Novel pyrimidines as acid pump antagonists (APAs)

Article abstract of DOI:10.1016/j.bmcl.2010.08.007

A series of pyrimidine derivatives as acid pump antagonists (APAs) was synthesized and the inhibitory activities against H⁺/K⁺ ATPase isolated from hog gastric mucosa were determined. After elaborating on substituents at C2 and C4 po

Full text of DOI:10.1016/j.bmcl.2010.08.007

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5735–5738
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel pyrimidines as acid pump antagonists (APAs)
⇑
Young Ae Yoon, Chan Sun Park, Myung Hun Cha, Hyunho Choi, Jae Young Sim, Jae Gyu Kim
Yuhan Research Institute, 416-1, Gongse-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-902, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 February 2010
Revised 20 July 2010
Accepted 3 August 2010
Available online 6 August 2010
A series of pyrimidine derivatives as acid pump antagonists (APAs) was synthesized and the inhibitory
activities against H+/K+ ATPase isolated from hog gastric mucosa were determined. After elaborating
on substituents at C2 and C4 position of the pyrimidine scaffold, we have observed that the compound
7h is a potent APA with H+/K+ ATPase, IC50 = 52 nM.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Gastroesophageal reflux disease
Ulcer
H+/K+ ATPase
Acid pump antagonist (APA)
Gastroesophageal reflux disease (GERD) is a common acid-re-
lated disorder with a prevalence estimated to range between 10%
and 20% in Western countries and about 5% in Asia when it is de-
fined as at least weekly heartburn and/or acid regurgitation.1 The
severity of the symptoms and esophageal mucosal damage were
correlated well with the exposure of the esophagus to gastric acid.2
Therefore, acid suppression is considered as the first-line therapy
for GERD, and many drugs that suppress the acid secretion are
now available including histamine 2 receptor antagonists (H2RAs)
and proton pump inhibitors (PPIs). Presently, PPIs are recognized
as the ‘treatment of choice’ in most countries.3 However, PPIs still
continue to have their set of limitations. The currently available
PPIs require around 3–5 days to achieve maximum acid inhibition
at existent therapeutic doses, primarily due to their chemical
structures and irreversible inhibition of H+/K+ ATPase.4 Failure to
demonstrate a sustained acid inhibition throughout the day and
night, in spite of twice daily administration and nocturnal acid
breakthrough (NAB) are found to be common in patients taking
PPIs.5 Therefore, many novel strategies to address the unmet needs
of existent PPI therapy have been investigated, and acid pump
antagonists (APAs) could play a promising role, as they provide fas-
ter onset and longer duration of action than conventional PPIs by
virtue of their ability to reversibly bind to the proton pump.6
APAs are lipophilic and weak bases that have diverse structures
such as imidazopyridines, pyrimidines, imidazonaphthyridines, or
quinolines, etc.6 Revaprazan (1), the representative agent based
on pyrimidine scaffold, was shown to inhibit gastric acid secretion
by reversibly binding to H+/K+ ATPase, and it also displayed excel-
lent antisecretory properties both in animals and human beings
(Fig. 1).7–9 Revaprazan (1) was launched in Korea in 2007 for the
treatment of duodenal ulcer, gastric ulcer and gastritis. It is also
undergoing phase III clinical studies for the treatment of GERD.
In this paper, we described our extended work to the corre-
sponding pyrimidines related to revaprazan (1). Pyrimidine deriv-
atives (2) that have substituent at C2 other than aniline group were
prepared and their inhibitory activities against H+/K+ ATPase were
evaluated (Fig. 2). We were able to identify pyrimidine APAs that
have inhibitory activity superior to that of revaprazan (1).
