Nicotinamide derivatives as a new class of gastric H+/K+-ATPase inhibitors. 1. Synthesis and structure-activity relationships of N- substituted 2-(benzhydryl- and benzylsulfinyl)nicotinamides

Article abstract of DOI:10.1021/jm9605593

A new series of N-Substituted 2-(benzhydryl- and benzylsulfinyl)nicotinamides 7 and 8 were synthesized. Upon acid activation in the acidic environment of the parietal cell, these compounds are converted into their active forms, 2,3-dihydro-3-oxoisothiazolo[5,4-b]pyridines 5, which inhibit gastric H⁺/K⁺-ATPase. Inhibitory activities against [¹⁴C]aminopyrine accumulation stimulated by dibutyryl cAMP in isolated rabbit parietal cells in vitro and histamine-induced gastric acid secretion in pylorus-ligated rats by intraduodenal administration in vivo were evaluated, and the structure-activity relationships were examined. Among the compounds synthesized, 2-[(2,4-dimethoxybenzyl)sulfinyl]-N-(4- pyridyl)nicotinamide (8b) showed potent inhibitory activities in vitro and in vivo equivalent to those of omeprazole, a typical H⁺/K⁺-ATPase inhibitor. Moreover, 8b was much more stable at neutral and weakly acidic pH than omeprazole, lansoprazole, and pantoprazole. Compound 8b is considered to be a promising agent for treating acid-related gastrointestinal disorders.

Full text of DOI:10.1021/jm9605593

J . Med. Chem. 1997, 40, 313-321
313
Nicotin a m id e Der iva tives a s a New Cla ss of Ga str ic H+/K+-ATP a se In h ibitor s. 1.
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of N-Su bstitu ted
2-(Ben zh yd r yl- a n d ben zylsu lfin yl)n icotin a m id es
Hideo Terauchi,* Akihiko Tanitame, Keiko Tada, Keiji Nakamura, Yasuhiro Seto, and Yoshinori Nishikawa
Discovery Research Laboratories, Dainippon Pharmaceutical Company, Ltd., Enoki 33-94, Suita/ Osaka 564, J apan
Received J uly 29, 1996X
A new series of N-Substituted 2-(benzhydryl- and benzylsulfinyl)nicotinamides 7 and 8 were
synthesized. Upon acid activation in the acidic environment of the parietal cell, these
compounds are converted into their active forms, 2,3-dihydro-3-oxoisothiazolo[5,4-b]pyridines
5, which inhibit gastric H+/K+-ATPase. Inhibitory activities against [14C]aminopyrine ac-
cumulation stimulated by dibutyryl cAMP in isolated rabbit parietal cells in vitro and histamine-
induced gastric acid secretion in pylorus-ligated rats by intraduodenal administration in vivo
were evaluated, and the structure-activity relationships were examined. Among the com-
pounds synthesized, 2-[(2,4-dimethoxybenzyl)sulfinyl]-N-(4-pyridyl)nicotinamide (8b) showed
potent inhibitory activities in vitro and in vivo equivalent to those of omeprazole, a typical
H+/K+-ATPase inhibitor. Moreover, 8b was much more stable at neutral and weakly acidic
pH than omeprazole, lansoprazole, and pantoprazole. Compound 8b is considered to be a
promising agent for treating acid-related gastrointestinal disorders.
Sch em e 1
Peptic ulcer has been generally thought to result from
an imbalance between the aggressive factors of acid and
pepsin and defensive forces of resistance. Consequently,
antiulcer therapy has been directed toward these fac-
tors. Since the discovery of the histamine H2 receptor
antagonist cimetidine and its clinical success, the
inhibition of gastric acid secretion has been the major
focus of antiulcer therapy. Subsequently, gastric H+/
K+-ATPase, which is located in the apical membrane
of the parietal cell and plays a major role in acid
secretion, has become the next target for numerous
investigations. Among studies on H+/K+-ATPase in-
hibitors, 2-[(2-pyridylmethyl)sulfinyl]benzimidazoles
(PSBs) such as omeprazole (1a ),1 lansoprazole (1b),2 and
pantoprazole (1c)3 (Scheme 1) have been found to have
superior properties responsible for complete suppression
of gastric acid secretion, and 1a -c have recently been
introduced as clinically useful agents. The PSBs act as
prodrugs, being chemically transformed into the highly
thiophilic sulfenic acid 2 and the cyclic sulfenamide 3
in an acidic environment such as the apical membrane
of the parietal cell (Scheme 1). The thiophilic sulfenic
acid 2 and the sulfenamide 3 react readily with thiol
groups on the enzyme to form an enzyme-inhibitor
complex (4) with a tight S-S bond.4 However, there is
another possibility of this successive reaction taking
place not only within the acid compartment of the
parietal cells but also at weakly acidic pH conditions
outside the parietal cells.3 All known irreversible H+/
K+-ATPase inhibitors do not exhibit activity by the
mechanism described above, but they are structurally
similar to the PSBs5,6 and their analogues.7-14 On the
basis of the mechanism of pH-dependent inhibition as
observed in the PSBs, we aimed to find out potent and
more selective inhibitors of H+/K+-ATPase in vivo,
which have a different structure from PSBs.
(Table 1); however, they did not exhibit inhibitory
activity against gastric acid secretion in vivo by in-
traduodenal (id) administration. The potent in vitro
activities of the isothiazolopyridines 5 are due to their
high-thiophilic property; compound 5a reacted rapidly
with N-acetyl-L-cysteine to give 6 in high yield due to
its thiophilic property as shown in Scheme 2. So, we
speculated that the compounds reacted with thiol groups
on other proteins before they reached the target enzyme,
gastric H+/K+-ATPase. To get good in vivo efficacy, it
appeared necessary to find prodrugs which are con-
On the basis of our random screening, we have found
that N-substituted 2,3-dihydro-3-oxoisothiazolo[5,4-b]-
pyridines 5a ,b inhibited the H+/K+-ATPase in vitro
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
S0022-2623(96)00559-6 CCC: $14.00 © 1997 American Chemical Society

314 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Terauchi et al.
Ta ble 1. Inhibitory Activity against Gastric H+/K+-ATPase of
N-Substituted 2,3-Dihydro-3-oxoisothiazolo[5,4-b]pyridines 5
(25) via alkylation with chloromethyl methyl ether
(procedure C) (Scheme 3).
The nicotinamides 7a -d ,f-t and 8a -1,n -p listed in
Tables 3 and 4 were prepared from the corresponding
nicotinic acids 11-24 and 26-28 with the appropriate
amines by (i) the 1-ethyl-3-[3-(dimethylamino)propyl]-
carbodiimide hydrochloride method and (ii) the acid
chloride method followed by oxidation with m-chlorop-
erbenzoic acid (mCPBA) (procedures D and E, respec-
tively), as reported previously15 (Scheme 3). The nico-
tinamide 7e was prepared starting from 11, which
was converted to 2-(benzhydrylthio)-N-(4-nitrophenyl)-
nicotinamide (29) by condensation with 4-nitroaniline
by the use of oxalyl chloride, followed by catalytic
reduction and subsequent oxidation with mCPBA (pro-
cedure F). The nicotinamide 8m was prepared by
condensation of 4-aminopyridine with 2-[[4-methoxy-2-
(2-acetoxyethoxy)benzyl]thio]nicotinic acid derived from
the nicotinic acid 25 by acetylation followed by cleavage
of the acetyl group with K2CO3 and subsequent oxida-
tion with mCPBA (procedure G). In the case of 7d ,g,
oxidation of the respective 2-(benzhydrylthio)nicotina-
mides with mCPBA was performed in the presence of
hydrochloric acid to avoid formation of the N-oxide
derivatives.
inhibition of porcine
gastric H+/K+-ATPase,
compd
R
IC50 (µM)a,b
5a
5b
5c
isobutyl
phenyl
4-pyridyl
0.53
0.19
0.22
a
b
See the Experimental Section. IC50 values were calculated
from the regression lines.
