Synthesis of N-(trifluoromethyl-2-pyridinyl)arenesulfonamides as an inhibitor of secretory phospholipase A2

Article abstract of DOI:10.1248/cpb.59.783

A series of N-(trifluoromethyl-2-pyridinyl)alkane- and arenesulfonamides 2-5 have been synthesized by the substitution reaction of 2- chloro(trifluoromethyl)pyridines 6 with alkane- and arenesulfonamides 7. Their inhibitory activities against secretory ph

Full text of DOI:10.1248/cpb.59.783

June 2011
Note
Chem. Pharm. Bull. 59(6) 783—786 (2011)
783
Synthesis of N-(Trifluoromethyl-2-pyridinyl)arenesulfonamides as an
Inhibitor of Secretory Phospholipase A2
Hitoshi NAKAYAMA, ,a,b Yuka MORITA,a Hirohiko KIMURA,b Keiichi ISHIHARA,c Satoshi AKIBA,c and
*
a
Jun’ichi UENISHI
a Department of Pharmaceutical Chemistry, Kyoto Pharmaceutical University; Misasagi, Yamashina, Kyoto 607–8412,
Japan: b Ishihara Sangyo Kaisha, Ltd.; 2–3–1 Nishi-shibukawa, Kusatsu 525–0025, Japan: and c Department of
Pathological Biochemistry, Kyoto Pharmaceutical University; 5 Misasagi-Nakauchi-cho, Yamashina-ku, Kyoto 607–8414,
Japan. Received January 31, 2011; accepted March 13, 2011; published online March 17, 2011
A series of N-(trifluoromethyl-2-pyridinyl)alkane- and arenesulfonamides 2—5 have been synthesized by the
substitution reaction of 2-chloro(trifluoromethyl)pyridines 6 with alkane- and arenesulfonamides 7. Their in-
hibitory activities against secretory phospholipase A2 of porcine pancreas were examined and the analog N-[4,5-bis-
(trifluoromethyl)-2-pyridinyl]-4-trifluoromethylbenzenesulfonamide 4i was shown to have the highest inhibitory
activity, with an IC50 value of 0.58 mM.
Key words bistrifluoromethylpyridine; sulfonamide; secretory phospholipase A2 inhibitor; structure–activity relationship
Phospholipase A2 (PLA2) is an enzyme that catalyzes the methyl) groups, as compounds, 2a—d, 3a—d, 4a—k, and
hydrolysis of membrane glycerophospholipids at the 2 posi- 5a—f, respectively.
tion to produce free fatty acids, in particular arachidonic
The formation of the amide bond was carried out simply
acid, and lysophospholipids. Among a number of PLA2 by heating of 2-chloropyridine 6a—f and alkane- or arene-
isozymes identified so far, several isozymes including secre- sulfonamide 7a—j with K2CO3 in dimethyl sulfoxide
tory forms are involved in the release of arachidonic acid, a (DMSO) at 115—150 °C. For example, the reaction of 2-
precursor for prostaglandins and leukotrienes.1) Since the chloro-5-trifluoromethylpyridine (6a) and ethanesulfonamide
eicosanoids are potent mediators of inflammation, the inhibi- (7a) (1.5 eq) in DMSO in the presence of K2CO3 (2.5 eq) at
tion of PLA2 is a prime target for designing new anti-inflam- 130 °C for 2 h gave N-(5-trifluoromethyl-2-pyridinyl)ethane-
matory agents.2—7) In fact, a number of its inhibitors such as sulfonamide (2a) in 65% yield. Other molecules, with the ex-
varespladib,8) pyrrophenone,9) efipladib10) and methyl in- ception of 4k, were similarly prepared and the results are as
doxam11) have been reported. N-[2-(Ethanesulfonylamino)-5- shown in Table 1. The compound 4k was prepared in 85%
trifluoromethyl-3-pyridinyl]cycloalkanecarboxamide (1) hav- yield from 3a by the standard nitration.
ing a trifluoromethyl (CF3)-pyridine ring was reported to
All new sulfonamides were evaluated for their inhibiting
possess a moderate inhibitory activity against pancreatic se- activity against porcine pancreas originated sPLA2. The ac-
cretory phospholipase A2 (sPLA2) in in vitro experiments.12) tivity was assessed by measuring free fatty acids released
Trifluoromethyl-substituted pyridines are useful ring unit13,14) from phospholipids, as a substrate, with photometric assay
and have shown interesting profiles as essential structures in using the acyl-CoA synthetase/acyl-CoA oxidase (ACS–
many kinds of biological molecules including agrochemical ACO) method.19) We have screened all the sulfonamides at a
and medicinal candidates.15—18) We have re-investigated the fixed concentration (0.5 mM), and some of them were also
above molecule and attempted to simplify and modify its
structure in order to improve the inhibitory activity for PLA2
(Fig. 1).
