Novel imidazo[1,2-a]pyrazine derivatives as potent reversible inhibitors of the gastric H+/K+-ATPase

Article abstract of DOI:10.1016/j.bmc.2007.09.009

A series of novel 6-substituted imidazo[1,2-a]pyrazines were synthesized via palladium catalyzed amino- or alkoxycarbonylation as key step. The anti-secretory activity of these compounds has been assessed in a binding assay against H⁺/K⁺

Full text of DOI:10.1016/j.bmc.2007.09.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 536–541
Novel imidazo[1,2-a]pyrazine derivatives as potent
reversible inhibitors of the gastric H+/K+-ATPaseq
Peter Jan Zimmermann,* Christof Brehm, Wilm Buhr, Andreas Marc Palmer,
Jurgen Volz and Wolfgang-Alexander Simon
¨
Nycomed GmbH, Byk-Gulden-Str. 2, D-78467 Konstanz, Germany
Received 3 April 2007; accepted 4 September 2007
Available online 12 September 2007
Abstract—A series of novel 6-substituted imidazo[1,2-a]pyrazines were synthesized via palladium catalyzed amino- or alkoxycarb-
onylation as key step. The anti-secretory activity of these compounds has been assessed in a binding assay against H+/K+-ATPase
from hog gastric mucosa. Some of the compounds proved to be potent inhibitors of the gastric acid pump.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
O
NH2
R
N
N
N
The treatment of gastro-oesophageal reflux disease
(GERD) and other acid related diseases relies primarily
on inhibition of the gastric H+/K+-ATPase which effects
the final step of acid secretion in the stomach. Although
irreversible inhibitors of the H+/K+-ATPase (PPIs) are
considered the treatment of choice, there is still potential
for improvement in GERD and ulcer therapy.1 The new
reversible potassium competitive acid blockers (P-
CABs) may offer some therapeutic advantages such as
faster and better symptom control and more rapid
healing.2
N
N
R'
N
N
O
NH
SCH 32651 (1)
2
Figure 1. Structure of SCH 32651 (1) and target compound 2.
Over the past two decades, lead optimization of P-CABs
has been mainly focused on the structural class of substi-
tuted imidazo[1,2-a]pyridines3–6 or imidazonaphthyri-
dines.7 The closely related imidazo[1,2-a]pyrazines
have been studied less extensively.8,9 As part of our
continuing efforts to identify novel P-CABs, we envis-
aged the known inhibitor SCH 32651 (1)8 as a suitable
starting point for further structural modifications. Here-
in, we report on the synthesis and biological evaluation
of some new 6-carboxamide-substituted imidazo[1,2-
a]pyrazines 2 and certain derivatives thereof (Fig. 1).10
2. Chemistry
The most prevalent synthetic route toward imi-
dazo[1,2-a]pyrazines comprises the condensation of
aminopyrazines
with
a-halogenocarbonyl
com-
pounds.11 In order to have access to the target com-
pounds 2 and derivatives thereof, we planned to
introduce the amide functionality at a late stage of
the synthesis, after the remaining substituents had al-
ready been established. For this approach, we consid-
ered the palladium catalyzed carbonylation of an
appropriate substituted 6-halogeno-imidazo[1,2-a]pyra-
zine to be the method of choice. Starting from com-
mercially available aminopyrazine (3), bromination
Keywords: Anti-ulcer; P-CAB; H+/K+-ATPase; Carbonylation; Imi-
dazo[1,2-a]pyrazine.
q
Design of novel P-CABs, III. -Part II: Palmer, A. M.; Grobbel, B.;
Brehm, C.; Zimmermann, P. J.; Buhr, W.; Feth, M. P.; Holst, H. C.;
Simon, W. A. doi:10.1016/j.bmc.2007.08.065.
*
Corresponding author. Tel.: +49 0 7531 84 4783; fax: +49 0 7531 84
94783; e-mail: pjz@lycos.de
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.009

P. J. Zimmermann et al. / Bioorg. Med. Chem. 16 (2008) 536–541
537
with N-bromosuccinimide gave 2-amino-3,5-dibromo-
O
pyrazine (4).12 Initial attempts on the condensation
of 4 with 3-bromo-2-butanone failed, in accordance
with the literature reports on related reactions of
2-amino-3,5-dibromopyrazine (4) with a-halogenoke-
tones.13 Therefore, we decided to introduce the
3-aminobenzyl group first:14 Reaction of 2-ethyl-6-
methylbenzylamine with compound 4 was best accom-
plished under microwave-assisted conditions to yield
the benzylamino derivative 5 on a multigram scale
with high regioselectivity (>95%) and in 68% yield.
Subsequent condensation of compound 5 with 3-bro-
mo-2-butanone proceeded smoothly to give the 6-bro-
mo-imidazo[1,2-a]pyrazine 6 which was isolated as its
salt with oxalic acid in 70% yield (Scheme 1).
