Novel acid-type cyclooxygenase-2 inhibitors: Design, synthesis, and structure-activity relationship for anti-inflammatory drug

Article abstract of DOI:10.1016/j.ejmech.2012.01.053

Cyclooxygenase (COX) is a key rate-limiting enzyme for prostaglandin (PG) production cascades in the human body. The mechanisms of both the anti-inflammation effects and the side-effects of traditional COX inhibitors are associated with the existence of two COX isoforms. Thus while COX-1 is predominantly expressed ubiquitously and constitutively, and it serves a housekeeping role in processes such as gastrointestinal (GI) mucosa protection, COX-2 is absent or exhibits a low level of expression in most tissues, and is highly upregulated in response to endotoxin, virus, inflammatory or tissue-injury stimuli/signals, and tumour promoter in the various types of organs, tissues, and cells. Furthermore, COX-2 contribution to PGE₂ and PGI₂ production evokes and sustains systemic or peripheral inflammatory disease, but it is not involved in the COX-1-mediated GI tract events. Also, hypersensitivity of aspirin owing to its inhibitory action against COX-1 is a significant concern clinically. Consequently, highly selective COX-2 inhibitors have been needed for the treatment of inflammatory- and inflammation related-diseases that include pyrexia, inflammation, pain, rheumatoid arthritis, osteoarthritis, and cancers. In this study, a series of novel [2-{[(4-substituted or 4,5-disubstituted)-pyridin-2-yl]carbonyl}-(5- or 6-substituted or 5,6-disubstituted)-1H-indol-3-yl]acetic acid analogues was designed, synthesized, and evaluated to identify potent and selective COX-2 inhibitors as potential agents against inflammatory diseases. As significant findings, the present study clarified unique structure-activity relationship of the analogues toward potent and selective COX-2 inhibition in vitro, and identified 2-{6-fluoro-2-[4-methyl-2-pridinyl)carbonyl]-1H-indol-3-yl}acetic acid as a potent and selective COX-2 inhibitor in vitro that demonstrated orally potent anti-inflammation efficacy against carrageenan-induced oedema formation in the foot of SPF/VAF male SD rats as a peripheral inflammation model in vivo.

Full text of DOI:10.1016/j.ejmech.2012.01.053

European Journal of Medicinal Chemistry 50 (2012) 179e195
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel acid-type cyclooxygenase-2 inhibitors: Design, synthesis, and
structureeactivity relationship for anti-inflammatory drug
*
Shigeo Hayashi , Naomi Ueno, Akio Murase, Yoko Nakagawa, Junji Takada
Pfizer Global Research & Development, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo, Aichi 470-2393, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclooxygenase (COX) is a key rate-limiting enzyme for prostaglandin (PG) production cascades in the
human body. The mechanisms of both the anti-inflammation effects and the side-effects of traditional
COX inhibitors are associated with the existence of two COX isoforms. Thus while COX-1 is predomi-
nantly expressed ubiquitously and constitutively, and it serves a housekeeping role in processes such as
gastrointestinal (GI) mucosa protection, COX-2 is absent or exhibits a low level of expression in most
tissues, and is highly upregulated in response to endotoxin, virus, inflammatory or tissue-injury stimuli/
signals, and tumour promoter in the various types of organs, tissues, and cells. Furthermore, COX-2
contribution to PGE2 and PGI2 production evokes and sustains systemic or peripheral inflammatory
disease, but it is not involved in the COX-1-mediated GI tract events. Also, hypersensitivity of aspirin
owing to its inhibitory action against COX-1 is a significant concern clinically. Consequently, highly
selective COX-2 inhibitors have been needed for the treatment of inflammatory- and inflammation
related-diseases that include pyrexia, inflammation, pain, rheumatoid arthritis, osteoarthritis, and
cancers. In this study, a series of novel [2-{[(4-substituted or 4,5-disubstituted)-pyridin-2-yl]carbonyl}-
(5- or 6-substituted or 5,6-disubstituted)-1H-indol-3-yl]acetic acid analogues was designed, synthesized,
and evaluated to identify potent and selective COX-2 inhibitors as potential agents against inflammatory
diseases. As significant findings, the present study clarified unique structureeactivity relationship of the
analogues toward potent and selective COX-2 inhibition in vitro, and identified 2-{6-fluoro-2-[4-methyl-
2-pridinyl)carbonyl]-1H-indol-3-yl}acetic acid as a potent and selective COX-2 inhibitor in vitro that
demonstrated orally potent anti-inflammation efficacy against carrageenan-induced oedema formation
in the foot of SPF/VAF male SD rats as a peripheral inflammation model in vivo.
Received 3 November 2011
Received in revised form
26 January 2012
Accepted 27 January 2012
Available online 6 February 2012
Keywords:
NSAID
COX-2/COX-1 selectivity
Orally active COX-2 inhibitor
Carrageenan-induced oedema
Inflammation
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
COX catalyzes conversion of arachidonic acid (AA) into prosta-
glandin (PG) H2 (PGH2) that is further metabolized into various
Cyclooxygenase (COX, or prostaglandin G/H synthase or endo-
peroxidase; EC 1.14.99.1) is a key rate-limiting enzyme for PG
production cascades in the human body (and in the animals). Thus,
types of prostanoids (eicosanoids), namely, PGs such as PGE2, PGD2,
and PGF2a, prostacyclin (PGI2), and thromboxane (TX) A2 (TXA2)
[1,2]. These individual prostanoids are closely involved in various
regulations of human physiology and pathophysiology, respec-
tively. Notably, PGE2 and PGI2 play critical roles in the inflammatory
process to cause increases in body temperature, vascular perme-
ability, and oedema as inflammatory-disease mediators [1e6]. Also,
the mechanisms of both the anti-inflammation effects and the side-
effects for traditional non-steroidal anti-inflammatory drugs
(NSAIDs) with COX inhibition are associated with the existence of
two COX isoforms, namely, COX-1 (EC 1.14.99.1) and COX-2 (EC
1.14.99.1). COX-1 is predominantly expressed ubiquitously and
constitutively, and it serves a housekeeping role in processes such
as gastrointestinal (GI) mucosa protection. By contrast, COX-2 is
absent or exhibits a low level of expression in most tissues, and is
highly upregulated in response to endotoxin, virus, inflammatory
Abbreviations: AA, arachidonic acid; COX, cyclooxygenase (or endoperoxidase,
or prostaglandin G/H synthase, EC 1.14.99.1); PG, prostaglandin; PGH2, prosta-
glandin H2; PGG2, prostaglandin G2; PGE2, prostaglandin E2; PGI2, prostacyclin; 6-
keto-PGF1a, 6-keto-prostaglandin F1a; TXA2, thromboxane A2; TXB2, thromboxane
B2; NSAID, non-steroidal anti-inflammatory drug; SPF, specific pathogen free; VAF,
virus antibody free; SD rat, SpragueeDawley rat; TNF-
a, tumour necrosis factor-a;
IL-1 , interleukin-1 ; IL-6, interleukin-6; LDA, lithium diisopropylamide; DBU, 1,8-
b
b
diazabicyclo[5.4.0]undec-7-ene; LPS, lipopolysaccharide; HUVEC, human umbilical
vein endothelial cell; HWP, human washed platelet; HWB, human whole blood; po,
oral administration; OA, osteoarthritis; RA, rheumatoid arthritis; HBD, hydrogen
bond donor; HBA, hydrogen bond acceptor.
* Corresponding author.
E-mail address: Shigeo_Hayashi@nifty.com (S. Hayashi).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.053

180
S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
or tissue-injury stimuli/signals, proinflammatory cytokines,
carcinogens/tumour-promoters, and hyperglycemia-induced
mitochondrial reactive-oxygen species in the various types of
organs, tissues, and cells [1,5,7e16] (see also Ref. [17] by Hayashi
et al.). COX-2 contribution to PGE2 and PGI2 production evokes and
sustains systemic/peripheral inflammatory diseases or syndrome
but it is not involved in the COX-1-mediated GI tract events
[18e20]. In fact, the major issues of clinically used traditional
NSAIDs, that are nonselective COX inhibitors or rather COX-1
selective inhibitors, are GI side-effects such as GI tract bleeding,
ulcer, and perforation, owing to their COX-1 inhibitory activities
[1,18e21]. In addition, the inhibitory actions of aspirin (acetylsali-
cylic acid) and other traditional NSAIDs against COX-1 can be
crucial problems in clinical pharmacotherapy, such as respiratory-
and cutaneous-hypersensitivities and cross-reactivities [22].
Besides, it has been reported that COX-2 inhibitors or traditional
NSAIDs showed no or respective agent-dependent potential
cardiovascular risk in long-term studies [23,24], showing impor-
tance of safety index assessment for COX-2 inhibitor in long-term
use. Taken together, highly selective COX-2 inhibitor is significant
for the clinical treatments of various inflammatory diseases/
syndromes or inflammation- and/or COX-2 action-derived diseases
such as pyrexia, inflammation, pain, OA, RA [1e3,5,9,11,25e27] or
cancers/tumours [28e35], as well as coronary artery disease and
endotoxin-induced cardiovascular- or liver-disease [36,37] without
the clinical issues related to COX-1 inhibition [11,21,38].
Scheme 1. Convergent synthesis of [2-{[(4-substituted or 4,5-disubstituted)-pyridin-
2-yl]carbonyl}-(5- or 6-substituted or 5,6-disubstituted)-1H-indol-3-yl]acetic acids
consisted of three independent parts (1)e(3).
[42,43] to afford methyl (2E)-3-(2-amino-4-substituted-phenyl)
acrylates 5 and 6, respectively. Subsequently, arylsulfonylation of
the amino group of the acrylates 5 and 6 with arylsulfonyl chloride
afforded the corresponding methyl 3-{2-[(arylsulfonyl)amino]
phenyl}acrylates 7 and 8, respectively (Scheme 2).
The aim of the present drug-discovery study is to identify orally
potent and selective COX-2 inhibitor for the treatment of inflam-
matory diseases. In this COX-2 inhibitor study, it was crucial to
explore and identify key factors for potency of COX-2 inhibition
and selectivity of COX-2 inhibition over COX-1 inhibition since
COX-2 has approximately 60% similarity to COX-1 in coding-
sequence comparison between the isozymes [1,7,39]. As reported
herein, novel [2-{[(4-substituted or 4,5-disubstituted)-pyridin-2-
yl]carbonyl}-(5- or 6-substituted or 5,6-disubstituted)-1H-indol-3-
yl]acetic acid analogues were designed, synthesized, and evaluated
to discover novel COX-2 inhibitors as potential anti-inflammatory
drugs, including with the viewpoints.
As for the multistep preparation of 3-(4-ethylphenyl)acrylate
derivative, 4-bromo-3-nitroacetophenone
9
was used for
HeckeMiziroki reaction to give methyl (2E)-3-(4-acetyl-2-
nitrophenyl)acrylate 10. Next, an approach for direct reduction of
the acetyl group to form ethyl group using triethylsilane in TFA at
room temperature gave decomposed products, therefore sequential
chemoselective reductions for the moiety were conducted. Thus,
after the acetyl group was reduced with sodium borohydride to
afford methyl (2E)-3-[4-(1-hydroxyethyl)-2-nitrophenyl]acrylate
11, dehydroxylation of the 1-hydroxyethyl group with TMSI,
produced in situ from TMSCl and NaI [44], was performed to afford
the desired methyl (2E)-3-(4-ethyl-2-nitrophenyl)acrylate 12. Then
the nitro group of the acrylate 12 was reduced with iron(0) to form
methyl (2E)-3-(2-amino-4-ethylphenyl)acrylate 13, which was
arylsulfonylated to give the desired compound 14 in the same way
as mentioned above (Scheme 3).
2. Results and discussion
2.1. Chemistry
When 2-bromoanilines 15e18 were used for HeckeMiziroki
reaction, the corresponding methyl (2E)-3-(2-aminophenyl)
For this study, various [2-{[(4-substituted or 4,5-disubstituted)-
pyridin-2-yl]carbonyl}-(5- or 6-substituted or 5,6-disubstituted)-
1H-indol-3-yl]acetic acid derivatives were prepared, indeed.
Scheme 1 illustrates the convergent synthesis consisted of three
independent parts, similar to our reported method for some {2-
[(pyridin-2-yl)carbonyl]-1H-indol-3-yl}acetic acid derivatives by
Hayashi et al. [17], that is, (1) synthesis of methyl 3-{2-[(arylsul-
fonyl)amino]phenyl}acrylates, (2) synthesis of 2-bromo-1-(heter-
oaryl)ethanones, and (3) synthesis of the desired 3-[2-
(heteroaroyl)-1H-indol-3-yl]acetic acid derivatives from the above
substrates synthesized in (1) and (2).
(1) First, various methyl 3-{2-[(arylsulfonyl)amino]-(4- or 5-
substituted or 4,5-disubstituted)-phenyl}acrylate derivatives were
prepared using various synthetic methods with respective starting
materials as shown in Schemes 2e5.
In case 1-halogeno-2-nitrobenzenes 1 and 2 were available as
starting materials, HeckeMizoroki cross-coupling reaction with
methyl acrylate was performed in the presence of palladium(II)
catalyst, electron-donative triarylphosphine ligand, and triethyl-
amine [40,41], to convert into methyl (2E)-3-(4-substituted-2-
nitrophenyl)acrylates 3 and 4, respectively. Then the nitro group
of the acrylates 3 and 4 was reduced with low-valent iron metal
Scheme 2. Synthesis of methyl 3-{2-[(4-methylphenyl)amino]phenyl}acrylates 7 and
8
from 1-halogeno-2-nitro-(4-substituted)-benzenes. Reagents and conditions: (a)
methyl acrylate, Pd(OAc)2, PPh3, Et3N, DMF, 130 ꢀC, 4e9 h; (b) Fe, NH4Cl, EtOH/H2O,
reflux, 2 h; (c) p-toluenesulfonyl chloride, pyridine, CH2Cl2, 10e24 h.

S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
181
Scheme 3. Synthesis of methyl 3-(4-fluoro-2-{[(4-methylphenyl)sulfonyl]amino}phenyl)acrylate 14. Reagents and conditions: (a) methyl acrylate, Pd(OAc)2, PPh3, Et3N, DMF,
100 ꢀC, 6 h, then 130 ꢀC, 21 h; (b) NaBH4, MeOH, 10 min; (c) TMSI from TMSCleNaI (in situ), hexane/toluene/CH3CN, 48 h; (d) Fe, NH4Cl, EtOH/H2O, reflux, 1 h; (e) p-toluenesulfonyl
chloride, pyridine, CH2Cl2, 0 ꢀC to room temp, 16 h.
acrylates 19e22 afforded directly, which were converted into the
corresponding arylsulfonylamino compounds 23e26, respectively
(Scheme 4).
vicinal coupling, respectively. As well, at the NMR study of
compound 50, an acetyl peak ( 2.69 ppm) was generated, and
ortho-proton ( 8.35 ppm) and meta-proton ( 7.75 ppm) peaks had
d
d
d
Also, using 4- or 5-substituted 2-nitrobenzaldehydes 27e29
with methyl (triphenylphosphoranylidene)acetate for Wittig reac-
tion, the corresponding 3-(2-nitrophenyl)acrylate derivatives
30e32 were prepared, which were reduced to amino compounds
33e35 and then led to the corresponding methyl arylsulfonylamino
compounds 36e38 as usual [17,45] (Scheme 5; for details, see
Scheme S1 in Appendix, Supplementary material).
no orthoemeta vicinal coupling, respectively. Hence, it was
confirmed that the regioselectively-acetylated compounds 47 and
50 were successfully obtained, respectively (see 4.1.12.2. and
4.1.13.1. in the Experimental section, respectively). Their ethanone
portions of compounds 47 and 50 were then 2-brominated to afford
the desired 2-bromoethanones 48 and 51, respectively (Scheme 6).
(3) Finally, Michael addition reaction of the methyl 3-{2-[(aryl-
sulfonyl)amino]-(4- or 5-substituted or 4,5-disubstituted)-phenyl}
acrylates 7, 8, 14, 23e26, and 36e38 and the 2-bromo-1-[(4-
substituted or 4,5-disubstituted)-pyridin-2-yl]ethanones 43, 44,
48, and 51 with K2CO3 to form indoline skeleton and the subse-
quent aromatization of the indolines via dephenylsulfonation with
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave various methyl 3-
[2-(heteroaroyl)-1H-indol-3-yl] acetates 52e63. The methyl ester
portions were then hydrolyzed to afford the requisite {2-[(4-
substituted or 4,5-disubstituted)-pyridin-2-yl)carbonyl]-(5- or 6-
substituted or 5,6-disubstituted)-1H-indol-3-yl}acetic acids
64e75, respectively (Scheme 7).
(2) Besides, various 2-bromo-1-[(4-substituted or 4,5-
disubstituted)-pyridin-2-yl]ethanones were prepared. (a) Multi-
step bromoacetylation was performed for 4-substituted pyridine
derivatives. After 2-nitrilation of 1-oxo-pyridines 39 and 40 [46],
conversion of the nitrile group into acetyl group with methyl
Grignard reagent to afford 2-acetylpyridines 41 and 42, respec-
tively, and the subsequent 2-bromination of the acetyl group
afforded 2-bromo-1-[(4-alkyl)pyridin-2-yl]ethanones 43 and 44,
respectively, similar to the reported method [17,47] (Scheme S2 in
Appendix, Supplementary material). (b) Direct radical acetylation
at ortho-position of 4-alkyl substituted pyridine with pyruvic acid,
silver catalyst, and persulfate in two-phase redox systems [48] was
applied for 4,5-disubstituted pyridine analogues. Thus, after
4-position of 3-chloropyridine 45 was regioselectively lithiated
in situ with lithium diisopropylamide (LDA) as reported [49],
4-ethylation with EtI was performed to afford 3-chloro-4-
ethylpyridine 46, then the radical ortho-acetylation was per-
formed to afford 1-(5-chloro-4-ethylpyridin-2-yl)ethanone 47.
Also, 5,6,7,8-tetrahydroisoquinoline 49 was converted into 1-
(5,6,7,8-tetrahydroisoquinolin-3-yl)ethanone 50 by the radical
acetylation. Indeed, compared with NMR spectra before and after
radical acetylation, one peak of ortho-protons of compound 46 was
In Experimental section, stepwise synthetic procedures for the
respective final test compounds 64e75 were described. All of these
compounds 64e75 were used for pharmacological evaluations as
free acids, respectively.
2.2. Structureeactivity relationship for COX-2 inhibitor
2.2.1. In vitro study
As drug design, synthesis, and structureeactivity relationship
(SAR) studies, various types of [2-{[(4-substituted or 4,5-
disubstituted)-pyridin-2-yl]carbonyl}-(5- or 6-substituted or 5,6-
disubstituted)-1H-indol-3-yl]acetic acid analogues were investi-
gated to seek and identify potent and selective COX-2 inhibitors and
their requisite/appropriate characters of pharmacophores in vitro.
disappeared at compound 47 with generating an acetyl peak (
2.70 ppm). And remained ortho-proton ( 8.55 ppm) and meta-
proton ( 7.94 ppm) peaks of compound 47 had no orthoemeta
d
d
d
Scheme 4. Synthesis of methyl 3-{2-[(arylsulfonyl)amino]phenyl}acrylates 23e26 from 1-halogeno-(4- or 5-substituted or 4,5-disubstituted)-2-anilines. Reagents and conditions:
(a) methyl acrylate, Pd(OAc)2, P(o-Tol)3, Et3N, CH3CN, reflux, w9 h; (b) p-toluenesulfonyl chloride for compounds 23 and 25 or benzenesulfonyl chloride for compounds 24 and 26,
pyridine, CH2Cl2, 0 ꢀC to room temp, w8 h.

