Suramin analogues with a 2-phenylbenzimidazole moiety as partial structure; potential anti HIV- and angiostatic drugs, 2: Sulfanilic acid-, benzenedisulfonic acid-, and naphthalenetrisulfonic acid analogues.

Article abstract of DOI:10.1002/(SICI)1521-4184(199803)331:3<97::AID-ARDP97>3.0.CO;2-F

The synthesis of suramin analogues bearing a 2-phenyl-benzimidazole moiety is described. Aminoarene sulfonic acids 2a-e are acylated with 3,4-dinitrobenzoyl chloride 3 yielding the amides 4a-e which are hydrogenated to the corresponding diamines 5a-e. The

Full text of DOI:10.1002/(SICI)1521-4184(199803)331:3<97::AID-ARDP97>3.0.CO;2-F

Suramin Analogues
97
Suramin Analogues with a 2-Phenylbenzimidazole Moiety as Partial Structure; Potential anti HIV- and
1)
Angiostatic Drugs, 2 :
Sulfanilic Acid-, Benzenedisulfonic Acid-, and Naphthalenetrisulfonic
Acid Analogues✩
a)
a)
a)
a)
b)
Annett Kreimeyer , Guido Müller , Matthias Kassack , Peter Nickel *, and Antonio R.T. Gagliardi
a) Pharmazeutisches Institut der Universität Bonn, An der Immenburg 4, D-53121 Bonn, Germany
b) VA Medical Center and Departments of Obstetrics/Gynecology and Physiology, University of Kentucky College of Medicine, Lexington, KY, USA
Key Words: Suramin; benzimidazole analogues synthesis; in vitro anti HIV activity; in vitro antitumor activity; angiogenesis
inhibition
logues in which the enframed parts of the suramin molecule
(see Fig. 1) have been replaced by a 2-phenylbenzimidazole
moiety. The rationale for this insertion was the fact that
2-phenylbenzimidazole was the lead compound for the devel-
Summary
The synthesis of suramin analogues bearing a 2-phenyl-benzimid-
azole moiety is described. Aminoarene sulfonic acids 2a–e are
acylated with 3,4-dinitrobenzoyl chloride 3 yielding the amides
4a–e which are hydrogenated to the corresponding diamines 5a–e.
These are treated with 3-nitrobenzaldehyde, yielding the azo-
methines 7a–e and their isomers 8a–e and 9a–e. Key step in the
synthesis of the target compounds 12a–e is the oxidation of the
azomethines with oxygen to the benzimidazoles 10a–e. These are
hydrogenated to the amines 11a–e reacting with phosgene to yield
the symmetric ureas 12a–e. Results of the anti-HIV, cytostatic, and
antiangiogenic screening are presented.
[9]
opment of a series of potent anthelmintics . Originally, our
main interest in the suramin field was the discovery of new
macrofilaricidal compounds. Therefore, we tried to combine
structural elements of different anthelmintic agents in one
molecule. Furthermore, an anti-HIV activity of suramin and
[10]
its analogues seems to require a flat and rigid structure
.
The replacement of the enframed parts of the suramin mole-
cule by 2-phenylbenzimidazole will lead to compounds with
molecular dimensions that are very similar to those of the
suramin molecule, but they will have an increased rigidity.
With the aim of studying the influence of this replacement on
the biological activities of suramin, we synthesized a series
of suramin analogues replacing the enframed parts of 1 by
Introduction
Suramin 1 was developed by Bayer as a drug for the
treatment of African sleeping sickness, a trypanosome infec-
benzimidazole and varying the trisulfonaphthyl residues of
tion, and has been used for the treatment of this disease since
[11, 12]
[1, 2]
suramin
. This report is the continuation of our research
1920
. In 1947, its activity against Onchocerca volvulus,
on the synthesis of suramin analogues with 2-phenylbenz-
the causative agent of Onchocerciasis (river blindness), was
[13]
[3]
imidazole as partial structure
.
reported . To this day, it is the only approved macrofilari-
cidal drug available for the treatment of Onchocerciasis. The
interest in new macrofilaricidal agents has declined during
the last decade due to the great success of the WHO Oncho-
cerciasis Control Program (OCP) since 1987, when large
[4]
scale prophylactic Ivermectine treatment was introduced
.
However, further biological activities of suramin stimulated
the continuation of the synthetic work in the suramin field.
Suramin is a potent inhibitor of the reverse transcriptase of
[5]
[1]
retro-viruses and has an anti HIV activity . Because of
severe toxic side effects, however, suramin is not recom-
Figure 1. Suramin.
[1]
mended for the treatment of AIDS . Recently, it has been
shown that suramin is an anticancer drug with a unique mode
of action, it inhibits tumor growth factors . Therefore,
suramin has become an interesting lead for the development
of new cytostatic agents.
The replacement of benzene rings by heterocyles in phar-
macologically active compounds is a common method ap-
plied in investigations of structure activity relationships.
Suramin analogues in which benzene rings have been re-
placed by furan
literature. Here we describe the synthesis of suramin ana-
Chemistry
[6]
The four step synthetic pathway to the targets 12a–e is
outlined in Scheme 1. Aqueous solutions of the aminoarene-
sulfonic acids 2a–e were treated at pH 4.5 with a solution of
3,4-dinitrobenzoyl chloride (3)
dinitrobenzamides 4a–e in high yields. 4a–e were catalyti-
cally hydrogenated to the corresponding diamines 5a–e .
Using palladium catalysts, deep-red reaction products were
obtained. The TLC showed several coloured by-products,
presumably azo and azoxy derivatives. The hydrogenation
conditions have been systematically varied. Best results were
[14]
in toluene giving the
[7]
[8]
or pyrrole
rings are described in the
———
1) Part 1 of this series see ref.[13]
.
obtained using PtO as the catalyst and a mixture of metha-
2
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0365-6233/98/0303/0097 $17.50 +.50/0

98
Kreimeyer, Müller, Kassack, Nickel, and Gagliardi
Scheme 2. Formation of the 1-(3-nitrobenzyl)benzimidazole derivatives
14 a–e and their reduction to 15 a–e.
