Structure-activity relationships of analogues of NF449 confirm NF449 as the most potent and selective known P2X1 receptor antagonist

Article abstract of DOI:10.1016/j.ejmech.2004.01.007

NF449 [4,4′,4″,4′′′-(carbonylbis(imino-5,1,3- benzenetriyl-bis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid-octasodiumsalt)] was recently described to inhibit recombinant rP2X ₁ receptors (Naunyn Schmiedeberg's Arch. Pharmacol. 364 (2001

Full text of DOI:10.1016/j.ejmech.2004.01.007

European Journal of Medicinal Chemistry 39 (2004) 345–357
www.elsevier.com/locate/ejmech
Original article
Structure–activity relationships of analogues of NF449 confirm NF449 as
the most potent and selective known P2X receptor antagonist
1
a,
b
b
a
a
Matthias U. Kassack *, Kirsten Braun , Matthias Ganso , Heiko Ullmann , Peter Nickel ,
a
a
b
Barbara Böing , Gregor Müller , Günter Lambrecht
a
Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
b
Department of Pharmacology, Biocentre Niederursel, University of Frankfurt, Marie-Curie-Strasse 9, 60439 Frankfurt/Main, Germany
Received 17 July 2003; received in revised form 19 January 2004; accepted 19 January 2004
Abstract
NF449 [4,4′,4″,4′′′-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid-octasodiumsalt)] was
recently described to inhibit recombinant rP2X receptors (Naunyn Schmiedeberg’s Arch. Pharmacol. 364 (2001) 285). The purpose of this
1
study was to examine structure–activity-relationships at P2 receptors of a series of NF449 analogues. Thus, compounds containing various
arylaminemono-, di-, or trisulfonic acids and a replacement of the central urea bridge were synthesized. NF449 displayed a pIC50 at P2X1
receptors (rat vas deferens) of 6.31 ± 0.04 being at least 19-fold more potent at P2X than at P2X , P2Y , P2Y , or P2Y . Any deletion or
1
3
1
2
11
change of position of sulfonic acid groups or replacing the central urea bond by the bisamide of terephthalic acid reduced the potency at P2X1
by at least 90%. All compounds were very weak antagonists at P2Y or P2Y receptors (pIC50 < 4.5). In conclusion, NF449 remains the most
2
11
potent and selective P2X antagonist known. Potential lead compounds among the suramin class for P2X (16d) and P2Y (16a) receptors
1
3
1
were identified.
2004 Elsevier SAS. All rights reserved.
©
Keywords: P2X; P2Y; NF449; NF279; NF023; Suramin
1
.. Introduction
profile (CNS and periphery) and are involved in a variety of
physiological functions such as neurotransmission, secre-
tion, vasodilation, urinary bladder control, nociception,
thrombosis induction, proliferation and cell death. Thus, P2
receptors emerge as interesting potential therapeutic targets
[5,7,8]. Three subunits may assemble to build one functional
ATP-gated P2X ion channel [5]. P2X receptors consist of
A series of adenine and uracil 5′-nucleotide (P2) receptors
have been cloned [1–3]. P2 receptors can be divided into
different classes according to their structure (ionotropic P2X
and metabotropic G-protein coupled P2Y receptors) or to
their responsiveness to different nucleotides, i.e., purine or
pyrimidine nucleotides [4,5]. So far, seven P2X and eight
P2Y receptors (including the UDP-glucose receptor) have
been cloned [2,3,5,6]. P2 receptors display a wide expression
homo- or heterooligomers (except P2X which builds heter-
6
omers only) [9] and can be divided into three groups [5]:
P2X and P2X which show rapid activation upon agonist
1
3
stimulation and equally rapid inactivation. The second group
P2X , P2X2/3, P2X2/6, and P2X ) displays rapid activation
(
2
5
Abbreviations: PPADS, pyridoxal-5′-phosphate-6-azophenyl-2′,4′-
disulfonic acid; PPNDS, pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-
nitro-4′,8′-disulfonate); IsoPPADS, pyridoxal-5′-phosphate-6-azophenyl-
but slow inactivation. The third group (P2X1/5, P2X , P2X4/6,
4
and P2X ) are rapidly activated and show both, fast and slow
7
desensitization. P2Y receptors can be divided into subgroups
by structural similarity (P2Y1, 2, 4, 6, 11 and P2Y12, 13, 14) [10],
or by activation by uracil 5′-nucleotides (P2Y2, 4, 6, 14) or
adenine 5′-nucleotides (P2Y1, 2, 11, 12, 13) [5,11] or by cou-
pling to different G-proteins (Gq/11: P2Y1, 2, 4, 6; G and G :
2′,5′-disulfonic acid; BzATP, 2′,3′-O-(4-benzoyl-benzoyl)-ATP; TNP-ATP,
trinitrophenyl-ATP; Ip5I, diinosinepentaphosphate pentasodiumsalt;
abMeATP, a,b-methylene-ATP; ADPbS, adenosine-5′-O-(2-thiodi-
phosphate); ATPcS, adenosine-5′-O-(3-thiotriphosphate); cAMP, cyclic
3′,5′-adenosinemonophosphate.
q
s
*
Corresponding author.
P2Y ; G : P2Y12, 13) [5].
E-mail address: kassack@uni-bonn.de (M.U. Kassack).
11
i
©
2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.01.007

346
M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
SO Na
3
SO Na
3
Cl
O
SO3Na
SO3Na
SO Na
3
NaO3S
NaO3S
NaO S
3
SO Na
3
3
NaO S
a
b
+
Suramin
NO2
NaO3S
N
O
NaO3S
N
O
NaO3S
NH2
H
H
NaO3S
N
O
O
N
SO3Na
H
H
1
a
2 (3-NO2)
(4-NO2)
4 (3-NO2)
5 (4-NO2)
6 (3-NH2)
7 (4-NH2)
3
O
H
N
H
N
O
NO2
NH2
N
H
N
H
SO Na
3
SO Na
3
CH3
O
CH3
NaO3S
NaO3S
N
C
O
1
)
a
b
7
+ 3
NaO3S
N
H
O
NaO S
3
N
H
O
c
H
SO3Na
SO3Na
2)
H
NaO3S
SO Na
3
H
H
N
8
NH2
9
N
NF023
N
N
O
NaO3S
N
O
O
N
SO3Na
O
O
H
H
SO3Na
SO3Na
O
NaO3S
SO3Na
N
N
H
H
COCl2
d
NaO3S
N
O
O
N
SO3Na
2
x 6
H
H
SO Na
3
SO3Na
O
NaO S
3
SO3Na
10 (NF023)
N
N
H
H
NF279
SO3Na
SO3Na
NaO3S
N
H
O
O
N
H
SO3Na
H
H
NaO S
3
3
SO Na
H
H
N
N
COCl2
d
NaO3S
N
H
O
O
N
H
SO3Na
2
x 8
H
H
N
O
N
H
N
H
N
11 (NF279)
O
O
SO3Na
N
O
N
O
O
SO3Na
Fig. 2. Syntheses of compounds 9, NF023 (10), and NF279 (11).
NF449
Reagents/reaction conditions: (a) H O, pH 3.8 (4), pH 3.0 (5 and nitropre-
2
NaO3S
SO Na
3
cursor of 8); 2, 3 dissolved in toluene, 82.6% (4), 85% (5), 75% (nitropre-
N
O
O
N
H
H
O
cursor of 8). (b) Palladium (10%) on charcoal, H O, 98.2% (6), 85% (7),
2
7
4% (8). (c) H O, triethylamine, phenylisocyanate, diethylether, 70.8% (9).
NaO3S
H
N
O
H
N
SO3Na
2
(d) H O, pH 3.5, phosgene (20%) in toluene, 84.6% (10), 84.6% (11).
2
N
H
N
H
O
NaO3S
SO3Na
similarity (rapid activation and inactivation) and may be of
therapeutic importance as drug targets [5], the antagonistic
properties of NF449 analogues were tested at these P2X
receptor subtypes. Further, the selectivity of the test com-
pounds was evaluated at adenine- and uracil-5′- nucleotide
sensitive P2Y receptors, i.e., P2Y , P2Y , and P2Y . Be-
Fig. 1. Structural formula of suramin, NF023 (10), NF279 (11), and NF449
16b).
(
The physiological function of P2 receptors has been ex-
plored with pharmacological and genetic approaches [1,5].
However, this evaluation has been hampered by a lack of
subtype selective ligands, especially selective antagonists.
Our group has provided an important advance by introducing
1
2
11
sides the pharmacological results of the new NF449 ana-
logues at P2X and P2Y receptors, the synthesis and analyti-
cal data of NF023, NF279, NF449 and analogues are
presented which have not been published before.
the potent and selective P2X antagonist NF449 and its
1
“
first-runners” NF023 and NF279 (Fig. 1), as well as PPADS
and PPNDS [12–22]. Other potent P2X antagonists are
1
TNP-ATP and Ip I [5]. The suramin analogues NF023,
2. Chemistry
5
NF279, and NF449 are at least 10-fold selective antagonists
for P2X receptors compared to P2X whereas PPADS is
1
3
Compounds derived from suramin and NF449 (16b) were
synthesized. Variations in the arylsulfonic acid moieties
mono-, di-, or trisulfonic acids of benzene or naphthalene
about twofold selective for P2X , and TNP-ATP is a nano-
1
molar, approximately sevenfold selective antagonist for
(
P2X vs. P2X [5].Very recently, a novel potent and selective
3
1
residues) and of the central urea bridge (replacement of the
urea against the bisamide of terephthalic acid) have been
introduced. Four types of compounds were synthesized: first,
the suramin-like but asymmetric urea 9 (Fig. 2); second, the
suramin-like symmetric ureas NF023 (10) and NF279 (11)
(Fig. 2) which were described earlier but without providing
analytical data [24]; third, NF449 (16b) and other symmetric
ureas containing various arylmono-, di-, or trisulfonic acid
nonnucleotide antagonist of P2X receptors has been re-
3
ported [23]. Except for P2Y and P2Y receptors, no selec-
1
12
tive and potent antagonists are available for P2Y receptors
5,11].
The purpose of this study was a systematic analysis of
[
structural analogues of NF449 at various P2X and P2Y
receptors. Since P2X and P2X receptors show functional
1
3

