Novel inhibitors of the sodium-calcium exchanger: Benzene ring analogues of N-guanidino substituted amiloride derivatives

Article abstract of DOI:10.1016/S0223-5234(01)01247-8

A series of N-guanidino substituted 2,4-diamino-5-carbonylguanidine molecules related to amiloride were synthesised and evaluated for their ability to inhibit the sodium-calcium exchanger in rat insulinoma cells (RINm5F) and human platelets. Specific chemical pathways were used to prepare the benzene derivatives designed as bioisosteric analogues of the pyrazine derivatives of amiloride. Several so-called 'simplified analogues', where some substituents of amiloride were omitted or replaced, were also prepared and included in the biological evaluation. The inhibitory potency of the sodium-calcium exchanger was screened on both cell types by measuring their effect on ⁴⁵Ca²⁺ uptake. Among the most active compounds, N-(2-amino-5-chloro-4-nitrobenzoyl)-N′-(1-naphtylmethyl)guanidine (IC₅₀ = 3.4 μM) was found more active than amiloride (IC₅₀ = 690 μM) and 3,4-dichlorobenzamil (IC₅₀ = 15.2 μM), the reference inhibitor.

Full text of DOI:10.1016/S0223-5234(01)01247-8

Eur. J. Med. Chem. 36 (2001) 597–614
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01247-8/FLA
Original article
Novel inhibitors of the sodium–calcium exchanger: benzene ring analogues of
N-guanidino substituted amiloride derivatives
Franc¸oise Rogistera, Didier Laeckmanna, Pierre-Olivier Plasmanb, Franc¸oise Van Eylenb,
Marianne Ghyootb, Carine Maggettob, Jean-Franc¸ois Lie´geoisa, Joseph Ge´czyc,
Andre´ Herchuelzb, Jacques Delargea, Bernard Masereeld,*
aLaboratory of Medicinal Chemistry, University of Lie`ge, 1 avenue de l’Hoˆpital (B36), B-4000 Lie`ge 1, Belgium
bLaboratory of Pharmacology, Free University of Brussels, School of Medicine, Route de Lennik, 808-Baˆtiment GE,
B-1070 Brussels, Belgium
cTherabel Research S.A., 108 rue E. Van Ophem, B-1180 Brussels, Belgium
dDepartment of Pharmacy, University of Namur, 61 rue de Bruxelles, B-5000 Namur, Belgium
Received 4 April 2001; revised 11 June 2001; accepted 12 June 2001
Abstract – A series of N-guanidino substituted 2,4-diamino-5-carbonylguanidine molecules related to amiloride were synthesised and
evaluated for their ability to inhibit the sodium–calcium exchanger in rat insulinoma cells (RINm5F) and human platelets. Specific
chemical pathways were used to prepare the benzene derivatives designed as bioisosteric analogues of the pyrazine derivatives of
amiloride. Several so-called ‘simplified analogues’, where some substituents of amiloride were omitted or replaced, were also
prepared and included in the biological evaluation. The inhibitory potency of the sodium–calcium exchanger was screened on both
cell types by measuring their effect on 45Ca2+ uptake. Among the most active compounds, N-(2-amino-5-chloro-4-nitrobenzoyl)-N%-
(1-naphtylmethyl)guanidine (IC50=3.4 mM) was found more active than amiloride (IC50=690 mM) and 3,4-dichlorobenzamil
´
(IC50=15.2 mM), the reference inhibitor. © 2001 Editions scientifiques et me´dicales Elsevier SAS
sodium–calcium exchanger / amiloride / RINm5F / human platelets / substituted guanidines
1. Introduction
cium exchanger have been described. The first
exchanger type (NCX) operates with a stoichiometry
Calcium plays an important second messenger role
of 3 Na+ for 1 Ca2+ [2]. Three genes coding for three
in most cell types. This role involves a tight control of
different isoforms (NCX1, NCX2, and NCX3) have
been identified in mammals [3–5]. NCX1 is by far the
most extensively studied and characterised isoform; it
free cytosolic Ca2+ ([Ca2+]i). Two processes located at
the plasma membrane appear to mediate Ca2+ extru-
sion from cells and take part in the control of [Ca2+]i:
is widely distributed [6]. Further variability in this
the Ca2+-ATPase and the electrogenic sodium–cal-
group results from alternative splicing of NCX1, and
cium exchanger [1]. Two major types of sodium–cal-
tissue-specific variants have been identified [7, 8].
The second sodium–calcium exchanger type (RetX
or NCKX) was identified and cloned in rod outer
segments [9, 10]. It differs from NCX in both func-
tional and molecular terms: for instance, its stoi-
chiometry is different from that of the former because
4 Na+ are exchanged for 1 Ca2+ plus 1 K+. Two
exchanger isoforms NCKX1 and NCKX2 have been
identified [11].
Abbreviations: Ac2O: acetic anhydride; 3,4-DCB: 3,4-dichlorobenza-
mil; ADP: adenosine 5%-diphosphate; U-46619: 9,11-dideoxy-9a,-11a-
methanoepoxy prostaglandin F2a; PMA: phorbol 12-myristate
13-acetate; HEPES: N-2-hydroxyethylpiperazine-N%-2-ethane-sulfonic
acid; DCC: dicyclohexylcarbodiimide; EGTA: ethylene glycol-bis(b-
aminoethyl ether)-N,N,N%,N%-tetraacetic acid; Tris: tris(hydroxy-
methyl)aminomethane; BSA: bovine serum albumin.
* Correspondence author
E-mail address: bernard.masereel@fundp.ac.be (B. Masereel).

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F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
Although the sodium–calcium exchange activity
through the pharmacomodulation of the 5-amino
moiety, the 6-chloro group or the 2-carbonyl-
guanidine function. At the physiological pH (7.4) the
acylguanidine moiety of amiloride 1 is positively
charged (pKa =8.7) [35]. The structural features re-
quired for potent inhibition of the sodium–calcium
exchanger are the presence of an aryl or an aralkyl on
the distal nitrogen of the guanidine function, hydro-
phobic substituents on the 5-amino moiety, and a
chlorine atom on the 6-position of the pyrazine ring.
These structural features led to compounds able to
inhibit ion transporters like sodium channels and
voltage-operated calcium channels (L- and T-type)
[28, 29, 35, 37, 38]. For instance, 3,4-dichlorobenzamil
(2) is the most widely used reference inhibitor of the
sodium–calcium exchanger. It was 37 times more
potent than amiloride on the sodium–calcium ex-
changer (figure 1).
Despite its lack of specificity, 3,4-dichlorobenzamil
(2) and its related compounds showed a positive in-
otropic effect combined with a negative chronotropic
activity which was likely the result of the inhibition of
sodium–calcium exchange activity [39, 40]. Hence
such derivatives might represent a new type of in-
otropic agent [41, 42].
Searching novel, more potent and more selective
inhibitors of the sodium–calcium exchanger, we have
explored the bioisosteric replacement of the pyrazine
ring of amiloride by a phenyl. The structural require-
ments described to inhibit the sodium–calcium ex-
changer were preserved: a carbonylguanidino group
with a distal nitrogen substituted by an aralkyl group,
two amino moieties and a chloro group in the corre-
sponding positions compared with the structure of
amiloride.
The novel and reference compounds were screened
(10 mM) for their inhibitory potency on the sodium–
calcium exchanger. Their activity was estimated as the
inhibition of the intracellular Na+-dependent 45Ca2+
uptake by rat insulinoma cells (RINm5F) and human
platelets. Dose–response curves were performed for
the most active molecules.
participates in calcium homeostasis in a wide variety
of cell types [12], it exerts a prominent role in several
cells subject to high Ca2+ levels [13]. For instance in
the heart [14] and in the pancreatic b-cells [15],
sodium–calcium exchange appears to be the main
mechanism for Ca2+ extrusion. It has also been pro-
posed to play a key role in Ca2+ homeostasis of many
cell types like neuronal [16], vascular [17] or renal cells
[18]. In addition to its major involvement in the Ca2+
extrusion mechanism, the sodium–calcium exchange
may play a crucial role in cardiac arrhythmogenesis
[19–21]. This transport system may be important for
the regulation of blood pressure and may there-
by play a role in the pathogenesis of salt-depen-
dent essential hypertension [22, 23]. Moreover, the
sodium–calcium exchanger may prevent the platelet
integrin aIIbb3 activation and consequently the platelet
aggregation induced by various agonists such as
ADP, collagen, thrombin, epinephrine, U46619 and
PMA [24, 25]. Increased sodium–calcium exchanger
activity has also been reported in brain tissue from
Alzheimer’s disease subjects [26].
Due to the lack of specific inhibitors of this ex-
changer, investigation of the sodium–calcium ex-
changer remains difficult. Among the identified
non-specific inhibitors, amiloride and its derivatives
constitute
a chemically homogeneous series of
molecules, with known qualitative structure–activity
relationships, as a starting point for further develop-
ment [27–34] (figure 1).
Amiloride,
3,5-diamino-6-chloropyrazinoyl-
guanidine (1), was initially demonstrated to inhibit
the Na+ channels present in urinary epithelia and was
used as a K+-sparing diuretic [33]. At higher concen-
trations, amiloride and its analogues were subse-
quently shown to inhibit other ion transporters such
as the sodium–calcium exchanger [33]. The structure
of amiloride has been extensively studied, mainly
2. Chemistry
2.1. Synthesis of amiloride and its N-guanidino
substituted pyrazine derivatives
To prepare amiloride 1 and its described pyrazine
derivatives 2–8 [43], the required N-substituted
Figure 1. Structure and inhibitory potency of amiloride and
3,4-dichlorobenzamil on ion transporters.

