1-(2-Mercaptobenzenesulfonyl)-3-hydroxyguanidines - Novel potent antiproliferatives, synthesis and in vitro biological activity

Article abstract of DOI:10.1016/j.ejmech.2012.07.042

Twenty four 1-[2-alkylthio-5-(azol-2 or 5-yl)-4-chlorobenzenesulfonyl]-3- hydroxyguanidines 6a-x have been synthesized in order to evaluate their biological activity. Compounds 6a, 6c, 6d, 6f, 6g, 6i-p, 6r-t, and 6v-x were tested for their in vitro anticancer activity at the US National Cancer Institute. The highest in vitro anticancer activity was found for compounds 6d, 6g and 6k with GI₅₀ average value in the range 1.62-1.86 μM, and TGI mean values 3.72-4.47 μM, whereas the remaining compounds showed broad spectrum of anticancer activity at low micromolar GI₅₀ level against all tested cancer cell lines. These results were subjected to CoMSIA analysis to establish quantitative structure-activity relationships. The results evidence that potency of these compounds correlates mainly with hydrophobic and polar surface properties of substituents located both at 2 and 5 positions of 1-(4-chlorobenzenesulfonyl) moiety of investigated 3-hydroxyguanidine series.

Full text of DOI:10.1016/j.ejmech.2012.07.042

European Journal of Medicinal Chemistry 55 (2012) 384e394
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1-(2-Mercaptobenzenesulfonyl)-3-hydroxyguanidines e Novel potent
antiproliferatives, synthesis and in vitro biological activity
*
_
ꢀ
Kamil Brozewicz, Jaros1aw S1awinski
ꢀ
ꢀ
Department of Organic Chemistry, Medical University of Gdansk, Al. Gen. J. Hallera 107, 80-416 Gdansk, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty four 1-[2-alkylthio-5-(azol-2 or 5-yl)-4-chlorobenzenesulfonyl]-3-hydroxyguanidines 6aex have
been synthesized in order to evaluate their biological activity. Compounds 6a, 6c, 6d, 6f, 6g, 6iep, 6ret,
and 6vex were tested for their in vitro anticancer activity at the US National Cancer Institute. The highest
in vitro anticancer activity was found for compounds 6d, 6g and 6k with GI50 average value in the range
Received 19 March 2012
Received in revised form
20 July 2012
Accepted 27 July 2012
Available online 4 August 2012
1.62e1.86 mM, and TGI mean values 3.72e4.47 mM, whereas the remaining compounds showed broad
spectrum of anticancer activity at low micromolar GI50 level against all tested cancer cell lines. These
results were subjected to CoMSIA analysis to establish quantitative structureeactivity relationships. The
results evidence that potency of these compounds correlates mainly with hydrophobic and polar surface
properties of substituents located both at 2 and 5 positions of 1-(4-chlorobenzenesulfonyl) moiety of
investigated 3-hydroxyguanidine series.
Keywords:
2-Mercaptobenzenesulfonamide
Hydroxyguanidine
Synthesis
Ó 2012 Elsevier Masson SAS. All rights reserved.
Anticancer activity
1. Introduction
Our systematic study of 2-mercaptobenzenesulfonamide deriva-
tives led us to discover novel anticancer [12e18], anti-HIV [19e21]
N-Hydroxyguanidine derivatives represent a unique group of
compounds which has found limited attention in synthetic
medicinal chemistry. Most of them are considered as NOS
substrates, thus being nitric oxide donors (NO) [1e3]. Recently
cytotoxicity studies of simple 1-aryl-3-hydroxyguanidine deriva-
tives revealed that this kind of compounds can selectively kill
tumor cells via NO dependent mechanism [4]. There is limited
evidence about antitumor activity of N-sulfonylated hydrox-
yguanidine derivatives. Closely related Eli Lily’s diarylsulfonylureas
(Fig. 1A) have been identified with high therapeutic efficacy in
various in vitro and in vivo preclinical anticancer assays [5,6] and
reached clinical trials [7,8]. Earlier studies implicate mitochondria
as a potential site of action [9], although their primary molecular
target remains unknown. Due to discovered sulfonylurea instability
in physiological conditions, bioisosteric hydroxyguanidine deriva-
tives were developed which showed interesting activity on several
human cancer cell lines (Fig. 1B) [10]. Another researchers reported
tricyclic analogs with built-in 1-sulfonyl-3-hydroxyguanidine
moiety (Fig. 1C) as potent antiproliferatives against KB, Colo 205,
HeLa and Hepa-2 cell lines [11].
and antibacterial [22] agents. Our ongoing research in the synthesis
and biological activity of novel 2-mercaptobenzenesulfonamides
(MBSA) with five-membered heterocycles located in 5 position of
MBSA scaffold [17,18] led us to an assumption that incorporation of
hydroxyguanidine moiety into developed structures will be of high
value to understand more about the chemistry and biological
activity of this class of compounds. This paper herein describes
a novel class of highly potent antitumor agents derived from
MBSA scaffold, bearing the common core of 1-(4-chloro-2-
mercaptobenzenesulfonyl)-3-hydroxyguanidine.
