Synthesis and biological evaluation of aroylguanidines related to amiloride as inhibitors of the human platelet Na+/H+ exchanger

Article abstract of DOI:10.1016/S0968-0896(02)00022-6

Pyridine and benzene bioisosteres of amiloride were synthesized and evaluated for their inhibitory potency against the sodium-hydrogen exchanger (NHE) involved in intracellular pH regulation. The inhibition of NHE was determined by using the platelet swelling assay (PSA) in which the swelling of human platelets was induced by their incubation in an acid buffer (pH 6.7). Additionally, the inhibitory potency of the most active compounds was assessed by measuring the inhibition of the EIPA-sensitive ²²Na⁺ uptake (UIA) by human platelets after intracellular acidosis. The results indicated that several benzene derivatives and compounds bearing an carbonylguanidine moiety in the meta position of the pyridine nitrogen were much more potent than amiloride (PSA:IC₅₀=43.5 μM; UIA:IC₅₀=100.1 μM), but less than EIPA, a pyrazine NHE inhibitor (PSA:IC₅₀=0.08 μM; UIA:IC₅₀=0.5 μM). In both biological assays (2-amino-5-bromo-pyridine-3-carbonyl)guanidine (32) was the most active molecule (PSA:IC₅₀=0.8 μM, UIA:IC₅₀=0.8 μM). Our investigations demonstrated that the replacement of the pyrazine ring of amiloride by a pyridine or a phenyl ring improved the NHE inhibitory potency (phenyl >pyridine >pyrazine).

Full text of DOI:10.1016/S0968-0896(02)00022-6

Bioorganic & Medicinal Chemistry10 (2002) 1793–1804
Synthesis and Biological Evaluation of Aroylguanidines
Related to Amiloride as Inhibitors of the Human Platelet
Na+/H+ Exchanger
Didier Laeckmann,a,* Francoise Rogister,b Jean-Victor Dejardin,a
Christelle Prosperi-Meys,c Joseph Geczy,b Jacques Delargea and Bernard Masereeld
a
´ `
Department of Medicinal Chemistry, Natural and Synthetic Drugs Research Center, Universite de Liege, CHU,
`
1avenue de l’Hoˆpital, B 36, tour 4, B-4000 Liege, Belgium
bTherabel Research S.A., 108 rue Egide Van Ophem, B-1180 Bruxelles, Belgium
c
´
´
`
`
Service de Physique Experimentale, Institut de Physique, B 5, Universite de Liege, B-4000 Liege, Belgium
dDepartment of Pharmacy, University of Namur, FUNDP, 61rue de Bruxelles, B-5000 Namur, Belgium
Received 16 October 2001; accepted 9 January2002
Abstract—Pyridine and benzene bioisosteres of amiloride were synthesized and evaluated for their inhibitory potency against the
sodium–hydrogen exchanger (NHE) involved in intracellular pH regulation. The inhibition of NHE was determined by using the
platelet swelling assay(PSA) in which the swelling of human platelets was induced bytheir incubation in an acid buffer (pH 6.7).
Additionally, the inhibitory potency of the most active compounds was assessed by measuring the inhibition of the EIPA-sensitive
22Na+ uptake (UIA) byhuman platelets after intracellular acidosis. The results indicated that several benzene derivatives and
compounds bearing an carbonylguanidine moiety in the meta position of the pyridine nitrogen were much more potent than
amiloride (PSA:IC50=43.5 mM; UIA:IC50=100.1 mM), but less than EIPA, a pyrazine NHE inhibitor (PSA:IC50=0.08 mM;
UIA:IC50=0.5 mM). In both biological assays (2-amino-5-bromo-pyridine-3-carbonyl)guanidine (32) was the most active molecule
(PSA: IC50=0.8 mM, UIA : IC50=0.8 mM). Our investigations demonstrated that the replacement of the pyrazine ring of amiloride
by a pyridine or a phenyl ring improved the NHE inhibitory potency (phenyl >pyridine >pyrazine). # 2002 Elsevier Science Ltd.
All rights reserved.
using the transmembrane Na+ gradient established by
Introduction
Na+/K+ATPase.8 The structure and the functional
implications of NHE-1 and other isoforms have been
In manycell types, including platelets, several mechan-
isms take part in the maintenance of intracellular acid-
base homeostasis.1,2 One of the predominant mechan-
ism bywhich cells adapt to intracellular acidosis is
through the Na+/H+ antiport system (NHE). To date,
six mammalian isoforms of the Na+/H+ exchanger
have been identified and characterized (NHE-1–NHE-
6).3ꢀ6 The most widelydistributed subtpye is the
amiloride-sensitive Na+/H+ antiporter (NHE-1) which
is involved in intracellular pH (pHi) homeostasis, cell
volume control, and in cell signaling.7 Isoform NHE-1,
the main Na+/H+ exchanger in platelets, mediates the
electroneutral exchange of intracellular H+ for extra-
cellular Na+ with a 1:1 stoichiometric relationship
9,10
recentlyreviewed.
The activityof the Na +/H+ anti-
porter, especiallyNHE-1, can be modulated bya num-
ber of stimuli such as growth factors, hormones and
neurotransmitters, as well as bychanges in cell volume
and mechanical stimuli.11 Although the activation of
NHE is essential for the restoration of physiological
pH, an excessive stimulation of NHE results in an
increase of intracellular Na+ concentration and a sub-
sequent activation of Na+/K+ATPase, with a con-
secutive increase of energyconsumption. Since cellular
Na+ and Ca2+ transport is coupled via the Na+/Ca2+
exchanger, which depends on the Na+ gradient, the
high intracellular Na+ levels lead to raised intracellular
Ca2+ 8,12,13 This cellular Ca2+ overload is assumed to
.
be involved in ischemic and reperfusion injuries, like
arrhythmias, myocardial contracture, stunning and tis-
sue necrosis.14,15 In addition, the abnomalities in the
*Corresponding author. Tel.: +32-4-3664381; fax: +32-4-3664362;
e-mail: d.laeckmann@ulg.ac.be
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00022-6