Sch em e 2
verted into the active isothiazolopyridines 5 only within
the acid compartment of the parietal cells.
P h a r m a cologica l Resu lts a n d Discu ssion
Compounds 7a -t and 8a -p were first evaluated for
their ability to inhibit [14C]aminopyrine (AP) accumula-
tion stimulated by dibutyryl cyclic AMP (dbcAMP) in
isolated rabbit parietal cells. The compounds which
significantly inhibited the AP accumulation were fur-
ther evaluated for the ability to inhibit histamine-
induced gastric acid secretion in pylorus-ligated rats by
id administration. The results are summarized in
Tables 5 and 6.
We focused our initial effort on the SARs associated
with substitution on the nitrogen atom of the carbamoyl
moiety of the 2-(benzhydrylsulfinyl)nicotinamides 7
(Table 5). Most compounds 7 were supposed to be
readily converted into the isothiazolopyridines 5 at room
temperature at low pH as described in our previous
report:15 Half-lives at pH 1 of the compounds 7j,k were
less than 0.02 h. The N-(1-benzyl-4-piperidyl) (7d ) and
the N-phenyl derivatives bearing an amino (7e), dim-
ethylamino (7f), or 2-(dimethylamino)ethyl (7g) group
at the para position exhibited inhibitory activity against
the AP accumulation, whereas the N-isobutyl (7a ), the
N-phenyl (7b), and the N-naphthyl (7c) derivatives did
not inhibit the accumulation. It was deduced from this
finding that the basic part of a substituent on the
carbamoyl moiety was necessary for inhibitory activity
against the AP accumulation.
The effect of substitution of heteroaromatic rings
having a basic nitrogen atom on the nitrogen atom of
the carbamoyl moiety was examined (Table 5). The
4-pyridyl (7j,k ), 5-quinolyl (7m ), 6-quinolyl (7n ), and
2-naphthyridinyl (7p ) derivatives exhibited inhibitory
activity against the AP accumulation; particularly,
compounds 7j,k bearing the 4-pyridyl group showed the
greatest activity in this series. The 4-pyridyl group was
therefore selected as the optimal substituent on the
nitrogen atom of the carbamoyl moiety. Compounds
such as 7j,k which inhibited the AP accumulation,
however, did not show significant inhibitory activity
Recently, we reported a convenient method for pre-
paring 5 from 2-(benzhydrylsulfinyl)nicotinamides 7 or
2-(benzylsulfinyl)nicotinamides 8 substituted with alkoxy
groups at the ortho and para positions of the benzyl
group at room temperature in a diluted hydrochloric
acid-methanol solution in high yields.15 We considered
that the mechanism for the conversion of 7 and 8 might
involve the sulfonium salt 9 as an intermediate followed
by elimination of the leaving group R1, as shown in
Scheme 2, and that the conversion rate might depend
on the stability of the carbonium ion of the leaving group
R1. This finding suggested the possibility of seeking
compounds acting as prodrugs with a desirable chemical
profile of being converted into the isothiazolopyridines
5 at low pH but being more stable at both neutral and
weakly acidic pH than the PSBs to avoid interaction
with thiol groups on proteins except H+/K+-ATPase in
the body.
The present study was focused on finding a new class
of potent gastric H+/K+-ATPase inhibitors which possess
in vivo gastric antisecretory activity by id administra-
tion and are more stable at both neutral and weakly
acidic pH than the PSBs. The present paper deals with
syntheses, chemical stability, and antisecretory activi-
ties of N-substituted 2-(benzhydryl- and benzylsulfinyl)-
nicotinamides; the structure-activity relationships
(SARs) of these compounds are also discussed.
Ch em istr y
The requisite nicotinic acids 11-27 (Table 2) were
prepared by condensation of 2-mercaptonicotinic acid
(10) with the corresponding benzyl or benzhydryl chlo-
ride (procedure A) or with the corresponding benzyl
alcohols under acidic condition (procedure B) according
to the method reported previously15 (Scheme 3). 2-[[4-
Methoxy-2-[2-(methoxymethoxy)ethoxy]benzyl]thio]-
nicotinic acid (28) (Table 2) was prepared from 2-[[4-
methoxy-2-(2-hydroxyethoxy)benzyl]thio]nicotinic acid

Nicotinamides as Gastric H+/ K+-ATPase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 315
Ta ble 2. Nicotinic Acids 11-28
compd
R2
phenyl
4-methylphenyl 4-CH3
R3
procedurea
mp, °C
recrystn solventb yield, %
formulac
11d
12d
13
14d
15
16
17
18d
19
20
21
22
23
24
25
26
27
28
H
A
A
A
B
B
B
B
B
A
B
B
B
B
A
B
B
B
C
209-211
211-214
215-217
189-192
224-225
185-187
181-183
189-192
214-215
214-216
215-218
209-211
160-162
140-142
124-129
152-154
149-162
98-100
A
60
92
81
96
92
85
98
99
85
98
94
97
85
95
97
91
77
93
A
A
B
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-OCH3
2-OCH3, 4-OCH3
2-OCH3, 6-OCH3
C14H13NO3S
C-D
C15H15NO4S
C16H17NO5S
C16H17NO5S
2-OCH3, 3-OCH3, 4-OCH3
2-OCH3, 4-OCH3, 5-OCH3
2-OCH3, 4-OCH3, 6-OCH3
3-OCH3, 4-OCH3, 5-OCH3
2-OCH3, 3-CH3, 4-OCH3
2-OCH3, 4-OCH3, 6-CH3
2-OCH3, 4-OCH3, 5-Br
2-OC2H5, 4-OCH3
A-C-D
A-B-C
B-D
F
C16H17NO5S
C16H17NO5S
C16H17NO5S
C15H14BrNO4S
C16H17NO4S‚0.35H2O
C17H19NO4S
A-B-C
B-D
B
A
A
B
A
A
B
2-OC2H5, 4-OC2H5
2-OCH2CH2OH, 4-OCH3
2-OCH2CH2F, 4-OCH3
2-OCH2OCH3, 4-OCH3
2-OCH2CH2OCH2OCH3, 4-OCH3
f
e
C16H17NO5S‚0.25H2O
g
a
b
Capital letters refer to the procedures in the Experimental Section. A ) CH3CN, B ) CH3OH, C ) acetone, D ) H2O, E ) CHCl3,
F ) toluene, G ) (C2H5)2O, H ) C2H5OH, I ) n-hexane, J ) (iC3H7)2O. c All compounds were analyzed for C, H, N, S, and halogen;
analytical results were within (0.4% of the theoretical values. These compounds were previously prepared.15 e MS (SIMS): m/z 338
d
(MH+). f MS (APCI): m/z 336 (MH+). SIMS: m/z 380 (MH+).
g
Sch em e 3
trimethoxy (8g) . 4-methoxy (8a ) . 2,6-dimethoxy (8c)
> 2,3,4-trimethoxy (8d ) > 2,4-dimethoxy-5-methyl (8h )
> 2,4-dimethoxy-5-bromo (8j) > 2,4-dimethoxy (8b),
2,4,5-trimethoxy (8e), 2,4,6-trimethoxy (8f), and 2,4-
dimethoxy-6-methyl (8i). Compounds 8a,c-e,g,h showed
weak inhibitory activity against the AP accumulation,
whereas 8b,f,i,j inhibited the accumulation potently.