We have designed new molecules by the following direc-
tions; i) removal of the 3-cycloalkanecarboxamide moiety
from 1, ii) change of the position of the CF3 group and intro-
duction of an additional CF3 group or other electron with-
drawing group, iii) replacement of ethanesulfonamide with
other alkane- and arenesulfonamides. In this paper, we de-
scribe the preparation of compounds 2—5, designed by the
above direction, and evaluation of their structure–activity re-
lationships with respect to the inhibiting activity against
PLA2 Group IB (GIB).
The structure of designed compounds are listed in Fig. 2.
We categorized the analogs by 5-trifluoromethyl, 4-trifluo-
romethyl, 4,5-bis(trifluoromethyl), and 4,6-bis(trifluoro-
Fig. 2. Structures of Compounds 2—5
© 2011 Pharmaceutical Society of Japan
Fig. 1. Structure of 1 (RꢀCycloalkanecarbonyl)
∗ To whom correspondence should be addressed. e-mail: hi-nakayama@iskweb.co.jp

784
Vol. 59, No. 6
screened at 3 mM concentration. IC50 values were determined pounds 2c and 2d (entries 3 and 4) provided inhibitors that
for four compounds. These results are summarized in Table were slightly active than compounds 2a and 2b. While, the
1.
corresponding 4-trifluoromethyl derivatives 3c and 3d (en-
If a functional substituent on the pyridine ring was re- tries 7 and 8) were less active than 3a and 3b. It is noted that
placed with hydrogen without loss of biological activity, it benzenesulfonamides 2b, 2d, 3b and 3d were more potent
would reduce the number of synthetic steps. In fact, for the than ethanesulfonamides 2a, 2c, 3a and 3c, respectively.
synthesis of compounds 2—5, the synthesis would become
Since the CF3 substituent at C-4 showed a better inhibitory
much simpler in comparison with that of 1. Replacing the C- profile than that at C-5, we examined 4,5-bis(trifluoro-
3 carboxamide moiety in 1 with hydrogen afforded 5-trifluo- methyl)pyridine derivatives 4. In the cases of 2a—d and
romethyl substituted ethanesulfonamide 2a (entry 1), which 3a—d, benzenesulfonamides were more potent than ethane-
inhibited sPLA2 by 4.6%. Compound 3a in which a CF3 sulfonamides. A little but similar trend was observed in 4,5-
group located at the C-4 position instead of at C-5, inhibited bis(triflluoromethyl)pyridine derivatives 4a and 4b (entries 9
by 20.6%. In comparison of compounds 2a and 3a (entries 1 and 10). N-[4,5-Bis(trifluoromethyl)-2-pyridinyl]ethanesul-
and 5), in both of which the C-3 position is unsubstituted, the fonamide (4a) and the corresponding benzenesulfonamide
inhibitory activity of 3a exceeded that of 2a, indicating the 4b, each at 0.5 mM inhibited PLA2GIB by 24.5% and 26.4%,
importance of the CF3 substituent at the C-4 site. When respectively. Other alkanesulfonamides, the respective
ethanesulfonamides in 2a and 3a were replaced with ben- methyl-, propyl-, and isopropyl analogs, 4c, 4d, and 4e, as
zenesulfonamides gave 2b and 3b, the inhibitory activity en- well as trifluoromethanesulfonamide 4f (entries 11—14), all
hanced to 15.4% and 25.0% in both cases (entries 2 and 6). showed inferior activity than 4a.
The presence of a chloro group at the C-6 position, com-
Next, we examined the effect of the substituent on the aro-
matic ring of the arenesulfonamide in the series of N-[4,5-
bis(trifluoromethyl)-2-pyridinyl] derivatives. 4-Methoxyben-
zenesulfonamide 4g (entry 15) was slightly active to compare
with the phenyl analogue 4b, while the 4-chloro-, and 4-tri-
fluoromethylbenzenesulfonamides 4h and 4i (entries 16 and
17) were 1.4 fold more active than 4b. 2-Naphtharenesulfon-
amide 4j (entry 18) was a little less potent than 4h and 4i.