M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
347
R
N
R
N
O
O
HO
O
Cl
O
O
O
H
O
H
O
R-NH2
SOCl2
a
(1a-e)
H
N
c
H
N
SOCl2
a
15b
OH
Cl
HO
Cl
b
NO2
NO2
R
NO2
R
NH2
HO
Cl
b
O
O
12
13
14a-e
15a-e
O
O
R
R
N
O
O
N
17
18
H
O
H
COCl2
d
H
O
H
2
x 15a-e
N
N
R
N
N
R
R
H
H
O
O
N
H
O
16a-e
R
R
O
H
N
SO3Na
SO3Na
O
H
H
N
N
R
NaO3S
a
d
R
H
N
H
NaO3S
O
SO3Na
SO3Na
19b
b
c
e
N
O
SO3Na
R
Fig. 4. Synthesis of the NF449 (16b) analogue 19b. For residue “R”, refer to
table in Fig. 3. 18 was synthesized from 17 according to standard procedures
and not isolated. Reagents/reaction conditions: (a) 17 (23.3 g, 140 mmol),
toluene (50 ml), DMF (0.1 ml), thionylchloride (40 ml, 550 mmol), heating
under reflux, excessive thionylchloride was removed under vacuum. (b)
NaO3S
SO3Na
Fig. 3. Syntheses of NF449 (16b) and analogues with variations of the
sulfonic acid residues. 13 was synthesized from 12 according to standard
procedures and not isolated. Reagents/reaction conditions: (a) 12 (29.6 g,
H O, pH 4.5, 18 dissolved in toluene, 22.8 % (19b).
2
1
6
40 mmol), toluene (60 ml), DMF (0.2 ml), thionylchloride (50 ml,
90 mmol), heating under reflux, excessive thionylchloride was removed
published [28]. Due to the hygroscopic character of the
synthesized compounds, the water content was estimated by
Karl-Fischer titration analysis. Further, the sodium chloride
content was estimated by titration analysis because sodium
chloride may be present from the synthesis of the carboxam-
ides and ureas. Sodium chloride content in all compounds
was <5% except in the precursor compounds 14a and 15a.
Water content estimated by Karl-Fischer titration was sub-
tracted to evaluate elemental analysis data. Then, all elemen-
tal analysis data were within ± 0.4%, and HPLC purity
was >95% for all compounds.
under vacuum. (b) H O, pH 3.5 (14a, 14b, and 14c), pH 5.0 (14d and 14e),
1
7
2
3 dissolved in toluene, 76% (14a), 33.2% (14b), 58.7% (14c), 85% (14d),
9.6% (14e). (c) Palladium (10%) on charcoal, H O, 76% (15a), 82.5%
2
(15b), 97.7% (15c), 89.4% (15d), 80.8% (15e). (d) H O, pH 4.0 (16a, 16b,
2
16d, and 16e), pH 3.7 (16c), phosgene (20%) in toluene, 54.9% (16a),
5
4.6% (16b), 93.8% (16c), 87.5% (16d), 66.7% (16e).
groups (16a–e, Fig. 3); fourth, replacement of the central
urea bridge in NF449 (16b) by terephthalic acid (17) result-
ing in the bisamide 19b (Fig. 4). Syntheses of carboxylic acid
chlorides (13, 18), carboxamides (4, 5, 8, 14a–e, 19b),
reduction of nitro- to amine-groups (6, 7, 8, 15a–e), and
syntheses of the ureas NF023 (10), NF279 (11), and 16a–e by
use of phosgene in toluene were all performed according to
methods previously published [24,25]. The asymmetric urea
3. Pharmacology
9
was synthesized according to Findeisen et al. [26]and
All target compounds (ureas and bisamide of terephthalic
acid) and the nitro- and amineprecursors of 16a–c (14a–c,
15a–c, Fig. 3) were tested for blockade of various P2X and
P2Y receptors. Test systems included the abmeATP (10 µM)
Petersen et al. [27].
Identity of compounds was confirmed by nuclear mag-
1
13
netic resonance (NMR, H, and C), and IR spectroscopy.
1
Protons and carbons were assigned to their respective H and
-stimulated rat vas deferens (native P2X receptors), the
1
13
C NMR signals as previously performed and published for
abmeATP (1 µM) -stimulated guinea pig ileum (native
other suramin analogues [25]. Further, electron spray mass
spectrometry was performed. All synthesized compounds
were obtained as sodium salts. Electron spray mass spectra
were however monitored in a 5 mM ammonium acetate
P2X ), the ADPbS (10 µM) -stimulated guinea-pig ileum
3
(native P2Y ), UTP or ATP (3 µM) -stimulated HEK293
1
wildtype cells (native P2Y ), and ATPcS (3 µM) -stimulated
2
CHO cells recombinantly expressing human P2Y11 recep-
tors. The P2X , P2X , and P2Y test systems have been
solution in methanol/H O (1:1). Thus, various anions (1–
2
1
3
1
4
negative charges) with up to four sodium ions could be
widely used for the characterization of various test com-
pounds including the suramin analogues NF023 (10), NF279
(11), and NF449 (16b) [13,14,29,30]. The used agonist con-
centrations of abmeATP and ADPbS were their approxima-
tive corresponding EC50 values. HEK293 cells are known to
identified in the mass spectra of all compounds. Purity of
compounds was demonstrated by elemental analysis (C, H,
N) and by using thin layer chromatography (TLC) and a
high-performance liquid chromatography method previously

348
M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
Table 1
1
00
%
Inhibition ± S.E.M. by precursors 14a–c and 15a–c and urea 16e at
various P2X and P2Y receptors
80
60
40
20
0
a
b
c
d
e
Compound P2X1
P2X3
P2Y1
P2Y2
NE
NE
NE
NE
NE
NE
NE
P2Y11
NE
f
1
1
1
4a
4b
4c
9.0 ± 4.9
NE
15.4 ± 1.3 47.7 ± 11
5.8 ± 5.8
5.1 ± 5.0
14.3 ± 4.9
7.8 ± 3.5
NE
UTP
ATP
3.2 ± 1.7
3.6 ± 2.0
1.8 ± 0.4
NE
NE
g
15a
32.9 ± 2.9 35.6 ± 8.5
NE
1
1
1
5b
5c
6e
NE
NE
ND
14.4 ± 1.5
6.5 ± 3.3
16.9 ± 5.9
NE
NE
3.3 ± 2.9
NE
-
10
-9
-8
-7
-6
-5
-4
-3
NE means “no effect at 10 (P2X /P2X ) or 100 (P2Y /P2Y11) µM of test
1
3
2
compound”.
log [UTP or ATP], M
A
ND means “not determined”.
a
Rat vas deferens, 10 µM abmeATP as agonist, 10 µM test compound.
Guinea-pig ileum, 1 µM abmeATP as agonist, 10 µM test compound.
Guinea-pig ileum, 10 µM ADPbS as agonist, 10 µM test compound.
HEK293 wildtype cells, 3 µM UTP or ATP as agonists, 100 µM test
1
00
b
c
d
80
60
40
20
0
compound.
e
Human recombinant receptors expressed in CHO cells, 3 µM ATPcS as
agonist, 100 µM test compound.
f
pIC50 = 5.05 ± 0.06.
pIC50 = 5.03 ± 0.19.
g
showed weak antagonist potency only (Table 1) when tested
at a concentration of 10 µM (P2X , P2Y ) or 100 µM (P2Y ,
1
1
2
P2Y ). pIC values of the corresponding ureas 16a–c, the
1
1
50
-
9
-8
-7
-6
-5
-4
benzene-4-sulfonic acid derivative of NF449, 16d, the
terephthalic acid bisamide analogue of NF449, 19b, along
with pIC50 values of suramin and the suramin analogues
NF023 (10), NF279 (11), and the asymmetric urea 9 are
listed in Table 2. All synthesized compounds (precursors,
ureas, or bisamide of terephthalic acid) were inactive or very
weak antagonists at P2Y or P2Y receptors (Tables 1 and
log [ATPγS], M
B
Fig. 5. Concentration–response curves of UTP and ATP in HEK293
wildtype cells (A) and of ATPcS in CHO cells recombinantly expressing
hP2Y11 receptors (B) using a recently described calcium assay [32]. Maxi-
mum increase in intracellular calcium was set as 100%. EC50 values were as
follows (mean ± S.D.): UTP: 0.96 ± 0.20 µM; ATP: 1.13 ± 0.62 µM; ATPcS:
2
11
0
.41 ± 0.07 µM. Slopes of all curves were not significantly different from
2
). The most potent compound at P2Y11 was suramin
unity.
(pIC50 = 4.73) which is however a non-selective and weak
antagonist. Starting from NF023 (10), the selectivity and
express P2Y and P2Y receptors [31]. However, P2Y re-
ceptors are about 10-fold more sensitive to ATP than P2Y1
1
2
2
potency of the compounds for P2X was increased with
1
NF279 (11) and NF449 (16b) (Table 2) [13,14,16]. For the
sake of a better comparison of potencies, Table 3 displays the
relative potencies of the ureas and bisamide 19b from Table 2
receptors, and UTP is selective for P2Y receptors [5].
2
Fig. 5A shows that ATP and UTP were equipotent at native
P2Y receptors in HEK293 wildtype cells increasing intra-
2
normalized to the activity of NF449 (16b) at P2X receptors.
1
cellular calcium concentration. The same calcium assay [32]
was used to monitore functional activation of P2Y11 recep-
tors recombinantly expressed in CHO cells after agonist
stimulation with ATPcS. Fig. 5B shows a concentration–
response curve of ATPcS at P2Y11 receptors.
Formal exchange of one half of the NF279 (11) molecule by
a benzene residue gave the asymmetric urea 9, and reduced
the activity at P2X by eightfold and further resulted in a loss
1
of P2X vs. P2Y selectivity (Tables 2 and 3). Among the
1
1
analogues of NF449 (16b) with different arylsulfonic acid
moieties (16a, 16c–e), none achieved the potency and selec-
tivity for P2X of NF449 (16b) (Tables 1–3). NF449 dis-
1
4. Results
played a pIC50 at P2X receptors of 6.31 (using 10 µM
1
abmeATP, Table 2) and 7.15 (using 1 µM abmeATP) [13],
Nitro- and amine-precursors 14a–c and 15a–c (10 µM) of
thus being at least 19-fold more potent at P2X than at P2X ,
1
3
the ureas 16a–c did not display appreciable activities at any
of the receptors under investigation (Table 1) except for the
naphthalenetrisulfonic acid derivatives 14a and 15a which
blocked the P2Y receptor with pIC values of 5.05 and
P2Y , P2Y , or P2Y receptors (Table 3). Any deletion or
change of position of sulfonic acid groups (16c–e) led to a
1 2 11
decreased potency at P2X receptors (Tables 1 and 2). How-
1
ever, introducing a naphthalenetrisulfonic acid residue (16a)
instead of benzenemono- or disulfonic acids, led to a certain
P2Y selectivity [pIC (P2Y ): 5.95; pIC (P2X ): 5.32]
1
50
5.03, respectively (Table 1). Even 100 µM 14a–c and 15a–c
did not show any effect at P2Y or P2Y receptors (Table 1).
2
11
1
50
1
50
1
Also, urea 16e containing a benzene-3- sulfonic acid residue
whereas introducing a benzene-4-sulfonic acid moiety in-

M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
349
Table 2
pIC50 ± S.E.M. values of suramin, the asymmetric urea 9, NF023 (10), NF279 (11), NF449 (16b), and analogues of NF449 (16a, 16c–d, and 19b) at various P2X
and P2Y receptors
a
b
c
d
e
Compound
Suramin
9
P2X1
P2X3
P2Y1
P2Y2
P2Y11
h
h
4.68 ± 0.02
4.80 ± 0.01
4.93 ± 0.03
5.71 ± 0.09
5.32 ± 0.02
6.31 ± 0.04
4.40 ± 0.02
4.80 ± 0.01
5.47 ± 0.04
4.36 ± 0.06
4.85 ± 0.08
4.81 ± 0.01
4.52 ± 0.05
4.42 ± 0.01
5.95 ± 0.13
4.85 ± 0.16
5.08 ± 0.04
3.55 ± 0.09
4.39 ± 0.06
4.32 ± 0.15
4.73 ± 0.27
f
ND
<4
<4
<4
h
h
10 (NF023)
4.07 ± 0.20
4.71 ± 0.07
4.87 ± 0.02
5.04 ± 0.08
4.13 ± 0.01
5.77 ± 0.12
5.50 ± 0.02
3.98 ± 0.04
g
g
g
g
g
g
g
11 (NF279)
~4
4.32 ± 0.01
1
1
1
1
1
6a
<4
<4
<4
<4
<4
<4
g
h
6b (NF449)
4.47 ± 0.22
6c
6d
9b
a
<4
<4
<4
Rat vas deferens, 10 µM abmeATP as agonist.
Guinea-pig ileum, 1 µM abmeATP as agonist.
Guinea-pig ileum, 10 µM ADPbS as agonist.
HEK293 wildtype cells, 3 µM UTP or ATP as agonists.
Human recombinant receptors expressed in CHO cells, 3 µM ATP cS as agonist.
ND means “not determined”.
b
c
d
e
f
g
h
Data taken from Damer et al. [14], Braun et al. [13], and Lambrecht et al. [20].
±S.D. instead of S.E.M. values.
stead of aryldi-, or trisulfonic acids, yielded a P2X prefer-
have not yet been published [12–14,16]. In this study, struc-
3
ence of 16d [pIC50 (P2X ): 5.77; pIC (P2X ): 4.80]
tural variations of NF449 (16b), the so far most potent an-
3
50
1
(
Table 2). Replacing the central urea bridge in NF449 (16b)
by the bisamide of terephthalic acid resulting in compound
9b, reduced the activity at P2X by sevenfold and also led to
tagonist at P2X receptors, have been synthesized and bio-
1
logically evaluated at various P2 receptors to unravel
structure–activity relationships. Furthermore, the synthesis
of NF023 (10), NF279 (11), and an asymmetric urea deriva-
tive of NF279, compound 9, and their analytical data are
presented. NF449 (16b) derivatives include comprehensive
variations of the benzene- or naphthalene-mono-, di-, or
trisulfonic acid moieties (Fig. 3) and one variation of the urea
bridge (replacement by the bisamide of terephthalic acid) but
retaining the essential benzenedisulfonic acid residue of
NF449, namely 1b, resulting in compound 19b (Fig. 4).
1
1
a loss of selectivity for P2X vs. P2X receptors (Tables 2 and
3
1
3
).
5
. Discussion
Suramin has long been known to inhibit various P2 recep-
tors [20,33]. However, suramin is neither potent nor selective
for any of the P2X or P2Y receptor subtypes [5,20]. Our
group has introduced NF023 (10), NF279 (11), and NF449
Evaluation at P2 receptors of compounds 16a–e with
arylsulfonic acid variations and the NF449-like linker (5-
nitroisophthalic acid, urea bridge) revealed that even slight
variations of the aniline-2,4-disulfonic acid residue (deletion
or change of position of sulfonic acid groups) resulted in a
massive loss of potency at P2X and/or selectivity for P2X
(16b) as selective and increasingly potent P2X receptor
1
antagonists (Table 2) but analytical data for these compounds
Table 3
1
1
Relative potencies of the ureas from Table 2 normalized to the potency of
1
over P2X or P2Y receptors (Table 3). Furthermore, the
3
1
6b (NF449) at P2X which was set as 100
1
linker region of the NF449 molecule is also sensitive to
P2X1 a
P2X3 b
P2Y1 c
P2Y2 d
P2Y11 e
Compound
Suramin
9
1
1
1
1
1
1
1
variations in terms of P2X potency and selectivity: com-
1
2.3
1.1
3.5
1.0
2.6
pound 19b contains the aniline-2,4-disulfonic acid but the
urea linker from NF449 (16b) has been exchanged against
the bisamide of terephthalic acid (17). This exchange re-
f
3.1
ND
3.2
1.6
1.3
43.7
3.5
5.9
0.2
1.2
<0.5
<0.5
~0.5
<0.5
1.4
<0.5
~0.5
1.0
0 (NF023)
1 (NF279)
6a
4.2
0.6
25.1
10.2
100
1.2
2.5
sulted in a sevenfold loss of potency at P2X and a loss of
1
3.6
<0.5
<0.5
<0.5
<0.5
<0.5
selectivity for P2X vs. P2X : 19b was about equipotent at
6b (NF449)
5.4
1
3
P2X and P2X receptors (pIC = 5.47 and 5.50, respec-
6c
6d
9b
a
0.7
<0.5
<0.5
<0.5
1
3
50
3.1
28.8
15.5
tively, Table 2). Thus, two regions in the NF449 (16b) mol-
ecule have been identified in this study that are critical for a
potent and selective antagonism at P2X1 receptors: the
benzene-2,4-disulfonic acid moieties and the urea linker.
14.5
Rat vas deferens, 10 µM abbmeATP as agonist.
Guinea-pig ileum, 1 µM abmeATP as agonist.
Guinea-pig ileum, 10 µM ADPbS as agonist.
b
c
d
e
A further question to be addressed was whether symmet-
ric ureas or bisamides with two in the case of NF023 (10) and
NF279 (11) or four in the case of NF449 (16b) arylsulfonic
acid residues have to be present to antagonize potently P2X
HEK293 wildtype cells, 3 µM UTP or ATP as agonists.
Human recombinant receptors expressed in CHO cells, 3 µM ATPcS as
agonist.
f
ND means “not determined”.