2. Results and discussion
2.1. Chemistry
The synthetic route for the preparation of target 1-[2-alkylthio-
5-(azol-2 or 5-yl)-4-chlorobenzenesulfonyl]-3-hydroxyguanidines
6aex are shown in Schemes 1 and 2.
Thus, the essential intermediates 3aed for the synthesis of 3-
amino-1,1-dioxo-1,4,2-benzodithiazines 4aed were obtained accord-
ing to the methods described previously [17]. Subsequent new inter-
mediates 3eeh for the synthesis of novel 3-aminobenzodithiazines
4eeh were synthesized as depicted in Scheme 1.
Synthesis and antitumor activity of unsubstituted 1-
benzenesulfonyl-3-hydroxyguanidines appear to be unknown.
Thus, the starting 6-chloro-3-methylthio-1,1-dioxo-1,4,2-
benzodithiazine-7-carboxylic acid 1 [23] could be easily converted
* Corresponding author. Tel.: þ48 58 349 10 98; fax: þ48 58 349 12 77.
ꢀ
E-mail address: jaroslaw@gumed.edu.pl (J. S1awinski).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.042

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K. Brozewicz, J. Sławinski / European Journal of Medicinal Chemistry 55 (2012) 384e394
385
concentration of test agent) nineteen compounds were subjected to
further studies in 5-dose assay (Table 1). Most of 1-(2-
mercaptobenzenesulfonyl)-3-hydroxyguanidine derivatives (6a,
6c, 6d, 6f, 6g, 6iep, 6ret, and 6vex) show broad spectrum of
cytostatic activity overall tested cancer cell lines, and compound 6k
seems to be the most potent against tested cell lines (Tables 1e3).
Represented mostly in 5-dose screen of the series compounds
with incorporated (1,3-benzodioxol-5-yl)methylthio moiety in 2
position (6iep: mean GI50
TGI ¼ 3.72e75.86 M) and 5-aryl-1,3,4-thiadiazol-5-yl moiety in 5
position (6c, 6d, 6k, 6l, 6s, 6t: mean GI50 ¼ 1.62e18.62 M, mean
TGI ¼ 3.72e70.79 M) (Table 1) suggest that these groups increase
¼
1.62e11.22
mM, mean
m
Fig. 1. (A) Diarylsulfonylureas (DSUs); (B) 1-benzenesulfonyl-2-hydroxyguanidines;
(C) 1-benzenesulfonyl-3-hydroxyguanidine heterocyclic anticancer agents.
m
m
invitroanticanceractivityof1-benzenesulfonyl-3-hydroxyguanidine
derivatives against cell lines in NCI-60 panel. Considering rather high
efficacy toward cancer cell lines of this series of compounds, it’s
worth mentioning that CCRF-CEM (leukemia) is the most sensitive
cell line in the panel, which growth is suppressed by ca. 70% of tested
1-benzenesulfonyl-3-hydroxyguanidine derivatives (6a, 6c, 6d, 6f,
6g, 6l, 6oes, 6t, 6w) at low micromolar concentrations
to thioamide 2 by three-step reaction sequence which involves reac-
tion with an excess of thionyl chloride, then 12% aqueous ammonia
solution in benzene mixture and finally thionation with Lawesson’s
reagent (LR) to give thioamide 2 in 78% yield. Subsequent reactions of
2 with the appropriate fenacyl bromides afforded the expected thia-
zoles 3e and 3f ingood yields. Inturn, desired1,2,4-oxadiazoles 3g and
3h were synthesized in one-pot procedure starting from 6-chloro-3-
methylthio-1,1-dioxo-1,4,2-benzodithiazine-7-carboxylic acid chlo-
ride with the appropriate commercially available N0-hydrox-
ybenzamidines by sequential O-acylation and intramolecular
dehydration reaction in toluene (Scheme 1).
3-Aminobenzodithiazines 4aed [17] and 4eeh were in turn ob-
tained by the reaction of the corresponding 3-methylthio-
benzodithiazines 3aed [17] and 3eeh with 25% aqueous ammonia
solution in methanol or methanol/toluene mixture at ambient
temperature for several hours (Scheme 2). Subsequent reactions of
4aeh with halomethyl electrophiles such as corresponding benzyl
chloride derivatives (Scheme 2) in dry THF in the presence of an
excess anhydrous potassium carbonate (K2CO3) furnished desired N-
(benzenesulfonyl)cyanamide potassium salts 5aed [17] and 5eex in
57e99% yields. Finally, when potassium salts 5aex were subjected to
the reactions with 3 molar equiv of hydroxylamine hydrochloride in
refluxing dry acetonitrile, the desired 1-[2-alkylthio-5-(azol-2 or 5-
(GI50 ¼ 0.55e1.58
mM, TGI ¼ 3.23e7.77
mM).
In some cases was observed a slight decrease in cytostatic
activity when 4-chlorophenyl substituted heterocycles are present
in 5 position of the core structure (6i vs. 6j, 6m vs. 6n, and 6s vs. 6t).
On the other hand presence of a chlorine atom in 1,2,4-oxadiazoles
increases their overall activity (6o vs. 6p, 6w vs. 6x). Careful anal-
ysis in the series of sulfur-containing azoles with Ar2 ¼ 1,3-
benzodioxol-5-yl substituent (6ken) shows that chlorine intro-
duction decreases their cytostatic effect toward SF-268, U251,
MDA-MB-435, UACC-62, IGROV1, and MDA-MB-231/ATCC cell lines
(Table 2). The influence of a chlorine as a modifier of the activity is
more pronounced in 1,3,4-thiadiazoles (6k and 6l) contrary to
1,3,4-oxadiazoles (6i and 6j) (Table 1).