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D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
activityof the NHE are implicated in the pathogenesis
and/or pathophysiological processes such as essential
hypertension,16ꢀ18 diabetes mellitus,19,20 postischemic
dysfunction, and cellular death. Moreover, raised NHE
activityin kidneyand vascular smooth muscle could
Since the nature of groups R and R0 (Fig. 1) has been
extensivelystudied in the amiloride famil,y we have
examined the influence of bioisosteric replacement of
the pyrazine ring by a pyridine or a phenyl, and the
effect of other substitutions on the aromatic ring (Fig.
2).
20ꢀ22
playa critical role in the etiologyof hypertension,
vascular smooth muscle cell proliferation23 and renal
disorders.24 Recently, the inhibition of the NHE has
been considered as an useful approach to limit Ca2+
influx and avoid its deleterious consequences during
cardiac ischemia and reperfusion.25
Chemistry
In a previous work,35 we reported the preparation of
derivatives of amiloride where their pyrazine ring was
substituted bya phenyl. Here, pyridine compounds ( 12,
14, 28, 30–33) structurallyrelated to amiloride were
prepared (Table 1). The strictlypyridine bioisosteres of
amiloride (12, 28) differ bythe position of the pyridinic
nitrogen. The preparation of 12 started from 2-amino-6-
methylpyridine (Scheme 1). The nitration of this pro-
duct led to 6-methyl-2-nitraminopyridine 1.36,37 The
one-pot nitration of 1 with an excess of nitric acid (3
equivalents) gave 2-amino-6-methyl-3,5-dinitropyridine
2.38 The synthesis of 2-hydroxy-6-methyl-3,5-di-
Consequently, to attenuate the harmful consequences of
an excessive NHE activation, several inhibitors were
developed (Fig. 1). Beside its inhibitorypotencyagainst
the Na+/Ca2+ exchanger and Na+ channels, amiloride
is a NHE inhibitor. Pharmacomodulation of amiloride
generated more potent and more specific NHE inhibi-
tors: the 5-N-substituted pyrazine derivatives closely
related to the structure of amiloride (Fig. 1). Later,
benzoylguanidines were developed (e.g., cariporide,
HOE 694, EMD-96785 and FR-183998). These drugs
are highlypotent and markedlyselective inhibitors of
NHE-1 without acting on other ion carrier or pH
nitropyridine 3 bydiazotation of 2 was carried out
39
under the conditions used byBerrie et al.
This step
26,27
regulatorysystems.
was required bythe presence of a high chloride con-
centration which could not directlygenerate 2-chloro-6-
methyl-3,5-dinitropyridine 4.40 This latter was obtained
bychlorination of 3 with a mixture of PCl5/OPCl3
according to Czuba method.41 After reduction of both
nitro groups,42 the 3,5-diamino-2-chloro-6-methylpyr-
idine 5 was acetylated (6), and then oxidized byKMnO 4
Pharmacological development of NHE inhibitors for
cardiac therapyfocused on selective NHE-1 inhibitors.
These agents, as well the earlier nonspecific amiloride
derivatives, have shown excellent cardioprotective
properties. Thus, the NHE inhibitors were expected to
be efficient tools for preventing high intracellular Na+
levels and concomitant Ca2+ overload.28ꢀ30
to
acid 7.
3,5-diacetylamino-6-chloro-pyridine-2-carboxylic
The success of NHE inhibitors in experimental studies
has led to clinical trials in high risk patients with cor-
onaryarterydisease as well in patients with acute myo-
cardial infarction. These NHE inhibitors attenuated
both acute and chronic responses to myocardial post-
infarction resulting in the evolution of heart failure.31ꢀ34
These data suggest a potentiallyeffective new ther-
apeutic perspective for the treatment of heart failure
with NHE-1 inhibitors.
In a first procedure, this intermediate gave the methyl
ester 8 or 9, which could not lead to the desired com-
pound 12. In a second approach, 7 was hydrolyzed in
alkaline conditions, providing the 3,5-diamino-6-chloro-
pyridine-2-carboxylic acid 10 with high yields (70–
75%). The carboxylic derivative 10 was then esterified
bymeans of N-tert-butyl-5-methylisoxazolium perchlo-
rate (NBI)43 to give the crotonamide intermediate 11
which finallyreacted with guanidine to give 12. A third
Figure 1. Inhibitors of the Na+/H+ exchanger.

D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
1795
tion of amino groups have also been investigated but
did not succeed to reach final compound 12.
The synthesis of the other pyridine bioisostere 28 started
from 16 obtained from 2-methylglutaronitrile following
Fritz procedure (Scheme 2).45
Although, the preparation of intermediates 17 and 18
was previouslydescribed, 46 a mixture of both molecules
was always generated, and required fractional crystal-
lization. The 2,5,6-trichloropyridine-3-carboxylic acid
21 was produced bythree different ways: the oxidation
of 17 byKMnO 4, the catalytic oxidation of a mixture of
bromide derivatives (19 and 20), and the oxydative
process of 23 which was obtained from 2-methyleneglu-
taronitrile according to Mutterer procedure.47 In the
last two cases, the yields were of 50 and 80%, respec-
tively. Indeed, the conversion of a polyhalogenated
pyridine to its carboxylic acid derivative by KMnO4 is
Figure 2. Pyridine and benzene derivatives of amiloride.
pathwayinvolved the cyclization of 7 into N-(6-chloro-2
-methyl-4-oxo-4H-pyrido[3,2-d]-1,3-oxazin-7-yl)-aceta-
mide 13, which reacted with guanidine to generate the
diacetylated and acetylated carbonylguanidine com-
pounds 14 and 15, respectively. This latter underwent
acidic hydrolysis to give the pyridine bioisostere 12.
However, this hydrolysis occurred with poor yields (25–
30%). Alternative pathways44 such as the synthesis of
N-hydroxysuccinimide esters or trifluoroacetic protec-
Scheme 1. Reagents: (i) H2SO4/HNO3,ꢀ20 ꢁC; (ii) H2SO4; (iii) NaNO2/H2SO4; (iv) PCl5/OPCl3; (v) SnCl2/HCl; (vi) Ac2O; (vii) KMnO4; (viii)
CH2N2; (ix) CH3OH/H2SO4; (x) NaOH; (xi) Et3N/DMF/NBI; (xii) guanidine.HCl/tert-ButOK; (xiii) Ac2O/EtOAc; (xiv) guanidine:HCl/Na; (xv)
guanidine.HCl/Na/HCl; (xvi) HCl/H2O/EtOH.

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D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
known to give low yields.48,49 As observed,47 an attempt
to prepare the acid 27 byreaction of 21 with NH4OH
led to decarboxylation (22). The carboxamide 25 was
generated from the acyl chloride 24, and the substitu-
tion of both chlorine atoms gave the 2,6-diamino-5-
chloro-pyridine-3-carboxamide 26 with satisfactory
yields (65%). Alkaline hydrolysis led to the key inter-
mediate 27. This latter was then converted byreaction
with N,N’-carbonyldiimidazole (CDI) into its corre-
sponding ester, which reacted with guanidine to provide
the pyridine bioisostere 28.
Finally, some ‘simplified’ pyridine (33) and benzene (40,
42) derivatives were prepared according to known
procedures.52,53
Results and Discussion
To measure the effect of the synthesized compounds on
the NHE activity, the human platelet swelling assay
(PSA) was used.54,55 Addition of platelet-rich plasma
(PRP) to an acid propionate buffer led to the activation
of the NHE which uptakes extracellular Na+ and
extrudes cytoplasmic H+. To restore iso-osmotic con-
ditions, the cytosolic accumulation of Na+ is accom-
panied bya cell swelling due to the influx of water. This
Some compounds, related to 28, where the amino
groups are substituted bychlorine were snythesized
(Scheme 3).
platelet swelling induced a decrease of the optical den-
16,18
Hence, the 2,6-dichloropyridine-3-carboxylic acid 29
was prepared by ortho-lithiation according to the pro-
cedure reported byRadinov. The carboxylic acids 21
sity(OD) of the platelet suspension.
This OD
decrease is related to the value of extracellular pH.
Indeed, at pH 6.7, EIPA (10 mM), a specific NHE inhi-
bitor, completelyabolished the decrease of OD and
prevented the platelet swelling (Fig. 3). Other molecules
acting on Na+ carriers such as ouabain (inhibitor of
Na+/K+ATPase pump) or furosemide (inhibitor of
the symport Na+K+2Clꢀ) were investigated, and failed
to modifythe OD decrease, and thus the platelet
swelling.
50
and 29 were transformed bycondensation with CDI
into their corresponding esters, which then reacted with
guanidine to provide the desired molecules 31 and 30,
respectively. An attempt to obtain 31 via the methyl
ester was unsuccessful. Compound 32 was obtained by
bromination of (2-amino-pyridine-3-carbonyl)guanidine
51
as previouslydescribed byCragoe.
Table 1. Chemical structures and inhibitorypotencyof the platelet Na +/H+ exchanger
Compound
X
Y
N
R1
R2
R3
Cl
R
H
IC50 (mM)ÆSEM
PSAa
22Na+ uptakeb
12
CH
NH2
NH2
24.0Æ2.0
14
28
32
31
30
33
CH
N
N
N
N
N
NHCOCH3
NHCOCH3
Cl
Cl
Br
Cl
H
H
H
H
H
H
H
9.9Æ0.9
1.2Æ0.5
0.85Æ0.05
2.8Æ0.4
NA
CH
CH
CH
CH
CH
NH2
NH2
Cl
Cl
H
NH2
H
Cl
Cl
H
1.3Æ0.4
0.8Æ0.1
5.3Æ0.6
N
H
NA
34d
35d
36d
37d
38d
39d
40
41
42
43
44
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
NH2
NH2
NH2
NH2
NO2
Cl
H
Cl
H
NH2
NH2
H
H
NO2
H
Cl
H
H
NO2
NO2
H
Cl
Cl
Cl
H
Cl
NO2
Cl
NH2
H
Cl
Cl
H
H
C2H5
H
H
H
H
H
H
H
0.8Æ0.08
NA
16.0Æ3.8
1.8Æ0.4
0.5Æ0.04
NA
1.1Æ0.3
0.7Æ0.4
1.3Æ0.5
NA
10.6Æ0.1
5.6Æ2.1
2.7Æ0.5
NA
NHCOCH3
NHCOCH3
NHCOCH3
H
C2H5
H
0.11Æ0.1
24.4Æ0.2
66.1Æ4.0
3.6Æ2.2
45
Amiloride (1)
EIPA
DMA
HMA
Cariporide
EMD-96785
N
N
N
N
CH
CH
N
N
N
N
CH
CH
NH2
NH2
NH2
NH2
H
NH2
(C2H5)NCH(CH3)2
N(CH3)2
Cl
Cl
Cl
Cl
H
H
H
H
H
H
43.5Æ4.5
0.08Æ0.03
0.8Æ0.04
0.2Æ0.01
0.12c
100.1Æ32
0.5Æ0.1
N(CH2)6
(CH3)2CH
pyrrole
3.3Æ0.4
SO2CH3
SO2CH3
CH3
0.03c
aDrug concentration to achieve half-maximal inhibition of acid-induced swelling of human platelets.
bDrug concentration to achieve half-maximal inhibition of the EIPA-sensitive 22Na+ uptake byhuman platelets. NA=no activityat 10 mM.
cData from ref. 56.
dSynthesis of compounds 34–39, 41, 43–45 previouslydescribed in ref 35.

