 5.1 Hz, CH2), 3.78 (3H, s, CH3O), 4.15 (2H, q, J ¼ 7.2 Hz, CH2),
4.52e4.58 (1H, m, CH), 5.95 (1H, d, J ¼ 5.4 Hz, NH), 6.81 (2H, d,
J ¼ 7.2 Hz, phenyl), 7.10 (2H, d, J ¼ 7.2 Hz, phenyl); MS (MALDI-TOF):
m/z ¼ 279.3 [M þ H].
4.1.1.11. Ethyl
2-(3-(3-phenoxyphenyl)propanamido)propanoate
(10k). Following the general procedure for the synthesis of 10aeo,
coupling reaction of 8 (3.0 g, 19 mmol) and 3-(3-phenoxyphenyl)
propionic acid (5.0 g, 21 mmol) afforded 10k (6.5 g). Yield 96%; 1H
NMR (CDCl3, 400 MHz)
d
1.27 (3H, t, J ¼ 7.6 Hz, CH3), 1.34 (3H, d,
4.1.1.5. 2-[3-(3-Methoxy-phenyl)-propionylamino]-propionic
acid
J ¼ 7.2 Hz, CH3), 2.48 (2H, t, J ¼ 7.2 Hz, CH2), 2.94 (2H, t, J ¼ 7.2 Hz,
CH2), 4.18 (2H, q, J ¼ 7.6 Hz, CH2), 4.53e4.56 (1H, m, CH), 5.99 (1H,
d, J ¼ 6.0 Hz, NH), 6.85 (2H, s, phenyl), 6.95 (2H, d, J ¼ 7.6 Hz,
phenyl), 7.10 (1H, t, J ¼ 7.6 Hz, phenyl), 7.23e7.35 (4H, m, phenyl);
MS (ESI): m/z ¼ 342.1 [M þ H].
ethyl ester (10e). Following the general procedure for the synthesis
of 10aeo, coupling reaction of 8 (7.1 g, 46 mmol) and 3-(3-
methoxyphenyl)propionic acid (10 g, 56 mmol) afforded 10e
(8.6 g). Yield 67%; 1H NMR (CDCl3, 300 MHz)
d
1.22 (3H, t, J ¼ 7.2 Hz,
CH3), 1.35 (3H, d, J ¼ 7.2 Hz, CH3), 2.51 (2H, t, J ¼ 6.9 Hz, CH2), 2.93
(2H, t, J ¼ 6.9 Hz, CH2), 3.80 (3H, s, CH3O), 4.17 (2H, q, J ¼ 7.2 Hz,
CH2), 4.54e4.59 (1H, m, CH), 5.99 (1H, d, J ¼ 5.4 Hz, NH), 6.73e6.80
(2H, m, phenyl), 7.17e7.22 (2H, m, phenyl); MS (ESI): m/z ¼ 280.3
[M þ H].
4.1.1.12. Ethyl
2-(3-phenoxybenzamido)propanoate
(10l).
Following the general procedure for the synthesis of 10aeo,
coupling reaction of 8 (2.6 g, 17 mmol) and 3-phenoxybenzoic acid
(4.0 g, 19 mmol) afforded 10l (5.9 g). Yield 99%; 1H NMR (CDCl3,
400 MHz)
d
1.29 (3H, t, J ¼ 7.2 Hz, CH3), 1.48 (3H, d, J ¼ 7.6 Hz, CH3),
4.1.1.6. Ethyl 2-(3-(2-methoxyphenyl)propanamido)propanoate (10f).
Following the general procedure for the synthesis of 10aeo,
coupling reaction of 8 (3.0 g, 20 mmol) and 3-(2-methoxyphenyl)
propionic acid (4.1 g, 22 mmol) afforded 10f (4.6 g). Yield 82%; 1H
4.22 (2H, q, J ¼ 7.2 Hz, CH2), 4.70e4.78 (1H, m, CH), 6.69 (1H, d,
J ¼ 6.1 Hz, NH), 7.00 (2H, d, J ¼ 7.6 Hz, phenyl), 7.11 (2H, t, J ¼ 7.6 Hz,
phenyl), 7.33e7.41 (3H, m, phenyl), 7.45 (2H, t, J ¼ 7.6 Hz, phenyl);
MS (ESI): m/z ¼ 314.1 [M þ H].
NMR (CDCl3, 300 MHz)
d
1.22 (3H, t, J ¼ 7.2 Hz, CH3), 1.35 (3H, d,
J ¼ 7.2 Hz, CH3), 2.53 (2H, t, J ¼ 6.9 Hz, CH2), 2.88 (2H, t, J ¼ 6.9 Hz,
CH2), 3.79 (3H, s, CH3O), 4.15 (2H, q, J ¼ 7.2 Hz, CH2), 4.52e4.59 (1H,
m, CH), 5.96 (1H, d, J ¼ 5.4 Hz, NH), 6.82e7.19 (4H, m, phenyl); MS
(ESI): m/z ¼ 280.4 [M þ H].
4.1.1.13. Ethyl 2-(2-(4-phenoxyphenyl)acetamido)propanoate (10m).
Following the general procedure for the synthesis of 10aeo,
coupling reaction of 8 (0.6 g, 4.0 mmol) and 4-phenoxyphenylacetic
acid (1.0 g, 4.4 mmol) afforded 10m (1.4 g). Yield 99%; 1H NMR
(CDCl3, 400 MHz)
d
1.26 (3H, t, J ¼ 7.2 Hz, CH3), 1.36 (3H, d,
4.1.1.7. 4-[2-(1-Ethoxycarbonyl-ethylcarbamoyl)-ethyl]-benzoic acid
methyl ester (10g). Following the general procedure for the syn-
thesis of 10aeo, coupling reaction of 8 (4.9 g, 32 mmol) and 3-(4-
(methoxycarbonyl)phenyl)propionic acid (7.9 g, 38 mmol) afforded
J ¼ 7.2 Hz, CH3), 3.55 (2H, s, CH2), 4.18 (2H, q, J ¼ 7.2 Hz, CH2), 4.54e
4.61 (1H, m, CH), 5.99 (1H, d, J ¼ 6.0 Hz, NH), 6.97e7.02 (4H, m,
phenyl), 7.10 (1H, t, J ¼ 7.2 Hz, phenyl), 7.23e7.35 (4H, m, phenyl);
MS (ESI): m/z ¼ 342.1 [M þ H].

820
J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
4.1.1.14. Ethyl 2-(2-(2-phenoxyphenyl)acetamido)propanoate (10n).
Following the general procedure for the synthesis of 10aeo,
coupling reaction of 8 (3.0 g, 19 mmol) and 2-phenoxyphenylacetic
acid (5.0 g, 21 mmol) afforded 10n (6.8 g). Yield 95%; 1H NMR
4.1.2.5. 5-Ethoxy-2-[2-(3-methoxy-phenyl)-ethyl]-4-methoxy-oxa-
zole (11e). Following the general procedure for the synthesis of
11aeo, the compound 10e (3.9 g, 14 mmol) was reacted with
phosphorous pentoxide (7.9 g, 28 mmol) to afford 11e (3.6 g). Yield
(CDCl3, 400 MHz)
d
1.21 (3H, t, J ¼ 7.2 Hz, CH3), 1.28 (3H, d,
99%; 1H NMR (CDCl3, 300 MHz)
d
1.34 (3H, t, J ¼ 6.9 Hz, CH3), 2.04
J ¼ 7.2 Hz, CH3), 3.62 (2H, s, CH2), 4.13 (2H, q, J ¼ 7.2 Hz, CH2), 4.46e
4.53 (1H, m, CH), 6.34 (1H, d, J ¼ 6.8 Hz, NH), 6.89 (1H, d, J ¼ 8.0 Hz,
phenyl), 6.99 (2H, d, J ¼ 8.0 Hz, phenyl), 7.10 (2H, q, J ¼ 6.4 Hz,
phenyl), 7.23e7.25 (1H, m, phenyl), 7.31e7.39 (3H, m, phenyl); MS
(ESI): m/z ¼ 328.8 [M þ H].
(3H, s, CH3), 2.90 (2H, t, J ¼ 6.9 Hz, CH2), 3.01 (2H, t, J ¼ 6.9 Hz, CH2),
3.81 (3H, s, CH3O), 4.31 (2H, q, J ¼ 6.9 Hz, CH2), 6.74e6.81 (3H, m,
phenyl), 7.17e7.24 (1H, m, phenyl); MS (ESI): m/z ¼ 262.3 [M þ H].
4.1.2.6. 5-Ethoxy-2-(2-methoxyphenethyl)-4-methyl-oxazole (11f).
Following the general procedure for the synthesis of 11aeo, the
compound 10f (4.6 g, 16 mmol) was reacted with phosphorous
pentoxide (9.3 g, 32 mmol) to afford 11f (4.1 g). Yield 95%; 1H NMR
4.1.1.15. Ethyl
2-(2-(benzyloxy)acetamido)propanoate
(10o).
Following the general procedure for the synthesis of (10aeo),
coupling reaction of 8 (4.2 g, 27 mmol) and benzyloxyacetic acid
(5.0 g, 30 mmol) afforded 10o (6.0 g). Yield 83%; 1H NMR (CDCl3,
(CDCl3, 300 MHz)
d
1.32 (3H, t, J ¼ 6.9 Hz, CH3), 2.03 (3H, s, CH3),
2.90 (2H, t, J ¼ 6.9 Hz, CH2), 3.02 (2H, t, J ¼ 6.9 Hz, CH2), 3.92 (3H, s,
CH3O), 4.32 (2H, q, J ¼ 6.9 Hz, CH2), 6.76e7.32 (4H, m, phenyl); MS
(MALDI-TOF): m/z ¼ 262.3 [M þ H].
400 MHz)
d
1.29 (3H, t, J ¼ 6.8 Hz, CH3), 1.43 (3H, d, J ¼ 7.2 Hz, CH3),
4.00 (2H, s, CH2), 4.23 (2H, q, J ¼ 6.8 Hz, CH2), 4.57e4.66 (3H, m, CH2
& CH), 7.13 (1H, d, J ¼ 4.8 Hz, NH), 7.33e7.41 (5H, m, phenyl); MS
(ESI): m/z ¼ 266.1 [M þ H].
4.1.2.7. 4-[2-(5-Ethoxy-4-methyl-oxazole-2-yl)-ethyl]-benzoic acid
methyl ester (11g). Following the general procedure for the syn-
thesis of 11aeo, the compound 10g (5.8 g, 19 mmol) was reacted
with phosphorous pentoxide (11 g, 38 mmol) to afford 11g (2.1 g).
4.1.2. General procedure for the preparation compounds (11aeo)
A mixture of 10aeo (1.0 equiv) and phosphorous pentoxide
(2.0 equiv) in anhydrous chloroform was refluxed for 4 h. After
reaction mixture was cooled to room temperature, it was treated
with 20% KOH aqueous solution and stirred vigorously for 30 min.
The mixture was extracted with dichloromethane three times.
The organic layers were combined and treated with anhydrous
Na2SO4. After filtered off, organic layers were concentrated in
vacuum and the residue was purified by silica gel column chro-
matography with hexanes: ethyl acetate ¼ 3:1 to afford 11aeo as
sticky oil.
Yield 39%; 1H NMR (CDCl3, 300 MHz)
d
1.32 (3H, t, J ¼ 6.0 Hz, CH3),
2.01 (3H, s, CH3), 2.91 (2H, t, J ¼ 5.4 Hz, CH2), 3.04 (2H, t, J ¼ 5.4 Hz,
CH2), 3.91 (3H, s, COOCH3), 4.06 (2H, q, J ¼ 6.0 Hz, CH2), 7.27 (2H, d,
J ¼ 8.1 Hz, phenyl), 7.95 (2H, d, J ¼ 8.1 Hz, phenyl); MS (MALDI-TOF):
m/z ¼ 290.4 [M þ H].
4.1.2.8. 5-Ethoxy-4-methyl-2-(naphthalen-2-ylmethyl)oxazole (11h).
Following the general procedure for the synthesis of 11aeo, the
compound 10h (4.1 g, 14 mmol) was reacted with phosphorous
pentoxide (7.9 g, 28 mmol) to afford 11h (3.8 g). Yield 98%; 1H NMR
4.1.2.1. 2-Benzyl-5-ethoxy-4-methyl-oxazole (11a). Following the
general procedure for the synthesis of 11aeo, the compound 10a
(27 g, 0.11 mol) was reacted with phosphorous pentoxide (62 g,
0.22 mol) to afford 11a (25 g). Yield 99%; 1H NMR (CDCl3, 300 MHz)
(CDCl3, 400 MHz)
d
1.29 (3H, t, J ¼ 6.8 Hz, CH3), 2.01 (3H, s, CH3),
4.04 (2H, q, J ¼ 6.8 Hz, CH2), 4.10 (2H, s, CH2), 7.39e7.46 (3H, m,
naphthyl), 7.71e7.81 (4H, m, naphthyl); MS (MALDI-TOF): m/
z ¼ 268.4 [M þ H].
d
1.29 (3H, t, J ¼ 7.2 Hz, CH3), 2.01 (3H, s, CH3), 3.94 (2H, s, CH2), 4.05
(2H, q, J ¼ 7.2 Hz, CH2), 7.23e34 (5H, m, phenyl); MS (MALDI- TOF):
m/z ¼ 216.9 [M þ H].
4.1.2.9. 2-(2,2-Diphenylethyl)-5-ethoxy-4-methyl-oxazole
(11i).