N
N
i
N
N
6
NH
ii
7
O
O
R1
N
R2
N
RO
N
iv (R = H)
v (R = Et)
N
N
N
N
NH
NH
Having obtained the key intermediate 6, elaboration of
the carboxamide had to be accomplished. At first, as
shown in Scheme 2, we examined the direct aminocarb-
onylation of the 6-bromo-imidazo[1,2-a]pyrazine 6
according to standard procedures described in the litera-
ture.15 To our delight, heating a mixture of bromide 6,
palladium acetate (15 mol %), triphenylphosphine, and
dimethylamine (2 M solution in tetrahydrofuran) under
a pressure of 6 bars carbon monoxide for 16 h furnished
the N,N-dimethylcarboxamide 7 in 66% yield. Although
the preparation of amide 7 according to this procedure
proceeded well, we considered the alkoxycarbonylation
of the 6-bromo precursor 6 as more viable, since the
resulting ester 8 would allow the convenient synthesis
of a variety of amides. In fact, reaction of 6 under similar
conditions applied for the preparation of amide 7, but
using triethylamine in ethanol rather than dimethyl-
amine in tetrahydrofuran, delivered ethyl ester 8 in excel-
lent yield. After saponification of ethyl ester 8 to the
carboxylic acid 9, the carboxamides 10–12 were obtained
in good to excellent yields by reaction of 9 with the
respective amine and TBTU16 as coupling reagent. Alter-
natively, heating ethyl ester 8 with 2-aminoethanol or
10: R1 = R2 = H
11: R1 = CH3, R2 = H
12: R1, R2 = (CH2)4
13: R1 = 2-hydroxyethyl, R2 = H
14: R1 = 2-methoxyethyl, R2 = H
8: R = Et
9: R = H
iii
Scheme 2. Reagents and conditions: (i) Pd(OAc)2, PPh3, 2 M Me2NH
in THF, 6 bars CO, 120 ꢁC, 16 h, 66%; (ii) Pd(OAc)2, PPh3, EtOH,
Et3N, 10 bars CO, 100 ꢁC, 14 h, 89%; (iii) 2 N NaOH, dioxane, 80 ꢁC,
1 h, 95%; (iv) TBTU, CH2Cl2; 10: NH3 (g), rt to reflux, 3 h, 66%; 11:
8 M MeNH2 in EtOH, rt, 16 h, 94%; 12: pyrrolidine, rt, 7 h, 78%; (v)
13: 2-aminoethanol, 80–100 ꢁC, 1.5 h, 91%, 14: 2-methoxyethylamine,
reflux, 20 h, 49%.
2-methoxyethylamine provided directly the correspond-
ing carboxamides 13 and 14, respectively (Scheme 2).
In addition to the imidazo[1,2-a]pyrazine-6-carboxam-
ides 10–14 thus prepared, we were interested in the 6-
alkoxymethyl-substituted analogues of 2 to enlarge the
structure–activity relation. Consequently, ethyl ester 8
was subjected to lithium aluminium hydride reduction
to give the hydroxymethyl derivative 15, which in turn
was alkylated to yield the corresponding methyl ether
16 (Scheme 3).
Br
N
N
Br
NH2
N
N
i
ii
NH
N
N
NH2
NH2
O
Br
R
O
N
EtO
N
5
3
4
i
N
N
N
N
Br
NH
NH
N
O
N
N
iii
5
+
NH
Br
8
15: R = H
.
6
(COOH)2
ii
.
16 HCl: R = CH3
Scheme 1. Reagents and conditions: (i) N-bromosuccinimide, CH2Cl2,
63%; (ii) 2-ethyl-6-methylbenzylamine, Et3N, CH3CN, microwave
180 ꢁC, 40 min, 68%; (iii) (1)dioxane, 100 ꢁC; (2)oxalic acid, acetone,
70%.
Scheme 3. Reagents and conditions: (i) LiAlH4, THF, 0 ꢁC, 1 h, 87%;
(ii) (1)NaH, CH3I, DMF, rt, 1 h; (2)1.5 M HCl in Et2O, CH2Cl2, rt,
21%.

538
P. J. Zimmermann et al. / Bioorg. Med. Chem. 16 (2008) 536–541
Table 1. Inhibition of H+/K+-ATPase by compounds 1, 7, and 10–16
(15) or methoxymethyl group (16) led to derivatives with
an activity comparable to the monoalkyl-substituted
carboxamides 14 and 11, respectively.
R
N
N
N
4. Conclusions
NH
In summary, we have reported a general route for the
preparation of 6,8-disubstituted imidazo[1,2-a]pyra-
zines. This new approach has been exemplified by the
convenient synthesis of some 6-carboxamides and re-
lated derivatives. Two compounds of this series (7, 12)
were shown to have a superior in vitro activity as antag-
onists of the gastric H+/K+-ATPase compared to the
known P-CAB SCH 32651 (1).
Compound
R
H+/K+-ATPase ꢀlog IC50
5.9
SCH 32651 (1)
O
10
11
n.i.a
5.5
H2N
5. Experimental
O
(H3C)HN
(H3C)2N
5.1. General methods
O
All chemicals and solvents were commercially available
and used without further purification. The reactions
were monitored by thin-layer chromatography (TLC)
7
6.4
using silica plates (HPTLC-plates Nano-SIL 20 UV254
,
O
N
Macherey-Nagel) and spots were visualized in UV-light
at k = 254 nm. Column chromatography was performed
on Merck silica gel 60. Melting points (mp) were deter-
12
13
6.9
5.0
mined on a Buchi B-540 melting point apparatus and are
¨
uncorrected. H NMR spectra were recorded on a Bru-
O
O
1
HO
N
H
ker 200 MHz spectrometer with TMS as an internal
standard at 300 K. HPLC–HRMS spectra were re-
corded in positive ion mode on an electrospray quadru-
pole-time of flight mass spectrometer (micrOTOF-Q,
Bruker Daltonics, Bremen) that is coupled to an Agilent
1100 series HPLC. The spectra were calibrated exter-
nally with sodium formate cluster ions. Microwave syn-
theses were carried out with a Personal Chemistry
Emrys Optimizer.