F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
599
Figure 2. Pyrazine analogues of amiloride.
guanidine reacted with the methyl ester of 3,5-diamino-
6-chloropyrazine-2-carboxylic acid [43] or with N-tert-
butyl-3-[(3,5-diamino-6-chloropyrazine-2-carbonyl)oxy]-
crotonamide [44, 45] (figure 2). This latter was much
easier to handle and gave higher yields. The aralkyl-
guanidines were obtained by using the well-known reac-
tion of the desired amine with a methylthiopseudourea
salt or of an aminium salt with cyanamide [46].
2.2. Synthesis of benzene analogues of amiloride and its
N-guanidino substituted derivatives
As the methyl ester of 3,5-diamino-6-chloropyrazine-
2-carboxylic acid was the key intermediate in the pyra-
zine series, the preparation of benzene ring analogues of
amiloride was planned by a similar reaction between the
methyl ester of 2,4-diamino-5-chlorobenzoic acid and

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F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
the required guanidine under related conditions. Several
variations of the benzoic ester structure had also been
foreseen, like amino-protected or nitro compounds, im-
plying additional steps for reduction, protection and
deprotection processes. The use of several activated
esters was also considered in conjunction with protec-
tion/deprotection reactions of the amino substituents.
Moreover, many attempts were carried out, often with
relatively simple benzoic acid analogues. In particular,
the reactions of 2-aminobenzoic acid and 2-amino-5-
chlorobenzoic acid were studied and led to molecules
which were also included in the pharmacological testing.
The most satisfactory chemical pathway involved the
synthesis of a benzoxazinone derivative 14 as key inter-
mediate [47] (figure 3).
The synthesis of the benzene bioisosteres of amiloride
derivatives (17–22) started from 3-chlorotoluene (9)
(figure 3a). After nitration and reduction of both nitro
groups, 2,4-diamino-5-chlorotoluene analogue 11 was
acetylated (12), and then oxidised by KMnO4 to give
Figure 3. Synthesis of benzene analogues of amiloride 16–22, 27–30, and 36–37.

F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
601
2-Acetylamino-5-chloro-4-nitrobenzoic acid (34) was
used in the same sequence of reactions leading to com-
pounds 36 and 37 (figure 3c). The conditions of the
reaction involving benzoxazinone 35 and the guanidine
moiety were very similar. Several alternative synthetic
pathways were also investigated to prepare other ben-
zoylguanidines (figure 4). They included the reaction
between isatoic anhydride 38 and guanidine (figure 4a).
Generally, isatoic anhydride reacts with nucleophilic
reagents but steric hindrance influences the outcome of
the reaction [48]. Indeed, the only positive result was
obtained with unsubstituted guanidine 39. The usual
preparation of guanidine derivatives from cyanamide
and aminium salt was transposed to the acylcyanamide
2,4-diacetylamino-5-chlorobenzoic acid (13). By heating
this latter in the presence of acetic anhydride, cyclo-de-
hydration occurred to give N-(6-chloro-2-methyl-4-oxo-
4H-benzo[d][1,3]oxazin-7-yl)-acetamide (14). This key
intermediate reacted with the appropriate guanidine
leading to the corresponding acetylated carbonyl-
guanidines (15, 16) which underwent acid hydrolysis,
generating the benzene amiloride derivatives substituted
on the distal nitrogen of the guanidine moiety (17–22).
2-Aminobenzoic acid 23 and 2-amino-5-chlorobenzoic
acid 24 were also employed in a reaction with acetic
anhydride in order to produce the corresponding ben-
zoxazinones 25 and 26 which finally reacted with the
various guanidines to give molecules 27–30 (figure 3b).
Figure 4. Synthetic pathways of compounds 39–54.

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F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
Figure 4. (Continued)
40 intermediate [49–52] (figure 4b). This approach gave
the benzoylguanidines 41–43. N,N%-carbonyldiimidazole
(CDI), a carboxylate activating agent, was used for its
high reactivity towards nucleophilic residues [53, 54].
The activated ester reacted with ethylguanidine and led
to compound 45 (figure 4c). The reaction between 2,4-di-
ethoxycarbonylamino-5-chlorobenzoic acid (47) and
ethylguanidine gave the benzoylguanidine 48, but its
deacylation was unsuccessful (figure 4d). N-Hydroxysuc-
cinimide was also used as activating agent of 49 [55], the
resulting activated ester reacted with guanidine and gave
51 (figure 4e). Finally, the Woodward reagent, N-tert-
butyl-5-methylisoxazolium perchlorate (NBI), employed
as activating agent in peptide synthesis [56–58], was
successfully used when it reacted with 2-amino-5-chloro-
4-nitro-benzoic acid (52), but it gave multiple by-prod-
ucts when 2,4-diamino-5-chlorobenzoic acid (46) was the
starting molecule (figure 4f).These alternative methods
were generally less convenient than the ‘benzoxazinone’
pathway (figure 3), and could not be applied to a wide
variety of N-substituted guanidine moieties.
evaluated on RINm5F cells and on human platelets.
The RINm5F cells are rat clonal insulin-producing
cells which express two isoforms of the sodium–cal-
cium exchanger type-1, namely NCX1.3 and NCX1.7
[59]. Human platelets exhibit a sodium–calcium ex-
change activity supported by a K+-dependent carrier
where K+ is transported with Ca2+ [60]. This platelet
exchanger is identical to the sodium–calcium retinal
exchanger RetX [11]. In both cell types, the sodium–
calcium exchange activity was evaluated by measuring
the intracellular Na+-dependent 45Ca2+ uptake in con-
ditions where the sodium–calcium exchanger worked
in reverse mode. The potential inhibitory activity of
the tested compounds was assessed at the concentra-
tion of 10 mM. For the most active molecules in
human platelets, it was determined that their concen-
tration required a decrease of 50% the 45Ca2+ uptake
(IC50 values).
4. Results and discussion
In RINm5F cells (table I), amiloride (1) showed a
weak inhibitory potency on 45Ca2+ uptake. Its pyra-
zine derivatives bearing a hydrophobic substituent on
the guanidine side-chain (2–8) are more potent. The
3. Pharmacology
The sodium–calcium exchanger inhibitory potency
of the novel compounds and the reference drugs were

F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
603
Table I. Inhibition of sodium–calcium exchange mediated 45Ca2+ uptake by RINm5F cells in presence of pyrazine and novel
benzene derivatives (10 mM).
Compound
X
R2
R4
R5
R
Inhibition of 45Ca2+ uptake at 10 mM (%) a
1
N
N
N
N
N
N
N
N
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NHCOCH3
NH2
NH2
NH2
NH2
NH2
NH2
NHCOCH3
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NHCOCH3
NH2
NH2
NH2
NH2
NH2
NH2
H
H
H
H
NO2
NO2
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
11.294.5
48.894.0
30.092.1
37.591.6
25.593.4
54.8925.8
44.194.9
14.593.8
8.892.2
NA
17.394.5
15.395.1
55.1914.1
68.594.8
96.992.4
NA
2
3,4-ClC6H3CH2
C6H5CH2
2,4-ClC6H3CH2
C6H5CH2CH2
1-naphtylCH2
cC8H15
(C6H5)2CH
C2H5
H
3
4
5
6
7
8
16
17
18
19
20
21
22
27
28
29
30
36
37
39
41
42
43
45
48
51
54
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
C2H5
C6H5CH2CH2
C6H5CH2
3,4-ClC6H3CH2
1-naphtylCH2
C2H5
H
NA
C6H5CH2
1-naphtylCH2
H
C2H5
H
C6H5
C6H5CH2
C6H5
C2H5
C2H5
17.692.5
100.094.2
16.696.1
NA
NH2
NHCOCH3
NHCOCH3
NH2
NHCOCH3
NHCOCH3
NH2
NHCOOC2H5
NHCOOC2H5 NHCOOC2H5 Cl
NO2
NH2
NA
37.092.0
31.094.3
16.492.8
NA
11.795.7
8.992.2
100.096.3
H
H
NO2
NO2
Cl
Cl
H
1-naphtylCH2
a Complete inhibition of Na0-stimulated 45Ca2+ uptake is considered as 100%. Mean9S.E.M.
NA: not active at 10 mM.
ing a benzyl (20), a 3,4-dichlorobenzyl (21) or a
highest inhibitory activity was observed when the
substituent was a 3,4-dichlorobenzyl (2), a 2,4-
dichlorobenzyl (4), a 1-naphthylmethyl (6) or a cy-
clooctyl (7). This is in agreement with published
qualitative structure–activity relationships [5]. The
absence of chlorine atoms on the benzyl moiety did
not result in a total loss of inhibition (3). The bioisos-
teric replacement of the pyrazine by a benzene ring
did not disrupt the established structure–activity rela-
tionships, and a similar range of activity was observed
(table I). The benzene counterpart of amiloride 17
was inactive at 10 mM. The benzene derivatives bear-
1-naphthylmethyl (22) were more potent than their
pyrazine counterpart (compare 2–21, 3–20 and 6–
22). A short alkyl group on the guanidine side-chain
(18) strongly reduced the biological activity. These
results indicated that, to preserve the inhibitory activ-
ity, the distal nitrogen of the guanidine had to be
substituted with a hydrophobic aromatic or cy-
clooctyl ring. When using a linker, a methylene was
better than an ethylene link which strongly reduced
the inhibitory potency whatever the nature of tetra-
substituted ring (compare 3–5 and 19–20).