It was also found that in a series of 1,2,4-oxadiazole (6o, 6p, 6w,
6x) as well as 1,3,4-thiadiazole (6k, 6l, 6s, 6t) derivatives changing
Ar2
¼
1,3-benzodioxol-5-yl to Ar2
¼
3,4,5-trimethoxyphenyl
substituent results in substantial decrease in antiproliferative
activity toward selected cell lines (Table 3).
yl)-4-chlrobenzenesulfonyl]-3-hydroxyguanidines
6aex
were
formed in moderate to excellent yields (51e99%) (Scheme 2).
The structure of the new compounds was evidenced by IR, 1H
and 13C and MS spectra as well as elemental analyses. Inspection of
the 1H NMR spectra revealed that in DMSO solution compounds
6aex exist as a stable sulfonylamino tautomers (see Scheme 2).
These spectra provided evidences for the confirmation of this sul-
fonylamino tautomeric form, since four separate signals of the
chemically non-equivalent (D2O exchangeable) protons of NeH and
3. Conclusions
We developed 1-(2-mercaptobenzenesulfonyl)-3-hyroxyguani-
dine derivatives 6aex and discovered their antiproliferative activity.
Most of the evaluated compounds exhibit remarkable in vitro anti-
cancer activity over DTP NCI-60 test panel represented by drug
sensitive and multi-drug resistant cell lines of main human cancer
types (leukemia, non-small cell lung cancer, melanoma, colon, CNS,
breast, ovary, prostate, and renal cancers). The COMPARE analysis of
several derivatives revealed that their mean-graph screening profiles
correlate with dactinomycin, bruceantin, chromomycin A3, and
OeH groups were found. Two singlets in the region d 7.12e7.29 and
7.34e7.40 ppm are assigned to the C]N2H and ]CeN3He protons,
respectively. Whereas singlet signals attribute to N3eOeH and
SO2eN1He groups were found downfield in the regions
d
9.44e9.47 and 10.04e10.09 ppm, respectively.
echinomycin profiles
e
nucleic acid interfering agents.
Structureeactivity relationship analysis implicates that potencyof the
series correlates mostly with hydrophobic and polar surface proper-
ties of substituents located both in 2 and 5 position of 1-(4-
chlorobenzenesulfonyl)-3-hydroxyguanidine core. The most potent
1-benzenesulfonyl-3-hydroxyguanidine derivatives have (1,3-
benzodioxol-5-yl)methylthio moiety in 2 position and/or 5-aryl-
2.2. Antiproliferative activity
The antiproliferative activity of 6aex was assessed according to
the NIH Developmental Therapeutics Program [26,27]. Identified by
the preliminary studies of one dose screen test results (10
mM
Scheme 1. Reactions conditions: (a) SOCl2 (excess), benzene, reflux [24]; (b) 12% NH3 (aq)/benzene, 5e10 ꢀC [25]; (c) 0.5 molar equiv Lawesson’s reagent, toluene, 110 ꢀC; (d)
1 molar equiv Ar1eC(]O)CH2Br, MeOH, reflux; (e) 1.1 molar equiv Ar1eC(]NeOH)NH2, toluene, 20 ꢀC, then 110 ꢀC.

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386
Scheme 2. Reactions conditions: (a) 1.5 molar equiv 25% NH4OH, MeOH or MeOH/toluene, 20 ꢀC; (b) 1e1.3 molar equiv CleCH2eAr2, 5 molar equiv K2CO3, dry THF, 65 ꢀC; (c)
3 molar equiv NH2OH ꢃ HCl, dry MeCN, 83 ꢀC.
1,3,4-thiadiazol-2-yl moiety at 5 position and render as promising
antiproliferatives with broad spectrum of cytostatic activity.
filtered out from the reaction mixture while hot and reacted with
subsequent portion of Lawesson’s reagent (1.36 g, 3.36 mmol) in
30 ml of fresh dry toluene for another 1 h at reflux, then filtered out.
Combined filtrates were slowly cooled to ꢂ20 ꢀC and the precipi-
tated yellow solid was filtered off and purified by crystallization
4. Experimental protocols
4.1. Synthesis
from toluene. Yield: 3.17 g (78%): m.p. 194e196 ꢀC; IR (KBr)
3311, 3165, 2924, 2853, 1618, 1580, 1493 cmꢂ1; 1H NMR (500 MHz,
DMSO-d6): 2.73 (s, 3H, SCH3), 7.94 (s, 1H, H-5), 8.10 (s, 1H, H-8),
n 3426,
Melting points were determined with Boëtius apparatus and are
uncorrected. The IR spectra were taken using Thermo Mattson
Satellite FTIR spectrophotometer, 1H and 13C NMR were taken with
a Varian Gemini 200 MHz or Varian Unity Plus 500 MHz apparatus.
d
10.25 (br s, 2H, NH2); Anal. C9H7ClN2O2S4 (C, H, N).