Replacement of an electron-withdrawing CF3 group at the C-
5 position with other electron-withdrawing nitro group, in 4k
(entry 19), decreased the inhibitory potency by 25%, in com-
parison to 4a. These results indicated that the 4,5-bis(trifluo-
romethyl) substituent pattern positively influenced the in-
hibitory activity.
Chart 1. Synthesis of Sulfonamides 2—5
Table 1. Reaction Conditions, Yields and sPLA2-Inhibiting Activities of N-(Trifluoromethyl-2-pyridinyl)alkane- and Arenesulfonamides 2—5
Chloro-
pyridine
6
Sulfon-
amide
7
Position
of
(CF3)n
Inhibition (%)
Yielda)
(%)
IC50
(mM)
Entry
Product
X
R
at 0.5 mM
at 3 mM
85.3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
6a
6a
6b
6b
6c
6c
6d
6d
6e
6e
6e
6e
6e
6e
6e
6e
6e
6e
—
6f
7a
7b
7a
7b
7a
7b
7a
7b
7a
7b
7e
7f
7g
7h
7c
7d
7i
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
65
43
56
70
37
71
76
32
83
92
93
71
89
50
53
46
68
61
—
81
75
87
90
81
75
5-
5-
5-
5-
4-
4-
4-
4-
4,5-
4,5-
4,5-
4,5-
4,5-
4,5-
4,5-
4,5-
4,5-
4,5-
4-
4,6-
4,6-
4,6-
4,6-
4,6-
4,6-
H
H
6-Cl
6-Cl
H
Et
Ph
Et
Ph
Et
Ph
Et
Ph
Et
Ph
Me
n-Pr
i-Pr
4.6
15.4
15.4
25.6
20.6
25.0
8.9
1.0
H
6-Cl
6-Cl
H
H
H
H
H
H
H
H
H
H
5-NO2
H
H
H
H
H
16.1
24.5
26.4
16.5
4.3
87.5
3.1
CF3
13.3
28.4
36.7
36.1
30.8
18.0
23.0
25.6
20.0
14.4
28.1
34.6
4g
4h
4i
4-MeOPh
4-ClPh
4-CF3Ph
2-Naphthyl
Et
89.6
98.3
85.0
0.66
0.58
7j
4j
—
7a
7b
7c
7d
7i
4k
5a
5b
5c
5d
5e
5f
Et
Ph
6f
6f
6f
6f
91.1
4-MeOPh
4-ClPh
4-CF3Ph
2-Naphthyl
83.2
91.2
6f
7j
H
0.68
a) Isolated yields. The yields were not optimized.

June 2011
785
7.15 (1H, dd, Jꢀ8.1, 0.75 Hz), 7.85 (1H, dd, Jꢀ8.1, 0.6 Hz). IR (KBr) cmꢂ1
1345, 1160. Anal. Calcd for C8H8ClF3N2O2S: C, 33.29; H, 2.79; N, 9.70.
Found: C, 33.36; H, 2.53; N, 9.75.
:
We have also examined 4,6-bis(trifluoromethyl)pyridine
derivatives 5. Ethane- and benzenesulfonamides 5a and 5b
(entries 20 and 21) showed similar potency as same as 4a and
4b. 4-Methoxy- and 4-trifluoromethylbenzenesulfonamides
5c and 5e (entries 22 and 24) were less active than the corre-
sponding 4g and 4i. However, somehow the 4-chloroben-
zenesulfonamide 5d (entry 23) was 1.8-fold less active than
5b. 2-Naphtharenesulfonamide 5f (entry 25) was the most
active of the 4,6-bis(trifluoromethyl)pyridine derivatives.
N-(6-Chloro-5-trifluoromethyl-2-pyridinyl)benzenesulfonamide (2d)
1
Reaction condition, 120 °C, 3.5 h. Pale yellow solid; mp 104—105 °C. H-
NMR (CDCl3) d: 7.32 (1H, d, Jꢀ8.4 Hz), 7.51—7.57 (2H, m), 7.64 (1H, m),
7.83 (1H, bs), 7.90 (1H, d, Jꢀ8.4 Hz), 7.96—7.99 (2H, m). IR (KBr) cmꢂ1
:
1313, 1146. Anal. Calcd for C12H7Cl2F3N2O2S: C, 38.83; H, 1.90; N, 7.55.