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F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
Among the other benzene derivatives (27–54), the
For human platelets (table II), the complete inhibi-
tion of the RetX type sodium–calcium exchanger was
considered as the inhibition of the 45Ca2+ uptake in
presence of 10 mM 3,4-dichlorobenzamil (2), the refer-
ence inhibitor. In the pyrazine series, the best results
occurred with amiloride derivatives bearing on the
guanidine side-chain a 2,4-dichlorobenzyl (4), a 1-
naphthylmethyl (6) or a diphenylmethyl (8). Except
for 8 and the benzyl derivative (3), these results are
consistent with those obtained with the RINm5F
cells. At 10 mM, amiloride (1) was not active on
platelet 45Ca2+ uptake.
two molecules bearing a 1-naphtylmethyl (30 and 54)
completely inhibited the 45Ca2+ uptake. When com-
pared with the potency of 22, this suggested that the
R4-amino is not required to keep a high inhibitory
potency. Indeed, it could be omitted (30) or replaced
by a nitro (54). Here again, the replacement of the
R-phenyl (41) or the R-benzyl (42) by an ethyl (27)
deleted the biological activity. Other molecules bear-
ing an ethyl- or an unsubstituted-guanidine side-chain
appeared as poor inhibitors (36, 48, 51) or are inac-
tive (28, 37, 39, 45).
Table II. Inhibition of sodium–calcium exchange mediated 45Ca2+ uptake by human platelets in presence of pyrazine and novel
benzene derivatives (10 mM).
Compound
X
R2
R4
R5
R
Inhibition of 45Ca2+ uptake at 10 mM (%) a
1
N
N
N
N
N
N
N
N
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH-COCH3
NH2
NH2
NH2
NH2
NH2
NH2
H
H
H
H
NO2
NO2
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
NA
100.093.4
NA
2
3,4-ClC6H3CH2
C6H5CH2
2,4-ClC6H3CH2
C6H5CH2CH2
1-naphtylCH2
cC8H15
(C6H5)2CH
C2H5
H
3
4
124.1942.0
23.693.6
91.694.6
21.393.0
118.093.5
NA
13.491.8
35.591.0
23.793.0
47.692.7
25.193.5
NA
5
6
7
8
16
17
18
19
20
21
22
27
28
29
30
36
37
39
41
42
43
45
48
51
54
CH NHCOCH3
CH NH2
CH NH2
CH NH2
CH NH2
CH NH2
CH NH2
CH NHCOCH3
CH NH2
CH NH2
C2H5
C6H5CH2CH2
C6H5CH2
3,4-ClC6H3CH2
1-naphtylCH2
C2H5
NA
H
13.490.3
13.293.2
69.493.0
NA
NA
NA
9.091.3
NA
25.390.9
NA
NA
NA
C6H5CH2
1-naphtylCH2
H
C2H5
H
C6H5
C6H5CH2
C6H5
C2H5
C2H5
CH NH2
CH NHCOCH3
CH NHCOCH3
CH NH2
CH NHCOCH3
CH NHCOCH3
CH NH2
CH NHCOOC2H5
CH NHCOOC2H5 NH-COOC2H5 Cl
CH NO2
CH NH2
H
H
NO2
NO2
Cl
Cl
H
1-naphtylCH2
110.0913.6
a Inhibition of 45Ca2+ uptake with 10 mM of 3,4-dichlorobenzamil (2) was considered as 100%. Other drugs were used at 10 mM.
Mean9S.E.M. NA=not active at 10 mM.

F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
605
extracellular Na+ (table III). In the pyrazine series,
the 2,4-dichlorobenzyl (4, IC50 =11.2 mM) and the
1-naphtylmethyl derivatives (6, IC50 =15.1 mM) were
as potent as 3,4-dichorobenzamil (2, IC50 =15.2 mM).
The diphenylmethyl compound (8, IC50 =25.5 mM)
was less active than 3,4-dichlorobenzamil, but 27
times more potent than amiloride (IC50 =690 mM).
The benzene derivative 54, bearing a 1-naphtylmethyl
on the guanidine side-chain and where the R4-amino
was substituted by a nitro, was the most potent in-
hibitor (IC50 =3.4 mM), four times more active than
3,4-dichlorobenzamil, the reference compound.
Figure 5. Concentration-response relationships of 3,4-
dichlorobenzamil (2, ꢀ) and 57 (ꢁ).
5. Conclusion
In conclusion, among the novel derivatives related
to the structure of amiloride, several compounds in-
hibited the sodium–calcium exchange activity. Many
structures were found to be active as inhibitors of the
sodium–calcium exchanger of both NCX and RetX
types. According to the pKa value of the acyl-
guanidine moiety of amiloride (pKa =8.7), 3 (pKa =
8.1) and 5 (pKa =8.2) [36] which are weak inhibitors,
the protonated acylguanidine side-chain is probably
not the biologically active species at physiological pH
(7.4). The results obtained also highlighted that small
structural differences in the new compounds allowed
modulation of the inhibitory activity of both ex-
changer types. For instance, the replacement of the
NO2 (54) by a NH2 (22) in position 4 did not modify
the biological response of the NCX sodium–calcium
exchanger (table I), while it did suppress the activity
on the RetX subtype (table II). This substitution
reduced the ability of the drug to behave like a
H-bond donor for the RetX exchanger, and probably
could explain this loss of activity. These novel phar-
macological tools will certainly be useful to elucidate
the structure–activity relationships and to lead to the
discovery of specific inhibitors of the sodium–calcium
exchanger.
Table III. Drug concentration reducing of 50% (IC50) the
3,4-dichlorobenzamil-sensitive 45Ca2+ uptake by human
platelets.
a
Compound
IC50 (mmol)
1
690919
15.292.6
11.291.0
15.192.9
25.592.2
3.490.7
2
4
6
8
54
a Mean9S.E.M.
The replacement of the pyrazine by a benzene ring
resulted in a loss of activity for the 3,4-dichlorobenzyl
and for the 1-naphtylmethyl derivatives (compare 2–
21 and 6–22). Similar sodium–calcium exchanger in-
hibitory activity of the other benzene derivatives
(16–54) was observed with RINm5F cells and human
platelets. While many compounds were equally active
on both cellular models, some of them (e.g. 22)
showed a preferential activity for one subtype ex-
changer NCX or RetX. Such differences between the
two cellular models could be attributed to differences
in the molecular structures of the two sodium–cal-
cium exchanger type.
The most active compounds (4, 6, 8, 54) on human
platelets were selected to determine their concentra-
tion-response potency and compared to both refer-
ences, amiloride (1) and 3,4-dichlorobenzamil (2)
(figure 5). These curves led to determination of the
concentration which decreased by 50% the 45Ca2+
uptake (IC50) by human platelets in the absence of
6. Experimental protocols
6.1. Chemistry
Melting points were determined on a Bu¨chi–Tottoli
apparatus and were uncorrected. IR spectra were
recorded as KBr pellets on a Perkin–Elmer 1750 FT