14
15
16
6.0
5.9
5.5
O
HO
O
N
H
a No inhibition, i.e. ꢀlog IC50 < 4.
5.1.1. 2-Ethyl-6-methylbenzylamine. To a suspension of
LiAlH4 (10.4 g, 275 mmol) in dried Et2O (200 mL) was
slowly added a solution of 2-ethyl-6-methylbenzonitrile
(20.0 g, 138 mmol) in Et2O (60 mL) at ꢀ10 ꢁC. After
1 h at 0 ꢁC and 1 h at room temperature, the reaction
mixture was carefully hydrolyzed with water (4 mL)
and 6 N NaOH (4 mL). After 2 h at room temperature,
anhydrous MgSO4 was added and the reaction mixture
was filtered through Celite. Evaporation of the solvent
yielded 2-ethyl-6-methylbenzylamine (15.5 g, 80%) as a
colorless oil which was used without further purification
3. Results and discussion
All imidazo[1,2-a]pyrazines 7 and 10–16 as well as SCH
32651 (1)17 were evaluated in a competitive binding as-
say against H+/K+-ATPase from hog gastric mucosa.
The results are summarized in Table 1. The unsubstitut-
ed carboxamide 10 showed an approximately 100-fold
reduction in activity (ꢀlogIC50 < 4) compared to the
reference compound 1. On the other hand, the in vitro
activity of compound 10 was clearly improved when
one or two alkyl substituents were introduced on the
amide nitrogen. A substantial increase in potency was
observed when one of the amide hydrogens of 10 was re-
placed by a methyl group (11) or an extended alkyl chain
(13, 14). The increase in activity was even more pro-
nounced when both of the amide hydrogens of 10 were
substituted by methyl groups (7) or when the amide
nitrogen was replaced by a pyrrolidino group (12). The
substitution of the amide moiety by a hydroxymethyl
1
in the next step. H NMR (200 MHz, CDCl3) d (ppm):
1.16–1.32 (m, 5H, CH2CH3, NH2), 2.40 (s, 3H, CH3),
2.73 (q, J = 7.5 Hz, 2H, CH2CH3), 3.88 (s, 2H,
CH2NH), 6.99–7.14 (m, 3H, C6H3).
5.1.2. 5-Bromo-N3-(2-ethyl-6-methylbenzyl)pyrazine-2,3-
diamine (5). A mixture of 2-amino-3,5-dibromopyrazine
(4)12 (1.26 g, 5 mmol), 2-ethyl-6-methylbenzylamine
(1.5 g, 10 mmol), Et3N (1.5 mL), and CH3CN (3.5 mL)
in a sealed tube was irradiated in a microwave-oven
for 40 min at 180 ꢁC. The crude reaction mixtures of
10 such runs were combined, treated with saturated

P. J. Zimmermann et al. / Bioorg. Med. Chem. 16 (2008) 536–541
539
aqueous NaHCO3, and extracted with EtOAc. The or-
ganic phase was dried over anhydrous MgSO4 and evap-
orated. The residue was purified by column
chromatography (light petroleum ether/EtOAc, 4:1).
Crystallization from dioxane yielded 5 (10.2 g, 68%) as
a colorless solid. Mp 155 ꢁC; 1H NMR (200 MHz,
DMSO-d6) d (ppm): 1.15 (t, J = 7.5 Hz, 3H, CH2CH3),
2.33 (s, 3H, CH3), 2.67 (q, J = 7.5 Hz, 2H, CH2CH3),
4.40 (d, J = 4.0 Hz, 2H, CH2NH), 6.24 (br s, 2H,
NH2), 6.44 (br t, 1H, CH2NH), 7.06–7.22 (m, 4H,
C6H3, 6-H); HRMS [M+H]+: 321.0690, C14H18BrN4 re-
quires 321.0709.
N-(2-ethyl-6-methylbenzyl)-2,3-dimethylimidazo[1,2-a]-
pyrazin-8-amine oxalate (6ÆCOOH, 10.0 g, 22 mmol)
was treated with saturated aqueous NaHCO3 and ex-
tracted with EtOAc. The organic phase was separated,
dried over anhydrous MgSO4 and evaporated to give
crude 6 as a colorless oil. After dissolving 6 in EtOH
(80 mL) and Et3N (16 mL), Pd(OAc)2 (0.50 g, 2.2 mmol)
and PPh3 (1.64 g, 6.2 mmol) were added. The mixture
was transferred to an autoclave and carbonylated
(10 bars CO, 100 ꢁC) for 14 h. The reaction mixture
was filtered and evaporated to leave an orange oil which
was dissolved in CH2Cl2, and extracted with water. The
organic phase was dried over anhydrous MgSO4 and
evaporated. Crystallization of the residue from EtOAc/
n-heptane yielded 8 (7.2 g, 89%) as a colorless solid.