182
S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
showed less potent inhibition of TXB2 production in HWP or in
HWB, that is, compounds 65, 66, 70, 74, and 75 exhibited less than
50% inhibition of TXB2 production at a concentration of 10 M in
m
human washed platelets, and compounds 65, 69, 70, and 73
exhibited less than 50% inhibition of TXB2 production at a concen-
tration of 100 mM in human whole blood; also the other compounds
exhibited less potencies of COX-1 inhibition in the assays compared
to that of COX-2 inhibition, respectively.
In detail, SAR study around 5- and/or 6-substituted indole core
gave important results. In comparison of functional group effects at
6- or 5-position of indole core for COX-2 inhibition, the rank of the
orders of the inhibitory activities in HUVEC assay for {2-[(4-
methylpyridin-2-yl)carbonyl]-1H-indol-3-yl}acetic acid analogues
was fluoro (64) > methoxy (65) > ethyl (66) for 6-substituent, and
fluoro (72) > ethyl (68) > trifluoromethoxy (69) > tert-butyl (67) >
methoxy (73) for 5-substituent, respectively. At the respective
position of indole core for COX-2 inhibition, electron-withdrawing
group such as halogen group was prefer to rather electron-
donating/electron-rich groups such as alkyl group and alkoxy
group (see also other examples in our previous report [17]). As well,
trifluoromethoxy group (69) was prefer to methoxy group (73) for
COX-2 inhibition, thus, as modification of alkoxy group, electron-
negative trifluoromethyl group was more effective than electron-
donating methyl group. In a viewpoint of substituent size, bulker
group such as 5-tert-butyl group (67) was less favourable to smaller
group such as 5-ethyl group (68), which would indicate influence of
bulkiness for this portion on van der Waals interactions between
the compound and enzyme (COX-2 as well as COX-1).
Scheme 5. Synthesis of methyl 3-{2-[(arylsulfonyl)amino]phenyl}acrylates 36e38
from 2-nitro-(4- or 5-substituted)-benzaldehydes. Reagents and conditions: (a) see
text in this article and Scheme S1 in Appendix, Supplementary material; see also [17]
by Hayashi et al.
Thus, the in vitro inhibitory activities and selectivities for the
compounds against enzymatic activities of COX isozymes, inducible
COX-2 and constitutional COX-1, on human cellular and on human
whole blood were evaluated with our reported procedures [17].
Briefly, the activities of COX isozymes on human cellular were
determined (i) by 6-keto-prostaglandin F1 (6-keto-PGF1a, spon-
a
taneously degraded stable form of PGI2) production in human
umbilical vein endothelial cells (HUVECs) that were expressing
COX-2 induced by interleukin-1
b (IL-1b) for COX-2, and (ii) by
thromboxane B2 (TXB2, spontaneously degraded stable form of
TXA2) production in human washed platelets (HWP) for COX-1,
respectively [50,51]. As well, the activities of COX isozymes on
human whole blood (HWB) were determined (i) by PGE2 synthesis
in HWB that was expressing COX-2 induced by lipopolysaccharide
(LPS) for COX-2, and (ii) by TXB2 production in HWB for COX-1,
respectively [50,52e54]. For selectivity indices of human COX-2
inhibitors in the present study in vitro, COX-2-inhibition selec-
tivity was estimated as IC50 against COX-2 over IC50 against COX-1
in the human cellular and HWB studies, respectively. As described
Compared to the COX-2 inhibitory activities in HUVEC assay
between 5- and 6-substituents on indole core, 5-fluoro- or 5-ethyl-
substituted analogue showed more potent activity than that of
corresponding 6-substituted analogue, that is, the IC50 values were
already [17], the above 6-keto-PGF1 production in HUVEC and
0.00568
mM for 5-fluoro analogue 72 and 0.00981
mM for 6-fluoro
a
PGE2 production in HWB in our study conditions are rationalized as
COX-2 induction-dependent responses.
analogue 64, and 0.0157
for 6-ethyl analogue 66, respectively. Besides, 5- and 6-methoxy
m
M for 5-ethyl analogue 68 and 0.414 mM
Table 1 shows the results of in vitro SAR study for [2-{[(4-
substituted or 4,5-disubstituted)-pyridin-2-yl]carbonyl}-(5- or 6-
substituted or 5,6-disubstituted)-1H-indol-3-yl]acetic acids
64e75. In general, all these compounds displayed potent and
highly selective COX-2 inhibitory activities against the production
of COX-2-derived inflammatory mediators in vitro. Thus, these
analogues showed the same COX-2 inhibitory activities in the
assays, that is, the IC50 values were 0.128 mM for both 5-methoxy
analogue 73 and 6-methoxy analogue 65, respectively. Also, as
5,6-disubstituted indole analogue, 6-chloro-5-fluoro analogue 71
showed potent COX-2 inhibitory activity (IC50 ¼ 0.00677
mM),
which was comparative to that of 5-fluoro analogue 72.
compounds exhibited highly potent inhibition of 6-keto-PGF1
As for the comparison of the selectivities of COX-2 inhibition over
COX-1 inhibition in human cellular assays between 5- and
6-substituents, 6-fluoro or 6-methoxy substituted analogue showed
higher COX-2 selectivity than that of the corresponding 5-
a
synthesis in COX-2-induced HUVEC (IC50s: 0.00568e0.421
and highly potent inhibition of PGE2 synthesis in COX-2-induced
HWB (IC50s: 0.100e5.70 M). Alternatively, these compounds
mM),
m
Scheme 6. Synthesis of 2-bromo-1-(5-chloro-4-ethylpyridin-2-yl)ethanone 48 and 2-bromo-1-(5,6,7,8-tetrahydroisoquinolin-3-yl)ethanone 51. Reagents and conditions: (a) LDA/
heptanes/THF/EtPh, THF, ꢁ78 ꢀC, 1 h; then EtI, ꢁ78 ꢀC to room temp, 20 min; (b) MeCOCO2H, AgNO3, (NH4)2S2O8, H2SO4, H2O/CH2Cl2, 40 ꢀC, 3.5e12 h; (c) Br2, HBr/AcOH, 0 ꢀC to
room temp, 3e4 h.

S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
183
Scheme 7. Synthesis of [2-{[(4-substituted or 4,5-disubstituted)-pyridin-2-yl]carbonyl}-(5- or 6-substituted or 5,6-disubstituted)-1H-indol-3-yl]acetic acid derivatives 64e75.
Reagents and conditions: (a) K2CO3, acetone, w48 h; then DBU, w16.5 h; (b) 2 N NaOH, THF/MeOH or THF/EtOH, reflux, w6 h; then acidified by 2 N HCl, room temp.
substituted analogue, respectively, that is, the respective COX-2
inhibition selectivities were 86.5-fold for 6-fluoro analogue 64 and
26.4-fold for 5-fluoro analogue 72, and greater than 78.1-fold for 6-
methoxy analogue 65 and 23.8-fold for 5-methoxy analogue 73,
respectively. Besides, compared to 5-fluoro analogue 72, 5-fluoro-6-
chloro analogue 71 showed lower COX-2 selectivity (9.73-fold).
These results indicate electronic effect for the substituents and/
pyridine portion would contribute to (a) pharmacophore-
interactions (and their potencies) between this portion (respec-
tive substituent(s) or pyridine nucleus) and enzyme, or (b)
conformational or torsion-angle flexibility/strain/stability of this
portion in the moleculeeenzyme complex (for example, intra-
molecular interaction of pyridine portion with 2-carbonyl portion
by electronic-repulsion/dipole-moment effect). As well, 2-(5,6,7,8-
tetrahydroisoquinoline) analogue 75 showed potent activity as
or for p-electron density of the indole core as well as steric effect,
which would contribute to the dynamism of the formation (coor-
dination) and stabilization processes of compoundeenzyme
complex, for example (i) the potency of pharmacophore-
interactions and/or molecular (frontier) orbital interactions for
each substituent portion, indole core, and whole molecule with
enzyme protein structure, (ii) molecular electrostatic potentials of
the compounds in the biological catalytic reaction field, (iii) van der
Waals interactions between compound and enzyme (COX-2 or
COX-1), and (iv) integrated whole molecular properties.
a COX-2 inhibitor, that is, the IC50 value was 0.0192 mM. Notably,
the selectivities of COX-2 inhibition over COX-1 inhibition in
human cellular assays for the 4,5-disubstituted pyridine analogues
74 and 75 were very high, that is, greater than 326-fold for 5-
chloro-4-ethylpyridine analogue 74 and greater than 521-fold for
2-(5,6,7,8-tetrahydroisoquinoline) analogue 75, respectively. Their
steric effects for this portion might indicate potential potency and
selectivity of COX-2 inhibition for further analogues around these
compounds.
Next, SAR study around 4-substituted and 4,5-disubstituted
pyridine portion of 2-carbonylindole gave unique results as
well. Compared to COX-2 inhibitory activities in HUVEC assay for
4-alkyl substituent on the pyridine portion, 4-methyl group
showed more potent than 4-ethyl group for {2-[(4-alkylpyridin-
Taken together, the present SAR study of these compounds with
modification of substituents at 6- or 5-position of the indole core
and at 4- or 5-position of the pyridine portion led to highly potent
and selective human COX-2 inhibitors in vitro. Notably, the present
in vitro SAR study revealed the characteristics of the substituents/
compounds which contribute to or affect potency and selectivity of
COX-2 inhibition for the compounds in the respective manners
such as electron density/negativity, size, shape, and the substitu-
tion mode that is the position of substituent and/or the number of
substituent in the respective analogues. Besides, the time-
dependent mechanisms of PGH2 biosynthesis from AA in the COX
protein are estimated that (i) AA is bound to cyclooxygenase
channel and the entity (shape and conformation) of AA fits with the
channel cavity surrounded by amino acid residues that containing
aromatic, aliphatic, phenolic, and basic groups, owing to or assisted
with variously-classified interactions such as van der Waals, elec-
trostatic, hydrogen bonding, molecular orbital, hydrophobic, and
ionic interactions between AA and the channel, and (ii) two O2
(oxygen) molecules are added onto AA cooperated with tyrosyl
radical 385 in the appropriate orientation and conformation of AA
2-yl)carbonyl]-5-(trifluoromethoxy)-1H-indol-3-yl}acetic
analogues, that is, the IC50 values were 0.0396 M for 4-methyl
analogue 69 and 0.421 M for 4-ethyl analogue 70, respectively.
acid
m
m
As for {2-[(4,5-disubstituted-pyridin-2-yl)carbonyl]-6-chloro-
1H-indol-3-yl}acetic acid analogues, 5-chloro-4-ethylpyridine
analogue 74 showed potent COX-2 inhibition, that is, the IC50
value was 0.0307 mM, which was more potent than corresponding
5-ethoxy-4-ethylpyridine analogue (of which 5-chloro group of
compound 74 was replaced by ethoxy group as an apparent
electron-donating/electron-rich group), {6-chloro-2-[(5-ethoxy-4-
ethylpyridin-2-yl)carbonyl]-1H-indol-3-yl}acetic acid (whose IC50
value against COX-2 in IL-1
and whose IC50 value against COX-1 in HWP was greater than
10.0 M, further data not shown). Hence electronic effect for the
substituent and/or for -electron-density or basicity of the
b-stimulated HUVEC was 0.598 mM,
m
p

184
S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
Table 1
2.2.2. In vivo and physicochemical studies
In vitro structureeactivity relationships of inhibitory effects of [2-{[(4-substituted or
4,5-disubstituted)-pyridin-2-yl]carbonyl}-(5- or 6-substituted or 5,6-disubsti-
tuted)-1H-indol-3-yl]acetic acid analogues 64e75 against COX-2/COX-1 activities in
human cellular and HWB assays.
Next, as an oral anti-inflammatory efficacy for COX-2 inhibitor in
peripheral-inflammation model, in vivo oral inhibitory activity
against oedema-swelling stimulated by intraplantarly injected-
carrageenan in the hind paw of specific pathogen free and virus
antibody free (SPF/VAF) male SpragueeDawley (SD) rats was
studied [17,57e59]. Thus, as
a representative of this study,
compound 64 was evaluated in the in vivo model per os (p.o.)
encouraged by its highly potent COX-2 inhibitory activity and high
COX-2 selectivity in vitro in the above study. Indeed, compound 64 at
a dosing of 10 mg/kg (p.o.) exhibited 70% inhibition against oedema
formation by carrageenan in the foot of SPF/VAF SD rats as expected.
Besides, Table 2 shows physicochemical properties of compound
64 as well as the other analogues 65e75 to be considered (poten-
tial) in vivo activity and its mechanisms in the present study. Thus,
for compounds 64e75, molecular weight (MW) values are lower
than 400, topological polar surface area (TPSA) values are
83.05e92.08 Å2, and lipophilicity values calculated as ACD log D at
pH 7.4 (ACD log D7.4) are ꢁ2.10 to 0.06, respectively. As well, their
numbers of hydrogen bond donor (HBD) are two, which are
multisite HBD functionalities (for hydrophilic functionalities), and
their numbers of hydrogen bond acceptor (HBA) are four or five.
Compounds
Human cell assay
COX-2a COX-1b
IC50 M) IC50
HWB assay
COX-2c
No Substituent
COX-1d
R1
R2
R3
R4
(m
(
m
M) IC50
(
m
M) IC50
33.5
(mM)
64
65
66
H
H
H
F
Me
H
H
H
H
H
H
H
H
H
H
Cl
0.00981
0.128
0.414
0.0415
0.0157
0.0396
0.421
0.849
>10.0
>10.0
1.51
0.882
1.00
0.155
1.90
NTe
MeO Me
>100
NT
NT
47.0
>100
>100
7.70
21.4
>100
NT
Et
H
H
H
H
Cl
H
H
Cl
Cl
Me
Me
Me
Me
Et
Me
Me
Me
Et
67 tert-Bu
68 Et
69 F3C-O
70 F3C-O
NT
In carrageenan-stimulated peripheral inflammation model,
oedema formation as inflammatory response is developed in multi-
phase mechanisms as reported, thus, (i) proinflammatory cytokines
1.60
1.90
5.70
>10.0
71
72
F
F
0.00677
0.00568
0.128
0.0659 0.100
0.150
3.05
such as IL-6, IL-1b, and TNF-a are released by the peripheral
0.150
2.20
NT
stimulus, (ii) COX-2 mRNA and COX-2 protein are inducted and/or
upregulated, and (iii) COX-2-dependent induction/upregulation of
PGE2 and PGI2 causes increases of vasodilation, vascular perme-
ability, extravasation of plasma proteins, and leakage to water
73 MeO
74
75
H
H
0.0307
e(CH2)4e 0.0192
>10.0
>10.0
NT
NT
a
IC50 values of compounds against COX-2 activity were determined as inhibitory
activity against 6-keto-prostaglandin F1 synthesis in human umbilical vein endo-
a
thelial cells (HUVECs) that were expressing COX-2 by interleukin-1b-stimulation.
b
IC50 values of compounds against COX-1 activity were determined as inhibitory
activity against thromboxane B2 production in human washed platelets (HWPs).
Table 2
Physicochemical properties of compounds 64e75.
c
IC50 values of compounds against COX-2 activity were determined as inhibitory
activity against prostaglandin E2 synthesis in human whole blood (HWB) that was
expressing COX-2 by lipopolysaccharide-stimulation.
d
IC50 values of compounds against COX-1 activity were determined as inhibitory
activity against thromboxane B2 production in HWB.
e
NT: not tested.
within the site to form PGG2, and then (iii) the hydroperoxide
group of PGG2 is reduced (PGG2 is subjected to the reduction of
two electrons) to form PGH2 at peroxidase site where haem
(ironeporphyrin complex) is bound and histidine groups are linked
[1,55,56]. Also, there are structural differences between COX-2 and
COX-1 originated from respective amino acid sequence, for example,
COX-2 has valine 523 and COX-1 has isoleucine 523 in the corre-
sponding active-sites [1,7,39]. In the present in vitro study, the
designed compounds competed against or interfered with the
sequential enzymatic reactions as COX-2 inhibitors with respective
molecule-dependent potencies and selectivities. Hence the molec-
ular characteristics of the inhibitors described above would be
significant to determine pharmacophore-recognition/-interaction
mode, binding mode, mutual induced-fitting ability and/or various
stressestrain responses between respective compounds and
respective isozymes in terms of orientation, conformation, and
potency. Moreover, these novel acid-type COX-2 inhibitors with the
present findings might be useful to elucidate detailed mechanisms
underlying COX-2 selective potent inhibitory activity in vitro for
further COX-2 inhibitor study. As well, the significant various
substituent-effects toward potent and selective COX-2 inhibition
clarified in the present SAR study in vitro might be useful for
designing further COX-2 inhibitors.
Compounds
Physicochemical properties
No Substituent
Size
TPSA Lipophilicity HBDa
HBAb
R1
R2
R3
R4 MW
Å2
ACD
log D7.4
Number Number
c
64
65
66
H
H
H
F
Me
H
H
H
H
H
H
H
H
H
H
Cl
312.30 83.05 ꢁ1.96
324.33 92.28 ꢁ2.10
322.36 83.05 ꢁ0.92
350.41 83.05 ꢁ0.19
322.36 83.05 ꢁ0.90
378.30 92.28 ꢁ1.08
392.33 92.28 ꢁ0.55
346.74 83.05 ꢁ1.23
312.30 83.05 ꢁ1.85
324.33 92.28 ꢁ1.99
2
2
2
2
2
2
2
2
2
2
2
2
4
5
4
4
4
5
5
4
4
5
4
4
MeO Me
Et
H
H
H
H
Cl
H
H
Cl
Cl
Me
Me
Me
Me
Et
Me
Me
Me
Et
67 tert-Bu
68 Et
69 F3C-O
70 F3C-O
71
72
F
F
73 MeO
74
75
H
H
377.22 83.05
0.06
e(CH2)4e 368.81 83.05 ꢁ0.19
a
HBD, hydrogen bond donor.
HBA, hydrogen bond acceptor.
Lipophilicity values calculated as ACD log D at pH 7.4, predicted by ACD/Labo-
b
c
ratories 9.0.