606
F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
spectrophotometer. The 1H-NMR spectra were recorded
on a Bruker AW-80 (80 MHz) instrument in CDCl3 or
in DMSO-d6 with HMDS as an internal standard or on
a Bruker AW-400 (400 MHz) instrument in DMSO-d6
with TMS as an internal standard. Chemical shifts are
reported in l values (ppm) relative to internal HMDS or
TMS. The abbreviations, e.g. s=singlet, d=doublet,
t=triplet, q=quadruplet, m=multiplet, and br. s=
broad signal are used throughout. For most amiloride
analogues, the high number of protons bound to nitro-
gen atoms and the hydrate form increase the occurrence
of exchange and greatly influence the shift values which
depend on the sample concentration and temperature.
Elemental analyses (C, H, N) were carried out on a
Carlo-Erba EA 1108-elemental analyser and were within
0.4% of the theoretical values. All reactions were rou-
tinely checked by TLC on silica gel Merck 60F 254
plates.
6.1.3. 2,4-Diacetylamino-5-chlorotoluene (12)
The title compound was prepared from 2,4-diamino-
5-chlorotoluene (11) according to a procedure previ-
ously described [61] (yield: 80%): m.p. 260 °C; 1H-NMR
(80 MHz, DMSO-d6): l 2.04 (s, 3H, CH3), 2.09 (s, 3H,
CH3), 2.15 (s, 3H, CH3-phenyl), 7.18 (s, 1H, 6H-
phenyl), 7.80 (s, 1H, 3H-phenyl), 9.01 (s, 1H, NH), 9.17
(s, 1H, NH); IR (KBr): 3231, 1661, 1526, 1282, 880, 729
cm−1. Anal.: C11H13N2ClO2: (C, H, N).
6.1.4. 2,4-Diacetylamino-5-chlorobenzoic acid (13)
In a round-bottom flask, a mixture of MgSO4 (3 g,
0.025 mol) and 12 (10 g, 0.04 mol) in H2O (200 ml) was
stirred and heated at 80 °C. KMnO4 (16 g, 0.1 mol) was
added portionwise and the mixture was stirred at 85–
90 °C for 1 h. The exceeding KMnO4 was then reduced
using NaHSO3; insoluble MnO2 was removed by filtra-
tion of the hot mixture and washed with hot H2O (2×50
ml). The combined filtrates were acidified with 20%
H2SO4, and the crude compound 13 precipitated. After
standing at 4 °C overnight, it was filtered, washed with
H2O and dried. Crystallisation from ethyl alcohol gave
the title compound as white crystals (6.75 g, 60%): m.p.
The main pathways leading to amiloride 1 and its
pyrazine derivatives (2–8) have been described [43–45],
and the preparation of aralkylguanidine compounds has
previously been reported [46].
6.1.1. 5-Chloro-2,4-dinitrotoluene (10)
1
The title compound was prepared as previously de-
263–265 °C; H-NMR (80 MHz, CDCl3+DMSO-d6): l
1
scribed [61, 62] (yield: 45%): m.p. 88–89 °C; H-NMR
2.13 (s, 3H, CH3), 2.16 (s, 3H, CH3), 7.99 (s, 1H,
3H-phenyl), 9.23 (s, 2H, 6H-phenyl+NH), 11.23 (s, 1H,
NH); IR (KBr): 3416, 3133, 2938, 2686, 1835, 1610,
1583, 1535, 1494, 1401, 877, 823, 793 cm−1. Anal.:
C11H11N2ClO4: (C, H, N).
(80 MHz, CDCl3) l 3.70 (s, 3H, CH3), 7.60 (s, 1H,
6H-phenyl), 8.61 (s, 1H, 3H-phenyl); IR (KBr) 3422,
1607, 1583, 1525, 1373, 1343, 834, 740 cm−1. Anal.:
C7H5N2ClO4: (C, H, N).
6.1.2. 2,4-Diamino-5-chlorotoluene (11) (adapted from
Ref. [61])
6.1.5. N-(6-Chloro-2-methyl-4-oxo-4H-benzo[d][1,3]-
oxazin-7-yl)-acetamide (14)
Portionwise 5-chloro-2,4-dinitrotoluene (10) (15 g,
0.07 mol) was added to stirred HCl 12N (100 ml). Tin
chloride (100 g) was dissolved in HCl 12N (210 ml) and
added dropwise to the hydrochloric solution of 10 at
40 °C for about 90 min. After an additional 1 h stirring
at 70–75 °C, most of the hydrochloric acid was evapo-
rated under reduced pressure and the residue was di-
luted with 30% NaOH (50 ml). The alkaline suspension
was continuously extracted by CHCl3 overnight. The
organic phase was washed with H2O, dried, filtered and
the solvent was evaporated under reduced pressure to
yield a brownish residue. Crystallisation from toluene
afforded the title compound 11 as yellow needles (5.4 g,
A mixture of ethyl acetate (5 ml), acetic anhydride (10
ml) and 13 (1 g, 0.004 mol) was refluxed for 3.5 h. The
mixture was then evaporated under reduced pressure.
The white residue formed was crystallised from ethyl
acetate to give the title compound 14, which was filtered,
washed with petroleum ether 40–60 °C, and dried (0.65
1
g, 70%): m.p. 193–195 °C; H-NMR (80 MHz, DMSO-
d6) l 2.18 (s, 3H, CH3), 2.30 (s, 3H, CH3), 8.14 (s, 1H,
8H), 8.25 (s, 1H, 5H), 9.69 (s, 1H, NH amide); IR
(KBr) 3047, 1769, 1646, 1541, 1270, 1212 cm−1. Anal.:
C11H9N2ClO3: (C, H, N).
1
50%): m.p. 119–120 °C; H-NMR (80 MHz, CDCl3) l
6.1.6. (2,4-Diacetylamino-5-chlorobenzoyl)guanidine (15)
Under nitrogen atmosphere and stirring, NaH (0.8 g
of a 60% dispersion, 0.02 mol) was added to a mixture
of guanidine hydrochloride (1.91 g, 0.02 mol) and
1.96 (s, 3H, CH3), 3.44 (br. s, 4H, NH2), 6.04 (s, 1H,
3H-phenyl), 6.80 (s, 1H, 6H-phenyl); IR (KBr) 3452,
3361, 1629, 1585, 1433, 1296, 1251 cm−1. Anal.:
C7H9ClN2: (C, H, N).

F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
607
dimethylformamide (10 ml), and the resulting mixture
6.1.9. N-(2,4-Diamino-5-chlorobenzoyl)-N%-
ethylguanidine dihydrochloride hydrate (18)
was heated at 50 °C for 20 min. After cooling to room
temperature (r.t.), N-(6-chloro-2-methyl-4-oxo-4H-ben-
zo[d][1,3]oxazin-7-yl)-acetamide (14) (2.53 g, 0.01 mol)
was added and the mixture stirred for 30 min. After-
wards, it was diluted with H2O (60 ml) and crude
product 15 precipitated. It was collected, washed with
H2O, and dried. It was dissolved in HCl 6N with
charcoal. The suspension was filtered and the filtrate
diluted with 10% NaOH. After standing at 4 °C for 1 h,
the crystalline compound 15 was filtered, washed with
H2O, and dried (1.71 g, 55%): m.p. 263–265 °C (decom-
A suspension of 16 (1 g, 0.003 mol) in methanol (25
ml) was bubbled with HCl and stirred for 1.5 h. Then,
diethyl ether (50 ml) was added and a precipitate was
formed. After cooling for 2 h, the crystalline compound
18 was collected, washed with diethyl ether, and dried
1
(0.3 g, 30%): m.p. 192–197 °C; H-NMR (400 MHz,
DMSO-d6) l 1.18 (t, 3H, CH2ꢀCH3), 3.34 (m, 2H,
CH2ꢀCH3), 5.90 (br. s, guanidine), 6.11 (s, 1H, 3H-
phenyl), 8.06 (s, 1H, 6H-phenyl), 8.72 (br. s, 2H, 4-
NH2), 9.02 (br. s, 2H, 2-NH2), 9.49 (s, 1H, CONH); IR
(KBr) 3347, 3292, 3024, 1681, 1631, 1610, 1542, 1475,
1264, 889 cm−1. Anal.: C10H14N5ClO·2HCl·H2O: (C, H,
N).
1
position); H-NMR (80 MHz, DMSO-d6) l 2.02 (s, 3H,
CH3), 2.05 (s, 3H, CH3), 7.42 (br. s, 4H, guanidine), 8.04
(s, 1H, 3H-phenyl), 8.84 (s, 1H, 6H-phenyl), 9.41 (s, 1H,
NH amide), 12.79 (s, 1H, NH amide); IR (KBr) 3398,
3360, 1656, 1590, 1545, 1339, 1304, 869 cm−1. Anal.:
C12H14N5ClO3: (C, H, N).
6.1.10. N-(2,4-Diamino-5-chlorobenzoyl)-N%-
(2-phenylethyl)guanidine dihydrochloride hydrate (19)
Under nitrogen atmosphere and stirring, NaH (0.2 g
of a 60% dispersion, 0.005 mol) was added to a mixture
of (2-phenylethyl)guanidine hydrochloride (1 g, 0.005
mol) and tetrahydrofuran (30 ml), and the resulting
mixture was heated at 50 °C for 20 min. After cooling
to r.t., N-(6-chloro-2-methyl-4-oxo-4H-benzo[d][1,3]-
oxazin-7-yl)-acetamide (14) (1.26 g, 0.005 mol) was
added, then the insoluble matter was discarded by filtra-
tion and the filtrate was evaporated under reduced
pressure. The residue was dissolved in methanol (25 ml)
and HCl bubbled. The precipitate formed (19) was
collected, washed with diethyl ether, and dried (0.31 g,
6.1.7. N-(2,4-Diacetylamino-5-chlorobenzoyl)-
N%-ethylguanidine (16)
The title compound was prepared from 14 and ethyl-
guanidine sulphate as described for 15. The crude
product 16 was dissolved in a mixture of ethyl alcohol–
dimethylformamide (3:1), and stirred with charcoal. Af-
ter filtration, 16 was precipitated by water addition. 16
was filtered, washed with H2O, and dried (yield 25–
35%): m.p. 226–228 °C; 1H-NMR (80 MHz, DMSO-d6)
l 1.07 (t, 3H, CH2ꢀCH3), 2.02 (s, 3H, CH3), 2.05 (s, 3H,
CH3), 3.18 (m, 2H, CH2ꢀCH3), 7.76 (br. s, 4H,
guanidine), 8.08 (s, 1H, 3H-phenyl), 8.84 (s, 1H, 6H-
phenyl), 9.41 (s, 1H, NH amide), 10.54 (s, 1H, NH
amide); IR (KBr): 3384, 3264, 1677, 1602, 1582, 1502,
1333, 1261 cm−1. Anal.: C14H18N5ClO3: (C, H, N).
1
15%): m.p. 172–177 °C; H-NMR (400 MHz, DMSO-
d6) l 2.90 (t, 2H, NHꢀCH2ꢀCH2-phenyl), 3.56 (m, 2H,
NHꢀCH2ꢀCH2-phenyl), 5.37 (br. s, guanidine), 6.09 (s,
1H, 3H-phenyl), 7.22–7.41 (m, 5H, NHꢀCH2ꢀCH2-
phenyl), 8.02 (s, 1H, 6H-phenyl), 8.82 (br. s, 2H, 4-
NH2), 9.04 (br. s, 2H, 2-NH2), 9.49 (s, 1H, CONH); IR
(KBr) 3329, 3289, 3028, 1683, 1629, 1607, 1543, 1520,
1466, 1263, 893, 699 cm−1
. Anal.: C16H18N5ClO·
6.1.8. (2,4-Diamino-5-chlorobenzoyl)guanidine
dihydrochloride (17)
2HCl·H2O: (C, H, N).
A suspension of 15 (1 g, 0.003 mol) in methanol (25
ml) was bubbled with HCl and stirred for 1 h. After
cooling for 2 h, the crystalline compound 17 was col-
lected, washed with diethyl ether, and dried (0.43 g,
6.1.11. N-(2,4-Diamino-5-chlorobenzoyl)-N%-
benzylguanidine dihydrochloride (20)
The title compound was prepared from 14 and ben-
zylguanidine hydrochloride as described for 19 (yield
1
45%): m.p.>300 °C; H-NMR (400 MHz, DMSO-d6) l
1
6.12 (s, 1H, 3H-phenyl), 7.04 (br. s, guanidine), 8.00 (s,
1H, 6H-phenyl), 8.39 (br. s, 2H, 4-NH2), 8.62 (br. s, 2H,
2-NH2); IR (KBr) 3376, 3306, 1676, 1634, 1572, 1538,
1501, 1273, 894, 721 cm−1. Anal.: C8H10N5ClO·2HCl:
(C, H, N).
10–25%): m.p. 300–302 °C (decomposition); H-NMR
(400 MHz, DMSO-d6) l 4.60 (d, 2H, NHꢀCH2-phenyl),
6.10 (s, 1H, 3H-phenyl), 6.31 (br. s, guanidine), 7.31–
7.46 (m, 5H, NHꢀCH2-phenyl), 8.05 (s, 1H, 6H-phenyl),
8.93 (br. s, 2H, 4-NH2), 9.17 (br. s, 2H, 2-NH2), 9.85