D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
1797
Scheme 2. Reagents: (i) H2SO4/CH3COOH; (ii) PCl5; (iii) KMnO4; (iv) NBS/CCl4/UV; (v) H2SO4/HNO3/HgNO3/CuSO4.5H2O; (vi) NH4OH; (vii)
SOCl2/DMF; (viii) NH4OH; (ix) NH4OH/NH3(g); (x) NaOH; (xi) guanidine.HCl/tert-ButOK/CDI.
The inhibitorypotencyof the novel compounds was
evaluated in the PSA and compared to amiloride and to
other potent and specific NHE inhibitors such as EIPA,
HMA and DMA (Fig. 1). Concentration–response
curves (Fig. 4) were drawn to determine the concentra-
tion required to prevent of 50% the platelet swelling
(IC50) induced bya propionate buffer (pH 6.7) (Table 1).
amino moeities bya chlorine atom ( 31) preserved the
activityif a halide was present in R3 (compare 30 and
31). The unsubstituted (pyridyl-3-carbonyl)guanidine 33
was inactive.
The benzene counterpart of amiloride (34) was 54 times
more potent (IC50=0.8 mM) than its parent. It was as
active as the pyridine bioisostere 28 and DMA
(IC50=0.8 mM), a pyrazine NHE inhibitor. Compared
to 34, the compound with an ethyl group on the distal
nitrogen of the guanidine (35) showed a complete loss of
inhibitoryactivit.y In the benzene series ( 34–45), the
removal of the R2 amino group (36) also maintained the
activity(IC 50=0.5 mM) if a chlorine atom was present
in R3 (e.g., 36 and 37). The deletion of both amino
moieties and their replacement bytwo nitro ( 38) or one
chlorine (39, 40) preserved the potency(0.7 <IC50 <1.3
The first pyridine bioisostere of amiloride bearing the
carbonylguanidine side chain in ortho of the pyridine
nitrogen (12, IC50=24.0 mM) and its N-diacetyl deriva-
tive (14, IC50=9.9 mM) were more active than amiloride
which was confirmed to be a poor inhibitor of NHE (1,
IC50=43.5 mM). The second pyridine bioisostere bearing
the carbonylguanidine moeity in the meta position (28)
was much more efficient (IC50=1.2 mM). The deletion
of the R2 amino group (32) or the replacement of both
Scheme 3. Reagents: (i) n-BuLi/diisopropylamine/CO2; (ii) guanidine.HCl/tert-butOK/CDI.

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D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
Figure 3. Effect of extracellular pH (left panel) and EIPA (right panel) on the platelet swelling expressed as the decrease of the relative optical
density(OD at 680 nm) of a platelet suspension.
mM). Once more, the replacement of the nitro group
(39) or of the chlorine (40) in R3 byan amino ( 41) led to
a marked loss of activity. The unsubstituted benzoyl-
guanidine (42) was inactive.
late the concentration required to reduce of 50% (IC50)
the so-called EIPA-sensitive 22Na+ uptake (Fig. 5).
The meta-carbonylguanidine pyridine derivatives (28,
31, 32) were much more potent (0.8<IC50<5.3 mM)
than amiloride (IC50=100.1 mM). As observed in the
platelet swelling assay, 32 remained the most active
molecule in this series. The benzene derivatives (34, 36,
38, 39, 40, 43), active in the swelling assay, reduced
dose-dependentlythe EIPA-sensitive 22Na+ uptake. As
observed for EIPA and HMA, their IC50 value was 2–30
times higher than in the PSA. In contrast to the results
obtained in the PSA, the benzene bioisostere of amilo-
ride (34) was less active than the pyridine bioisostere 28.
With respect to these considerations, and as observed
for the pyridine derivatives, a chloro or a nitro group
was required in R3 to inhibit the NHE (c.f., 36, 37 and
41). As shown in the pyridine series, an acetylamino in
R1 (43–45) enhanced the potency(c.f., 14 and 43). The
substitution on the distal nitrogen of the guanidine with
an ethyl strongly reduced the biological response (c.f.,
43 and 44). These results indicated that the nature of the
aromatic ring and the position of the heterocyclic
nitrogen stronglyinfluenced the NHE inhibitory
potencyfollowing the sequence phenyl ( 34)>pyridine
(28>12)>pyrazine (amiloride).
Conclusion
For the reference drugs, and for the most active com-
pounds (IC50 <3 mM), the inhibitorypotencyof NHE
inhibitors was also evaluated bytheir abilityto reduce
the EIPA-sensitive 22Na+ uptake byhuman platelets
suspended at pH 6.7. After 5 and 10 min of incubation
with EIPA (10 mM), the platelet 22Na+ uptake was
reduced by31 and 33%, respectively. The EIPA con-
centration–response curve was drawn and led to calcu-
In conclusion, this studydemonstrated that the repla-
cement of the pyrazine ring of amiloride by a pyridine
or a phenyl ring improved the NHE inhibitory potency.
Figure 5. Human platelet sodium uptake inhibited byEIPA ( *) or 32
(*). Each point is the mean and SEM of the response in at least three
different experiments.
Figure 4. EIPA (*) or 34 (*) reduced the NHE activityexpressed as
the human platelet swelling induced byan acid buffer (pH 6.7).