Following the general procedure for the synthesis of 11aeo, the
compound 10i (4.1 g, 12 mmol) was reacted with phosphorous
pentoxide (7.1 g, 24 mmol) to afford 11i (4.0 g). Yield 99%; 1H NMR
4.1.2.2. 5-Ethoxy-4-methyl-2-phenethyl-oxazole (11b). Following the
general procedure for the synthesis of 11aeo, the compound 10b
(7.0 g, 28 mmol) was reacted with phosphorous pentoxide (16 g,
56 mmol) to afford 11b (6.3 g). Yield 97%; 1H NMR (CDCl3, 300 MHz)
(CDCl3, 400 MHz)
d
1.20 (3H, t, J ¼ 7.2 Hz, CH3), 1.92 (3H, s, CH3),
3.33 (2H, d, J ¼ 8.4 Hz, CH2), 3.93 (2H, q, J ¼ 7.2 Hz, CH2), 4.53 (1H, t,
J ¼ 8.4 Hz, CH), 7.15e7.28 (10H, m, phenyl); MS (ESI): m/z ¼ 307.8
[M þ H].
d
1.34 (3H, t, J ¼ 6.0 Hz, CH3), 2.02 (3H, s, CH3), 2.45 (2H, t, J ¼ 6.0 Hz,
CH2), 2.90 (2H, t, J ¼ 6.0 Hz, CH2), 4.02 (2H, q, J ¼ 6.0 Hz, CH2), 7.18e
7.32 (5H, m, phenyl); MS (MALDI-TOF): m/z ¼ 232.0 [M þ H].
4.1.2.10. 5-Ethoxy-4-methyl-2-(3-phenoxybenzyl)oxazole
(11j).
Following the general procedure for the synthesis of 11aeo, the
compound 10j (7.2 g, 22 mmol) was reacted with phosphorous
pentoxide (13 g, 44 mmol) to afford 11j (6.2 g). Yield 92%; 1H NMR
4.1.2.3. 5-Ethoxy-4-methyl-2-styryl-oxazole (11c). Following the
general procedure for the synthesis of 11aeo, the compound 10c
(7.8 g, 32 mmol) was reacted with phosphorous pentoxide (18 g,
64 mmol) to afford 11c (6.4 g). Yield 90%; 1H NMR (CDCl3, 300 MHz)
(CDCl3, 400 MHz)
d
1.31 (3H, t, J ¼ 7.2 Hz, CH3), 2.01 (3H, s, CH3),
3.92 (2H, s, CH2), 4.07 (2H, q, J ¼ 7.2 Hz, CH2), 6.87e6.95 (3H, m,
phenyl), 7.02 (2H, d, J ¼ 7.6 Hz, phenyl), 7.12 (1H, t, J ¼ 7.2 Hz,
phenyl), 7.25e7.35 (3H, m, phenyl); MS (ESI): m/z ¼ 310.3 [M þ H].
d
1.36 (3H, t, J ¼ 7.2 Hz, CH3), 2.11 (3H, s, CH3), 4.30 (2H, q, J ¼ 7.2 Hz,
CH2), 6.45 (1H, d, J ¼ 15.9 Hz, ]CH), 6.76 (1H, d, J ¼ 15.9 Hz, ]CH),
7.28e7.41 (3H, m, phenyl), 7.52e7.54 (2H, m, phenyl); MS (ESI): m/
z ¼ 230.3 [M þ H].
4.1.2.11. 5-Ethoxy-4-methyl-2-(3-phenoxyphenethyl)oxazole (11k).
Following the general procedure for the synthesis of 11aeo, the
compound 10k (6.5 g, 20 mmol) was reacted with phosphorous
pentoxide (12 g, 40 mmol) to afford 11k (5.6 g). Yield 86%; 1H NMR
4.1.2.4. 5-Ethoxy-2-(4-methoxyphenethyl)-4-methyl-oxazole (11d).
Following the general procedure for the synthesis of 11aeo, the
compound 10d (7.5 g, 27 mmol) was reacted with phosphorous
pentoxide (15 g, 54 mmol) to afford 11d (6.4 g). Yield 91%; 1H NMR
(CDCl3, 400 MHz)
d
1.33 (3H, t, J ¼ 7.2 Hz, CH3), 2.00 (3H, s, CH3),
2.87e2.91 (2H, m, CH2), 2.99e3.02 (2H, m, CH2), 4.07 (2H, q,
J ¼ 7.2 Hz, CH2), 6.85e6.87 (2H, m, phenyl), 6.94 (1H, d, J ¼ 7.6 Hz,
phenyl), 6.98 (2H, d, J ¼ 7.6 Hz, phenyl), 7.11 (1H, t, J ¼ 7.2 Hz,
phenyl), 7.23e7.26 (2H, m, phenyl), 7.32 (1H, t, J ¼ 7.6 Hz, phenyl);
MS (ESI): m/z ¼ 324.1 [M þ H].
(CDCl3, 300 MHz)
d
1.31 (3H, t, J ¼ 7.2 Hz, CH3), 2.01 (3H, s, CH3),
2.83 (2H, t, J ¼ 6.0 Hz, CH2), 2.90 (2H, t, J ¼ 6.0 Hz, CH2), 4.05 (3H, s,
OCH3), 4.32 (2H, q, J ¼ 7.2 Hz, CH2), 6.81 (2H, d, J ¼ 8.7 Hz, phenyl),
7.10 (2H, d, J ¼ 8.7 Hz, phenyl); MS (ESI): m/z ¼ 262.4 [M þ H].

J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
821
4.1.2.12. 5-Ethoxy-4-methyl-2-(3-phenoxyphenyl)oxazole
(11l).
(3H, s, CH3O), 7.20e7.24 (3H, m, phenyl), 7.26e7.29 (2H, m, phenyl);
MS (ESI): m/z ¼ 330.4 [M þ H].
Following the general procedure for the synthesis of 11aeo, the
compound 10l (5.9 g, 19 mmol) was reacted with phosphorous
pentoxide (11 g, 38 mmol) to afford 11l (2.4 g). Yield 43%; 1H NMR
4.1.3.3. 5-Hydroxy-6-methyl-2-styryl-pyridine-3,4-dicarboxylic acid
dimethyl ester (12c). Following the general procedure for the syn-
thesis of 12aeo, DielseAlder reaction of 11c (4.0 g, 19 mmol) and
dimethyl maleate (4.7 mL, 38 mmol) afforded 12c (1.4 g). Yield 23%;
(CDCl3, 400 MHz)
d
1.38 (3H, t, J ¼ 7.2 Hz, CH3), 2.10 (3H, s, J ¼ 7.6 Hz,
CH3), 4.21 (2H, q, J ¼ 7.2 Hz, CH2), 7.02 (3H, d, J ¼ 8.0 Hz, phenyl),
7.12 (1H, t, J ¼ 7.6 Hz, phenyl), 7.33e7.39 (3H, m, phenyl), 7.57 (1H,
m, phenyl), 7.66 (1H, d, J ¼ 7.6 Hz, phenyl); MS (ESI): m/z ¼ 296.0
[M þ H].
1H NMR (CDCl3, 300 MHz)
d
2.49 (3H, s, CH3), 3.83 (6H, s, 2ꢃ CH3O),
6.82 (1H, d, J ¼ 15.4 Hz, ]CH), 7.19e7.25 (3H, m, phenyl), 7.38 (2H,
d, J ¼ 5.4 Hz, phenyl), 7.72 (1H, t, J ¼ 15.4 Hz, ]CH); MS (ESI): m/
z ¼ 328.2 [M þ H].
4.1.2.13. 5-Ethoxy-4-methyl-2-(4-phenoxybenzyl)oxazole
(11m).
Following the general procedure for the synthesis of 11aeo, the
compound 10m (1.4 g, 4.3 mmol) was reacted with phosphorous
pentoxide (2.4 g, 8.6 mmol) to afford 11m (1.1 g). Yield 84%; 1H NMR
4.1.3.4. 5-Hydroxy-2-[2-(4-methoxy-phenyl)-ethyl]-6-methyl-pyri-
dine-3,4-dicarboxylic acid dimethyl ester (12d). Following the gen-
eral procedure for the synthesis of 12aeo, DielseAlder reaction of
11d (6.0 g, 23 mmol) and dimethyl maleate (5.8 mL, 46 mol)
(CDCl3, 400 MHz)
d
1.34 (3H, t, J ¼ 7.2 Hz, CH3), 2.02 (3H, s, CH3),
3.92 (2H, s, CH2), 4.01 (2H, q, J ¼ 7.2 Hz, CH2), 6.94e7.01 (4H, m,
phenyl), 7.11 (1H, t, J ¼ 7.6 Hz, phenyl), 7.23 (2H, d, J ¼ 8.4 Hz,
phenyl), 7.28 (2H, t, J ¼ 7.6 Hz, phenyl); MS (ESI): m/z ¼ 310.1
[M þ H].
afforded 12d (3.1 g). Yield 37%; 1H NMR (CDCl3, 300 MHz)
d 3.02
(3H, s, CH3), 3.04 (2H, t, J ¼ 4.8 Hz, CH2), 3.32 (2H, t, J ¼ 4.8 Hz, CH2),
3.79 (3H, s, CH3O), 4.00 (3H, s, CH3O), 4.08 (3H, s, CH3O), 6.83 (2H, d,
J ¼ 5.4 Hz, phenyl), 7.27 (1H, d, J ¼ 5.4 Hz, phenyl); MS (ESI): m/
z ¼ 360.4 [M þ H].
4.1.2.14. 5-Ethoxy-4-methyl-2-(2-phenoxybenzyl)oxazole
(11n).
Following the general procedure for the synthesis of 11aeo, the
compound 10n (6.8 g, 21 mmol) was reacted with phosphorous
pentoxide (12 g, 42 mmol) to afford 11n (4.0 g). Yield 59%; 1H NMR
4.1.3.5. 5-Hydroxy-2-[2-(3-methoxy-phenyl)-ethyl]-6-methyl-pyri-
dine-3,4-dicarboxylic acid dimethyl ester (12e). Following the gen-
eral procedure for the synthesis of 12aeo, Diels-Alder reaction of 11e
(2.0 g, 7.6 mmol) and dimethyl maleate (2.0 mL, 15 mmol) afforded
(CDCl3, 400 MHz)
d
1.26 (3H, t, J ¼ 7.2 Hz, CH3), 1.96 (3H, s, CH3),
3.97e4.03 (4H, m, 2ꢃ CH2), 6.89 (1H, d, J ¼ 8.4 Hz, phenyl), 6.92 (2H,
d, J ¼ 7.6 Hz, phenyl), 7.04e7.11 (2H, d, J ¼ 7.6 Hz, phenyl), 7.20 (1H,
m, phenyl), 7.23 (3H, t, J ¼ 8.4 Hz, phenyl); MS (ESI): m/z ¼ 309.8
[M þ H].
12e (0.86 g). Yield 31%; 1H NMR (CDCl3, 300 MHz)
d 3.00 (3H, s, CH3),
3.10 (2H, t, J ¼ 3.9 Hz, CH2), 3.35 (2H, t, J ¼ 3.9 Hz, CH2), 3.86 (3H, s,
CH3O), 3.98 (3H, s, CH3O), 4.10 (3H, s, CH3O), 6.78 (1H, dd, J ¼ 7.5 Hz,
8.1 Hz, phenyl), 6.90 (1H, d, J ¼ 7.5 Hz, phenyl), 7.00 (1H, s, phenyl),
7.21 (1H, t, J ¼ 8.1 Hz, phenyl); MS (ESI): m/z ¼ 360.1 [M þ H].
4.1.2.15. 2-((Benzyloxy)methyl)-5-ethoxy-4-methyl-oxazole
(11o).
Following the general procedure for the synthesis of 11aeo, the
compound 10o (5.9 g, 23 mmol) was reacted with phosphorous
pentoxide (13 g, 45 mmol) to afford 11o (4.4 g). Yield 79%; 1H
4.1.3.6. Dimethyl 5-hydroxy-2-(2-methoxyphenethyl)-6-
methylpyridine-3,4-dicarboxylate (12f). Following the general pro-
cedure for the synthesis of 12aeo, DielseAlder reaction of 11f
(1.5 g, 5.7 mmol) and dimethyl maleate (1.4 mL, 11 mmol) afforded
NMR (CDCl3, 400 MHz)
d
1.34 (3H, t, J ¼ 7.2 Hz, CH3), 2.04 (3H, s,
CH3), 4.16 (2H, q, J ¼ 7.2 Hz, CH2), 4.44 (2H, s, CH2), 4.59 (2H, s,
CH2), 7.35e7.37 (5H, m, phenyl); MS (MALDI-TOF): m/z ¼ 248.3
[M þ H].
12f (0.49 g). Yield 24%; 1H NMR (CDCl3, 300 MHz)
d 2.58 (3H, s,
CH3), 2.92 (2H, t, J ¼ 7.2 Hz, CH2), 2.99 (2H, t, J ¼ 7.2 Hz, CH2), 3.83
(3H, s, CH3O), 3.86 (3H, s, CH3O), 3.94 (3H, s, CH3O), 6.84e7.21 (4H,
m, phenyl), 10.55 (1H, s, OH); MS (ESI): m/z ¼ 340.4 [M þ H].
4.1.3. General procedure for the preparation of compounds (12aeo)
To a neat of 11aeo (1.0 equiv) was added dropwise dimethyl
maleate (2.0 equiv). The mixture was refluxed for 5 h with stirring
and cooled to room temperature. The mixture was diluted using
ethanol and saturated with HCl gas and concentrated in vacuum.
Water was added to the mixture and mixture was extracted with
dichloromethane three times. The organic layers were combined
and dried with anhydrous Na2SO4. After Na2SO4 was filtered, sol-
vent was removed in vacuum and the residue was purified by silica
gel column chromatography with hexanes: ethyl acetate ¼ 7:1 to
afford 12aeo as sticky oil.
4.1.3.7. 5-Hydroxy-2-[2-(4-methoxycarbonyl-phenyl)-ethyl]-6-
methyl-pyridine-3,4-dicarboxylic acid dimethyl ester (12g).
Following the general procedure for the synthesis of 12aeo, Dielse
Alder reaction of 11g (2.1 g, 7.1 mmol) and dimethyl maleate
(1.8 mL, 14 mmol) afforded 12g (0.92 g). Yield 33%; 1H NMR (CDCl3,
300 MHz)
d
2.57 (3H, s, CH3), 2.90 (2H, t, J ¼ 9.3 Hz, CH2), 3.04 (2H, t,
J ¼ 9.3 Hz, CH2), 3.86 (3H, s, CH3O), 3.91 (3H, s, CH3O), 3.95 (3H, s,
COOCH3), 7.25 (2H, d, J ¼ 8.7 Hz, phenyl), 7.94 (2H, d, J ¼ 8.7 Hz,
phenyl); MS (ESI): m/z ¼ 388.4 [M þ H].