350
M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
receptors. There is a report from Bültmann et al. [29] that the
compound BSt101, differing from NF023 (10) by removal of
the three sulfonic acid groups from one of the naphthalene
rings, is about equipotent to NF023 (10). The authors con-
clude that a second naphthalene-trisulfonic acid moiety as
present in NF023 (10) may not be a precondition for “rela-
tively high affinity” [29]. However, since both compounds,
NF023 (10) and BSt101, are rather weak antagonists at P2X1
receptors (pIC s < 5), the results of Bültmann et al. may be
9
8
.5
.5
9.5
8.5
7.5
6.5
5.5
4.5
3.5
7.5
6
5
.5
.5
4.5
3.5
5
0
limited to NF023 (10) and BSt101. Furthermore, Lambrecht
et al. [17,30] have introduced the P2 antagonists PPADS and
SB9, hybrid compounds containing arylsulfonic acids and
pyridoxal-5′-phosphate also bearing two anionic moieties. In
the present study, we also found that among our compounds
only those with at least two moieties containing anionic
charges achieved a potent and selective blockade of P2X1
receptors. The replacement of one half of NF279 (11) by a
simple benzene moiety, resulting in the asymmetric urea 9,
led—compared to NF279 (11)—to a eightfold decrease in
P2X potency and loss of selectivity for P2X vs. P2Y
RVD
P2X1
GPI
P2X3
XLO
P2X1
XLO
P2X3
Suramin
NF023 (10)
NF279 (11)
NF449 (16b)
Fig. 6. Comparison of pIC50 values of suramin, NF023 (10), NF279 (11),
and NF449 (16b) at native (RVD, GPI) vs. recombinant (XLO) rat P2X and
rat P2X receptors. Data for RVD/P2X and GPI/P2X are from Table 2, data
1
3
1
3
for XLO/P2X
and P2X are taken from Refs. [13,21,22,34].
3
1
1
1
1
selectivity for P2Y over P2X receptors with a low µM-
potency [pIC50 (P2Y ): 5.95, Table 2]. P2Y antagonists with
nanomolar potency and highly selective for P2Y receptors
are already available: MRS2179 and MRS2279 [5]. How-
ever, so far selective and potent P2Y antagonists among
suramin derivatives have not yet been identified [5,20]. A
P2X -selective compound was also found among the deriva-
tives of NF449 (16b) namely 16d (ninefold selective for
P2X over P2X , 144-fold selective for P2X over P2Y
receptors, pIC50 (P2X ): 5.77, Tables 2 and 3). Besides TNP-
ATP which is about sevenfold selective for P2X over P2X
receptors [5,20], compound A-317491 was recently de-
scribed as a novel potent and selective non-nucleotide an-
tagonist of P2X and P2X receptors (selectivity over other
P2-receptors >100-fold) [23]. PPADS and isoPPADS are
antagonists in the micromolar range, however they show no
selectivity for P2X but rather a small selectivity for P2X
receptors [19]. Thus, 16d can serve as a lead compound for
receptors (Table 3). Furthermore, the precursor compounds
4a–c and 15a–c lacking a second anionic moiety, i.e., aryl-
sulfonic acid residue, compared to their corresponding ureas
6a–c or bisamide 19b turned out to be inactive or very weak
inhibitors at P2X or P2Y receptors (pIC s ≤ 5, Table 1). In
1
3
1
1
1
1
1
1
50
contrast to Bültmann et al., in our study not only the sulfonic
acid groups were removed from one naphthalene ring in
NF279 but also the naphthalene and a further benzene resi-
due resulting in compound 9, and three aromatic residues and
the urea bridge were missing in case of the precursor com-
pounds 14a–c and 15a–c. The removed aromatic residues
may however contribute to P2 receptor blockade. Thus,
asymmetric NF449-derived ureas containing benzene in-
stead of benzene-2,4-disulfonic acid residues or a NF279
analogue where only three sulfonic acid groups are removed
from one of the naphthalene rings are necessary to clarify
whether at least two anionic moieties in a distinct distance
3
3
1
3
1
3
3
1
3
2/3
3
1
are compulsory for potent and selective P2X receptor block-
1
the development of P2X selective antagonists derived from
ade.
3
the suramin/NF449 (16b) family. All compounds tested for
inhibition of ecto-nucleotidases (ecto-N), namely suramin,
NF023 (10), NF279 (11), 16a, NF449 (16b), 16c, and 16d
were weak inhibitors of ecto-N (less than 45% inhibition of
ecto-N at a concentration of 300 µM in folliculated Xenopus
NF023 (10) was the first P2X -selective suramin analogue
1
displaying a sevenfold selectivity for P2X over P2X and a
1
3
2
[
.6-fold selectivity for P2X over P2Y receptors (Table 3)
1 1
16]. An improvement in terms of P2X selectivity and po-
1
tency was achieved by introducing NF279 (11) (Table 3)
14]. NF449 (16b) showed a further improved potency at
P2X receptors and comparable selectivity to NF279 (11)
laevis oocytes) [20], thus confirming the P2X (NF449-16b),
[
1
P2X (16d), or P2Y (16a) selectivity of these three com-
3
1
1
pounds.
(
Table 3) [13]. All suramin analogues tested in this study are
very weak or inactive at UTP and/or ATP preferring
P2Y /P2Y receptors (Tables 1 and 2). At P2Y receptors
P2X and P2X potencies and selectivities for suramin,
1 3
NF023 (10), NF279 (11), and NF449 (16b) have previously
2
11
1
which prefer nucleoside-diphosphates (ADP) over nucle-
osidetriphosphates as agonists, some of the tested com-
pounds displayed an appreciable potency (Tables 2 and 3).
The symmetrical urea 16a with a urea spacer identical to that
of NF449 (16b) but a naphthalene-trisulfonic acid residue
like suramin, NF023 (10) and NF279 (11), showed a surpris-
ing fourfold selectivity for P2Y over P2X and 12-fold
also been evaluated at rat and human P2X and rat P2X
1
3
receptors recombinantly expressed in Xenopus laevis oo-
cytes (XLO) [12,13,21,22,34]. All compounds were 10–
1000-fold more potent in the recombinant XLO assays than
in the native test systems, rat vas deferens (RVD) for P2X1,
and guinea pig ileum (GPI) for P2X (Fig. 6). With increas-
3
ing potency at recombinant rat P2X receptors, the selectivity
1
1
1

M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
351
for P2X over P2X increased as well (Fig. 6). Whereas
500 (500 MHz) spectrometer (Karlsruhe, Germany) using
1
3
1
3
suramin remained approximately equipotent at rat P2X and
DMSO-d as a solvent and D O for H–D exchange. C-
1
6 2
rat P2X receptors in both the physiological RVD/GPI and
NMR spectra were recorded by use of a Varian XL 300
3
the recombinant XLO assays, the selectivity for P2X over
(75 MHz) spectrometer using DMSO-d as a solvent. NMR
1
6
P2X increased in the recombinant system for NF023 (10)
experiments were carried out at 15 °C. NMR chemical shifts
3
(
(
(
RVD/GPI: sevenfold, XLO: 35-fold), NF279 (11)
RVD/GPI: 10-fold, XLO: 85-fold), and for NF449 (16b)
RVD/GPI: 19-fold, XLO: 8710-fold) (Fig. 6) [13,21,22,34].
are reported as d values (ppm) downfield relative to Me Si
4
which was used as internal standard (0 ppm). The following
abbreviations are used: s (singlet), d (doublet), dd (double of
doublet), pt (pseudotriplet), m (multiplet), ar (aromatic), br
The effect of increased potencies at recombinant P2 recep-
tors may be due to depletion of suramin analogues in the
RVD/GPI tissues used for estimation of pIC50 values at
native P2 receptors [13]. However, further experiments are
needed to clarify this issue. A similar apparent loss in po-
tency has been observed for TNP-ATP and IP I which can be
attributed to metabolic breakdown of these nucleotides in
whole tissues [19].
(broad), ex (exchangeable with D O), J (coupling constant in
2
hertz). IR spectra were recorded with
a
FT-IR-
spectrophotometer “Paragon 1000” from Perkin Elmer. Pu-
rity of compounds was checked by a previously published
HPLC method [28]. Briefly, a Hewlett-Packard 1050 series
HPLC apparatus equipped with a Hewlett-Packard MOS-
Hypersil RP-C8 analytical column (5 µM, 100 × 2.1 mm) and
a Hewlett-Packard MOS-Hypersil RP-C8 as precolumn
5
(5 µM, 20 × 2.1 mm) was used. Temperature of the column
was kept at 37 °C. The gradient solvent system consisted of a
mixture of 6.25 mM tetrabutylammonium hydrogensulfate in
6. Conclusion
0
8
4
0
.02 M phosphate buffer pH 6.5 and methanol starting at
0:20. A linear gradient was applied reaching a mixture of
6:54 within 8 min. The flow rate was maintained at
.6 ml/min. Peaks were detected by UV absorption using a
In conclusion, in this study we present the synthesis and
analytical data of the previously described P2X -selective
1
antagonists NF023 (10), NF279 (11), and NF449 (16b)
Figs. 2 and 3). Furthermore, a series of NF449 (16b) ana-
(
Hewlett-Packard 1040A diode array detector.All compounds
tested for biological activity showed ≥95% purity in the
HPLC analysis. Thin layer chromatography (TLC) was per-
formed with all compounds on 20 × 20 cm aluminium sheets
precoated with silica gel 60 F254 (Merck, Darmstadt, Ger-
many). Elution solvent mixture was 2-propanol:ammonia
logues has been synthesized and evaluated at P2X , P2X ,
1
3
P2Y , P2Y , and P2Y receptors (Figs. 3 and 4, Tables 1–3).
1
2
11
New pharmacological data for NF023 (10), NF279 (11), and
NF449 (16b) at P2Y and P2Y receptors are also presented
2
11
(
Table 2). NF449 (16b) remains the most potent so far known
P2X antagonist with submicromolar potency at rat vas def-
1
(25%) = 5:2. TLC confirmed ≥95% purity for all compounds.
erens P2X receptors (Table 2) and with subnanomolar po-
1
Low-resolution ES (electron spray) mass spectra were
tency at recombinant rat P2X receptors (Fig. 6). Any dele-
1
carried out with API2000 Applied Biosystems/MDS SCIEX
LC/MS mass spectrometer fromApplied Biosystems (Darm-
stadt, Germany). Solvent for the measurement was a solution
tions or shifts of the sulfonic acid residues or exchange of the
central urea bridge by a bisamide of terephthalic acid in
NF449 (16b) led to decreased activity at P2X receptors
1
of 5 mM ammonium acetate in a mixture of H O:methanol
2
and/or a loss of P2X selectivity (Tables 2 and 3). Com-
1
(1:1; pH 7). Elemental analyses (C, H, N) were performed on
pounds 16a and 16d differing from NF449 (16b) only in the
sulfonic acid residues showed a fourfold selectivity for P2Y1
a Vario EL apparatus from Elementar (Hanau, Germany).
Melting points of all compounds were greater than 300 °C
(
16a) and a ninefold selectivity for P2X (16d) compared to
3
(Mettler FP 61 apparatus, Giessen, Germany). NaCl content
P2X receptors, respectively (Tables 2 and 3). Thus, potential
1
was estimated by potentiometric titration analysis on a titro-
processor 672 from Metrohm (Herisau, Switzerland) and
found to be between 0.7% and 28.1%. Water content was
measured by Karl-Fischer titration analysis using a Titrino
lead compounds for P2Y and P2X receptors related to
1
3
suramin were identified.
7
01 KT from Metrohm (Herisau, Switzerland). Between
7. Experimental protocols
2 and 16 mol H O per mol of compound were found. Yields
2
are calculated for the pure compounds (without NaCl).
7.1. Chemistry
7
.2. General acylation procedure. Synthesis of nitro
derivatives 4, 5, nitroprecursor of 8, and 14a–e
Suramin, arylaminemono-, di-, and trisulfonic acids were
gifts from Bayer AG (Leverkusen, Germany). All other re-
agents were purchased from Fluka, Aldrich, or Sigma (all:
Taufkirchen, Germany).
7.2.1. 8-(3-Nitrobenzamido)-naphthalene-1,3,5-trisulfonic
acid trisodium salt × 2 H O (4)
2
1
H-NMR spectra were obtained with a Varian T 60
1a (42.7 g, 100 mmol) was dissolved in water (400 ml)
(60 MHz), a Varian XL 300 (300 MHz), and a Bruker DRX
and the pH was adjusted to 3.8. To the stirred solution,