S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
185
through the vessel walls [1e3,6,59e65]. On the other hand,
compound 64 demonstrated strong inhibitory effects against COX-2
moisture-sensitive reactants and solvents were carried out under
nitrogen atmosphere unless noted otherwise. Flash column chro-
matography (medium pressure liquid chromatography) purifica-
tions were carried out using Merck silica gel 60 (230e400 mesh
ASTM) (Merck KGaA, Darmstadt, Germany). The structures of all
isolated compounds were ensured by NMR, IR, MS or elementary
analysis. 1H nuclear magnetic resonance (1H NMR) data were
determined at 270 MHz on a JNM-LA 270 (JEOL Ltd., Akishima,
Tokyo, Japan) spectrometer. Chemical shifts are expressed in
induction-derived syntheses of PGE2 and of 6-keto-PGF1 (that is
a
degraded stable form of PGI2) in the present in vitro study as
described. Also, the above representative physicochemical proper-
ties of compound 64 would be appropriate for oral activity in vivo
and for peripheral selectivity in the oral/systemic-administration
condition, which have been already described in the previous
reports for orally potent COX-2 inhibitors [17] and other systemi-
cally/orally potent compounds [66,67a,67b] by Hayashi et al. in
detail. Consequently, the potent in vivo efficacy of compound 64
would be due to its intrinsic potent and selective inhibitory activity
against COX-2-derived production of inflammatory disease medi-
ators such as PGE2 and PGI2 in the peripheral inflammatory site
after oral administration of the compound, which was also indi-
cated in our previous study report [17]. As well, other analogues
that have potent intrinsic COX-2 inhibitory activities in the present
study in vitro might have oral anti-inflammatory activities in vivo
because of their appropriate physicochemical properties for
potential in vivo activity mentioned above.
Overall, the present in vivo study demonstrated that compound
64 per se might be a potential anti-inflammatory drug that showed
potent anti-oedematous effect in the peripheral inflammatory
model. As well, these findings of the drug-discovery study might
show that compound 64 and other compounds, identified as potent
and selective COX-2 inhibitors with their unique pharmacophores,
might be useful tools to explore further potential of COX-2 inhibitor
for the treatment of inflammatory diseases/syndromes or inflam-
mation- and/or COX-2 action-derived diseases, and helpful to eluci-
date detailed mechanisms of COX-2 dependent pathophysiology.
d
(ppm). 1H NMR chemical shifts were determined relative to tet-
ramethylsilane (TMS) as internal standard. NMR data are reported
as follows: chemical shift, number of atoms, multiplicities (s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, double
doublet; br, broadened), and coupling constants. Infrared spectra
were measured by an IR-470 (Shimadzu Co., Kyoto, Japan) infrared
spectrometer. Low-resolution mass spectral data (EI) were obtained
on an Automass 120 (JEOL Ltd., Akishima, Tokyo, Japan) mass
spectrometer. Melting points were obtained using an Exstar 6000
(Seiko Instruments Inc., Chiba, Japan) and were uncorrected. All
final compounds 64e75 and synthetic intermediates were
synthesized by us at Pfizer Global Research & Development, Nagoya
Laboratories (Aichi, Japan). All general chemicals were the highest
available grade.
4.1.2. 2-{6-Fluoro-2-[(4-methyl-2-pridinyl)carbonyl]-1H-indol-3-yl}
acetic acid (64)
4.1.2.1. Step 1. Methyl (2E)-3-(4-fluoro-2-nitrophenyl)acrylate (3). A
solution of 4-fluoro-1-iodo-2-nitrobenzene 1 (Oakwood Products,
2.403 g, 9.00 mmol), triphenylphosphine (1.416 g, 5.40 mmol), dry
Et3N (1.88 mL, 13.5 mmol), Pd(OAc)2 (303.1 mg, 1.35 mmol), and
methyl acrylate (1.62 mL, 18.0 mmol) in dry DMF (24.0 mL) was
stirred at 130 ꢀC under N2 for 9 h. The reaction solution was cooled
to room temperature, and concentrated in vacuo. The residue was
partitioned between AcOEt/toluene solution (1:2, 60 mL) and H2O
(50 mL). The organic layer was separated and the aqueous layer was
extracted with AcOEt/toluene solution (1:3, 40 mL ꢂ 3). The organic
layers were combined, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, hexane/AcOEt ¼ 5:1) to afford 286.7 mg
of the title product 3 in 14% yield as a yellow crystalline solid. 1H
3. Conclusions
In conclusion, various types of novel [2-{[(4-substituted or 4,5-
disubstituted)-pyridin-2-yl]carbonyl}-(5- or 6-substituted or 5,6-
disubstituted)-1H-indol-3-yl]acetic acids were designed, synthe-
sized, and biologically evaluated, which led to the discovery of potent
and selective human COX-2 inhibitors in vitro, as significant findings.
Furthermore, these studies revealed that the various substituents at
5- and/or 6-position of the indole core and at 4- and/or 5-position of
the pyridine portion have great influences on the potent and selec-
tive intrinsic COX-2 inhibitory activities in human cellular and HWB
assays in vitro, respectively. Especially, 2-{6-fluoro-2-[(4-methyl-2-
pridinyl)carbonyl]-1H-indol-3-yl}acetic acid 64 was identified as
a highly effective COX-2 inhibitor that demonstrates potent and
selective COX-2 inhibition in vitro and potent anti-inflammation
efficacy against carrageenan-induced oedema formation in the foot
of SPF/VAF male SD rats that is a peripheral inflammation model
in vivo. These results demonstrated the unique and significant
potential for the series of compounds as potent and selective COX-2
inhibitors in vitro and in vivo with their appropriate physicochemical
properties for in vivo activities. As well, the significant factors
regarding the mechanisms of interactions between small drug and
COX-2 were found out in the present study. Consequently, this study
might be useful or helpful for further studies to develop COX-2
inhibitors as potential anti-inflammatory drugs and to elucidate
detailed mechanisms for drug and COX-2 interactions.
NMR (270 MHz, CDCl3)
d 8.07 (1H, d, 15.8 Hz), 7.78 (1H, dd,
J ¼ 8.07 Hz, J ¼ 2.65 Hz), 7.68e7.63 (1H, m), 7.43e7.38 (1H, m), 6.34
(1H, d, J ¼ 15.8 Hz), 3.84 (3H, s).
4.1.2.2. Step 2. Methyl (2E)-3-(2-amino-4-fluorophenyl)acrylate
(5). A mixture of methyl (2E)-3-(4-fluoro-2-nitrophenyl)acrylate 3
(286.7 mg, 1.27 mmol), Fe powder (313.9 mg, 5.62 mmol), and
NH4Cl (30.1 mg, 0.563 mmol) in EtOH (47.0 mL)eH2O (23.5 mL)
was stirred under reflux conditions under N2 for 2 h. The reaction
mixture was cooled to room temperature, filtered through a Celite
pad with EtOH, and the resulting elute was concentrated in vacuo.
The residue was partitioned between AcOEt (40 mL) and H2O
(40 mL). The organic layer was separated and the aqueous layer was
extracted with AcOEt (30 mL ꢂ 4). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo to afford 244.8 mg of the title product 5 in 99% yield as
a brown solid. This compound was used for the next step without
further purification. 1H NMR (270 MHz, CDCl3)
d 7.75 (1H, d,
4. Experimental section
J ¼ 15.8 Hz), 7.34 (1H, dd, J ¼ 8.72 Hz, J ¼ 6.43 Hz), 6.47 (1H, ddd,
J ¼ 8.72 Hz, J ¼ 8.24 Hz, J ¼ 2.48 Hz), 6.39 (1H, dd, J ¼ 10.4 Hz,
J ¼ 2.48 Hz), 6.28 (1H, d, J ¼ 15.6 Hz), 4.13 (2H, br s), 3.80 (3H, s).
4.1. Chemistry
4.1.1. General
4.1.2.3. Step 3. Methyl (2E)-3-(4-fluoro-2-{[(4-methylphenyl)sul-
fonyl]amino}phenyl)acrylate (7). To a stirred solution of methyl
(2E)-3-(2-amino-4-fluorophenyl)acrylate 5 (244.8 mg, 1.25 mmol)
In general, reagents, solvents, and other chemicals were used as
purchased without further purification. All reactions with air- or

186
S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
in dry CH2Cl2 (7.0 mL) was added dry pyridine (380
m
L, 4.70 mmol)
afford 136.4 mg of the title product 52 in 76% yield as a yellow solid.
1H NMR (270 MHz, CDCl3)
12.49 (1H, br s), 8.61 (1H, d,
J ¼ 4.94 Hz), 8.17 (1H, s), 7.64 (1H, dd, J ¼ 8.88 Hz, J ¼ 5.27 Hz), 7.35
(1H, d, J ¼ 5.10 Hz), 7.18e7.13 (1H, m), 6.98e6.78 (1H, m), 4.31 (2H,
s), 3.73 (3H, s), 2.47 (3H, s).
followed by 4-toluenesulfonyl chloride (400.4 mg, 2.10 mmol) at
room temperature under N2. The reaction mixture was stirred at
room temperature under N2 for 24 h, and then dry MeOH (5 mL)
was added. The mixture was concentrated in vacuo, and the residue
was partitioned between AcOEt (25 mL) and 2 N HCl (25 mL). The
organic layer was separated and the aqueous layer was extracted
with AcOEt (25 mL). The organic layers were combined, washed
with saturated aqueous NaHCO3 (25 mL) and brine (25 mL), dried
over anhydrous MgSO4, filtered, and concentrated in vacuo to afford
417.6 mg of the title product 7 in 96% yield as a brown solid. 1H NMR
d
4.1.2.6. Step 6. 2-{6-Fluoro-2-[(4-methyl-2-pridinyl)carbonyl]-1H-
indol-3-yl}acetic acid (64). To a stirred solution of methyl 6-fluoro-
2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-3-yl}acetate
52
(136.4 mg, 0.418 mmol) in THF (10.0 mL)eMeOH (20.0 mL) was
added 2 N NaOH (1.1 mL, 2.2 mmol) at room temperature under N2.
The reaction mixture was warmed up to reflux conditions using an
oil bath, stirred under N2 for 2.5 h, cooled to room temperature, and
concentrated in vacuo. The residue was partitioned between Et2O
(40 mL) and H2O (40 mL). After the ethereal layer was separated to
remove impurity, the resulting aqueous layer was acidified by
adding 2 N HCl (3.0 mL), extracted with AcOEt (40 mL ꢂ 3). The
combined extracts were dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The residue was recrystallized from
AcOEtehexane to afford 103.0 mg of the title product 64 in 79%
yield as a lemon-yellow solid. Mp: 208 ꢀC. 1H NMR (270 MHz,
(270 MHz, CDCl3)
d
7.64 (1H, d, J ¼ 8.40 Hz), 7.46e7.19 (6H, m),
6.94e6.87 (1H, m), 6.77 (1H, br s), 6.13 (1H, d, J ¼ 15.8 Hz), 3.79 (3H,
s), 2.38 (3H, s).
4.1.2.4. Step 4. 2-Bromo-1-(4-methylpyridin-2-yl)ethanone hydro-
bromide (43). To a stirred solution of 2-nitrile-4-methylpyridine
{31.19 g, 229 mmol; that was prepared by us from the corre-
sponding 4-methylpyridine 1-oxide according to the reported
procedure [17,46], 1H NMR (270 MHz, CDCl3)
d 8.57 (1H, d,
J ¼ 5.1 Hz), 7.53e7.52 (1H, m), 7.35e7.32 (1H, m), 2.44 (3H, m)} in
dry benzene (300 mL)eanhydrous Et2O (230 mL) was added
dropwise 2 M solution of MeMgI/Et2O (149.0 mL, 298 mmol) at 0 ꢀC
under N2 over 1 h. The reaction mixture was warmed to room
temperature, stirred under N2 for 1 h, and then cooled to 0 ꢀC. After
aqueous NH4Cl (120 mL) was added dropwise to the mixture at
0 ꢀC, the resulting mixture was warmed to room temperature and
stirred for 1 h. The aqueous layer was removed, and the organic
layer was washed with water, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography (silica gel, hexane/AcOEt ¼ 8:1) to
afford 16.20 g of 1-(4-methylpyridin-2-yl)ethanone 41 as a yellow
DMSO-d6)
d 12.28 (1H, br s), 12.26 (1H, br s), 8.70 (1H, d,
J ¼ 4.91 Hz), 7.95 (1H, s), 7.80 (1H, dd, J ¼ 8.88 Hz, J ¼ 5.59 Hz), 7.57
(1H, d, J ¼ 4.94 Hz), 7.42 (1H, dd, J ¼ 10.2 Hz, J ¼ 2.16 Hz), 6.99 (1H,
ddd, J ¼ 10.2 Hz, J ¼ 9.23 Hz, J ¼ 2.16 Hz), 4.09 (2H, s), 2.47 (3H, s). IR
(KBr): 3238, 1701, 1638, 1597, 1533, 1398, 1281, 1211, 1132,
1003 cmꢁ1. MS (EI direct) m/z: Mþ 312. Anal. (C17H13FN2O3) C, H, N.
4.1.3. {6-Methoxy-2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-3-yl}
acetic acid (65)
4.1.3.1. Step 1. Methyl (2E)-3-(4-methoxy-2-nitrophenyl)acrylate
(4). A mixture of 1-bromo-4-methoxy-2-nitrobenzene 2 (Lancaster
Synthesis, 4.177 g, 18.0 mmol), methyl acrylate (3.24 mL,
36.0 mmol), Pd(OAc)2 (606.1 mg, 2.70 mmol), triphenylphosphine
(1.416 g, 5.40 mmol), and dry Et3N (3.76 mL, 27.0 mmol) in dry DMF
(45.0 mL) was stirred at 130 ꢀC under N2 for 4 h, cooled to room
temperature, and concentrated in vacuo. The residue was parti-
tioned between AcOEt/toluene solution (3:1, 40 mL) and H2O
(30 mL). The organic layer was separated and the aqueous layer was
extracted with AcOEt (30 mL ꢂ 2). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo to afford 6.420 g of the title product 4 as a brown oil
(crude). This compound was used for the next step without further
oil in 52% yield. 1H NMR (CDCl3, 270 MHz)
d
8.54 (1H, d, J ¼ 4.9 Hz),
7.88e7.87 (1H, m), 7.30e7.27 (1H, m), 2.72 (3H, s), 2.43 (3H, m).
To a stirred solution of the above 1-(4-methylpyridin-2-yl)
ethanone 41 (16.20 g, 120 mmol) in 25% HBr/AcOH (220 mL) was
added bromine (21.2 g, 132 mmol) in glacial AcOH (30.0 mL) at 0 ꢀC
under N2 over 30 min. The resulting yellow mixture was stirred at
room temperature under N2 for 2 h, then cooled to 0 ꢀC. After
anhydrous Et2O (1.0 L) was added to the cooled mixture, the
resulting solid was collected by filtration, followed by vacuum
drying to afford 28.78 g of the titled compound 43 as a slight yellow
crystalline solid in 81% yield. 1H NMR (270 MHz, DMSO-d6)
d 8.62
(1H, d, J ¼ 5.1 Hz), 7.93 (1H, s), 7.60e7.58 (1H, m), 5.02 (2H, s), 2.45
purification. 1H NMR (270 MHz, CDCl3)
d 8.07e7.15 (4H, m), 6.31
(1H, d, J ¼ 15.8 Hz), 3.90 (3H, s), 3.82 (3H, s).
(3H, s).
4.1.2.5. Step 5. Methyl 6-fluoro-2-[(4-methylpyridin-2-yl)carbonyl]-
1H-indol-3-yl}acetate (52). A mixture of methyl (2E)-3-(4-fluoro-2-
4.1.3.2. Step 2. Methyl (2E)-3-(2-amino-4-methoxyphenyl)acrylate
(6). A mixture of methyl (2E)-3-(4-methoxy-2-nitrophenyl)acry-
late 4 (crude, 6.420 g), Fe powder (5.027 g, 90.0 mmol), and NH4Cl
(481.4 mg, 9.00 mmol) in EtOH (80.0 mL)eH2O (40.0 mL) was stir-
red under reflux conditions under N2 for 2 h, then cooled to room
temperature. The mixture was filtered through a Celite pad with
EtOH, then the resulting elute was concentrated in vacuo. The
residue was partitioned between AcOEt (50 mL) and H2O (30 mL).
The organic layer was separated and the aqueous layer was
extracted with AcOEt (30 mL ꢂ 2). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo to afford 4.476 g of the title product 6 as a brown solid
(crude). This compound was used for the next step without further
{[(4-methylphenyl)sulfonyl]amino}phenyl)acrylate
7
(191.2 mg,
0.547 mmol), 2-bromo-1-(4-methylpyridin-2-yl)ethanone hydro-
bromide 43 (274.4 mg, 0.930 mmol), and anhydrous K2CO3
(907.2 mg, 6.56 mmol) in dry acetone (5.5 mL) was stirred at room
temperature under N2 for 22.5 h, then the hydrobromide 43
(274.4 mg) and anhydrous K2CO3 (907.2 mg) were added. The
reaction mixture was stirred for 16.5 h, the hydrobromide 43
(137.2 mg) and anhydrous K2CO3 (453.6 mg) were added, stirred for
7 h, then 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (Wako Pure
Chemical Industries, 254 mL, 1.70 mmol) was added. The reaction
mixture was further stirred at room temperature under N2 for 16 h,
and concentrated in vacuo. The residue was partitioned between
AcOEt (40 mL) and H2O (40 mL). The organic layer was separated
and the aqueous layer was extracted with AcOEt (40 mL ꢂ 3). The
organic layers were combined, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography (silica gel, hexane/AcOEt ¼ 2:1) to
purification. 1H NMR (270 MHz, CDCl3)
d 7.80e6.21 (5H, m), 4.03
(2H, br s), 3.79 (6H, s).
4.1.3.3. Step 3. Methyl (2E)-3-(4-methoxy-2-{[(4-methylphenyl)sul-
fonyl]amino}phenyl)acrylate (8). To a stirred solution of methyl
(2E)-3-(2-amino-4-methoxyphenyl)acrylate 6 (crude, 1.500 g) in