[16]
genolysis
. Therefore, we expected that catalytic hydro-
genation of 14a–e will lead to a reduction of the nitro groups
and a hydrogenolytic debenzylation yielding 11a–e
(Scheme 2). This reaction was intensely investigated with
14c using different solvents and catalysts (Pd, Pt). Surpris-
ingly, no debenzylation could be observed. 15c was the main
hydrogenation product of 14c.
Treatment of aqueous solutions of the arylamines 11b–e
with a solution of phosgene in toluene at pH 4.5 resulted in
the target ureas 12b–e. The sulfanilic acid derivative 11a was
not soluble in water at this pH. Therefore, the reaction with
11a was carried out in suspension according to previous
[13]
experiments
yielding 12a.
Spectroscopic data collected for the synthesized com-
pounds were in agreement withthe proposed structures. How-
13
ever, in the C-NMR spectra of the benzimidazole
derivatives 10a–e no resonances were detected for the car-
bons 3a, 4, 7, and 7a of the heterocycle. This could be
explained by the tautomeric character of the imidazole. As
expected, addition of one equivalent of trifluoroacetic acid to
the sample resulted in the occurrence of the previously miss-
ing C resonances.
Benzimidazoles are amphoteric compounds. The NH-
acidic character of benzimidazoles allows to determine the
13
Scheme 1. Synthesis of the new benzene sulfonic, benzene disulfonic, and
naphthalenetrisulfonic acid analogues of suramin.
nol/water as the solvent. 5a–e are very sensitive towards
oxidation by air. Therefore, they have not been isolated but
were immediately treated with 3-nitrobenzaldehyde (6).
When equimolar amounts of 5a–e and 6 were used, 5a–e
could be detected by TLC even after a reaction time of 12 h
at room temperature (RT). Therefore, an excess of6 was used.
It can be assumed that the regioisomeric azomethines 7a–e
and 9a–e are formed together with the dihydrobenzimidazole
derivatives 8a–e. No attempts were made to isolate one of
complete conversion of 7–9 to 10 by titration of 10 in DMF
[17]
with 0.1 N aqueous NaOH
. The purity of all compounds
screened biologically was determined by HPLC using our
recently published method for the determination of
[18]
suramin
.
Biological Screening
The in-vitro anti-HIV screening and the cytostatic screen-
ing were carried out in the Department of Health and Human
Services, National Cancer Institute, Developmental Thera-
peutics Program, Bethesda, Maryland 20892, USA. The anti-
angiogenic screening was done at the VA Medical Center and
Departments of Obstetrics/Gynecology and Physiology, Uni-
versity of Kentucky College of Medicine, Lexington, KY,
USA. The results of the biological screening are shown in
Tables 1 – 3. In these tables the new compounds are cited
according to their numbers from Scheme 1 and in parentheses
with their NF codes (N stands for Nickel and F for Filariasis;
these isomers. The mixture of isomers was directly oxidized
[15]
with oxygen
to the corresponding benzimidazoles 10a–e
[13]
according to our recently developed strategy
.
By-products in this reaction were the 1-(3-nitrobenzyl)-
benzimidazole derivatives 14a–e resulting from the interme-
diate formation of the bis(benzylidenimino) derivatives
13a–e during the reaction of 5 with 6 (Scheme 2). 14a–e
could be separated from 10a–e by recrystallisation.
Catalytic hydrogenation (Pd/C) of the nitro compounds
10a–e gave the corresponding aromatic amines 11a–e. N-
Benzyl groups can be smoothly removed by catalytic hydro- originally our main interest in the suramin field was the
Arch. Pharm. Pharm. Med. Chem. 331, 97–103 (1998)

Suramin Analogues
99
Table 2. In vitro cytostatic activity of the benzimidazole analogues of
suramin.
finding of new filaricidal compounds). This is for comparison
[22, 23, 25]
purposes because in previous publications
suramin analogues have NF codes.
our
——————————————————————————————
Anti HIV Screening: The sreening method is described in
Tumor
cell line
Type of
cancer
–log(GI50) a)
[19]
b)
detail in ref.
. Briefly, the test compound is dissolved in
Cmpnd
NF
Suramin
S
DMSO. The DMSO solution is diluted with culture medium.
T4 lymphocytes (CEM cell line) are added and after a brief
interval HIV-1 is added. Uninfected cells with the compound
serve as toxicity controls, and uninfected cells without the
compound serve as basic controls. The results of the test are
described by the expressions IC50, EC50, TI50. Eight differ-
ent concentrations of the test compound are used for the
calculations of IC50 and EC50. The highest concentration of
the test compounds in this screening system is normally
100 µM. IC50 >100µM means: no cell toxicity observed at
this concentration. The results of the anti HIV screening are
shown in Table 1.
——————————————————————————————
12a
(NF503) K-562
Leukemia
4.07
<4.00
>1.17
12d
MCF7/ADR-RES
Breast C.
Colon C.
Colon C.
Colon C.
Leukemia
Leukemia
Melanoma 4.27
Renal C.
Renal C.
4.10
4.18
4.40
4.16
4.24
4.12
<4.00
<4.00
4.04
4.11
<4.00
4.09
4.13
<4.00
4.59
>1.26
1.51
2.29
1.12
>1.74
1.07
1.38
1.29
0.35
(NF506) COLO 205
KM 12
SW-620
CCRF-CEM
HL-60 (TB)
LOX IMVI
ACHN
4.11
4.13
UO-31
17
MDA-MB-231/ATCC Breast C.
4.33
4.53
4.61
4.71
<4.00
<4.00
4.09
>2.14
>3.39
3.31
Table 1. Anti HIV activity of the benzimidazole analogues of suramin.