352
M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
3
7
-nitrobenzoylchloride 2 (26.2 g, 141 mmol) dissolved in
5 ml of toluene was slowly added. The reaction mixture was
C–C), 130.8 (ar, C–C), 129.2 (ar, 2 C–H), 128.6 (ar, 2 C–H),
126.5 (ar, C–H), 125.4 (ar, C–H), 124.7 (ar, C–H), 123.2 (ar,
2 C–H), 123.0 (ar, C–C), 122.2 (ar, C–H), 119.2 (ar, 2 C–H).
kept at a constant pH of 3.8 by automatic addition of a 2 M
Na CO solution.After separating the toluene from the water
phase, pH was adjusted to 2.0, and the aqueous phase was
extracted four times with 70 ml diethylether, respectively.
After neutralisation (pH 7.0), the water was removed under
–
1
IR mmax (KBr, cm ): 3448, 1665, 1534, 1333, 1233, 1195,
041. NaCl: 24.6%. TLC: R 0.33. Anal. C H N Na O S
3 13 3
2
3
1
f
24 14
3
(C, H, N).
vacuum. The crude product was purified by recrystallization
7.2.4. 8,8′-(5-Nitro-1,3-benzenediyl-bis(carbonylimino))-
bis(naphthalene-1,3,5-trisulfonic acid) hexasodium salt ×
1
from methanol.Yield: 49.4 g (82.6%). H NMR (DMSO-d ):
6
d 12.91 (br s,1H, NH, ex), 9.41 (d, 1H, ar, J = 1.9 Hz), 8.91
4.5 H O (14a)
2
(
1
pt, 1H, ar, J = 1.9 Hz), 8.62 (d, 1H, ar, J = 2.2 Hz), 8.60 (m,
H, ar), 8.41 (dd, 1H, ar, J = 8.0, 2.2 Hz), 8.09 (d, 1H, ar,
J = 8.2 Hz), 8.04 (d, 1H, ar, J = 7.9 Hz), 7.82 (pt, 1H, ar,
14a was synthesized from 1a and 13 according to Section
1
7.2.1. pH was kept at 3.5.Yield: 76%. H NMR (DMSO-d ):
6
d 12.98 (br s, 2H, NH, ex), 9.43 (d, 2H, ar, J = 2.2 Hz), 9.24
(d, 2H, ar, J = 1.3 Hz), 9.03 (pt, 1H, ar, J = 1.3 Hz), 8.64 (d,
2H, ar, J = 1.9 Hz), 8.12 (d, 2H, ar, J = 8.2 Hz), 8.06 (d, 2H,
1
3
J = 7.9 Hz). C NMR (DMSO-d ): d 163.6 (C–O), 147.9 (ar,
C–N), 143.0 (ar, C–S), 142.8 (ar, C–S), 141.3 (ar, C–S),
6
1
3
ar, J = 8.2 Hz). C NMR (DMSO-d ): d 163.4 (2 C–O),
1
37.4 (ar, C–C), 134.5 (ar, C–H), 133.8 (ar, C–N), 131.5 (ar,
C–C), 130.0 (ar, C–H), 127.0 (ar, C–H), 126.1 (ar, C–H),
25.7 (ar, C–H), 124.9 (ar, C–H), 123.4 (ar, C–C), 123.1 (ar,
6
1
(
47.8 (ar, C–N), 143.0 (ar, 2 C–N), 142.7 (ar, 2 C–S), 141.4
ar, 2 C–S), 137.4 (ar, 2 C–C), 134.3 (ar, 2 C–S), 133.8 (ar,
C–H), 131.5 (ar, 2 C–C), 127.0 (ar, 2 C–H), 126.1 (ar,
1
–1
C–H), 123.0 (ar, C–H). IR mmax (KBr, cm ): 3468, 1632,
534, 1353, 1245, 1193, 1044. NaCl: 1.4%. TLC: R 0.47.
2
2
1
0
C–H), 125.1 (ar, 2 C–H), 124.9 (ar, 2 C–H), 123.5 (ar,
1
f
–
1
C–C), 123.4 (ar, 2 C–H). IR mmax (KBr, cm ): 3444, 3099,
652, 1532, 1342, 1197, 1149, 1044. NaCl: 22.8%. TLC: Rf
.14. Anal. C H N Na O S (C, H, N).
Anal. C H N Na O S (C, H, N).
1
7
9
2
3 12 3
2
8
13
3
6 22 6
7
.2.2. 8-(4-Nitrobenzamido)-naphthalene-1,3,5-trisulfonic
acid trisodium salt × 3 H O (5)
2
7
.2.5. 4,4′-(5-Nitro-1,3-benzenediyl-bis(carbonylimino))-
Compound
5
was synthesized from 1a and
bis(benzene-1,3-disulfonic acid) tetrasodium salt × 3 H O
2
4-nitrobenzoylchloride 3 according to Section 7.2.1. pH was
(
14b)
14b was synthesized from 1b and 13 according to Section
.2.1. pH was kept at 3.5. Purification was done by recrystal-
kept constant at 3.0. Purification of the crude product was
done by washing twice with ethanol (50 ml). Yield: 85%. H
1
7
NMR (DMSO-d ): d 12.86 (br s, 1H, NH, ex), 9.32 (d, 1H, ar,
6
1
lization from water. Yield: 33.2%. H NMR (DMSO-d ): d
1
2
6
J = 1.9 Hz), 8.58 (d, 1H, ar, J = 2.0 Hz), 8.31 (dd, 2H, ar,
J = 8.8, 2.2 Hz), 8.21 (dd, 2H, ar, J = 9.0, 2.2 Hz), 8.13 (d, 1H,
2.00 (br s, 2H, NH, ex), 8.94 (pt, 1H, ar, J = 1.6 Hz), 8.91 (d,
H, ar, J = 1.3 Hz), 8.47 (d, 2H, ar, J = 8.5 Hz), 8.06 (d, 2H,
13
ar, J = 8.3 Hz), 8.01 (d, 1H, ar, J = 8.1 Hz). C NMR
DMSO-d ): d 163.7 (C–O), 148.6 (ar, C–N), 142.5 (ar,
13
ar, J = 2.2 Hz), 7.65 (dd, 2H, ar, J = 8.5, 2.2 Hz). C NMR
(
6
(DMSO-d ): d 161.1 (2 C–O), 148.4 (ar, C–N), 143.0 (ar,
6
C–S), 142.2 (ar, C–C), 141.3 (ar, C–S), 141.0 (ar, C–S),
33.5 (ar, C–N), 131.1 (ar, C–C), 129.2 (ar, 2 C–H), 126.6
ar, C–H), 125.6 (ar, C–H), 124.6 (ar, C–H), 123.0 (ar, C–C),
2
2
2
C–N), 136.6 (ar, 2 C–S), 134.7 (ar, 2 C–S), 134.4 (ar,
C–C), 132.1 (ar, C–H), 127.0 (ar, 2 C–H), 124.8 (ar,
1
(
C–H), 124.2 (ar, 2 C–H), 119.0 (ar, 2C–H). IR m (KBr,
–
1
max
122.9 (ar, 2 C–H), 122.6 (ar, C–H). IR mmax (KBr, cm ):
–1
cm ): 3468, 1696, 1625, 1591, 1540, 1229, 1187, 1040.
NaCl: 1.7%. TLC: R 0.40. Anal. C H N Na O S (C, H,
N).
3468, 1680, 1538, 1511, 1357, 1208, 1045. NaCl: 15.3%.
f
20 11
3
4 16 4
TLC: R 0.43. Anal. C H N Na O S (C, H, N).
f
17
9
2
3
12
3
7
1
.2.3. 8-(4-(4-Nitrobenzamido)-benzamido)-naphthalene-
,3,5-trisulfonic acid trisodium salt × 4 H O (nitroprecur-
7
.2.6. 2,2′-(5-Nitro-1,3-benzenediyl-bis(carbonylimino))-
2
bis(benzene-1,4-disulfonic acid) tetrasodium salt × 3 H O
2
sor of 8)
The nitroprecursor of 8 was synthesized from 7 and
-nitrobenzoylchloride 3 according to Section 7.2.1. pH was
kept at 3.0. The crude product was purified by washing twice
(
14c)
1
4c was synthesized from 1c and 13 according to Section
4
7
.2.1. pH was kept at 3.5. Purification was done by recrystal-
1
lization in water. Yield: 58.7%. H NMR (DMSO-d ): d
11.89 (br s, 2H, NH, ex), 8.94 (pt, 1H, J = 1.6 Hz), 8.92 (d,
2H, ar, J = 1.3 Hz), 8.81 (d, 2H, ar, J = 1.6 Hz), 7.71 (d, 2H,
6
1
with ethanol (70 ml). Yield: 75%. H NMR (DMSO-d ): d
6
1
1
2.59 (br s, 1H, NH, ex), 10.90 (br s, 1H, NH, ex), 9.41 (d,
H, ar, J = 2.0 Hz), 8.64 (d, 1H, ar, J = 2.0 Hz), 8.37 (dd, 2H,
1
3
ar, J = 7.9 Hz), 7.41 (dd, 2H, ar, J = 7.9, 1.6 Hz). C NMR
ar, J = 9.0, 2.3 Hz), 8.26 (dd, 2H, ar, J = 8.8, 2.2 Hz), 8.17 (dd,
(DMSO-d ): d 161.2 (2 C–O), 149.7 (ar, C–N), 148.6 (ar,
6
2
1
H, ar, J = 9.0, 2.3 Hz), 8.10 (d, 1H, ar, J = 8.0 Hz), 8.01 (d,
2 C–S), 137.0 (ar, 2 C–N), 135.9 (ar, 2 C–S), 134.1 (ar,
2 C–C), 132.4 (ar, C–H), 126.7 (ar, 2 C–H), 124.3 (ar,
1
3
H, ar, J = 8.3 Hz), 7.95 (dd, 2H, ar, J = 8.8, 2.4 Hz).
C
NMR (DMSO-d ): d 164.7 (C–O), 163.9 (C–O), 149.0 (ar,
C–N), 142.3 (ar, C–S), 141.4 (ar, C–S), 141.2 (ar, C–N),
2 C–H), 120.8 (ar, 2 C–H), 117.7 (ar, 2 C–H). IR m
cm ): 3468, 1692, 1579, 1531, 1407, 1221, 1190. NaCl:
(KBr,
6
max
–
1
141.0 (ar, C–S), 140.2 (ar, C–C), 134.3 (ar, C–N), 131.1 (ar,
1.3%. TLC: R 0.43. Anal. C H N Na O S (C, H, N).
f
20 11
3
4 16 4