S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
187
dry CH2Cl2 (23.0 mL) was added dry pyridine (1.55 mL, 19.2 mmol)
followed by 4-toluenesulfonyl chloride (1.83 g, 9.60 mmol) at
room temperature under N2. The reaction mixture was stirred at
room temperature under N2 for 10 h, and then dry MeOH was
added. The mixture was concentrated in vacuo, and the residue
was partitioned between AcOEt (30 mL) and 2 N HCl (30 mL). The
organic layer was separated, washed with saturated aqueous
NaHCO3 (30 mL) and brine (30 mL), dried over anhydrous MgSO4,
filtered, and concentrated in vacuo to afford 2.007 g of the title
product 8 as an orange solid (crude). 1H NMR (270 MHz, CDCl3)
partitioned between AcOEt/toluene solution (4:1, 30 mL) and H2O
(30 mL). The organic layer was separated and the aqueous layer was
extracted with AcOEt/toluene solution (4:1, 30 mL ꢂ 3). The organic
layers were combined, washed with H2O (40 mL ꢂ 2), dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash column chromatography (silica gel, hexane/
AcOEt ¼ 2:1) to afford 479.8 mg of the title product 10 in 23% yield
as a yellow crystalline solid. This compound was used for the next
step without further purification. 1H NMR (270 MHz, CDCl3)
d
8.58e8.10 (3H, m), 7.77e7.74 (1H, m), 6.45 (1H, d, J ¼ 15.8 Hz),
d
7.62e7.20 (6H, m), 6.98 (1H, d, J ¼ 2.65 Hz), 6.76 (1H, dd,
3.85 (3H, s), 2.68 (3H, s).
J ¼ 8.91 Hz, J ¼ 2.65 Hz), 6.73 (1H, br s), 6.07 (1H, d, J ¼ 15.8 Hz),
3.80 (3H, s), 3.77 (3H, s), 2.37 (3H, s).
4.1.4.2. Step 2. Methyl (2E)-3-[4-(1-hydroxyethyl)-2-nitrophenyl]
acrylate (11). To a stirred solution of methyl (2E)-3-(4-acetyl-2-
nitrophenyl)acrylate 10 (462.2 mg, 1.85 mmol) in dry MeOH
(55.0 mL) was added NaBH4 (176.3 mg, 4.66 mmol) at room
temperature under N2. The reaction solution was stirred under N2
for 10 min, and concentrated in vacuo. The residue was diluted with
CH2C12 (20 mL) then poured into brine (20 mL). The organic layer
was separated and the aqueous layer was extracted with CH2C12
(20 mL ꢂ 2). The organic layers were combined, dried over anhy-
drous MgSO4, filtered, concentrated in vacuo to afford 442.3 mg of
the titled product 11 (crude). This compound was used for the next
step without further purification. 1H NMR (270 MHz, CDCl3)
4.1.3.4. Step 4. Methyl {6-methoxy-2-[(4-methylpyridin-2-yl)car-
bonyl]-1H-indol-3-yl}acetate (53). A mixture of methyl (2E)-3-(4-
methoxy-2-{[(4-methylphenyl)sulfonyl]amino}phenyl)acrylate
8
(crude, 361.4 mg), 2-bromo-1-(4-methylpyridin-2-yl)ethanone
hydrobromide 43 (442.5 mg, 1.50 mmol), and anhydrous K2CO3
(1.382 g, 10.0 mmol) in dry acetone (10.0 mL) was stirred at room
temperature under N2 for 9 h, then DBU (449 mL, 3.00 mmol) was
added. The reaction mixture was further stirred at room tempera-
ture under N2 for 10 h, and concentrated in vacuo. The residue was
partitioned between AcOEt (40 mL) and H2O (30 mL). The organic
layer was separated and the aqueous layer was extracted with
AcOEt (40 mL ꢂ 2). The organic layers were combined, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash column chromatography (silica gel, hexane/
AcOEt ¼ 2:1) to afford 184.4 mg of the title product 53 in 9.0% yield
(four steps from 2) as a yellow solid. 1H NMR (270 MHz, CDCl3)
d
8.12e7.60 (4H, m), 6.36 (1H, d, J ¼ 15.8 Hz), 5.06e4.94 (1H, m),
3.84 (3H, s), 1.53 (3H, d, J ¼ 6.43 Hz).
4.1.4.3. Step 3. Methyl (2E)-3-(4-ethyl-2-nitrophenyl)acrylate (12).
To a stirred mixture of NaI (1.668 g, 11.1 mmol) and dry CH3CN
(581 mL, 11.1 mmol) was added TMSCI (1.4l mL, 11.1 mmol) at room
d
12.33 (1H, br s), 8.61 (1H, d, J ¼ 4.94 Hz), 8.18 (1H, m), 7.57 (1H, d,
temperature under N2. After the mixture was stirred at the
temperature under N2 for 10 min, a solution of methyl (2E)-3-[4-(1-
hydroxyethyl)-2-nitrophenyl]acrylate 11 (crude, 442.3 mg) in dry
hexane (2.0 mL)edry toluene (2.0 mL)edry CH3CN (2.0 mL) was
added. The reaction mixture was stirred at room temperature
under N2 for 48 h, diluted with AcOEt/toluene solution (15:10,
25 mL), then poured into H2O (25 mL). The organic layer was
separated and the aqueous layer was extracted with Et2O
(15 mL ꢂ 2). The organic layers were combined, washed with
aqueous Na2S2O3 and with brine, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo to afford 457.0 mg of the titled
product 12 as a brown oil (crude). This compound was used for the
next step without further purification. 1H NMR (270 MHz, CDCl3)
J ¼ 8.91 Hz), 7.34e7.32 (1H, m), 6.90 (1H, d, J ¼ 1.97 Hz), 6.84 (1H,
dd, J ¼ 8.91 Hz, J ¼ 2.13 Hz), 4.31 (2H, s), 3.89 (3H, s), 3.71 (3H, s),
2.47 (3H, s).
4.1.3.5. Step 5. {6-Methoxy-2-[(4-methylpyridin-2-yl)carbonyl]-1H-
indol-3-yl}acetic acid (65). To a mixture of methyl {6-methoxy-2-
[(4-methylpyridin-2-yl)carbonyl]-1H-indol-3-yl}acetate
53
(165.7 mg, 0.490 mmol) in MeOH (30.0 mL)eTHF (15.0 mL) was
added 2 N NaOH (1.4 mL, 2.8 mmol) at room temperature under N2.
The reaction mixture was warmed up to reflux conditions, stirred
under N2 for 5 h, cooled to room temperature, acidified by adding
2 N HCl (2 mL), and concentrated in vacuo. The residue was parti-
tioned between AcOEt/THF solution (3:10, 130 mL) and H2O
(20 mL). The organic layer was separated, dried over anhydrous
Mg2SO4, and concentrated in vacuo. The residue was recrystallized
from AcOEtehexane to afford 124.2 mg of the title product 65 in
78% yield as a yellow solid. Mp: 203.6 ꢀC. 1H NMR (270 MHz, DMSO-
d
8.12e6.32 (5H, m), 3.83 (3H, s), 2.76 (2H, q, J ¼ 7.59 Hz),1.29 (3H, t,
J ¼ 7.59 Hz).
4.1.4.4. Step 4. Methyl (2E)-3-(2-amino-4-ethylphenyl)acrylate (13).
A mixture of methyl (2E)-3-(4-ethyl-2-nitrophenyl)acrylate 12
(crude, 457.0 mg), Fe powder (519.4 mg, 9.30 mmol), and NH4Cl
(49.8 mg, 0.931 mmol) in EtOH (9.0 mL)eH2O (4.5 mL) was stirred
under reflux conditions for 1 h, then cooled to room temperature.
The mixture was filtered through a Celite pad with EtOH, then the
resulting elute was concentrated in vacuo. The residue was parti-
tioned between AcOEt (20 mL) and H2O (10 mL). The organic layer
was separated and the aqueous layer was extracted with Et2O
(10 mL ꢂ 3). The organic layers were combined, dried over anhy-
drous MgSO4, filtered, and concentrated in vacuo to afford 297.2 mg
of the titled product 13 (crude). This compound was used for the
next step without further purification. 1H NMR (270 MHz, CDCl3)
d6) d 12.11 (2H, br s), 8.68 (1H, d, 4.94 Hz), 7.94 (1H, m), 7.62 (1H, d,
J ¼ 8.88 Hz), 7.56e7.52 (1H, m), 7.14 (1H, d, J ¼ 2.30 Hz), 6.76 (1H,
dd, J ¼ 8.88 Hz, J ¼ 2.30 Hz), 4.05 (2H, s), 3.82 (3H, s), 2.47 (3H, s). IR
(KBr): 3198, 1709, 1630, 1593, 1531, 1460, 1423, 1334, 1275, 1213,
1161, 1136, 1039, 1001 cmꢁ1. MS (EI direct) m/z: Mþ 324. Anal.
(C18H16N2O4) C, H, N.
4.1.4. {6-Ethyl-2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-3-yl}
acetic acid (66)
4.1.4.1. Step 1. Methyl (2E)-3-(4-acetyl-2-nitrophenyl)acrylate (10).
A solution of 1-(4-bromo-3-nitrophenyl)ethanone 9 (Lancaster
Synthesis, 2.000 g, 8.20 mmol), triphenylphosphine (43.0 mg,
0.164 mmol), dry Et3N (1.50 mL, 10.8 mmol), Pd(OAc)2 (18.4 mg,
0.0820 mmol), and methyl acrylate (1.10 mL, 12.2 mmol) in dry
DMF (20.0 mL) was stirred at 100 ꢀC under N2 for 6 h, warmed up to
130 ꢀC, and stirred for 21 h. The solution was cooled to room
temperature, and concentrated in vacuo at 40 ꢀC. The residue was
d
7.84e6.29 (5H, m), 3.82 (2H, br s), 3.80 (3H, s), 2.56 (2H, q,
J ¼ 7.59 Hz), 1.21 (3H, t, J ¼ 7.59 Hz).
4.1.4.5. Step 5. Methyl (2E)-3-(4-ethyl-2-{[(4-methylphenyl)sulfonyl]
amino}phenyl)acrylate (14). To a stirred solution of methyl (2E)-3-
(2-amino-4-ethylphenyl)acrylate 13 (crude, 294.1 mg) in dry

188
S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
CH2Cl2 (4.0 mL) was added dry pyridine (267
m
L, 3.30 mmol) fol-
and tri-o-tolylphosphine (584.5 mg) were added. The reaction
mixture was additionally stirred under reflux conditions for 5 h,
cooled to room temperature, and concentrated in vacuo. The
residue was partitioned between AcOEt (40 mL) and H2O (40 mL).
The organic layer was separated and the aqueous layer was
extracted with AcOEt (40 mL ꢂ 2). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by flash column chromatography
(silica gel, hexane/AcOEt ¼ 3:1) to afford 1.394 g of the title product
lowed by 4-toluenesulfonyl chloride (314.6 mg, 1.65 mmol) at 0 ꢀC
under N2. The reaction mixture was warmed to room temperature,
stirred at room temperature under N2 for 16 h, and then dry MeOH
(2 mL) was added. The mixture was stirred for 5 min, and
concentrated in vacuo. The residue was partitioned between AcOEt
(20 mL) and 2 N HCl (20 mL). The organic layer was separated and
the aqueous layer was extracted with AcOEt (15 mL). The organic
layers were combined, washed with 2 N HCl (10 mL), saturated
aqueous NaHCO3 (20 mL), and brine (20 mL), dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was puri-
fied by flash column chromatography (silica gel, hexane/
AcOEt ¼ 5:2) to afford 129.2 mg of the title product 14 in 20% yield
(four steps from 10) as a slight yellow solid. 1H NMR (270 MHz,
19 in 75% yield as a brown oil. 1H NMR (270 MHz, CDCl3)
d 7.85 (1H,
d, J ¼ 15.8 Hz), 7.38 (1H, d, J ¼ 2.30 Hz), 7.23 (1H, dd, J ¼ 8.56 Hz,
J ¼ 2.30 Hz), 6.67 (1H, d, J ¼ 8.56 Hz), 6.37 (1H, d, J ¼ 15.8 Hz), 3.87
(2H, br s), 3.81 (3H, s), 1.28 (9H, s).
CDCl3)
d
7.57 (2H, d, J ¼ 8.40 Hz), 7.46 (1H, d, J ¼ 15.8 Hz), 7.40e7.04
4.1.5.2. Step 2. Methyl (2E)-3-(5-tert-butyl-2-{[(4-methylphenyl)
sulfonyl]amino}phenyl)acrylate (23). To a stirred solution of methyl
(2E)-3-(2-amino-5-tert-butylphenyl)acrylate 19 (1.392 g, 5.97
mmol) in dry CH2C12 (16.0 mL) was added dry pyridine (1.45 mL,
17.9 mmol) followed by 4-toluenesulfonyl chloride (1.706 g,
8.95 mmol) at 0 ꢀC under N2. The reaction mixture was warmed to
room temperature, stirred under N2 for 5 h, and then dry MeOH
(10 mL) was added. The mixture was concentrated in vacuo, and the
residue was partitioned between AcOEt (20 mL) and 2 N HCl
(20 mL). The organic layer was separated, and the aqueous layer
was washed with saturated aqueous NaHCO3 (20 mL) and brine
(20 mL), dried over anhydrous MgSO4, filtered, and concentrated in
vacuo to afford 2.314 g of the titled compound 23 in 100% yield. This
compound was used for the next step without further purification.
(5H, m), 6.43 (1H, br s), 6.14 (1H, d, J ¼ 15.8 Hz), 3.78 (3H, s), 2.62
(2H, q, J ¼ 7.59 Hz), 2.37 (3H, s), 1.18 (3H, t, J ¼ 7.59 Hz).
4.1.4.6. Step 6. Methyl {6-ethyl-2-[(4-methylpyridin-2-yl)carbonyl]-
1H-indol-3-yl}acetate (54). A mixture of methyl (2E)-3-(4-ethyl-2-
{[(4-methylphenyl)sulfonyl]amino}phenyl)acrylate 14 (127.0 mg,
0.353 mmol), 2-bromo-1-(4-methylpyridin-2-yl)ethanone hydro-
bromide 43 (156.3 mg, 0.530 mmol), and anhydrous K2CO3
(488.4 mg, 3.53 mmol) in dry acetone (3.5 mL) was stirred at room
temperature under N2 for 9 h, then DBU (168 mL, 1.12 mmol) was
added. The reaction mixture was further stirred at room tempera-
ture under N2 for 12 h, and concentrated in vacuo. The residue was
cooled to 0 ꢀC, poured into 2 N HCl (20 mL), then the mixture was
extracted with AcOEt (30 mL ꢂ 3). The combined extracts were
washed with saturated aqueous NaHCO3 (20 mL) and brine
(20 mL), dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The residue was purified by flash column chromatography
(silica gel, hexane/AcOEt ¼ 2:1) to afford 90.6 mg of the title
product 54 in 76% yield as a yellow solid. 1H NMR (270 MHz, CDCl3)
1H NMR (270 MHz, CDCl3)
d 7.60e7.56 (2H, m), 7.51 (1H, d,
J ¼ 16.0 Hz), 7.45 (1H, d, 2.13 Hz), 7.39e7.20 (4H, m), 6.53 (1H, br s),
6.19 (1H, d, J ¼ 15.8 Hz), 3.79 (3H, s), 2.38 (3H, s), 1.29 (9H, s).
4.1.5.3. Step 3. Methyl {5-tert-butyl-2-[(4-methylpyridin-2-yl)
carbonyl]-1H-indol-3-yl}acetate (55). A mixture of methyl (2E)-3-
(5-tert-butyl-2-{[(4-methylphenyl)sulfonyl]amino}phenyl)acrylate
23 (465.0 mg, 1.20 mmol), 2-bromo-1-(4-methylpyridin-2-yl)
ethanone hydrobromide 43 (530.9 mg, 1.80 mmol), and anhydrous
K2CO3 (1.659 g, 12.0 mmol) in dry acetone (12.0 mL) was stirred at
d
12.30 (1H, br s), 8.62 (1H, d, J ¼ 4.94 Hz), 8.19 (1H, m), 7.62 (1H, d,
J ¼ 8.40 Hz), 7.36e7.32 (2H, m), 7.06e7.02 (1H, m), 4.33 (2H, s), 3.72
(3H, s), 2.79 (2H, q, J ¼ 7.43 Hz), 2.48 (3H, s), 1.31 (3H, t, J ¼ 7.56 Hz).
4.1.4.7. Step 7. {6-Ethyl-2-[(4-methylpyridin-2-yl)carbonyl]-1H-
room temperature under N2 for 19 h, then DBU (538 mL, 3.60 mmol)
indol-3-yl}acetic acid (66). To a stirred solution of methyl {6-ethyl-
was added. The reaction mixture was further stirred at room
temperature under N2 for 9 h, and concentrated in vacuo. The
residue was partitioned between AcOEt (20 mL) and H2O (20 mL).
The organic layer was separated and the aqueous layer was
extracted with AcOEt (20 mL ꢂ 3). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by flash column chromatography
(silica gel, hexane/AcOEt ¼ 5:1 to 4:1) to afford 365.5 mg of the title
product 55 in 84% yield as a yellow solid. 1H NMR (270 MHz, CDCl3)
2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-3-yl}acetate
54
(89.1 mg, 0.265 mmol) in EtOH (15.0 mL) was added 2 N NaOH
(0.70 mL, 1.4 mmol) at room temperature under N2. The reaction
mixture was warmed up to reflux conditions using an oil bath,
stirred under N2 for 3 h, cooled to room temperature, acidified by
adding 2 N HCl (1.5 mL), and concentrated in vacuo. The residue was
partitioned between AcOEt (30 mL) and H2O (15 mL). The residue
was recrystallized from AcOEtehexane to afford 78.3 mg of the title
product 66 in 92% yield as a yellow solid. Mp: 204e207 ꢀC. 1H NMR
d
12.33 (1H, br s), 8.63 (1H, d, J ¼ 4.94 Hz), 8.18 (1H, m), 7.62e7.32
(270 MHz, DMSO-d6)
d
12.05 (2H, br s), 8.67 (1H, d, J ¼ 4.94 Hz),
(4H, m), 4.33 (2H, s), 3.74 (3H, s), 2.47 (3H, s), 1.40 (9H, s).
7.92 (1H, s), 7.62 (1H, d, J ¼ 8.40 Hz), 7.55e7.52 (1H, m), 7.44 (1H, s),
6.97 (1H, d, J ¼ 8.40 Hz), 4.03 (2H, s), 2.71 (2H, q, J ¼ 7.56 Hz), 2.45
(3H, s), 1.23 (3H, t, J ¼ 7.56 Hz). IR (KBr): 3240, 1709, 1636, 1593,
1531, 1394, 1275, 1204, 1144, 1001 cmꢁ1. MS (EI direct) m/z: Mþ 322.
Anal. (C19H18N2O3) C, H, N.
4.1.5.4. Step 4. {5-tert-Butyl-2-[(4-methylpyridin-2-yl)carbonyl]-1H-
indol-3-yl}acetic acid (67). To a stirred solution of methyl {5-tert-
butyl-2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-3-yl}acetate 55
(301.9 mg, 0.828 mmol) in MeOH (30.0 mL)eTHF (10.0 mL) was
added 2 N NaOH (2.3 mL, 4.6 mmol) at room temperature under N2.
The reaction mixture was warmed up to reflux conditions, stirred
under N2 for 6 h, cooled to room temperature, 2 N HCl (2.3 mL) was
added. The mixture was stirred at room temperature for several
minutes, and concentrated in vacuo. The residue was dissolved in
anhydrous THF (40 mL). The resulting solution was dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue
was recrystallized from AcOEt to afford 245.6 mg of the title
product 67 in 85% yield as a yellow solid. Mp: 190.9 ꢀC. 1H NMR
4.1.5. {5-tert-Butyl-2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-
3-yl}acetic acid (67)
4.1.5.1. Step 1. Methyl (2E)-3-(2-amino-5-tert-butylphenyl)acrylate
(19). A mixture of 2-bromo-4-tert-butylaniline 15 (Wako Pure
Chemical Industries, 1.825 g, 8.00 mmol), methyl acrylate (1.80 mL,
20.0 mmol), Pd(OAc)2 (215.5 mg, 0.960 mmol), tri-o-tolylphos-
phine (1.169 g, 3.84 mmol), and dry Et3N (4.30 mL, 30.9 mmol) in
dry CH3CN (16.0 mL) was stirred under reflux conditions under N2
for 4 h, then more methyl acrylate (1.0 mL), Pd(OAc)2 (107.9 mg),
(270 MHz, DMSO-d6)
d
12.10 (2H, br s), 8.70 (1H, d, J ¼ 5.10 Hz), 7.95