Found: C, 38.70; H, 1.95; N, 7.50.
N-(4-Trifluoromethyl-2-pyridinyl)ethanesulfonamide (3a) Reaction
condition, 120 °C, 4 h. Pale yellow solid; mp 140.5—141 °C. 1H-NMR
Comparison of the inhibition data between 5-CF3, 6-CF3, (CDCl3) d: 1.39 (3H, t, Jꢀ7.5 Hz), 3.31 (2H, q, Jꢀ7.5 Hz), 7.27 (1H, d,
Jꢀ4.8 Hz), 7.63 (1H, s), 8.63 (1H, d, Jꢀ4.8Hz). IR (KBr) cmꢂ1: 1335, 1145.
Anal. Calcd for C8H9F3N2O2S: C, 37.79; H, 3.57; N, 11.02. Found: C, 37.51;
H, 3.54; N, 10.86.
4,5-bis-CF3 and 4,6-bis-CF3 derivatives indicate that the 4,5-
bis(trifluoromethyl) as well as 4,6-bis(trifluoromethyl)sub-
stituent patterns effectively show better inhibitory activities.
N-(4-Trifluoromethyl-2-pyridinyl)benzenesulfonamide (3b) Reaction
condition, 140 °C, 3 h. White prisms; mp 139—140 °C. 1H-NMR (CDCl3) d:
7.21 (1H, d, Jꢀ5.4 Hz), 7.45—7.50 (2H, m), 7.59 (1H, m), 7.67 (1H, s),
Inhibitory activities of several selected compounds were
tested at the 3 mM concentration. These results were consis-
tently supported the results obtained at the 0.5 mM concentra-
tion. The IC50 values for compounds 2d, 4h, 4i, and 5f are
listed in Table 1. Particularly, N-[4,5-bis(trifluoromethyl)-2-
pyridinyl]-4-trifluoromethylbenzenesulfonamide 4i exhibited
the most potent IC50 value (at 0.58 mM) in the inhibition of
sPLA2. Although they are lacking a carboxamide moiety at
the C-3 position on the pyridine ring, their activities are al-
most similar or even better than those of the compounds 1.12)
In conclusion, we have demonstrated a simple CF3-substi-
tuted 2-pyridinylarenesulfonamides exhibited an inhibitory
activity of sPLA2. Although their acivitiy is moderate, N-
[bis(trifluoromethyl)-2-pyridinyl]arenesulfonamides 4h, 4i,
and 5f showed excellent profile in the inhibition. Particularly,
7.85—7.88 (2H, m), 8.62 (1H, d, Jꢀ5.4 Hz), 10.82 (1H, bs). IR (KBr) cmꢂ1
:
1335, 1171. Anal. Calcd for C12H9F3N2O2S: C, 47.68; H, 3.00; N, 9.27.
Found: C, 47.41; H, 2.97; N, 9.07.
N-(6-Chloro-4-trifluoromethyl-2-pyridinyl)ethanesulfonamide (3c)
1
Reaction condition, 120 °C, 1.5 h. White solid; mp 100—101 °C. H-NMR
(CDCl3) d: 1.45 (3H, t, Jꢀ7.3 Hz), 3.41 (2H, q, Jꢀ7.3 Hz), 7.27 (1H, s),
7.37 (1H, s), 7.65 (1H, bs). IR (KBr) cmꢂ1: 1316, 1147. Anal. Calcd for
C8H9F3N2O2S: C, 33.29; H, 2.79; N, 9.70. Found: C, 33.36; H, 2.74; N, 9.69.
N-(6-Chloro-4-trifluoromethyl-2-pyridinyl)benzenesulfonamide (3d)
Reaction condition, 130 °C, 2.5 h. White prisms; mp 138—139 °C. 1H-NMR
(CDCl3) d: 7.20 (1H, s), 7.47 (1H, s), 7.51—7.57 (2H, m), 7.63 (1H, m),
7.75 (1H, bs), 7.95—7.97 (2H, m). IR (KBr) cmꢂ1: 1338, 1170, 687. Anal.
Calcd for C12H8ClF3N2O2S: C, 42.80; H, 2.39; N, 8.32. Found: C, 43.00; H,
2.20; N, 8.32.