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F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
(br. s, 1H, CONH); IR (KBr) 3300, 3290, 3023, 1677,
mol) in dimethylformamide (10 ml), NaH (0.8 g of a
60% dispersion, 0.02 mol) was added, and the mixture
was heated at 50 °C for 20 min. After cooling to r.t.,
2-methyl-4H-3,1-benzoxazin-4-one (25) (1.61 g, 0.01
mol) was added and the mixture stirred for 1 h. After-
wards, it was poured on H2O (40 ml), the precipitate
formed (27) was collected, washed with H2O, and dried.
It was purified by dissolution in ethyl alcohol with
charcoal. After filtration, 27 precipitated by water addi-
tion (1 g, 40%); m.p. 174–178 °C; IR (KBr) 3343, 3184,
1672, 1617, 1593, 1301, 756 cm−1. Anal.: C12H16N4O2:
(C, H, N).
1632, 1609, 1539, 1465, 1262, 880 cm−1
. Anal.:
C15H16N5ClO·2HCl: (C, H, N).
6.1.12. N-(2,4-Diamino-5-chlorobenzoyl)-N%-
(3,4-dichlorobenzyl)guanidine dihydrochloride hydrate
(21)
The title compound was prepared from 14 and (3,4-
dichlorobenzyl)guanidine hydrochloride as described for
19 (yield 15–20%): m.p. 298–300 °C (decomposition);
1H-NMR (400 MHz, DMSO-d6)
l 4.62 (d, 2H,
NHꢀCH2ꢀC6H3Cl2(3,4)), 5.80 (br. s, guanidine), 6.11 (s,
1H, 3H-phenyl), 7.40 (q, 1H, 6%HꢀC6H3Cl2(3,4)), 7.67
(d, 1H, 5%HꢀC6H3Cl2(3,4)), 7.70 (d, 1H, 2%HꢀC6H3Cl2-
(3,4)), 8.05 (s, 1H, 6H-phenyl), 8.98 (br. s, 2H, 4-NH2),
9.16 (br. s, 2H, 2-NH2), 9.94 (s, 1H, CONH); IR (KBr)
3341, 3296, 3024, 1693, 1632, 1609, 1544, 1476, 1263,
885, 824, 713 cm−1. Anal.: C15H14N5Cl3O·2HCl·H2O:
(C, H, N).
6.1.17. (2-Amino-5-chlorobenzoyl)guanidine
dihydrochloride (28)
Under nitrogen atmosphere and stirring, NaH (0.8 g
of a 60% dispersion, 0.02 mol) was added to a mixture
of guanidine hydrochloride (1.91 g, 0.02 mol) and
dimethylformamide (10 ml), and the resulting mixture
was heated at 50 °C for 20 min. After cooling to r.t.,
6-chloro-2-methyl-4H-3,1-benzoxazin-4-one (26) (1.97 g,
0.01 mol) was added and the mixture stirred for 30 min.
Afterwards, it was diluted with H2O (80 ml), the precip-
itate formed was filtered, washed with H2O, and dried
(1.36 g, 50%). Crude (2-acetylamino-5-chloroben-
zoyl)guanidine hydrate was suspended in methanol (15
ml) and HCl was bubbled. After 2 h, the precipitate 28
was collected, washed with diethyl ether, and dried
(yield 80–85%): m.p.>300 °C; IR (KBr) 3277, 2807,
6.1.13. N-(2,4-Diamino-5-chlorobenzoyl)-N%-
(1-naphtylmethyl)guanidine dihydrochloride hydrate (22)
The title compound was prepared from 14 and (1-
naphthylmethyl)guanidine hydrochloride as described
for 19 (yield 15–25%): m.p. 184–188 °C (decomposi-
1
tion); H-NMR (400 MHz, DMSO-d6) l 5.08 (d, 2H,
NHꢀCH2-naphtyl), 5.66 (br. s, guanidine), 6.10 (s, 1H,
3H-phenyl), 7.52–8.07 (m, 8H, napthyl+6H-phenyl),
9.05 (br. s, 2H, 4-NH2), 9.26 (br. s, 2H, 2-NH2), 9.81 (s,
1H, CONH); IR (KBr) 3307, 3154, 2926, 1691, 1624,
1540, 1520, 1269, 888, 724 cm−1. Anal.: C19H20N5ClO·
2HCl·H2O: (C, H, N).
1721, 1695, 1623, 1544, 1291, 837 cm−1
. Anal.:
C8H9N4ClO·2HCl: (C, H, N).
6.1.14. 2-Methyl-4H-3,1-benzoxazin-4-one (25)
The title compound was prepared from 2-aminoben-
zoic acid (23) according to a procedure previously de-
scribed [63] (yield 75%): m.p. 72–75 °C; IR (KBr) 2932,
1757, 1694, 1646, 1299, 1266, 1230, 781 cm−1. Anal.:
C9H7NO2: (C, H, N).
6.1.18. N-(2-Amino-5-chlorobenzoyl)-N%-benzylguanidine
hydrochloride hemihydrate (29)
The title compound was prepared from 26 and ben-
zylguanidine hydrochloride as described for 28 (yield
30–35%). It was crystallised from a mixture of iso-
propanol–methanol (1:2): m.p. 126–131 °C; IR (KBr)
3438, 3032, 1687, 1619, 1575, 1453, 1243, 837 cm−1
.
6.1.15. 6-Chloro-2-methyl-4H-3,1-benzoxazin-4-one
(26)
Anal.: C15H15N4ClO·HCl·0.5H2O: (C, H, N).
The title compound was prepared from 2-amino-5-
chlorobenzoic acid (24) according to a procedure previ-
ously described [64] (yield 75%): m.p. 119–121 °C; IR
(KBr) 3475, 1705, 1657, 1371, 1293, 1230 cm−1. Anal.:
C9H6NClO2: (C, H, N).
6.1.19. N-(2-Amino-5-chlorobenzoyl)-N%-
(1-naphtylmethyl)guanidine hydrochloride (30)
Under nitrogen atmosphere and stirring, potassium
tert-butyl oxide (0.62 g, 0.0055 mol) was added to a
mixture of (1-naphthylmethyl)guanidine hydrochloride
(1.30 g, 0.0055 mol) and dioxane (15 ml), and the
resulting mixture was heated at 50 °C for 15 min. After
6.1.16. N-(2-Acetylaminobenzoyl)-N%-ethylguanidine (27)
To a solution of ethylguanidine sulphate (2.72 g, 0.01