4.1.3.1. 2-Benzyl-5-hydroxy-6-methyl-pyridine-3,4-dicarboxylic acid
dimethyl ester (12a). Following the general procedure for the syn-
thesis of 12aeo, Diels-Alder reaction of 11a (9.4 g, 40 mmol) and
dimethyl maleate (11 mL, 80 mmol) afforded 12a (8.0 g). Yield 59%;
4.1.3.8. Dimethyl
5-hydroxy-6-methyl-2-(naphthalen-1-ylmethyl)
pyridine-3,4-dicarboxylate (12h). Following the general procedure
for the synthesis of 12aeo, Diels-Alder reaction of 11h (3.8 g,
14 mmol) and dimethyl maleate (3.6 mL, 28 mmol) afforded 12h
1H NMR (CDCl3, 300 MHz)
d
2.84 (3H, s, CH3), 3.76 (3H, s, CH3O),
(2.7 g). Yield 52%; 1H NMR (CDCl3, 400 MHz)
d 2.54 (3H, s, CH3), 3.72
4.00 (3H, s, CH3O), 4.42 (2H, s, CH2), 7.24e7.31 (5H, m, phenyl); MS
(MALDI-TOF): m/z ¼ 314.9 [M þ H].
(3H, s, CH3O), 3.92 (3H, s, CH3O), 4.21 (2H, s, CH2), 7.35e7.48 (3H, m,
naphthyl), 7.62e7.78 (4H, m, naphthyl), 10.57 (1H, s, OH); MS (ESI):
m/z ¼ 366.4 [M þ H].
4.1.3.2. 5-Hydroxy-6-methyl-2-phenethyl-pyridine-3,4-dicarboxylic
acid dimethyl ester (12b). Following the general procedure for the
synthesis of 12aeo, DielseAlder reaction of 11b (2.8 g, 12 mmol)
and dimethyl maleate (3.0 mL, 24 mmol) afforded 12b (1.8 g). Yield
4.1.3. 9. Dimethyl 2-(2,2-diphenylethyl)-5-hydroxy-6-
methylpyridine-3,4-dicarboxylate (12i). Following the general pro-
cedure for the synthesis of 12aeo, Diels-Alder reaction of 11i (4.0 g,
13 mmol) and dimethyl maleate (3.3 mL, 26 mmol) afforded 12i
47%; 1H NMR (CDCl3, 300 MHz)
d
2.64 (3H, s, CH3), 3.10 (2H, t,
J ¼ 7.2 Hz, CH2), 3.35 (2H, t, J ¼ 7.2 Hz, CH2), 4.00 (3H, s, CH3O), 4.10
(1.1 g). Yield 21%; 1H NMR (CDCl3, 400 MHz)
d 2.46 (3H, s, CH3), 3.37

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J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
(2H, d, J ¼ 8.0 Hz, CH2), 3.75 (3H, s, CH3O), 3.88 (3H, s, CH3O), 4.51
(1H, t, J ¼ 8.0 Hz, CH), 7.10e7.24 (10H, m, phenyl), 10.47 (1H, s, OH);
MS (ESI): m/z ¼ 406.0 [M þ H].
water to dissolve the KCl salt. The solid product was filtered with
excess water and chloroform. After that, solid product was dried to
afford 13aeo as a white solid.
4.1.3.10. Dimethyl
5-hydroxy-6-methyl-2-(3-phenoxybenzyl)pyri-
4.1.4.1. 2-Benzyl-5-hydroxy-6-methyl-pyridine-3,4-dicarboxylic acid
(13a). Following the general procedure for the synthesis of 13aeo,
the compound 12a (1.0 g, 3.2 mmol) was reacted with aqueous 20%
KOH (15 mL) to afford 13a (0.60 g). Yield 66%; 1H NMR (DMSO-d6,
dine-3,4-dicarboxylate (12j). Following the general procedure for
the synthesis of 12aeo, Diels-Alder reaction of 11j (5.5 g, 18 mmol)
and dimethyl maleate (4.5 mL, 36 mmol) afforded 12j (2.3 g). Yield
32%; 1H NMR (CDCl3, 400 MHz)
d
2.52 (3H, s, CH3), 3.78 (3H, s,
300 MHz) d 2.28 (3H, s, CH3), 3.91 (2H, s, CH2), 7.11e20 (5H, m,
CH3O), 3.93 (3H, s, CH3O), 4.02 (2H, s, CH2), 6.81 (1H, d, J ¼ 8.0 Hz,
phenyl), 6.91e6.99 (4H, m, phenyl), 7.09 (1H, t, J ¼ 6.8 Hz, phenyl),
7.20 (1H, t, J ¼ 7.6 Hz, phenyl), 7.32 (2H, t, J ¼ 8.0 Hz, phenyl), 10.57
(1H, s, OH); MS (ESI): m/z ¼ 408.4 [M þ H].
phenyl); MS (ESI): m/z ¼ 286.1 [M þ H].
4.1.4.2. 5-Hydroxy-6-methyl-2-phenethyl-pyridine-3,4-dicarboxylic
acid (13b). Following the general procedure for the synthesis of
13aeo, the compound 12b (40 mg, 0.12 mmol) was reacted with
aqueous 20% KOH (2.5 mL) to afford 13b (26 mg). Yield 70%; 1H
4.1.3.11. Dimethyl 5-hydroxye6-methyl-2-(3-phenoxyphenethyl)pyr-
idinee3,4-dicarboxylate (12k). Following the general procedure for
the synthesis of 12aeo, DielseAlder reaction of 11k (2.0 g,
6.2 mmol) and dimethyl maleate (1.6 mL, 12 mmol) afforded 12k
NMR (DMSO-d6, 300 MHz)
CH2), 7.18e7.28 (5H, m, phenyl); MS (ESI): m/z ¼ 300.1 [M þ H].
d
2.31 (3H, s, CH3), 2.84e2.87 (4H, m, 2ꢃ
(0.80 g). Yield 31%; 1H NMR (CDCl3, 400 MHz)
d
2.54 (3H, s, CH3),
4.1.4.3. 5-Hydroxy-6-methyl-2-styryl-pyridine-3,4-dicarboxylic acid
(13c). Following the general procedure for the synthesis of 13aeo,
the compound 12c (0.25 g, 0.76 mmol) was reacted with aqueous
20% KOH (8.0 mL) to afford 13c (0.18 g). Yield 79%; 1H NMR (DMSO-
2.92 (2H, m, CH2), 2.94 (2H, m, CH2), 3.83 (3H, s, CH3O), 3.94 (3H, s,
CH3O), 6.82 (2H, m, phenyl), 6.94e7.00 (3H, m, phenyl), 7.10 (1H, t,
J ¼ 7.2 Hz, phenyl), 7.22 (1H, t, J ¼ 8.0 Hz, phenyl), 7.33 (2H, t,
J ¼ 8.0 Hz, phenyl), 10.56 (1H, s, OH); MS (ESI): m/z ¼ 422.1 [M þ H].
d6, 300 MHz)
d
2.42 (3H, s, CH3), 7.11 (1H, t, J ¼ 16.5 Hz, ]CH), 7.30
(1H, t, J ¼ 16.5 Hz, ]CH), 7.31e38 (3H, m, phenyl), 7.55 (2H, d,
J ¼ 7.2 Hz, phenyl); MS (ESI): m/z ¼ 298.1 [M þ H].
4.1.3.12. Dimethyl
5-hydroxy-6-methyl-2-(3-phenoxyphenyl)pyri-
dine-3,4-dicarboxylate (12l). Following the general procedure for
the synthesis of 12aeo, Diels-Alder reaction of 11l (1.4 g, 4.7 mmol)
and dimethyl maleate (1.2 mL, 9.4 mmol) afforded 12l (0.46 g).
4.1.4.4. 5-Hydroxy-2-[2-(4-methoxy-phenyl)ethyl]-6-methyl-pyri-
dine-3,4-dicarboxylic acid (13d). Following the general procedure
for the synthesis of 13aeo, the compound 12d (90 mg, 0.27 mmol)
and aqueous 20% KOH (3.0 mL) to afford 13d (52 mg). Yield 65%; 1H
Yield 24%; 1H NMR (CDCl3, 400 MHz)
d 2.61 (3H, s, CH3), 3.67 (3H, s,
CH3O), 3.96 (3H, s, CH3O), 7.02 (3H, m, phenyl), 7.11 (1H, t, J ¼ 7.6 Hz,
phenyl), 7.17 (1H, m, phenyl), 7.33e7.39 (4H, m, phenyl), 10.77 (1H,
s, OH); MS (ESI): m/z ¼ 392.3 [M ꢄ H].
NMR (DMSO-d6, 300 MHz)
d
2.25 (3H, s, CH3), 2.80e2.95 (4H, m, 2ꢃ
CH2), 3.80 (3H, s, CH3O), 6.91 (2H, d, J ¼ 8.7 Hz, phenyl), 7.21 (2H, d,
J ¼ 8.7 Hz, phenyl); 13C NMR (DMSO-d6, 400 MHz) 19.07, 35.48,
37.68, 55.43, 114.12, 120.40, 128.32, 129.68, 135.06, 142.99, 144.39,
157.83, 166.88, 171.98, 175.15; MS (ESI): m/z ¼ 332.3 [M þ H].
4.1.3.13. Dimethyl
5-hydroxy-6-methyl-2-(4-phenoxybenzyl)pyri-
dine-3,4-dicarboxylate (12m). Following the general procedure for
the synthesis of 12aeo, Diels-Alder reaction of 11m (0.50 g,
1.6 mmol) and dimethyl maleate (0.40 mL, 3.2 mmol) afforded 12m
4.1.4.5. 5-Hydroxy-2-[2-(3-methoxy-phenyl)-ethyl]-6-methyl-pyri-
dine-3,4-dicarboxylic acid (13e). Following the general procedure
for the synthesis of 13aeo, the compound 12e (0.50 g, 1.4 mmol)
was reacted with aqueous 20% KOH (10 mL) to afford 13e (0.25 g).
(0.25 g). Yield 38%; 1H NMR (CDCl3, 400 MHz)
d 2.55 (3H, s, CH3),
3.91 (3H, s, CH3O), 3.94 (3H, s, CH3O), 4.03 (2H, s, CH2), 6.89 (2H, d,
J ¼ 8.4 Hz, phenyl), 6.97(2H, d, J ¼ 7.6 Hz, phenyl), 7.09 (1H, t,
J ¼ 7.6 Hz, phenyl), 7.19(2H, d, J ¼ 8.8 Hz, phenyl), 7.30 (2H, t,
J ¼ 8.8 Hz, phenyl), 10.56 (1H, s, OH); MS (ESI): m/z ¼ 407.9 [M þ H].
Yield 54%; 1H NMR (DMSO-d6, 300 MHz)
d 2.34 (3H, s, CH3), 2.87e
2.91 (4H, m, 2ꢃ CH2), 3.74 (3H, s, CH3O), 6.76e6.87 (2H, m, phenyl),
7.23e7.25 (2H, m, phenyl); MS (ESI): m/z ¼ 332.3 [M þ H].
4.1.3.14. Dimethyl
5-hydroxy-6-methyl-2-(2-phenoxybenzyl)pyri-
dine-3,4-dicarboxylate (12n). Following the general procedure for
the synthesis of 12aeo, Diels-Alder reaction of 11n (3.9 g, 12 mmol)
and dimethyl maleate (3.1 mL, 24 mmol) afforded 12n (2.0 g). Yield
4.1.4.6. 5-Hydroxy-2-[2-(2-methoxy-phenyl)-ethyl]-6-methyl-pyri-
dine-3,4-dicarboxylic acid (13f). Following the general procedure
for the synthesis of 13aeo, the compound 12f (0.30 g, 0.84 mmol)
was reacted with aqueous 20% KOH (6.0 mL) to afford 13f (0.25 g).
40%; 1H NMR (CDCl3, 400 MHz)
d 2.47 (3H, s, CH3), 3.70 (3H, s,
CH3O), 3.91 (3H, s, CH3O), 4.12 (2H, s, CH2), 6.86 (1H, d, J ¼ 8.4 Hz,
phenyl), 6.89 (2H, d, J ¼ 8.0 Hz, phenyl), 7.01 (2H, t, J ¼ 8.0 Hz,
phenyl), 7.13e7.18(2H, m, phenyl), 7.24e7.29 (2H, m, phenyl), 10.46
(1H, s, OH); MS (ESI): m/z ¼ 407.7 [M þ H].
Yield 92%; 1H NMR (DMSO-d6, 300 MHz)
d 2.59 (3H, s, CH3), 2.86
(2H, t, J ¼ 6.9 Hz, CH2), 2.89 (2H, t, J ¼ 6.9 Hz, CH2), 3.77 (3H, s,
CH3O), 6.88e6.96 (2H, m, phenyl), 7.11e7.19 (2H, m, phenyl); MS
(ESI): m/z ¼ 332.3 [M þ H].
4.1.3.15. Dimethyl
2-(benzyloxymethyl)-5-hydroxy-6-methyl-pyri-
4.1.4.7. 2-[2-(4-Carboxy-phenyl)-ethyl]-5-hydroxy-6-methyl-pyri-
dine-3,4-dicarboxylic acid (13g). Following the general procedure
for the synthesis of 13aeo, the compound 12g (15 mg, 0.040 mmol)
was reacted with aqueous 20% KOH (1.3 mL) to afford 13g (7.2 mg).
dine-3,4-dicarboxylate (12o). Following the general procedure for
the synthesis of 12aeo, DielseAlder reaction of 11o (0.50 g, 2.0 mmol)
and dimethyl maleate (0.50 mL, 4.0 mmol) afforded 12o (0.33 g). Yield
47%; 1H NMR (CDCl3, 400 MHz)
d
2.56 (3H, s, CH3), 3.71 (3H, s, CH3O),
Yield 54%; 1H NMR (DMSO-d6, 300 MHz)
d 2.38 (3H, s, CH3), 2.94e
3.95 (3H, s, CH3O), 4.52 (2H, s, CH2), 4.64 (2H, s, CH2), 7.30e7.34 (5H,
m, phenyl), 10.66 (1H, s, OH); MS (ESI): m/z ¼ 346.1 [M þ H].