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]ethanesulfonamide (4a) Re-
1
action condition, 120 °C, 1.5 h. Pale yellow prisms; mp 174—175 °C. H-
compound 4i was found to be the most potent. It is noted that NMR (CDCl3) d: 1.46 (3H, t, Jꢀ7.3 Hz), 3.46 (2H, q, Jꢀ7.3 Hz), 7.55 (1H,
s), 7.74 (1H, bs), 8.77 (1H, s). IR (KBr) cmꢂ1: 1321, 1146. Anal. Calcd for
C9H8F6N2O2S: C, 33.55; H, 2.50; N, 8.69. Found: C, 33.81; H, 2.47; N, 8.57.
all these compounds are able to prepare quite easily by a sin-
gle coupling of 6 with sulfonamide 7.
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]benzenesulfonamide (4b)
Experimental
Reaction condition, 120 °C, 1.5 h. White prisms; mp 164—165 °C. 1H-NMR
(CDCl3) d: 7.53—7.58 (2H, m), 7.64 (1H, m), 7.68 (1H, s), 7.96—7.98 (2H,
m), 8.15 (1H, bs), 8.68 (1H, s). IR (KBr) cmꢂ1: 1326, 1142. Anal. Calcd for
C13H8F6N2O2S: C, 42.17; H, 2.18; N, 7.57. Found: C, 42.31; H, 2.07; N,
Melting points were determined on a Yanagimoto micro-melting point ap-
1
paratus and were not corrected. H-NMR spectra were recorded on a JEOL
JNM-GSX 400 (400 MHz) or JNM-AL 300 (300 MHz) spectrometers in
CDCl3 or DMSO-d6 with tetramethylsilane as an internal standard. IR spec- 7.55.
tra were recorded on JASCO FT/IR-410 instrument. Column chromatogra-
phy was carried out using Merck silica gel 60 (70—230 mesh).
Typical Reaction for Coupling of 2-Chloropyridines 6a—f and Sulfon-
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]methanesulfonamide (4c)
1
Reaction condition, 130 °C, 3 h. Pale yellow needles; mp 163—164 °C. H-
NMR (CDCl3) d: 3.35 (3H, s), 7.26 (1H, s), 7.51 (1H, s), 8.81 (1H, s). IR
(KBr) cmꢂ1: 1322, 1153. Anal. Calcd for C8H6F6N2O2S: C, 31.18; H, 1.96;
N, 9.09. Found: C, 30.99; H, 1.87; N, 9.16.
amides 7a—j To
a mixture of 7 (7.5 mmol) and K2CO3 (1.73 g,
12.5 mmol) in dimethyl sulfoxide (DMSO) (20 ml) was added 6 (5 mmol) in
DMSO (2 ml) portionwise at 95 °C. The mixture was heated at 115—150 °C
for 1.5—17 h. After cooling, water (50—100 ml) was added to the reaction
mixture, and the mixture was washed with ether. The pH of the aqueous
layer was adjusted to a range between 4 to 6 with 2 N hydrochloric acid to
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]propanesulfonamide (4d)
1
Reaction condition, 120 °C, 2 h. Pale yellow needles; mp 145—146 °C. H-
NMR (CDCl3) d: 1.09 (3H, t, Jꢀ7.4 Hz), 1.89—1.99 (2H, m), 3.39 (2H, t,
Jꢀ7.9 Hz), 7.57 (1H, s), 7.96 (1H, bs), 8.79 (1H, s). IR (KBr) cmꢂ1: 1321,
form a precipitate, which was collected and recrystallized from a mixture of 1146. Anal. Calcd for C10H10F6N2O2S: C, 35.72; H, 3.00; N, 8.33. Found: C,
EtOAc and hexane to give 2—5. When precipitate was not formed, the aque-
ous layer was extracted with EtOAc (50 mlꢁ2), the combined organic layer
was washed with brine, dried over Na2SO4, and condensed. The residual liq-
uid was purified by silica gel column chromatography. For the synthesis of
3c and 3d, reverse addition was taken.
35.96; H, 2.77; N, 8.38.
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]isopropanesulfonamide (4e)
Reaction condition, 150 °C, 2 h. Pale yellow needles; mp 167—168 °C. H-
NMR (CDCl3) d: 1.47 (6H, d, Jꢀ6.8 Hz), 3.63 (1H, m), 7.67 (1H, s), 8.15
(1H, bs), 8.77 (1H, s). IR (KBr) cmꢂ1: 1325, 1280, 1138. Anal. Calcd for
C10H10F6N2O2S: C, 35.72; H, 3.00; N, 8.33. Found: C, 35.79; H, 2.78; N,
8.57.