S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
189
(1H, m), 7.65e7.55 (3H, m), 7.46 (1H, dd, J ¼ 8.91 Hz, J ¼ 1.84 Hz),
4.09 (2H, s), 2.46 (3H, s),1.35 (9H, s). IR (KBr): 2963,1717,1645,1597,
1533, 1437, 1281, 1211, 1080, 1032, 1003 cmꢁ1. MS (EI direct) m/z:
Mþ 350. Anal. (C21H22N2O3) C, H, N.
for the synthesis of compound 64 (that is, preparation method of
arylsulfonylamide derivative 7, see 4.1.2.3.),1.40 gof the title product
24 was prepared in quantitative yield as a yellow solid, from methyl
(2E)-3-(2-amino-5-ethylphenyl)acrylate 20 (788 mg, 3.84 mmol)
and benzenesulfonyl chloride (735
instead of compound 5 and 4-toluenesulfonyl chloride in the
procedure. 1H NMR (270 Hz, CDCl3)
7.70e7.48 (4H, m), 7.42e7.36
mL, 5.76 mmol) that were used
4.1.6. {5-Ethyl-2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-3-yl}
acetic acid (68)
d
4.1.6.1. Step 1. 2-Bromo-4-ethylaniline (16). A mixture of 4-
ethylaniline (Tokyo Chemical Industry, 1.00 g, 8.25 mmol) and
glacial AcOH (5.0 mL) was stirred under reflux conditions under N2
for 5 h, cooled to room temperature, neutralized by adding anhy-
drous K2CO3 with stirring, and then the mixture was extracted with
AcOEt. The extracts were dried over anhydrous MgSO4, filtered, and
concentrated in vacuo to afford 1.35 g of N-(4-ethylphenyl)acet-
amide in quantitative yield as a brown oil (crude). This compound
was used for the next step without further purification. 1H NMR
(2H, m), 7.29e7.26 (2H, m), 7.20e7.17 (2H, m), 6.14 (1H, d,
J ¼ 15.8 Hz), 3.76 (3H, s), 2.62 (2H, q, J ¼ 7.6 Hz), 1.21 (3H, t,
J ¼ 7.6 Hz).
4.1.6.4. Step 4. Methyl {5-ethyl-2-[(4-methylpyridin-2-yl)carbonyl]-
1H-indol-3-yl}acetate (56). According to the procedure described in
step 5 for the synthesis of compound 64 (that is, preparation
method of methyl 6-fluoro-2-[(4-methylpyridin-2-yl)carbonyl]-
1H-indol-3-yl}acetate 52, see 4.1.2.5.), 220 mg of the title product
56 was prepared in 56% yield as a yellow solid from methyl (2E)-3-
{5-ethyl-2-[(phenylsulfonyl)amino]phenyl}acrylate 24 (400 mg,
(270 MHz, CDCl3)
d
7.90 (1H, br s), 7.40 (2H, d, J ¼ 8.4 Hz), 7.11 (2H, d,
J ¼ 8.4 Hz), 2.59 (2H, q, J ¼ 7.6 Hz), 2.12 (3H, s),1.20 (3H, t, J ¼ 7.6 Hz).
To a stirred solution of the above N-(4-ethylphenyl)acetamide
(1.32 g, 8.09 mmol) in glacial AcOH (15.0 mL) was added dropwise
1.16 mmol)
hydrobromide 43 (512 mg, 1.74 mmol). 1H NMR (270 Hz, CDCl3)
12.32 (1H, br s), 8.59e8.57 (1H, m), 8.14 (1H, m), 7.47e7.39 (2H,
and
2-bromo-1-(4-methylpyridin-2-yl)ethanone
a solution of bromine (468
m
L, 9.08 mmol) in glacial AcOH (3.0 mL)
d
at 0 ꢀC under N2. The reaction mixture was stirred at room
temperature under N2 for 30 min, warmed up to 55 ꢀC, stirred for
3 h, and then cooled to room temperature. The mixture was poured
into H2O and then extracted with AcOEt. The extracts were washed
with saturated aqueous NaHCO3, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo to afford 1.88 g of N-(2-bromo-
4-ethylphenyl)acetamide in 96% yield as a slight brown oil. 1H NMR
m), 7.30e7.21 (2H, m), 4.32 (2H, s), 3.73 (3H, s), 2.75 (2H, q,
J ¼ 7.6 Hz), 2.43 (3H, s), 1.27 (3H, t, J ¼ 7.6 Hz).
4.1.6.5. Step 5. {5-Ethyl-2-[(4-methylpyridin-2-yl)carbonyl]-1H-
indol-3-yl}acetic acid (68). According to the procedure described in
step 6 for the synthesis of compound 64 (the final step, see 4.1.2.6.),
190 mg of the title product 68 in 90% yield as a yellow solid was
(270 MHz, CDCl3)
d
8.15 (1H, d, J ¼ 8.4 Hz), 7.55 (1H, br s), 7.36 (1H,
prepared
from
methyl
{5-ethyl-2-[(4-methylpyridin-2-yl)
m), 7.14e7.10 (1H, m), 2.58 (2H, q, J ¼ 7.6 Hz), 2.21 (3H, s),1.20 (3H, t,
carbonyl]-1H-indol-3-yl}acetate 56 (220 mg, 0.654 mmol). Mp:
215 ꢀC. 1H NMR (270 Hz, DMSO-d6) 12.07 (1H, br s), 8.67 (1H, d,
J ¼ 4.94 Hz), 7.92 (1H, s), 7.56e7.49 (3H, m), 7.19 (1H, d, J ¼ 8.59 Hz),
4.04 (2H, s), 2.67 (2H, q, J ¼ 7.59 Hz), 2.44 (3H, s), 1.21 (3H, t,
J ¼ 7.59 Hz). IR (KBr): 1701, 1638, 1597, 1535, 1443, 1420, 1398, 1331,
1279, 1236, 1205 cmꢁ1. Anal. (C19H18N2O3) C, H, N.
J ¼ 7.6 Hz). MS (EI direct) m/z: Mþ 241.
To a stirred solution of the above N-(2-bromo-4-ethylphenyl)
acetamide (1.88 g, 7.77 mmol) in dry EtOH (2.8 mL) was added
concentrated HCl(2.5 mL)at 0 ꢀC under N2. Thereaction mixturewas
stirred under reflux conditions under N2 for 3.5 h. The mixture was
cooled to room temperature, and concentrated in vacuo. The result-
ing solid was washed with cooled EtOH to give 2-amino-1-bromo-5-
ethylbenzene hydrochloride as a slight brown solid, which was
neutralized by adding 2 N NaOH and then extracted with AcOEt. The
extracts were dried over anhydrous MgSO4, filtered, and concen-
trated in vacuo to afford 1.42 g of the title product 16 in 91% yield as
4.1.7. {2-[(4-Methylpyridin-2-yl)carbonyl]-5-(trifluoromethoxy)-
1H-indol-3-yl}acetic acid (69)
4.1.7.1. Step 1. Methyl (2E)-3-[2-amino-5-(trifluoromethoxy)phenyl]
acrylate (21). A mixture of 2-bromo-4-(trifluoromethoxy)aniline
17 (Maybridge, 2.048 g, 8.00 mmol), methyl acrylate (1.80 mL,
20.0 mmol), Pd(OAc)2 (215.5 mg, 0.960 mmol), tri-o-tolylphos-
phine (1.169 g, 3.84 mmol), and dry Et3N (4.30 mL, 30.9 mmol) in
dry CH3CN (16.0 mL) was stirred under reflux conditions under N2
for 4 h, then concentrated in vacuo. The residue was partitioned
between H2O (30 mL) and AcOEt (30 mL). The organic layer was
separated and the aqueous layer was extracted with AcOEt
(30 mL ꢂ 2). The organic layers were combined, dried over anhy-
drous MgSO4, filtered, and concentrated in vacuo to afford 3.630 g
of the title product 21 (crude). This compound was used for the
next step without further purification. 1H NMR (270 MHz, CDCl3)
a brown oil.1H NMR (270 MHz, CDCl3)
d
7.25 (1H, d, J ¼ 2.00 Hz), 6.94
(1H, dd, J ¼ 8.07 Hz, J ¼ 1.97 Hz), 6.70 (1H, d, J ¼ 8.07 Hz), 3.93 (2H, br
s), 2.52 (2H, q, J ¼ 7.59 Hz), 1.18 (3H, t, J ¼ 7.59 Hz).
4.1.6.2. Step 2. Methyl (2E)-3-(2-amino-5-ethylphenyl)acrylate
(20). A mixture of 2-bromo-4-ethylaniline 16 (1.02 g, 5.10 mmol),
methyl acrylate (1.15 mL, 12.8 mmol), Pd(OAc)2 (137 mg,
0.610 mmol), tri-o-tolylphosphine (745 mg, 2.45 mmol), and dry
Et3N (2.5 mL, 18 mmol) in dry CH3CN (10.0 mL) was stirred under
reflux conditions under N2 for 2 h, then more methyl acrylate
(0.60 mL), Pd(OAc)2 (69 mg), tri-o-tolylphosphine (372 mg), and
dry Et3N (1.3 mL) were added. The reaction mixture was stirred
under reflux conditions for 7 h, and concentrated in vacuo. The
residue was diluted with AcOEt, washed with H2O, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash column chromatography (silica gel, hexane/
AcOEt ¼ 5:1 to 4:1) to afford 793 mg of the title product 20 in 76%
d
7.74 (1H, d, J ¼ 15.8 Hz), 7.23e7.02 (2H, m), 6.68 (1H, d,
J ¼ 8.72 Hz), 6.36 (1H, d, J ¼ 15.8 Hz), 4.00 (2H, br s), 3.81 (3H, s).
4.1.7.2. Step 2. Methyl (2E)-3-[2-{[(4-methylphenyl)sulfonyl]amino}-
5-(trifluoromethoxy)phenyl]acrylate (25). To a stirred solution of
methyl (2E)-3-[2-amino-5-(trifluoromethoxy)phenyl]acrylate 21
(crude, 3.627 g) in dry CH2C12 (23.0 mL) was added dry pyridine
(1.55 mL, 19.2 mmol) followed by 4-toluenesulfonyl chloride
(1.830 g, 9.60 mmol) at 0 ꢀC under N2. The reaction mixture was
warmed to room temperature, stirred under N2 for 8 h, and then
dry MeOH (18 mL) was added. The mixture was stirred for 5 min
and concentrated in vacuo. The residue was partitioned between
AcOEt (30 mL) and 2 N HCI (30 mL). The organic layer was sepa-
rated, washed with saturated aqueous NaHCO3 (30 mL) and brine
yield as a yellow solid. 1H NMR (270 Hz, CDCl3)
d 7.83 (1H, d,
J ¼ 15.8 Hz), 7.21 (1H, m), 7.04e7.01 (1H, m), 6.64 (1H, d, J ¼ 8.2 Hz),
6.36 (1H, d, J ¼ 15.8 Hz), 3.87 (2H, br s), 3.80 (3H, s), 2.51 (2H, q,
J ¼ 7.6 Hz), 1.19 (3H, t, J ¼ 7.6 Hz).
4.1.6.3. Step 3. Methyl (2E)-3-{5-ethyl-2-[(phenylsulfonyl)amino]
phenyl}acrylate (24). According to the procedure described in step 3

190
S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
(30 mL), dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by flash column chromatog-
raphy (silica gel, hexane/AcOEt ¼ 3:1) to afford 1.338 g of the titled
compound 25 in 40% yield (two steps from 17) as a pink solid. 1H
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash column chromatography (silica gel, hexane/
AcOEt ¼ 5:1) to afford 200.0 mg of the title product 58 in 62% yield
as a lemon-yellow solid. 1H NMR (270 MHz, CDCl3)
d 12.64 (1H, br s),
NMR (270 MHz, CDCl3)
d
7.58 (2H, d, J ¼ 8.24 Hz), 7.48e7.19
8.66 (1H, d, J ¼ 5.10 Hz), 8.22 (1H, m), 7.55 (1H, m), 7.52 (1H, d,
J ¼ 9.07 Hz), 7.41e7.22 (2H, m), 4.30 (2H, s), 3.74 (3H, s), 2.79 (2H, q,
J ¼ 7.72 Hz), 1.32 (3H, t, J ¼ 7.59 Hz).
(6H, m), 6.76 (1H, br s), 6.14 (1H, d, J ¼ 15.8 Hz), 3.80 (3H, s),
2.38 (3H, s).
4.1.7.3. Step 3. Methyl {2-[(4-methylpyridin-2-yl)carbonyl]-5-(tri-
fluoromethoxy)-1H-indol-3-yl}acetate (57). A mixture of methyl
(2E)-3-[2-{[(4-methylphenyl)sulfonyl]amino}-5-(trifluoromethoxy)
phenyl]acrylate 25 (332.3 mg, 0.800 mmol), 2-bromo-1-(4-
4.1.8.2. Step 2. {2-[(4-Ethylpyridin-2-yl)carbonyl]-5-(trifluorometh-
oxy)-1H-indol-3-yl}acetic acid (70). To a stirred solution of methyl
{2-[(4-ethylpyridin-2-yl)carbonyl]-5-(trifluoromethoxy)-1H-indol-
3-yl}acetate 58 (170.0 mg, 0.418 mmol) in MeOH (15.0 mL)eTHF
(5.0 mL) was added 2 N NaOH (1.2 mL, 2.4 mmol) at room temper-
ature under N2. The reaction mixture was warmed up to reflux
conditions, stirred under N2 for 6 h, cooled to room temperature,
acidified by adding 2 N HCl (1.2 mL), and concentrated in vacuo. The
residue was dissolved in anhydrous THF, and the resulting solution
was concentrated in vacuo. The residue was recrystallized from
AcOEt to afford 153.4 mg of the title product 70 in 94% yield as
methylpyridin-2-yl)ethanone
hydrobromide
43
(354.0 mg,
1.20 mmol), and anhydrous K2CO3 (1.106 g, 8.00 mmol) in dry
acetone (8.0 mL) was stirred at room temperature under N2 for 8 h,
then DBU (359 mL, 2.40 mmol) was added. The reaction mixture
was further stirred at room temperature under N2 for 16 h, and
concentrated in vacuo. The residue was partitioned between AcOEt
(40 mL) and H2O (30 mL). The organic layer was separated and
concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, hexane/AcOEt ¼ 3:1) to afford 209.7 mg
of the title product 57 in 67% yield as a yellow solid. 1H NMR
a yellow solid. Mp: 222.6 ꢀC. 1H NMR (270 MHz, DMSO-d6)
d 12.41
(1H, br s), 8.74 (1H, d, J ¼ 4.94 Hz), 7.99 (1H, m), 7.80 (1H, m), 7.77
(1H, d, J ¼ 8.88 Hz), 7.63e7.30 (2H, m), 4.10 (2H, s), 2.79 (2H, q,
7.59 Hz), 1.26 (3H, t, J ¼ 7.59 Hz). IR (KBr): 3271, 1697, 1645, 1597,
1539, 1423, 1400, 1337, 1258, 1198, 1117, 1028, 1003 cmꢁ1. MS (EI
direct) m/z: Mþ 392. Anal. (C19H15F3N2O4) C, H, N.
(270 MHz, CDCl3)
8.19 (1H, m), 7.55e7.23 (4H, m), 4.30 (2H, s), 3.74 (3H, s), 2.49
d
12.60 (1H, br s), 8.64 (1H, d, J ¼ 5.13 Hz),
(3H, s).
4.1.7.4. Step 4. {2-[(4-Methylpyridin-2-yl)carbonyl]-5-(trifluoro-
methoxy)-1H-indol-3-yl}acetic acid (69). To a stirred solution of
methyl {2-[(4-methylpyridin-2-yl)carbonyl]-5-(trifluoromethoxy)-
1H-indol-3-yl}acetate 57 (187.5 mg, 0.478 mmol) in MeOH
(20.0 mL)eTHF (9.0 mL) was added 2 N NaOH (1.3 mL, 2.6 mmol)
at room temperature under N2. The reaction mixture was warmed
up to reflux conditions, stirred under N2 for 4 h, cooled to room
temperature, acidified by adding 2 N HCl (2.0 mL), and concen-
trated in vacuo. The residue was partitioned between AcOEt/THF
solution (3:10, 130 mL) and H2O (20 mL). The organic layer was
separated, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was recrystallized from AcOEtehexane to
afford 161.1 mg of the title product 69 in 89% yield as a greenish-
yellow solid. Mp: 235e238 ꢀC. 1H NMR (270 MHz, DMSO-d6)
4.1.9. {6-Chloro-5-fluoro-2-[(4-methylpyridin-2-yl)carbonyl]-1H-
indol-3-yl}acetic acid (71)
4.1.9.1. Step 1. 2-Bromo-5-chloro-4-fluoroaniline (18). To a stirred
solution of 3-chloro-4-fluoroaniline (Tokyo Chemical Industry,
5.00 g, 34.4 mmol) and dry pyridine (3.50 mL, 43.3 mmol) in dry
CH2Cl2 (17.0 mL) was added dropwise a solution of bromine
(1.90 mL, 36.9 mmol) in CH2Cl2 (10.0 mL) at 0 ꢀC under N2. The
reaction mixture was stirred at the temperature under N2 for 1 h,
warmed up to room temperature, and stirred for 1 h. The mixture
was poured into H2O and then extracted with AcOEt. The extracts
were dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The residue was purified by flash column chromatography
(silica gel, hexane/AcOEt ¼ 10:1) to afford 2.76 g of the title product
18 in 36% yield as a brown solid. 1H NMR (270 MHz, CDCl3)
(1H, d, J ¼ 8.6 Hz), 6.77 (1H, d, J ¼ 6.4 Hz), 3.84 (2H, br s).
d 7.22
d
12.38 (1H, br s), 12.23 (1H, br s), 8.69 (1H, d, J ¼ 4.94 Hz),
7.95e7.94 (1H, m), 7.77 (1H, s), 7.74 (1H, d, J ¼ 9.07 Hz), 7.57e7.54
(1H, m), 7.31e7.27 (1H, m), 4.07 (2H, s), 2.45 (3H, s). IR (KBr): 3248,
1701, 1645, 1597, 1537, 1447, 1420, 1333, 1259, 1203, 1115,
1034, 1003 cmꢁ1. MS (EI direct) m/z: Mþ 378. Anal. (C18H13F3N2O4)
C, H, N.
4.1.9.2. Step 2. Methyl (2E)-3-(2-amino-4-chloro-5-fluorophenyl)
acrylate (22). A mixture of 2-bromo-5-chloro-4-fluoroaniline 18
(1.00 g, 4.46 mmol), methyl acrylate (1.00 mL, 11.1 mmol), Pd(OAc)2
(120 mg, 0.535 mmol), tri-o-tolylphosphine (651 mg, 2.14 mmol),
and dry Et3N (2.50 mL, 18.0 mmol) in dry CH3CN (8.0 mL) was
stirred under reflux conditions under N2 for 2 h, more methyl
acrylate (0.50 mL), Pd(OAc)2 (60 mg), tri-o-tolylphosphine
(325 mg), and dry Et3N (1.20 mL) were added, stirred under reflux
conditions for 7 h, and then cooled to room temperature. The
mixture was concentrated in vacuo. The residue was diluted with
AcOEt, washed with H2O, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, hexane/AcOEt ¼ 6:1 to 4:1) to afford
197 mg of the title product 22 in 19% yield as a yellow solid. 1H NMR
4.1.8. {2-[(4-Ethylpyridin-2-yl)carbonyl]-5-(trifluoromethoxy)-1H-
indol-3-yl}acetic acid (70)
4.1.8.1. Step 1. Methyl {2-[(4-ethylpyridin-2-yl)carbonyl]-5-(tri-
fluoromethoxy)-1H-indol-3-yl}acetate (58). A mixture of methyl
(2E)-3-[2-{[(4-methylphenyl)sulfonyl]amino}-5-(trifluoromethoxy)
phenyl]acrylate 25 (332.3 mg, 0.800 mmol), 2-bromo-1-(4-
ethylpyridin-2-yl)ethanone 44 {salt free, 370.0 mg, 1.62 mmol; that
was synthesized by us according to the reported procedure by
Hayashi et al. [17], 1H NMR (CDCl3, 270 MHz)
d 8.56 (1H, d,
J ¼ 4.94 Hz), 7.95e7.94 (1H, m), 7.37e7.34 (1H, m), 4.86 (2H, s), 2.74
(2H, q, J ¼ 7.56 Hz), 1.29 (3H, t, J ¼ 7.56 Hz)}, and anhydrous K2CO3
(1.106 g, 8.00 mmol) in dry acetone (8.0 mL) was stirred at room
(270 Hz, CDCl3)
d
7.69 (1H, dd, J ¼ 15.8 Hz, J ¼ 0.84 Hz), 7.15 (1H, d,
J ¼ 9.72 Hz), 6.74 (1H, d, J ¼ 6.43 Hz), 6.31 (1H, d, J ¼ 15.7 Hz), 3.90
(2H, br s), 3.81 (3H, s).
temperature under N2 for 18 h, then DBU (359 mL, 2.40 mmol) was
added. The reaction mixture was further stirred at room tempera-
ture under N2 for 7 h, and concentrated in vacuo. The residue was
partitioned between AcOEt (15 mL) and H2O (15 mL). The organic
layer was separated and the aqueous layer was extracted with AcOEt
(15 mL ꢂ 3). The organic layers were combined, dried over
4.1.9.3. Step 3. Methyl (2E)-3-{4-chloro-5-fluoro-2-[(phenylsulfonyl)
amino]phenyl}acrylate (26). To a stirred solution of methyl (2E)-3-
(2-amino-4-chloro-5-fluorophenyl)acrylate 22 (197 mg, 0.858
mmol) in dry CH2Cl2 (3.0 mL) was added dry pyridine (208
2.57 mmol) followed by benzenesulfonyl chloride (164
m
m
L,
L,