(NF417) CCRF-CEM
Leukemia
Leukemia
Leukemia
——————————————————————————————
HL-60 (TB)
SR
4.16
3.55
Compound
EC50 µM
IC50 µM
TI50
M14
IGRO VI
Melanoma 4.34
Ovarian C. 4.35
4.26
4.39
1.20
2.19
——————————————————————————————
——————————————————————————————
GI50: Growth Inhibition, concentration M of the test compound which
Suramin
33.5
39.0
34.6
35.6
>174
115
>174
>174
>5.2
2.9
>5.0
>4.9
a)
decreases the tumor cell growth to 50% of the untreated tumor cell culture.
b)
S: Selectivity factor GI50(Suramin)/GI50(NF), values > 1 indicate that
the NF-compound is more active than suramin.
12a
(NF503)
3.4
8.6
58.5
60.0
>150
>150
>45
>17
Antiangiogenic screening: Suramin has a unique mode of
cytostatic action; it inhibits tumor growth factors . The
inhibition of the angiogenesis growth factor is a main target
for the development of new anti tumor drugs. The antiangio-
[6]
12d
(NF506)
37.0
29.9
29.0
30.0
64.8
1.8
genic activity of suramin and of suramin analogues is de-
[20–22]
60.9
72.7
82.4
2.0
2.5
2.8
scribed in the literature
. The antiangiogenic activity
was investigated in the chick egg chorioallantoic membrane
(CAM) assay. The method is described in detail in the litera-
[20, 22]
ture
. Briefly, fertile chick eggs are incubated for 72 h
at 37 °C. After 72 h the egg shells are broken and the egg
contents are placed in petri dishes. Each compound to be
tested is dissolved in an aqueous 0.45% solution of methyl-
cellulose. A 10 µl aliquot of this solution is air dried on a
17
(NF417)
2.2
23.2
51.3
81.4
147.0
>1000
>457
>200
>1000
>200
>8.6
>19.5
>2.5
>200
>1.4
Table 3. Inhibition of angiogenesis by the benzimidazole analogues of
suramin.
——————————————————————————————
——————————————————————————————
IC50 µM: Toxicity, concentration of the test compound which decreases the
cell growth of the uninfected cell culture to 50% of the untreated cell culture.
EC50 µM: Efficiency, concentration of the test compound which increases
the cell growth of the infected cell culture to 50% of the uninfected, untreated
cell culture
Compound
# Embryos % Inhib-
ition
ID50
nmol/disk
——————————————————————————————
Suramin
12c
(NF504)
67
23
20
21
64
33
58
60
75
60
70
65
TI50: Therapeutic index, IC50/EC50
Cytostatic Screening: In the NCI program, the cytostatic
activity of the test compounds against 60 tumor cell lines is
investigated. The cell cultures are incubated with five differ-
ent concentrations of the test compounds. The highest con-
centration tested is normally 100 µM. The results of the test
are described by the expressions GI50, and TGI (Total
Growth Inhibition). None of the compounds described in this
paper was able to totally inhibit tumor cell growth at the
highest tested concentration. The results of the NCI cytostatic
screening are shown in Table 2.
12d
(NF506)
12e
(NF507)
——————————————————————————————
# Embryos: number of eggs tested
% Inhibition: percentage of the investigated eggs in which an inhibition of
angiogenesis was observed on treatment with approximatively
70 nmol/disk of the compound
ID50: dose in nmol/disk that induces 50 % inhibition of angiogenesis in
the CAM assay
Arch. Pharm. Pharm. Med. Chem. 331, 97–103 (1998)

100
Kreimeyer, Müller, Kassack, Nickel, and Gagliardi
Teflon-coated metal tray forming a disk around 2 mm diame- concentration. All cell lines against which the compounds
ter. This disk is implanted on the outer third of six-day CAM
showed an activity at concentrations ≤100 µM are shown.
where capillaries are intensively growing. The zone around 12a (NF503), a disulfonate shows a marginal activity against
the methylcellulose disk is examined 48 h after implantation. only one leukemia cell line (K-562). Incontrast, 12d (NF506)
A positive inhibition of angiogenesis is indicated by an avas- which is a close analogue of suramin inhibits 9 cell lines.
cular area of ≥ 4 mm. Standard dose of the test compound is However, the activities are also marginal. The activities are
70 nmol/disk. The results of the anti angiogenic screening are compared with the activity of suramin against these cell lines.
shown in Table 3.
Suramin is inactive against 4 cell lines at the highest investi-
gated concentration (100 µM). Against 5 cell lines,
12d (NF506) and suramin show comparable low activities.
Here again, the activity of the dicarboxylic acid analogue
17 (NF417) is of special interest. It is more active than the
sulfanilic acid analogue 12a (NF503). Against 7 cell lines, it
shows an activity comparable or slightly higher than that of
suramin. However, the spectrum of activity of 17 (NF417)
differs markedly from that of suramin. It shows the highest
activity against three leukemia cell lines against which
suramin and 12a (NF503) are completely inactive.
Figure 2. Compound 17 (NF417).
Discussion
Inhibition of angiogenesis: Suramin inhibits tumor growth
[6]
factors . The most interesting target of suramin analogues
Anti HIV activity: Simian reverse transcriptase
SIVagmTYO-7 RT was inhibited by 12d with an IC50 of
is the inhibition of angiogenesis. This activity can not be
tested in a cell culture. The three benzimidazole analogues
12c (NF504), 12d (NF506), and 12e (NF507) show activi-
ties comparable to that of suramin. The tetrasulfonate
[23]
[11]
0.5 µM
. The batch of 12d
used in this experiment was
not very pure. This high inhibition of SIV-RT could later not
be reproduced with a very pure sample (HPLC purity > 99 %)
12c (NF504) has the lowest ID .
[12]
50
of 12d (NF506)
. This disappointing result could be due
to the high non-selective binding of 12d (NF506) to proteins,
[24]
e.g. to serum albumin
. The repeated investigation of
Conclusion
12d (NF506) was done with a different batch of the enzyme.