1
Chemical yields of the products are listed in Table 1.
N-(5-Trifluoromethyl-2-pyridinyl)ethanesulfonamide (2a) Reaction
condition, 130 °C, 2 h. Pale yellow powder; mp 164—166 °C. 1H-NMR
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]trifluoromethanesulfonamide
1
(CDCl3) d: 1.41 (3H, t, Jꢀ7.4 Hz), 3.35 (2H, q, Jꢀ7.4 Hz), 7.45 (1H, d, (4f) Reaction condition, 150 °C, 2 h. White prisms; mp 173—174 °C. H-
Jꢀ8.8 Hz), 7.93 (1H, dd, Jꢀ8.8, 2.4 Hz), 8.67 (1H, s), 9.56 (1H, bs). IR
NMR (CDCl3) d: 7.98 (1H, d, Jꢀ3.6 Hz), 8.26 (1H, s). IR (KBr) cmꢂ1
:
(KBr) cmꢂ1: 1335, 1132. Anal. Calcd for C8H9F3N2O2S: C, 37.79; H, 3.57; 1341, 1203. Anal. Calcd for C8H3F9N2O2S: C, 26.53; H, 0.83; N, 7.73.
N, 11.02. Found: C, 37.88; H, 3.50; N, 10.99.
Found: C, 26.79; H, 0.64; N, 7.90.
N-(5-Trifluoromethyl-2-pyridinyl)benzenesulfonamide (2b) Reaction
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]-4ꢀ-methoxybenzenesulfon-
amide (4g) Reaction condition, 120 °C, 2 h. Pale yellow prisms; mp 156—
condition, 130 °C, 3 h. Pale yellow solid; mp 161—163 °C. 1H-NMR
(CDCl3) d: 7.47—7.54 (3H, m), 7.61 (1H, m), 7.85—7.91 (3H, m), 8.74 157 °C. 1H-NMR (CDCl3) d: 3.87 (3H, s), 6.99 (2H, d, Jꢀ9.2 Hz), 7.64 (1H,
(1H, s), 11.36 (1H, bs). IR (KBr) cmꢂ1: 1328, 1167. Anal. Calcd for
s), 7.91 (2H, d, Jꢀ9.2Hz), 8.65 (1H, s). IR (KBr) cmꢂ1: 1328, 1156. Anal.
C12H9F3N2O2S: C, 47.68; H, 3.00; N, 9.27. Found: C, 47.71; H, 2.78; N, Calcd for C14H10F6N2O3S: C, 42.01; H, 2.52; N, 7.00. Found: C, 41.85; H,
9.18.
2.45; N, 6.92.
N-(6-Chloro-5-trifluoromethyl-2-pyridinyl)ethanesulfonamide (2c)
Reaction condition, 115 °C, 17 h. White prisms; mp 105—107 °C. H-NMR
(CDCl3) d: 1.47 (3H, t, Jꢀ7.5 Hz), 3.75 (2H, q, Jꢀ7.5 Hz), 7.14 (1H, bs),
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]-4ꢀ-chlorobenzenesulfonamide
(4h) Reaction condition, 120 °C, 1.5 h. Pale yellow needles; mp 147—
148 °C. 1H-NMR (CDCl3) d: 7.53 (2H, d, Jꢀ8.6 Hz), 7.61 (1H, s), 7.93 (2H,
1

786
Vol. 59, No. 6
d, Jꢀ8.6 Hz), 8.66 (1H, s). IR (KBr) cmꢂ1: 1328, 1159. Anal. Calcd for
C13H7ClF6N2O2S: C, 38.58; H, 1.74; N, 6.92. Found: C, 38.39; H, 1.76; N,
6.82.
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]-4ꢀ-trifluoromethylbenzene-
sulfonamide (4i) Reaction condition, 120 °C, 3 h. Yellow powder; mp
187—188 °C. 1H-NMR (CDCl3) d: 7.60 (1H, s), 7.82 (2H, d, Jꢀ8.2 Hz),
8.12 (2H, d, Jꢀ8.2 Hz), 8.67 (1H, s). IR (KBr) cmꢂ1: 1325, 1155. Anal.