M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
353
–
1
7.2.7. 4,4′-(5-Nitroisophthaloylbisimino)-
(KBr, cm ): 3436, 1652, 1528, 1489, 1338, 1199, 1043.
bis(benzenesulfonic acid) disodium salt × 5 H O (14d)
NaCl: 1.3%. TLC: R 0.38. Anal. C H N Na O S (C, H,
2
f
17 11
2
3 10 3
N).
14d was synthesized from 1d and 13 according to Section
7
.2.1. pH was kept at 5. During the reaction, a precipitate of
1
pure 14d occurred. Yield: 85%. H NMR (DMSO-d ): d
7.3.2. 8-(4-Aminobenzamido)-naphthalene-1,3,5-trisulfonic
acid trisodium salt × 1.5 H O (7)
6
1
2
4
0.77 (br s, 2H, NH, ex), 9.00 (pt, 1H, ar, J = 1.6 Hz), 8.94 (d,
2
H, ar, J = 1.6 Hz), 7.75 (dd, 4H, ar, J = 8.7, 1.8 Hz), 7.62 (dd,
7 was synthesized from 5 according to Section 7.3.1.
13
1
H, ar, J = 8.7, 1.8 Hz). C NMR (DMSO-d ): d 163.1
Yield: 85.1%. H NMR (DMSO-d ): d 12.24 (br s, 1H, NH,
6
6
(
2
4
2 C–O), 147.9 (ar, C–N), 143.4 (ar, 2 C–S), 139.1 (ar,
ex), 9.28 (d, 1H, ar, J = 2.0 Hz), 8.57 (d, 1H, ar, J = 2.0 Hz),
8.09 (d, 1H, ar, J = 8.2 Hz), 7.92 (d, 1H, ar, J = 8.4 Hz), 7.75
C–N), 136.5 (ar, 2 C–C), 133.0 (ar, C–H), 126.2 (ar,
C–H), 125.2 (ar, 2 C–H), 119.8 (ar, 4 C–H). IR m (KBr,
(dd, 2H, ar, J = 8.7, 1.9 Hz), 6.65 (dd, 2H, ar, J = 8.7, 1.9 Hz),
max
–1
13
cm ): 3440, 1670, 1590, 1530, 1400, 1230, 1190. NaCl:
5.60 (br s, 2H, NH, ex). C NMR (DMSO-d ): d 165.2
6
1
.0%. TLC: R 0.59. Anal. C H N Na O S (C, H, N).
(C–O), 151.3 (ar, C–N), 142.1 (ar, C–S), 141.3 (ar, C–S),
f
20 13
3
2 10 2
1
2
1
2
40.7 (ar, C–S), 135.0 (ar, C–N), 131.0 (ar, C–C), 129.6 (ar,
C–H), 126.4 (ar, C–H), 125.2 (ar, C–H), 124.7 (ar, C–H),
22.9 (ar, C–C), 122.2 (ar, C–C), 121.7 (ar, C–H), 112.2 (ar,
7
.2.8. 3,3′-(5-Nitroisophthaloylbisimino)-
bis(benzenesulfonic acid) disodium salt × 3.5 H O (14e)
2
–
1
C–H). IR mmax (KBr, cm ): 3527, 1624, 1508, 1287, 1204,
14e was synthesized from 1e and 13 according to Section
1
1188, 1043. NaCl: 14.9%. TLC:
C H N Na O S (C, H, N).
R
f
0.24. Anal.
7
.2.1. pH was kept constant at 5. Yield: 79.6%. H NMR
1
7
11
2
3 10 3
(DMSO-d ): d 10.84 (br s, 2H, NH, ex), 9.09 (pt, 1H, ar,
6
J = 1.3 Hz), 8.96 (d, 2H, ar, J = 1.6 Hz), 8.09 (pt, 2H, ar,
J = 1.3 Hz), 7.88 (dd, 2H, ar, J = 8.1, 1.3 Hz), 7.40 (dd, 2H, ar,
7
.3.3. 8-(4-(4-Aminobenzamido)-benzamido)-naphthalene-
13
1,3,5-trisulfonic acid trisodium salt × 8 H O (8)
2
J = 7.6, 1.3 Hz), 7.34 (pt, 2H, ar, J = 7.6 Hz). C NMR
DMSO-d ): d 162.8 (2 C–O), 148.5 (ar, 2 C–S), 147.9 (ar,
8
was synthesized from the nitroprecursor of 8 according
(
6
1
to Section 7.3.1. Yield: 74%. H NMR (DMSO-d ): d 12.53
C–N), 138.1 (ar, 2 C–N), 136.4 (ar, 2 C–C), 132.9 (ar, C–H),
28.0 (ar, 2 C–H), 125.1 (ar, 2 C–H), 121.5 (ar, 2 C–H), 120.7
6
(br s, 1H, NH, ex), 10.01 (br s, 1H, NH, ex), 9.32 (d, 1H, ar,
1
–1
J = 2.0 Hz), 8.60 (d, 1H, ar, J = 1.8 Hz), 8.12 (d, 1H, ar,
J = 8.2 Hz), 8.01 (dd, 2H, ar, J = 8.8, 2.4 Hz), 7.99 (d, 1H, ar,
J = 8.2 Hz), 7.79 (dd, 2H, ar, J = 8.8, 2.4 Hz), 7.70 (dd, 2H, ar,
J = 8.6, 2.7 Hz), 6.63 (dd, 2H, ar, J = 8.8, 2.6 Hz), 5.78 (br s,
(ar, 2 C–H), 118.0 (ar, 2 C–H). IR mmax (KBr, cm ): 3450,
1680, 1600, 1540, 1430, 1200, 1040. NaCl: 9.5%. TLC: Rf
0
.66. Anal. C H N Na O S (C, H, N).
2
0
13
3
2 10 2
1
3
2
H, NH, ex). C NMR (DMSO-d ): d 165.3 (C–O), 164.8
6
(
1
C–O), 152.1 (ar, C–N), 142.3 (ar, C–N), 142.2 (ar, C–S),
41.3 (ar, C–S), 141.2 (ar, C–S), 134.4 (ar, C–N), 131.1 (ar,
C–C), 129.5 (ar, C–C), 129.3 (ar, 2 C–H), 128.5 (ar, 2 C–H),
26.5 (ar, C–H), 125.4 (ar, C–H), 124.7 (ar, C–H), 123.0 (ar,
7
.3. General hydrogenation procedure. Synthesis of amino
derivatives 6, 7, 8, and 15a-e
1
7.3.1. 8-(3-Aminobenzamido)-naphthalene-1,3,5-trisulfonic
C–C), 122.2 (ar, C–H), 120.7 (ar, C–C), 118.7 (ar, 2 C–H),
112.4 (ar, 2 C–H). IR mmax (KBr, cm ): 3436, 1636, 1513,
acid trisodium salt × 3.5 H O (6)
–1
2
To a neutral solution of the nitro derivative 4 (31.3 g,
1232, 1188, 1044. NaCl: 25.2%. TLC: R 0.25. Anal.
f
52 mmol) in 300 ml of water, palladium (10%) on charcoal
C H N Na O S (C, H, N).
2
4
16
3
3 11 3
was added as catalyst (350 mg; general: between 1% and 5%
of the weight of the nitro compound). Under heavy stirring,
the reaction mixture was hydrogenated under pressure
7
.3.4. 8,8′-(5-Amino-1,3-benzenediyl-bis(carbonylimino))-
bis(naphthalene-1,3,5-trisulfonic acid) hexasodium salt ×
H O (15a)
(4.0 bar) in a Parr apparatus (Bonn, Germany). After the end
4
2
of hydrogen absorption, the catalyst was removed by filtra-
1
5a was synthesized from 14a according to Section 7.3.1.
tion. The aqueous phase was evaporated under vacuum yield-
1
Yield: 76%. H NMR (DMSO-d ): d 12.49 (br s, 2H, NH,
ex), 9.39 (d, 2H, ar, J = 1.9 Hz), 8.61 (d, 2H, ar, J = 1.9 Hz),
6
1
ing 29.2 g (98.2%) of 6. H NMR (DMSO-d ): d 12.44 (br s,
6
1H, NH, ex), 9.37 (d, 1H, ar, J = 1.9 Hz), 8.59 (d, 1H, ar,
8
.06 (d, 2H, ar, J = 8.2 Hz), 7.96 (d, 2H, ar, J = 8.2 Hz), 7.75
J = 1.9 Hz), 8.03 (d, 1H, ar, J = 7.9 Hz), 7.98 (d, 1H, ar,
J = 8.2 Hz), 7.32 (dd, 1H, ar, J = 7.9, 1.6 Hz), 7.24 (pt, 1H, ar,
J = 1.9 Hz), 7.09 (pt, 1H, ar, J = 7.9 Hz), 6.71 (dd, 1H, ar,
J = 7.9, 2.2 Hz), 5.13 (br s, 2H, NH, ex). C NMR (DMSO-
d6): d 166.4 (C–O), 148.5 (ar, C–N), 142.8 (ar, C–S), 141.8
(
pt, 1H, ar, J = 1.6 Hz), 7.44 (d, 2H, ar, J = 1.3 Hz), 5.25 (br
1
3
s, 2H, NH, ex). C NMR (DMSO-d ): d 166.5 (2 C–O),
1
(ar, 2 C–S), 136.5 (ar, 2 C–C), 134.9 (ar, 2 C–S), 131.4 (ar,
2 C–C), 126.8 (ar, 2 C–H), 125.7 (ar, 2 C–H), 125.1 (ar,
2 C–H), 123.5 (ar, 2 C–C), 123.1 (ar, 2 C–H), 116.3 (ar,
C–H), 116.2 (ar, 2 C–H). IR mmax (KBr, cm ): 3435, 1651,
1601, 1530, 1337, 1188, 1044. NaCl: 23.2%. TLC: R 0.11.
Anal. C H N Na O S (C, H, N).
6
48.2 (ar, C–N), 142.7 (ar, 2 C–N), 141.9 (ar, 2 C–S), 141.5
13
(
1
ar, C–S), 141.6 (ar, C–S), 136.6 (ar, C–C), 134.9 (ar, C–N),
31.4 (ar, C–C), 128.4 (ar, C–H), 126.8 (ar, C–H), 125.7 (ar,
C–H), 124.9 (ar, C–H), 123.3 (ar, C–H), 122.6 (ar, C–C),
16.6 (ar, C–H), 115.8 (ar, C–H), 114.0 (ar, C–H). IR mmax
–
1
f
1
28
15
3
6 20 6