The purity of the enzyme can have an important influence on
the test result.
The title compounds, suramin analogues with 2-phenyl-
benzimidazole as partial structure, show biological activities
comparable to those shown by suramin in the cited test
systems. These results differ significantly from those ob-
served with trypanosomes. Very small variations of the
suramin structure, e.g. the substitution of the two methyl
Similar confusing results were obtained with compounds
[13]
12a (NF503) and 17 (NF417)
in the NCI HIV test shown
in Table 1. The high activities and selectivities of both com-
pounds could not be reproduced when the experiments were
repeated after several months. But, in contrast to the experi-
ments with SIV-RT, all results cited in Table 1 were done
with the same batches of very pure compounds. With suramin
and with 12d (NF506), the results were reproducible when
the experiments were repeated after some months.
12d (NF506) seems to be slightly more active but also more
toxic than suramin. Therefore, 12d (NF506) has no advan-
tage compared with suramin. 12d (NF506) is like suramin a
hexasulfonate. It is highly soluble in water. The results of the
four different tests do not vary significantly. In contrast,
12a (NF503) and 17 (NF417) have a very low solubility in
water. In the NCI screening, the compounds are dissolved in
DMSO. These solutions are added to the aqueous medium of
the cell cultures. We assume that the compounds precipitate
partially in the medium. Only the dissolved compounds can
lead to a biological activity. The consequence of this (cer-
tainly not reproducible) precipitation will be a strong vari-
groups by hydrogen or other substituents, lead to a nearly
[27]
complete loss of trypanocidal activity
.
Acknowledgment
The authors are very grateful to Prof. Dr. John P. Bader, Chief Antiviral
Evaluation Branch, National Cancer Institute, Bethesda, Maryland 20892,
USA for evaluation of the in-vitro anti HIV and cytostatic tests. Thanks are
also due to the members of NCI staff.
Experimental
The benzenedisulfonic acids 2b,c were a gift from Hoechst, the naphthale-
netrisulfonic acids 2d–e were a gift from Bayer. Oxidations were performed
in: Laborautoclave HR 500 (Berghof) with heating equipment HT 20 (Horst)
and magnetic stirring equipment. pH-Stat equipment: Metrohm 718
STAT Titrino. Titrations: Metrohm Titroprozessor 672; determination of the
equivalent mass: about 0.1 mmol of the compound was dissolved in 5.0 ml
of DMF and titrated with aqueous 0.1 N NaOH (glass electrode). TLC:
Merck aluminum sheets with silica gel 60F254, solvent 2-propanol/NH3 25%,
5:2. NMR (solvent as internal standard): Varian XL 300, chemical shifts are
given in ppm; pt: pseudotriplet; coupling constants are given in Hertz (Hz).
FAB-MS: Kratos concept 1H. IR: (KBr) Perkin-Elmer 1420. UV/VIS: Spec-
trophotometer HP 8451 A. The purity of all suramin analogues described in
ation of the results. The activity of the dicarboxylic acid 17
[13]
(NF417)
is quite interesting. In all earlier screenings of
suramin analogues, the activities were completely lost when
sulfonate substituents were replaced by carboxylate substi-
this paper was determined by the HPLC method described in ref. [18]
.
[26, 27]
tuents
.
Experimental details are described only for the synthesis of the naphtha-
lene-1,3,6-trisulfonic acid derivative 12e. The sulfanilic acid (a), benzene-
1,3-disulfonic acid (b), benzene-1,4-disulfonic acid (c) and naphthalene-
1,3,5-trisulfonic acid (d) derivatives were prepared in a similar manner
starting from 2a, 2b, 2c, or 2d, respectively instead of 2e (see Table 4).
Cytostatic activity: In Table 2, the cytostatic activities of
3 new suramin analogues are presented. Neither suramin nor
any one of the investigated suramin analogues was able to
inhibit totally the tumor cell growth at the highest tested
Arch. Pharm. Pharm. Med. Chem. 331, 97–103 (1998)

Suramin Analogues
101
Table 4. Synthetic and analytical data of the sulfanilic acid (4a–12a), benzene disulfonic acid (4b,c–12b,c), and naphthalenetrisulfonic acid (4d,e–12d,e)
derivatives .
—————————————————————————————————————————————————————————————–
Molecular mass
Compd.
Starting
material
yield
%
TLC
calcd.