3.0 (4H, m, 2ꢃ CH2), 7.22 (2H, d, J ¼ 8.1 Hz, phenyl), 7.72 (2H, d,
J ¼ 8.1 Hz, phenyl); 13C NMR (DMSO-d6, 400 MHz) 18.79, 36.46,
37.50, 122.78, 127.80, 129.61, 136.69, 137.48, 143.05, 144.36, 171.21,
175.58; MS (ESI): m/z ¼ 346.3 [M þ H].
4.1.4. General procedure for the preparation of compounds (13aeo)
To a solution of aqueous 20% KOH was added 12aeo with
vigorous stirring for 6 h. The mixture was acidified with concen-
trated HCl solution. Generated solid product was diluted with
4.1.4.8. 5-Hydroxy-6-methyl-2-(naphthalene-1-ylmethyl)pyridine-
3,4-dicarboxylic acid (13h). Following the general procedure for the

J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
823
synthesis of 13aeo, the compound 12h (0.18 g, 0.49 mmol) was
reacted with aqueous 20% KOH (6.0 mL) to afford 13h (0.12 mg).
synthesis of 13aeo, the compound 12o (100 mg, 0.28 mmol) was
reacted with aqueous 20% KOH (4.0 mL) to afford 13o (30 mg). Yield
Yield 73%; 1H NMR (DMSO-d6, 400 MHz)
d
2.36 (3H, s, CH3), 4.22
27%; 1H NMR (MeOH-d4, 400 MHz)
d 2.41 (2H, s, CH3), 4.56 (2H, s,
(2H, s, CH2), 7.03e7.18 (4H, m, naphthyl), 7.23e7.26 (3H, m, naph-
thyl); MS (ESI): m/z ¼ 337.3 [M þ H].
CH2), 4.59 (2H, s, CH2), 7.22e7.37 (5H, m, phenyl); MS (ESI): m/
z ¼ 318.1 [M þ H].
4.1.4.9. 2-(2,2,-Diphenylethyl)5-hydroxy-6-methylpyridine-3,4-
dicarboxylic acid (13i). Following the general procedure for the
synthesis of 13aeo, the compound 12i (0.10 g, 0.25 mmol) was
reacted with aqueous 20% KOH (4.0 mL) to afford 13i (40 mg). Yield
4.1.5. General procedure for the preparation of (E)-ethyl 4-oxopent-
2-enoate (16)
A solution of 14, (carbethoxymethylene)triphenylphosphorane,
(21 g, 61 mmol) in a small volume of dichloromethane was slowly
added aqueous 15, pyruvalehyde, (47 mL, 300 mmol) at room
temperature. The mixture was diluted with N,N-dimethylforma-
mide (30 mL), and it was stirred for 20 h until the starting material
was disappeared by TLC monitoring. The reaction mixture was
added water and extracted with dichloromethane. The organic
layer was washed successively with water and brine, dried over
MgSO4 and evaporated under reduced pressure to give the product
as sticky oil. 1H NMR spectroscopy showed the product a mixture of
E and Z isomers (9:1). The isomers were separated by flash column
chromatography, eluting with n-hexanes-ethyl acetate ¼ 3:1. E
isomer, compound 16 (8.9 g): Rf 0.80 (hexanes-ethyl acetate ¼ 1:1);
Yield 80%; 1H NMR (acetone-d6, 300 MHz) 1.4 (3H, t, J ¼ 7.2 Hz,
CH3), 2.3 (3H, s, CH3CO), 4.15 (2H, q, J ¼ 7.2 Hz, CH2), 6.6 (1H, d,
J ¼ 15.0 Hz, ]CH), 6.9 (1H, d, J ¼ 15.0 Hz, ]CH); Z isomer (0.90 g):
Rf 0.74 (hexanes-ethyl acetate ¼ 1:1); Yield 8.1%; 1H NMR (acetone-
d6) 1.3 (3H, t, J ¼ 7.2 Hz, CH3), 2.3 (3H, s, CH3CO), 4.15 (2H, q,
J ¼ 7.2 Hz, CH2), 5.9 (1H, d, J ¼ 12.0 Hz, ]CH), 6.4 (1H, d, J ¼ 12.0 Hz,
]CH).
43%; 1H NMR (DMSO-d6, 400 MHz)
d 2.36 (3H, s, CH3), 3.38 (2H, d,
J ¼ 8.0 Hz, CH2), 4.74 (H, t, J ¼ 8.0 Hz, CH), 7.03e7.26 (10H, m,
phenyl); MS (ESI): m/z ¼ 377.4 [M þ H].
4.1.4.10. 5-Hydroxy-6-methyl-2-(3-phenoxybenzyl)pyridine-3,4-
dicarboxylic acid (13j). Following the general procedure for the
synthesis of 13aeo, the compound 12j (0.47 g, 0.060 mmol) was
reacted with aqueous 20% KOH (10 mL) to afford 13j (0.33 g). Yield
72%; 1H NMR (DMSO-d6, 400 MHz) 2.57 (3H, s, CH3), 4.17 (2H, s,
CH2), 6.81 (1H, d, J ¼ 8.0 Hz, phenyl), 6.81 (1H, d, J ¼ 8.4 Hz, phenyl),
6.88 (1H, s, phenyl), 6.93 (2H, d, J ¼ 8.0 Hz, phenyl), 7.08 (1H, t,
J ¼ 7.6 Hz, phenyl), 7.24 (1H, t, J ¼ 8.4 Hz, phenyl), 7.32 (2H, t,
J ¼ 7.6 Hz, phenyl); 13C NMR (DMSO-d6, 400 MHz) 14.79, 36.99,
116.75, 118.60, 118.90, 123.14, 129.54, 130.84, 139.45, 140.02, 148.04,
155.31, 157.64, 168.15; MS (ESI): m/z ¼ 380.3 [M þ H].
4.1.4.11. 5-Hydroxy-6-methyl-2-(3-phenoxyphenethyl)pyridine-3,4-
dicarboxylic acid (13k). Following the general procedure for the
synthesis of 13aeo, the compound 12k (0.30 g, 0.74 mmol) was
reacted with aqueous 20% KOH (8.5 mL) to afford 13k (0.14 g). Yield
4.1.6. General procedure for the preparation of compounds (17aef)
The oxazole 11aef (1.0 equiv) and 16, ethyl 4-oxopent-2-enoate,
(1.5 equiv) were mixed well and heated to 55e60 ꢀC for 10e14 h.
The reaction mixture containing intermediate (Diels-Alder adduct)
was dissolved in ethanol and cooled to 0 ꢀC. HCl gas was passed
through the reaction mixture for 20 min and the reaction mixture
was stirred at 0e10 ꢀC for about 30 min and then at room tem-
perature for 1 h. The reaction mixture was poured into 8% aqueous
sodium bicarbonate solution and extracted with chloroform. The
organic layer concentrated and the crude product was purified by
silica gel column to afford 17aef as sticky oil.
50%; 1H NMR (DMSO-d6, 400 MHz)
d 2.55 (3H, s, CH3), 2.92e2.95
(2H, m, CH2), 2.98e3.01 (2H, m, CH2), 6.82 (2H, t, J ¼ 8.0 Hz, phenyl),
6.93e6.99 (3H, m, phenyl), 7.10 (1H, t, J ¼ 7.2 Hz, phenyl), 7.26e7.36
(3H, m, phenyl); 13C NMR (DMSO-d6, 400 MHz) 16.66, 34.36, 35.83,
117.00, 118.60, 119.08, 123.67, 126.43, 130.02, 130.65, 132.29, 138.12,
143.36,149.82,157.21,167.63,168.19; MS (ESI): m/z ¼ 394.2 [M þ H].
4.1.4.12. 5-Hydroxy-6-methyl-2-(3-phenoxyphenyl)pyridine-3,4-
dicarboxylic acid (13l). Following the general procedure for the
synthesis of 13aeo, the compound 12l (180 mg, 0.45 mmol) was
reacted with aqueous 20% KOH (5.0 mL) to afford 13l (60 mg). Yield
35%; 1H NMR (DMSO-d6, 400 MHz)
d
2.42 (3H, s, CH3), 6.98 (3H, d,
4.1.6.1. Ethyl 4-acetyl-2-benzyl-5-hydroxy-6-methylnicotinate (17a).
Following the general procedure for the synthesis of 17aef, Diels-
Alder reaction of the compound 11a (1.0 g, 4.6 mmol) with 16 (1.3 g,
9.2 mmol) afforded 17a (0.86 g). Yield 30%; 1H NMR (CDCl3,
J ¼ 8.0 Hz, phenyl), 7.09e7.13 (2H, m, phenyl), 7.23 (1H, d, J ¼ 8.0 Hz,
phenyl), 7.35e7.42 (3H, m, phenyl); MS (ESI): m/z ¼ 366.3 [M þ H].
4.1.4.13. 5-Hydroxy-6-methyl-2-(4-phenoxybenzyl)pyridine-3,4-
dicarboxylic acid (13m). Following the general procedure for the
synthesis of 13aeo, the compound 12m (100 mg, 0.24 mmol) was
reacted with aqueous 20% KOH (4.0 mL) to afford 13m (60 mg).
300 MHz)
d
1.17 (3H, t, J ¼ 6.9 Hz, CH3), 2.55 (3H, s, CH3), 2.59 (3H, s,
CH3CO), 4.40 (2H, s, CH2), 4.43 (2H, q, J ¼ 6.9 Hz, CH2), 7.17e7.45
(5H, m, phenyl), 11.34 (1H, s, OH); MS (FABþ): m/z ¼ 314.3 [M þ H].
Yield 66%; 1H NMR (DMSO-d6, 400 MHz)
d
2.39 (3H, s, CH3), 4.06
4.1.6.2. Ethyl 4-acetyl-5-hydroxy-6-methyl-2-phenethylnicotinate
(17b). Following the general procedure for the synthesis of 17ae
f, Diels-Alder reaction of the compound 11b (2.0 g, 8.6 mmol) with
16 (2.4 g, 17 mmol) afforded 7b (0.65 g). Yield 23%; 1H NMR (CDCl3,
(2H, s, CH2), 6.82 (2H, d, J ¼ 8.4 Hz, phenyl), 6.91 (2H, d, J ¼ 8.4 Hz,
phenyl), 7.04 (1H, t, J ¼ 7.2 Hz, phenyl), 7.32 (4H, t, J ¼ 7.6 Hz,
phenyl); MS (ESI): m/z ¼ 379.9 [M þ H].
300 MHz)
d
1.28 (3H, t, J ¼ 6.9 Hz, CH3), 2.52 (3H, s, CH3), 2.56 (3H, s,
4.1.4.14. 5-Hydroxy-6-methyl-2-(2-phenoxybenzyl)pyridine-3,4-
dicarboxylic acid (13n). Following the general procedure for the
synthesis of 13aeo, the compound 12n (0.50 g, 1.2 mmol) was
reacted with aqueous 20% KOH (10 mL) to afford 13n (0.29 g). Yield
CH3CO), 3.00 (2H, t, J ¼ 7.2 Hz, CH2), 3.23 (2H, t, J ¼ 7.2 Hz, CH2), 4.41
(2H, q, J ¼ 6.9 Hz, CH2), 7.18e7.30 (5H, m, phenyl), 11.42 (1H, s, OH);
MS (MALDI-TOF): m/z ¼ 328.4 [M þ H].
62%; 1H NMR (MeOH-d4, 400 MHz)
d
2.34 (3H, s, CH3), 4.26 (2H, s,
4.1.6.3. (E)-Ethyl 4-acetyl-5-hydroxy-6-methyl-2-styrylnicotinate
(17c). Following the general procedure for the synthesis of 17aef,
Diels-Alder reaction of the compound 11c (4.0 g, 17 mmol) with 16
(4.7 g, 34 mmol) afforded 17c (1.0 g). Yield 18%; 1H NMR (CDCl3,
CH2), 6.70 (2H, d, J ¼ 8.0 Hz, phenyl), 6.81 (1H, d, J ¼ 8.0 Hz, phenyl),
6.98 (1H, t, J ¼ 7.2 Hz, phenyl), 7.10 (1H, t, J ¼ 7.6 Hz, phenyl), 7.17e
7.25 (4H, m, phenyl); MS (ESI): m/z ¼ 379.7 [M þ H].
300 MHz)
d
1.20 (3H, t, J ¼ 7.2 Hz, CH3), 1.89 (3H, s, CH3), 2.66 (3H, s,
4.1.4.15. 2-(Benzyloxymethyl)-5-hydroxy-6-methylpyridine-3,4-
dicarboxylic acid (13o). Following the general procedure for the
CH3CO), 4.10 (2H, q, J ¼ 7.2 Hz, CH2), 7.26e7.40 (3H, m, phenyl), 7.65
(1H, d, J ¼ 7.2 Hz, phenyl), 7.67 (1H, d, J ¼ 7.2 Hz, phenyl), 7.96 (1H,

824
J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
d, J ¼ 15.9 Hz, ]CH), 8.01 (1H, d, J ¼ 15.9 Hz, ]CH); MS (ESI): m/
z ¼ 326.2 [M þ H].
J ¼ 7.2 Hz, phenyl), 7.96 (1H, d, J ¼ 15.9 Hz, ]CH), 8.01 (1H, d,
J ¼ 15.9 Hz, ]CH); MS (ESI): m/z ¼ 298.2 [M þ H].