Calcd for C14H7F9N2O2S: C, 38.37; H, 1.61; N, 6.39. Found: C, 38.09; H,
1.41; N, 6.40.
N-[4,5-(Bistrifluoromethyl)-2-pyridinyl]-2ꢀ-naphthalenesulfonamide
(4j) Reaction condition, 120 °C, 2 h. Pale yellow prisms; mp 193—194 °C.
1H-NMR (CDCl3) d: 7.60—7.74 (2H, m), 7.74 (1H, s), 7.85—7.95 (2H, m),
7.97—8.00 (2H, m), 8.58 (1H, s), 8.63 (1H, s), 10.2 (1H, bs). IR (KBr)
cmꢂ1: 1324, 1160. Anal. Calcd for C17H10F6N2O2S: C, 48.58; H, 2.40; N,
6.66. Found: C, 48.65; H, 2.39; N, 6.67.
cholate, 10 mM CaCl2, 94 mM NaCl, 1 mg/ml bovine serum albumin (BSA),
94 mM Tris–HCl, pH 8, and the test sample at various concentrations. The
reaction was started by addition of the substrate to a mixture of PLA2 and a
test sample, and incubation was continued at 37 °C for 30 min. The produc-
tion of free fatty acids was measured by the ACS–ACO method.19) After the
incubation, 50 ml of colorization reagent A (NEFA C-Test Wako; 1.46 mM
coenzyme A, 9 mM ATP, 3 mM 4-aminoantipyrine, 0.54 U/ml ACS, 5.4 U/ml
ascorbate oxidase, 50 mM phosphonate, pH 7) was added to the reaction
mixture and incubation was continued at 37 °C for another 10 min. Then
100 ml of colorization reagent B (NEFA C Test Wako; 5.5 U/ml ACO,
6.8 U/ml peroxidase, 1.2 mM 3-methyl-N-ethyl-N-(b-hydroxyethyl)aniline)
was added and incubation was continued at 37 °C for another 10 min. The
production of dye was evaluated by measuring the absorbance at 595 nm.
Three replicates were used for each determination of sPLA2 activity.
Acknowledgement The authors are very grateful to Drs. T. Haga, T.
Koyanagi, S. Mizukoshi, F. Kato and Mr. S. Yotsuya for their kind sugges-
tions on this work.
N-[4,6-(Bistrifluoromethyl)-2-pyridinyl]ethanesulfonamide (5a) Re-
action condition, 115 °C, 3 h. White prisms; mp 91—93 °C. 1H-NMR
(CDCl3) d: 1.48 (3H, t, Jꢀ7.5 Hz), 3.51 (2H, q, Jꢀ7.5 Hz), 7.59 (2H, s),
7.90 (1H, bs). IR (KBr) cmꢂ1: 1334, 1140. Anal. Calcd for C9H8F6N2O2S: C,
33.55; H, 2.50; N, 8.69. Found: C, 33.81; H, 2.47; N, 8.77.
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N-[4,6-(Bistrifluoromethyl)-2-pyridinyl]benzenesulfonamide (5b)
Reaction condition, 120 °C, 3 h. White solid; mp 99—100 °C. 1H-NMR
(CDCl3) d: 7.54 (1H, m), 7.51 (1H, s), 7.54 (1H, s), 7.61—7.66 (2H, m),
8.01—8.04 (2H, m). IR (KBr) cmꢂ1: 1336, 1166. Anal. Calcd for
C13H8F6N2O2S: C, 42.17; H, 2.18; N, 7.57. Found: C, 42.25; H, 1.96; N,
7.87.
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N-[4,6-(Bistrifluoromethyl)-2-pyridinyl]-4ꢀ-methoxybenzenesulfon-
amide (5c) Reaction condition, 140 °C, 2 h. White needles; mp 127—
128 °C. H-NMR (DMSO-d6) d: 3.83 (3H, s), 7.12 (2H, d, Jꢀ8.7 Hz), 7.43
(1H, s), 7.83 (1H, s), 7.95 (2H, d, Jꢀ8.7 Hz), 11.96 (1H, bs). IR (KBr)
cmꢂ1: 1317, 1185. Anal. Calcd for C14H10F6N2O3S: C, 42.01; H, 2.52; N,
7.00. Found: C, 42.04; H, 2.61; N, 7.02.
1
N-[4,6-(Bistrifluoromethyl)-2-pyridinyl]-4ꢀ-chlorobenzenesulfonamide
(5d) Reaction condition, 120 °C, 3.5 h. White needles; mp 136—138 °C.