D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
1799
To preserve the biological activity, a nitro or a halide
group was required in the R3 position. For the pyridine
derivatives, the nitrogen of the heterocycle should be in
the meta position of the carbonylguanidine side–chain.
Taking into account these results, and as compared to
the structure of EIPA and other 5-N-substituted
amiloride derivatives, the synthesis of pyrid-3-yl carbonyl-
guanidine compounds bearing a N-substituted nitrogen
in R2 should improve the inhibitorypotencyof NHE.
Finally, further functional experiments are warranted to
confirm their therapeutic interest.
solid brown residue. The crude product 5 was crystal-
lized from toluene. The precipitate was filtered, washed
with petroleum ether 40–60 ꢁC, and dried (3.3 g, 60%):
mp 170–172 ꢁC; IR 3460, 3375, 3301, 1618, 1477, 1449,
1
1427, 1245, 1212, 871, 741 cmꢀ1; H NMR (80 MHz,
CDCl3+DMSO-d6, HMDS), d 2.26 (s, 3H, CH3), 3.49
(s, 2H, 3-NH2), 3.74 (s, 2H, 5-NH2), 6.36 (s, 1H, 4H-
pyridine). Anal. calcd for C6H8N3Cl: C 45.73, H 5.12, N
26.66; found: C 46.05, H 5.23, N 26.57.
3,5-Diacetylamino-2-chloro-6-methylpyridine (6).
A
solution of 3,5-diamino-2-chloro-6-methylpyridine 5
(7.5 g, 48 mmol) in acetic anhydride (100 mL, 1.06 mol)
was stirred for 30 min at room temperature until for-
mation of a white precipitate. The medium was then
poured on ice (100 g) and stirred for 30 min. After-
wards, it was neutralized with 30% NaOH to pH 5, and
then to pH 7 with saturated NaHCO3. After cooling
overnight, the crude compound 6 was filtered, washed
with water, and dried. Crystallization from toluene gave
the title compound (10.3 g, 90%): mp 238–240 ꢁC;
IR 3306, 3270, 3016, 1667, 1536, 1499, 1432, 1373, 1241,
Experimental
Drugs and reagents
Stock solutions (0.5 mM) of DMA, EIPA, HMA (all
from Sigma, Belgium) or amiloride derivatives were
prepared in DMSO. The final concentration of DMSO
was minimized (maximum:2%) so that it did not affect
the measurements. 22NaCl (37 Mbq) was purchased
from NEN (Brussels, Belgium) and ouabain from Sigma
(Brussels, Belgium). Furosemide was a generous gift
from Pharmachim (Jemeppe, Belgium).
681 cmꢀ1
;
1H NMR (80 MHz, CDCl3+DMSO-d6,
HMDS), d 2.07 (s, 6H, NH–CO–CH3), 2.33 (s, 3H,
CH3), 8.26 (s, 1H, 4H-pyridine), 9.48 (s, 2H, NH–CO–
CH3). Anal. calcd for C10H12N3O2Cl: C 49.70, H 5.00,
N 17.39; found: C 49.45, H 5.26, N 17.66.
Chemistry
Melting points were determined on a Buchi-Tottoli
apparatus in open capillarytubes and are uncorrected.
IR spectra were recorded as KBr pellets on a Perkin–
3,5-Diacetylamino-6-chloro-pyridine-2-carboxylic
acid
(7). A mixture of 3,5-diacetylamino-2-chloro-6-methyl-
pyridine 6 (10 g, 41 mmol) and MgSO4 (3 g, 25 mmol) in
water (200 mL) was stirred and heated at 80 ꢁC. KMnO4
(16 g, 101 mmol) was added portionwise, and the
resulting mixture was stirred for 1 h. Afterwards, the
1
Elmer 1750 FT spectrophotometer. The H NMR spec-
tra were taken on either a Bruker AW-80 (80 MHz) in
CDCl3 or in DMSO-d6 with HMDS as the internal
standard and a Bruker AW-400 (400.13 MHz) instru-
ment in DMSO-d6 with TMS as the internal standard,
at 25 ꢁC; chemical shifts are reported in d values (ppm)
relative to internal HMDS or TMS. The abbreviation
s=singlet, d=doublet, t=triplet, q=quadruplet,
m=multiplet, and br. s=broad singulet are used
throughout. For most amiloride analogues, the high
number of protons bound to nitrogen atoms increases
the occurence of exchange and greatlyinfluence the shift
values. The off-decoupling 13C NMR spectra were
measured with a Bruker DRX 400 Avance spectrometer
(100.62 MHz) in DMSO-d6, at 25 ꢁC. Chemical shifts
are reported in d values (ppm) with TMS as the internal
standard. Elemental analyses (C, H, N, S in%) were
performed with a Carlo-Erba EA 1108-elemental ana-
lyser and were withinÆ0.4% of the theoretical values.
All reactions were routinelychecked byTLC on silica
gel Merck 60F 254 plates.
excess of KMnO4 was reduced byNaHSO , the man-
3
ganese dioxide (MnO2) filtered, and washed with hot
water (2Â50 mL). The combined filtrates were adjusted
to pH 1 with 20% H2SO4. Left overnight at 4 ꢁC, the
formed precipitate was collected, washed with water,
and dried. Crystallization from ethanol gave the title
compound 7 (6.75 g, 60%): mp 212–215 ꢁC; IR 3533,
3346, 3245, 1712, 1690, 1584, 1537, 1475, 1371, 1247,
1228, 1194, 723, 667 cmꢀ1
;
1H NMR (80 MHz,
CDCl3+DMSO-d6, HMDS), d 2.19 (s, 3H, 5-NH–CO–
CH3), 2.33 (s, 3H, 3-NH–CO–CH3), 5.36 (s,
COOH+H2O), 9.35 (s, 1H, 5-NH–CO–CH3), 9.68 (s,
1H, 4H-pyridine), 11.07 (s, 1H, 3-NH–CO–CH3). Anal.
calcd for C10H10N3O4Cl: C 44.21, H 3.71, N 15.47;
found: C 43.96, H 3.98, N 15.76.
Methyl 3,5-diacetylamino-6-chloro-pyridine-2-carboxylic
ester (8). The decomposition of methylnitrosourea (4.1
g, 40 mmol) into NaOH 5 N gave diazomethane, which
was extracted with diethyl ether (10 mL). The diazo-
methane solution was then added dropwise to a solution
3,5-Diamino-2-chloro-6-methylpyridine (5). Portionwise
2-chloro-6-methyl-3,5-dinitropyridine
4
(7.5 g, 35
mmol) was added to a suspension of tin chloride (42 g,
186 mmol) in HCl 12 N (110 mL). After stirring for 90
min at room temperature, the mixture was heated at
100 ꢁC for 45 min. After cooling, it was concentrated
under reduced pressure, the residue brought to pH 12
of
3,5-diacetylamino-6-chloro-pyridine-2-carboxylic
acid 7 (2.7 g, 9.9 mmol) in a mixture ethanol–dimethyl-
formamide (1:1, 100 mL), and the temperature was kept
under 10 ꢁC. After warming to room temperature, the
medium was concentrated under reduced pressure, and
diluted with water (100 mL). The pure compound 8 was
filtered, washed with water, and dried (1.85 g, 65%): mp
197–199 ꢁC; IR 3321, 3277, 1710, 1688, 1586, 1523,
with NaOH 5N, and continouslyextracted with CHCl
overnight. The organic layer was washed with water,
dried and evaporated under reduced pressure to afford a
3