found
purity
HPLC
Method
Rf
Formula
MS
titrationa)
—————————————————————————————————————————————————————————————–
4a
4b
4c
2a, 3
2b, 3
2c, 3
A
A
A
69
72
49
0.68
0.52
0.58
C13H8N3NaO8S
C13H9N3O11S2
C13H7N3Na2O10S2
C13H10N3O10S2
389.27
447.36
491.32
447.34
577.48
577.48
460.40
438.42
562.44
562.44
714.55
714.55
430.42
532.46
532.46
684.57
684.57
886.83
1090.92
1090.92
1395.13
1395.13
388(M-H)
446(M-H)
446(M-H)
576(M-H)
576(M-H)
4d
4e
2d, 3
A
A
B
78
72
68
0.51
0.55
0.70
C17H11N3O14S3
2e, 3
C17H11N3O14S3
10a
7a - 9a
C20H13N4NaO6S
C20H14N4NaO6S
C20H12N4Na2O9S2
C20H12N4Na2O9S2
C24H13N4Na3O12S3
463.3
437(M-H)
10b
10c
10d
10e
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
7b–9b
7c–9c
7d–9d
7e–9e
10a
B
B
B
B
C
C
C
C
C
D
D
D
D
D
40
52
36
39
61
66
49
53
53
51
26
18
34
36
0.56
0.58
0.41
0.55
0.66
0.48
0.55
0.47
0.47
0.53
0.44
0.41
0.40
0.51
539(M+Na-2H)
539(M-Na)
571.6
565.1
691(M-Na)
C
24H13N4Na3O12S3
691(M+H-2Na)
429(M-H)
704.7
422.5
527.4
566.0
679.9
679.9
450.6
532.4
496.8
C20H15N4NaO4S
10b
C20H14N4Na2O7S2
487(M+H-2Na)
487(M+H-2Na)
661(M-Na)
b)
10c
C
20H14N4Na2O7S2
C20H15N4Na3O10S3
20H15N4Na3O10S3
10d
10e
C
661(M-Na)
11a
C41H28N8Na2O9S2
C41H26N8Na4O15S4
885(M-H)
11b
1067(M-Na)
1045(M+H-2Na)
1371(M-Na)
1327(M+2H-3Na)
~100%
~95%
>99%
>99%
11c
C41H26N8Na4O15S4
11d
C49H28N8Na6O21S6
C49H28N8Na6O21S6
b)
11e
716.7
—————————————————————————————————————————————————————————————–
a) titration in DMF with aqueous 0.1 N NaOH
b) contains 2 mol H2O
A) 8-(3,4-Dinitrobenzamido)-1,3,6-naphthalenetrisulfonic acid trisodium
salt (4e)
(C-8), 133.75 (C-6′), 132.04 (C-4a), 128.10 (C-4), 126.45 (C-5), 125.77
(C-2), 124.91 (C-5′), 123.63 (C-2′), 122.52 (C-8a), 122.49 (C-3). FAB MS
(Glycerol/DMSO) m/z: 496 [M-SO3H]–, 560 [M-OH]–, 576 [M-H]–, 668
[M+Glyc-H]–, 690 [M+Glyc+Na-2H]–, 760 [M+2Glyc-H]–, 852 [M+3Glyc-
H]–, 944 [M+4Glyc-H]–.
Toa solution of 2e (21.4 g, 50 mmol) in water (150 ml), 3,4-dinitrobenzoyl
chloride (3, 23 g, 100 mmol), dissolved in 100 ml of toluene, was added
under vigorous stirring. During the reaction time (3 h), a pH of 4.5 was
maintained by automatic addition of 1 M NaHCO3. The aqueous phase was
separated. The organic phase was washed twice with water. The combined
aqueous phases were evaporated in vacuo to dryness and the residue was
twice recrystallized from EtOH/H2O (3 : 1). 4e was obtained as a yellow
powder (23.6 g, 78 %).
C
17H8N3Na3O14S3 (643.42), C17H11N3O14S3 (577.48).
In a similar way, compounds 4a–d were synthesized; for analytical data
see Table 4.
B) 8-[2-(3-Nitrophenyl)benzimidazole-5-carboxamido]-1,3,6-naphthalene-
trisulfonic acid trisodium salt (10e)
TLC: Rf 0.55. IR (cm–1): 3470, 3280, 1670, 1550, 1530, 1390, 1350, 1220,
A solution of 4e (12.8 g, 20 mmol) in MeOH (300 ml) and H2O (200 ml)
was hydrogenated at a hydrogen pressure of 5 bar with PtO2 as the catalyst.
After the hydrogen absorption had come to an end, the reaction mixture was
mixed immediately with a solution of 3-nitrobenzaldehyde (6, 4.5 g,
30 mmol) in MeOH (60 ml). After 10 min the mixture was filtrated. The
filtrate was stirred for 12 h at RT, evaporated to a volume of 100 ml, and
oxidized in an autoclave at 120 °C for 12 h under an oxygen pressure of 6 bar.
The reaction mixture was evaporated to dryness and the residue was recrys-
1185, 1040, 845, 675. UV/VIS (H2O): λmax (logε): 194 (4.56), 238 (4.48),
300 (3.90). 1H-NMR (300 MHz [d6]DMSO): δ = 8.14 (d, J = 2.0 Hz, 1H,
5-H), 8.26 (d, J = 2.0 Hz, 1H, 7-H), 8.42 (d, J = 8.5 Hz, 1H, 5′-H), 8.64 (d,
J = 2.0 Hz, 1H, 2-H), 8.64 (d, J = 2.0 Hz, 1H, 2′-H), 8.69 (dd, J = 8.5 Hz,
J = 2.0 Hz, 1H, 6′-H), 8.92 (d, J = 2.0 Hz, 1H, 4-H), 12.85 (s, 1H, -NH-CO-).
13C-NMR (75 MHz [d6]DMSO): δ = 161,75 (C-9), 144.75 (C-1), 143.38
(C-3), 143.01 (C-4′), 141.49 (C-3′), 141.26 (C-6), 140.60 (C-1′), 134.38
Arch. Pharm. Pharm. Med. Chem. 331, 97–103 (1998)

102
Kreimeyer, Müller, Kassack, Nickel, and Gagliardi
tallized from MeOH/H2O (1:1) yielding 6.5 g (39 %) of 10e as a beige
powder.
J = 2.0 Hz, 1H, 7-H), 8.41 (s, 1H, 2′′-H), 8.53 (d, J = 1.8 Hz, 1H, 2-H), 8.55
(s, 1H, 4′-H), 8.67 (d, J = 2.0 Hz, 1H, 4-H), 9.55 (s, 1H, -NH-CO-), 12.7 (s,
1H, -NH-CO-). 13C-NMR (75 MHz [d6]DMSO): δ = 151.70 (C-10), 165.55
(C-9), 152.67 (C-2′), 144.36 (C-1), 142.98 (C-3), 141.56 (C-6), 140.48
(C-3′′), 136.99 (C-3′), 134.80 (C-8), 134.75 (C-7a′), 133.36 (C-4a), 131.83
(C-1′′), 130.29 (C-5′′), 128.86 (C-4), 126.56 (C-5), 126.32 (C-5′), 124.98
(C-2), 123.48 (C-7), 123.16 (C-8a), 122.82 (C-6′′), 122.61 (C-6′), 121.63
(C-4′′), 117.42 (C-2′′), 114.88 (C-4′), 114.23 (C-7′). FAB MS (Glyc-
erol/DMSO) m/z: 1269 [M+H SO3Na Na]–, 1327 [M+2H 3Na]–.