1H-NMR (CDCl3) d: 7.51 (2H, d, Jꢀ6.8 Hz), 7.53 (1H, s), 7.57 (1H, s), 7.96
(2H, d, Jꢀ6.8 Hz). IR (KBr) cmꢂ1
: 1345, 1155. Anal. Calcd for
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Seno K., Biochem. J., 363, 727—735 (2002).
C13H7ClF6N2O2S: C, 38.58; H, 1.74; N, 6.92. Found: C, 38.62; H, 1.72; N,
7.01.
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Behnke M. L., Hu B., Sum F., Tam S., Hu Y., Chen L., Kirincich S. J.,
Michalak R., Thomason J., Ipek M., Wu K., Wooder L., Ramarao M.
K., Murphy E. A., Goodwin D. G., Albert L., Xu X., Donahue F., Ku
M. S., Keith J., Nickerson-Nutter C. L., Abraham W. M., Williams C.,
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N-[4,6-(Bistrifluoromethyl)-2-pyridinyl]-4ꢀ-trifluoromethylbenzene-
sulfonamide (5e) Reaction condition, 140 °C, 2 h. White needles; mp
1
158—159 °C. H-NMR (CDCl3) d: 7.56 (1H, s), 7.61 (1H, s), 7.81 (2H, d,
Jꢀ8.1 Hz), 7.92 (1H, bs), 8.16 (2H, d, Jꢀ8.1 Hz). IR (KBr) cmꢂ1: 1420,
1330, 1170. Anal. Calcd for C14H7F9N2O2S: C, 38.37; H, 1.61; N, 6.39.
Found: C, 38.42; H, 1.75; N, 6.37.
N-[4,6-(Bistrifluoromethyl)-2-pyridinyl]-2ꢀ-naphthalenesulfonamide
1
(5f) Reaction condition, 120 °C, 4 h. White prisms; mp 126—127 °C. H-
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NMR (CDCl3) d: 7.47 (1H, s), 7.60—7.70 (2H, m), 7.72 (1H, s), 7.88—8.00
(4H, m), 8.64 (1H, s). IR (KBr) cmꢂ1: 1333, 1139. Anal. Calcd for
C17H10F6N2O2S: C, 48.58; H, 2.40; N, 6.66. Found: C, 48.85; H, 2.15; N,
6.93.
Preparation of N-(5-Nitro-4-trifluoromethyl-2-pyridinyl)ethanesulfon-
amide (4k) To a stirred solution of 3a (16 mmol) in acetic acid (15 ml)
was added dropwise fumic nitric acid (2.1 ml, 48 mmol) at 100 °C and the
reaction was continued for an additional 2.5 h at 115 °C. After cooling the
mixture was poured into an ice water (70 ml) to form solid. The solid were
collected by filtration, washed with water and dried to give crude product,
which was purified by silica gel column chromatography eluted with EtOAc
1
to give 4k as pale yellow solid. mp 201—202 °C; H-NMR (DMSO-d6) d:
1.27 (3H, t, Jꢀ6.9 Hz), 3.60 (2H, q, Jꢀ6.9 Hz), 7.38 (1H, s), 9.18 (1H, s),
11.83 (1H, bs). IR (KBr) cmꢂ1: 1543, 1342, 1145. Anal. Calcd for
C8H8F3N3O4S: C, 32.11; H, 2.69; N, 14.04. Found: C, 32.40; H, 2.72; N,
13.90.
Assay of PLA2 Activity The ability of the synthetic compounds to in-
hibit sPLA2 activity was determined in an assay system using a substrate of
mixed micelles of phosphatidylcholine and cholate, according to the proce-
dure reported by Volwerk et al.20)
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1799—1803 (1981).
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1726—1733 (1986).
Dipalmitoyl phosphatidylcholine (13.6 mmol) and 0.5 M sodium cholate
(120 ml) were suspended in 1880 ml of buffer containing 250 mM NaCl and
250 mM Tris–HCl, pH 8, and used as the substrate. The reaction mixture
(total volume of 50 ml) contained (final concentrations) 0.2 mg/ml porcine
pancreatic PLA2 (Sigma-Aldrich), 2.7 mM dipalmitoyl-PC, 12 mM sodium