Treating the commercially available 4-fluorophenylacetonitrile
3a with iodomethane using potassium t-butoxide and 18-crown-
6 at ꢀ78 °C afforded 3b in good yield (92%). Also the reaction of
3a with 1-bromo-2-chloroethane using N,N,N-trimethylbenzylam-
monium chloride as a phase transfer catalyst led to 3c (42%). Sub-
sequently, amidine derivatives of structure 4 can be readily
obtained by following the procedure as described in the litera-
ture.10 Nitrile derivatives 3 were added to the mixture of trimeth-
ylaluminum and ammonium chloride in toluene at 0 °C, and the
solution was heated to 80 °C for 22 h to afford 4 in good yield
H Cl
N
F
N
N
N
Revaprazan(1)
⇑
Corresponding author.
E-mail address: jagkim@yuhan.co.kr (J.G. Kim).
Figure 1. Structure of pyrimidine APA, revaprazan (1).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.007

5736
Y. A. Yoon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5735–5738
R'
dimethylpyrimidine 8 (Scheme 2). Chlorination of 8 with phospho-
rus oxychloride using N,N-dimethylaniline (DMA) as a catalyst
yielded 2,4-dichloro-5,6-dimethylpyrimidine 9 (85%). Substitution
by respective 1,2,3,4-tetrahydroisoquinoline derivatives occurred
mainly at C4 position of pyrimidine ring with C2 substituted one
as a by-product. Reaction of 9 and respective 1,2,3,4-tetrahydroiso-
quinoline derivatives with triethylamine (TEA) in DMF at room
temperature led to 10 in 44–56% yield. 2-Stannylpyrimidine deriv-
atives of structure 11 were prepared by stannyl anion substitution
in 2-chloropyrimidines 10 using lithium diisopropylamide (LDA)
and tributyltin hydride (15–42%).12 Stille coupling of 11 with 4-flu-
orobenzoyl chloride and subsequent crystallization of the interme-
diate from a solution of hydrochloric acid in ethyl acetate afforded
corresponding 2-(4-fluorobenzoyl) substituted pyrimidine deriva-
tives 12 (44–92%, over two-steps).12 Also reduction of 2-(4-fluor-
obenzoyl) substituted pyrimidine derivatives 12 with sodium
borohydride and subsequent transformation with (diethyl-
amino)sulfur trifluoride (DAST) provided 13 and 14 in good yield
(88% and 73%, respectively).
Table 1 enlists the inhibitory activities of pyrimidine derivatives
7, 12, 13 and 14 against H+/K+ ATPase isolated from hog gastric
mucosa.13 The inhibitory activity against H+/K+ ATPase of 2-(4-flu-
orobenzyl) substituted pyrimidines with various 1-methyl-1,2,3,4-
tetrahydroisoquinoline substituents at C4 was evaluated. It was
noteworthy that the electron-donating substituents, methyl
(R = CH3, R0 = H or R = H, R0 = CH3) and methoxy groups (R = H,
R0 = OCH3), at 1-methyl-1,2,3,4-tetrahydroisoquinoline were more
favorable than the substituents with electron-withdrawing proper-
ties (R = H, R0 = F, Cl); 7g (R = H, R0 = 6-OCH3), 7h (R = H, R0 = 7-
OCH3), 7k (R = H, R0 = 6-CH3), and 7l (R = H, R0 = 7-CH3) were more
R
N
N
H Cl
F
N
R1'
R1
2
R = H, CH3
R' = H, F, Cl, CH3, OCH3
R1 = R1' = H
R1 = R1' = CH3
R1 = H, R1' = F, OH
R1-R1' : cyclopropyl, =O
Figure 2. Structure of pyrimidine APAs (2).
(65–69%). Condensation of amidine derivatives of 4 with ethyl 2-
methylacetoacetate in methanol using sodium methoxide pro-
vided 4-hydroxy pyrimidine derivatives 5 in satisfactory yield
(51–65%). After chlorination of 5 with phosphorus oxychloride
(6), substitution with respective 1,2,3,4-tetrahydroisoquinoline
derivatives11 and subsequent crystallization of the intermediate
from a solution of hydrochloric acid in ethyl acetate afforded cor-
responding pyrimidine derivatives
Scheme 1).