The result suggested that the ready conversion into 5c
at pH 1 tended to enhance the potency in inhibition of
the AP accumulation. Compounds 8b,f,i which potently
inhibited the AP accumulation exhibited potent inhibi-
tory activity against the histamine-induced gastric acid
secretion in vivo with the exception of 8j; particularly,
the activity of 8b (ED50 ) 4.2 mg/kg id) was comparable
to that of omeprazole. In addition, 8b was much more
stable at weakly acidic pH (pH 5.0) than 8f,i.
The effect of replacement of the methoxy groups of
8b by other alkoxy groups was examined (Table 6).
Replacement of the methoxy groups by an ethoxy group
(8k ,l) had little effect on inhibition of the AP accumula-
tion but resulted in a considerable decrease in inhibitory
activity against the histamine-induced gastric acid
secretion in vivo. Replacement by other alkoxy groups
(8m -p) caused a decrease in inhibitory activities against
both the AP accumulation in vitro and the histamine-
induced secretion in vivo. As shown in Table 6, there
was not a correlation between the AP accumulation
assay and in vivo in every case. This may be because
there was a disparity in the ability of the compounds to
reach the acidic compartments of the parietal cell.
Structure-activity relationship studies on N-(4-py-
ridyl)nicotinamides 7 and 8 revealed that decreasing the
hydrophobicity tended to enhance the inhibitory activity
against the histamine-induced gastric acid secretion in
pylorus-ligated rats.
against the histamine-induced gastric acid secretion in
pylorus-ligated rats by id administration. On the other
hand, intravenous administration led 7k to exhibit the
inhibitory activity significantly: Its inhibition percents
at doses of 3 and 10 mg/kg were 46.1% and 73.5%,
respectively. So, we assumed that the weak inhibitory
activities of these compounds in vivo could be attributed
to the highly hydrophobic benzhydryl groups.
Accordingly, it appeared that replacement of the
benzhydryl group by less hydrophobic benzyl groups was
required to improve the bioavailability. Since 2-(ben-
zylsulfinyl)-N-(4-pyridyl)nicotinamide is not readily con-
verted into its active form, N-(4-pyridyl)-2,3-dihydro-3-
oxoisothiazolo[5,4-b]pyridine (5c) (Table 1), at room
temperature under acidic conditions as reported previ-
ously,15 it was necessary to introduce an electron-
donating alkoxy group into the ortho and/or para
position(s) of the phenyl ring of the benzyl group to raise
the conversion rate. The inhibitory activities against
the AP accumulation in vitro and the histamine-induced
gastric acid secretion in vivo as well as half-lives (t1/2
)
of conversion at pH 5.0, 3.0, and 1.0 for 8a -p are
summarized in Table 6. Concerning the effect of
substitution of methoxy groups on the phenyl ring of
8a -j, decreasing order of t1/2 at pH 1 was 3,4,5-
To clarify whether the compounds 8 are mainly
converted into the isothiazolopyridine 5c in various

316 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Ta ble 3. N-Substituted 2-(Benzhydrylsulfinyl)nicotinamides 7
Terauchi et al.
compd
R
R3
procedurea
mp, °C
recrystn solventb yield,c %
formulad
7a
7b
7c
7d
7e
7f
isobutyl
phenyl
1-naphthyl
1-benzyl-4-piperidyl
4-aminophenyl
4-(dimethylamino)phenyl
4-[2-(dimethylamino)ethyl]phenyl
2-pyridyl
3-pyridyl
4-pyridyl
4-pyridyl
2-methoxy-3-pyridyl
5-quinolyl
H
H
H
H
H
H
H
H
H
H
CH3
H
CH3
H
H
H
H
H
E
D
D
E
F
138-140
179-183
152-154
168-170
104-106
155-157
119-121
160-163
165-166
175-178
159-163
177-173
157-159
165-168
145-150
141-145
260-265
153-156
164-165
172-175
J
E
B
B
77
51
22
27
27
33
22
16
52
23
23
40
59
40
53
51
9
C23H24N2O2S
C
C
C
C
C
C
C
C
25H20N2O2S
29H22N2O2S
31H31N3O2S
25H21N3O2S‚0.6CH3CN
27H25N3O2S
29H29N3O2S‚0.25H2O
24H19N3O2S
24H19N3O2S
A-E-I
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
A
G
A
B
B
A
B
7g
7h
7i
7je
7k e
7l
C
25H21N3O3S
7m
7n
7o
7p
7q
7r
A
H
F
A
A
A
B-E
A
C30H25N3O2S
6-quinolyl
C
C
C
C
C
C
C
28H21N3O2S
29H23N3O2S
29H24N4O2S
32H23N3O2S
23H18N4O2S
23H18N4O2S
22H17N3O2S2
2-methyl-8-quinolyl
5,7-dimethyl-2-naphthyridinyl
9-acridinyl
pyrimidinyl
pyrazinyl
18
49
32
7s
7t
H
H
2-thiazolyl
a
b
See footnote a in Table 2. See footnote b in Table 2. c Total yields (%) of the N-substituted 2-(benzhydrylsulfinyl)nicotinamides were
based on the corresponding nicotinic acids. See footnote c in Table 2. c See footnote d in Table 2.
d
Ta ble 4. 2-(Benzylsulfinyl)-N-(4-pyridyl)nicotinamides 8
compd
R3
procedurea
mp, °C
recrystn solventb
yield,c %
formulad
8a
8be
8c
8d
8e
8fe
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
4-OCH3
2-OCH3, 4-OCH3
2-OCH3, 6-OCH3
D
D
D
D
D
D
D
D
D
D
E
D
G
E
D
E
210-212
190-192
243-246
164-166
103-106
182-185
207-210
105-108
195-198
209-211
200-202
165-166
182-184
163-164
189-191
177-180
A
A
A
A
A
A
A
A
A
A
A
A
C
A
A
A-E
68
33
23
26
61
35
36
41
39
42
8
10
16
55
18
64
C19H17NO3S‚0.25H2O
C20H19N3O4S
C21H21N3O5S
C21H21N3O5S
2-OCH3, 3-OCH3, 4-OCH3
2-OCH3, 4-OCH3, 5-OCH3
2-OCH3, 4-OCH3, 6-OCH3
3-OCH3, 4-OCH3, 5-OCH3
2-OCH3, 3-CH3, 4-OCH3
2-OCH3, 4-OCH3, 6-CH3
2-OCH3, 4-OCH3, 5-Br
2-OC2H5, 4-OCH3
C21H21N3O5S
C21H21N3O4S
C21H21N3O4S
C20H18BrN3O4S
C21H21N3O4S
C22H23N3O4S
C21H21N3O5S
C21H20FN3O4S
C21H21N3O5S
C23H25N3O6S
2-OC2H5, 4-OC2H5
2-OCH2CH2OH, 4-OCH3
2-OCH2CH2F, 4-OCH3
2-OCH2OCH3, 4-OCH3
2-OCH2CH2OCH2OCH3, 4-OCH3
a
b
d
See footnote a in Table 2. See footnote b in Table 2. c See footnote c in Table 3. See footnote c in Table 2. e See footnote d in Table
2.
acidic conditions and whether their conversion rates
ridine 5c (IC50 ) 0.22 µM) in the acidic environment of
the parietal cell and then the formed 5c inhibited the
H+/K+-ATPase.
depend on pH, the concentrations of the representative
8b and the formed 5c were determined at pH 2.0, 3.0,
and 4.0 in the conversion. As shown in Figure 1, 8b
was efficiently converted into 5c depending on pH; 8b
was immediately converted into 5c at pH 1 (t1/2 < 0.02
h). Moreover, 8 prepared in the present study with the
exception of 8f did not inhibit the H+/K+-ATPase in vitro
for themselves; the activity of 8f seems to be due to a
little 5c formed during the assay because of its ready
conversion. We deduced from these findings that the
nicotinamides 8 were converted into the isothiazolopy-
The stability of 8b at pH 5.0 and 7.0 was compared
with those of the reference compounds omeprazole (1a ),
lansoprazole (1b), and pantoprazole (1c) (Table 7).