5.1.3. 6-Bromo-N-(2-ethyl-6-methylbenzyl)-2,3-dimethyl-
imidazo[1,2-a]pyrazin-8-amine oxalate (6ÆCOOH). To a
suspension of 5 (10 g, 31 mmol) in dioxane (60 mL)
was added 3-bromo-2-butanone (4.90 mL, 46.7 mmol)
and the resulting mixture was heated to 100 ꢁC. After
1
Mp 144 ꢁC; H NMR (200 MHz, DMSO-d6) d (ppm):
1.13 (t, J = 7.5 Hz, 3H, CH2CH3), 1.34 (t, J = 7.1 Hz,
3H, OCH2CH3), 2.28 (s, 3H, CH3), 2.39 (s, 3H, CH3),
2.45 (s, 3H, CH3), 2.83 (q, J = 7.5 Hz, 2H, CH2CH3),
4.32 (q, J = 7.1 Hz, 2H, OCH2CH3), 4.70 (d,
J = 5.4 Hz, 2H, CH2NH), 6.99–7.15 (m, 4H, C6H3,
CH2NH), 8.19 (s, 1H, 5-H); HRMS [M+H]+:
367.2110, C21H27N4O2 requires 367.2129.
2 h,
additional
3-bromo-2-butanone
(4.90 mL,
46.7 mmol) was added and heating was continued for
16 h. The mixture was cooled, diluted with CH2Cl2,
and extracted with saturated aqueous NaHCO3. The or-
ganic phase was dried over anhydrous MgSO4 and evap-
orated. Purification of the residue by column
chromatography (light petroleum ether/EtOAc, 4:1)
provided a colorless oil which was dissolved in acetone
and treated with a solution of oxalic acid dihydrate
(3.91 g, 31 mmol) in acetone. The precipitate was col-
lected and washed with n-heptane to yield 6ÆCOOH
5.1.6. 8-[(2-Ethyl-6-methylbenzyl)amino]-2,3-dimethylim-
idazo[1,2-a]pyrazine-6-carboxylic acid (9). To a solution
of 8 (4.00 g, 10.9 mmol) in dioxane (40 mL) was added
2 N NaOH (8 mL). After 1 h at 80 ꢁC, the reaction mix-
ture was evaporated to half of its volume and the pH
was adjusted to pH 6 with 6 N HCl. The thick precipi-
tate was collected, washed with water, and dried in va-
cuo over P2O5 to yield 9 (3.52 g, 95%) as a colorless
1
(10 g, 70%) as a colorless solid. Mp 163 ꢁC; H NMR
(200 MHz, DMSO-d6) d (ppm): 1.14 (t, J = 7.5 Hz,
3H, CH2CH3), 2.26 (s, 3H, CH3), 2.32 (s, 3H, CH3),
2.41 (s, 3H, CH3), 2.78 (q, J = 7.5 Hz, 2H, CH2CH3),
4.61 (d, J = 4.8 Hz, 2H, CH2NH), 7.00–7.17 (m, 3H,
C6H3), 7.75 (s, 1H, 5-H); HRMS [M+H]+: 373.1013,
C18H22BrN4 requires 373.1022.
1
solid. Mp 230 ꢁC; H NMR (200 MHz, DMSO-d6) d
(ppm): 1.13 (t, J = 7.5 Hz, 3H, CH2CH3), 2.27 (s, 3H,
CH3), 2.40 (s, 6H, 2· CH3), 2.78 (q, J = 7.5 Hz, 2H,
CH2CH3), 4.73 (d, J = 5.1 Hz, 2H, CH2NH), 6.92–7.17
(m, 4H, C6H3, CH2NH), 8.20 (s, 1H, 5-H), 12.50 (br s,
1H, COOH); HRMS [M+H]+: 339.1797, C19H23N4O2
requires 339.1816.
5.1.4. 8-[(2-Ethyl-6-methylbenzyl)amino]-N,N,2,3-tetra-
methylimidazo[1,2-a]pyrazine-6-carboxamide (7). To a
solution of 6 (2.3 g, 5.1 mmol, liberated from 6ÆCOOH
as described in 5.1.5) in dimethylamine (50 mL, 2 M
solution in THF) were added Pd(OAc)2 (0.17 g,
0.76 mmol) and PPh3 (0.80 g, 3.1 mmol). The mixture
was transferred to an autoclave and carbonylated
(6 bars CO, 120 ꢁC) for 16 h. The reaction mixture was
evaporated and the residue was dissolved in CH2Cl2.
The organic phase was extracted with saturated aqueous
NH4Cl, dried over anhydrous MgSO4, and evaporated.
Purification of the residue by column chromatography
(light petroleum ether/EtOAc, 1:1) yielded 7 (1.22 g,
66%) as a colorless solid. Mp 174 ꢁC; 1H NMR
(200 MHz, DMSO-d6) d (ppm): 1.12 (t, J = 7.5 Hz,
3H, CH2CH3), 2.27 (s, 3H, CH3), 2.35 (s, 3H, CH3),
2.37 (s, 3H, CH3), 2.74 (q, J = 7.5 Hz, 2H, CH2CH3),
3.03 [br s, 6H, N(CH3)2], 4.65 (d, J = 5.1 Hz, 2H,
CH2NH), 6.94–7.17 (m, 4H, C6H3, CH2NH), 7.75 (s,
1H, 5-H); HRMS [M+H]+: 366.2271, C21H28N5O
requires 366.2288; Anal. Calcd for C21H27N5O: C,
69.01; H, 7.45; N, 19.16. Found: C, 68.95; H, 7.43; N,
19.11.