1800
D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
1
1476, 1229, 1190, 1117, 741 cmꢀ1; H NMR (80 MHz,
CDCl3+DMSO-d6, HMDS), d 2.03 (s, 3H, 5-NH–CO–
CH3), 2.10 (s, 3H, 3-NH–CO–CH3), 3.74 (s, 3H, CH3
ester), 9.25 (s, 1H, 4H-pyridine), 9.51 (s, 1H, 5-NH–
CO–CH3), 10.41 (s, 1H, 3-NH–CO–CH3). Anal. calcd
for C11H12N3O4Cl: C 46.25, H 4.23, N 14.71; found: C
46.03, H 4.32, N 15.02.
a mixture of guanidine hydrochloride (0.48 g, 5 mmol)
and potassium tert-butyl oxide (0.56 g, 5 mmol) in
dioxane (10 mL) was heated at 50 ꢁC for 20 min. After
cooling to room temperature, N-tert-butyl-3-[(3,5-di-
amino-6-chloro-pyridine-2-carbonyl)-oxy]crotonamide
11 (0.82 g, 2.5 mmol) was added, and the resulting mix-
ture was refluxed for 1 h. After cooling, dioxane was
evaporated under reduced pressure and water (30 mL)
was added. After stirring for 30 min, the crude product
12 was collected, washed with water, and dried. It was
purified bydissolution in methanol or isopropanol with
charcoal. The suspension was filtered, and the filtrate
diluted with a HCl saturated diethyl ether solution to
give the title compound. It was filtered, washed with
diethyl ether, and dried (0.21 g, 30%)
Methyl 3,5-diamino-6-chloro-pyridine-2-carboxylic ester
(9). Under nitrogen atmosphere, H2SO4 (d=1.84; 1.8
mL, 34 mmol) was added dropwise to a suspension of
3,5-diacetylamino-6-chloro-pyridine-2-carboxylic acid 7
(1 g, 3.7 mmol) in methanol (20 mL).The reaction mix-
ture was refluxed for 3 h. After cooling, methanol was
evaporated under reduced pressure and crushed ice (30
g) was added. The medium was then neutralized to pH 7
with saturated NaHCO3. After standing at 4 ꢁC for 2 h,
the precipitate formed was filtered, washed with water,
and dried. Crystallization from toluene gave the title
compound 9 (0.19 g, 25%): mp 211–213 ꢁC; IR 3475,
3436, 3367, 3337, 3040, 1676, 1619, 1434, 1273, 1122,
Method 2. A mixture of (3-amino-5-acetylamino-6-
chloro-pyridine-2-carbonyl)guanidine
hydrochloride
hydrate 15 (0.94 g, 3 mmol), ethanol (4 mL) and HCl
6 N (6 mL) was refluxed for 2 h. After cooling, ethanol
was evaporated under reduced pressure, water (5 mL)
added, and the solution heated at 60 ꢁC with charcoal.
After filtration, the filtrate was kept at 4 ꢁC for 3 h, and
the precipitate formed 12 was filtered, washed with die-
thyl ether, and dried (0.25 g, 30%): mp 184–185 ꢁC; IR
3390, 3336, 3287, 3187, 1692, 1674, 1628, 1536, 1244,
1066, 696 cmꢀ1
;
1H NMR (80 MHz, DMSO-d6,
HMDS), d 3.65 (s, 3H, CH3 ester), 5.96 (s, 2H, 5-NH2),
6.31 (s, 1H, 4H–pyridine), 6.50 (s, 2H, 3-NH2). Anal.
calcd for C7H8N3O2Cl: C 41.70, H 4.00, N 20.84; found:
C 41.58, H 4.26, N 21.12.
3,5-Diamino-6-chloro-pyridine-2-carboxylic acid (10). A
solution of 3,5-diacetylamino-6-chloro-pyridine-2-car-
boxylic acid 7 (2 g, 7.4 mmol) in NaOH 1 N (60 mL)
was refluxed for 3 h. After cooling to room temperature,
pH was adjusted to 5 with acetic acid 2 N. The pre-
cipitate formed 10 was collected, washed with water,
and dried (1.0 g, 75%): mp 192–194 ꢁC; IR 3481, 3442,
3350, 2925, 1688, 1614, 1430, 1255, 690 cmꢀ1; 1H NMR
(400 MHz, DMSO-d6+CF3COOD, TMS), d 6.37 (s,
1H, 4H–pyridine); 13C NMR (400 MHz, DMSO-d6,
TMS), d 105.82 (C4), 116.04 (C6), 124.18 (C2), 145.84
(C5), 149.84 (C3), 168.45 (C¼O). Anal. calcd for
C6H6N3O2Cl: C 38.42, H 3.22, N 22.40; found: C 38.76,
H 3.49, N 22.11.
709 cmꢀ1 1H NMR (400 MHz, DMSO-d6, TMS), d
;
6.45 (s, 1H, 4H–pyridine), 6.92 (br. s, guanidine), 8.53
(br. s, 4H, NH2); 13C NMR (400 MHz, DMSO-d6,
TMS), d 104.99 (C4), 114.77 (C6), 124.73 (C2), 147.07
(C5), 150.48 (C3), 155.54 (C¼NH), 166.62 (C¼O). Anal.
calcd for C7H12N6O2Cl2: C 29.70, H 4.27, N 29.68;
found: C 29.39, H 4.58, N 29.92.
N-(6-Chloro-2-methyl-4-oxo-4H-pyrido[3,2-d]-1,3-oxazin-
7-yl)-acetamide (13). A mixture of 3,5-diacetylamino-6-
chloro-pyridine-2-carboxylic acid 7 (1.36 g, 5 mmol),
acetic anhydride (20 mL, 213 mmol) and ethyl acetate
(10 mL) was refluxed for 4 h. After cooling, ethyl ace-
tate was evaporated under reduced pressure, and the
solution was kept at ꢀ18 ꢁC for 2 h. The precipitate
formed was filtered, washed with ethyl acetate, and
dried. Crystallization from the mixture ethyl acetate–
petroleum ether 40–60 ꢁC (1:3) gave the title compound
13 (0.85 g, 70%): mp 215–218 ꢁC; IR 3454, 3354, 3086,
1764, 1713, 1642, 1555, 1506, 1451, 1383, 1358, 1228,
N-tert-Butyl-3-[(3,5-diamino-6-chloro-pyridine-2-carbonyl)-
oxy]crotonamide (11). NBI (1.2 g, 5 mmol) was added to
a mixture of 3,5-diamino-6-chloro-pyridine-2-carboxylic
acid 10 (0.94 g, 5 mmol) and triethylamine (0.5 g, 5
mmol) in dimethylformamide (5 mL), and the resulting
mixture was stirred for 2 h at room temperature. After-
wards, it was poured into ice-water (60 mL), and the
precipitate formed was filtered, washed with water, and
dried. Crystallization from acetonitrile afforded the title
compound 11 (1.14 g, 70%): mp 121–122 ꢁC; IR 3451,
3399, 3344, 3311, 3069, 1708, 1677, 1632, 1614, 1543,
1
1202, 1180 cmꢀ1; H NMR (80 MHz, CDCl3+DMSO-
d6, HMDS), d 2.15 (s, 3H, CH3), 2.34 (s, 3H, CH3), 8.50
(s, 1H, 8H), 9.70 (s, 1H, CONH). Anal. calcd for
C9H8N3O3Cl: C 44.74, H 3.34, N 17.39; found: C 45.03,
H 3.63, N 17.56.
1364, 1275, 1242, 1218, 1154, 1092, 1058, 735, 704 cmꢀ1
;
(3,5-Diacetylamino-6-chloro-pyridine-2-carbonyl)guan-
idine hydrate (14). Guanidine base was prepared by
consecutive addition of Na (0.63 g, 27.5 mmol) and
guanidine hydrochloride (2.64 g, 27.5 mmol) to dry
methanol (50 mL), and the solution was refluxed for
30 min under nitrogen atmosphere. After cooling at
room temperature, N-(6-chloro-2-methyl-4-oxo-4H-pyr-
ido[3,2-d]-1,3-oxazin-7-yl)-acetamide 13 (1.37 g, 55
mmol) was added, and the resulting mixture was stirred
for 45 min at room temperature. Afterwards, methanol
1H NMR (80 MHz, DMSO-d6, HMDS), d 1.09 (s, 9H,
N-tert-butyl), 1.86 (s, 3H, C(CH3)¼CH), 5.42 (s, 1H,
C(CH3)¼CH), 6.03 (s, 2H, 5-NH2), 6.34 (s, 1H, 4H–
pyridine), 6.55 (s, 2H, 3-NH2), 7.00 (s, 1H, CONH).
Anal. calcd for C14H19N4O3Cl: C 51.46, H 5.86, N
17.14; found: C 51.29, H 5.98, N 17.36.
(3,5 - Diamino - 6 - chloro - pyridine - 2 - carbonyl)guanidine
hydrochloride hydrate (12). Method 1. Under nitrogen,