2-(4-Fluorobenzoyl) substituted pyrimidine derivatives were
obtained starting from commercially available 2,4-dihydroxy-5,6-
7 in good yield (46–86%,
F
NC
3b
F
NC
a
3c
f
OH
Cl
N
F
F
a
F
F
b
NH
N
N
c
NC
H2N
N
R1 R1'
H Cl
R1'
R1'
R1
R1
3a
4
5
6
R'
R
7a : R = CH3, R' = H, R1 = R1' = H
7b : R = H, R' = 8-CH3, R1 = R1'= H
7c : R = H, R' = 5-Cl, R1 = R1' = H
7d : R = H, R' = 7-F, R 1 = R1' = H
7e : R = H, R' = 6-Cl, R1 = R1' = H
7f : R = H, R' = 7-Cl, R1 = R1' = H
7g : R = H, R' = 6-OCH3, R1 = R1' = H
7h : R = H, R' = 7-OCH3, R1 = R1' = H
7i : R = H, R' = 7-OCH3, R1 = R1' = CH3
7j : R = H, R' = 7-OCH3, R1-R1' = cPro
7k : R = H, R' = 6-CH3, R1 = R1' = H
7l : R = H, R' = 7-CH3, R1 = R1' = H
7m : R = H, R' = 6-CH3, R1 = R1' = CH3
7n : R = H, R' = 6-CH3, R1-R1' = cPro
N
N
H Cl
F
d,e
N
R1'
R1
7
Scheme 1. Reagents and conditions: (a) NH4Cl, Al (CH3)3, toluene, 80 °C, 22 h, 65–69%; (b) ethyl 2-methylacetoacetate, NaOMe, MeOH, reflux, 51–65%; (c) POCl3, reflux, 2 h,
62–97%; (d) 1,2,3,4-tetrahydroisoquinoline derivatives, TEA, DMF, reflux, 7 h; (e) HCl(g), EtOAc, 0 °C, 46–86% over two-steps; (f) iodomethane (3b), t-BuOK, 18-crown-6, THF,
ꢀ78 °C, 2 h, 92%; N,N,N-trimethylbenzylammonium chloride, 1-bromo-2-chloroethane (3c), 50% NaOH, 45 °C, 6 h, 42%.

Y. A. Yoon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5735–5738
5737
Table 1
OH
Cl
H+/K+ ATPase inhibition assay results for 7, 12, 13 and 14
a
b
N
N
R'
N
OH
R'
N
Cl
R'
R
8
9
N
N
H Cl
F
N
R1 R1'
c
d,e
N
N
7, 12, 13, 14
N
N
0
Compds
R
R0
H
8-CH3
5-Cl
7-F
6-Cl
7-Cl
6-OCH3
7-OCH3
7-OCH3
7-OCH3
6-CH3
7-CH3
6-CH3
6-CH3
6-CH3
7-OCH3
6-CH3
6-CH3
R1
R1
H+/K+ ATPase IC50
(lM)
N
Cl
N
Sn(n-Bu)3
1
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
12a
12b
13
14
0.35
0.61
0.70
1.99
1.24
0.94
1.63
0.17
0.052
34.2%a
1.4%a
0.23
0.30
1.76
1.29
1.91
1.12
1.51
1.13
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
10
11
R'
R'
g
H Cl
f
H Cl
N
N
N
CH3
CH3
F
F
cPro
N
N
H
H
CH3
H
H
N
CH3
O
OH
cPro
=O
=O
12
13
(R' = 6-CH3)
12a : R' = 6-CH3
H
H
OH
F
12b : R' = 7-OCH3
R'
a
Inhibition percentage at 4 lM.