4.1.6.4. Ethyl 4-acetyl-5-hydroxy-2-(4-methoxyphenethyl)-6-
methylnicotinate (17d). Following the general procedure for the
synthesis of 17aef, Diels-Alder reaction of the compound 11d (1.6 g,
6.1 mmol) with 16 (1.7 g, 12 mmol) afforded 17d (1.1 g). Yield 51%;
4.1.7.4. 4-Acetyl-5-hydroxy-2-(4-methoxyphenethyl)-6-methy-
lnicotinic acid (18d). Following the general procedure for the syn-
thesis of 18aef, the compound 17d (0.22 g, 0.62 mmol) was reacted
with methanolic KOH (15 mL) to afforded 18d (0.18 g). Yield 88%; 1H
1H NMR (CDCl3, 300 MHz)
d
1.34 (3H, t, J ¼ 7.2 Hz, CH3), 2.52 (3H, s,
NMR (DMSO-d6, 300 MHz) d 2.52 (3H, s, CH3), 2.56 (3H, s, CH3CO),
CH3), 2.56 (3H, s, CH3CO), 2.94(2H, d, J ¼ 7.2 Hz, CH2), 3.03 (2H, d,
J ¼ 7.2 Hz, CH2), 3.80 (3H, s, CH3O), 4.35 (2H, q, J ¼ 7.2 Hz, CH2), 6.82
(2H, d, J ¼ 8.4 Hz, phenyl), 7.11 (2H, d, J ¼ 8.4 Hz, phenyl), 11.38 (1H,
s, OH); MS (ESI): m/z ¼ 358.3 [M þ H].
2.94(2H, d, J ¼ 7.2 Hz, CH2), 3.03 (2H, d, J ¼ 7.2 Hz, CH2), 3.80 (3H, s,
CH3O), 6.82 (2H, d, J ¼ 8.4 Hz, phenyl), 7.11 (2H, d, J ¼ 8.4 Hz, phenyl),
11.38 (1H, s, OH); MS (ESI): m/z ¼ 330.4 [M þ H].
4.1.7.5. 4-Acetyl-5-hydroxy-2-(3-methoxyphenethyl)-6-methy-
lnicotinic acid (18e). Following the general procedure for the syn-
thesis of 18aef, the compound 17e (0.38 g, 1.1 mmol) was reacted
with methanolic KOH (20 mL) to afforded 18e (0.18 g). Yield 50%; 1H
4.1.6.5. Ethyl 4-acetyl-5-hydroxy-2-(3-methoxyphenethyl)-6-
methylnicotinate (17e). Following the general procedure for the
synthesis of 17aef, Diels-Alder reaction of the compound 11e (2.0 g,
7.7 mmol) with 16 (2.2 g, 15 mmol) afforded 17e (0.85 g). Yield 31%;
NMR (DMSO-d6, 300 MHz)
d 1.85 (3H, s, CH3), 2.60 (3H, s, CH3CO),
1H NMR (CDCl3, 300 MHz)
d
1.21 (3H, t, J ¼ 7.2 Hz, CH3), 2.45 (3H, s,
2.98 (2H, t, J ¼ 7.2 Hz, CH2), 3.43 (2H, t, J ¼ 7.2 Hz, CH2), 3.78 (3H, s,
CH3O), 6.79e6.84 (3H, m, phenyl), 7.31 (1H, s, phenyl), 11.28 (1H, br
s, OH); MS (ESI): m/z ¼ 330.2 [M þ H].
CH3), 2.60 (3H, s, CH3CO), 2.98 (2H, t, J ¼ 7.2 Hz, CH2), 3.43 (2H, t,
J ¼ 7.2 Hz, CH2), 3.78 (3H, s, CH3O), 4.32 (2H, q, J ¼ 7.2 Hz, CH2),
6.79e6.84 (3H, m, phenyl), 7.31 (1H, s, phenyl), 11.28 (1H, br s, OH);
MS (ESI): m/z ¼ 357.4 [M þ H].
4.1.7.6. 4-Acetyl-5-hydroxy-2-(2-methoxyphenethyl)-6-methy-
lnicotinic acid (18f). Following the general procedure for the syn-
thesis of 18aef, the compound 17f (40 mg, 0.62 mmol) was reacted
with methanolic KOH (20 mL) to afforded 18f (31 mg). Yield 84%; 1H
4.1.6.6. Ethyl 4-acetyl-5-hydroxy-2-(2-methoxyphenethyl)-6-
methylnicotinate (17f). Following the general procedure for the
synthesis of 17aef, Diels-Alder reaction of the compound 11f (1.6 g,
6.1 mmol) with 16 (1.7 g, 12 mmol) afforded 17f (0.93 g). Yield 43%;
NMR (DMSO-d6, 300 MHz)
d 2.51 (3H, s, CH3), 2.56 (3H, s, CH3CO),
2.96 (2H, t, J ¼ 6.9 Hz, CH2), 3.40 (2H, t, J ¼ 6.9 Hz, CH2), 3.81 (3H, s,
CH3O), 6.85e7.26 (4H, m, phenyl), 11.35 (1H, br s, OH); MS (ESI):
330.4 [M þ H].
1H NMR (CDCl3, 300 MHz)
d
1.34 (3H, t, J ¼ 7.2 Hz, CH3), 2.51 (3H, s,
CH3), 2.56 (3H, s, CH3CO), 2.96 (2H, t, J ¼ 6.9 Hz, CH2), 3.40 (2H, t,
J ¼ 6.9 Hz, CH2), 3.81 (3H, s, CH3O), 4.38 (2H, q, J ¼ 7.2 Hz, CH2),
6.85e7.26 (4H, m, phenyl), 11.35 (1H, br s, OH); MS (ESI): m/
z ¼ 357.4 [M þ H].
4.1.8. General procedure for the preparation of compounds (20 and
21)
Following the general procedure for the synthesis of 12aeo,
DielseAlder reaction of the 19 (4-methyl-oxazole-2-amine, 1.0 g,
10 mmol) with dimethyl maleate (2.5 mL, 20 mmol) afforded 20
(0.15 g) and 21 (0.21 g) were obtained as sticky oil.
4.1.7. General procedure for the preparation of compounds (18aef)
Compounds 17aef were dissolved in 5% methanolic KOH and
stirred at room temperature for 2e3 h. The reaction mixture was
then acidified with concentrated HCl and extracted with chloro-
form. Organic layer was dried over anhydrous Na2SO4 and
concentrated. Crude product was purified by silcagel column using
ethyl acetate/n-hexanes solvent system to afford 18aef as white
solid.
4.1.8.1. Dimethyl 2-amino-6-methylpyridine-3,4-dicarboxylate (20).
Yield 13%; 1H NMR (CDCl3, 400 MHz)
d 2.41 (3H, s, CH3), 3.84 (3H, s,
CH3O), 3.89 (3H, s, CH3O), 6.30 (2H, s, NH2), 6.51 (1H, s, pyridine);
MS (ESI): m/z ¼ 225.1 [M þ H].
4.1.7.1. 4-Acetyl-2-benzyl-5-hydroxy-6-methylnicotinic acid (18a).
Following the general procedure for the synthesis of 18aef, the
compound 17a (1.1 g, 3.4 mmol) was reacted with methanolic KOH
(50 mL) to afforded 18a (0.80 g). Yield 84%; 1H NMR (DMSO-d6,
4.1.8.2. Dimethyl 2-amino-5-hydroxy-6-methylpyridine-3,4-
dicarboxylate (21). Yield 17.2%; 1H NMR (CDCl3, 400 MHz)
d 2.43
(3H, s, CH3), 3.84 (3H, s, CH3O), 3.91 (3H, s, CH3O), 5.31 (2H, s, NH2),
8.04 (1H, s, OH); MS (ESI): m/z ¼ 241.1 [M þ H].
300 MHz) d 2.55 (3H, s, CH3), 2.59 (3H, s, CH3CO), 4.42 (2H, s, CH2),
7.17e7.45 (5H, m, phenyl), 11.30 (1H, s, OH); MS (FABþ): m/
4.1.9. Dimethyl 2-amino-5-(benzyloxy)-6-methylpyridine-3,4-
dicarboxylate (22)
z ¼ 286.2 [M þ H].
A suspension of 21 (0.15 g, 0.61 mmol), K2CO3 (0.25 g, 1.8 mmol),
4.1.7.2. 4-Acetyl-5-hydroxy-6-methyl-2-phenethylnicotinic
acid
and benzyl bromide (87 mL, 0.73 mmol) in dry acetone (10 mL) was
(18b). Following the general procedure for the synthesis of 18ae
f, the compound 17b (0.43 g, 1.3 mmol) was reacted with
methanolic KOH (20 mL) to afforded 18b (0.24 g). Yield 62%; 1H
refluxed for 2 h. After reaction mixture was cooled down, it was
neutralized with NH4Cl and extracted with dichloromethane twice.
The organic layers were combined and dried with anhydrous
Na2SO4. After Na2SO4 was removed, organic layer was concentrated
in vacuum and purified by silica gel column chromatography with
n-hexanes/ethyl acetate ¼ 3:1 to afford 22 (0.21 g) as yellow sticky
NMR (DMSO-d6, 300 MHz)
d 2.52 (3H, s, CH3), 2.56 (3H, s,
CH3CO), 3.00 (2H, t, J ¼ 7.2 Hz, CH2), 3.23 (2H, t, J ¼ 7.2 Hz, CH2)
7.18e7.30 (5H, m, phenyl), 11.42 (1H, s, OH); MS (ESI): m/
z ¼ 300.2 [M þ H].
oil. Yield 99%; 1H NMR (CDCl3, 400 MHz)
d 2.41 (3H, s, CH3), 3.85
(3H, s, CH3O), 3.86 (3H, s, CH3O), 4.85 (2H, s, CH2), 6.31 (2H, s, NH2),
4.1.7.3. (E)-4-Acetyl-5-hydroxy-6-methyl-2-styrylnicotinic
acid
7.35e7.43 (5H, m, phenyl); MS (ESI): m/z ¼ 330.7 [M þ H].
(18c). Following the general procedure for the synthesis of 18aef,
the compound 17c (50 mg, 0.15 mmol) was reacted with meth-
anolic KOH (8.0 mL) to afforded 18c (20 mg). Yield 37%; 1H NMR
4.1.10. Dimethyl 2-benzamido-5-(benzyloxy)-6-methylpyridine-
3,4-dicarboxylate (23a)
(DMSO-d6, 300 MHz)
d
1.89 (3H, s, CH3), 2.66 (3H, s, CH3CO), 7.26e
A respective 22 (0.10 g, 0.28 mmol) was dissolved in dry toluene
7.40 (3H, m, phenyl), 7.65 (1H, d, J ¼ 7.2 Hz, phenyl), 7.67 (1H, d,
(3.0 mL). Benzoyl chloride (39 mL, 0.34 mmol) was added to solution

J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
825
of 22 and then diisopropylethylamine (59
m
L, 0.34 mmol) was
J ¼ 7.6 Hz, phenyl), 7.88 (1H, s, OH); MS (MALDI-TOF): m/z ¼ 409.1
[M þ H].
added to the reaction mixture. After being stirred for 2 h at 80 ꢀC,
the mixture was neutralized with saturated aqueous NH4Cl and
extracted with dichloromethane (3 times). The organic layers were
combined and treated with anhydrous Na2SO4. After Na2SO4 was
filtered off, organic layer was concentrated in vacuum and purified
by silica gel column chromatography with n-hexanes/ethyl
acetate ¼ 3:1 to afford 23a (0.10 g) as a sticky oil. Yield 77%; 1H NMR
4.1.16. Dimethyl 2-benzamido-6-methylpyridine-3,4-dicarboxylate
(26)
Following the procedure for the synthesis of 23a, the compound
20 (90 mg, 0.27 mmol) was reacted with benzoyl chloride (63
0.54 mmol) to afford 26 (49 mg) as a sticky oil. Yield 67%; 1H NMR
(CDCl3, 400 MHz) 2.64 (3H, s, CH3), 3.89 (3H, s, CH3O), 3.93 (3H, s,
mL,
(CDCl3, 400 MHz)
d
2.62 (3H, s, CH3), 3.87 (3H, s, CH3O), 3.90 (3H, s,
d
CH3O), 7.39e7.58 (8H, m, phenyl), 8.02 (2H, m, phenyl), 10.80 (1H, s,
NH), MS (ESI): m/z ¼ 435.4 [M þ H].
CH3O), 7.13 (1H, s, pyridine), 7.52 (2H, t, J ¼ 7.6 Hz, phenyl), 7.57 (1H,
t, J ¼ 7.2 Hz, phenyl), 7.98 (2H, d, J ¼ 7.6 Hz, phenyl), 10.32 (1H, s,
NH); MS (ESI): m/z ¼ 329.0 [M þ H].
4.1.11. Dimethyl 5-(benzyloxy)-6-methyl-2-(3-phenoxybenzamido)
pyridine-3,4-dicarboxylate (23b)
4.1.17. 2-Benzamido-6-methyl-pyridine-3,4-dicarboxylic acid (27)
Following the general procedure for the synthesis of 13aeo, the
compound 26 (0.12 g, 0.38 mmol) was reacted with aqueous 20%
KOH (6.0 mL) to afford 27 (40 mg) as a white solid. Yield 35%; 1H
Following the procedure for the synthesis of 23a, the compound
22 (0.14 g, 0.42 mmol) was reacted with 3-phenoxybenzoyl chlo-
ride (0.12 g, 0.50 mmol) to afford 23b (0.17 g) as a sticky oil. Yield
77%; 1H NMR (CDCl3, 400 MHz)
d
2.61 (3H, s, CH3), 3.87 (3H, s,
NMR (DMSO-d6) d 2.47 (3H, s, CH3, 400 MHz), 7.29 (1H, s, pyridine),
CH3O), 3.89 (3H, s, CH3O), 4.94 (2H, s, CH2), 7.06 (2H, d, J ¼ 8.0 Hz,
phenyl), 7.16 (1H, t, J ¼ 7.6 Hz, phenyl), 7.20e7.23 (1H, m, phenyl),
7.36e7.43 (7H, m, phenyl), 7.47 (1H, t, J ¼ 8.0 Hz, phenyl), 7.64e7.71
(2H, m, phenyl), 10.82 (1H, s, NH); MS (ESI): m/z ¼ 526.6 [M þ H].
7.45 (2H, t, J ¼ 7.6 Hz, phenyl), 7.54 (1H, t, J ¼ 7.2 Hz, phenyl), 7.93
(2H, d, J ¼ 7.6 Hz, phenyl), 10.87 (1H, s, NH); MS (ESI): m/z ¼ 229.1
[M ꢄ H].