5.1.7. 8-[(2-Ethyl-6-methylbenzyl)amino]-2,3-dimethylim-
idazo[1,2-a]pyrazine-6-carboxamide (10). To a suspen-
sion of 9 (1.02 g, 3 mmol) in CH2Cl2 (20 mL) was
added TBTU16 (1.61 g, 5 mmol). After 30 min, NH3
was passed over the mixture. After 1 h, additional
TBTU (1.0 g, 3.1 mmol) was added. Stirring was con-
tinued for 1 h at room temperature and finally 1 h un-
der reflux. The reaction mixture was extracted with 2 N
NaOH and the organic phase was separated, dried over
anhydrous MgSO4, and evaporated. Purification of the
residue by column chromatography (CH2Cl2/MeOH,
20:1) and crystallization from EtOAc/n-heptane yielded
10 (0.67 g, 66%) as a colorless solid. Mp 227 ꢁC; 1H
NMR (200 MHz, DMSO-d6)
d
(ppm): 1.12 (t,
J = 7.5 Hz, 3H, CH2CH3), 2.27 (s, 3H, CH3), 2.35 (s,
3H, CH3), 2.39 (s, 3H, CH3), 2.73 (q, J = 7.5 Hz, 2H,
CH2CH3), 4.78 (d, J = 5.1 Hz, 2H, CH2NH), 6.93 (t,
J = 5.1 Hz, 1H, CH2NH), 7.01–7.18 (m, 3H, C6H3),
7.55 (br s, 1H of NH2), 7.84 (br s, 1H of NH2), 8.06
(s, 1H, 5-H); HRMS [M+H]+: 338.1960, C19H24N5O
requires 338.1975. Anal. Calcd for C19H23N5O: C,
67.63; H, 6.87; N, 20.76. Found: C, 67.62; H, 6.86;
N, 20.83.
5.1.5. Ethyl 8-[(2-ethyl-6-methylbenzyl)amino]-2,3-dime-
thylimidazo[1,2-a]pyrazine-6-carboxylate (8). 6-Bromo-

540
P. J. Zimmermann et al. / Bioorg. Med. Chem. 16 (2008) 536–541
5.1.8. 8-[(2-Ethyl-6-methylbenzyl)amino]-N,2,3-trimeth-
ylimidazo[1,2-a]pyrazine-6-carboxamide (11). To a sus-
pension of 9 (1.02 g, 3 mmol) in CH2Cl2 (20 mL) was
added TBTU (1.61 g, 5 mmol). After 30 min, methyl-
amine (1.5 mL, 12 mmol, 8 M solution in EtOH) was
added and stirring was continued for 16 h. The reaction
mixture was extracted with 2 N NaOH and the organic
phase was separated, dried over anhydrous MgSO4, and
evaporated. Purification of the residue by column chro-
matography (light petroleum ether/EtOAc, 1:1) and
crystallization from EtOAc/n-heptane yielded 11
(0.99 g, 94%) as a colorless solid. Mp 120 ꢁC; 1H
5.1.11. 8-[(2-Ethyl-6-methylbenzyl)amino]-N-(2-methoxy-
ethyl)-2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide
(14). A solution of 8 (1.00 g, 2.73 mmol) in 2-methoxy-
ethylamine (10 mL) was refluxed for 20 h. The reaction
mixture was diluted with water and extracted with
CH2Cl2. The organic phase was dried over anhydrous
MgSO4 and evaporated. Purification of the residue by
column chromatography (light petroleum ether/EtOAc,
1:1) and crystallization from diisopropyl ether yielded
14 (0.53 g, 49%) as a colorless solid. Mp 111 ꢁC; 1H
NMR (200 MHz, DMSO-d6)
d
(ppm): 1.12 (t,
J = 7.5 Hz, 3H, CH2CH3), 2.27 (s, 3H, CH3), 2.37 (s,
3H, CH3), 2.39 (s, 3H, CH3), 2.74 (q, J = 7.5 Hz, 2H,
CH2CH3), 3.29 (s, 3H, OCH3), 3.48 (m, 4H, 2· CH2),
4.80 (d, J = 5.1 Hz, 2H, CH2NH), 7.02–7.18 (m, 4H,
C6H3, CH2NH), 8.05 (s, 1H, 5-H), 8.37 (br t, 1H,
CONHCH2); HRMS [M+H]+: 396.2383, C22H30N5O2
requires 396.2394. Anal. Calcd for C22H29N5O2: C,
66.81; H, 7.39; N, 17.71. Found: C, 66.89; H, 7.39; N,
17.86.
NMR (200 MHz, DMSO-d6)
d
(ppm): 1.12 (t,
J = 7.5 Hz, 3H, CH2CH3), 2.26 (s, 3H, CH3), 2.34 (s,
3H, CH3), 2.39 (s, 3H, CH3), 2.73 (q, J = 7.5 Hz, 2H,
CH2CH3), 2.85 (d, 3H, J = 4.9 Hz, NHCH3), 4.81 (d,
J = 5.1 Hz, 2H, CH2NH), 6.96 (t, J = 5.1 Hz, 1H,
CH2NH), 7.02–7.18 (m, 3H, C6H3), 8.03 (s, 1H, 5-H),
8.40 (q, J = 4.9 Hz, 1H, NHCH3); HRMS [M+H]+:
352.2136, C20H26N5O requires 352.2132. Anal. Calcd
for C20H25N5OÆH2O: C, 65.02; H, 7.37; N, 18.96.