D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
1801
was evaporated under reduced pressure and water (100
mL) was added. The precipitate formed was filtered,
washed with water, and dried. It was purified bydis-
solution in methanol with charcoal. The suspension was
filtered, and the filtrate diluted with water to give the
title compound 14 (2.92 g, 32%): mp 244–247 ꢁC
(decomposition); IR 3346, 3245, 1712, 1690, 1584, 1537,
1249, 1203, 1185 cmꢀ1. Anal. calcd for C6H9NO2: C
56.68, H 7.13, N 11.02; found: C 56.72, H 7.08, N 11.07.
2,5,6-Trichloro-3-methylpyridine (17). A well-powdered
mixture of 3-methyl-2,6-piperidinedione 16 (23.3 g, 183
mmol) and phosphorus pentachloride (240 g, 1.2 mol)
was quicklyintroduced in a flask fitted with a large
reflux condenser and slowlyheated to 150 ꢁC for 2 h,
until a viscous liquid was formed. The hot mixture was
then poured on crushed ice (1 kg), and kept at 4 ꢁC
overnight. The precipitate formed 17 was filtered,
washed with iced water, and dried (16.2 g, 45%). An
analytical sample of 17 was obtained byfractional
crystallization from a mixture ethanol–petroleum ether
40–60 ꢁC (1 :8): mp 94–96 ꢁC; IR 3446, 3053, 2968, 2628,
1810, 1631, 1566, 1532, 1442, 1387, 1336, 1235, 1212,
1177, 1102, 1038, 1009, 927, 907, 825, 720, 707, 669,
1475, 1371, 1247, 1228, 1194, 723 cmꢀ1 1H NMR
;
(400 MHz, DMSO-d6, TMS), d 2.19 (s, 3H, 5-NH–CO–
CH3), 2.33 (s, 3H, 3-NH–CO–CH3), 6.83 (s, 3H, ami-
dine), 9.35 (s, 1H, 5-NH–CO–CH3), 9.68 (s, 1H, 4H–
pyridine), 10.41 (s, 1H, 3-NH–CO–CH3), 11.07 (s, 1H,
CO–NH). Anal. calcd for C11H15N6O4Cl: C 39.95, H
4.57, N 25.41; found: C 40.12, H 4.68, N 25.13.
(3-Amino-5-acetylamino-6-chloro-pyridine-2-carbonyl)-
guanidine hydrochloride hydrate (15). Guanidine base
was prepared byconsecutive addition of Na (0.23 g, 10
mmol) and guanidine hydrochloride (0.96 g, 10 mmol)
to drymethanol (20 mL), and the solution was refluxed
for 30 min under nitrogen atmosphere. After cooling at
room temperature, N-(6-chloro-2-methyl-4-oxo-4H-pyr-
ido[3,2-d]-1,3-oxazin-7-yl)-acetamide 13 (0.50 g, 20
mmol) was added, and the resulting mixture was
refluxed for 75 min. Afterwards, methanol was evapo-
rated under reduced pressure, water (40 mL) added, and
the mixture triturated for 15 min. The precipitate
formed was collected, and washed with water. It was
dissolved in HCl 1N (50 mL) at 50 ꢁC with charcoal.
The suspension was filtered, and the filtrate kept at 4 ꢁC
for 2 h. The precipitate formed 15 was filtered, washed
with water, and dried (0.13 g, 40%): mp 255 ꢁC; IR 3355,
3192, 1700, 1651, 1579, 1550, 1495, 1442, 1418, 1376,
1228, 778 cmꢀ1; 1H NMR (400 MHz, DMSO-d6, TMS),
d 2.11 (s, 3H, 5-NH–CO–CH3), 6.92 (s, 2H, 3-NH2),
8.22 (s, 1H, 4H-pyridine), 8.83 (s, 3H, amidine), 10.66 (s,
1H, 5-NH–CO–CH3), 10.97 (s, 1H, CONH). Anal.
calcd for C9H14N6O3Cl2: C 33.24, H 4.34, N 25.85;
found: C 33.13, H 4.16, N 26.05.
1
536, 520 cmꢀ1; H NMR (80 MHz, CDCl3, HMDS), d
2.28 (s, 3H, CH3), 6.55 (s, 1H, 4H–pyridine). Anal.
calcd for C6H4NCl3: C 36.68, H 2.05, N 7.13; found: C
36.72, H 1.81, N 7.19.
2,5,6-Trichloro-3-bromomethyl-pyridine (19) and 2,5,6-
Trichloro-3-dibromomethyl-pyridine (20). A vigorously
stirred mixture of 2,5,6-trichloro-3-methylpyridine 17
(20 g, 102 mmol), N-bromosuccinimide (50.9 g, 286
mmol), carbon tetrachloride (200 mL) was refluxed and
illuminated with a 60-watt photoflood lamp for 4 h. The
succinimide formed was then removed byfiltration. The
filtrate was evaporated under reduced pressure and the
yellow oil residue was purified by flash column
chromatographyon silica gel with n-hexane as eluent to
give 19 and 20. 19: mp 51–52 ꢁC; IR 3468, 3028, 1567,
1532, 1438, 1392, 1350, 1220, 1174, 1129, 1092, 940,
927, 878, 744, 719, 670, 590, 552 cmꢀ1 1H NMR
;
(80 MHz, DMSO-d6, HMDS), d 3.28 (s, 2H, CH2Br),
8.38 (s, 1H, 4H–pyridine). Anal. calcd for C6H3NCl3Br:
C 26.17, H 1.10, N 5.09; found: C 26.47, H 1.30, N 5.07.
20: mp 47–48 ꢁC; IR 3011, 1566, 1525, 1385, 1347, 1215,
1187, 1174, 1150, 1084, 941, 905, 753, 735, 703, 671,
3-Methyl-2,6-piperidinedione (16). A mixture of H2SO4
(d=1.84; 160 mL, 3 mol), acetic acid (d=1.049; 1000
mL, 17.5 mol) and a-methylglutaronitrile (85% purity;
257 g, 2.4 mol) was stirred at room temperature, and a
solution of acetic acid (d=1.049; 200 mL, 3.5 mol) in
water (63 mL) was then added dropwise for 1 h. The
reaction medium was heated to 130 ꢁC and this tem-
perature was sustained for an additional hour. After-
wards, it was cooled at 4 ꢁC overnight, (NH4)2SO4 was
filtered off, washed with acetic acid, and the filtrate was
concentrated under reduced pressure. The residue
formed (470 g) was collected, poured into water (1.5 L),
and the solution was adjusted to pH 5 with Na2CO3
(120 g). The first crop of crude product 16 was filtered,
and dried (96.5 g). The filtrate was extracted with
CHCl3 (3Â300 mL) and washed with water. The com-
bined organic layers were dried and evaporated under
reduced pressure to give a second crop of 16 (269 g).
The different crops were crystallized from water or from
a mixture of toluene–petroleum ether 40–60 ꢁC (1:5) to
give the title compound 16 (yield: 60–65%): mp 91–
92 ꢁC; IR 3197, 3094, 2993, 2974, 2960, 2937, 2883,
1713, 1703, 1649, 1639, 1463, 1444, 1364, 1339, 1315,
662, 595, 550 cmꢀ1 1H NMR (80 MHz, DMSO-d6,
;
HMDS), d 7.26 (s, 1H, CHBr2), 8.44 (s, 1H, 4H–pyri-
dine). Anal. calcd for C6H2NCl3Br2: C 20.34, H 0.57, N
3.95; found: C 20.64, H 0.79, N 3.99. Generally, the
residue (19 and 20) was used without anyfurther
purification.
2,5,6-Trichloro-pyridine-3-carboxylic acid (21). Method
1. Portionwise KMnO4 (7.6 g, 48 mmol) was added to a
suspension of 2,5,6-trichloro-3-methylpyridine 17 (3.9 g,
20 mmol) in water (250 mL), and the resulting mixture
was heated at 90 ꢁC for 6.5 h. Afterwards, the manga-
nese dioxide (MnO2) was filtered off and washed with
hot water (2Â50 mL). The combined filtrates were con-
centrated to 20 mL and adjusted to pH 1 with HCl
12 N. After standing at 4 ꢁC overnight, the crude pro-
duct was collected, washed with a minimum amount of
iced water, and dried. Crystallization from water gave
the title compound 21 (1.37 g, 30%). The unreacted
starting material was recovered from MnO2 cake by
extraction with acetone (150 mL). The yield of deriva-
tive 21 was then calculated on the basis of the quantity
of reacted starting material