Compound 8b was much more stable than the reference
compounds 1a -c at both neutral and weakly acidic pH
(pH 5.0). This stability profile has the potential advan-
tage of minimizing a risk of activation at weakly acidic
pH found outside the parietal cells, for example, lyso-
somes.16

Nicotinamides as Gastric H+/ K+-ATPase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 317
Sch em e 4a
a
(a) R1C1, N(C2H5)3; (b) R1OH, concentrated HCl; (c) chloromethyl methyl ether, diisopropylethylamine; (d) (1) RNH2, 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide, (2) mCPBA; (e) (1) RNH2, (COCl)2, (2) mCPBA; (f) (1) (CH3CO)2O, (2) 4-aminopyridine, 1-ethyl-3-
[3-(dimethylamino)propyl]carbodiimide, (3) K2CO3, (4) mCPBA; (g) 4-nitroaniline, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; (h)
(1) H2-Pd/C, (2) mCPBA.
Ta ble 5. Antisecretory Activities of N-Substituted
2-(Benzhydrylsulfinyl)nicotinamides 7
were recorded on a Shimazu FTIR-8200PC spectrophotometer.
1H NMR spectra were taken at 200 MHz with a Varian
Gemini-200 spectrometer in (CH3)2SO-d6. Chemical shifts are
expressed in δ (ppm) values with tetramethylsilane as the
internal standard. The abbreviations used are as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
Mass spectra were obtained on a J OEL J MS D-300 mass
spectrometer for electron impact-mass spectra (EIMS), Hitachi
M-80-B mass spectrometer for secondary-ion mass spectra
(SIMS) and fast atom bombardment mass spectra (FABMS),
or Hitachi M-1000 LC API mass spectrometer for atmospheric
pressure chemical ionization mass spectra (APCIMS). The
HPLC was carried out on Shimazu LC-10A system. Organic
extracts were dried over anhydrous MgSO4. 2-(Meth-
oxymethoxy)-4-methoxybenzyl alcohol was prepared according
to the cited literature.17
inhibtn of gastric
[
14C]AP
acid secretion in
accumulation,a,b
pylorus-ligated rats,b %
inhibtn at 100 mg/kg id
compd
% inhibtn at 10 µM
7a
7b
7c
7d
7e
7f
1.3
5.0
NEc
84.3d
NE
57.4d
58.3d
7g
93.5d
7.4 (at 1 µM)
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
8.0
43.7d
NE
30.2
48.1
55.0 (at 1 µM)d
IC50 ) 1.5 µMe
NE
2-(Acet yla m in o)-3-[3-(isob u t ylca r b a m oyl)-2-p yr id yl]-
d ith iop r op a n oic Acid (6). To a stirred solution of the
N-isobutyl-2,3-dihydro-3-oxoisothiazolo[5,4-b]pyridine15 (6.0 g,
0.029 mol) in CH3OH (200 mL) was added 4.7 g (0.029 mol) of
N-acetyl-L-cysteine at room temperature. The resulting mix-
ture was stirred at the same temperature for 3 min and
concentrated to dryness in vacuo. The residue was recrystal-
lized from C2H5OH to give 7.6 g (71%) of 6: mp 175-177 °C;
1H NMR δ 0.90 (6H, d, J ) 6.2 Hz), 1.86 (3H, s), 4.01 (1H, m),
7.33 (1H, dd, J ) 5.1, 7.9 Hz), 7.94 (1H, dd, J ) 2.0, 7.9 Hz),
8.63 (1H, dd, J ) 2.0, 5.1 Hz), 12.80 (1H, s); SIMS m/z 372
(MH+); Anal. (C15H21N3O4S2) C, H, N, S.
74.1d
30.4
21.7
61.8d
24.4d
68.6d
NE
47.6d
7r
7s
NE
NE
7t
14.7
omeprazole
IC50 ) 0.37 µMe
a
Inhibition of [14C]aminopyrine (AP) accumulation determined
b
in isolated rabbit parietal cells after dbcAMP stimulation. See
d
the Experimental Section. c NE: not effective. p < 0.01. e See
2-(2-Hyd r oxyeth oxy)-4-m eth oxyben zyl Alcoh ol. A mix-
ture of 2-hydroxy-4-methoxybenzaldehyde (20 g, 0.13 mol),
acetic acid 2-chloroethyl ester (17 mL, 0.16 mol), K2CO3 (27 g,
0.20 mol), and dimethylformamide (DMF) (200 mL) was stirred
at 150 °C for 3 days and concentrated to dryness in vacuo.
The residue was taken up in 50 mL of water, and the aqueous
mixture was extracted with two 200-mL portions of (C2H5)2O.
The combined extracts were dried and concentrated to dryness
in vacuo to give 31 g (99%) of crude 2-(2-acetoxyethoxy)-
4-methoxybenzaldehyde: APCIMS m/z 239 (MH+).
footnote b in Table 1.
As a result of the present study, compound 8b was
found to be a selective inhibitor of H+/K+-ATPase; its
inhibitory activities against the [14C]aminopyrine ac-
cumulation stimulated by dbcAMP in isolated rabbit
parietal cells and against the histamine-induced gastric
acid secretion in pylorus-ligated rats by id administra-
tion were comparable to those of omeprazole. In addi-
tion, 8b was readily converted into the active form 5c
at low pH and was much more stable at both neutral
and weakly acidic pH than pantoprazole. Therefore,
this compound, having a different structure from the
PSBs, seems to be a promising agent for treating acid-
related gastrointestinal disorders.
To a stirred solution of the crude 2-(2-acetoxyethoxy)-4-
methoxybenzaldehyde (31 g, 0.13 mol) in CH3OH (500 mL) was
added slowly 4.5 g (0.12 mol) of NaBH4. The resulting mixture
was stirred at room temperature for 30 min and concentrated
to dryness in vacuo. The residue was taken up in 50 mL of
water, and the aqueous mixture was extracted with two 200-
mL portions of CHCl3. The combined extracts were washed
with brine, dried, and concentrated to dryness in vacuo to give
25 g (97%) of crude 2-(2-hydroxyethoxy)-4-methoxybenzyl
alcohol: 1H NMR δ 3.65-3.75 (2H, m), 3.73 (3H, s), 3.98 (2H,
t, J ) 5.2 Hz), 4.43 (2H, d, J ) 5.7 Hz), 4.78 (1H, t, J ) 5.2
Hz), 4.85 (1H, t, J ) 5.4 Hz), 6.44-6.53 (2H, m), 7.22 (1H, m).
Exp er im en ta l Section
All melting points were determined on
a Yanagimoto
micromelting point apparatus and are uncorrected. IR spectra

318 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Terauchi et al.