Found: C, 64.99; H, 7.30; N, 19.00.
5.1.12. {8-[(2-Ethyl-6-methylbenzyl)amino]-2,3-dimeth-
ylimidazo[1,2-a]pyrazin-6-yl}methanol (15). To a suspen-
sion of LiAlH4 (0.31 g, 8.2 mmol) in dried THF (10 mL)
was slowly added a solution of 8 (1.0 g, 2.7 mmol) in
THF (20 mL) at 0 ꢁC. After 1 h at 0 ꢁC, the reaction
mixture was successively hydrolyzed with water
(0.2 mL), 6 N NaOH (0.4 mL), and water (1 mL). After
1 h at room temperature, anhydrous MgSO4 was added
and the reaction mixture was filtered through Celite. On
evaporation of the filtrate, a precipitate was obtained
which was collected, washed with Et2O, and dried in va-
cuo to yield 15 (0.77 g, 87%) as a colorless solid. Mp
5.1.9.
N-(2-Ethyl-6-methylbenzyl)-2,3-dimethyl-6-
(pyrrolidin-1-ylcarbonyl)imidazo[1,2-a]pyrazin-8-amine (12).
To a suspension of 9 (0.50 g, 1.5 mmol) in CH2Cl2
(10 mL) was added TBTU (0.70 g, 2.2 mmol). After
30 min, pyrrolidine (0.5 mL, 6 mmol) was added and
the mixture was stirred for 7 h. The reaction mixture
was extracted with 2 N NaOH and the organic phase
was separated, dried over anhydrous MgSO4, and evap-
orated. Purification of the residue by column chroma-
tography (CH2Cl2/MeOH, 20:1) and crystallization
from EtOAc/n-heptane yielded 12 (0.45 g, 78%) as a col-
1
166 ꢁC; H NMR (200 MHz, DMSO-d6) d (ppm): 1.13
1
orless solid. Mp 197 ꢁC; H NMR (200 MHz, DMSO-
(t, J = 7.5 Hz, 3H, CH2CH3), 2.24 (s, 3H, CH3), 2.32
(s, 3H, CH3), 2.38 (s, 3H, CH3), 2.76 (q, J = 7.5 Hz,
2H, CH2CH3), 4.43 (d, J = 5.6 Hz, 2H, CH2OH), 4.63
(d, J = 5.1 Hz, 2H, CH2NH), 5.20 (t, J = 5.6 Hz, 1H,
OH), 6.55 (t, J = 5.1 Hz, 1H, CH2NH), 7.00–7.17 (m,
3H, C6H3), 7.39 (s, 1H, 5-H); HRMS [M+H]+:
325.2014, C19H25N4O requires 325.2023. Anal. Calcd
for C19H24N4O: C, 70.34; H, 7.46; N, 17.27. Found:
C, 70.22; H, 7.45; N, 17.29.
d6) d (ppm): 1.12 (t, J = 7.5 Hz, 3H, CH2CH3), 1.84
(m, 4H, 2· CH2), 2.27 (s, 3H, CH3), 2.36 (s, 6H, 2·
CH3), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 3.50 (m, 2H,
CH2), 3.77 (m, 2H, CH2), 4.67 (d, J = 5.0 Hz, 2H,
CH2NH), 6.89 (t, J = 5.0 Hz, 1H, CH2NH), 7.01–7.18
(m, 3H, C6H3), 7.89 (s, 1H, 5-H); HRMS [M+H]+:
392.2431, C23H30N5O requires 392.2445. Anal. Calcd
for C23H29N5O: C, 70.56; H, 7.47; N, 17.89. Found:
C, 70.60; H, 7.47; N, 17.61.
5.1.13. N-(2-Ethyl-6-methylbenzyl)-6-(methoxymethyl)-
2,3-dimethylimidazo[1,2-a]pyrazin-8-amine (16ÆHCl). To
a suspension of 15 (0.50 g, 1.5 mmol) in dried DMF
(5 mL) was added NaH (0.18 g, 4.5 mmol, 60% disper-
sion in mineral oil) in portions at room temperature.