1802
D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
Method 2. A mixture of 2,5,6-trichloro-3-bromomethyl-
cipitate formed 27 was collected, washed with cold
water, and dried (4.9 g, 90%): mp 159–160 ꢁC; IR 3487,
3470, 3333, 3288, 3147, 2393, 1633, 1603, 1580, 1545,
1428, 1364, 1338, 1300, 1233, 1126, 1062, 1034, 941,
830, 796, 702, 598 cmꢀ1; 1H NMR (80 MHz, DMSO-d6,
HMDS), d 6.46 (br. s, 2H, 6-NH2), 6.90 (br. s, 3H, 2-
NH2+HN+), 7.65 (s, 1H, 4H–pyridine). Anal. calcd for
C6H6N3O2Cl: C 38.42, H 3.22, N 22.40; found: C 38.57,
H 3.37, N 22.30.
pyridine 19 and 2,5,6-trichloro-3-dibromomethyl-pyri-
dine 20 (30.9 g), HgNO3 (1.51 g, 5.7 mmol), CuSO4.
5H2O (0.67 g, 2.7 mmol) in H2SO4 (d=1.84; 73 mL, 1.4
mole) was stirred and heated at 110 ꢁC. Dropwise
HNO3 (d=1.52; 40 mL, 0.97 mol) was added for 90 min
and the temperature was maintained at 130 ꢁC. 15 min
later, the medium was poured on crushed ice (Æ300 g),
and the precipitate formed was filtered, and dried. It
was dissolved in saturated NaHCO3 (200 mL) with
charcoal. The suspension was filtered, and the filtrate
acidified with HCl 12 N to give the crude product 21. It
was collected, washed with iced water, and dried. Crys-
tallization from water afforded the title compound 21
(yield 40–50%): mp 160–162 ꢁC; IR 3070, 2631, 1711,
1694, 1566, 1520, 1380, 1329, 1271, 1235, 1176, 1156,
(2,6 - Diamino - 5 - chloro - pyridine - 3 - carbonyl)guanidine
(28). Guanidine base was prepared byconsecutive
addition of potassium tert-butyl oxide (0.28 g, 2.5
mmol) and guanidine hydrochloride (0.24 g, 2.5 mmol)
to drymixture of dimethylformamide–dioxane (1:1; 10
mL). Under nitrogen atmosphere, the mixture was
heated at 50 ꢁC for 20 min, and then KCl filtered off.
2,6-Diamino-5-chloro-pyridine-3-carboxylic acid 27
(0.47 g, 2.5 mmol) was added to a solution of CDI (0.41
g, 2.5 mmol) in dimethylformamide (10 mL), and the
mixture was stirred at room temperature for 1 h. This
latter was added to the guanidine filtrate, and the final
mixture was stirred at room temperature for 5 h. After-
wards, the solvents were evaporated under reduced
pressure, and the residue suspended in cold water (20
mL). The crystalline solid formed was filtered, washed
with water, and dried. It was purified byfractional cris-
tallization from methanol to give the title compound 28
(0.13 g, 22%): mp >300 ꢁC; IR 3466, 3419, 3390, 3350,
3264, 3168, 1644, 1591, 1520, 1395, 1367, 1309, 1232,
1
1086, 920, 783, 759, 718, 675, 643, 503 cmꢀ1; H NMR
(80 MHz, CDCl3+CF3COOD, TMS), d 8.40 (s, 1H,
4H–pyridine); 13C NMR (400 MHz, DMSO-d6, TMS), d
128.58 (C5), 129.39 (C3), 142.91 (C4), 145.87 (C6), 149.06
(C2), 164.37 (C¼O). Anal. calcd for C6H2NO2Cl3: C
31.82, H 0.89, N 6.18; found: C 31.91, H 1.13, N 6.26.
2,5,6-Trichloro-pyridine-3-carboxamide (25). A mixture
of 2,5,6-trichloro-pyridine-3-carboxylic acid 21 (8.6 g,
38 mmol), thionyl chloride (4.9 mL, 7.9 g, 67 mmol),
benzene (16 mL) and dimethylformamide (0.08 mL) was
refluxed for 2.5 h. Afterwards, the reaction medium was
evaporated under reduced pressure, the residue dis-
persed in drytoluene (25 mL) and the solvent elimi-
nated under reduced pressure to give 24 (7.6 g, 81%).
This step was repeated twice. A solution of 2,5,6-tri-
chloropyridine-3-carboxylic acid chloride 24 (7.6 g, 31
mmol) in drydioxane (10 mL) was then added to 10%
NH4OH (500 mL), and the mixture was stirred at 4 ꢁC
for 1 h. The precipitate formed 25 was filtered, washed
with cold water, and dried (6.3 g, 90%): mp 151–153 ꢁC;
IR 3391, 3181, 1662, 1567, 1522, 1423, 1374, 1321, 1172,
1085, 911, 715, 659, 600 cmꢀ1. Anal. calcd for
C6H3N2OCl3: C 31.96, H 1.34, N 12.42; found: C 31.99,
H 1.05, N 12.40.
1
1105, 1033, 945, 894, 805, 730, 603, 519, 474 cmꢀ1; H
NMR (400 MHz, DMSO-d6+D2O, TMS), d 8.02 (s,
1H, 4H–pyridine). Anal. calcd for C7H9N6OCl: C 36.77,
H 3.97, N 36.76; found: C 36.42, H 3.79, N 36.48.
2,6-Dichloro-3-pyridine-carboxylic acid (29). A solution
of n-butyllithium (0.25 mol) in hexane (100 mL) was
added dropwise to a cold (ꢀ80 ꢁC) and freshlyprepared
solution of diisopropylamine (35 mL, 0.25 mol) in tet-
rahydrofuran (200 mL), and the medium was stirred for
1 h. A solution of 2,6-dichloropyridine (37 g, 0.25 mole)
in tetrahydrofuran (100 mL) was then added by por-
ꢁ
2,6-Diamino-5-chloro-pyridine-3-carboxamide (26).
A
tions, and the resulting mixture was stirred at ꢀ80 C
mixture of 2,5,6-trichloro-pyridine-3-carboxamide 25
(2.0 g, 44 mmol) and a 25% NH4OH solution (60 mL)
saturated with NH3 was placed in a stainless-steel auto-
clave and heated at 130 ꢁC for 12 h. After cooling at
room temperature, the solid residue formed was filtered,
washed with water, and dried. Crystallization from ace-
tone gave the title compound 26 (5.35 g, 65%): mp 181–
184 ꢁC; IR 3495, 3425, 3346, 3215, 1698, 1646, 1607,
1526, 1482, 1396, 1314, 1265, 1087, 1033, 788, 671, 563
;
cmꢀ1 1H NMR (400 MHz, DMSO-d6, TMS), d 6.41
(br. s, 2H, CONH2), 7.16 (br. s, 4H, NH2), 7.85 (s, 1H,
4H–pyridine). Anal. calcd for C6H7N4OCl: C 38.62, H
3.78, N 30.02; found: C 38.61, H 3.89, N 29.78.
for 30 min. After warming at 0 ꢁC, it was quickly
poured on a large excess of carbogene in diethyl ether (1
L), left until CO2 production ceased, and then water was
added (500 mL). The aqueous layer was extracted with
diethyl ether (2Â250 mL) and mixed with charcoal. The
suspension was filtered, and the filtrate adjusted to pH 1
with HCl 12 N. After standing at 4 ꢁC overnight, the
precipitate formed was collected and dried. Crystal-
lization from toluene afforded the title compound 29
(28.8 g, 60%): mp 145–147 ꢁC; IR 3427, 3090, 2970,
2652, 2550, 1722, 1578, 1542, 1454, 1438, 1406, 1342,
1283, 1245, 1171, 1144, 1052, 904, 851, 780, 756, 668,
603, 569 cmꢀ1. Anal. calcd for C6H3NO2Cl2: C 37.53, H
1.57, N 7.29; found: C 37.24, H 1.88, N 7.27.
2,6-Diamino-5-chloro-pyridine-3-carboxylic acid (27). A
suspension of 2,6-diamino-5-chloro-pyridine-3-carbox-
amide 26 (5.35 g, 29 mmol) in NaOH 4 N (80 mL) was
refluxed for 1 h. After cooling at room temperature, the
medium was diluted with water (40 mL), adjusted to pH
1 with HCl 6 N and kept at 4 ꢁC overnight. The pre-
(2,6-Dichloro-pyridine-3-carbonyl)guanidine
hydrate
(30). The title compound was prepared from 2,6-
dichloro-3-pyridine-carboxylic acid 29 (1 g, 5 mmol) as
described for 28. It was crystallized in dioxane (0.3 g,
24%): mp >300 ꢁC; IR 3430, 3074, 2156, 1670, 1615,