Ta ble 6. Antisecretory Activities and Half-Lives of Conversion of 2-(Benzylsulfinyl)-N-(4-pyridyl)nicotinamides 8
inhibtn of gastric acid secretion
inhibtn of
H+/K+-ATPase,b
IC50 (µM)
t1/2 of conversion (h)b
[
14C]AP accumulation,a,b
in pylorus-ligated rats,b
d
compd
IC50c or % inhibtn (µM)
ED50 or % inhibtn (id dose mg/kg)e
pH 1.0
pH 3.0
pH 5.0
8a
8b
8c
33.1% (10)f
0.59
26
44.4% (100)f
4.2 [1.4-12.2]
60.7% (100)f
12.7% (10)
2.14
<0.02
0.66
0.84
38.51
38.51
>100
>100
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
19.6% (10)
32.5% (10)f
1.2
11.3% (10)
16.6
0.46
1.9
0.45
0.40
59
1.7
2.2
2.0
0.45
<0.02
<0.02
7.70
0.12
<0.02
0.07
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
115.52
0.14
<0.03
21.7% (100)
6.8 [2.0-22.7]
>100
0.29
4.28
7.3
38.5% (10)g
11.55
0.09
28.88
0.71
0.49
1.78
3.30
8.25
2.75
>100
>100
5.7 [1.0-31.2]
34.8% (100)
19 [7.3-49.4]
11.2 [2.9-42.7]
41.6% (10)g
8.1 [1.1-59.1]
37.7% (100)
76.6% (10)f
>100
>100
>100
14.4% (3)
omeprazole
0.37
4.1 [1.5-10.9]
a
b
d
See footnote a in Table 5. See the Experimental Section. c See footnote b in Table 1. ED50 values were calculated from the regression
lines; 95% confidence limits in brackets. e n ) 4-7 at each dose. f p < 0.01. 0.01 < p < 0.05.
g
were dried and concentrated to dryness in vacuo to give 12 g
(96%) of crude methyl 2-(2-fluoroethoxy)-4-methoxybenzoate:
SIMS m/z 229 (MH+).
To a stirred solution of the crude benzoate (12 g, 0.053 mol)
in (C2H5)2O (150 mL) was added slowly 1.9 g (0.049 mol) of
LiAlH4 at 0 °C. After the resulting mixture was stirred at the
same temperature for 30 min, the remaining LiAlH4 was
allowed to decompose by the cautious addition of 10 mL of CH3-
OH and 5 mL of water. The organic solvent was removed by
distillation in vacuo, and the resulting aqueous mixture was
extracted with two 100-mL portions of CHCl3. The combined
extracts were washed with brine, dried, and concentrated to
dryness in vacuo to give 10 g (92%) of crude 2-(2-fluoroethoxy)-
4-methoxybenzyl alcohol: 1H NMR δ 3.74 (3H, s), 4.13-4.87
(5H, m), 4.44 (2H, d, J ) 5.6 Hz), 6.52-6.58 (2H, m), 7.26 (1H,
F igu r e 1. Kinetics of conversions of compound 8b in aqueous
solution (pH 2.0, 3.0, 4.0) at 37 °C. Compound 8b was
incubated at each pH; the concentrations of 8b (pH 2.0, b; pH
3.0, 2; pH 4.0, 9) and compound 5c (pH 2.0, O; pH 3.0, 4; pH
4.0, 0) were determined at the time points indicated using
HPLC.
m).
Crude 2,4-dimethoxy-6-methylbenzyl alcohol was prepared
from ethyl 2,4-dihydroxy-6-methylbenzoate in a manner simi-
lar to that described above; the overall yield was 97%. The
crude benzyl alcohols, without further purification, were used
for the preparation of the corresponding nicotinic acids.
2-(Ben zylth io)n icotin ic Acid s 11-28 (Ta ble 2). P r o-
ced u r e A (1). 2-[(4-Meth oxyben zyl)th io]n icotin ic Acid
(13). This compound was prepared from 2-mercaptonicotinic
acid (10) and 4-methoxybenzyl chloride in a manner similar
to that described previously:15 1H NMR δ 3.72 (3H, s), 4.30 (2H,
s), 6.85 (2H, m), 7.23 (1H, dd, J ) 5, 7 Hz), 7.31 (2H, m), 8.20
(1H, dd, J ) 2, 7 Hz), 8.65 (1H, dd, J ) 2, 5 Hz); EIMS m/z
275 (M+); IR (KBr) 1681 cm-1 (CdO).
Ta ble 7. pH-Dependent Chemical Stability of Compound 8b
and Reference Compounds
a
t1/2 (h)
compd
pH 5
pH 7
>100
8b
38.51
0.32
0.28
1.17
omeprazole
lansoprazole
pantoprazole
10.50
6.08
P r oced u r e A (2). 2-[(2,4-Dieth oxyben zyl)th io]n ico-
tin ic Acid (24). To a stirred solution of the crude 2,4-
diethoxybenzyl alcohol (20 g, 0.11 mol) in toluene (400 mL)
was added 60 mL of concentrated HCl at room temperature.
The resulting mixture was stirred at the same temperature
for 30 min, washed successively with water and brine, dried,
and concentrated to dryness in vacuo. The residue was
dissolved in 500 mL of CH2Cl2, and then a suspension of
2-mercaptonicotinic acid (14 g, 0.089 mol) in CH2Cl2 (500 ml)
was added. To the stirred resulting mixture was added slowly
14 g (0.14 mol) of triethylamine at 0 °C. The mixture was
stirred at room temperature for 6 h, taken up in 100 ml of
water, and extracted with two 100-mL portions of CHCl3. The
combined extracts were dried and concentrated to dryness in
vacuo to give 28 g (95%) of 24: 1H NMR δ 1.29 (3H, t, J ) 6.8
Hz), 1.30 (3H, t, J ) 6.8 Hz), 3.99 (2H, q, J ) 6.8 Hz), 4.04
(2H, q, J ) 6.8 Hz), 4.27 (1H, s), 6.42 (1H, m), 6.52 (1H, m),
7.17-7.26 (2H, m), 8.17 (1H, dd, J ) 1.9, 7.6 Hz), 8.63 (1H,
dd, J ) 1.9, 4.9 Hz); SIMS m/z 333 (M+); IR (KBr) 1695 cm-1
(CdO).
23.11
a
See the Experimental Section.
2,4-Diethoxybenzyl alcohol was prepared starting from 2,4-
dihydroxybenzaldehyde in a manner similar to that described
above; the overall yield was 81%. Crude 5-bromo-2,4-
dimethoxybenzyl alcohol and 2-ethoxy-4-methoxybenzyl alco-
hol were prepared from 5-bromo-2,4-dimethoxybenzaldehyde
and 2-ethoxy-4-methoxybenzaldehyde, respectively, by reduc-
tion with NaBH4 in a manner similar to that described above;
the yields were 97% and 85%. The crude benzyl alcohols,
without further purification, were used for the preparation of
the corresponding nicotinic acids.