4.2. General procedure for the synthesis of 7-(4-arylthiazol-2-yl)-6-
Chemical shifts are reported in ppm (
d
). The mass spectra were
chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazines 3e and 3f
acquired on a Bruker BIFLEX III MALDI-TOF spectrometer after
deposition on a 2,5-dihydroxybenzoic acid (DHB) matrix. The
results of elemental analyses for C, H and N were in agreement with
the calculated values within ꢁ0.4% range (see Supplementary data).
A suspension of 2 (1.36 g, 4.0 mmol) and the appropriate
-bromoacetophenone (4.0 mmol) in MeOH (20 ml) was refluxed
for 4e5 h. The precipitated solid was filtered off and washed with
MeOH. The crude product was purified by crystallization from DMF.
u
4.1.1. 6-Chloro-3-methylthio-7-thiocarbamoyl-1,1-dioxo-1,4,2-
benzodithiazine (2)
4.2.1. 6-Chloro-3-methylthio-7-(4-phenylthiazol-2-yl)-1,1-dioxo-
The suspension of 6-chloro-3-methylthio-7-carbamoyl-1,1-
dioxo-1,4,2-benzodithiazine [25] (3.88 g, 12.0 mmol), Lawesson’s
reagent (2.66 g, 6.6 mmol) in dry toluene (60 ml) was refluxed for
1 h. The unreacted amide (about 1.92 g, 6.0 mmol) was quickly
1,4,2-benzodithiazine (3e)
Starting from
(66%): m.p. 300e302 ꢀC; IR (KBr)
1163 cmꢂ1 1H NMR (500 MHz, DMSO-d6):
u
-bromoacetophenone (0.80 g). Yield: 1.16 g
3100, 2926, 1497, 1479, 1335,
2.76 (s, 3H, SCH3);
n
;
d

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K. Brozewicz, J. Sławinski / European Journal of Medicinal Chemistry 55 (2012) 384e394
387
Table 1
solution was concentrated under reduced pressure to dryness and
MeOH (5 ml) was added. Precipitated solid was filtered off, and
purified by crystallization from EtOH.
Overview of in vitro antiproliferative activity of 1-(2-mercaptobenzenesulfonyl)-3-
hydroxyguanidines 6a, 6c, 6d, 6f, 6g, 6iep, 6ret, and 6vex.
TGIb
MGeMIDc No.d Ranged
[mM]
b
Compd No. of
tested
GI50
[
mM]
MGeMIDc No.d Ranged
4.3.1. 6-Chloro-3-methylthio-7-(3-phenyl-1,2,4-oxadiazol-5-yl)-
1,1-dioxo-1,4,2-benzodithiazine (3g)
cell linesa
6a
6c
6d
6f
6g
6i
6j
6k
6l
6m
6n
6o
6p
6r
6s
6t
6v
6w
6x
60
2.88
2.40
1.86
2.24
1.86
2.00
2.14
1.62
3.39
2.51
11.22
2.04
1.95
2.04
3.16
18.62
3.39
4.90
3.02
54 1.01e5.99
55 1.20e3.50
55 0.83e2.57
53 1.07e3.70
57 1.19e3.56
55 0.55e2.92
55 1.41e3.78
56 0.24e2.41
52 1.55e7.52
56 1.57e4.63
9.77
6.76
4.07
6.03
4.47
5.13
5.50
3.72
9.55
6.46
55
56
53
52
55
52
50
56
58
56
2.99e23.50
2.97e33.50
2.85e4.81
2.84e9.95
2.71e9.03
2.87e9.78
2.87e9.83
0.80e6.61
3.13e34.40
3.02e17.50
Starting from benzamidoxime (0.42 g). Yield: 0.87 g (66%): m.p.
59
57
59
57
57
57
57
59
58
55
57
56
57
58
59
58
59
59
196e198 ꢀC; IR (KBr)
n
1595, 1506, 1445, 1337, 1169 cmꢂ1; 1H NMR
(200 MHz, DMSO-d6):
d 2.75 (s, 3H, SCH3), 7.61e7.64 (m, 3H, Ar),
8.10e8.15 (m, 2H, Ar), 8.37 (s, 1H, H-5), 8.74 (s, 1H, H-8); Anal.
C16H10ClN3O3S3 (C, H, N).
4.3.2. 6-Chloro-7-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]-3-
methylthio-1,1-dioxo-1,4,2-benzodithiazine (3h)
50 2.84e75.40 75.86
55 19.80e100.00
Starting from 4-chlorobenzamidoxime (0.55 g). Yield: 0.88 g
54 1.24e2.86
55 1.02e2.98
53 1.29e2.65
5.50
4.47
5.13
54
53
53
52
16
48
50
56
2.85e17.30
2.46e7.70
2.81e9.83
2.87e45.90
4.33e95.50
3.76e29.50
2.86e28.00
2.95e34.70
(60%): m.p.154e156 ꢀC; IR (KBr)
n
2928, 1587, 1508, 1473, 1345,
1171 cmꢂ1
;
1H NMR (200 MHz, DMSO-d6):
d
2.75 (s, 3H, SCH3),
7.66e7.71 (m, 2H, Ar), 8.11e8.15 (m, 2H, Ar), 8.37 (s, 1H, H-5), 8.74
(s, 1H, H-8); Anal. C16H9Cl2N3O3S3 (C, H, N).