C49H28N8Na6O21S6 (1395.13).
TLC: Rf 0.45. Titration: equiv. mass calcd. 714.6; found 704.6. IR (cm–1):
3460, 1660, 1635, 1535, 1390, 1360, 1220, 1200, 1150, 715, 680. UV/VIS
(H2O): λmax (logε): 192 (4.36), 240 (4.75) 316 (4.47). 1H-NMR (300 MHz
[d6]DMSO): δ = 7.91 (dd, J = 8.7 Hz, J = 0.5 Hz, 1H, 7′-H), 7.99 (pt,
J = 8.2 Hz, 1H, 5′′-H), 8.01 (d, J = 2.0 Hz, 1H, 5-H), 8.17 (d, J = 1.8 Hz, 1H,
7-H), 8.27 (dd, J = 8.5 Hz, J = 1.8 Hz, 1H, 6′-H), 8.48 (ddd, J = 8.2 Hz,
J = 2.0 Hz, J = 1.0 Hz, 1H, 6′′-H), 8.59 (d, J = 2.0 Hz, 1H, 2-H), 8.63 (d,
J = 1.8 Hz, 1H, 4-H), 8.65 (ddd, J = 8.2 Hz, J = 2.0 Hz, J = 1.0 Hz, 1H,
4′′-H), 8.8 (dd, J = 1.8 Hz, J = 0.5 Hz, 1H, 4′-H), 9.13 (pt, J = 2.0 Hz, 1H,
2′′-H), 12.75 (s, 1H, -NH-CO-). 13C-NMR (75 MHz [d6]DMSO): δ = 164.48
(C-9), 149.04 (C-2′), 148.09 (C-3′′), 144.71 (C-1), 143.26 (C-3), 141.45
(C-6), 135.85 (C-3a′), 134.34 (C-8), 133.62 (C-7a′), 133.39 (C-6′′), 133.01
(C-1′′), 132.63 (C-4a), 131.01 (C-5′′), 127.85 (C-4), 126.67 (C-5′), 126.39
(C-2′′), 126.12 (C-5), 125.53 (C-2), 122.62 (C-7), 122.31 (C-8a), 122.23
(C-4′′), 121.94 (C-6′), 114.43 (C-4′), 114.07 (C-7′). FAB MS (Glyc-
erol/DMSO) m/z: 567 [M+2H-2Na-SO3Na]–, 589 [M+H-Na-SO3Na]–, 631
[M+3H-3Na-OH]–, 647 [M+2H-3Na]–, 669 [M+H-2Na]–.
In a similar way, compounds 12a–d were synthesized; for analytical data
see Table 4.
E) 3-[1-(3-Nitrobenzyl)-2-(3-nitrophenyl)-benzimidazole-5-carboxamido]-
1,4-benzene disulfonic acid disodium salt (14c)
A solution of 4c (5.0 g, 10.5 mmol) in MeOH (150 ml) and H2O (60 ml)
was hydrogenated and treated with 3-nitrobenzaldehyde (6, 2.28 g,
15.1 mmol) according to method B. The reaction mixture was oxidized in an
autoclave at 120 °C for 12 hr under an oxygen pressure of 6 bar. 10c (3.1 g,
52 %) precipitated and was isolated by filtration. The filtrate was evaporated
to dryness, and the residue was recrystallised from MeOH/H2O (8 + 1)
yielding 1.42 g (19 %) of 14c as a beige powder.
C24H13N4Na3O13S3 (714.55).
In a similar way, compounds 10a–d were synthesized; for analytical data
see Table 4.
C) 8-[2-(3-Aminophenyl)benzimidazole-5-carboxamido]-1,3,6-naphthal-
enetrisulfonic acid trisodium salt (11e)
TLC: Rf 0.58. IR (cm–1): 3485, 1660, 1535, 1410, 1350, 1275, 1220, 1185,
1015, 820, 730. UV/VIS (0.1 N NaOH): λmax (log ε): 298 (4.49). 1H-NMR
(300 MHz [d6]DMSO): δ = 5.9 (s, 2H, -CH2-), 7.32 (dd, J = 8.0 Hz,
J = 1.6 Hz, 1H, 6-H), 7.54 (ddd, J = 7.8 Hz, J = 1.8 Hz, J = 1.3 Hz, 1H,
4′′′-H), 7.61 (dd, J = 8.0 Hz, J = 7.8 Hz, 1H, 5′′′-H), 7.67 (d, J = 8.0 Hz, 1H,
5-H), 7.85 (dd, J = 8.5 Hz, J = 8.0 Hz, 1H, 5′′-H), 7.93 (dd, J = 9.0 Hz,
J = 1.2 Hz, 1H, 6′-H), 7.94 (dd, J = 2.5 Hz, J = 1.8 Hz, 1H, 2′′′-H), 7.96 (d,
J = 9.0 Hz, 1H, 7′-H), 8.15 (ddd, J = 8.0 Hz, J = 2.5 Hz, J = 1.3 Hz, 1H,
6′′′-H), 8.23 (ddd, J = 8.0 Hz, J = 1.9 Hz, J = 1.0 Hz, 1H, 4′′-H), 8.26 (d,
J = 1.2 Hz, 1H, 4′-H), 8.39 (ddd, J = 8.5 Hz, J = 2.3 Hz, J = 1.0 Hz, 1H,
6′′-H), 8.51 (dd, J = 2.3 Hz, J = 1.9 Hz, 1H, 2′′-H), 8.79 (d, J = 1.6 Hz, 1H,
2-H), 8.83 (d, J = 1.9 Hz, 1H, 2-H), 11.45 (s, 1H, -NH-CO-). FAB MS
(Glycerol) m/z: 572 [M+H-Na-SO3Na]–, 594 [M-SO3Na]–, 652 [M+H-
2Na]–, 674 [M-Na]–, 744 [M+Gly+H-2Na]–, 766 [M+Gly-Na]–, 788
[M+Gly-H]–.