2-(2-F lu or oeth oxy)-4-m eth oxyben zyl Alcoh ol. A mix-
ture of methyl 2-hydroxy-4-methoxybenzoate (10 g, 0.055 mol),
1-chloro-2-fluoroethane (4.7 mL, 0.066 mol), K2CO3 (11.4 g,
0.079 mol), and DMF (100 mL) was stirred at 90 °C for 2 days
and concentrated to dryness in vacuo. The residue was taken
up in 50 mL of water, and the aqueous mixture was extracted
with two 50-mL portions of (C2H5)2O. The combined extracts

Nicotinamides as Gastric H+/ K+-ATPase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 319
P r oced u r e B. 2-[(2,4-Dim eth oxy-6-m eth ylben zyl)th io]-
n icotin ic Acid (21). Compound 21 was derived from 2-mer-
captonicotinic acid (10) that was condensed with 2,4-dimethoxy-
6-methylbenzyl alcohol in the presence of concentrated HCl
in a manner similar to that described previously:15 1H NMR δ
2.28 (3H, s), 3.74 (3H, s), 3.76 (3H, s), 4.24 (2H, s), 6.42 (2H,
m), 7.18 (1H, dd, J ) 4.8, 7.9 Hz), 8.15 (1H, dd, J ) 2.0, 7.9
Hz), 8.59 (1H, dd, J ) 2.0, 4.8 Hz); EIMS m/z 319 (M+); IR
(KBr) 1772 cm-1 (CdO).
0.016 mol) was added. The reaction mixture was stirred at
room temperature overnight and concentrated to dryness in
vacuo. The residue was taken up in 100 mL of water, and the
aqueous mixture was extracted with two 300-mL portions of
CHCl3. The combined extracts were dried and concentrated
to dryness in vacuo. The residue was chromatographed on
silica gel, eluted with CHCl3-CH3OH (50:1), and crystallized
from CH3CN to give 3.5 g (56%) of 2-(benzhydrylthio)-
N-(2-pyrimidinyl)nicotinamide: mp 155-159 °C; 1H NMR δ
6.41 (1H, s), 7.89 (1H, dd, J ) 1.9, 8.1 Hz), 8.46 (1H, dd, J )
1.9, 5.1 Hz), 8.67 (2H, d, J ) 4.1 Hz); FABMS m/z 399 (MH+);
IR (KBr) 1666 cm-1 (CdO).
P r oced u r e C. 2-[[4-Meth oxy-2-[2-(m eth oxym eth oxy)-
eth oxy]ben zyl]th io]n icotin ic Acid (28). A mixture of 25
(7.0 g, 0.021 mol), chloromethyl methyl ether (4.8 mL, 0.063
mol), diisopropylethylamine (18 mL, 0.10 mol), and CH2Cl2
(200 mL) was stirred at room temperature for 16 h and
concentrated to dryness in vacuo. The residue was taken up
in 100 mL of water, and the aqueous mixture was extracted
with two 200-mL portions of CH3CO2C2H5. The combined
extracts were dried and concentrated to dryness in vacuo. The
residue was dissolved in 400 mL of CH3OH, and then 60 mL
of 1 N NaOH was added. The resulting mixture was stirred
at reflux temperture for 1 h. The reaction mixture was
neutralized with 1 N HCl and concentrated to dryness in
vacuo. The residue was taken up in 50 mL of water, and the
aqueous mixture was extracted with two 200-mL portions of
CHCl3. The combined extracts were washed with brine, dried,
and concentrated to dryness in vacuo to give 7.4 g (93%) of
28: 1H NMR δ 3.18 (3H, s), 3.73 (3H, s), 3.77 (2H, m), 4.15
(2H, m), 4.28 (2H, s), 4.59 (2H, s), 6.48 (1H, dd, J ) 2.2, 8.7
Hz), 6.58 (1H, d, J ) 2.2 Hz), 8.19 (1H, dd, J ) 1.9, 7.6 Hz),
8.65 (1H, J ) 1.9, 4.8 Hz); IR (KBr) 1682 cm-1 (CdO).
2-(Ben zh yd r ylsu lfin yl)n icotin a m id es 7a -t a n d 2-(Ben -
zylsu lfin yl)n icotin a m id es 8a -p (Ta bles 3 a n d 4). P r o-
ced u r e D. 2-(Ben zh yd r ylsu lfin yl)-N-p h en yln icot in a -
m id e (7b). 2-(Benzhydrylthio)-N-phenylnicotinamide was
derived from 2-(benzhydrylthio)nicotinic acid (11) in 82% yield
by condensation with aniline by the use of 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride in a man-
ner similar to that described previously:15 mp 156-159 °C
To a stirred solution of the 2-(benzhydrylthio)-N-(2-pyrim-
idinyl)nicotinamide (2.0 g, 5.0 mmol) in CH2Cl2 (50 mL) was
added dropwise a solution of 80% mCPBA (1.3 g, 6.0 mmol) in
CH2Cl2 (20 mL) at 0 °C. The resulting mixture was stirred at
the same temperature for 3 min, washed with saturated
aqueous NaHCO3 (30 mL), and dried. The solvent was
removed by distillation in vacuo. The residue was chromato-
graphed on silica gel with CHCl3-CH3OH (30:1) as the eluent
and recrystallized from CH3CN to give 0.66 g (32%) of 7r : 1H
NMR δ 5.81 (1H, s), 7.52 (1H, dd, J ) 5.0, 7.8 Hz), 8.03 (1H,
dd, J ) 1.9, 7.8 Hz), 8.61 (1H, dd, J ) 4.9, 5.0 Hz), 8.66 (2H,
d, J ) 4.9 Hz), 11.30 (1H, s); SIMS m/z 415 (MH+); IR (KBr)
1045 (SdO), 1683 (CdO) cm-1
.
Compound 7s was prepared in a manner similar to that
described above.
P r oced u r e F . N-(4-Am in op h en yl)-2-(ben zh yd r ylsu lfi-
n yl)n icotin a m id e (7e). 2-(Benzhydrylthio)-N-(4-nitrophe-
nyl)nicotinamide (29) was prepared from 2-(benzhydrylthio)-
nicotinic acid (10), which was chlorinated with oxalyl chloride
followed by condensation with 4-nitroaniline in a manner
similar to that described previously.15 The overall yield was
55%: mp 166-167 °C; 1H NMR δ 6.44 (1H, s), 7.16-7.48 (10H,
m), 8.28 (2H, m), 8.52 (1H, dd, J ) 1.8, 4.8 Hz), 11.09 (1H, s);
SIMS m/z 441 (M+); IR (KBr) 1658 cm-1 (CdO). Anal.
(C25H19N3O3S) C, H, N, S.
Compound 29 (1.0 g, 2.3 mmol) was hydrogenated in 100
mL of CH3CO2C2H5 containing 0.1 g of 10% Pd/C at 40 °C
under atmospheric pressure. After removal of the catalyst
by filtration, the solvent was removed in vacuo to give
0.90 g (97%) of crude 2-(benzhydrylthio)-N-(4-nitrophenyl)-
nicotinamide: SIMS m/z 411 (M+).
1
(CH3OH); H NMR δ 6.58 (1H, s), 7.87 (1H, dd, J ) 1.9, 7.9
Hz), 8.00 (1H, s), 8.46 (1H, dd, J ) 1.9, 5.1 Hz); EIMS m/z 396
(M+); IR (KBr) 1645 cm-1 (CdO).
Compound 7b was derived from 2-(benzhydrylthio)-N-phe-
nylnicotinamide in 62% yield by oxidation with mCPBA in a
manner similar to that described previously:15 1H NMR δ 5.87
(1H, s), 7.59 (1H, dd, J ) 4.8, 5.2 Hz), 8.11 (1H, dd, J ) 1.7,
8.2 Hz), 8.66 (1H, dd, J ) 1.7, 4.8 Hz); SIMS m/z 413 (MH+);
The crude nicotinamide was dissolved in 50 mL of CH2Cl2-
CH3OH (2:1), and then a solution of 80% mCPBA (0.40 g, 1.9
mmol) in CH2Cl2 (50 mL) was added dropwise at 0 °C. The
resulting mixture was stirred at the same temperature for 3
min, washed with saturated aqueous NaHCO3 (20 mL), and
dried. The solvent was removed by distillation in vacuo. The
residue was chromatographed on silica gel with CHCl3-CH3-
OH (20:1) as the eluent and recrystallized from n-hexane-
CHCl3 to give 0.37 g (51%) of 7e: 1H NMR δ 5.01 (2H, br),
5.83 (1H, s), 6.57 (2H, m), 7.55 (1H, dd, J ) 4.5, 8.1 Hz), 8.07
(1H, dd, J ) 1.8, 8.1 Hz), 8.62 (1H, J ) 1.8, 4.5 Hz), 10.16
(1H, s); SIMS m/z 427 (M+); IR (KBr) 1036 (SdO), 1661 (CdO)
IR (KBr) 1043 (SdO), 1668 (CdO) cm-1
.