4.1.18. Dimethyl 5-(benzyloxy)-6-methyl-2-(naphthalen-1-
ylmethyl)pyridine-3,4-dicarboxylate (28h)
Following the procedure for the synthesis of 22, the compound
12h (1.0 g, 2.7 mmol) was reacted with benzyl bromide (0.40 mL,
3.3 mmol) to afford 28h (1.3 g) as yellow sticky oil. Yield 96%; 1H
4.1.12. Dimethyl 2-benzamido-5-hydroxy-6-methylpyridine-3,4-
dicarboxylate (24a)
A solution of 23a (100 mg, 0.21 mmol) in methanol was added
10% Pd/C (10 mg, 0.010 mmol) and stirred for 0.5 h under H2 at-
mosphere. The mixture was filtered through celatum bed. The
filtrate was evaporated in vacuum and purified by silica gel column
chromatography with chloroform/methanol ¼ 20:1 to afford 24a
NMR (DMSO-d6, 300 MHz)
d 2.56 (3H, s, CH3), 3.72 (3H, s, CH3O),
3.80 (3H, s, CH3O), 4.52 (2H, s, CH2), 4.96 (2H, s, benzyl-CH2), 7.34e
7.44 (8H, m, naphthyl & phenyl), 7.61e7.78 (4H, m, naphthyl); MS
(ESI): m/z ¼ 455.9 [M þ H].
(60 mg) as a sticky oil. Yield 76%; 1H NMR (CDCl3, 400 MHz)
d 2.54
(3H, s, CH3), 3.76 (3H, s, CH3O), 3.89 (3H, s, CH3O), 7.43 (2H, t,
J ¼ 7.6 Hz, phenyl), 7.53 (1H, t, J ¼ 7.6 Hz, phenyl), 7.90 (2H, d,
J ¼ 7.6 Hz, phenyl), 10.51 (1H, s, NH); MS (ESI): m/z ¼ 345.3 [M þ H].
4.1.19. Dimethyl 5-(benzyloxy)-6-methyl-2-(3-phenoxybenzyl)
pyridine-3,4-dicarboxylate (28j)
Following the procedure for the synthesis of 22, the compound
12j (2.3 g, 5.6 mmol) was reacted with benzyl bromide (0.80 mL,
6.8 mmol) to afford 28j (2.4 g) as yellow sticky oil. Yield 85%; 1H
4.1.13. Dimethyl 5-hydroxy-6-methyl-2-(3-phenoxybenzamido)
pyridine-3,4-dicarboxylate (24b)
Following the procedure for the synthesis of 24a, the compound
23b (100 mg, 0.19 mmol) was reacted with 10% Pd/C (21 mg,
0.20 mmol) to afford 24b (44 mg) as sticky oil. Yield 53%; 1H NMR
NMR (DMSO-d6, 300 MHz) d 2.53 (3H, s, CH3), 3.73 (3H, s, CH3O),
3.81 (3H, s, CH3O), 4.32 (2H, s, CH2), 4.95 (2H, s, benzyl-CH2), 6.80e
7.09 (7H, m, phenyl), 7.29e7.37 (7H, m, phenyl); MS (ESI): m/
z ¼ 498.0 [M þ H].
(CDCl3, 400 MHz) d 2.56 (3H, s, CH3), 3.85 (3H, s, CH3O), 3.97 (3H, s,
CH3O), 7.03 (2H, d, J ¼ 7.6 Hz, phenyl), 7.15e7.21 (2H, m, phenyl),
7.35 (2H, t, J ¼ 7.6 Hz, phenyl), 7.42 (1H, t, J ¼ 7.6 Hz, phenyl), 7.53
(1H, s, phenyl), 7.59 (1H, d, J ¼ 7.6 Hz, phenyl), 8.57 (1H, s, OH), 9.87
(1H, s, NH); MS (ESI): m/z ¼ 436.6 [M þ H].
4.1.20. (5-(Benzyloxy)-6-methyl-2-(naphthalen-1-ylmethyl)
pyridine-3,4-diyl)dimethanol (29h)
A solution of 28h (1.6 g, 3.5 mmol) in diethyl ether was added a
suspension of LiAlH4 (0.90 mL, 21 mmol) in dry diethyl ether
(20 mL) dropwise over a period of 0.5 h in ice-bath. After the
addition was complete, the reaction mixture was stirred at 0 ꢀC
under N2 atmosphere for 1 h. The mixture was neutralized with
saturated aqueous NH4Cl and extracted with dichloromethane 3
times. The organic layers were combined and dried with anhydrous
Na2SO4. After Na2SO4 was removed, organic layer was concentrated
in vacuum and purified by silica gel column chromatography with
n-hexanes/ethyl acetate ¼ 2:1 to afford 29h (0.76 g) as a yellow
4.1.14. 2-Benzamido-5-hydroxy-6-methylpyridine-3,4-dicarb-
oxylic acid (25a)
Following the general procedure for the synthesis of 13aeo, the
compound 24a (60 mg, 0.13 mmol) was reacted with aqueous 20%
KOH (5.0 mL) to afford 25a (11 mg) as a white solid. Yield 27%; 1H
NMR (DMSO-d6, 400 MHz)
d
2.37 (3H, s, CH3), 7.44 (2H, t, J ¼ 7.6 Hz,
phenyl), 7.56 (1H, t, J ¼ 6.8 Hz, phenyl), 7.91 (2H, d, J ¼ 7.6 Hz,
phenyl), 10.53 (1H, s, NH)); 13C NMR (DMSO-d6, 400 MHz) 19.76,
121.41, 128.32, 129.81, 130.63, 132.34, 134.41, 140.04, 146.73, 149.57,
166.41, 166.82, 167.83; MS (ESI): m/z ¼ 315.0 [M ꢄ H].
sticky oil. Yield 69%; 1H NMR (CDCl3, 400 MHz)
d 2.56 (3H, s, CH3),
4.45 (2H, s, CH2), 4.71 (2H, s, CH2OH), 4.72 (2H, s, CH2OH), 4.96 (2H,
s, benzyl-CH2), 7.39e7.44 (8H, m, naphthyl & phenyl), 7.61e7.78
(4H, m, naphthyl); MS (ESI): m/z ¼ 399.8 [M þ H].
4.1.15. 5-Hydroxy-6-methyl-2-(3-phenoxybenzamido)pyridine-3,4-
dicarboxylic acid (25b)
Following the general procedure for the synthesis of 13aeo, the
compound 24b (44 mg, 0.10 mmol) was reacted with aqueous 20%
KOH (4.0 mL) to afford 25b (12 mg) as a white solid. Yield 37%; 1H
4.1.21. (5-(Benzyloxy)-6-methyl-2-(3-phenoxybenzyl)pyridine-3,4-
diyl)dimethanol (29j)
Following the procedure for the synthesis of 29h, the compound
28j (1.0 g, 2.1 mmol) was reacted with LiAlH4 (1.0 mL, 25 mmol) to
afford 29j (0.80 g) as yellow sticky oil. Yield 88%; 1H NMR (CDCl3,
NMR (MeOH-d4, 400 MHz)
phenyl), 7.12e7.20 (2H, m, phenyl), 7.35 (2H, t, J ¼ 7.6 Hz, phenyl),
d
2.50 (3H, s, CH3), 7.01 (2H, d, J ¼ 7.6 Hz,
7.46 (1H, d, J ¼ 8.0 Hz, phenyl), 7.55 (1H, s, phenyl), 7.66 (1H, d,
400 MHz) d 2.53 (3H, s, CH3), 4.26 (2H, s, CH2), 4.68 (2H, s, CH2OH),

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J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
4.72 (2H, s, CH2OH), 4.93 (2H, s, benzyl-CH2), 6.80e7.09 (7H, m,
phenyl), 7.29e7.37 (7H, m, phenyl); MS (ESI): m/z ¼ 441.5 [M þ H].
CH3), 4.43 (2H, s, CH2), 5.10 (2H, s, CH2OH), 6.81e7.33 (9H, m,
phenyl), 10.38 (H, s, CHO); MS (ESI): m/z ¼ 389.9 [M þ H].
4.1.22. (5-Hydroxy-6-methyl-2-(naphthalen-1-ylmethyl)pyridine-
3,4-diyl)dimethanol (30h)
4.1.28. (E)-Ethyl 3-(2,2,8-trimethyl-6-(naphthalen-1-ylmethyl)-4H-
[1,3]dioxino[4,5-c]pyridin-5-yl)acrylate (33h)
Following the procedure for the synthesis of 24a, the compound
29h (0.33 g, 0.82 mmol) was reacted with 10% Pd/C (30 mg,
0.28 mmol) to afford 30h (0.21 g) as sticky oil. Yield 82%; 1H NMR
To a suspension of NaH (13 mg, 0.53 mmol) in dry THF was
added dropwise triethyl phosphonoacetate (0.10 mL, 0.53 mmol).
The mixture color was changed to clear yellow solution with H2 gas
release. After 5 min being stirred, 32h (0.18 g, 0.48 mmol) in dry
THF was added to the reaction mixture and was stirred for 1 h. The
mixture was extracted with dichloromethane and washed with
brine. The organic layer was dried over Na2SO4, filtered, concen-
trated, and purified by silica gel column chromatography with n-
hexanes/ethyl acetate ¼ 3:1 to afford 33h (0.20 g) as a sticky oil.
(CDCl3, 400 MHz)
d 2.47 (3H, s, CH3), 4.37 (2H, s, CH2), 4.57 (2H, s,
CH2OH), 4.87 (2H, s, CH2OH), 7.30e7.43 (4H, m, naphthyl), 7.69e
7.76 (3H, m, naphthyl); MS (ESI): m/z ¼ 309.8 [M þ H].
4.1.23. (5-Hydroxy-6-methyl-2-(3-phenoxybenzyl)pyridine-3,4-
diyl)dimethanol (30j)
Following the procedure for the synthesis of 24a, the compound
29h (1.5 g, 3.4 mmol) was reacted with 10% Pd/C (0.15 g, 1.4 mmol)
to afford 30j (1.1 g) was sticky oil. Yield 92%; 1H NMR (CDCl3,
400 MHz) d 2.41 (3H, s, CH3), 4.13 (2H, s, CH2), 4.46 (2H, s, CH2OH),
4.90 (2H, s, CH2OH), 6.74e7.27 (9H, m, phenyl); MS (ESI): m/
z ¼ 351.9 [M þ H].
Yield 96%; 1H NMR (CDCl3, 400 MHz)
d
1.28 (3H, t, J ¼ 7.2 Hz, CH3),
1.54 (6H, s, 2ꢃ CH3), 2.48 (3H, s, CH3), 4.19 (2H, q, J ¼ 7.2 Hz, CH2),
4.32 (2H, s, CH2), 4.77 (2H, s, CH2OH), 5.85 (1H, d, J ¼ 16.4 Hz, ]CH),
7.35e7.57 (4H, m, naphthyl), 7.67e7.76 (4H, m, naphthyl & ]CH);
MS (ESI): m/z ¼ 418.1 [M þ H].
4.1.29. (E)-Ethyl 3-(2,2,8-trimethyl-6-(3-phenoxybenzyl)-4H-[1,3]
dioxino[4,5-c]pyridin-5-yl)acrylate (33j)
4.1.24. (2,2,8-Trimethyl-6-(naphthalen-1-ylmethyl)-4H-[1,3]
dioxino[4,5-c]pyridin-5-yl)methanol (31h)
Following the procedure for the synthesis of 33h the compound
32j (0.56 g, 1.4 mmol) was reacted with triethyl phosphonoacetate
(0.29 mL, 1.5 mmol) to afford 33j (0.59 g) as a sticky oil. Yield 92%;
To
a mixture of 30h (0.20 g, 0.65 mmol) and 2,2-
dimethoxypropane (1.3 mL, 11 mmol) was added p-toluene-
sulfonic acid (0.50 g, 2.6 mmol) in dry acetone. This mixture was
stirred for 12 h at room temperature and neutralized with saturated
aqueous NaHCO3 and extracted with dichloromethane 3 times. The
organic layers were combined and dried with anhydrous Na2SO4.
After Na2SO4 was removed, the organic layer were concentrated in
vacuum and purified by silica gel column chromatography with
chloroform/methanol ¼ 30:1 to afford 31h (0.22 g) as a sticky oil.
1H NMR (CDCl3, 400 MHz)
d
1.29 (3H, t, J ¼ 7.2 Hz, CH3), 1.54 (6H, s,
2ꢃ CH3), 2.40 (3H, s, CH3), 4.20 (2H, q, J ¼ 7.2 Hz, CH2), 4.41 (2H, s,
CH2), 4.76 (2H, s, CH2OH), 5.84 (1H, d, J ¼ 16.4 Hz, ]CH), 6.83e7.29
(9H, m, phenyl), 7.61 (1H, d, J ¼ 16.4 Hz, ]CH); MS (ESI): m/
z ¼ 460.1 [M þ H].
4.1.30. (E)-3-(2,2,8-Trimethyl-6-(naphthalen-1-ylmethyl)-4H-[1,3]
dioxino[4,5-C]pyridin-5-yl)acrylic acid (34h)
Yield 99%; 1H NMR (CDCl3, 400 MHz)
d
1.39 (6H, s, 2ꢃ CH3), 2.49
(3H, s, CH3), 4.22 (2H, s, CH2), 4.71 (2H, s, CH2OH), 4.91 (2H, s,
CH2OH), 7.25e7.27 (4H, m, naphthyl), 7.70e7.73 (3H, m, naphthyl);
MS (ESI): m/z ¼ 350.9 [M þ H].
Compounds 33h (90 mg, 0.21 mmol) was dissolved in 5%
methanolic KOH (7.0 mL) and stirred at room temperature for 2 h.
The reaction mixture was then acidified with 1 N HCl and solvent
was evaporated. KCl salt was removed by filtration. After being
concentrated, the crude product was purified by silcagel column
using chloroform/methanol ¼ 10:1 to afford 34h (60 mg) as a sticky
4.1.25. (2,2,8-Trimethyl-6-(3-phenoxybenzyl)-4H-[1,3]dioxino[4,5-
c]pyridin-5-yl)methanol (31j)
Following the procedure for the synthesis of 31h, the compound
30j (1.2 g, 3.4 mmol) was reacted with 2,2-dimethoxypropane
(6.7 mL, 54 mmol) to afford 31j (0.58 g) as a sticky oil. Yield 96%;
oil. Yield 71%; 1H NMR (CDCl3, 400 MHz)
d
1.54 (6H, s, 2ꢃ CH3), 2.46
(3H, s, CH3), 4.31 (2H, s, CH2), 4.77 (2H, s, CH2OH), 5.86 (1H, d,
J ¼ 16.4 Hz, ]CH), 7.25e7.55 (4H, m, naphthyl), 7.69e7.73 (3H, m,
naphthyl), 7.79 (1H, d, J ¼ 16.4 Hz, ]CH); MS (ESI): m/z ¼ 390.8
[M þ H].