A suspension of 10e (10 g, 14 mmol) in MeOH (200 ml) and H2O (200 ml)
was hydrogenated at a hydrogen pressure of 5 bar with Pd/C (10% Pd,
200 mg) as the catalyst. 10e dissolved during the reaction course. The
reaction mixture was filtrated, and the filtrate evaporated in vacuo to dryness.
Recrystallization in EtOH/H2O (4:3) gave 5.0 g (52 %) of 11e as a yellow-
green powder.
TLC: Rf 0.77. Titration: equiv. mass calcd. 684.6; found 733.7. IR (cm–1):
3440, 3060, 1655, 1625, 1590, 1565, 1545, 1485, 1385, 1350, 1220, 1175,
1035, 715, 670. UV/VIS (H2O): λmax (logε): 236 (4.83), 316 (4.47). 1H-NMR
(300 MHz [d6]DMSO): δ = 7.14 (ddd, J = 8.3 Hz, J = 2.3 Hz, J = 1.2 Hz,
1H, 6′′-H), 7.45 (pt, J = 8.3 Hz, 1H, 5′′-H), 7.58 (m, 1H, 2′′-H), 7.59 (m, 1H,
4′′-H), 7.85 (d, J = 8.8 Hz, J = 0.5 Hz, 1H, 7′-H), 7.9 (s, 2H, -NH2), 8.09 (d,
J = 2.0 Hz, 1H, 5-H), 8.22 (dd, J = 8.8 Hz, J = 1.8 Hz, 1H, 6′-H), 8.24 (d,
J = 2.0 Hz, 1H, 7-H), 8.50 (d, J = 2.0 Hz, 1H, 2-H), 8.58 (dd, J = 1.8 Hz,
J = 0.8 Hz, 1H, 4′-H), 8.63 (d, J = 2.0 Hz, 1H, 4-H), 12.75 (s, 1H, -NH-CO-).
13C-NMR (75 MHz [d6]DMSO): δ = 164.45 (C-9), 150.97 (C-2′), 144.66
(C-1), 144.40 (C-3′′), 143.22 (C-3), 141.44 (C-6), 134.73 (C-3a′), 134.35
(C-8), 133.02 (C-7a′), 132.68 (C-1′′), 132.60 (C-4a), 130.24 (C-5′′), 127.90
(C-4), 126.11 (C-5), 125.46 (C-2), 124.78 (C-5′), 122.68 (C-7), 122.36
(C-8a), 122.01 (C-6′), 120.96 (C-6′′), 118.60 (C-4′′), 114.92 (C-4′), 114.05
(C-7′), 113.55 (C-2′′). FAB MS (Glycerol/DMSO) m/z: 617 [M+2H-3Na]–.
C24H15N4Na3O10S3 (684.57).
C27H17N5Na2O11S2 (697.57).
F) 3-[1-(3-Aminobenzyl)-2-(3-aminophenyl)-benzimidazole-5-carbox-
amido]-1,4-benzene disulfonic acid disodium salt (15c)
14c (1.0 g, 1.29 mmol) was dissolved in MeOH (170 ml) and H2O (30 ml)
and hydrogenated at a hydrogen pressure of 5 bar with Pd/C (10% Pd, 70 mg)
as the catalyst. The reaction mixture was filtrated, and the filtrate evaporated
in vacuo to dryness. Recrystallization of the residue in EtOH/H2O (8 + 1)
gave 0.34 g (41.3 %) of 15c as a green powder. 11c was not detectable by
TLC.
In a similar way, compounds 11a–d were synthesized; for analytical data
see Table 4.
D) 8,8-[Carbonylbis(imino-3,1-phenylene-(2,5-benzimidazolylene)-car-
TLC: Rf 0.55. IR (cm–1): 3420 3060, 1660, 1605, 1575, 1530, 1450, 1400,
1275, 1215, 1185, 1015, 660. UV/VIS (0.1 N NaOH): λmax (logε): 230
(4.68), 304 (4.40). 1H-NMR (300 MHz [d6]DMSO): δ = 5.0 (s, 4H,
2 × –NH2), 5.43 (s, 2H, -CH2-), 6.20 (ddd, J = 7.7 Hz, J = 1.8 Hz,
J = 1.1 Hz, 1H, 6′′′-H), 6.43 (ddd, J = 8.0 Hz, J = 2.2 Hz, J = 1.0 Hz, 1H,
4′′′-H), 6.48 (dd, J = 2.2 Hz, J = 1.8 Hz, 1H, 2′′′-H), 6.76 (ddd, J = 8.0 Hz,
J = 2.3 Hz, J = 1.0 Hz, 1H, 6′′-H), 6.87 (ddd, J = 7.8 Hz, J = 1.8 Hz,
J = 1.0 Hz, 1H, 4′′-H), 6.93 (dd, J = 8.0 Hz, J = 7.7 Hz, 1H, 5′′′-H), 7.08 (dd,
J = 2.3 Hz, J = 1.8 Hz, 1H, 2′′-H), 7.18 (dd, J = 8.0 Hz, J = 7.8 Hz, 1H,
5′′-H), 7.33 (dd, J = 8.3 Hz, J = 1.9 Hz, 1H, 6-H), 7.69 (d, J = 8.3 Hz, 1H,
5-H), 7.80 (d, J = 8.6 Hz, 1H, 7′-H), 7.86 (dd, J = 8.6 Hz, J = 1.8 Hz, 1H,
6′-H), 8.10 (d, J = 1.8 Hz, 1H, 4′-H), 8.83 (d, J = 1.9 Hz, 1H, 2-H), 11.3 (s,
1H, -NH-CO-). FAB MS (Glycerol) m/z: 512 [M+H-Na-SO3Na]–, 592
[M+H-Na]–, 614 [M-Na]–, 636 [M-H]–, 706 [M+Gly-Na]–, 798 [M+2Gly-
Na]–.