100
80
60
40
20
H Cl
N
F
N
N
F
14
(R' = 6-CH3)
1
Scheme 2. Reagents and conditions: (a) POCl3, DMA, 130 °C, 2 h, 85%; (b) 1,2,3,4-
tetrahydroisoquinoline derivatives, TEA, DMF, rt, 1 h, 44–56%; (c) LDA, Bu3SnH, THF,
0 °C, 3 h, 15–42%; (d) 4-fluorobenzoyl chloride, THF, ꢀ78 °C, 3 h; (e) HCl(g), EtOAc,
0 °C, 44–92%(over two-steps); (f) NaBH4, IPA, reflux, 14 h; HCl(g), EtOAc, 0 °C, 88%;
(g) DAST, MC, ꢀ78 °C, 1 h; HCl(g), EtOAc, 0 °C, 73%.
7h
0
-9
-8
-7
-6
-5
Log(M)
than three-fold potent compared to the compounds 7c (R = H,
R0 = 5-Cl), 7d (R = H, R0 = 7-F), 7e (R = H, R0 = 6-Cl), and 7f (R = H,
R0 = 7-Cl). And 7g (R = H, R0 = 6-OCH3), 7h (R = H, R0 = 7-OCH3), 7k
(R = H, R0 = 6-CH3), and 7l (R = H, R0 = 7-CH3) were even more po-
tent than revaprazan (1). Especially, 7h (R = H, R0 = 7-OCH3) was
shown to have nanomolar inhibitory concentration (IC50 = 52 nM)
in a dose-dependent manner (Fig. 3). Although 7a (R = CH3,
R0 = H) and 7b (R = H, R0 = 8-CH3) were more potent than the com-
pounds that have electron-withdrawing substituents at 1-methyl-
1,2,3,4-tetrahydroisoquinoline (7c, 7d, 7e, and 7f), they were less
favored than those with methyl substituent at the phenyl ring of
1-methyl-1,2,3,4-tetrahydroisoquinoline (7k and 7l). Among the
compounds with electron-withdrawing substituents at 1-methyl-
1,2,3,4-tetrahydroisoquinoline, 7c (R = H, R0 = 5-Cl) was shown to
have lowest inhibitory activity against H+/K+ ATPase. Therefore, it
was evident that 1-methyl-1,2,3,4-tetrahydroisoquinoline deriva-
tives substituted by electron-donating groups at C6 or C7 are fa-
vored at C4 of pyrimidines.
Figure 3. Dose-dependent inhibitions of H+/K+ ATPase by pyrimidine APA 1 and 7h.
After exploring the substituents at C4 of pyrimidines to improve
inhibitory activity against H+/K+ ATPase, we chose to more extend
C2 substituents with 7-methoxy-1-methyl-1,2,3,4-tetrahydroiso-
quinoline (R = H, R0 = 7-OCH3, as seen in 7h) or 1,6-dimethyl-
1,2,3,4-tetrahydroisoquinoline (R = H, R0 = 6-CH3 as seen in 7k) at
C4 fixed. Replacement of 4-fluorobenzyl group with 4-fluorophe-
nylcyclopropyl (7j and 7n) or 2-methyl-2-(4-fluorophenyl)ethyl
(7i and 7m) led to the reduction in the inhibitory activity against
H+/K+ ATPase compared to the 2-(4-fluorobenzyl) group substi-
tuted ones (7h and 7k). Same trend was also observed in 2-(4-flu-
orobenzoyl) substituted pyrimidines 12a and 12b, and in hydroxy
and fluoro group substituted pyrimidines (13 and 14) instead of
carbonyl group at C2 indicating the 4-fluorobenzyl group at C2 of
pyrimidines is the most favored for inhibiting H+/K+ ATPase.

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Y. A. Yoon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5735–5738
40.0
30.0
20.0
10.0
0.0
References and notes
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3. Robinson, M. Eur. J. Gastroenterol. Hepatol. 2001, 13, S43.
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M.; Briving, C.; Wallmark, B.; Hersey, S. Annu. Rev. Pharmacol. Toxicol. 1995, 35,
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1998, 12, 1235; (b) Katz, P. O.; Hatlebakk, J. G.; Castell, D. O. Aliment. Pharmacol.
Ther. 2000, 14, 709; (c) Katz, P. O.; Anderson, C.; Khoury, R.; Castell, D. O.