S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
191
1.29 mmol) at room temperature under N2. The reaction mixture
(585 mg, 3.00 mmol) and benzenesulfonyl chloride (574
4.50 mmol) that was instead of 4-toluenesulfonyl chloride in the
procedure. 1H NMR (270 Hz, CDCl3)
7.68e7.65 (2H, m), 7.55e7.49
mL,
was stirred at room temperature under N2 for 5 h, and then dry
MeOH (2 mL) was added. The mixture was concentrated in vacuo,
and the residue was partitioned between AcOEt and 2 N HCl. The
organic layer was separated, washed with saturated aqueous
NaHCO3, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The residue was purified by flash column chromatography
(silica gel, hexane/AcOEt ¼ 5:1 to 4:1) to afford 236 mg of the title
product 26 in 74% yield as a brown solid. 1H NMR (270 Hz, CDCl3)
d
(2H, m), 7.44e7.33 (3H, m), 7.15 (1H, dd, J ¼ 9.21 Hz, J ¼ 2.97 Hz),
7.10e7.03 (1H, m), 6.10 (1H, d, J ¼ 15.8 Hz), 3.78 (3H, s).
4.1.10.4. Step 4. Methyl 5-fluoro-2-[(4-methylpyridin-2-yl)carbonyl]-
1H-indol-3-yl}acetate (60). According to the procedure described in
step 5 for the synthesis of compound 64 (that is, preparation method
of methyl 6-fluoro-2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-3-
yl}acetate 52, see 4.1.2.5.), 365 mg of the title product 60 was
prepared in 75% yield as a yellow solid from methyl (2E)-3-{5-fluoro-
2-[(phenylsulfonyl)amino]phenyl}acrylate 36 (500 mg, 1.49 mmol)
and 2-bromo-1-(4-methylpyridin-2-yl)ethanone hydrobromide 43
d
7.71e7.67 (2H, m), 7.57e7.40 (6H, m), 7.23 (1H, d, J ¼ 9.40 Hz), 6.09
(1H, d, J ¼ 15.8 Hz), 3.77 (3H, s).
4.1.9.4. Step 4. Methyl {6-chloro-5-fluoro-2-[(4-methylpyridin-2-yl)
carbonyl]-1H-indol-3-yl}acetate (59). According to the procedure
described in step 3 for the synthesis of compound 64 (that is,
preparation method of arylsulfonylamide derivative 7, see 4.1.2.3.),
106 mg of the title product 59 in 83% yield as a yellow solid was
prepared from methyl (2E)-3-{4-chloro-5-fluoro-2-[(phenyl-
sulfonyl)amino]phenyl}acrylate 26 (130 mg, 0.352 mmol) and 2-
(660 mg, 2.24 mmol). 1H NMR (270 Hz, CDCl3)
d 12.46 (1H, br s),
8.58e8.56 (1H, m), 8.12 (1H, s), 7.43e7.39(1H, m), 7.33e7.26 (2H, m),
7.14e7.06 (1H, m), 4.26 (2H, s), 3.74 (3H, s), 2.45 (3H, s).
4.1.10.5. Step 5. {5-Fluoro-2-[(4-methylpyridin-2-yl)carbonyl]-1H-
indol-3-yl}acetic acid (72). According to the procedure described in
step 6 for the synthesis of compound 64 (the final step, see 4.1.2.6.),
310 mg of the title product 72 was prepared in 89% yield as a yellow
solid from methyl 5-fluoro-2-[(4-methylpyridin-2-yl)carbonyl]-
1H-indol-3-yl}acetate 60 (365 mg, 1.12 mmol). Mp: 221e223 ꢀC. 1H
bromo-1-(4-methylpyridin-2-yl)ethanone
hydrobromide
43
(156 mg, 0.529 mmol). 1H NMR (270 Hz, CDCl3)
d
12.54 (1H, br s),
8.58 (1H, d, J ¼ 4.9 Hz), 8.14 (1H, m), 7.56e7.53 (1H, m), 7.39e7.34
(2H, m), 4.25 (2H, s), 3.75 (3H, s), 2.48 (3H, s).
4.1.9.5. Step 5. {6-Chloro-5-fluoro-2-[(4-methylpyridin-2-yl)carbonyl]-
1H-indol-3-yl}acetic acid (71). According to the procedure described
in step 6 for the synthesis of compound 64 (the final step, see
4.1.2.6.), 88.0 mg of the title product 71 in 86% yield as a yellow solid
was prepared from methyl {6-chloro-5-fluoro-2-[(4-methylpyridin-
2-yl)carbonyl]-1H-indol-3-yl}acetate 59 (106 mg, 0.294 mmol). Mp:
NMR (270 MHz, DMSO-d6) d 12.26 (1H, br s), 8.68 (1H, d,
J ¼ 4.94 Hz), 7.93 (1H, s), 7.66 (1H, dd, J ¼ 9.07 Hz, J ¼ 4.78 Hz),
7.58e7.50 (2H, m), 7.19 (1H, ddd, J ¼ 9.37 Hz, J ¼ 9.07 Hz,
J ¼ 2.32 Hz), 4.04 (2H, s), 2.48 (3H, s). IR (KBr): 1705, 1643, 1570,
1277, 1204, 1186 cmꢁ1. Anal. (C17H13FN2O3) C, H, N.
219.5 ꢀC. 1H NMR (270 Hz, DMSO-d6)
d
12.35 (1H, br s), 8.69 (1H, d,
4.1.11. {5-Methoxy-2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-
3-yl}acetic acid (73)
4.1.11.1. Step 1. 5-Methoxy-2-nitrobenzaldehyde (28). To a stirred
suspension of NaH (60%, 431 mg, 10.8 mmol) in dry DMF (30.0 mL)
J ¼ 4.94 Hz), 7.96 (1H, m), 7.86 (1H, d, J ¼ 6.59 Hz), 7.80 (1H, d,
J ¼ 9.88 Hz), 7.59e7.57 (1H, m), 4.05 (2H, s), 2.46 (3H, s). IR (KBr):
1732, 1709, 1647, 1597, 1529, 1279, 1252, 1204 cmꢁ1. MS (EI direct) m/
z: Mþ 346. Anal. (C17H12ClFN2O3) C, H, N.
was added
(SigmaeAldrich, 1.50 g, 8.98 mmol) in dry DMF (10.0 mL) drop-
wise followed by MeI (671
L, 10.8 mmol) at 0 ꢀC under N2. The
a
solution of 3-hydroxy-6-nitrobenzaldehyde
4.1.10. {5-Fluoro-2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-3-
yl}acetic acid (72)
m
reaction mixture was stirred under N2 at 0 ꢀC for 2 h, and concen-
trated in vacuo. The residue was partitioned between AcOEt and
H2O. The organic extracts were washed with 2 N HCl, saturated
aqueous NaHCO3, and brine, and concentrated in vacuo to afford
930 mg of the title product 28 in 57% yield. 1H NMR (270 Hz, CDCl3)
4.1.10.1. Step 1. Methyl 3-(5-fluoro-2-nitrophenyl)acrylate (30). A
mixture of 5-fluoro-2-nitrobenzaldehyde 27 (Marshallton Research
Laboratories, 700 mg, 4.14 mmol) and methyl (triphenylphosphor-
anylidene)acetate (1.45 g, 4.35 mmol) in dry toluene (15.0 mL) was
stirred under reflux conditions under N2 for 4 h, cooled to room
temperature, and concentrated in vacuo. The residue was purified
by flash column chromatography (silica gel, hexane/AcOEt ¼ 10:1 to
8:1) to afford 680 mg of the title product 30 in 73% yield as a white
d
10.48 (1H, s), 8.16 (1H, d, J ¼ 9.1 Hz), 7.33 (1H, d, J ¼ 2.8 Hz), 7.16
(1H, dd, J ¼ 9.1 Hz, J ¼ 2.8 Hz), 3.96 (3H, s).
4.1.11.2. Step 2. Methyl 3-(5-methoxy-2-nitrophenyl)acrylate (31). A
mixture of 5-methoxy-2-nitrobenzaldehyde 28 (930 mg,
5.13 mmol) and methyl (triphenylphosphoranylidene)acetate
(1.80 g, 5.38 mmol) in dry toluene (25 mL) was stirred under reflux
conditions under N2 for 3 h, then cooled to room temperature. The
mixture was concentrated in vacuo, and the residue was purified by
flash column chromatography (silica gel, hexane/AcOEt ¼ 4:1) to
afford 875 mg of the title product 31 in 72% yield. 1H NMR (270 Hz,
solid. 1H NMR (270 Hz, CDCl3)
d 8.17e8.10 (2H, m), 7.32e7.19 (2H,
m), 6.36 (1H, d, J ¼ 15.8 Hz), 3.84 (3H, s).
4.1.10.2. Step 2. Methyl 3-(2-amino-5-fluorophenyl)acrylate (33).
According to the procedure described in step 2 for the synthesis of
compound 64 (that is, preparation method of methyl 3-(2-
aminophenyl)acrylate derivative 5, see 4.1.2.2.), 592 mg of the
title product 33 was prepared in 100% yield as a yellow solid from
methyl (2E)-3-(5-fluoro-2-nitrophenyl)acrylate 30 (680 mg,
CDCl3)
d
8.21 (1H, d, J ¼ 15.8 Hz), 8.16e8.12 (1H, m), 7.00e6.96 (2H,
m), 6.30 (1H, d, J ¼ 15.8 Hz), 3.93 (3H, s), 3.83 (3H, s).
3.02 mmol). 1H NMR (270 Hz, CDCl3)
J ¼ 1.5 Hz), 7.10e7.06 (1H, m), 6.94e6.87 (1H, m), 6.68e6.63 (1H,
m), 6.33 (1H, d, J ¼ 15.8 Hz), 3.85 (2H, m), 3.81 (3H, s).
d
7.77 (1H, dd, J ¼ 15.8 Hz,
4.1.11.3. Step 3. Methyl 3-(2-amino-5-methoxyphenyl)acrylate
(34). According to the procedure described in step 2 for the
synthesis of compound 64 (that is, preparation method of methyl 3-
(2-aminophenyl)acrylate derivative 5, see 4.1.2.2.), 730 mg of the
title product 34 was prepared in 95% yield (crude) from methyl 3-
(5-methoxy-2-nitrophenyl)acrylate 31 (875 mg, 3.69 mmol). 1H
4.1.10.3. Step 3. Methyl 3-{5-fluoro-2-[(phenylsulfonyl)amino]
phenyl}acrylate (36). According to the procedure described in step
3 for the synthesis of compound 64 (that is, preparation method of
arylsulfonylamide derivative 7, see 4.1.2.3.), 1.04 g of the title
product 36 was prepared in quantitative yield (crude) as a yellow
solid, from methyl (2E)-3-(2-amino-5-fluorophenyl)acrylate 33
NMR (270 Hz, CDCl3)
d
7.83 (1H, d, J ¼ 15.8 Hz), 6.91 (1H, d,
J ¼ 2.81 Hz), 6.82 (1H, dd, J ¼ 8.72 Hz, J ¼ 2.81 Hz), 6.66 (1H, d,
J ¼ 8.59 Hz), 6.35 (1H, d, J ¼ 15.6 Hz), 3.80 (3H, s), 3.76 (3H, s).

192
S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
4.1.11.4. Step 4. Methyl 3-{5-methoxy-2-[(phenylsulfonyl)amino]
phenyl}acrylate (37). To a stirred solution of methyl 3-(2-amino-5-
methoxyphenyl)acrylate 34 (730 mg, 3.52 mmol) in dry CH2Cl2
4.1.12.2. Step 2. 1-(5-Chloro-4-ethylpyridin-2-yl)ethanone (47). A
mixture of 3-chloro-4-ethylpyridine 46 (crude, 8.61 g, 60.0 mmol),
pyruvic acid (15.85 g, 180 mmol), AgNO3 (815.4 mg, 4.80 mmol),
(NH4)2S2O8 (20.54 g, 90.0 mmol), and H2SO4 (3.20 mL, 60.0 mmol)
in H2O (300 mL)eCH2Cl2 (300 mL) was stirred at 40 ꢀC under N2 for
12 h, then cooled to room temperature. The mixture was basified by
adding 2 N NaOH, stirred for 20 min, and then filtered through
a Celite pad. The organic layer was separated, and the aqueous layer
was extracted with CH2Cl2 (200 mL). The organic layers were
combined, concentrated in vacuo. The residue was purified by flash
column chromatography (silica gel, hexane/AcOEt ¼ 11:1) to afford
1.341 g of the title product 47 in 12% yield as a white crystalline
(10.0 mL) was added dry pyridine (855
mL, 10.6 mmol) followed by
benzenesulfonyl chloride (674 L, 5.28 mmol) at room temperature
m
under N2. The reaction mixture was stirred at room temperature
under N2 for 1 h, and then dry MeOH (2 mL) was added. The
mixture was concentrated in vacuo, and the residue was partitioned
between AcOEt and 2 N HCl. The organic layer was separated,
washed with saturated aqueous NaHCO3, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was puri-
fied by flash column chromatography (silica gel, hexane/
AcOEt ¼ 4:1 to 3:1 to 2:1) to afford 715 mg of the title product 37 in
58% yield as a slight yellow crystalline solid. 1H NMR (270 Hz,
solid. 1H NMR (270 MHz, CDCl3)
d 8.55 (1H, s), 7.94 (1H, d, 0.49 Hz),
2.82 (2H, d, J ¼ 7.59 Hz), 2.70 (3H, s), 1.28 (3H, t, J ¼ 7.59 Hz).
CDCl3)
d 7.67e7.64 (2H, m), 7.54e7.37 (4H, m), 7.24 (1H, d,
J ¼ 8.7 Hz), 6.95 (1H, d, J ¼ 2.8 Hz), 6.89 (1H, dd, J ¼ 8.7 Hz,
J ¼ 2.8 Hz), 6.82 (1H, br s), 6.10 (1H, d, J ¼ 15.8 Hz), 3.81 (3H, s), 3.77
(3H, s).
4.1.12.3. Step 3. 2-Bromo-1-(5-chloro-4-ethylpyridin-2-yl)ethanone
(48). To
ethanone 47 (1.341 g, 7.30 mmol) in 25% HBr/AcOH (15.0 mL) was
added bromine (414 L, 8.04 mmol) in glacial AcOH (2.0 mL) at
a stirred solution of 1-(5-chloro-4-ethylpyridin-2-yl)
m
4.1.11.5. Step 5. Methyl {5-methoxy-2-[(4-methylpyridin-2-yl)
carbonyl]-1H-indol-3-yl}acetate (61). According to the procedure
described in step 5 for the synthesis of compound 64 (that is,
preparation method of methyl 6-fluoro-2-[(4-methylpyridin-2-yl)
carbonyl]-1H-indol-3-yl}acetate 52, see 4.1.2.5.), 197 mg of the title
product 61 was prepared in 81% yield as a yellow solid from methyl
0 ꢀC under N2 over 10 min. The reaction mixture was stirred at
room temperature under N2 for 4 h, and then some of the solvent
were removed in vacuo. The residue was cooled to 0 ꢀC, parti-
tioned between Et2O (100 mL) and saturated aqueous NaHCO3
(200 mL) at 0 ꢀC. The ethereal layer was separated, and the
aqueous layer was extracted with Et2O (50 mL). The ethereal layers
were combined, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo to afford 2.518 g of the titled compound 48
3-{5-methoxy-2-[(phenylsulfonyl)amino]phenyl}acrylate
37
(250 mg, 0.720 mmol) and 2-bromo-1-(4-methylpyridin-2-yl)
ethanone hydrobromide 43 (318 mg, 1.08 mmol). 1H NMR (270 Hz,
as a brown oil (crude). 1H NMR (270 MHz, CDCl3)
d 8.56 (1H, s),
CDCl3)
d
12.38 (1H, br s), 8.61 (1H, d, J ¼ 4.9 Hz), 8.17 (1H, s), 7.40
7.99 (1H, d, J ¼ 0.51 Hz), 4.79 (2H, s), 2.83 (2H, q, J ¼ 7.24 Hz), 1.30
(1H, d, J ¼ 8.7 Hz), 7.34e7.32 (1H, m), 7.08e7.03 (2H, m), 4.31 (2H,
(3H, t, J ¼ 7.24 Hz).
s), 3.88 (3H, s), 3.73 (3H, s), 2.47 (3H, s).
4.1.12.4. Step 4. Methyl {6-chloro-2-[(5-chloro-4-ethylpyridin-2-yl)
carbonyl]-1H-indol-3-yl}acetate (62). A mixture of methyl 3-{4-
chloro-2-[(phenylsulfonyl)amino]phenyl}acrylate 38 {2.287 g,
6.50 mmol; that was synthesized by us according to the reported
procedure by Hayashi et al. [17], 1H NMR (270 MHz, CDCl3)
4.1.11.6. Step 6. {5-Methoxy-2-[(4-methylpyridin-2-yl)carbonyl]-1H-
indol-3-yl}acetic acid (73). According to the procedure described in
the synthesis of compound 64 (the final step, see 4.1.2.6.), 172 mg of
the title product 73 was prepared in 91% yield as a yellow solid from
methyl {5-methoxy-2-[(4-methylpyridin-2-yl)carbonyl]-1H-indol-
3-yl}acetate 61 (197 mg, 0.582 mmol). Mp: 235 ꢀC. 1H NMR
d
7.75e7.72 (2H, m), 7.58e7.36 (6H, m), 7.20 (1H, dd, J ¼ 8.56 Hz,
J ¼ 2.13 Hz), 7.14 (1H, br s), 6.15 (1H, d, J ¼ 15.8 Hz), 3.78 (3H, s)}, 2-
bromo-1-(5-chloro-4-ethylpyridin-2-yl)ethanone 48 (crude,
(270 MHz, DMSO-d6)
d
12.10 (1H, br s), 8.68 (1H, d, J ¼ 4.94 Hz), 7.93
(1H, s), 7.57e7.54 (2H, m), 7.17 (1H, d, J ¼ 2.00 Hz), 6.99 (1H, dd,
J ¼ 9.05 Hz, J ¼ 2.32 Hz), 4.06 (2H, s), 3.78 (3H, s), 2.45 (3H, s). IR
(KBr): 1701, 1638, 1595, 1528, 1448, 1423, 1340, 1279, 1263, 1234,
1223, 1192, 1111 cmꢁ1. Anal. (C18H16N2O4) C, H, N.
2.518 g), and anhydrous K2CO3 (6.29 g, 45.5 mmol) in dry acetone
(60.0 mL) was stirred at room temperature under N2 for 48 h, then
DBU (3.89 mL, 26.0 mmol) was added. The reaction mixture was
further stirred at room temperature under N2 for 5 h, and
concentrated in vacuo. The residue was partitioned between AcOEt
(60 mL) and H2O (60 mL). The organic layer was separated and the
aqueous layer was extracted with AcOEt (50 mL ꢂ 2). The organic
layers were combined, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, hexane/AcOEt ¼ 5:1) to afford 347.0 mg
of the title product 62 in 14% yield as a lemon-yellow crystalline
4.1.12. {6-Chloro-2-[(5-chloro-4-ethylpyridin-2-yl)carbonyl]-1H-
indol-3-yl}acetic acid (74)
4.1.12.1. Step 1. 3-Chloro-4-ethylpyridine (46). To a stirred solution
of 3-chloropyridine 45 (SigmaeAldrich, 5.71 mL, 60.0 mmol) in
anhydrous THF (120.0 mL) was added dropwise lithium diisopro-
pylamide (LDA) (2 M in heptaneeTHFeEtPh, 36.0 mL, 72.0 mmol)
at ꢁ78 ꢀC under N2 over 10 min. After the solution was stirred
at ꢁ78 ꢀC under N2 for 1 h, EtI (9.60 mL, 120 mmol) was added
dropwise at ꢁ78 ꢀC over 5 min. The reaction solution was stirred at
the same temperature for 5 min, warmed up to room temperature,
and stirred for 20 min. The reaction solution was cooled to 0 ꢀC,
saturated aqueous NH4Cl (150 mL) was added at 0 ꢀC, and then
stirred at the temperature for 15 min. The aqueous layer was
separated, and the organic layer was washed with H2O
(100 mL ꢂ 2). The aqueous layer and the washings were combined,
then extracted with Et2O (150 mL ꢂ 2). The organic layers were
combined, washed with H2O (100 mL), dried over anhydrous
MgSO4, filtered, and concentrated in vacuo to afford 8.61 g of the
titled compound 46 in quantitative yield as a dark-red oil. 1H NMR
solid. 1H NMR (270 MHz, CDCl3)
d 12.20 (1H, br s), 8.68 (1H, s), 8.26
(1H, s), 7.63 (1H, d, J ¼ 9.07 Hz), 7.53e7.52 (1H, m), 7.16e7.12 (1H,
m), 4.30 (2H, s), 3.73 (3H, s), 2.87 (2H, q, J ¼ 7.56 Hz), 1.33 (3H, t,
J ¼ 7.56 Hz).
4.1.12.5. Step 5. {6-Chloro-2-[(5-chloro-4-ethylpyridin-2-yl)carbonyl]-
1H-indol-3-yl}acetic acid (74). To a mixture of methyl {6-chloro-2-
[(5-chloro-4-ethylpyridin-2-yl)carbonyl]-1H-indol-3-yl}acetate 62
(345.0 mg, 0.882 mmol) in MeOH (30.0 mL)eTHF (30.0 mL) was
added 2 N NaOH (2.5 mL, 5.0 mmol) at room temperature under N2.
The reaction mixture was warmed up to reflux conditions, stirred
under N2 for 4 h, cooled to room temperature, and then 2 N HCl
(2.5 mL) was added. The mixture was stirred at room temperature
for several minutes, and concentrated in vacuo. The residue was
dissolved in anhydrous THF. The resulting solution was dried over
(270 MHz, CDCl3)
d 8.51 (1H, s), 8.39 (1H, d, 4.9 Hz), 7.17 (1H, d,
4.9 Hz), 2.76 (2H, q, J ¼ 7.6 Hz), 1.25 (3H, t, J ¼ 7.6 Hz).