F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
609
cooling to r.t., 6-chloro-2-methyl-4H-3,1-benzoxazin-4-
(KBr) 3428, 1769, 1646, 1541, 1356, 1310, 1270 cm−1
Anal.: C9H5N2ClO4: (C, H, N).
.
one (26) (0.98 g, 0.005 mol) was added and the mixture
stirred for 20 min. Afterwards, the half volume of
solvent was evaporated under reduced pressure, the
residue diluted with cold H2O (80 ml), and the precipi-
tate formed was collected, washed with H2O, and dried.
The crude acetylated derivative was suspended in
methanol (20 ml) and HCl was bubbled. After 2 h., the
precipitate formed was collected, washed with diethyl
ether, and dried. Crystallisation from a mixture of iso-
propanol–methanol (1:1) gave the title compound 30
6.1.24. (2-Acetylamino-5-chloro-4-nitrobenzoyl)-
guanidine hydrochloride (36)
Under nitrogen atmosphere and stirring, potassium
tert-butyl oxide (1.12 g, 0.01 mol) was added to a
mixture of guanidine hydrochloride (0.96 g, 0.01 mol)
and dimethylformamide (6 ml), and the resulting mix-
ture was heated at 50 °C for 30 min. After cooling to
r.t., 6-chloro-2-methyl-7-nitro-4H-3,1-benzoxazin-4-one
(35) (1.2 g, 0.005 mol) was added and the mixture stirred
for 60 min. Afterwards, it was poured on cold H2O (120
ml), and the yellow precipitate formed was collected,
and washed with H2O. It was purified by dissolution in
HCl 6N with charcoal. After filtration, 36 precipitated
by 10% NaOH addition. 36 was filtered, washed with
H2O, and dried. Crystallisation from isopropanol gave
the title compound (0.25 g, 15%): m.p. 250–255 °C
(decomposition); 1H-NMR (400 MHz, DMSO-d6) l
2.21 (s, 3H, CH3), 7.18 (br. s, NH), 8.10 (br. s, NH),
8.30 (s, 1H, 6H-phenyl), 8.87 (br. s, NH), 9.16 (s, 1H,
3H-phenyl), 11.01 (s, 1H, CONH); IR (KBr) 3434,
3027, 1674, 1628, 1542, 1520, 1343 cm−1. Anal.:
C10H10N5ClO4·HCl: (C, H, N).
1
(yield 20–25%); m.p. 199–201 °C; H-NMR (400 MHz,
DMSO-d6) l 5.10 (d, 2H, NH-CH2-naphtyl), 6.87 (d,
1H, 3H-phenyl), 7.32 (q, 1H, 4H-phenyl), 7.52–8.08 (m,
8H, napthyl+6H-phenyl), 8.50 (br. s, guanidine), 9.22
(br. s, 2H, 2-NH2), 9.78 (s, 1H, CONH); IR (KBr) 3431,
3305, 3094, 1690, 1652, 1620, 1573, 1497, 1454, 1239,
824 cm−1. Anal.: C19H17N4ClO·HCl: (C, H, N).
6.1.20. 4-Chloro-2-methyl-5-nitroaniline (32)
The title compound was prepared from 4-chloro-2-
methylaniline (31) according to a procedure previously
described [65] (yield 45–50%): m.p. 121–123 °C; 1H-
NMR (80 MHz, CDCl3) l 2.16 (s, 3H, CH3), 3.84 (s,
2H, NH2), 7.12 (s, 1H, 3H-phenyl), 7.17 (s, 1H, 6H-
phenyl); IR (KBr) 3415, 3336, 1639, 1517, 1356, 1322,
882 cm−1. Anal.: C7H7N2ClO2: (C, H, N).
6.1.21. 4-Chloro-2-methyl-5-nitroacetanilide (33)
The title compound was prepared from 32 according
to a procedure previously described [65, 66] (yield 90–
6.1.25. N-(2-Acetylamino-5-chloro-4-nitrobenzoyl)-
N%-ethylguanidine hydrochloride (37)
1
95%): m.p. 133–137 °C; H-NMR (80 MHz, CDCl3+
Under nitrogen atmosphere and stirring, potassium
tert-butyl oxide (0.27 g, 0.004 mol) was added to a
mixture of ethylguanidine sulphate (0.33 g, 0.002 mol)
and dioxane (13 ml), and the resulting mixture was
heated at 50 °C for 15 min. After cooling to r.t.,
6-chloro-2-methyl-7-nitro-4H-3,1-benzoxazin-4-one (35)
(0.52 g, 0.0022 mol) was added and the mixture refluxed
for 2 h. Afterwards, the dioxane was evaporated under
reduced pressure, the residue was diluted with cold H2O
(75 ml), and the yellow precipitate formed was collected,
washed with H2O, and dried. It was dissolved in ethyl
alcohol, and a HCl saturated diethyl ether solution was
added to give the title compound 37. It was filtered,
washed with diethyl ether, and dried (0.24 g, 30%): m.p.
DMSO-d6) l 2.10 (s, 3H, 2-CH3), 2.25 (s, 3H, CH3
amide), 7.25 (s, 1H, 3H-phenyl), 8.28 (s, 1H, 6H-
phenyl), 8.97 (s, 1H, NH); IR (KBr) 3256, 1664, 1533,
1347, 1301, 894 cm−1. Anal.: C9H9N2ClO3: (C, H, N).
6.1.22. 2-Acetylamino-5-chloro-4-nitrobenzoic acid (34)
The title compound was prepared from 33 according
to a procedure previously described [66] (yield: 35–
1
40%): m.p. 239–243 °C (decomposition); H-NMR (80
MHz, CDCl3+DMSO-d6) l 2.09 (s, 3H, CH3), 8.08 (s,
1H, 6H-phenyl), 8.95 (s, 1H, 3H-phenyl), 11.01 (s, 1H,
NH); IR (KBr) 3247, 3187, 3119, 1684, 1648, 1544,
1520, 1364, 710 cm−1. Anal.: C9H7N2ClO5: (C, H, N).
1
157–158 °C; H-NMR (400 MHz, DMSO-d6) l 1.09 (t,
6.1.23. 6-Chloro-2-methyl-7-nitro-4H-3,1-
benzoxazin-4-one (35)
The title compound was prepared from 34 as de-
scribed for 14 (yield 65–70%): m.p. 149–154 °C; IR
3H, CH2ꢀCH3), 2.10 (s, 3H, CH3), 3.39 (m, 2H,
CH2ꢀCH3), 8.12 (s, 1H, 6H-phenyl), 8.45 (br. s, NH),
8.78 (br. s, NH), 9.18 (s, 1H, 3H-phenyl), 10.81 (s, 1H,