After 30 min, methyl iodide (0.12 mL, 2 mmol) was
slowly added. After 30 min, the reaction mixture was
carefully hydrolyzed with saturated aqueous NaHCO3
and extracted with CH2Cl2. The organic phase was dried
over anhydrous MgSO4 and evaporated. Purification of
the residue by column chromatography (light petroleum
ether/EtOAc, 4:1) yielded 0.18 g of a colorless oil which
was dissolved in CH2Cl2 and treated with HCl (1.5 M
solution in Et2O). Evaporation of all volatiles yielded
16ÆHCl (0.12 g, 21%) as a colorless solid. Mp 177 ꢁC;
1H NMR (200 MHz, DMSO-d6) d (ppm): 1.13 (t,
J = 7.5 Hz, 3H, CH2CH3), 2.24 (s, 3H, CH3), 2.32 (s,
3H, CH3), 2.39 (s, 3H, CH3), 2.77 (q, J = 7.5 Hz, 2H,
CH2CH3), 3.37 (s, 3H, OCH3), 4.33 (s, 2H, CH2OCH3),
5.1.10. 8-[(2-Ethyl-6-methylbenzyl)amino]-N-(2-hydroxy-
ethyl)-2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide
(13). A suspension of 8 (1.1 g, 3 mmol) in 2-aminoethanol
(10 mL) was heated at 80 ꢁC for 30 min. Then the reaction
mixture was diluted with 2-aminoethanol (10 mL) and the
temperature was raised to 100 ꢁC. After 1 h, the reaction
mixture was cooled and the precipitate was collected
and washed with water. The colorless solid was dried in
vacuo over P2O5 to yield 13 (1.04 g, 91%). Mp 229 ꢁC;
1H NMR (200 MHz, DMSO-d6) d (ppm): 1.13 (t,
J = 7.5 Hz, 3H, CH2CH3), 2.27 (s, 3H, CH3), 2.36 (s,
3H, CH3), 2.39 (s, 3H, CH3), 2.74 (q, J = 7.5 Hz, 2H,
CH2CH3), 3.36–3.59 (m, 4H, 2· CH2), 4.80 (m, 3H,
CH2NH, OH), 7.02–7.18 (m, 4H, C6H3, CH2NH), 8.05
(s, 1H, 5-H), 8.39 (t, J = 5.7 Hz, 1H, CONHCH2); HRMS
[M+H]+: 382.2220, C21H28N5O2 requires 382.2238. Anal.
Calcd for C21H27N5O2: C, 66.12; H, 7.13; N, 18.36.
Found: C, 66.13; H, 7.16; N, 18.29.

P. J. Zimmermann et al. / Bioorg. Med. Chem. 16 (2008) 536–541
541
R.; Sewell, J. Aliment. Pharmacol. Ther. 2005, 22, 79–94;
(c) Vakil, N. Aliment. Pharmacol. Ther. 2004, 19, 1041–
1049.
4.64 (d, J = 5.2 Hz, 2H, CH2NH), 6.65 (t, J = 5.2 Hz,
1H, CH2NH), 7.00–7.16 (m, 3H, C6H3), 7.45 (s, 1H, 5-
H); HRMS [M+H]+: 339.2158, C20H27N4O requires
339.2179. Anal. Calcd for C20H26N4OÆHCl: C, 64.07;
H, 7.26; N, 14.94; Cl, 9.46. Found: C, 63.85; H, 7.34;
N, 14.67; Cl, 9.40.
2. Andersson, K.; Carlsson, E. Pharmacol. Ther. 2005, 108,
294–307.
3. Kaminski, J. J.; Bristol, J. A.; Puchalski, C.; Lovey, R. G.;
Elliott, A. J.; Guzik, H.; Solomon, D. M.; Conn, D. J.;
Domalski, M. S.; Wong, S.-C.; Gold, E. H.; Long, J. F.;
Chiu, P. J. S.; Steinberg, M.; McPhail, A. T. J. Med.
Chem. 1985, 28, 876–892.
5.2. H+/K+-ATPase inhibition assay
The malachite green assay modified from Yoda and
Hokin18 was used for the determination of H+/K+-
ATPase IC50.19 Given data are the mean IC50 values
from two to three independent determinations. Pipes
[piperazine-1,4-bis(2-ethanesulfonic acid], sucrose, nige-
ricin, Na-ATP, and malachite green were purchased
from Sigma–Aldrich, Tris [tris(hydroxymethyl)amino-
methane], KCl, and ammoniumheptamolybdate tetra-
hydrate from Merck, and MgCl2 from Fluka. Final
assay concentrations: 4 mM Pipes/8 mM Tris buffer,
pH 7.4, 0.25 M sucrose, 1 mM KCl, 1 mM MgCl2,
0.5–1 lg/100 lL nigericin (1:1 ratio with enzyme),
0.5–1 lg/100 lL enzyme (dependent on K+-stimulated,
specific activity), and 1 mM Na-ATP (high grade),
reaction volume: 101 lL. Preparation of malachite
green reagent: two parts malachite green stock solution
(1.2 M in H2O, protected from light and used within
12 weeks) were mixed with one part ammoniumhepta-
molybdate tetrahydrate stock solution (42 g/L in 4 N
HCl) and kept for 30 min at room temperature prior
to use. Procedure: A Pipes/Tris buffer-based solution
with sucrose and MgCl2 was prepared. Nigericin and
enzyme were added to reach abovementioned final con-
centrations. Eighty microliters per well of this mixture
was placed into 96-well flat-bottommed plates (clear,
polystyrol, Greiner bio-one). Ten microliters per well
KCl (1 mM final) was used for stimulation of H+/K+-
ATPase activity. Test substances were dissolved as
10 mM solutions in 100% DMSO. One microliter of
substance solution was added in dilutions ranging from
10ꢀ4–10ꢀ9 M (final). The enzymatic reaction was
started by addition of 10 lL ATP (1 mM final). The as-
say was incubated for 30 min at room temperature.