1H NMR (CDCl3, 400 MHz)
d 1.54 (6H, s, 2CH3), 2.39 (3H, s, CH3), 4.17
(2H, s, CH2), 4.47 (2H, s, CH2OH), 4.93 (2H, s, CH2OH), 6.97e7.33
(9H, m, phenyl); MS (ESI): m/z ¼ 392.1 [M þ H].
4.1.31. (E)-3-(2,2,8-Trimethyl-6-(3-phenoxybenzyl)-4H-[1,3]
dioxino[4,5-c]pyridin-5-yl)acrylic acid (34j)
Following the procedure for the synthesis of 34h, the compound
33j (0.30 g, 0.65 mmol) was reacted with 5% methanolic KOH
(20 mL) to afford 34j (0.25 g) as a sticky oil. Yield 89%; 1H NMR
4.1.26. 2,2,8-Trimethyl-6-(naphthalen-1-ylmethyl)-4H-[1,3]dioxino
[4,5-c]pyridine-5-carbaldehyde (32h)
To a stirred solution of 31h (0.20 g, 0.57 mmol) in dry
dichloromethane was added pyridinium dichromate (0.26 g,
0.68 mmol) with vigorous stirring. After being stirred overnight,
the mixture was filtered through celatum bed with methanol. The
filtrate was evaporated under vacuum and purified by silica gel
column chromatography with n-hexanes/ethyl acetate ¼ 3:1 to
afford 32h (0.18 g) as a brown sticky oil. Yield 90%; 1H NMR (CDCl3,
(CDCl3, 400 MHz)
d
1.52 (6H, s, 2ꢃ CH3), 2.40 (3H, s, CH3), 4.13 (2H,
s, CH2), 4.78 (2H, s, CH2OH), 5.86 (1H, d, J ¼ 16.4 Hz, ]CH), 6.82e
7.31 (9H, m, phenyl), 7.70 (1H, d, J ¼ 16.4 Hz, ]CH); MS (ESI): m/
z ¼ 432.4 [M þ H].
400 MHz)
d
1.54 (6H, s, 2ꢃ CH3), 2.52 (3H, s, CH3), 4.62 (2H, s, CH2),
4.1.32. (E)-3-(5-Hydroxy-4-(hydroxymethyl)-6-methyl-2-
(naphthalen-1-ylmethyl)pyridin-3-yl)acrylic acid (35h)
5.11 (2H, s, CH2OH), 7.25e7.52 (4H, m, naphthyl), 7.74e7.76 (3H, m,
naphthyl), 10.48 (H, s, CHO); MS (ESI): m/z ¼ 348.4 [M þ H].
A solution 34h (55 mg, 0.14 mmol) in 10% formic acid (5.0 mL)
was refluxed for 3 h. When the mixture was cooled to room tem-
perature, it was added saturated NaHCO3 and extracted with
chloroform (3 times). The organic layers were combined, dried over
Na2SO4, filtered, concentrated, and purified by silica gel column
chromatography with chloroform/methanol ¼ 10:1 to afford 35h
(0.27 mg) as a white solid. Yield 54%; 1H NMR (MeOH-d4, 400 MHz)
4.1.27. 2,2,8-Trimethyl-6-(3-phenoxybenzyl)-4H-[1,3]dioxino[4,5-c]
pyridine-5-carbaldehyde (32j)
Following the procedure for the synthesis of 32h, the compound
31j (0.57 g, 1.5 mmol) was reacted with pyridinium dichromate
(0.67 g, 1.8 mmol) to afford 32j (0.52 g) as a brown sticky oil. Yield
90%; 1H NMR (CDCl3, 400 MHz)
d
1.53 (6H, s, 2ꢃ CH3), 2.46 (3H, s,
d 2.46 (3H, s, CH3), 4.28 (2H, s, CH2), 4.80(2H, s, CH2OH), 5.93 (1H, d,

J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
827
J ¼ 16.4 Hz, ]CH), 7.25e7.55 (4H, m, naphthyl), 7.69e7.73 (3H, m,
naphthyl), 7.88 (1H, d, J ¼ 16.4 Hz, ]CH); MS (ESI): m/z ¼ 350.3
[M þ H].
J ¼ 16.4 Hz, ]CH), 7.67e7.76 (3H, m, naphthyl); MS (ESI): m/
z ¼ 378.9 [M þ H].
4.1.38. (E)-Ethyl 3-(5-hydroxy-4-(hydroxymethyl)-6-methyl-2-(3-
phenoxybenzyl)pyridin-3-yl)acrylate (38j)
Following the procedure for the synthesis of 35h, the compound
33j (110 mg, 0.25 mmol) was reacted with 10% formic acid (5.0 mL)
to afford 38j (55 mg) as a white solid. Yield 52%; 1H NMR (CDCl3,
4.1.33. (E)-3-(5-Hydroxy-4-(hydroxymethyl)-6-methyl-2-(3-
phenoxybenzyl)pyridin-3-yl)acrylic acid (35j)
Following the procedure for the synthesis of 35h, the compound
34j (0.25 g, 0.60 mmol) was reacted with 10% formic acid (10 mL) to
afford 35j (0.19 g) as a white solid. Yield 82%; 1H NMR (MeOH-d4,
400 MHz)
d
1.28 (3H, t, J ¼ 7.2 Hz, CH3), 2.45 (3H, s, CH3), 4.04 (2H, s,
400 MHz)
d
2.41 (3H, s, CH3), 4.06 (2H, s, CH2), 4.76(2H, s, CH2OH),
CH2), 4.18 (2H, q, J ¼ 7.2 Hz, CH2), 4.90 (2H, s, CH2OH), 5.70 (1H, d,
J ¼ 16.4 Hz, ]CH), 6.77e7.31 (9H, m, phenyl), 7.55 (1H, d,
J ¼ 16.4 Hz, ]CH); MS (ESI): m/z ¼ 420.1 [M þ H].
5.87 (1H, d, J ¼ 16.4 Hz, ]CH), 6.66e7.31 (9H, m, phenyl), 7.58 (1H,
d, J ¼ 16.4 Hz, ]CH); MS (ESI): m/z ¼ 492.4 [M þ H].
4.1.34. (E)-3-(4-Formyl-5-hydroxy-6-methyl-2-(naphthalen-1-
ylmethyl)pyridin-3-yl)acrylic acid (36h)
4.1.39. Ethyl 3-(5-hydroxy-4-(hydroxymethyl)-6-methyl-2-
(naphthalen-1-ylmethyl)pyridin-3-yl)propanoate (39h)
To a solution of 35h (20 mg, 0.050 mmol) in dry dichloro-
methane was added activated manganese dioxide (30 mg,
0.35 mmol) with vigorous stirring for 2 h at room temperature
under N2 atmosphere. The mixture was filtered through celatum
bed with dichloromethane and the filtrate was evaporated in vac-
uum and purified by silica gel column chromatography with n-
hexanes/ethyl acetate ¼ 3:1 to afford 36h (14 mg) as a yellow solid.
Following the procedure for the synthesis of 24a, the compound
38h (60 mg, 0.16 mmol) was reacted with 10% Pd/C (10 mg,
0.090 mmol) to afford 39h (40 mg) as sticky oil. Yield 66%; 1H NMR
(CDCl3, 400 MHz)
d
1.10 (3H, t, J ¼ 6.8 Hz, CH3), 2.11 (2H, t, J ¼ 7.6 Hz,
CH2), 2.48 (3H, s, CH3), 2.77 (2H, t, J ¼ 7.6 Hz, CH2), 4.17 (2H, q,
J ¼ 6.8 Hz, CH2), 4.27 (2H, s, CH2), 4.89 (2H, s, CH2OH), 7.28e7.48
(4H, m, naphthyl), 7.68e7.76 (3H, m, naphthyl); MS (ESI): m/
z ¼ 379.4 [M þ H].
Yield 78%; 1H NMR (CDCl3, 400 MHz)
d 2.60 (3H, s, CH3), 4.32 (2H, s,
CH2), 5.92 (1H, d, J ¼ 16.4 Hz, ]CH), 7.29e7.56 (4H, m, naphthyl),
7.71e7.77 (3H, m, naphthyl), 7.98 (1H, d, J ¼ 16.4 Hz, ]CH), 10.13
(1H, s, CHO), 11.33 (1H, s, COOH); 13C NMR (DMSO-d6, 400 MHz)
18.38, 40.54, 116.82, 120.06, 124.94, 125.01, 127.96, 129.80, 131.75,
134.35, 147.47, 156.47, 157.13, 167.53, 196.93; MS (ESI): m/z ¼ 347.8
[M þ H].
4.1.40. Ethyl 3-(5-hydroxy-4-(hydroxymethyl)-6-methyl-2-(3-
phenoxybenzyl)pyridin-3-yl)propanoate (39j)
Following the procedure for the synthesis of 24a, the compound
38j (52 mg, 0.12 mmol) was reacted with 10% Pd/C (5.0 mg,
0.050 mmol) to afford 39j (35 mg) as sticky oil. Yield 66%; 1H NMR
(CDCl3, 400 MHz)
d
1.18 (3H, t, J ¼ 7.2 Hz, CH3), 2.16 (2H, t, J ¼ 7.6 Hz,
4.1.35. (E)-3-(4-Formyl-5-hydroxy-6-methyl-2-(3-phenoxybenzyl)
pyridin-3-yl)acrylic acid (36j)
CH2), 2.44 (3H, s, CH3), 2.78 (2H, t, J ¼ 7.6 Hz, CH2), 4.05 (2H, q,
J ¼ 7.2 Hz, CH2), 4.10 (2H, s, CH2), 4.92 (2H, s, CH2OH), 6.80e7.31
(9H, m, phenyl), MS (ESI): m/z ¼ 422.4 [M þ H].
Following the procedure for the synthesis of 36h, the compound
35j (0.18 g, 0.47 mmol) was reacted with manganese dioxide
(0.29 g, 3.4 mmol) to afford 36j (0.12 g) as a yellow solid. Yield 64%;
4.1.41. Ethyl 3-(4-formyl-5-hydroxy-6-methyl-2-(naphthalen-1-
ylmethyl)pyridin-3-yl)propanoate (40h)
1H NMR (CDCl3, 400 MHz)
d 2.55 (3H, s, CH3), 4.15 (2H, s, CH2), 5.92
(1H, d, J ¼ 16.0 Hz, ]CH), 6.79e7.31 (9H, m, phenyl), 7.92 (1H, d,
J ¼ 16.0 Hz, ]CH), 10.14 (1H, s, CHO), 11.39 (1H, s, COOH); 13C NMR
(DMSO-d6, 400 MHz) 18.83, 41.00, 116.78, 119.17, 123.34, 128.19,
129.81, 138.27, 140.92, 147.87, 150.82, 157.13, 167.20, 196.96; MS
(ESI): m/z ¼ 389.9 [M þ H].
Following the procedure for the synthesis of 36h, the compound
39h (37 mg, 0.10 mmol) was reacted with manganese dioxide
(60 mg, 0.70 mmol) to afford 40h (60 mg) as yellow sticky oil. Yield
79%; 1H NMR (CDCl3, 400 MHz)
d
1.13 (3H, t, J ¼ 7.2 Hz, CH3), 2.30
(2H, t, J ¼ 8.0 Hz, CH2), 2.54 (3H, s, CH3), 3.24 (2H, t, J ¼ 8.0 Hz, CH2),
4.02 (2H, q, J ¼ 7.2 Hz, CH2), 4.37 (2H, s, CH2), 7.32e7.42 (4H, m,
naphthyl), 7.71e7.78 (3H, m, naphthyl), 10.40 (1H, s, CHO); MS
(ESI): m/z ¼ 377.8 [M þ H].
4.1.36. (E)-3-(2-Carboxyvinyl)-5-hydroxy-6-methyl-2-(3-
phenoxybenzyl)isonicotinic acid (37j)
To a respective suspension of 36j (28 mg, 0.070 mmol) in DMF/
CAN (1:5) solution (2.0 mL) OxoneÒ (50 mg, 0.080 mmol) was
added. After the mixture was stirred for 2 h at room temperature,
the OxoneÒ was removed by filtration. The filtrate was concen-
trated in a vacuum and purified by silica gel column chromatog-
raphy with chloroform/methanol ¼ 5:1 to afford 37j (4.0 mg) as a
4.1.42. Ethyl 3-(4-formyl-5-hydroxy-6-methyl-2-(3-
phenoxybenzyl)pyridin-3-yl)propanoate (40j)
Following the procedure for the synthesis of 36h, the compound
39j (33 mg, 0.080 mmol) was reacted with manganese dioxide
(48 mg, 0.56 mmol) to afford 40j (27 mg) as yellow sticky oil. Yield
red solid. Yield 13%; 1H NMR (CDCl3, 400 MHz)
d
2.31 (3H, s, CH3),
86%; 1H NMR (CDCl3, 400 MHz)
d
1.21 (3H, t, J ¼ 7.2 Hz CH3), 2.37
4.15 (2H, s, CH2), 6.77e6.82 (2H, m, ]CH & phenyl), 6.89e6.94 (3H,
m, phenyl), 7.07 (1H, t, J ¼ 7.2 Hz, phenyl), 7.25e7.34 (4H, m,
phenyl), 7.63 (1H, d, J ¼ 16.4 Hz, ]CH); MS (ESI): m/z ¼ 406.1
[M þ H].
(2H, t, J ¼ 8.0 Hz, CH2), 2.49 (3H, s, CH3), 2.78 (2H, t, J ¼ 8.0 Hz, CH2),
4.09 (2H, q, J ¼ 7.2 Hz, CH2), 4.18 (2H, s, CH2), 6.78e7.32 (9H, m,
phenyl), 10.42 (1H, s, CHO); MS (ESI): m/z ¼ 420.4 [M þ H].