S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
193
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue
was washed with AcOEt followed by vacuum drying to afford
315.8 mg of the title product 74 in 95% yield as a yellow solid. Mp:
chloro-2-(5,6,7,8-tetrahydroisoquinolin-3-ylcarbonyl)-1H-indol-3-
yl]acetate 63 (41.0 mg, 0.107 mmol) in MeOH (20.0 mL)eTHF
(20.0 mL) was added 2 N NaOH (0.30 mL, 0.60 mmol) at room
temperature under N2. The reaction mixture was warmed up to
reflux conditions, stirred under N2 for 6 h, then more 2 N NaOH
(0.30 mL) was added. The mixture was stirred for 2 h under reflux
conditions, cooled to room temperature, and concentrated in vacuo.
The residue was dissolved in H2O (20 mL), and the resulting
aqueous solution was washed with Et2O (20 mL ꢂ 3). The aqueous
solution was acidified by adding 2 N HCl, then extracted with AcOEt
(30 mL ꢂ 3). The combined extracts were dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was
recrystallized from AcOEt to afford 23.3 mg of the title product 75
in 59% yield as a yellow solid. Mp: 225 ꢀC. 1H NMR (270 Hz, DMSO-
233 ꢀC. 1H NMR (270 MHz, DMSO-d6)
d 12.07 (1H, br s), 8.76 (1H, s),
8.07 (1H, s), 7.80 (1H, d, J ¼ 8.72 Hz), 7.67 (1H, d, J ¼ 1.81 Hz), 7.12
(1H, dd, J ¼ 8.72 Hz, J ¼ 1.81 Hz), 4.06 (2H, s), 2.86 (2H, q,
J ¼ 7.40 Hz), 1.26 (3H, t, J ¼ 7.40 Hz). IR (KBr): 3306, 1701, 1638, 1531,
1344, 1240, 1194, 1144, 1055, 916 cmꢁ1. MS (EI direct) m/z: Mþ 376.
Anal. (C18H14Cl2N2O3) C, H, N.
4.1.13. [6-Chloro-2-(5,6,7,8-tetrahydroisoquinolin-3-ylcarbonyl)-
1H-indol-3-yl]acetic acid (75)
4.1.13.1. Step 1. 1-(5,6,7,8-Tetrahydroisoquinolin-3-yl)ethanone (50).
A mixture of 5,6,7,8-tetrahydroisoquinoline 49 (Tokyo Chemical
Industry, 3.88 mL, 30.0 mmol), pyruvic acid (7.92 g, 90.0 mmol),
AgNO3 (407.7 mg, 2.40 mmol), (NH4)2S2O8 (10.27 g, 45.0 mmol),
and H2SO4 (1.60 mL, 30.0 mmol) in H2O (300 mL)eCH2Cl2 (300 mL)
was stirred at 40 ꢀC under N2 for 3.5 h. The reaction mixture was
cooled to room temperature, and then basified by adding 2 N NaOH.
The organic layer was separated and the aqueous layer was
extracted with CH2Cl2. The organic layers were combined, dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography (silica gel,
hexane/AcOEt ¼ 3:1) to afford 287.7 mg of the title product 50 in
d6)
d 12.28 (1H, br s), 8.50 (1H, s), 7.82 (1H, s), 7.76 (1H, d,
J ¼ 8.72 Hz), 7.72 (1H, d, J ¼ 1.65 Hz), 7.09 (1H, dd, J ¼ 8.56 Hz,
J ¼ 1.97 Hz), 4.06 (2H, s), 2.90e2.83 (4H, m), 1.85e1.77 (4H, m). IR
(KBr): 3422, 1699, 1645, 1537, 1319, 1200, 1061, 991 cmꢁ1. MS (EI
direct) m/z: Mþ 368. Anal. (C20H17ClN2O3) C, H, N.
4.2. Biology
4.2.1. Materials
Human umbilical vein endothelial cells (HUVECs) were
purchased from Morinaga Institute (Yokohama, Japan). Recombi-
5.5% yield as a brown oil. 1H NMR (270 MHz, CDCl3)
d 8.35 (1H, s),
7.75 (1H, s), 2.84e2.79 (4H, m), 2.69 (3H, s), 1.89e1.79 (4H, m).
nant human IL-1b was purchased from R&D Systems (Minneap-
olis, MN, USA). A23187 (calcium ionophore) and LPS (from
Escherichia coli 0111:B4) were purchased from SigmaeAldrich (St.
Louis, MO, USA). PGE2 and TXB2 were purchased from Cayman
Chemical (Ann Arbor, MI, USA). Sodium heparin (Novo-Heparin)
was purchased from Novo Nordisk A/S (Bagsværd, Denmark).
4.1.13.2. Step 2. 2-Bromo-1-(5,6,7,8-tetrahydroisoquinolin-3-yl)
ethanone (51). To
hydroisoquinolin-3-yl)ethanone 50 (287.7 mg, 1.64 mol) in 25%
HBr/AcOH (3.1 mL) was added a solution of bromine (93 L,
a
stirred solution of 1-(5,6,7,8-tetra-
m
1.8 mmol) in glacial AcOH (0.40 mL) at 0 ꢀC under N2 over 15 min.
The reaction mixture was stirred at room temperature under N2 for
3 h, then anhydrous Et2O (10 mL) was added. The mixture was
concentrated in vacuo, and the residue was cooled to 0 ꢀC, then
basified by adding saturated aqueous NaHCO3 (150 mL) at 0 ꢀC. The
mixture was extracted with Et2O (150 mL). The ethereal layer was
separated, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo to afford 524.9 mg of the title product 51 as a brown oil
(crude). This compound was used for the next step without further
l-Carrageenan (Picnin-A) was purchased from Zushikagaku
(Zushi, Kanagawa, Japan). Radioimmunoassay (RIA) kit was
purchased from Amersham (Buckinghamshire, United Kingdom).
Compounds 64e75 were used for pharmacological evaluations as
free acids, respectively. All other chemicals were obtained from
Wako Pure Chemical Industries (Osaka, Japan) unless stated
otherwise. All common chemicals were analytical or the highest
available grade.
purification. 1H NMR (270 MHz, CDCl3)
d
8.35 (1H, s), 7.89 (1H, s),
4.2.2. In vitro characterization of COX-2 inhibitors
4.87 (2H, s), 2.91e2.83 (4H, m), 1.90e1.82 (4H, m).
4.2.2.1. Human cell-based COX-1 assay. Platelets were prepared
from human peripheral blood obtained from healthy adult volun-
teers under informed consent. Fresh blood was collected into
vacutainers (Becton Dickinson, Franklin Lakes, NJ, USA) containing
1/10 volume of anticoagulant solution (3.8% sodium citrate), and
centrifuged at 200 ꢂ g for 10 min. The supernatant (platelet-rich
plasma) was mixed with 50% volume of 0.14 M NaCl containing
12 mM TriseHCl, 1.2 mM EDTA, pH 7.4. This mixture was centri-
fuged at 750 ꢂ g for 10 min, and the pellet was suspended in
platelet buffer (Ca2þ-free Hanks buffer containing 20 mM HEPES,
pH 7.4 and 0.2% BSA). After centrifugal washing with the platelet
buffer, the resulting pellet, referred to as human washed platelets
(HWPs), was resuspended in the platelet buffer at a cell concen-
tration of 2.85 ꢂ108 cells/mL, then stored at room temperature
4.1.13.3. Step 3. Methyl [6-chloro-2-(5,6,7,8-tetrahydroisoquinolin-3-
ylcarbonyl)-1H-indol-3-yl]acetate (63). A mixture of methyl 3-{4-
chloro-2-[(phenylsulfonyl)amino]phenyl}acrylate 38 (351.8 mg,
1.00 mmol), 2-bromo-1-(5,6,7,8-tetrahydroisoquinolin-3-yl)etha-
none 51 (crude, 524.9 mg), and anhydrous K2CO3 (1.3821 g,
10.0 mmol) in dry acetone (20.0 mL) was stirred at room temper-
ature under N2 for 16 h, then DBU (449 mL, 3.00 mmol) was added.
The reaction mixture was further stirred at room temperature
under N2 for 8 h, and concentrated in vacuo. The residue was par-
titioned between AcOEt (40 mL) and H2O (40 mL). The organic layer
was separated and the aqueous layer was extracted with AcOEt
(40 mL ꢂ 3). The organic layers were combined, dried over anhy-
drous MgSO4, filtered, and concentrated in vacuo. The residue was
purified by flash column chromatography (silica gel, hexane/
AcOEt ¼ 3:1) to afford 41.7 mg of the title product 63 in 11% yield as
until use. Immediately prior to assay, 10 mL of 12.6 mM CaCl2 was
added to 70
m
L HWPs suspension (2.0 ꢂ 107 cells/mL in a 96-well U
bottom plate). Platelets were preincubated in the absence or
a lemon-yellow crystalline solid. 1H NMR (270 MHz, CDCl3)
d
12.58
presence of 10
concentrations varying 0.01e10
0.1%) for 20 min before stimulation with 10
A23187 (final concentration, 10
m
L of test compound dissolved in DMSO at final
M (final concentration, less than
L of calcium ionophore
M). After further 15 min incuba-
(1H, br s), 8.43 (1H, s), 8.04 (1H, s), 7.64e7.60 (1H, m), 7.52 (1H, d,
J ¼ 1.81 Hz), 7.12 (1H, dd, J ¼ 8.72 Hz, J ¼ 1.81 Hz), 4.31 (2H, s), 3.72
(3H, s), 2.91e2.85 (4H, m), 1.92e1.85 (4H, m).
m
m
m
tion at 37 ꢀC with A23187, the reaction was stopped by the addition
of EDTA (final concentration, 7.7 mM), and the reaction medium
was quantitated for TXB2 by a radioimmunoassay (RIA) kit
4.1.13.4. Step 4. [6-Chloro-2-(5,6,7,8-tetrahydroisoquinolin-3-ylcar-
bonyl)-1H-indol-3-yl]acetic acid (75). To a mixture of methyl [6-

194
S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
(Amersham, Buckinghamshire, United Kingdom) according to the
manufacturer’s procedure [17,50].
volume for 3 h were calculated. Foot oedema was compared with
vehicle-control group, and the percent inhibition was calculated
taking the values in the control group as 0%.
4.2.2.2. Human cell-based COX-2 assay. The human cell-based COX-
2 assay was carried out as previously described [17,51]. Confluent
human umbilical vein endothelial cells (HUVECs) (2 ꢂ104 cells/
4.3. Physicochemistry
well) in a 96-well plate were washed with 100 mL of RPMI-1640
The lipophilicity values of COX-2 inhibitors were estimated as
values of ACD log D7.4, the octanolewater distribution coefficient
for ionizable compounds at pH 7.4, calculated by ACD software,
ACD/Laboratories 9.0 (Advanced Chemistry Development, Inc.,
Ontario, Canada).
containing 2% foetal calf serum (FCS), and incubated with 300 U/
mL of recombinant human IL-1
b
for 24 h at 37 ꢀC for induction of
COX-2. After washing with Hanks buffer containing 20 mM HEPES,
pH 7.4 and 0.2% BSA, HUVECs were preincubated in Hanks buffer
containing 20 mM HEPES, pH 7.4 and 0.2% BSA, with or without
10
m
L of test compound dissolved in DMSO at final concentrations
Appendix. Supplementary data
varying 1 nM to 1
mM (final concentration, less than 0.1%) for
20 min before stimulation with 30 mM of A23187. After further
The following are the Supplementary data related to this article:
Synthesis of methyl 3-{2-[(phenylsulfonyl)amino]phenyl}acrylates
36e38 from 2-nitro-(4- or 5-substituted)-benzaldehydes is shown
in Scheme S1. Synthesis of 2-bromo-1-(4-alkylpyridin-2-yl)etha-
nones 43 and 44 is shown in Scheme S2. Supplementary data
associated with this article can be found in the online version, at
doi:10.1016/j.ejmech.2012.01.053. These data include MOL files and
InChiKeys of the most important compounds described in this
article.
15 min incubation at 37 ꢀC with A23187, the reaction medium was
quantitated for 6-keto-prostaglandin F1 (6-keto-PGF1a), that is,
a
spontaneously degraded stable form of PGI2, by a RIA kit (Amer-
sham, Buckinghamshire, United Kingdom).
4.2.2.3. Human whole blood COX-1 assay. Human peripheral blood
obtained from healthy adult volunteers under informed consent
was collected into vacutainers containing 143 USP units of sodium
heparin. Aliquots of 80
in a 96-well U-bottom plate containing 10
dissolved in DMSO at final concentrations varying 0.1e100
(final concentration, less than 0.1%), and incubated with 10 L of
M) for 30 min at 37 ꢀC. The reac-
mL of the heparinized blood was dispensed
mL of test compound
References
mM
m
[1] Review: D.L. Simmons, R.M. Botting, T. Hla, Cyclooxygenase isozymes: the
biology of prostaglandin synthesis and inhibition, Pharmacol. Rev. 56 (2004)
387e437.
[2] Review: C.D. Funk, Prostaglandins and leukotrienes: advances in eicosanoid
biology, Science 294 (2001) 1871e1875.
A23187 (final concentration, 30
m
tion was stopped by centrifugation at 400 ꢂ g for 5 min at 4 ꢀC, and
the supernatant was quantitated for TXB2 by a RIA kit (Amersham,
Buckinghamshire, United Kingdom) [17,52,53].
[3] H.U. Zeilhofer, Prostanoids in nociception and pain, Biochem. Pharmacol. 73
(2007) 165e174.
[4] Review: H. Vanegas, H.-G. Schaible, Prostaglandins and cyclooxygenases in the
spinal cord, Prog. Neurobiol. 64 (2001) 327e363.
[5] Review: F. Kojima, M. Kapoor, S. Kawai, L.J. Crofford, New insights into
eicosanoid biosynthetic pathways: implication for arthritis, Expert Rev. Clin.
Immunol. 2 (2006) 277e291.
[6] Y. Sugimoto, S. Narumiya, A. Ichikawa, Distribution and function of prostanoid
receptors: studies from knockout mice, Prog. Lipid Res. 39 (2000) 289e314.
[7] T. Hla, K. Neilson, Human cyclooxygenase-2 cDNA, Proc. Natl. Acad. Sci. U.S.A
89 (1992) 7384e7388.
[8] G.P. O’Neill, A.W. Ford-Hutchinson, Expression of mRNA for cyclooxygenase-1
and cyclooxygenase-2 in human tissues, FEBS Lett. 330 (1993) 156e160.
[9] A.A. Romanovsky, M.C. Almeida, D.M. Aronoff, A.I. Ivanov, J.P. Konsman,
A.A. Steiner, V.F. Turek, Fever and hypothermia in systemic inflammation:
recent discoveries and revisions, Front. Biosci. 10 (2005) 2193e2216.
[10] P. Patrignani, M.R. Panara, A. Greco, O. Fusco, C. Natoli, S. Iacobelli,
F. Cipollone, A. Ganci, C. Créminon, J. Maclouf, C. Patrono, Biochemical and
pharmacological characterization of the cyclooxygenase activity of human
blood prostaglandin endoperoxide synthases, J. Pharmacol. Exp. Ther. 271
(1994) 1705e1712.
4.2.2.4. Human whole blood COX-2 assay. Human peripheral blood
obtained from healthy adult volunteers under informed consent
was collected into vacutainers (FalconÒ; Becton Dickinson, Franklin
Lakes, NJ, USA) containing 143 USP units of sodium heparin (Novo-
Heparin). Aliquots of 80
in a 96-well U-bottom plate containing 10
dissolved in DMSO at final concentrations varying 0.1e100
(final concentration, less than 0.1%), and incubated with 10 L of
lipopolysaccharide (LPS, from E. coli 0111:B4, final concentration,
m
L of the heparinized blood was dispensed
mL of test compound
mM
m
30 m
g/mL) for 24 h at 37 ꢀC for induction of COX-2. The reaction was
stopped by centrifugation at 400 ꢂ g for 5 min at 4 ꢀC, and the
supernatant was quantitated for PGE2 by RIA kit (Amersham,
Buckinghamshire, United Kingdom) [17,52e54].
[11] R.Y. Kang, J. Freire-Moar, E. Sigal, C.-Q. Chu, Expression of cyclooxygenase-2 in
human and an animal model of rheumatoid arthritis, Br. J. Rheumatol. 35
(1996) 711e718.
[12] F. Beiche, S. Scheuerer, K. Brune, G. Geisslinger, M. Goppelt-Struebe, Up-
regulation of cyclooxygenase-2 mRNA in the rat spinal cord following
peripheral inflammation, FEBS Lett. 390 (1996) 165e169.
[13] Review: S.A. Steer, J.A. Corbett, The role and regulation of COX-2 during viral
infection, Viral Immunol. 16 (2003) 447e460.
[14] J. Duque, M.D. Díaz-Muñoz, M. Fresno, M.A. Iñiguez, Up-regulation of
4.2.3. In vivo study
4.2.3.1. General. Animal experiment was conducted according to the
guideline of animal care and use, and its procedure was approved by
the Animal Ethics Committee in Pfizer Global Research & Develop-
ment, Nagoya Laboratories. The animal work was also approved by
the Pfizer Institutional Animal Care and Use Committee (IACUC).
cyclooxygenase-2 by interleukin-1
b in colon carcinoma cells, Cell. Signal. 18
(2006) 1262e1269.
[15] D.J. Kelley, J.R. Mestre, K. Subbaramaiah, P.G. Sacks, S.P. Schantz, T. Tanabe,
H. Inoue, J.T. Ramonetti, A.J. Dannenberg, Benzo[a]pyrene up-regulates
cyclooxygenase-2 gene expression in oral epithelial cells, Carcinogenesis 18
(1997) 795e799.
4.2.3.2. Carrageenan-induced foot-oedema formation in SPF/VAF
male SD rats. SPF/VAF male SpragueeDawley (SD) rats (Crj:CD(SD),
5 weeks old, 110e130 g, Charles River Laboratories, Japan Inc., Hino,
Japan) that were fasted overnight were injected intraplantarly with
[16] S. Kiritoshi, T. Nishikawa, K. Sonoda, D. Kukidome, T. Senokuchi, T. Matsuo,
T. Matsumura, H. Tokunaga, M. Brownlee, E. Araki, Reactive oxygen species
from mitochondria induce cyclooxygenase-2 gene expression in human
mesangial cells: potential role in diabetic nephropathy, Diabetes 52 (2003)
2570e2577.
[17] S. Hayashi, Y. Sumi, N. Ueno, A. Murase, J. Takada, Discovery of a novel COX-2
inhibitor as an orally potent anti-pyretic and anti-inflammatory drug: design,
synthesis, and structureeactivity relationship, Biochem. Pharmacol. 82 (2011)
755e768.
0.1 mL of a 1% (w/v) l-carrageenan (Picnin-A) suspension in saline
into the right hind paw as previously reported [17,57e59]. Either
the vehicle (5% (v/v) Tween 80 in distilled water) or a test
compound was dosed orally in a volume of 1 mL/100 g body weight
at 1 h before carrageenan injection. Foot volume was measured by
a water displacement plethysmometer (Unicom Co., Yachiyo, Japan)
before and 3 h after carrageenan injection. The increases in foot