610
F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
CONH); IR (KBr) 3466, 3350, 1679, 1647, 1564, 1533,
1349, 1303, 1226 cm−1. Anal.: C12H14N5ClO4·HCl: (C,
H).
toluene (5 ml), and the resulting mixture was refluxed
for 1.5 h. Afterwards, it was cooled to r.t. and allowed
to stand at 4 °C for 2 h. The precipitate formed was
filtered, washed with petroleum ether 40–60 °C, and
dried. Crystallisation from a mixture of ethyl alcohol–
H2O (1:5) afforded the title compound 41 (0.3 g, 40%):
m.p. 164–166 °C; 1H-NMR (80 MHz, DMSO-d6) l
1.63 (s, 3H, CH3), 7.10 (m, 3H, 3H+5H-phenyl+NH),
7.31 (s, 5H, phenyl), 8.20 (m, 3H, 4H+6H-phenyl+
NH), 9.47 (s, 1H, CONH), 12.32 (s, 1H, NH); IR (KBr)
3364, 3335, 3039, 1661, 1634, 1588, 1552, 1488, 1340,
755, 705 cm−1. Anal.: C16H16N4O2: (C, H, N).
6.1.26. (2-Aminobenzoyl)guanidine dihydrochloride (39)
Under nitrogen atmosphere and stirring, NaH (1.2 g
of a 60% dispersion, 0.03 mol) was added to a mixture
of guanidine hydrochloride (2.88 g, 0.03 mol) and
dimethylformamide (10 ml), and the resulting mixture
was heated at 50 °C for 20 min. After cooling to r.t.,
isatoic anhydride (38) (1.63 g, 0.01 mol) was added
portionwise and the mixture stirred for 3 h. Afterwards,
it was diluted with H2O (50 ml) and extracted three
times by a mixture of acetone–CHCl3 (1:1). The organic
phases were dried and evaporated under reduced pres-
sure. The residue was dissolved in methanol (10 ml) and
HCl was bubbled. After saturation of the solution, it
was diluted with diethyl ether (20 ml) and 39 precipi-
tated. It was collected, washed with diethyl ether, and
dried (0.38 g, 15%): m.p. 267–268 °C; 1H-NMR (80
MHz, DMSO-d6) l 6.05 (br. s, 5H, guanidine), 6.52 (m,
1H, 5H-phenyl), 6.80 (d, 1H, 3H-phenyl), 7.24 (m, 1H,
4H-phenyl), 7.94 (d, 1H, 6H-phenyl), 8.54 (s, 3H, NH+3);
IR (KBr) 3377, 3213, 3033, 1711, 1694, 1556, 1494,
1271, 741 cm−1. Anal.: C8H10N4O·2HCl: (C, H, N).
6.1.29. N-(2-Acetylaminobenzoyl)-N%-benzylguanidine
(42)
The title compound was prepared from benzylamine
as described for 41 (yield 20–25%). It was crystallised
from a mixture of methanol–H2O (1:4): m.p. 139–
142 °C; IR (KBr) 3429, 3345, 3027, 1672, 1631, 1556,
1495, 1352, 1304, 760, 693 cm−1. Anal.: C17H18N4O2: (C,
H, N).
6.1.30. N-(2-Aminobenzoyl)-N%-phenylguanidine
dihydrochloride (43)
Compound 41 (0.3 g, 0.001 mol) and 70% HClO4 (0.5
ml) were suspended in methanol (10 ml), and the sus-
pension was stirred at r.t. After one hour, it was diluted
with H2O (40 ml) and neutralised with NaHCO3. After-
wards, it was concentrated under reduced pressure and
extracted with a mixture of acetone–CHCl3 (1:1) (3×20
ml). The organic phases were dried and evaporated
under reduced pressure. The residue was dissolved in
methanol (15 ml) and HCl was bubbled until 43 precip-
itated. It was collected, washed with diethyl ether, and
dried (0.23 g, 70%): m.p. 224–226 °C; IR (KBr) 3244,
3099, 2875, 1698, 1641, 1598, 1572, 1496, 1305, 753
cm−1. Anal.: C14H14N4O·2HCl: (C, H, N, chlorides).
6.1.27. N-(2-Acetylaminobenzoyl)cyanamide (40)
Under nitrogen atmosphere and stirring, cyanamide
(1.26 g, 0.03 mol) was added to a solution of potassium
tert-butyl oxide (3.37 g, 0.03 mol) in dry tetrahydro-
furan (30 ml), and the resulting mixture was refluxed for
30 min. After cooling to r.t., 25 (1.61 g, 0.01 mol) was
added and the mixture stirred for 30 min. The precipi-
tate formed was collected, washed with tetrahydrofuran
and diethyl ether, and dried. It was dissolved in a
minimum amount of H2O, the solution was adjusted to
pH 1 with HCl 6N, and the precipitate formed (40) was
filtered, washed with H2O, and dried (1.32 g, 65%): m.p.
1
155–157 °C; H-NMR (80 MHz, CDCl3+DMSO-d6) l
6.1.31. N-(2-Ethoxycarbonylaminobenzoyl)-N%-
ethylguanidine (45)
Under nitrogen atmosphere, N-ethoxycarbonylan-
thranilic acid (44) (2.09 g, 0.01 mol) was added to a
solution of CDI (1.62 g, 0.01 mol) in dimethyl-
formamide (30 ml), and the mixture was stirred at r.t.
for 1 h.
2.14 (s, 3H, CH3), 7.06 (m, 1H, 5H-phenyl), 7.57 (m,
3H, 3H+4H-phenyl+NHCN), 8.48 (d, 1H, 6H-phenyl),
10.63 (s, 1H, NH); IR (KBr) 3478, 3154, 2988, 2256,
1666, 1589, 1501, 1431, 1370, 759 cm−1
C10H9N3O2: (C, H, N).
. Anal.:
6.1.28. N-(2-Acetylaminobenzoyl)-N%-phenylguanidine
(41)
Under nitrogen atmosphere and stirring, a solution of
aniline (0.23 g, 0.0025 mol) in dry toluene (5 ml) was
added to a suspension of 40 (0.51 g, 0.0025 mol) in dry
Under nitrogen atmosphere and stirring, NaH (0.8 g
of a 60% dispersion, 0.02 mol) was added to a solution
of ethylguanidine sulphate (2.72 g, 0.01 mol) in
dimethylformamide (15 ml), and the mixture was heated
at 50 °C for 20 min. and then allowed to cool to r.t.

F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
611
Both prepared solutions were mixed and stirred at r.t.
for 1 h. Afterwards, the final mixture was poured on
H2O (200 ml), the precipitate formed was collected and
washed with H2O. It was purified by dissolution in HCl
1N at 40 °C with charcoal. After filtration, the filtrate
was neutralised with NaHCO3. The precipitate formed
(45) was filtered, washed with H2O, and dried (1.11 g,
40%): m.p. 134–136 °C; IR (KBr) 3417, 3354, 3064,
2988, 2901, 1697, 1627, 1602, 1559, 1506, 1458, 1443,
1343, 1249, 762 cm−1. Anal.: C13H18N4O3: (C, H, N).
1330, 1248, 1216, 883, 713 cm−1. Anal.: C16H22N5ClO5:
(C, H, N).
6.1.35. 5-Chloro-2,4-dinitrobenzoic acid (49)
The title compound was prepared from 5-chloro-2,4-
dinitrotoluene (10) according to a procedure previously
described [67] (yield 35–40%): m.p. 180–181 °C; 1H-
NMR (80 MHz, CDCl3+DMSO-d6) l 8.05 (s, 1H,
6H-phenyl), 8.62 (s, 1H, 3H-phenyl); IR (KBr) 3436,
2881, 1719, 1542, 1347, 1301, 840 cm−1
C7H3N2ClO6: (C, H, N).
. Anal.:
6.1.32. 2,4-Diamino-5-chlorobenzoic acid (46)
A solution of 2,4-diacetylamino-5-chlorobenzoic acid
(13) (4 g, 0.015 mol) in NaOH 1N (60 ml) was refluxed
for 5 h. After cooling to r.t., the medium was adjusted
to pH 5 with acetic acid 2N, and a precipitate was
formed. After standing at 4 °C for one hour, it was
collected, washed with H2O, and dried. Crystallisation
from ethyl acetate gave the title compound 46 (2.52 g,
90%): m.p. 170–172 °C; 1H-NMR (80 MHz, DMSO-d6)
l 5.62 (br. s, 2H, 4-NH2), 5.96 (s, 1H, 3H-phenyl), 7.42
(s, 1H, 6H-phenyl), 8.15 (br. s, 3H, 2-NH+3); IR (KBr)
3496, 3382, 3343, 1680, 1626, 1588, 1422, 1290, 1261,
900 cm−1. Anal.: C7H7N2ClO2: (C, H, N).
6.1.36. 1-[(5-Chloro-2,4-dinitrobenzoyl)oxy]-
pyrrolidine-2,5-dione (50)
5-Chloro-2,4-dinitrobenzoic acid (49) (0.74 g, 0.003
mol) was added to a solution of N-hydroxysuccinimide
(0.38 g, 0.0033 mol) in dry tetrahydrofuran (8 ml), and
the mixture was stirred at temperature lowered to −5 to
−10 °C. A solution of DCC (0.68 g, 0.0033 mol) in dry
tetrahydrofuran (3 ml) was added and the resulting
mixture was stirred at r.t. overnight. The suspension was
filtered, the insoluble matter discarded, and the filtrate
evaporated under reduced pressure. The residue was
dissolved in saturated NaHCO3 and extracted with
CHCl3 (4×20 ml). The combined organic phases were
dried and evaporated under reduced pressure. The
residue was crystallised from petroleum ether 100–
140 °C to give the title compound 50 (0.46 g, 45%): m.p.
159–160 °C. Anal.: C11H6N3ClO8: (C, H, N).
6.1.33. 2,4-Diethoxycarbonylamino-5-chlorobenzoic acid
(47)
A mixture of 2,4-diamino-5-chlorobenzoic acid (46)
(1.87 g, 0.02 mol), K2CO3 (2.8 g, 0.02 mol), ethyl
chloroformate (5.7 ml, 0.06 mol) and dioxane (30 ml)
was refluxed for 2 h. Exceeding ethyl chloroformate was
evaporated under reduced pressure and insoluble potas-
sium salts were discarded by filtration. The filtrate was
diluted with H2O (90 ml), the precipitate formed was
collected, washed with H2O, and dried. Crystallisation
from a mixture of ethyl alcohol–H2O (5:1) gave the title
compound 47 (3.31 g, 50%): m.p. 208–212 °C; 1H-
NMR (80 MHz, CDCl3+DMSO-d6) l 1.31 (q, 4H,
CH2ꢀCH3), 4.20 (t, 6H, CH2ꢀCH3), 7.84 (s, 1H, 3H-
phenyl), 7.97 (s, 1H, 6H-phenyl), 9.20 (s, 2H,
NHCOOC2H5); IR (KBr) 3251, 3107, 1733, 1674, 1526,
1422, 1407, 880 cm−1. Anal.: C13H15N2ClO6: (C, H, N).
6.1.37. (5-Chloro-2,4-dinitrobenzoyl)guanidine
hydrochloride (51)
Na (0.115 g, 0.005 mol) was added to a suspension of
guanidine hydrochloride (0.48 g, 0.005 mol) in a mixture
of tetrahydrofuran–ethyl alcohol (1:1) (25 ml), and the
resulting mixture was stirred for 20 min at r.t. A solu-
tion of 1-[(5-chloro-2,4-dinitrobenzoyl)oxy]pyrrolidine-
2,5-dione (50) (1.7 g, 0.005 mol) in tetrahydrofuran (15
ml) was added dropwise, and the mixture refluxed for
1.5 h. Afterwards, the solvents were evaporated under
reduced pressure, the residue triturated with CHCl3 (30
ml), and the insoluble material discarded by filtration.
The filtrate was then extracted with HCl 1N (4×20 ml).
The combined acidic phases were evaporated under
reduced pressure and the residue was suspended in a
mixture of acetone–diethyl ether (3:1). The suspension
was filtered, and then the crystalline compound 51 was
dried (0.49 g, 30%); m.p. 248–249 °C; 1H-NMR (80
MHz, DMSO-d6) l 8.42 (s, 1H, 6H-phenyl), 8.84 (br. s,
6.1.34. N-(2,4-Diethoxycarbonylamino-5-chlorobenzoyl)-
N%-ethylguanidine (48)
The title compound was prepared from 2,4-diethoxy-
carbonylamino-5-chlorobenzoic acid (47) as described
for 45 (yield 20–25%): m.p. 180–184 °C; IR (KBr)
3450, 3410, 3358, 2985, 1727, 1617, 1557, 1486, 1434,