P r oced u r e E (1). 2-[[2-(2-F lu or oeth oxy)-4-m eth oxy-
ben zyl]su lfin yl]-N-(4-p yr id yl)n icotin a m id e (8n ). 2-[[2-(2-
Fluoroethoxy)-4-methoxybenzyl]thio]-N-(4-pyridyl)nicotina-
mide was prepared starting from 2-[[2-(2-fluoroethoxy)-4-
methoxybenzyl]thio]nicotinic acid (26), which was chlorinated
with oxalyl chloride followed by condensation with 4-aminopy-
ridine in a manner similar to that described previously.15 This
compound was obtained as an oily product. The overall yield
was 61%: 1H NMR δ 3.73 (3H, s), 4.16-4.82 (4H, m), 4.36
(2H, s), 6.47 (1H, dd, J ) 2.4, 8.6 Hz), 6.58 (1H, d, J ) 2.4
Hz), 7.65 (2H, m), 7.96 (1H, dd, J ) 1.9, 7.6 Hz), 8.47 (2H, m),
8.64 (1H, dd, J ) 1.9, 5.2 Hz), 10.79 (1H, s); APCIMS m/z 414
(MH+); IR (KBr) 1684 cm-1 (CdO).
cm-1
.
P r oced u r e G. 2-[[2-(2-Hyd r oxyeth oxy)-4-m eth oxyben -
zyl]su lfin yl]-N-(4-p yr id yl)n icotin a m id e (8m ). To a stirred
solution of 25 (1.0 g, 3.0 mmol) in pyridine (8 mL) was added
1 mL of (CH3CO)2O at room temperature. The resulting
mixture was stirred at same temperature for 30 min and at
reflux temperature for 1 h. The resulting mixture was
concentrated to dryness in vacuo and taken up in 100 mL of
water, and the aqueous mixture was extracted with two 200-
mL portions of CHCl3. The combined extracts were washed
with brine, dried, and concentrated to dryness in vacuo to give
1.1 g (98%) of crude 2-[[2-(2-acetoxyethoxy)-4-methoxybenzyl]-
thio]nicotinic acid.
2-[[2-(2-Acetoxyethoxy)-4-methoxybenzyl]thio]-N-(4-pyridyl)-
nicotinamide was derived from the crude 2-[[2-(2-acetoxy-
ethoxy)-4-methoxybenzyl]thio]nicotinic acid in 57% yield by
condensation with 4-aminopyridine by the use of 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride in a man-
ner similar to that described previously.15 This compound was
obtained as an oily product: 1H NMR δ 1.94 (3H, s), 3.72 (3H,
s), 4.19 (2H, m), 4.30 (2H, m), 4.34 (2H, s), 6.45 (1H, dd, J )
Compound 8n was derived from 2-[[2-(2-fluoroethoxy)-4-
methoxybenzyl]thio]-N-(4-pyridyl)nicotinamide in 90% yield by
oxidation with mCPBA in a manner similar to that described
previously:15 1H NMR δ 3.73 (3H, s), 4.19 (1H, d, J ) 12.0 Hz),
4.42 (1H, d, J ) 12.0 Hz), 6.47 (1H, dd, J ) 2.2, 8.3 Hz), 6.56
(1H, d, J ) 2.2 Hz), 7.02 (1H, d, J ) 8.3 Hz), 7.65 (2H, m),
7.71 (1H, dd, J ) 4.6, 8.0 Hz), 8.22 (1H, dd, J ) 1.9, 8.0 Hz),
8.85 (1H, dd, J ) 1.9, 4.6 Hz), 10.95 (1H, s); APCIMS m/z 430
(MH+); IR (KBr) 1037 (SdO), 1672 (CdO) cm-1
.
P r oced u r e E (2). 2-(Ben zh yd r ylsu lfin yl)-N-(2-p yr im -
id in yl)n icotin a m id e (7r ). Oxalyl chloride (10 g, 0.079 mol)
was added to a stirred suspension of 11 (5.0 g, 0.016 mol) in
dioxane (200 mL) at room temperature. The resulting mixture
was heated at 80 °C for 2 h with stirring. The mixture was
concentrated to dryness in vacuo. The residue was dissolved
in 300 mL of pyridine, and then 2-aminopyrimidine (1.5 g,

320 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Terauchi et al.
2.6, 8.6 Hz), 6.57 (1H, d, J ) 2.6 Hz), 7.65 (2H, m), 7.95 (1H,
dd, J ) 1.9, 7.6 Hz), 8.47 (2H, m), 8.64 (1H, dd, J ) 1.9, 4.9
Hz); APCIMS m/z 454 (M+); IR (KBr) 1684 cm-1 (CdO).
To a stirred solution of the crude 2-[[2-(2-acetoxyethoxy)-4-
methoxybenzyl]thio]-N-(4-pyridyl)nicotinamide (0.77 g, 1.7
mmol) in CH3OH (30 mL) was added 30 mL of 0.1 N K2CO3 at
room temperature. The resulting mixture was stirred at the
same temperature for 30 min, neutralized with 1 N HCl at 0
°C, and concentrated to about 30 mL. The aqueous mixture
was extracted with two 30-mL portions of CHCl3. The
combined extracts were washed with two 10-mL portions of
brine, dried, and concentrated to dryness in vacuo to give crude
2-[[2-(2-hydroxyethoxy)-4-methoxybenzyl]thio]-N-(4-pyridyl)-
nicotinamide (0.63 g, 96%): APCIMS m/z 412 (M+).
Refer en ce Com p ou n d s. Omeprazole and lansoprazole
were extracted from commercially available Omepral tablets
(Fujisawa Pharmaceutical Co. Ltd.) and Takepron capsules
(Takeda Chemical Industries Ltd.). Pantoprazole was pre-
pared according to known procedures.3
Biology. For the in vitro study, the test compounds were
dissolved in (CH3)2SO. All incubation mixtures contained less
than 1% (CH3)2SO.
H+/K+-ATP ase Activity in P or cin e Gastr ic Micr osom es.
Porcine gastric microsomes containing H+/K+-ATPase were
prepared as described by Nagaya.19 The membrane protein
(15 µg) was preincubated at 37 °C for 30 min in an assay
medium containing 50 mM Tris-HCl buffer, pH 7.4, 4 mM
MgCl2, 5 µg/mL gramicidin, and test compound, with or
without 20 mM KCl (total volume 1 mL). The enzyme reaction
was started by adding ATP to a final concentration of 2 mM.
After incubation for 20 min at 37 °C, the reaction was
terminated by adding 1 mL of 16% trichloroacetic acid.
Inorganic phosphate produced by hydrolysis of ATP was
determined according to the method of Sanui.20 The H+/K+-
ATPase activity was calculated by subtracting the base rate
(in the absent of K+) from the rate of hydrolysis of ATP in the
presence of K+.