S. Hayashi et al. / European Journal of Medicinal Chemistry 50 (2012) 179e195
195
[18] T.D. Warner, F. Giuliano, I. Vojnovic, A. Bukasa, J.A. Mitchell, J.R. Vane,
Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-
oxygenase-2 are associated with human gastrointestinal toxicity: a full in vi-
tro analysis, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 7563e7568.
[19] Z.A. Radi, N.K. Khan, Effects of cyclooxygenase inhibition on the gastrointes-
tinal tract, Exp. Toxicol. Pathol. 58 (2006) 163e173.
[20] L. Laine, GI risk and risk factors of NSAIDs, J. Cardiovasc. Pharmacol. 47 (Suppl.
1) (2006) S60eS66.
[21] Review: J.A. Mitchell, T.D. Warner, Cyclo-oxygenase-2: pharmacology, phys-
iology, biochemistry and relevance to NSAID therapy, Br. J. Pharmacol. 128
(1999) 1121e1132.
[47] F.H. Case, T.J. Kasper, The preparation of some substituted 2,6-bis-(2-pyridyl)-
pyridines, J. Am. Chem. Soc. 78 (1956) 5842e5844.
[48] F. Fontana, F. Minisci, M.C.N. Barbosa, E. Vismara, Homolytic acylation of
pyridines and pyrazines with
a-keto acids: the problem of monoacylation,
J. Org. Chem. 56 (1991) 2866e2869.
[49] G.W. Gribble, M.G. Saulnier, Regioselective ortho lithiation of halopyridines,
Tetrahedron Lett. 21 (1980) 4137e4140.
[50] D. Riendeau, M.D. Percival, S. Boyce, C. Brideau, S. Charleson, W. Cromlish,
D. Ethier, J. Evans, J.-P. Falgueyret, A.W. Ford-Hutchinson, R. Gordon, G. Greig,
M. Gresser, J. Guay, S. Kargman, S. Léger, J.A. Mancini, G. O’Neill, M. Ouellet,
I.W. Rodger, M. Thérien, Z. Wang, J.K. Webb, E. Wong, L. Xu, R.N. Young,
R. Zamboni, P. Prasit, C.-C. Chan, Biochemical and pharmacological profile of
a tetrasubstituted furanone as a highly selective COX-2 inhibitor, Br. J. Phar-
macol. 121 (1997) 105e117.
[22] M.L. Kowalski, J. Makowska, Use of nonsteroidal anti-inflammatory drugs in
patients with aspirin hypersensitivity. Safety of cyclo-oxygenase-2 inhibitors,
Treat. Respir. Med. 5 (2006) 399e406.
[23] W.B. White, G. Faich, J.S. Borer, R.W. Makuch, Cardiovascular thrombotic
events in arthritis trials of the cyclooxygenase-2 inhibitor celecoxib, Am. J.
Cardiol. 92 (2003) 411e418.
[24] B. Hinz, B. Renner, K. Brune, Drug insight: cyclo-oxygenase-2 inhibitorsda
critical appraisal, Nat. Clin. Pract. Rhum. 3 (2007) 552e560.
[25] A.A. Steiner, A.I. Ivanov, J. Serrats, H. Hosokawa, A.N. Phayre, J.R. Robbins,
J.L. Roberts, S. Kobayashi, K. Matsumura, P.E. Sawchenko, A.A. Romanovsky,
Cellular and molecular bases of the initiation of fever, PLoS Biol. 4 (2006)
1517e1524.
[51] P.F. Moore, D.L. Larson, I.G. Otterness, A. Weissman, S.B. Kadin, F.J. Sweeney,
J.D. Eskra, A. Nagahisa, M. Sakakibara, T.J. Carty, Tenidap, a structurally novel
drug for the treatment of arthritis: antiinflammatory and analgesic properties,
Inflamm. Res. 45 (1996) 54e61.
[52] C. Brideau, S. Kargman, S. Liu, A.L. Dallob, E.W. Ehrich, I.W. Rodger, C.-C. Chan,
A human whole blood assays for clinical evaluation of biochemical efficacy of
cyclooxygenase inhibitors, Inflamm. Res. 45 (1996) 68e74.
[53] J.M. Young, S. Panah, C. Satchawatcharaphong, P.S. Cheung, Human whole
blood assays for inhibition of prostaglandin G/H synthase-1 and -2 using
A23187 and lipopolysaccharide stimulation of thromboxane B2 production,
Inflamm. Res. 45 (1996) 246e253.
[26] Review: J.R. Vane, R.M. Botting, The mechanism of action of aspirin, Thromb.
Res. 110 (2003) 255e258.
[27] A.A. Khan, M. Iadarola, H.-Y.T. Yang, R.A. Dionne, Expression of COX-1 and
COX-2 in a clinical model of acute inflammation, J. Pain 8 (2007) 349e354.
[28] For example: K.K. Wu, Differential cyclooxygenase-2 transcriptional control in
proliferating versus quiescent fibroblasts, Prostaglandins Other Lipid Mediat.
83 (2007) 175e181.
[54] K. Glaser, M.-L. Sung, K. O’Neill, M. Belfast, D. Hartman, R. Carlson, A. Kreft,
D. Kubrak, C.-L. Hsiao, B. Weichman, Etodolac selectively inhibits human
prostaglandin G/H synthase 2 (PGHS-2) versus human PGHS-1, Eur. J. Phar-
macol. 281 (1995) 107e111.
[55] W.L. Smith, R.M. Garavito, D.L. DeWitt, Prostaglandin endoperoxide H syn-
thase (cyclooxygenase)-1 and -2, J. Biol. Chem. 271 (1996) 33157e33160.
[56] M.G. Malkowski, S.L. Ginell, W.L. Smith, R.M. Garavito, The productive
conformation of arachidonic acid bound to prostaglandin synthase, Science
289 (2000) 1933e1937.
[57] C.A. Winter, E.A. Risley, G.V. Nuss, Carrageenin-induced edema in hind paw of
the rat as an assay for antiinflammatory drugs, Proc. Soc. Exp. Biol. Med 111
(1962) 544e547.
[58] J.G. Lombardino, I.G. Otterness, E.H. Wiseman, Acidic anti-inflammatory
agents-correlations of some physical, pharmacological and clinical data, Arz-
neim. Forsch. 25 (1975) 1629e1635.
[29] O. Dormond, A. Foletti, C. Paroz, C. Rüegg, NSAIDs inhibit
aVb3 integrin-
mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration
and angiogenesis, Nat. Med. 7 (2001) 1041e1047.
[30] Q.-T. Zhao, S.-Q. Yue, Z. Cui, Q. Wang, X. Cui, H.-H. Zhai, L.-H. Zhang, K.-F. Dou,
Potential involvement of the cyclooxygenase-2 pathway in hepatocellular
carcinoma-associated angiogenesis, Life Sci. 80 (2007) 484e492.
[31] C. Iwata, M.R. Kano, A. Komuro, M. Oka, K. Kiyono, E. Johansson, Y. Morishita,
M. Yashiro, K. Hirakawa, M. Kaminishi, K. Miyazono, Inhibition of
cyclooxygenase-2 suppresses lymph node metastasis via reduction of lym-
phangiogenesis, Cancer Res. 67 (2007) 10181e10189.
[32] M.A. Hull, O.O. Faluyi, C.W.S. Ko, S. Holwell, D.J. Scott, R.J. Cuthbert,
R. Poulsom, R. Goodlad, C. Bonifer, A.F. Markham, P.L. Coletta, Regulation of
stromal cell cycloocygenase-2 in the ApcMin/þ mouse model of intestinal
tumorigenesis, Carcinogenesis 27 (2006) 382e391.
[59] J. Gracia Leme, L. Hamamura, M.P. Leite, M. Rocha e Silva, Pharmacological
analysis of the acute inflammatory process induced in the rat’s paw by
local injection of carrageenin and by heating, Br. J. Pharmacol. 48 (1973)
88e96.
[33] J.-H. Chen, C.-W. Wu, H.-L. Kao, H.-M. Chang, A.F.-Y. Li, T.-Y. Liu, C.-W. Chi,
Effects of COX-2 inhibitor on growth of human gastric cancer cells and its
relation to hepatocyte growth factor, Cancer Lett. 239 (2006) 263e270.
[34] B.S. Reddy, C.V. Rao, Novel approaches for colon cancer prevention by
cyclooxygenase-2 inhibitors, J. Environ. Pathol. Toxicol. Oncol. 21 (2002) 155e164.
[35] K. Krysan, K.L. Reckamp, S. Sharma, S.M. Dubinett, The potential and rationale
for COX-2 inhibitors in lung cancer, Anti-Cancer Agents Med. Chem. 6 (2006)
209e220.
[36] R. Chenevard, D. Hürlimann, M. Béchir, F. Enseleit, L. Spieker, M. Hermann,
W. Riesen, S. Gay, R.E. Gay, M. Neidhart, B. Michel, T.F. Lüscher, G. Noll,
F. Ruschitzka, Selective COX-2 inhibition improves endothelial function in
coronary artery disease, Circulation J. Am. Heart Assoc. 107 (2003) 405e409.
[37] K. Höcherl, F. Dreher, A. Kurtz, M. Bucher, Cyclooxygenase-2 inhibition
attenuates lipopolysaccharide-induced cardiovascular failure, Hypertension J.
Am. Heart Assoc. 40 (2002) 947e953.
[60] F. Nantel, D. Denis, R. Gordon, A. Northey, M. Cirino, K.M. Metters, C.C. Chan,
Distribution and regulation of cyclooxygenase-2 in carrageenan-induced
inflammation, Br. J. Pharmacol. 128 (1999) 853e859.
[61] S. Cuzzocrea, L. Sautebin, G. De Sarro, G. Costantino, L. Rombolá, E. Mazzon,
A. Ialenti, A. De Sarro, G. Ciliberto, M. Di Rosa, A.P. Caputi, C. Thiemermann,
Role of IL-6 in the pleurisy and lung injury caused by carrageenan, J. Immunol.
163 (1999) 5094e5104.
[62] M. Romano, R. Faggioni, M. Sironi, S. Sacco, B. Echtenacher, E. Di Santo,
M. Salmona, P. Ghezzi, Carrageenan-induced acute inflammation in the mouse
air pouch synovial model. Role of tumour necrosis factor, Mediat. Inflamm. 6
(1997) 32e38.
[63] S.S.V. Padi, N.K. Jain, S. Singh, S.K. Kulkarni, Pharmacological profile of para-
coxib: a novel, potent injectable selective cyclooxygenase-2 inhibitor, Eur. J.
Pharmacol. 491 (2004) 69e76.
[64] I. Posadas, M. Bucci, F. Roviezzo, A. Rossi, L. Parente, L. Sautebin, G. Cirino,
Carrageenan-induced mouse paw oedema is biphasic, age-weight dependent
and displays differential nitric oxide cyclooxygenase-2 expression, Br. J.
Pharmacol. 142 (2004) 331e338.
[38] P.S. Staats, Pain management and beyond: evolving concepts and treatments
involving cyclooxygenase inhibition, J. Pain Symptom Manage. 24 (1S) (2002)
S4eS9.
[39] W.L. Xie, J.G. Chipman, D.L. Robertson, R.L. Erikson, D.L. Simmons, Expression
of a mitogen-responsive gene encoding prostaglandin synthase is regulated
by mRNA splicing, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 2692e2696.
[40] B.A. Patel, C.B. Ziegler, N.A. Cortese, J.E. Plevyak, T.C. Zebovitz, M. Terpko,
R.F. Heck, Palladium-catalyzed vinylic substitution reactions with carboxylic
acid derivatives, J. Org. Chem. 42 (1977) 3903e3907.
[41] J.P. Knowles, A. Whiting, The HeckeMizoroki cross-coupling reaction:
a mechanistic perspective, Org. Biomol. Chem. 5 (2007) 31e44.
[42] A. Agrawal, P.G. Tratnyek, Reduction of nitro aromatic compounds by zero-
valent iron metal, Environ. Sci. Technol. 30 (1996) 153e160.
[65] R.F. Claudino, C.A.L. Kassuya, J. Ferreira, J.B. Calixto, Pharmacological and
molecular characterization of the mechanisms involved in prostaglandin
E2-induced mouse paw edema, J. Pharmacol. Exp. Ther. 318 (2006)
611e618.
[66] S. Hayashi, E. Nakata, A. Morita, K. Mizuno, K. Yamamura, A. Kato, K. Ohashi,
Discovery of {1-[4-(2-{hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl}-1H-benzimi-
dazol-1-yl)piperidin-1-yl]cyclooctyl}methanol, systemically potent novel
non-peptide agonist of nociceptin/orphanin FQ receptor as analgesic for the
treatment of neuropathic pain: design, synthesis, and structureeactivity
relationships, Bioorg. Med. Chem. 18 (2010) 7675e7699.
[43] T.-L. Ho, D.-G. Jou, Synthesis of cryptolepine and cryptoteckiene from
a common intermediate, Helv. Chim. Acta 85 (2002) 3823e3827.
[67] (a) S. Hayashi, A. Hirao, A. Imai, H. Nakamura, Y. Murata, K. Ohashi, E. Nakata,
Novel non-peptide nociceptin/orphanin FQ receptor agonist, 1-[1-(1-
methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole:
design, synthesis, and structureeactivity relationship of oral receptor occu-
pancy in the brain for orally potent antianxiety drug, J. Med. Chem. 52 (2009)
610e625;
[44] A.C. Cain, E.R. Holler, Extended scope of in situ iodotrimethylsilane mediated
selective reduction of benzylic alcohols, Chem. Commun. 13 (2001) 1168e1169.
[45] R.W. Carling, P.D. Leeson, K.W. Moore, J.D. Smith, C.R. Moyes, I.M. Mawer,
S. Thomas, T. Chan, R. Baker, A.C. Foster, S. Grimwood, J.A. Kemp,
G.R. Marshall, M.D. Tricklebank, K.L. Saywell, 3-Nitro-3,4-dihydro-2(1H)-qui-
nolones. Excitatory amino acid antagonists acting at glycine-site NMDA and
(b) S. Hayashi, A. Hirao, H. Nakamura, K. Yamamura, K. Mizuno, H. Yamashita,
Discovery of 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-
1H-benzimidazole: integrated drug-design and structureeactivity relation-
ships for orally potent, metabolically stable, and potential-risk reduced novel
non-peptide nociceptin/orphanin FQ receptor agonist as antianxiety drug,
Chem. Biol. Drug Des. 74 (2009) 369e381.
(RS)-
J. Med. Chem. 36 (1993) 3397e3408.
a
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid
receptors,
[46] R.T. Shuman, P.L. Ornstein, J.W. Paschal, P.D. Gesellchen, An improved
synthesis of homoproline and derivatives, J. Org. Chem. 55 (1990) 738e741.