53 1.16e7.89 11.48
36 1.15e28.80 70.79
57 0.44e6.60 16.22
59 0.64e15.70 16.22
58 1.36e9.78
9.55
4.4. General procedure for the synthesis of 3-amino-7-(azol-2 or 5-
yl)-6-chloro-1,1-dioxo-1,4,2-benzodithiazines 4eeh
a
Number of cell lines investigated.
Response parameter, describing molar concentration of the test agent causing
b
50% net cell growth inhibition (GI50) or total growth inhibition (TGI).
c
A suspension of the appropriate 3-methylthio derivative 3eeh
(2 mmol) in MeOH (10 ml) or MeOH/toluene (5/5 ml) was cooled
to 0 ꢀC and ammonium hydroxide solution (25%, 2.9 mmol, 0.20 g)
was added dropwise. The suspension was stirred at room temper-
ature until MeSH ceased to evolve (CAUTION: due to a high toxicity,
MeSH should be trapped into an aqueous NaOH solution). Precip-
itated crude product was filtered off and purified by crystallization
from the appropriate solvent.
Mean graph mid-point e response parameter average value for all tested cell
lines.
d
Response parameter range for selected no. of cell lines.
7.42e7.45 (m, 1H, Ar), 7.52e7.55 (m, 2H, Ar), 8.09e8.10 (m, 2H, Ar),
8.32 (s, 1H, H-5), 8.52 (s, 1H, H-8), 8.99 (s, 1H, Ar). Anal.
C17H11ClN2O2S4 (C, H, N).
4.4.1. 3-Amino-6-chloro-7-(4-phenylthiazol-2-yl)-1,1-dioxo-1,4,2-
benzodithiazine (4e)
4.2.2. 6-Chloro-7-[4-(4-chlorophenyl)thiazol-2-yl]-3-methylthio-
1,1-dioxo-1,4,2-benzodithiazine (3f)
Starting from 3e (0.88 g). Yield: 0.70 g (86%): m.p. >320 ꢀC (70%
Starting from
1.01 g (56%): m.p. 297e299 ꢀC; IR (KBr)
1339, 1165 cmꢂ1 1H NMR (500 MHz, DMSO-d6):
u
-bromo-4-chloroacetophenone (0.93 g). Yield:
3107, 1492, 1474, 1458,
2.76 (s, 3H,
DMF/H2O); IR (KBr):
1461, 1354, 1306, 1161 cmꢂ1
7.39e7.42 (m, 1H, Ar), 7.49e7.52 (m, 2H, Ar), 8.05e8.07 (m, 2H,
n
3398, 3314, 3199,1632,1583,1541,1531,1481,
n
;
1H NMR (500 MHz, DMSO-d6):
;
d
d
SCH3), 7.59e7.61 (m, 2H, Ar), 8.11e8.13 (m, 2H, Ar), 8.30 (s, 1H,
H-5), 8.56 (s, 1H, H-8), 8.97 (s, 1H, Ar); Anal. C17H10Cl2N2O2S4 (C,
H, N).
Ar), 8.20 (s, 1H, Ar), 8.45 (s, 1H, Ar), 8.86 (s, 1H, Ar), 9.31 (br s, 2H,
NH2); Anal. C16H10ClN3O2S3 (C, H, N).
4.4.2. 3-Amino-6-chloro-7-[4-(4-chlorophenyl)thiazol-2-yl]-1,1-
dioxo-1,4,2-benzodithiazine (4f)
4.3. Generalprocedureforthesynthesisof7-(3-aryl-1,2,4-oxadiazol-5-
yl)-6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodithiazines 3g and 3h
Starting from 3f (0.95 g). Yield: 0.71 (80%): m.p. >320 ꢀC (70%
DMF/H2O); IR (KBr):
n
3295, 3127, 1666, 1643, 1543, 1530, 1308,
7.55e7.60 (m, 2H,
1139 cmꢂ1 1H NMR (200 MHz, DMSO-d6):
;
d
A mixture of 6-chloro-3-methylthio-1,1-dioxo-1,4,2-benzodith-
iazine-7-carboxylic acid (1.00 g, 3.1 mmol) and thionyl chloride
(3 ml) in dry benzene (3 ml) was refluxed for 1 h. The reaction
mixture was concentrated to dryness and dry toluene (50 ml) was
added. The obtained solution was added dropwise at 0 ꢀC to the
suspension of the appropriate amidoxime (3.1 mmol) in dry
toluene (25 ml) within 1 h and then refluxed for 4e6 h. Obtained
Ar), 8.07e8.11 (m, 2H, Ar), 8.20 (s, 1H, H-5), 8.51 (s, 1H, H-8), 8.85
(s, 1H, Ar), 9.28e9.36 (m, 2H, NH2); Anal. C16H9Cl2N3O2S3 (C,
H, N).