354
M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
–
1
7.3.5. 4,4′-(5-Amino-1,3-benzenediyl-bis(carbonylimino))-
cm ): 3350, 1650, 1590, 1540, 1420, 1200, 1040. NaCl:
bis(benzene-1,3-disulfonic acid) tetrasodium salt × 2 H O
21.8%. TLC: R 0.60. Anal. C H N Na O S (C, H, N).
2
f
20 15
3
2 8 2
(15b)
1
5b was synthesized from 14b according to Section 7.3.1.
7.4. General phosgenation procedure. Synthesis of urea
derivatives 10, 11, and 16a–e
1
Yield: 82.5%. H NMR (DMSO-d ): d 11.36 (br s, 2H, NH,
6
ex), 8.42 (d, 2H, ar, J = 8.5 Hz), 8.04 (d, 2H, ar, J = 2.2 Hz),
7
7
.61 (pt, 1H, ar, J = 1.6 Hz), 7.59 (dd, 2H, ar, J = 8.5, 2.2 Hz),
.27 (d, 2H, ar, J = 1.6 Hz), 5.67 (br s, 2H, NH, ex). C NMR
7
.4.1. 8,8′-(Carbonylbis(imino-3,1-phenylenecarbonylimi-
13
no))bis(naphthalene-1,3,5-trisulfonic acid) hexasodium
salt × 10 H O (10)
(DMSO-d ): d 164.6 (2 C–O), 149.6 (ar, C–N), 142.5 (ar,
6
2
2
2
C–N), 136.4 (ar, 2 C–S), 135.3 (ar, 2 C–S), 134.8 (ar,
C–C), 127.0 (ar, 2 C–H), 125.0 (ar, 2 C–H), 119.1 (ar, 2
To a solution of 6 (27.5 g, 48 mmol) in water (300 ml; pH
was adjusted between 3.0 and 4.0 with 5 N NaOH) a solution
of phosgene (20% in toluene, 50 ml, 100 mmol) was slowly
added under heavy stirring at room temperature. The reaction
mixture was maintained at pH 3.5 by automatic addition of
C–H), 115.0 (ar, 2 C–H), 113.6 (ar, C–H). IR m
cm ): 3450, 1686, 1613, 1588, 1535, 1191, 1037. NaCl:
1
(KBr,
max
–1
.7%. TLC: R 0.37. Anal. C H N Na O S (C, H, N).
f 20 13 3 4 14 4
2
M Na CO solution. The aqueous phase was then neutra-
2 3
7
.3.6. 2,2′-(5-Amino-1,3-benzenediyl-bis(carbonylimino))-
lised with 2 M Na CO . Water was evaporated under
vacuum. NaCl was removed by stirring the crude product
three times in methanol (100 ml). 10 was nearly insoluble in
methanol. Yield: 23.6 g (84.6%). H NMR (DMSO-d ): d
12.62 (br s, 2H, NH, ex), 9.40 (d, 2H, ar, J = 1.9 Hz), 8.96 (br
s, 2H, NH, ex), 8.63 (d, 2H, ar, J = 1.9 Hz), 8.07 (d, 2H, ar,
J = 8.2 Hz), 8.04 (d, 2H, ar, J = 8.2 Hz), 7.98 (pt, 2H, ar,
J = 1.9 Hz), 7.85 (dd, 2H, ar, J = 8.2, 1.9 Hz), 7.82 (dd, 2H, ar,
2
3
bis(benzene-1,4-disulfonic acid) tetrasodium salt ×
.5 H O (15c)
3
2
1
1
5c was synthesized from 14c according to Section 7.3.1.
6
1
Yield: 97.7%. H NMR (DMSO-d ): d 11.25 (br s, 2H, NH,
ex), 8.78 (d, 2H, ar, J = 1.6 Hz), 7.68 (d, 2H, ar, J = 7.9 Hz),
6
7
7
.60 (pt, 1H, ar, J = 1.4 Hz), 7.34 (dd, 2H, ar, J = 7.9, 1.6 Hz),
.27 (d, 2H, ar, J = 1.6 Hz), 5.67 (br s, 2H, NH, ex). C NMR
13
1
3
(DMSO-d ): d 164.5 (2 C–O), 149.6 (ar, 2 C–S), 149.4 (ar,
6
J = 8.2, 1.9 Hz), 7.40 (pt, 2H, ar, J = 8.2 Hz). C NMR
DMSO-d ): d 165.6 (2 C–O), 152.8 (C–O), 142.8 (ar, 2 C–
C–N), 136.6 (ar, 2 C–N), 135.6 (ar, 2 C–S), 134.8 (ar, 2 C–C),
26.6 (ar, 2 C–H), 120.0 (ar, 2 C–H), 117.7 (ar, 2 C–H), 115.0
(
6
1
S), 142.1 (ar, 2 C–S), 141.5 (ar, 2 C–S), 139.7 (ar, 2 C–N),
136.5 (ar, 2 C–C), 134.6 (ar, 2 C–N), 131.5 (ar, 2 C–C), 128.6
–1
(ar, 2 C–H), 113.5 (ar, C–H). IR mmax (KBr, cm ): 3466,
1
1
614, 1574, 1537, 1406, 1279, 1191, 1049, 1022. NaCl:
.4%. TLC: R 0.40. Anal. C H N Na O S (C, H, N).
(
ar, 2 C–H), 126.9 (ar, 2 C–H), 125.9 (ar, 2 C–H), 125.0 (ar,
2 C–H), 123.4 (ar, 2 C–H), 122.9 (ar, 2 C–C), 121.7 (ar,
C–H), 121.0 (ar, 2 C–H), 118.3 (ar, 2 C–H). ES-MS:
f
20 13
3
4 14 4
2
–
7.3.7. 4,4′-(5-Aminoisophthaloylbisimino)-bis(benzene-
calcd./found (m/z): 1028.8/1028.6 [M–H] , 1050.9/1050.7
[M+Na–2H] , 514.0/514.2 [M–2H] . IR m
–
2–
–1
sulfonic acid) disodium salt × 6 H O (15d)
(KBr, cm ):
2
max
3
466, 1652, 1589, 1538, 1338, 1201, 1043. NaCl: 1.8%.
1
5d was synthesized from 14d according to Section 7.3.1.
1
Yield: 89.4%. H NMR (DMSO-d ): d 10.29 (br s, 2H, NH,
TLC: R 0.11. Anal. C35 Na (C, H, N).
f
H
20
N
4
O S
6 21 6
6
ex), 7.73 (dd, 4H, ar, J = 8.9, 1.8 Hz), 7.67 (pt, 1H, ar,
J = 1.3 Hz), 7.57 (dd, 4H, ar, J = 8.7, 1.8 Hz), 7.27 (d, 2H, ar,
7.4.2. 8,8′-(Carbonylbis(imino-4,1-
phenylenecarbonylimino-4,1-phenylenecarbonyl imino))bis
(naphthalene-1,3,5-trisulfonic acid) hexasodium salt ×
13
J = 1.6 Hz), 5.57 (br s, 2H, NH, ex). C NMR (DMSO-d ): d
6
1
2
4
65.8 (2 C–O), 148.9 (ar, C–N), 143.2 (ar, 2 C–S), 139.4 (ar,
C–N), 135.9 (ar, 2 C–C), 126.0 (ar, 4 C–H), 119.2 (ar,
8.5 H O (11)
2
C–H), 115.8 (ar, 2 C–H), 113.9 (ar, C–H). IR m
(KBr,
max
11 was synthesized from 8 according to Section 7.4.1.
–1
cm ): 3400, 1660, 1640, 1590, 1530, 1400, 1200, 1030.
1
Yield: 84.6%. H NMR (DMSO-d ): d 12.57 (br s, 2H, NH,
6
NaCl: 2.8%. TLC: R 0.49. Anal. C H N Na O S (C, H,
f
20 15
3
2
8
2
ex), 10.31 (br s, 2H, NH, ex), 9.53 (br s, 2H, NH, ex), 9.33 (d,
N).
2
H, ar, J = 1.8 Hz), 8.61 (d, 2H, ar, J = 2.0 Hz), 8.15 (d, 2H,
ar, J = 8.3 Hz), 7.94 (d, 4H, ar, J = 8.7 Hz), 7.91 (d, 2H, ar,
J = 8.4 Hz), 7.77 (d, 4H, ar, J = 8.7 Hz), 7.71 (d, 4H, ar,
7
.3.8. 3,3′-(5-Aminoisophthaloylbisimino)-bis(benzene-
1
3
sulfonic acid) disodium salt × 1.5 H O (15e)
2
J = 8.7 Hz), 7.36 (d, 4H, ar, J = 8.7 Hz). C NMR (DMSO-
d6): d 165.0 (2 C–O), 164.8 (2 C–O), 152.0 (C–O), 142.7 (ar,
2 C–N), 142.3 (ar, 2 C–S), 141.8 (ar, 2 C–N), 141.3 (ar,
2 C–S), 141.2 (ar, 2 C–S), 134.5 (ar, 2 C–N), 131.1 (ar,
2 C–C), 130.0 (ar, 2 C–C), 128.7 (ar, 4 C–H), 128.6 (ar,
4 C–H), 127.6 (ar, 2 C–C), 126.6 (ar, 2 C–H), 125.5 (ar,
2 C–H), 124.8 (ar, 2 C–H), 123.1 (ar, 2 C–C), 122.4 (ar,
2 C–H), 119.0 (ar, 4 C–H), 117.2 (ar, 4 C–H). ES-MS:
1
5e was synthesized from 14e according to Section 7.3.1.
1
Yield: 78%. H NMR (DMSO-d ): d 10.27 (br s, 2H, NH,
ex), 8.09 (s, 2H, ar), 7.79 (dd, 2H, ar, J = 8.1, 1.3 Hz), 7.73 (s,
1
J = 1.6 Hz), 7.28 (pt, 2H, ar, J = 7.6 Hz), 5.55 (br s, 2H, NH,
ex). C NMR (DMSO-d ): d 165.7 (2 C–O), 149.0 (ar,
C–H), 148.2 (ar, 2 C–S), 138.7 (ar, 2 C–N), 135.7 (ar, 2 C–C),
1
6
H, ar), 7.34 (dd, 2H, ar, J = 7.6, 1.3 Hz), 7.29 (d, 2H, ar,
1
3
6
–
27.7 (ar, 2 C–H), 120.8 (ar, 2 C–H), 120.5 (ar, 2 C–H), 117.8
calcd./found (m/z): 1267.0/1266.7 [M–H] , 1289.0/1288.6
–
2–
–1
(ar, 2 C–H), 115.9 (ar, 2 C–H), 114.0 (ar, C–H). IR mmax (KBr,
[M+Na–2H] , 633.0/633.0 [M–2H] . IR m
(KBr, cm ):
max