bonyl-imino)]bis-1,3,6-naphthalenetrisulfonic acid hexasodium salt (12e)
A 2 M solution of phosgene in toluene (20 ml, 40 mmol COCl2) was added
dropwise over 30 min at room temperature to a vigorously stirred solution of
11e (2.7 g, 4 mmol) in H2O (40 ml). During the reaction time, the mixture
was maintained at pH 4.5 by automatic addition of 2 N NaOH. Once the
addition was over, stirring was continued for 30 min and the pH was adjusted
to pH 6 with 2 M NaOH. The reaction mixture was evaporated to dryness
and the residue was recrystallized from EtOH/H2O (4 : 3) giving 11e as a
white powder (2.2 g), containing 2.1 % NaCl as measured by titration, thus
the yield of isolated 12e was 2.15 g (35 %).
TLC: Rf 0.51. Titration: equiv. mass calcd. 697.6; found 716.7. IR (cm–1):
3440, 3080, 1640, 1590, 1540, 1480, 1450, 1295, 1200, 1140, 1040, 715, 675.
UV/VIS (H2O): λmax (logε): 194 (4.82), 240 (5.01), 316 (4.73). 1H-NMR
(300 MHz [d6]DMSO): δ = 7.56 (pt, J = 8.0 Hz, 1H, 5′′-H), 7.7 (d,
J = 8.0 Hz, 1H, 6′′-H), 7.84 (d, J = 8.0 Hz, 1H, 4′′-H), 7.88 (d, J = 9.0, 1H,
7′-H), 8.135 (d, J = 1.8 Hz, 1H, 5-H), 8.18 (d, J = 9.0 Hz, 1H, 6′-H), 8.29 (d,
C27H21N5Na2O7S2 (637.61).
Arch. Pharm. Pharm. Med. Chem. 331, 97–103 (1998)

Suramin Analogues
103
[15] I.Y. Kvitko, L.I. Rudaya, E.S. Shcherbakha, S.S. Bogolyubova, De-
posited Doc VINITI. 1977, 5–10 (in Russian); Chem Abstr. 1979, 90,
123046k.
References
✩
Dedicated to Prof. Dr. W. Wiegrebe, Regensburg, on the occasion of
his 65th birthday.
[16] F. Möller, in Houben-Weyl, Methoden der organischen Chemie, Bd.
11/1, p. 968, Thieme, Stuttgart 1952.
[1] T.E. Voogd, E.L.M. Vansterkenburg, J. Wilting, L.H. Janssen, Phar-
macol. Rev. 1993, 45, 177–203.
[17] P. Nickel, W. Weber, Pharmaz. Ztg. (Scientific Edition) 1989, 134, 13.
[18] M. Kassack, P. Nickel, J. Chromatogr. B 1996, 686, 275 - 284.
[2] F. Hawking, Adv. Pharmacol. Chemother. 1987, 45, 177.
[19] O.W. Weislow, R. Kiser, D.L. Fine, J. Bader, R.H. Shoemaker,
[3] L. van Hoof, C. Henrard, E. Peel, N. Wanson, Ann. Soc. Belge Med.
Trop. 1947, 50, 3374.
M.R. Boyd, J. Natl. Cancer Inst. 1989, 81, 577.
[20] A.R. Gagliardi, H. Hadd, D. Collins, Cancer Res. 1992, 52, 5073.
[4] D.H. Molineux, J.B. Davies, Parasitol. Today 1997, 13, 418.
[5] E. DeClercq, Cancer Lett. 1979, 8, 9–22.
[21] A. Firsching, P. Nickel, P. Mora, B. Allolio, Cancer Res. 1995, 55,
4957.
[6] C.A. Stein, R.V. LaRocca, R. Thomas, N. McAtee, C.E. Myers,
[22] A.R. Gagliardi, M. Kassack, A. Kreimeyer,G. Müller, P. Nickel,
J. Clin. Oncol. 1989, 7, 499–508.
D. Collins, Cancer Chemother. Pharmacol. 1998, 41, 177.
[7] R. Rybka, Ph D. Thesis, Free University of Berlin 1988.
[23] R.U. Oesterle, E. Jurkiewicz, W. Lüke, P. Nickel, G. Hunsmann,
K.D. Jentsch, Antiviral Res. 1993, 22, 107–119.
[8] M. Ciomei, W. Pastori, M. Mariani, F. Sola, M. Grandi, N. Mongelli,
Biochem. Pharmacol. 1994, 47, 295.
[24] M. Oehlmann, Ph D. Thesis, University of Bonn 1996.
[25] K.D. Jentsch, G. Hunsmann, H. Hartmann, P. Nickel, J. Gen. Virol.
1987, 68, 2183.
[9] H. Loewe, Pharm. unserer Zt. 4, 79 (1975).
[10] P. Mohan, R. Singh, M. Baba, J. Med. Chem. 1991, 34, 212–217.
[11] E. Magor, Diplomarbeit, University of Bonn 1987.
[12] A. Kreimeyer, Ph D. Thesis, University of Bonn 1993
[13] A. Kreimeyer, E. Magor, P. Nickel, Pharmazie 1997, 52, 268.
[14] H. Goldstein,R. Voegeli, Helv. Chim. Acta 1943, 26, 475–481.
[26] P. Nickel, E. Schott, C. Gurgel, D. Düwel, W. Raether, Indian J. Chem.
1991, 30B, 182; Chem Abstr. 114, 229348q.
[27] P. Nickel., H.J. Haack, H. Widjaja, U. Ardanuy, C. Gurgel.,
D. Düwel., H. Loewe, W. Raether, Arzneim.-Forsch./Drug Res. 1986,
36, 1153.
Received: August 18, 1997 [FP246]
Arch. Pharm. Pharm. Med. Chem. 331, 97–103 (1998)