The reaction was stopped by addition of 150 lL mala-
chite green reagent and incubated for another 15 min
prior to photometric reading of the plate at 680 nm
in a PowerWave HT Microplate spectral photometer
(BioTek). The results were analyzed with GraphPad
Prism software (Version 4.02) to calculate IC50 values
by sigmoidal curve fitting. ‘Enzyme’ refers to H+/K+-
ATPase-containing vesicles prepared from hog gastric
mucosa.20
4. Wurst, W.; Hartmann, M. Yale J. Biol. Med. 1996, 69,
233–243.
¨
5. (a) Abelo¨, A.; Andersson, M.; Holmberg, A. A.; Karlsson,
M. Eur. J. Pharm. Sci. 2006, 29, 91–101; (b) Gedda, K.;
Briving, C.; Svensson, K.; Maxvall, I.; Andersson, K.
Biochem. Pharmacol. 2007, 73, 198–205.
6. Zimmermann, P. J.; Buhr, W.; Brehm, C.; Palmer, A. M.;
Feth, M. P.; Senn-Bilfinger, J.; Simon, W.-A. Bioorg. Med.
Chem. Lett. 2007, 17, 5374–5378.
7. (a) Senn-Bilfinger, J.; Ferguson, J. R.; Holmes, M. A.;
Lumbard, K. W.; Huber, R.; Zech, K.; Hummel, R.-P.;
Zimmermann, P. J. Tetrahedron Lett. 2006, 47, 3321–3323;
(b) Simon, W. A.; Herrmann, M.; Klein, T.; Shin, J. M.;
Huber, R.; Senn-Bilfinger, J.; Postius, S. J. Pharmacol.
Exp. Ther. 2007, 321, 866–874.
8. (a) Kaminski, J. J.; Hilbert, J. M.; Pramanik, B. M.;
Solomon, D. M.; Conn, D. J.; Rizvi, R. K.; Elliott, A. J.;
Guzik, H.; Lovey, R. G.; Domalski, M. S.; Wong, S.-C.;
Puchalski, C.; Gold, E. H.; Long, J. F.; Chiu, P. J. S.;
McPhail, A. T. J. Med. Chem. 1987, 30, 2031–2046; (b)
Kaminski, J. J.; Perkins, D. G.; Frantz, J. D.; Solomon, D.
M.; Elliott, A. J.; Chiu, P. J. S.; Long, J. F. J. Med. Chem.
1987, 30, 2047–2051; (c) Kaminski, J. J.; Doweyko, A. M.
J. Med. Chem. 1997, 40, 427–436.
9. Amin, K.; Dahlstro¨m, M.; Nordberg, P.; Starke, I.
WO1999/028322.
10. Zimmermann, P. J.; Buhr, W.; Chiesa, M. V.; Palmer,
A.; Brehm, C.; Grundler, G.; Senn-Bilfinger, J.; Simon,
W.-A.; Postius, S.; Kromer, W. WO2004/074289.
11. For a review see: Basiuk, V. A. Russ. Chem. Rev. 1997, 66,
187–204.
12. Jiang, B.; Yang, C.-G.; Xiong, W.-N.; Wang, J. Bioorg.
Med. Chem. 2001, 9, 1149–1154.
´ ´
13. Vitse, O.; Laurent, F.; Pocock, T. M.; Benezech, V.;
Zanik, L.; Elliott, K. R. F.; Subra, G.; Portet, K.;
Bompart, J.; Chapat, J.-P.; Small, R. C.; Michel, A.;
Bonnet, P.-A. Bioorg. Med. Chem. 1999, 7, 1059–
1065.
14. For related reactions of 2-amino-3,5-dibromopyrazine (4),
see e.g., (a) Barlin, G. B.; Ireland, S. J. Aust. J. Chem.
1984, 37, 1057–1064; (b) Bonnet, P. A.; Michel, A.;
Laurent, F.; Sablayrolles, C.; Rechencq, E.; Mani, J. C.;
Boucard, M.; Chapat, J. P. J. Med. Chem. 1992, 35,
3353–3358.
15. (a) Horino, H.; Sakaba, H.; Arai, M. Synthesis 1989, 715–
718; (b) Head, R. A.; Ibbotson, A. Tetrahedron Lett. 1984,
25, 5939–5942.
16. TBTU = O-(1H-benzotriazol-1-yl)-N,N,N0,N0-tetrameth-
yluronium tetrafluoroborate; see e.g., Montalbetti, C. A.
G. N.; Falque, V. Tetrahedron 2005, 61, 10827–10852.
17. 3-Amino-2-methyl-8-benzyloxy-imidazo[1,2-a]pyrazine
hydrochloride (1ÆHCl) was prepared as described in
Ref. 8a.
Acknowledgments
We are grateful to Ms. S. Chrismann, Ms. I. Hartig-
Beecken and Mr. K. Ha¨gele for technical assistance.
18. Yoda, A.; Hokin, L. E. Biochem. Biophys. Res. Commun.
1970, 40, 880–886.
References and notes
19. Lanzetta, P. A.; Alvarez, L. J.; Reinach, P. S.; Candia, O.
A. Anal. Biochem. 1979, 100, 95–97.
20. Rabon, E. C.; Im, W. B.; Sachs, G. Methods Enzymol.
1988, 157, 649–654.
1. (a) Parsons, M. E.; Keeling, D. J. Expert Opin. Invest.
Drugs 2005, 14, 411–421; (b) Fass, R.; Shapiro, M.; Dekel,