M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
355
3
1
436, 1655, 1594, 1529, 1321, 1236, 1185, 1042. NaCl:
7.4.6. 4,4′,4″,4′′′-(Carbonylbis(imino-5,1,3-benzenetriylbis-
(carbonylimino)))tetrakisbenzene-sulfonic acid tetraso-
.0%. TLC: R 0.06. Anal. C H N Na O S (C, H, N).
f
49 30
6
6 23 6
dium salt × 9.5 H O (16d)
2
7
.4.3. 8,8′,8″,8′′′-(Carbonylbis(imino-5,1,3-benzenetriyl-
16d was synthesized from 15d according to Section 7.4.1.
1
bis(carbonylimino))tetrakis-(naphthalene-1,3,5-trisulfonic
pH was kept at 4.0. Yield: 87.5%. H NMR (DMSO-d ): d
6
acid) dodecasodium salt × 9.5 H O (16a)
10.46 (br s, 4H, NH, ex), 9.30 (br s, 2H, NH, ex), 8.22 (d, 4H,
2
1
6a was synthesized from 15a according to Section 7.4.1.
ar, J = 1.3 Hz), 8.18 (pt, 2H, ar, J = 1.3 Hz), 7.75 (dd, 8H, ar,
J = 8.7, 1.8 Hz), 7.59 (dd, 8H, ar, J = 8.7, 1.8 Hz). C NMR
1
13
pH was kept at 4.0. Yield: 54.9%. H NMR (DMSO-d ): d
6
1
2.66 (br s, 4H, NH, ex), 9.42 (d, 4H, ar, J = 1.6 Hz), 9.17 (br
s, 2H, NH, ex), 8.63 (d, 4H, ar, J = 1.9 Hz), 8.38 (s, 4H, ar),
.29 (s, 2H, ar), 8.09 (d, 4H, ar, J = 8.2 Hz), 8.00 (d, 4H, ar,
(DMSO-d ): d 165.1 (4 C–O), 152.6 (C–O), 143.4 (ar, 4 C–
6
S), 139.9 (ar, 2 C–N), 139.2 (ar, 4 C–N), 135.7 (ar, 4 C–C),
126.0 (ar, 8 C–H), 120.6 (ar, 4 C–H), 120.1 (ar, 2 C–H), 119.3
(ar, 8 C–H). ES-MS: calcd./found (m/z): 1007.1/1006.9
8
1
3
J = 8.2 Hz). C NMR (DMSO-d ): d 166.4 (4 C–O), 153.5
6
–
–
(ar, C–O), 143.5 (ar, 2 C–N), 142.8 (ar, 4 C–N), 142.1 (ar,
[M–H] , 1029.1/1029.0 [M+Na–2H] , 503.0/503.1
2
–
–1
4
4
4
4
C–S), 140.1 (ar, 4 C–S), 137.2 (ar, 4 C–C), 135.3 (ar,
C–S), 132.2 (ar, 4 C–C), 127.6 (ar, 4 C–H), 126.6 (ar,
C–H), 125.8 (ar, 4 C–H), 124.3 (ar, 4 C–C), 124.0 (ar,
C–H), 122.6 (ar, 2 C–H), 121.4 (ar, 4 C–H). ES-MS:
[
M–2H] . IR m
(KBr, cm ): 3400, 1670, 1600, 1580,
max
1
540, 1400, 1330, 1195, 1130, 1040, 1010. NaCl: 1.8%.
TLC: R 0.40. Anal. C H N Na O S (C, H, N).
f
41 28
6
4 17 4
3–
calcd./found (m/z): 614.9/614.9 [M–3H] , 622.3/622.2
7
(
.4.7. 3,3′,3″,3′′′-(Carbonylbis(imino-5,1,3-benzenetriylbis-
carbonylimino)))tetrakisbenzene-sulfonic acid tetraso-
dium salt × 10 H O (16e)
3
–
3–
[M+Na–4H] , 629.6/629.5 [M+2Na–5H] . IR m
(KBr,
max
–1
cm ): 3446, 1653, 1533, 1339, 1199, 1047. NaCl: 0.7%.
TLC: R 0.01. Anal. C H N Na O S (C, H, N).
2
f
57 28
6
12 41 12
1
6e was synthesized from 15e according to Section 7.4.1.
pH was kept at 4.0. The product precipitated during the
7
(
.4.4. 4,4′,4″,4′′′-(Carbonylbis(imino-5,1,3-benzenetriylbis-
carbonylimino)))tetrakisbenzene-1,3-disulfonic acid octa-
sodium salt × 7 H O (16b)
1
reaction. Yield: 66.7%. H NMR (DMSO-d ): d 10.56 (br s,
6
4
(
H, NH, ex), 10.02 (br s, 2H, NH, ex), 8.29 (s, 2H, ar), 8.24
d, 4H, ar, J = 1.3 Hz), 8.13 (s, 4H, ar), 7.86 (dd, 4H, ar,
J = 8.2, 1.3 Hz), 7.37 (dd, 4H, ar, J = 7.8, 1.3 Hz), 7.31 (pt,
2
1
6b was synthesized from 15b according to Section 7.4.1.
1
pH was kept at 4.0. Yield: 54.6%. H NMR (DMSO-d ): d
13
6
4
1
4
4
2
1
5
H, ar, J = 7.9 Hz). C NMR (DMSO-d ): d 165.0 (4 C–O),
6
11.59 (br s, 4H, NH, ex), 9.43 (br s, 2H, NH, ex), 8.47 (d, 4H,
52.8 (C–O), 148.4 (ar, 4 C–S), 140.1 (ar, 2 C–N), 138.5 (ar,
C–N), 135.6 (ar, 4 C–C), 127.8 (ar, 4 C–H), 121.0 (ar,
C–H), 120.5 (ar, 4 C–H), 120.4 (ar, 4 C–H), 120.1 (ar,
C–H), 117.9 (ar, 4 C–H). ES-MS: calcd./found (m/z):
ar, J = 8.4 Hz), 8.24 (s, 4H, ar), 8.12 (s, 2H, ar), 8.05 (d, 4H,
13
ar, J = 2.2 Hz), 7.61 (dd, 4H, ar, J = 8.4, 2.2 Hz). C NMR
DMSO-d ): d 163.7 (4 C–O), 152.6 (C–O), 142.8 (ar, 4 C–
(
6
N), 140.6 (ar, 2 C–N), 136.4 (ar, 4 C–S), 135.2 (ar, 4 C–S),
34.8 (ar, 4 C–C), 127.1 (ar, 4 C–H), 125.0 (ar, 4 C–H), 120.0
ar, 4 C–H), 119.6 (ar, 4 C–H), 119.2 (ar, 2 C–H). ES-MS:
–
–
007.1/1006.8 [M–H] , 1029.1/1028.8 [M+Na–2H] ,
1
(
2
–
–1
03.0/503.2 [M–2H] . IR m
(KBr, cm ): 3440, 1660,
max
–
1590, 1535, 1200, 1030. NaCl: 28.1%. TLC: R 0.53. Anal.
f
calcd./found (m/z): 1326.9/1327.1 [M–H] , 1348.9/1348.8
–
2–
–1
C H N Na O S (C, H, N).
4
1
28
6
4 17 4
[M+Na–2H] , 662.9/663.2 [M–2H] . IR m
(KBr, cm ):
max
3
435, 1684, 1589, 1533, 1184, 1134, 1040. NaCl: 0.8%.
TLC: R 0.13. Anal. C H N Na O S (C, H, N).
7.5. Synthesis of 8-(4-(4-((N-Phenylcarbamoyl)-amino))-
benzamido)-benzamido)-naphthalene-1,3,5-trisulfonic acid
f
41 24
6
8 29 8
trisodium salt × 4.5 H O (9)
7
(
.4.5. 2,2′,2″,2′′′-(Carbonylbis(imino-5,1,3-benzenetriylbis-
carbonylimino)))tetrakisbenzene-1,4-disulfonic acid octa-
sodium salt × 9 H O (16c)
2
Compound 8 (0.5 g, 0.7 mmol) was dissolved in water
2
(
40 ml) and 0.5 ml triethylamine were added. Phenylisocy-
anate (0.9 ml, 8.4 mmol) was added in drops over a period of
6 h. Subsequently, the aqueous phase was exhaustively
1
6c was synthesized from 15c according to Section 7.4.1.
1
pH was kept at 3.7. Yield: 93.8%. H NMR (DMSO-d ): d
1.48 (br s, 4H, NH, ex), 9.44 (br s, 2H, NH, ex), 8.82 (d, 4H,
ar, J = 1.6 Hz), 8.24 (d, 4H, ar, J = 1.3 Hz), 8.14 (pt, 2H, ar,
J = 1.2 Hz), 7.72 (d, 4H, ar, J = 8.2 Hz), 7.38 (dd, 4H, ar,
J = 8.2 Hz). C NMR (DMSO-d ): d 163.6 (4 C–O), 152.6
6
3
1
extracted with diethylether. Compound was dried under
vacuum, and the crude product was stirred in methanol for
purification.Yield: 0.4 g of 9 (70.8%). H NMR (DMSO-d
1
3
1
):
6
6
(
C–O), 149.6 (ar, 4 C–S), 140.5 (ar, 4 C–N), 136.5 (ar,
d 12.57 (br s, 1H, NH, ex), 10.27 (br s, 1H, NH, ex), 9.28 (br
s, 1H, NH, ex), 9.04 (br s, 1H, NH, ex), 9.33 (d, 1H, ar,
J = 2.0 Hz), 8.61 (d, 1H, ar, J = 1.8 Hz), 8.13 (d, 1H, ar,
J = 8.3 Hz), 8.01 (dd, 2H, ar, J = 8.7, 2.0 Hz), 7.97 (d, 1H, ar,
J = 8.2 Hz), 7.86 (dd, 2H, ar, J = 8.7, 1.9 Hz), 7.79 (dd, 2H, ar,
J = 8.9, 2.0 Hz), 7.48 (dd, 2H, ar, J = 8.8, 1.8 Hz), 7.35 (dd,
2H, ar, J = 8.5, 1.2 Hz), 7.25 (pt, 2H, ar, J = 8.4 Hz), 6.99 (pt,
4
4
2
6
4
1
C–S), 135.7 (ar, 2 C–N), 134.6 (ar, 4 C–C), 126.6 (ar,
C–H), 120.3 (ar, 4 C–H), 120.0 (ar, 4 C–H), 119.7 (ar,
C–H), 117.8 (ar, 4 C–H). ES-MS: calcd./found (m/z):
2
–
2–
62.9/663.0 [M–2H] , 673.9/674.0 [M+Na–3H] ,
3
–
–1
41.6/441.8 [M–3H] . IR m
(KBr, cm ): 3444, 1678,
max
608, 1578, 1532, 1409, 1188, 1121, 1019. NaCl: 3.2%.
1
3
TLC: R 0.18. Anal. C H N Na O S (C, H, N).
1H, ar, J = 7.6 Hz). C NMR (DMSO-d ): d 164.9 (C–O),
f
41 24
6
8
29
8
6