D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
1803
1573, 1529, 1472, 1368, 1340, 1201, 1146, 1074, 906,
846, 815, 791, 678, 577 cmꢀ1 1H NMR (400 MHz,
according to the general procedure (yield: 18%): mp
211–214 ꢁC. Anal. calcd for C8H10N3OCl: C 48.13, H
5.05, N 21.05; found: C 48.33, H 5.24, N 21.35.
;
DMSO-d6, TMS), d 7.55 (d, 1H, 5H–pyridine), 8.03 (d,
1H, 4H–pyridine); 13C NMR (400 MHz, DMSO-d6,
TMS), d 123.84 (C5), 135.84 (C3), 141.89 (C4), 146.48
(C6), 148.65 (C2), 163.23 (C¼NH), 174.89 (C¼O). Anal.
calcd for C7H8N4O2Cl2: C 33.49, H 3.21, N 22.31;
found: C 33.76, H 3.25, N 21.95.
Pharmacology
Human platelet swelling assay (PSA). The NHE inhibi-
torypotencyof the novel drugs was assessed bytheir
abilityto reduce the human platelet swelling induced by
a propionate buffer (pH 6.7).16,18,27 A solution of the
tested compound (20 mL) was added to 1.73 mL of
propionate buffer (in mM: sodium propionate 140, glu-
cose 10, KCl 5, MgCl2 1, CaCl2 1, HEPES 20 qs ad pH
6.7) contained in a spectrophotometer cuvette. Then,
0.25 mL of human platelet rich plasma (PRP from the
Belgian Red Cross) containing 1–2Â108 cells/mL was
added. The suspension was stirred and the change in
optical density(OD) was recorded each 10 s for 5 min at
680 nm (Perkin-Elmer lambda 20 double-beam spectro-
photometer). During the experiment, the suspension
was maintained at 25 ꢁC. The decrease of OD corre-
sponded to a monoexponential curve following the
equation OD(t)=ODt=0 eꢀkt where t is the time (in s)
corresponding to the recorded OD and k is the decrease
rate constant.16 For each drug, the concentrations were
plotted against their corresponding k values. The max-
imum platelet swelling was measured in absence of any
drug. The minimum swelling was observed in presence
of EIPA (10 mM) and is the result of a completely
inhibited NHE. Sigmoidal curves were drawn bynon-
linear regression analysis (GraphPad Prism software).
(2,5,6-Trichloro-pyridine-3-carbonyl)guanidine (31). The
title compound was prepared from 2,5,6-trichloro-3-
pyridine-carboxylic acid 21 (0.72 g, 3 mmol) as descri-
bed for 28. It was crystallized in dioxane (97 mg, 12%):
mp >300 ꢁC; IR 3370, 1668, 1599, 1564, 1521, 1454,
1347, 1317, 1168, 1105, 970, 937, 836, 798, 656, 617
1
cmꢀ1; H NMR (400 MHz, DMSO-d6, TMS), d 6.95,
8.00 (br. s, 3H, guanidine), 8.35 (s, 1H, 4H–pyridine);
13C NMR (400 MHz, DMSO-d6, TMS), d 128.93 (C5),
136.52 (C3), 141.48 (C4), 144.61 (C6), 146.31 (C2),
163.13 (C¼NH), 173.12 (C¼O). Anal. calcd for
C7H5N4OCl3: C 31.43, H 1.88, N 20.94; found: C 31.23,
H 2.14, N 20.69.
(2-Amino-5-bromo-pyridine-3-carbonyl)guanidine (32).
The title compound was prepared bybromination of
(2-amino-pyridine-3-carbonyl)guanidine (1.5 g, 8.4
51
mmol) as previouslydescribed byCragoe
(1.0 g,
45%): mp >300 ꢁC; IR 3481, 3437, 3334, 1638, 1604,
1561, 1505, 1464, 1400, 1358, 1327, 1234, 918, 849, 806,
694 cmꢀ1 1H NMR (400 MHz, DMSO-d6, TMS), d
;
8.06 (d, 1H, 6H-pyridine), 8.28 (d, 1H, 4H–pyridine);
13C NMR (400 MHz, DMSO-d6, TMS), d 104.63 (C5),
116.52 (C3), 141.60 (C4), 151.33 (C6), 158.54 (C2),
162.92 (C¼NH), 175.01 (C¼O). Anal. calcd for
C7H8N5OBr: C 32.58, H 3.12, N 27.14; found: C 32.75,
H 3.29, N 26.96.
Theyled to calculate the drug concentration (IC
)
50
which decrease of 50% the platelet swelling. For each
molecule, the measurements were performed in tripli-
cate. The IC50 values are expressed as meanÆSEM.
22Na+ uptake inhibition assay (UIA). The 22Na+ uptake
56
inhibition assayused human washed platelets.
The
General procedure for the preparation of ‘simplified’
aroylguanidines
uptake of 22Na+ started when platelet-rich plasma (0.23
mL) was added to 0.75 mL of sodium propionate buffer
(pH 6.7) containing 1–2 mCi of 22Na+ (specific radio-
activity37 MBq) and to 0.02 mL of buffer containing
the drug. The incubation medium contained ouabain
(0.1 mM) to prevent sodium ejection through the Na+/
K+ ATPase. After 5 min. incubation at 25 ꢁC, 4 mL of
ice-cold buffer (containing in mM: choline chloride 100,
NaCl 15.4, Tris 45, HCl qs ad pH 7.4) was added, the
sample rapidlyfiltered through a glass-fibre filter
(Whatman GF/C), and the tube rinsed twice with ice-
cold buffer. The filter was placed in plastic scintillation
vial containing an emulsion-type scintillation mixture
(4 mL) and the radioactivitywas measured using a
b-counter (Packard Tricarb 460C). Three independant
22Na+-influx experiences were performed in quad-
ruplicate for each compound. The residual cell radio-
activitymeasured in presence of EIPA (10 mM) was
considered as non relevant of the sodium uptake
through the NHE. This EIPA-insensitive sodium uptake
was 153 pmol/108 platelets. The control was considered
as the sodium uptake in absence of drug (216Æ5 pmol/
108 platelets). The sodium uptake data were plotted
against drug concentrations. Sigmoidal curves were
drawn bya non-linear regression analysis (GraphPad
Compounds 33, 40, 42 were synthesized by condensing
the appropriate methyl ester with the guanidine under
reflux or byreaction between the desired acid chloride
and the guanidine at room temperature.52,53 Guanidine
base was always freshly prepared from the hydrochlo-
ride salt using Na, NaH or potassium tert-butyl oxide.
(Pyridine-3-carbonyl)guanidine dihydrochloride (33). The
title compound was prepared from methyl pyridine-3-
carboxylic ester according to the general procedure
(yield: 19%): mp 254–256 ꢁC (decomposition). Anal.
calcd for C7H10N4OCl2: C 35.46, H 4.25, N 23.63;
found: C 35.72, H 4.52, N 23.34.
(3,4-Dichlorobenzoyl)guanidine hydrochloride (40). The
title compound was prepared from (3,4-dichloro)-
benzoyl chloride according to the general procedure
(yield: 21%): mp 215–218 ꢁC. Anal. calcd for
C8H8N3OCl3: C 35.78, H 3.00, N 15.65; found: C 36.02,
H 3.32, N 15.52.
Benzoylguanidine hydrochloride (42). The tiltle com-
pound was prepared from methyl benzoic acid ester

1804
D. Laeckmann et al. / Bioorg. Med. Chem. 10 (2002) 1793–1804
Prism software). Theyled to determine the drug
concentration (IC50) required to decrease of 50% the
EIPA-sensitive sodium uptake induced byintracellular
acidosis (pH 6.7). The IC50 values are expressed as
meanÆSEM. (GraphPad Prism software).
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Acknowledgements
This studywas supported bya grant from the National
Bank of Belgium. We are grateful to M. O. Inarejos, M.
L. Pirard, S. Counerotte and A.-M. Murrayfor their
technical collaboration. We are also grateful to Ms.
Ferrauge (Croix Rouge de Namur) for her helpful co-
operation. Most of the products presented in this paper
were developed within the frame of a contract between
the Universite Libre de Bruxelles, the Universite de
Liege, Therabel Research and la Region Wallonne, of
which A. Herchuelz (Laboratoire de Pharmacodynamie et
de Therapeutique, Faculte de Medecine, Universite Libre
de Bruxelles) and J. Delarge were the projects leaders.
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