Aliment. Pharmacol. Ther. 1998, 12, 1231.
100nM
50nM
25nM
12.5nM
control
6. Andersson, K.; Carlsson, E. Pharmacol. Ther. 2005, 108, 294.
7. Park, S.; Ahn, B.; Lee, B.; Kang, H.; Song, K. Gut, 2003, 52, Abstr. MON-G-008.
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0
0.05
0.1
0.15
0.2
0.25
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Abstr. MON-G-009.
1/[KCl](mM -1
)
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11. (a) Larsen, R. D.; Reamer, R. A.; Corley, E. G.; Davis, P.; Grabowski, E. J. J.; Reider,
P. J.; Shinkai, I. J. Org. Chem. 1991, 56, 6034; (b) Hong, Y. W.; Lee, Y. N.; Kim, H.
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-10.0
Figure 4. Lineweaver–Burk plot showing H+/K+ ATPase activity versus K+ concen-
tration for various concentrations of 7h.
12. Sandosham, J.; Undheim, K. Tetrahedron 1994, 50, 275.
13. Ion-leaky membrane vesicle enriched in gastric H+/K+-ATPase was derived
from pig stomach as per the method described by Saccomani et al. with
suitable modifications (Saccomani, G.; Stewart, H. B.; Show, D.; Lewin, M.;
Sachs, G. Biochem. Biophy. Acta 1977, 465, 311). The inhibitory effects of the K+
specific H+/K+-ATPase activity was calculated based on the difference between
the activity of H+/K+-ATPase with and without K+ ion. The lyophilized vesicle in
5 mM Pipes/Tris buffer (pH 6.1) was pre-incubated in the presence of various
concentrations of compounds. After 5 min preincubation, negative and positive
buffers were, respectively, added to the previous reaction mixture. As the
substrate ATP was added to the reaction buffer, and incubated for 30 min at
37 °C. Enzymatic activity was stopped by adding colorimetric reagent and the
amount of mono phosphate (Pi) in the reaction was measured at 620 nm using
the microplate reader. The difference between Pi production with and without
The mechanism of inhibition by 7h was determined in relation
to the activation of H+/K+ ATPase activity by K+. Compound 7h
inhibited H+/K+ ATPase activity in a K+-competitive manner with
Ki = 16.5 nM (Fig. 4).14 The Lineweaver–Burk plot showed H+/K+
ATPase activity versus K+ concentration for various concentrations
of 7h, and demonstrated a common intercept with the Y-axis,
which is characteristic of competitive inhibition.
In summary, we have prepared a series of novel pyrimidines as
APAs. Optimization of substituents at C2 and C4 led to some potent
pyrimidine APAs. Especially, compound 7h was shown to have
excellent inhibitory activity against H+/K+ ATPase (IC50 = 52 nM).
Therefore, compound 7h is a promising lead for further develop-
ment as APAs, and this series of pyrimidine derivatives would be
explored for further optimization.
K+ was taken as K+ stimulated H+/K+-ATPase activity. The IC50
compounds were calculated from each inhibition value of compounds
s of test
%
using the method described by Litchfield–Wilcoxon (Litchfield, J. T.; Wilcoxon,
F. J. Pharmacol. Exp. Ther. 1949, 95).
14. The inhibition kinetics were determined in relation to the activation of H+/K+-
ATPase activity by K+. The lyophilized vesicle in 5 mM Pipes/Tris buffer (pH 6.1)
was pre-incubated in the presence of various concentrations of compounds.
Compounds were examined for its ability to inhibit the generation of inorganic
phosphate induced by various concentrations of KCl. The difference between
the Pi production with K+ and without K+ is taken as K+ stimulated H+/K+-
ATPase activity. The K+-stimulated H+/K+-ATPase activities were analyzed by
Lineweaver–Burk plot.
Acknowledgements
This study was supported by Ministry for Health, Welfare and
Family Affairs, Republic of Korea (A081052).