356
M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
1
1
64.7 (C–O), 152.1 (C–O), 142.9 (ar, C–N), 142.3 (ar, C–S),
41.7 (ar, C–N), 141.3 (ar, C–S), 141.2 (ar, C–S), 139.3 (ar,
experiments there was no significant change in tissue sensi-
tivity towards the nucleotide agonists. The antagonists were
allowed to equilibrate for 60–120 min, and preliminary ex-
periments indicated that these intervals were sufficient for
equilibration of the antagonist concentrations used. Each
concentration of antagonists to inhibit the contractile re-
sponses to abmeATP or ADPbS was tested at least three
times on rat vas deferens or guinea-pig ileum from at least
two different animals. abmeATP and ADPbS were obtained
from Sigma-Aldrich (Taufkirchen, Germany). All other
chemicals were of the highest grade available, and used as
purchased.
C–N), 134.4 (ar, C–N), 131.1 (ar, C–C), 130.0 (ar, C–C),
28.6 (ar, 2 C–H), 128.5 (ar, 4 C–H), 127.3 (ar, C–C), 126.5
ar, C–H), 125.4 (ar, C–H), 124.7 (ar, C–H), 123.0 (ar, C–C),
22.2 (ar, C–H), 121.8 (ar, C–H), 118.9 (ar, 2 C–H), 118.2
ar, 2 C–H), 116.9 (ar, 2 C–H). ES-MS: calcd./found (m/z):
1
(
1
(
–
–
7
3
1
39.1/739.2
[M–H] ,
761.0/761.2
[M+Na–2H] ,
2
–
-1
69.0/369.2 [M–2H] . IR m
652, 1596, 1531, 1512, 1326, 1187, 1046. NaCl: 4.9%.
(KBr, cm ): 3467, 1671,
max
TLC: R 0.27. Anal. C H N Na O S (C, H, N).
f
31 21
4
3 12 3
7
.6. Synthesis of 4,4′,4″,4′′′-(Terephthaloylbis(imino-5,1,3-
7
.7.2. Cell culture and measurements of intracellular
calcium
Wildtype HEK293 cells were cultured in Dulbecco’s
modified Eagle Medium Nutrient Mixture F-12 Ham
DMEM/F12 1:1 Mixture) (Sigma-Aldrich) containing
00 µg/ml streptomycin, 100 U/ml penicillin G, 10% fetal
bovine serum (Sigma-Aldrich), and 5 mM L-glutamine
Sigma-Aldrich). CHO-P2Y11 cells (chinese hamster ovary
benzenetriylbis-(carbonylimino-methylene)))tetrakis-
benzene-1,3-disulfonic acid octasodium salt × 16 H O
2
(19b)
(
1
15b (5.0 g, 6.8 mmol) was dissolved in 60 ml water.
Terephthaloyldichloride 18 (1.4 g, 7 mmol, dissolved in
toluene, 20 ml) was slowly dropped into the aqueous solution
of 15b, and the pH was kept at 4.5 by automatic addition of a
(
cells stably transfected with a plasmid containing the human
P2Y11 coding sequence) [36] were grown in Dulbecco’s
modified Eagle Medium supplemented with 10% fetal bo-
vine serum, 100 µg/ml streptomycin, 100 U/ml penicillin G,
2
M Na CO solution. After finishing of the reaction, the
2 3
water phase was separated from the toluene and extracted
four times with diethylether (each 20 ml) at pH 2. After
neutralisation, water was removed under vacuum. The crude
product was purified by recrystallization in water. Yield:
1
4
5
mM L-glutamine, and 200 µg/ml G418 (Sigma-Aldrich).
2
+
1
Cells were incubated at 37 °C in 5% CO . Ca fluorescence
2
.1 g of 19b (22.8%). H NMR (DMSO-d ): d 11.61 (br s,
6
measurements were performed as previously described using
a NOVOstar with a pipettor system (BMG LabTechnologies,
Offenburg, Germany) [32]. Briefly, cells were harvested with
H, NH, ex), 10.96 (br s, 2H, NH, ex), 8.56 (d, 4H, ar,
J = 1.3 Hz), 8.44 (d, 4H, ar, J = 8.7 Hz), 8.23 (s, 2H, ar), 8.21
s, 4H, ar), 8.05 (d, 4H, ar, J = 2.1 Hz), 7.62 (dd, 4H, ar,
(
0
.05% trypsin/0.02% EDTA (Sigma-Aldrich) and rinsed
1
3
J = 8.4, 2.1 Hz). C NMR (DMSO-d ): d 166.3 (2 C–O),
6
with culture medium containing 10% fetal bovine serum.
Pelleted cells were then resuspended in fresh medium, kept
under 5% CO at 37 °C for 30 min and vortexed every
164.6 (4 C–O), 143.6 (ar, 4 C–S), 140.9 (ar, 2 C–N), 138.1
(ar, 4 C–C), 137.1 (ar, 4 C–N), 136.0 (ar, 4 C–S), 135.7 (ar,
2
2
4
4
4
C–C), 129.0 (ar, 4 C–H), 128.0 (ar, 4 C–H), 125.9 (ar,
C–H), 123.5 (ar, 4 C–H), 121.4 (ar, 2 C–H), 120.2 (ar,
1
5 min. After two washes with Krebs-HEPES buffer, cells
were loaded with 3 µM Oregon Green 488 BAPTA-1/AM
Molecular Probes, Eugene, OR, USA) for 45 min at 25 °C in
2
–
C–H). ES-MS: calcd./found (m/e): 715.0/715.1 [M–2H] ,
(
3
–
3–
76.3/476.6 [M–3H] , 483.6/483.7 [M+Na–4H] . IR m
max
the same buffer containing 1% Pluronic F-127 (Sigma- Ald-
rich). Then, cells were rinsed three times with Krebs-HEPES
buffer, diluted, and evenly plated into 96 well plates (Greiner,
Frickenhausen, Germany) at a density of 35,000 cells/well.
Agonist concentration–response curves were obtained by
injection of increasing concentrations of UTP or ATP
–
1
(KBr, cm ): 3470, 1680, 1590, 1530, 1320, 1200, 1040.
NaCl: 0.9%. TLC: R 0.27. Anal. C H N Na O S (C, H,
N).
f
48 28
6
8 30 8
7
7
.7. Pharmacology
.7.1. Contractions in the rat vas deferens and guinea-pig
(HEK293 cells) or ATPcS (CHO-P2Y ) and monitoring
11
fluorescence intensity at 520 nm (bandwidth 35 nm) for 30 s
at 0.4 s intervals. Excitation wavelength was 485 nm (band-
width 12 nm). Concentration-inhibition curves of antagonists
were obtained by preincubating the cells with the compounds
ileum
The methods used have been described in detail previ-
ously [13,18,30,35]. Briefly, prostatic segments of vasa def-
erentia from rats were set up for isometric tension measure-
ment in modified Krebs solution (37 °C, pH 7.4, gassed with
for 30 min at 37 °C (HEK293 cells) and 25 °C (CHO-P2Y11
cells), respectively, prior to injection of agonist (3 µM UTP
or ATP in HEK293 cells or 3 µM ATPcS in CHO-P2Y11
95% O /5% CO ) as were strips of guinea-pig ileum longitu-
2 2
cells).
dinal smooth muscle. Contractions were elicited to single
doses of abmeATP in rat vas deferens (10 µM) or guinea-pig
ileum (1 µM) and to ADPbS in ileum (10 µM) leaving
7
.7.3. Data analysis
15–60 min before the next dose of agonist was added. Time-
Effects of single doses of antagonists (10 µM) were ex-
matched controls demonstrated that during the course of
pressed as a percentage of the agonist control responses.

M.U. Kassack et al. / European Journal of Medicinal Chemistry 39 (2004) 345–357
357
Antagonist IC50 values (pIC50 = –log IC ) represent the
[13] K. Braun, J. Rettinger, M. Ganso, M. Kassack, C. Hildebrandt, H. Ull-
mann, P. Nickel, G. Schmalzing, G. Lambrecht, Naunyn Schmiede-
bergs Arch. Pharmacol. 364 (2001) 285–290.
50
concentration needed to inhibit by 50% the effect elicited by
single doses of agonists. EC50 values for agonists and IC50
values for antagonists were derived from –log
concentration—effect (inhibition) curves fitted to the data by
logistic, non-linear regression analysis (Prism 3.0, GraphPad
Software, San Diego, CA, USA). All data are presented as
arithmetic means ± S.E.M. from at least three independent
experiments, unless otherwise stated. Differences between
mean values were assessed using Student’s t-test and consid-
ered significant when P < 0.05.
[
14] S. Damer, B. Niebel, S. Czeche, P. Nickel, U. Ardanuy, G. Schmalz-
ing, J. Rettinger, E. Mutschler, G. Lambrecht, Eur. J. Pharmacol. 350
(
1998) R5–R6.
[15] G. Lambrecht, J. Rettinger, H.G. Baumert, S. Czeche, S. Damer,
M. Ganso, C. Hildebrandt, B. Niebel, G. Spatz-Kümbel, G. Schmalz-
ing, E. Mutschler, Eur. J. Pharmacol. 387 (2000) R19–R21.
[16] R. Ziyal, A.U. Ziganshin, P. Nickel, U. Ardanuy, E. Mutschler,
G. Lambrecht, G. Burnstock, Br. J. Pharmacol. 120 (1997) 954–960.
[
17] G. Lambrecht, T. Friebe, U. Grimm, U. Windscheif, E. Bungardt,
C. Hildebrandt, H.G. Baumert, G. Spatz-Kümbel, E. Mutschler, Eur.
J. Pharmacol. 217 (1992) 217–219.
[
[
[
18] G. Lambrecht, S. Damer, B. Niebel, S. Czeche, P. Nickel, J. Rettinger,
G. Schmalzing, E. Mutschler, Prog. Brain Res. 120 (1999) 107–117.
Acknowledgements
19] G. Lambrecht, Naunyn Schmiedebergs Arch. Pharmacol. 362 (2000)
3
40–350.
This work was supported by DFG (Deutsche Forschungs-
gemeinschaft) grants GRK677/1, GRK137/2, GRK137/3,
and La350/7-3, as well as by the Fonds der Chemischen
Industrie (Germany). The authors gratefully acknowledge
the assistence of Mrs. M. Schneider in obtaining the mass
spectra. CHO-cells recombinantly expressing the P2Y11 re-
ceptor were kindly provided by Drs. D. Communi and J.M.
Boeynaems, Brussels, Belgium.
20] G. Lambrecht, K. Braun, M. Damer, M. Ganso, C. Hildebrandt,
H. Ullmann, M.U. Kassack, P. Nickel, Curr. Pharm. Des. 8 (2002)
2371–2399.
[21] J. Rettinger, G. Schmalzing, S. Damer, G. Muller, P. Nickel, G. Lam-
brecht, Neuropharmacology 39 (2000) 2044–2053.
[
22] F. Soto, G. Lambrecht, P. Nickel, W. Stuhmer, A.E. Busch, Neurop-
harmacology 38 (1999) 141–149.
[
23] M.F. Jarvis, E.C. Burgard, S. McGaraughty, P. Honore, K. Lynch,
T.J. Brennan, A. Subieta, T. Van Biesen, J. Cartmell, B. Bianchi,
W. Niforatos, K. Kage, H. Yu, J. Mikusa, C.T. Wismer, C.Z. Zhu,
K. Chu, C.H. Lee,A.O. Stewart, J. Polakowski, B.F. Cox, E. Kowaluk,
M. Williams, J. Sullivan, C. Faltynek, Proc. Natl. Acad. Sci. USA 99
References
(
2002) 17179–17184.
[
24] P. Nickel, H.J. Haack, H. Widjaja, U. Ardanuy, C. Gurgel, D. Duwel,
H. Loewe, W. Raether, Arzneimittelforschung 36 (1986) 1153–1157.
[
[
1] V. Ralevic, G. Burnstock, Pharmacol. Rev. 50 (1998) 413–492.
2] B.S. Khakh, G. Burnstock, C. Kennedy, B.F. King, R.A. North, P. Seg-
uela, M. Voigt, P.P. Humphrey, Pharmacol. Rev. 53 (2001) 107–118.
[25] A. Kreimeyer, G. Muller, M. Kassack, P. Nickel,A.R. Gagliardi,Arch.
Pharm. (Weinheim) 331 (1998) 97–103.
[
3] I. von Kügelgen, A. Wetter, Naunyn SchmiedebergsArch. Pharmacol.
[26] K. Findeisen, K. Koenig, R. Sundermann, fourth ed, Houben-Weyl
Methoden der organischen Chemie, 2, Georg Thieme Verlag, Stut-
tgart, New York, 1985, pp. 742–746.
3
62 (2000) 310–323.
[
[
4] M.P.Abbracchio, G. Burnstock, Pharmacol. Ther. 64 (1994) 445–475.
5] K.A. Jacobson, M.F. Jarvis, M. Williams, J. Med. Chem. 45 (2002)
[27] U. Petersen, fourth ed, Houben-Weyl Methoden der organischen Che-
mie, E4, Georg Thieme Verlag, Stuttgart, 1983, pp. 352–359.
4
057–4093.
[
6] J.K. Chambers, L.E. Macdonald, H.M. Sarau, R.S. Ames, K. Free-
man, J.J. Foley,Y. Zhu, M.M. McLaughlin, P. Murdock, L. McMillan,
J. Trill, A. Swift, N. Aiyar, P. Taylor, L. Vawter, S. Naheed, P. Szek-
eres, G. Hervieu, C. Scott, J.M. Watson, A.J. Murphy, E. Duzic,
C. Klein, D.J. Bergsma, S. Wilson, G.P. Livi, J. Biol. Chem. 275
[28] M. Kassack, P. Nickel, J. Chromatogr. B Biomed. Appl. 686 (1996)
275–284.
[29] R. Bültmann, H. Wittenburg, B. Pause, G. Kurz, P. Nickel, K. Starke,
Naunyn Schmiedebergs Arch. Pharmacol. 354 (1996) 498–504.
[
30] G. Lambrecht, M. Ganso, H.G. Baumert, G. Spatz-Kümbel, C. Hilde-
brandt, K. Braun, E. Mutschler, J. Auton. Nerv. Syst. 81 (2000)
171–177.
(
2000) 10767–10771.
[
[
7] G. Burnstock, Clin. Med. 2 (2002) 45–53.
8] F. DiVirgilio, P. Chiozzi, D. Ferrari, S. Falzoni, J.M. Sanz, A. Morelli,
M. Torboli, G. Bolognesi, O.R. Baricordi, Blood 97 (2001) 587–600.
[31] J.B. Schachter, S.M. Sromek, R.A. Nicholas, T.K. Harden, Neurop-
harmacology 36 (1997) 1181–1187.
[
[
9] R.A. North, A. Surprenant, Annu. Rev. Pharmacol. Toxicol. 40 (2000)
[32] M.U. Kassack, B. Hofgen, J. Lehmann, N. Eckstein, J.M. Quillan,
W. Sadee, J. Biomol. Screen. 7 (2002) 233–246.
5
63–580.
10] D. Communi, N.S. Gonzalez, M. Detheux, S. Brezillon, V. Lannoy,
M. Parmentier, J.M. Boeynaems, J. Biol. Chem. 276 (2001) 41479–
[33] P.M. Dunn, A.G. Blakeley, Br. J. Pharmacol. 93 (1988) 243–245.
[34] F. Soto, M. Garcia-Guzman, W. Stuhmer, J. Membr. Biol. 160 (1997)
91–100.
4
1485.
[
[
11] C.E. Müller, Curr. Pharm. Des. 8 (2002) 2353–2369.
[35] I. Kennedy, P.P. Humphrey, Eur. J. Pharmacol. 261 (1994) 273–280.
12] M. Hülsmann, P. Nickel, M. Kassack, G. Schmalzing, G. Lambrecht,
F. Markwardt, Eur. J. Pharmacol. 470 (2003) 1–7.
[36] D. Communi, B. Robaye, J.M. Boeynaems, Br. J. Pharmacol. 128
(1999) 1199–1206.