4.4.3. 3-Amino-6-chloro-7-(3-phenyl-1,2,4-oxadiazol-5-yl)-1,1-
dioxo-1,4,2-benzodithiazine (4g)
Starting from 3g (0.85 g). Yield: 0.64 (81%): m.p. 304e306 ꢀC
(20% DMF/MeOH); IR (KBr):
1565, 1539, 1521, 1474, 1350, 1302, 1159 cmꢂ1
DMSO-d6): 7.60e7.66 (m, 3H, Ar), 8.12e8.13 (m, 2H, Ar), 8.30 (s,
1H, Ar), 8.65 (s, 1H, Ar), 9.42 (br s, 2H, NH2); Anal. C15H9ClN4O3S2 (C,
H, N).
n
3416, 3283, 3188, 2924, 1620, 1596,
;
1H NMR (500 MHz,
Table 2
d
Comparison of cytostatic activity (GI50
chlorine introduction into 1,3-benzodioxol-5-yl derivatives 6k and 6m.
[mM]) showing decrease in activity after
Cell lines:
SF-268 U251 MDA
-MB-435 -62
1.40 1.38 1.70
UACC IGROV1 MDA
-MB-231
1.68
4.4.4. 3-Amino-6-chloro-7-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]-
1,1-dioxo-1,4,2-benzodithiazine(4h)
6k (Ar1 ¼ Ph)
1.61
2.04
1.86
1.68
3.47
6l (Ar1 ¼ 4-ClePh)
6m (Ar1 ¼ Ph)
10.72 2.63
1.95 2.00
11.22 3.89
2.14
2.14
7.76
5.13
2.34
Starting from 3h (0.92 g). Yield: 0.67 (78%): m.p. >320 ꢀC (20%
2.82
DMF/MeOH); IR (KBr):
n 3275, 3055, 2926, 1659, 1638, 1590, 1559,
6n (Ar1 ¼ 4-ClePh) 6.46
25.12
12.59
1519, 1467, 1338, 1311, 1173, 1156 cmꢂ1; 1H NMR (500 MHz, DMSO-

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K. Brozewicz, J. Sławinski / European Journal of Medicinal Chemistry 55 (2012) 384e394
388
Table 3
Influence of Ar2 substitution with 1,3-benzodioxol-5-yl and 3,4,5-trimethoxyphenyl moieties on cytostatic activity (GI50
and 1,3,4-thiadiazoles (6k, 6l, 6s, 6t) on selected cell lines.
[mM]) of the series of 1,2,4-oxadiazoles (6o, 6p, 6w, 6x)
Ar2
¼
1,2,4-oxadiazoles (X:Y:Z ¼ O:N:N)
1,3,4-thiadiazoles (X:Y:Z ¼ N:N:S)
Cell lines
6o
6p
6w
4.37
6x
6k
6l
6s
6t
NCI-H460
NCI-H522
HT29
SF-268
SNB-75
2.04
1.29
2.00
1.91
1.55
2.19
1.91
2.00
1.82
2.57
1.70
1.66
2.63
2.14
1.02
1.82
1.82
1.74
2.63
1.86
1.91
1.70
2.14
1.70
1.91
2.69
2.24
2.14
2.95
2.95
2.95
7.76
2.19
9.33
1.95
3.80
1.91
3.16
4.17
1.79
0.24
1.85
1.61
1.07
1.71
1.74
1.67
1.70
1.68
1.70
1.28
2.41
2.34
1.86
2.29
2.04
1.78
2.24
2.14
3.55
2.14
3.47
1.62
3.72
2.40
3.24
2.04
4.17
2.40
1.86
3.09
4.07
6.03
3.24
3.72
2.19
3.98
3.31
34.67
8.91
6.76
3.80
7.24
6.92
3.98
13.49
6.61
13.18
3.09
5.62
4.79
4.47
7.41
9.55
25.70
97.72
40.74
26.30
16.22
58.88
10.23
44.67
8.13
MALME-3M
M14
UACC-257
UACC-62
IGROV1
OVCAR-3
UO-31
HS 578T
d6):
d
7.68e7.69 (m, 2H, Ar), 8.12e8.14 (m, 2H, Ar), 8.30 (s, 1H, Ar),
4.5.3. N-[2-Benzylthio-4-chloro-5-(3-phenyl-1,2,4-oxadiazol-5-yl)
benzenesulfonyl]cyanamide potassium salt (5g)
8.64 (s, 1H, Ar), 9.41 (br s, 2H, NH2); Anal. C15H8Cl2N4O3S2 (C, H, N).
Starting from 4g (0.196 g) and benzyl chloride (0.076 g). Yield:
0.206 g (79%): m.p. 138e140 ꢀC; IR (KBr):
n
3433, 2924, 2854, 2176,
1737, 1594, 1529, 1495, 1360, 1143 cmꢂ1; 1H NMR (200 MHz, DMSO-
d6): 4.49 (s, 2H, SCH2), 7.32e7.44 (m, 5H, Ar), 7.51e7.54 (m, 2H,
4.5. General procedure for the synthesis of N-(benzenesulfonyl)
cyanamide potassium salts 5eex
d
Ar), 7.62e7.65 (m, 3H, Ar), 7.74 (s,1H, Ar), 8.11e8.16 (m, 2H, Ar), 8.60
Suspension of the appropriate 3-amino-1,1-dioxo-1,4,2-
benzodithiazine derivative 4aeh (0.5 mmol), anhydrous K2CO3
(1.5 mmol, 0.21 g) and the appropriate alkyl chloride (0.6 mmol) in
dry THF (5 ml) was heated at reflux for 20e24 h, then cooled in ice-
bath and filtered off. The crude product was suspended in 5 ml of
water, heated gently to ca. 50 ꢀC; and cooled with vigorous stirring
until granular precipitate appeared. Filtering off and washing with
cold water and diluted EtOH gave pure potassium salts.