Acid F or m a tion in Isola ted Ra bbit P a r ieta l Cells.
Rabbit parietal cells were isolated as described by Fryklund.21
Acid formation in the parietal cells was assessed by accumula-
tion of [14C]AP.22 The parietal cell-rich fraction (1-2 × 107
cells/300 µL) was suspended in 1.5 mL of Earle’s balanced salt
solution containing 5.6 kBq of [14C]AP, 25 mM HEPES-NaOH
buffer, pH 7.4, 0.2% bovine serum albumin, and test com-
pound. After dibutyryl cAMP (1 mM) was added, the reaction
mixture was incubated at 37 °C for 30 min under an atmo-
sphere of 95% O2 and 5% CO2. The cells were separated from
medium by brief centrifugation and digested with a tissue
solubilizer. After a liquid scintillator was added, radioactivity
was counted using a liquid scintillation counter. The radio-
activity accumulated by the cells in the presence of 0.1 mM
dinitrophenol was subtracted from all data to correct for
trapped [14C]AP.
Hista m in e-In d u ced Ga str ic Acid Secr etion in P ylor u s-
Liga ted Ra ts.2 Male stranded Wister rats weighing about
200 g were used. Rats were deprived of food but allowed free
access to water for 24 h prior to experiments. Each experiment
was performed using 4-7 rats/group. Under urethane anes-
thesia (1 g/kg), a midline laparotomy was performed and
ligature tightly secured around the pylorus. Either control
vehicle or drug was administered intraduodenally immediately
just after ligating the pylorus, and the abdominal incision was
closed. Thirty minutes later, histamine (30 mg/kg) was
injected sc. Three hours later, the stomach was removed, and
the gastric contents were collected. The volume of gastric juice
was measured, and the acid concentration of 1.0-mL sample
aliquots was determined by automatic titration to pH 7 with
0.01 N NaOH. The product of the gastric volume and acid
concentration was used to calculate the total acid output.
Total acid output during the 3-h period was compared with
that obtained in control animals, and results are expressed
as percent inhibition.
To a stirred solution of the crude 2-[[2-(2-hydroxyethoxy)-
4-methoxybenzyl]thio]-N-(4-pyridyl)nicotinamide (0.63 g, 1.5
mmol) in CH2Cl2 (50 mL) was added dropwise a solution of
80% mCPBA (0.36 g, 1.7 mmol) in CH2Cl2 (10 mL) at 0 °C.
The resulting mixture was stirred at the same temperature
for 3 min, washed with saturated aqueous NaHCO3 (10 mL),
and dried. The solvent was removed by distillation in vacuo.
The residue was chromatographed on silica gel with CHCl3-
CH3OH (10:1) as the eluent and recrystallized from acetone
to give 0.20 g (31%) of 8m : 1H NMR δ 3.66 (2H, m), 3.73 (3H,
s), 3.92 (2H, m), 4.24 (1H, d, J ) 12.3 Hz), 4.43 (1H, d, J )
12.3 Hz), 4.75 (1H, t, J ) 5.8 Hz), 6.44 (1H, dd, J ) 2.3, 8.1
Hz), 6.53 (1H, d, J ) 2.3 Hz), 6.99 (1H, d, J ) 8.1 Hz), 7.66
(2H, m), 7.72 (1H, dd, J ) 4.7, 7.8 Hz), 8.24 (1H, dd, J ) 1.8,
7.8 Hz), 8.50 (2H, m), 8.86 (1H, dd, J ) 1.8, 4.7 Hz), 10.97
(1H, s); APCIMS m/z 428 (MH+); IR (KBr) 1032 (CdO), 1676
(SdO) cm-1
.
Oxid a tion w ith mCP BA in Acid ic Con d ition s. 2-(Ben -
zh yd r ylsu lfin yl)-N-(1-b en zyl-4-p ip er id yl)n icot in a m id e
(7d ). 2-(Benzhydrylthio)-N-(1-benzyl-4-piperidyl)nicotinamide
was prepared from 2-(benzhydrylthio)nicotinic acid (11), which
was chlorinated with oxalyl chloride followed by condensation
with 4-amino-1-benzylpiperidine in a manner similar to that
described previously.15 The overall yield was 83%. This
compound was obtained as an oily product: 1H NMR δ 1.50-
1.69 (2H, m), 1.97-2.04 (2H, m), 2.11-2.25 (2H, m), 2.79-
2.88 (2H, m), 3.50 (2H, s), 3.92-4.09 (1H, m), 6.54 (1H, s), 6.99
(1H, dd, J ) 5, 7 Hz), 7.76 (1H, dd, J ) 1.9, 7.9 Hz), 8.40 (1H,
J ) 1.9, 4.9 Hz); EIMS m/z 493 (M+); IR (KBr) 1649 cm-1
(CdO).
To a stirred solution of 2-(benzhydrylthio)-N-(1-benzyl-4-
piperidyl)nicotinamide (2.8 g, 5.7 mmol) and 1.0 mL of 6 N
HCl in CH3OH (100 mL) was added dropwise a solution of 80%
mCPBA (1.3 g, 6.0 mM) in CH3OH (50 mL) at 0 °C. The
resulting mixture was stirred at the same temperature for 1
min, taken up in 200 mL of saturated aqueous NaHCO3,
extracted with two 300-mL portions of CHCl3, and dried. The
solvent was removed by distillation in vacuo. The residue was
chromatographed on silica gel with CHCl3-CH3OH (50:1) as
the eluent and recrystallized from CH3OH to give 1.0 g (35%)
of 7d : 1H NMR δ 1.47-1.63 (2H, m), 1.77-1.89 (2H, m), 2.01-
2.13 (2H, m), 2.79-2.87 (2H, m), 3.50 (2H, s), 3.70-3.85 (1H,
m), 5.79 (1H, s), 7.51 (1H, dd, J ) 5, 7 Hz), 7.96 (1H, dd, J )
2, 7 Hz), 8.60 (1H, dd, J ) 2, 5 Hz); SIMS m/z 510 (MH+); IR
(KBr) 1043 (SdO), 1651 (CdO) cm-1
.
p H-Dep en d en t Ch em ica l Sta bility of Com p ou n d s 8a -
p , Om ep r a zole, La n sop r a zole, a n d P a n t op r a zole. The
half-lives of each compound were determined in 100 mM HCl
(pH 1), 10 mM HCl (pH 2), and McIlvaine buffer18 (pH 3-7)
which had been adjusted to the appropriate pH. Each
compound was dissolved in (CH3)2SO at a concentration of 10
µM. Then 20 µL of the prepared solution was added to 1.96
mL of the appropriate buffer and 0.02 mL of 1% Triton
solution. The mixture was incubated at 37 °C for various
periods of time, and 20 µL of this solution was injected into
HPLC (Capcell Pack C18 SG120 4.6-250 mm, CH3CN/10 mM
phosphate buffer (pH 7.0), 1 mL/min, UV detector at 254 nm).
On the basis of peak area, the amounts of the nicotinamides,
the isothiazolopyridine, and the reference compounds were
estimated, and the half-life for each compound in different
media was determined from the liner regression of ln of the
concentration vs time.
Ackn owledgm en t. We are grateful to Dr. T. Karasa-
wa, Dr. S. Mishio, and Dr. K. Hino for encouragement
throughout this work. Thanks are also due to Dr. M.
Komiya and Mr. K. Kawashima for valuable discussion,
members of the Pharmacological Section for phamaco-
logical studies, and members of the Analytical Section
for elemental analyses and spectral measurements.
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