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F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
5H, guanidine+3H-phenyl), 9.19 (s, 1H, CONH); IR
(KBr) 3421, 3352, 3080, 1720, 1585, 1532, 1348, 1278,
891, 559 cm−1. Anal.: C8H6N5ClO5·HCl: (C, H, N).
3475, 3416, 1617, 1580, 1556, 1521, 1472, 1360, 1301,
775 cm−1. Anal.: C19H16N5ClO3: (C, H, N).
6.2. Pharmacology
6.1.38. 2-Amino-5-chloro-4-nitrobenzoic acid (52)
(adapted from Ref. [68])
Ouabain was purchased from Sigma (Brussels, Bel-
gium) and 45CaCl2 from NEN Products (Brussels,
Belgium).
A mixture of 2-acetylamino-5-chloro-4-nitrobenzoic
acid (33) (4 g, 0.015 mol), acetic acid (32 ml) and HCl
12N (32 ml) was refluxed for 1 h. After cooling to r.t.,
it was poured on H2O (200 ml), and the yellow precipi-
tate formed was filtered, washed with H2O, and dried.
Crystallisation from 10% sodium acetate gave the title
compound 52 (2.6 g, 80%); m.p. 268–270 °C; IR (KBr)
3501, 3387, 3069, 1680, 1622, 1583, 1528, 1429, 1363,
1254, 824, 710 cm−1. Anal.: C7H5N2ClO4: (C, H, N).
6.2.1. 45Calcium uptake by RINm5F cells
Cloned insulin-producing RINm5F cells were grown
in RPMI medium supplemented with 10% foetal calf
serum, 2 mM
L
-glutamine, 100 U ml−1 penicillin and
100 mg ml−1 streptomycin (from Life Technologies,
Merelbeke, Belgium).
The incubation medium was a Krebs–Ringer buffered
solution (pH 7.4, 37 °C) bubbled with oxygen (100%),
and containing (in mM): NaCl 139, KCl 5, MgCl2 1,
CaCl2 1, Hepes/NaOH 1. If required, NaCl was iso-os-
motically replaced by sucrose (241 mM) and HEPES/
NaOH by HEPES/KOH, and K+ concentration was
maintained at 5 mM.
6.1.39. N-tert-Butyl-3-[(2-amino-5-chloro-4-nitro-
benzenecarbonyl)oxy]crotonamide (53)
NBI (1.2 g, 0.005 mol) was added to a solution of
2-amino-5-chloro-4-nitrobenzoic acid (52) (1.08 g, 0.005
mol) and triethylamine (0.5 g, 0.005 mol) in dimethylfor-
mamide (5 ml), and the resulting mixture was stirred at
r.t. for 2 h. Afterwards, it was poured on iced water (60
ml), and the precipitate formed was collected, washed
with H2O, and dried. Crystallisation from acetonitrile
gave the title compound 53 (1.51 g, 85%): m.p. 164–
166 °C; IR (KBr) 3506, 3374, 3060, 2967, 2927, 1707,
1682, 1640, 1536, 1437, 1394, 1366, 1343, 1201, 1148,
874, 729 cm−1. Anal.: C15H18N3ClO5: (C, H, N).
Cells (3×106) were removed from plates by trypsine
and centrifuged (500 g, 3 min). The supernatant was
removed, and the cells were incubated at 37 °C in the
Krebs–Ringer buffer (1 ml) in the presence or absence
of drug. After 30 min, the cell suspension was cen-
trifuged (500 g, 3 min) and the pellet was incubated at
37 °C for five min in the same medium (1 ml) contain-
ing 45CaCl2 (200 mCi ml−1). Hence, the effect of these
drugs was evaluated after 30 min exposure. At the end
of the incubation, the uptake of 45Ca was stopped by
addition of 5 ml of the ice-cold Krebs–Ringer solution
containing LaCl3 (2 mM) and albumin (20 mg ml−1).
The pH was adjusted to pH 7.1. The suspension was
centrifuged again 20 min later (500 g, 3 min), and the
radioactive supernatant was discarded. The cells were
suspended in 1 ml of the same ice-cold stop solution.
Aliquots (0.1 ml) of this suspension were then placed in
polyethylene microcentrifuge tubes and centrifuged (30
s, Beckman Microfuge model B) to deposit the cells in
the tip of the tube. After addition of 0.2 ml of a mixture
of di-n-butylphthalate and di-n-nonyl phthalate (10:3), a
second centrifugation (30 s) was performed to separate
the cells from the medium. The bottom of the tube was
cut and transferred to a counting vial containing 5 ml of
scintillation fluid (Lumagel, Lumac, The Netherlands).
The 45Ca2+ content was evaluated using a b-counter
(Packard Tricarb 460C). The uptake of 45Ca2+ was
expressed as femtomoles of Ca2+/106 cells with the
6.1.40. N-(2-Amino-5-chloro-4-nitrobenzoyl)-
N%-(1-naphtylmethyl)guanidine (54)
Under nitrogen atmosphere and stirring, potassium
tert-butyl oxide (0.31 g, 0.00275 mol) was added to a
mixture of (1-naphthylmethyl)guanidine (0.65 g, 0,00275
mol) in dioxane (10 ml) and dimethylformamide (2 ml),
and the mixture was heated at 50 °C for 10 min. After
cooling to r.t., N-tert-butyl-3-[(2-amino-5-chloro-4-ni-
trobenzenecarbonyl)oxy]crotonamide (53) (0.89 g,
0.0025 mol) was added, and the resulting mixture
refluxed for 20 min. Afterwards, dioxane was evapo-
rated under reduced pressure and the residue was di-
luted with H2O (30 ml). The insoluble material was
collected, washed with H2O, and dried. Crystallisation
from ethyl alcohol gave the title compound 54 (0.45 g,
1
45%): m.p. 230–231 °C; H-NMR (400 MHz, DMSO-
d6) l 4.93 (br. s, 2H, CH2-naphtyl), 7.28 (s, 1H, 6H-
phenyl), 7.51–8.06 (m, 10H, naphtyl+3H-phenyl+NH),
8.67 (s, 2H, 2-NH2), 9.49 (s, 1H, CONH); IR (KBr)

F. Rogister et al. / European Journal of Medicinal Chemistry 36 (2001) 597–614
613
specific activity (45Ca/40Ca) of the incubation medium.
The complete inhibition of Na0-stimulated 45Ca2+ up-
take was considered as 100%. The results were expressed
as means S.E.M.
(GraphPad Prism software) and IC50 values were ex-
pressed as means S.E.M.
Acknowledgements
6.2.2. 45Calcium uptake by human washed platelets
Platelet-rich plasma (PRP) provided by the Belgian
Red Cross was washed three times by centrifugation at
1000 g for 10 min in a buffered solution (pH 7.4)
containing (in mM): NaCl 140, KCl 5, glucose 10,
EGTA 0.2, aspirin 0.1, Hepes 10. To the third washing
solution, BSA 0.1% was added, and EGTA was omitted.
Finally, washed platelets were suspended in a buffered
medium with the following composition (in mM): NaCl
137, KCl 2.7, glucose 5.6, MgCl2 1, CaCl2 0.2, NaH2PO4
3.3, HEPES 4, Tris ad pH 7.4. Aliquots (150 ml) of
washed platelets (1–2×106 cells mL−1) were incubated
for 30 min at 37 °C with 800 ml of buffered medium
This study was supported by the Re´gion Wallonne, by
Therabel Research and by a grant from the National
Bank of Belgium. Dr J.F. Lie´geois is Research Associate
of the FNRS (Fonds National de la Recherche Scien-
tifique). We are grateful to M.O. Inarejos, M.L. Pirard,
Y. Janssen, D. Dewalque, A. Van Praet and S.
Counerotte for their technical collaboration. We are
also grateful to Ms Ferrauge (Croix Rouge de Namur)
for her helpful co-operation.
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