(s, 1H, Ar); Anal. C22H14ClKN4O3S2 (C, H, N).
4.5.4. N-{2-Benzylthio-4-chloro-5-[3-(4-chlorophenyl)-1,2,4-
oxadiazol-5-yl]benzenesulfonyl}cyanamide potassium salt (5h)
Starting from 4h (0.214 g) and benzyl chloride (0.076 g). Yield:
0.236 g (85%): m.p. >320 ꢀC; IR (KBr):
n
2925, 2175, 1583,1560, 1503,
1470,1343,1298,1135 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
4.46 (s,
2H, SCH2), 7.28e7.31 (m,1H, Ar), 7.35e7.38 (m, 2H, Ar), 7.48e7.50 (m,
2H, Ar), 7.66e7.68 (m, 2H, Ar), 7.71 (s, 1H, Ar), 8.11e8.12 (m, 2H, Ar),
8.56 (s, 1H, Ar); Anal. C22H13Cl2KN4O3S2 (C, H, N).
4.5.1. N-[2-Benzylthio-4-chloro-5-(4-phenylthiazol-2-yl)
benzenesulfonyl]cyanamide potassium salt (5e)
Starting from 4e (0.204 g) and benzyl chloride (0.076 g). Yield:
4.5.5. N-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-(5-
phenyl-1,3,4-oxadiazol-2-yl)benzenesulfonyl}cyanamide potassium
salt (5i)
0.153 g (57%): m.p. 198e199 ꢀC; IR (KBr):
n
3092, 2926, 2170, 1602,
1578, 1528, 1494, 1476, 1452, 1350, 1284, 1140 cmꢂ1 1H NMR
(500 MHz, DMSO-d6): 4.40 (s, 2H, SCH2), 7.27e7.30 (m, 1H, Ar),
;
d
Starting from 4a (0.196 g) and 1,3-benzodioxol-5-ylmethyl
7.35e7.40 (m, 3H, Ar), 7.48e7.51 (m, 4H, Ar), 7.62 (s, 1H, Ar),
8.04e8.06 (m, 2H, Ar), 8.35 (s, 1H, Ar), 8.79 (s, 1H, Ar); Anal.
C23H15ClKN3O2S3 (C, H, N).
chloride (0.102 g). Yield: 0.172 g (61%): m.p. 278e280 ꢀC; IR
(KBr):
1H NMR (500 MHz, DMSO-d6):
n ;
2173, 1630, 1584, 1556, 1501, 1489, 1330, 1297, 1143 cmꢂ1
d
4.36 (s, 2H, SCH2), 6.00 (s, 2H,
OCH2O), 6.88e6.89 (m, 1H, Ar), 6.96e6.98 (m, 1H, Ar), 7.04 (s, 1H,
Ar), 7.62e7.76 (m, 4H, Ar), 8.07e8.08 (m, 2H, Ar), 8.46 (s, 1H, Ar);
Anal. C23H14ClKN4O5S2 (C, H, N).
4.5.2. N-{2-Benzylthio-4-chloro-5-[4-(4-chlorophenyl)thiazol-2-yl]
benzenesulfonyl}cyanamide potassium salt (5f)
Starting from 4f (0.221 g) and benzyl chloride (0.076 g). Yield:
0.217 g (76%): m.p. 228e230 ꢀC; IR (KBr):
n
2923, 2175, 1577, 1527,
1494, 1471, 1453, 1350, 1275, 1139 cmꢂ1; 1H NMR (500 MHz, DMSO-
d6): 4.40 (s, 2H, SCH2), 7.27e7.30 (m, 1H, Ar), 7.34e7.38 (m, 2H,
4.5.6. N-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-[5-(4-
chlorophenyl)-1,3,4-oxadiazol-2-yl]benzenesulfonyl}cyanamide
potassium salt (5j)
d
Ar), 7.48e7.49 (m, 2H, Ar), 7.55e7.57 (m, 2H, Ar), 7.62 (s, 1H, Ar),
8.06e8.08 (m, 2H, Ar), 8.41 (s, 1H, Ar), 8.77 (s, 1H, Ar); Anal.
C23H14Cl2KN3O2S3 (C, H, N).
Starting from 4b (0.214 g) and 1,3-benzodioxol-5-ylmethyl
chloride (0.102 g). Yield: 0.240 g (80%): m.p. 308e310 ꢀC; IR
(KBr): n 3436, 3086, 2924, 2172, 1602, 1585, 1543, 1502, 1486, 1327,

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K. Brozewicz, J. Sławinski / European Journal of Medicinal Chemistry 55 (2012) 384e394
389
1289, 1253, 1141 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
4.36 (s, 2H,
1147 cmꢂ1
;
1H NMR (500 MHz, DMSO-d6):
d
4.37 (s, 2H, SCH2),
SCH2), 6.01 (s, 2H, OCH2O), 6.88e7.04 (m, 5H, Ar), 7.68 (s, 1H, Ar),
7.70 (m, 2H, Ar), 8.08e8.09 (m, 2H, Ar), 8.47 (s, 1H, Ar); Anal.