4.1.43. 3-(4-Formyl-5-hydroxy-6-methyl-2-(naphthalen-1-
ylmethyl)pyridin-3-yl)propanoic acid (41h)
Following the procedure for the synthesis of 34h, the compound
40h (25 mg, 0.070 mmol) was reacted with 5% methanolic KOH
(5.0 mL) to afford 41h (13 mg) as a yellow solid. Yield 63%; 1H NMR
4.1.37. (E)-Ethyl 3-(5-hydroxy-4-(hydroxymethyl)-6-methyl-2-
(naphthalen-1-ylmethyl)pyridin-3-yl)acrylate (38h)
Following the procedure for the synthesis of 35h, the compound
33h (0.10 g, 0.24 mmol) was reacted with 10% formic acid (5.0 mL)
to afford 38h (64 mg) as a white solid. Yield 71%; 1H NMR (CDCl3,
(CDCl3, 400 MHz)
d
2.37 (2H, t, J ¼ 8.0 Hz, CH2), 2.56 (3H, s, CH3),
400 MHz)
d
1.22 (3H, t, J ¼ 7.2 Hz, CH3), 2.49 (3H, s, CH3), 4.17 (2H, q,
3.25 (2H, t, J ¼ 8.0 Hz, CH2), 4.37 (2H, s, CH2), 7.25e7.53 (4H, m,
naphthyl), 7.72e7.74 (3H, m, naphthyl), 10.40 (1H, s, CHO), 11.54
(1H, s, COOH); MS (ESI): m/z ¼ 349.8 [M þ H].
J ¼ 7.2 Hz, CH2), 4.20 (2H, s, CH2), 4.82 (2H, s, CH2OH), 5.68 (1H, d,
J ¼ 16.4 Hz, ]CH), 7.28e7.47 (4H, m, naphthyl), 7.57 (1H, d,

828
J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
4.1.44. 3-(4-Formyl-5-hydroxy-6-methyl-2-(3-phenoxybenzyl)
pyridin-3-yl)propanoic acid (41j)
(2.5 mL) to afford 45h (11 mg) as a white solid. Yield 53%; 1H NMR
(CDCl3, 400 MHz) 2.45 (3H, s, CH3), 4.32 (2H, s, CH2), 4.81 (2H, s,
d
Following the procedure for the synthesis of 34h, the compound
40j (27 mg, 0.060 mmol) was reacted with 5% methanolic KOH
(5.0 mL) to afford 41h (23 mg) was obtained as a yellow solid. Yield
CH2OH), 7.39e7.45 (3H, m, naphthyl), 7.67e7.74 (4H, m, naphthyl);
MS (ESI): m/z ¼ 324.2 [M þ H].
92%; 1H NMR (CDCl3, 400 MHz)
d
2.37 (2H, t, J ¼ 8.0 Hz, CH2), 2.49
4.1.51. 5-Hydroxy-4-(hydroxymethyl)-6-methyl-2-(3-
phenoxybenzyl)nicotinic acid (45j)
Following the procedure for the synthesis of 34h, the compound
44j (50 mg, 0.14 mmol) was reacted with 5% methanolic KOH (4 mL)
to afford 45j (30 mg) as a white solid. Yield 57%; 1H NMR (CDCl3,
(3H, s, CH3), 3.21 (2H, t, J ¼ 8.0 Hz, CH2), 4.18 (2H, s, CH2), 6.82e7.31
(9H, m, phenyl), 10.41 (1H, s, CHO), 11.56 (1H, s, COOH); 13C NMR
(DMSO-d6, 400 MHz) 18.45, 21.47, 35.34, 40.51, 116.72, 119.01,
120.80, 123.27, 129.83, 131.02, 141.66, 147.94, 149.40, 153.01, 157.09,
176.54, 196.63; MS (ESI): m/z ¼ 392.4 [M þ H].
400 MHz) d 2.38 (3H, s, CH3), 4.13 (2H, s, CH2), 4.85 (2H, s, CH2OH),
6.73 (1H, d, J ¼ 7.2 Hz, phenyl), 6.90 (3H, d, J ¼ 7.6 Hz, phenyl), 6.97
(1H, d, J ¼ 7.6 Hz, phenyl), 6.90 (1H, t, J ¼ 7.6 Hz, phenyl), 7.15 (1H, t,
J ¼ 7.2 Hz, phenyl), 7.26 (2H, t, J ¼ 7.2 Hz, phenyl); MS (ESI): m/
z ¼ 366.1 [M þ H].
4.1.45. 3-(2-Carboxyethyl)-5-hydroxy-6-methyl-2-(3-
phenoxybenzyl)isonicotinic acid (42j)
Following the procedure for the synthesis of 37j, the compound
41j (35 mg, 0.090 mmol)was reacted withOxoneÒ (61 mg, 0.10 mmol)
to afford 42j (9.9 mg) as a red solid. Yield 27%; 1H NMR (CDCl3,
4.1.52. 1,7-Dihydroxy-6-methyl-4-(naphthalen-1-ylmethyl)furo
[3,4-c]pyridin-3(1H)-one (46h)
300 MHz) d 2.06e2.12 (2H, m, CH2), 2.24 (3H, s, CH3), 3.15e3.21 (2H,
m, CH2), 4.12 (2H, s, CH2), 6.72e6.75 (2H, m, phenyl), 6.83 (1H, d,
J ¼ 8.1 Hz, phenyl), 6.94 (2H, d, J ¼ 8.1 Hz, phenyl), 7.09e7.21 (2H, m,
phenyl), 7.32 (2H, t, J ¼ 7.5 Hz, phenyl); MS (ESI): m/z ¼ 406.1 [M ꢄ H].
Following the procedure for the synthesis of 36h, the compound
45h (10 mg, 0.030 mmol) was reacted with manganese dioxide
(19 mg, 0.21 mmol) to afford 46h (2.1 mg) as yellow sticky oil. Yield
21%; 1H NMR (CDCl3, 400 MHz)
d 2.48 (3H, s, CH3), 4.50 (2H, s, CH2),
4.1.46. 2,2,8-Trimethyl-6-(naphthalen-1-ylmethyl)-4H-[1,3]dioxino
[4,5-c]pyridine-5-carboxylic acid (43h)
Following the procedure for the synthesis of 37j, the compound
32h (80 mg, 0.23 mmol) was reacted with OxoneÒ (140 mg,
0.25 mmol) to afford 43h (80 mg) as brown sticky oil. Yield 95%; 1H
7.35e7.45 (4H, m, naphthyl & CH), 7.68e7.73 (4H, m, naphthyl); MS
(ESI): m/z ¼ 322.1 [M þ H].
4.1.53. 1,7-Dihydroxy-6-methyl-4-(3-phenoxybenzyl)furo[3,4-c]
pyridin-3(1H)-one (46j)
NMR (CDCl3, 400 MHz)
(2H, s, CH2), 4.93 (2H, s, CH2OH), 7.27e7.34 (3H, m, naphthyl), 7.59e
7.65 (4H, m, naphthyl); MS (ESI): m/z ¼ 363.4 [M þ H].
d
1.50 (6H, s, 2ꢃ CH3), 2.44 (3H, s, CH3), 4.56
Following the procedure for the synthesis of 36h, the compound
45j (28 mg, 0.070 mmol) was reacted with manganese dioxide
(47 mg, 0.49 mmol) to afford 46j (13 mg) as yellow sticky oil. Yield
47%; 1H NMR (CDCl3, 400 MHz)
d 2.51 (3H, s, CH3), 4.38 (2H, s, CH2),
4.1.47. 2,2,8-Trimethyl-6-(3-phenoxybenzyl)-4H-[1,3]dioxino[4,5-c]
pyridine-5-carboxylic acid (43j)
Following the procedure for the synthesis of 37j, the compound
32j (0.25 g, 0.64 mmol) was reacted with OxoneÒ (0.43 g,
0.71 mmol) to afford 42j (0.15 g) as brown sticky oil. Yield 59%; 1H
6.73 (1H, d, J ¼ 7.6 Hz, phenyl), 6.92 (2H, d, J ¼ 8.0 Hz, phenyl), 7.00e
7.07 (4H, m, phenyl & CH), 7.13 (1H, t, J ¼ 8.0 Hz, phenyl), 7.28e7.32
(2H, m, phenyl); 13C NMR (CDCl3, 400 MHz) 19.54, 38.80, 93.08,
116.96, 119.14, 120.01, 123.47, 124.39, 129.84, 130.01, 141.56, 153.99,
157.41, 157.55, 168.42; MS (ESI): m/z ¼ 364.2 [M þ H].
NMR (CDCl3, 400 MHz)
d
1.50 (6H, s, 2ꢃ CH3), 2.40 (3H, s, CH3), 4.38
(2H, s, CH2), 4.90 (2H, s, CH2OH), 6.67 (1H, d, J ¼ 7.2 Hz, phenyl),
6.87 (4H, d, J ¼ 6.4 Hz, phenyl), 6.98 (1H, t, J ¼ 7.6 Hz, phenyl), 7.04
(1H, t, J ¼ 7.6 Hz, phenyl), 7.18 (2H, d, J ¼ 7.2 Hz, phenyl); MS (ESI):
m/z ¼ 406.0 [M þ H].
4.2. Biological methods
The cRNA of hP2X3 receptor was obtained by reverse tran-
scription of the cDNA of hP2X3 receptor, which was generously
provided by Dr. W. Stuhmer and Dr. F. Soto of the Max-Plank
Institute. The EST clones containing full-length cDNAs of mP2X1
(clone ID: 4189541) and hP2X7 receptor (clone ID: 5286944) were
purchased (Invitrogen, CA, U.S.A.), and their sequences were
confirmed by DNA sequencing.
4.1.48. 7-Hydroxy-6-methyl-4-(naphthalen-1-ylmethyl)furo[3,4-c]
pyridin-3(1H)-one (44h)
Following the procedure for the synthesis of 35h, the compound
43h (70 mg, 0.19 mmol) was reacted with 10% formic acid (5.0 mL)
to afford 44h (44 mg) as sticky oil. Yield 70%; 1H NMR (CDCl3,
400 MHz)
d
2.57 (3H, s, CH3), 4.63 (2H, s, CH2), 5.16 (2H, s, CH2OH),
4.2.1. Two-electrode voltage-clamp assay at recombinant mP2X1
and hP2X3 receptors
7.36e7.53 (3H, m, naphthyl), 7.70e7.82 (4H, m, naphthyl); MS (ESI):
m/z ¼ 306.2 [M þ H].
Xenopus oocytes were harvested and prepared as previously
described. mP2X1 and hP2X3 receptor cRNA (40 nL, 1
mg/mL),
4.1.49. 7-Hydroxy-6-methyl-4-(3-phenoxybenzyl)furo[3,4-c]
pyridin-3(1H)-one (44j)
Following the procedure for the synthesis of 35h, the compound
43j (0.14 g, 0.34 mmol) was reacted with 10% formic acid (7.0 mL) to
afford 44j (0.11 g) as sticky oil. Yield 92%; 1H NMR (CDCl3, 400 MHz)
respectively were injected cytosolically to defolliculated oocytes,
which were 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 experi-
ments. ATP-activated membrane currents (Vh ¼ ꢄ70 mV) were
recorded from cRNA-injected oocytes using the two-electrode
voltage-clamp technique (Axoclamp 2B amplifier). Voltage
d
2.55 (3H, s, CH3), 4.45 (2H, s, CH2), 5.18 (2H, s, CH2OH), 6.76 (1H, d,
J ¼ 6.8 Hz, phenyl), 6.96 (2H, d, J ¼ 7.6 Hz, phenyl), 7.05e7.13 (3H, m,
phenyl), 7.16 (1H, t, J ¼ 7.6 Hz, phenyl), 7.29 (2H, d, J ¼ 7.2 Hz,
phenyl); MS (ESI): m/z ¼ 348.0 [M þ H].
recording (1e2 M
U tip resistance) and current-recording micro-
electrodes (5 M tip resistance) were filled with 3.0 M KCl. Oocytes
U
were held in an electrophysiological chamber and superfused with
Ringer’s solution (5 mL/min, at 18 ꢀC) containing (mM) NaCl, 110;
4.1.50. 5-Hydroxy-4-(hydroxymethyl)-6-methyl-2-(naphthalen-1-
ylmethyl)nicotinic acid (45h)
Following the procedure for the synthesis of 34h, the compound
44h (20 mg, 0.070 mmol) was reacted with 5% methanolic KOH
KCl, 2.5; HEPES, 5; BaCl2, 1.8, adjusted to pH 7.5. 2 mM ATP was
superfused over the oocytes for 60e120 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. Compounds for testing were

J.-H. Cho et al. / European Journal of Medicinal Chemistry 70 (2013) 811e830
829
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max e I), where I is the current evoked by ATP in the presence of an
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I
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from different batches of oocytes.
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in a humidified atmosphere of 5% CO2. Cells were harvested with
treatment of Trypsin/EDTA solution and collected by centrifugation
(200 g for 5 min). The cells were resuspended at 2.5 ꢃ 106 cells/mL
in assay buffers, consisting of 10 nM HEPES, 140 mM KCl, 5 mM
glucose, 1 mM EDTA 1 (pH 7.4), and then 100 mM ethidium bromide
was added. Cell suspensions were added to 96 well plates con-
taining the P2X7 receptor agonist, 6
m
M BzATP, at 2 ꢃ 105 cells/well.
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anism, Long-Term Potentiation (LTP), was extracted from rat.
Voltage sensitive dye (Di-4-ANEPPS) was treated to slice of spinal
cords (0.5 mm) and changes in the optical signal in the dorsal horn
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v Faltynek,
We thank to Dr. W. Stuhmer and Dr. F. Soto in the Max-Plank
Institute for the generous providing of the cDNA of hP2X3 recep-
tor. This research was supported by a Basic Science Research Pro-
gram of the National Research Foundation of Korea (NRF) funded by
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the Ministry of Science, ICT
2013023642).
& Future Planning (Grant No.
Appendix A. Supplementary data
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