C23H13Cl2KN4O5S2 (C, H, N).
6.00 (s, 2H, OCH2O), 6.89 (d, J ¼ 7.82 Hz, 1H, Ar), 6.96 (d,
J ¼ 7.82 Hz, 1H, Ar), 7.02 (s, 1H, Ar), 7.66e7.68 (m, 2H, Ar), 7.69
(s, 1H, Ar), 8.11e8.12 (m, 2H, Ar), 8.56 (s, 1H, Ar); Anal.
C23H13Cl2KN4O5S2 (C, H, N).
4.5.7. N-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-(5-
phenyl-1,3,4-thiadiazol-2-yl)benzenesulfonyl}cyanamide potassium
salt (5k)
4.5.13. N-[4-Chloro-2-(3,4,5-trimethoxybenzylthio)-5-(5-phenyl-
1,3,4-oxadiazol-2-yl)benzenesulfonyl]cyanamide potassium salt
(5q)
Starting from 4c (0.204 g) and 1,3-benzodioxol-5-ylmethyl
chloride (0.102 g). Yield: 0.287 g (99%): m.p. 285e287 ꢀC; IR
Starting from 4a (0.196 g) and 3,4,5-trimethoxybenzyl chloride
(KBr):
n
3443, 2924, 2853, 2179, 1652, 1623, 1577, 1501, 1489, 1357,
4.35 (s, 2H, SCH2), 6.01
(0.130 g). Yield: 0.189 g (62%): m.p. 272e274 ꢀC; IR (KBr):
n
2926,
2851, 2180, 1591, 1552, 1507, 1452, 1331, 1294, 1145, 1126 cmꢂ1 1H
NMR (500 MHz, DMSO-d6):
1129 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
;
(s, 2H, OCH2O), 6.89 (d, J ¼ 7.81 Hz, 1H, Ar), 6.97 (d, J ¼ 7.81 Hz, 1H,
Ar), 7.05 (s, 1H, Ar), 7.58e7.60 (m, 3H, Ar), 7.68 (s, 1H, Ar), 8.07e8.08
(m, 2H, Ar), 8.69 (s, 1H, Ar); Anal. C23H14ClKN4O4S3 (C, H, N).
d
3.63 (s, 3H, OCH3), 3.77 (s, 6H, 2ꢃ
OCH3), 4.37 (s, 2H, SCH2), 6.84 (s, 2H, Ar), 7.63e7.66 (m, 3H, Ar), 7.73
(s, 1H, Ar), 8.07e8.08 (m, 2H, Ar), 8.46 (s, 1H, Ar); Anal.
C24H20ClKN4O6S2 (C, H, N).
4.5.8. N-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-[5-(4-
chlorophenyl)-1,3,4-thiadiazol-2-yl]benzenesulfonyl}cyanamide
potassium salt (5l)
4.5.14. N-{4-Chloro-5-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-2-
(3,4,5-trimethoxybenzylthio)benzenesulfonyl}cyanamide potassium
salt (5r)
Starting from 4d (0.222 g) and 1,3-benzodioxol-5-ylmethyl
chloride (0.102 g). Yield: 0.175 g (57%): m.p. 265e267 ꢀC; IR
Starting from 4b (0.214 g) and 3,4,5-trimethoxybenzyl chloride
(KBr):
n
2924, 2853, 2170, 1626, 1577, 1560, 1523, 1502, 1491, 1458,
4.34 (s, 2H,
(0.130 g). Yield: 0.258 g (80%): m.p. 313e315 ꢀC; IR (KBr):
n
3437,
2926, 2852, 2177, 1591, 1540, 1508, 1482, 1331, 1147, 1128 cmꢂ1; 1H
NMR (500 MHz, DMSO-d6):
1357, 1260, 1138 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
SCH2), 6.00 (s, 2H, OCH2O), 6.88e6.89 (m,1H, Ar), 6.96e6.97 (m,1H,
Ar), 7.03 (s, 1H, Ar), 7.64 (s, 1H, Ar), 7.66e7.67 (m, 2H, Ar), 8.09e8.11
(m, 2H, Ar), 8.69 (s, 1H, Ar); Anal. C23H13Cl2KN4O4S3 (C, H, N).
d
3.63 (s, 3H, OCH3), 3.77 (s, 6H, 2ꢃ
OCH3), 4.38 (s, 2H, SCH2), 6.84 (s, 2H, Ar), 7.70e7.72 (m, 2H, Ar), 7.73
(s, 1H, Ar), 8.07e8.09 (m, 2H, Ar), 8.46 (s, 1H, Ar); Anal.
C25H19Cl2KN4O6S2 (C, H, N).
4.5.9. N-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-(4-
phenylthiazol-2-yl)benzenesulfonyl}cyanamide potassium salt (5m)
Starting from 4e (0.204 g) and 1,3-benzodioxol-5-ylmethyl chlo-
4.5.15. N-[4-Chloro-2-(3,4,5-trimethoxybenzylthio)-5-(5-phenyl-
1,3,4-thiadiazol-2-yl)benzenesulfonyl]cyanamide potassium salt (5s)
Starting from 4c (0.204 g) and 3,4,5-trimethoxybenzyl chloride
ride (0.102 g). Yield: 0.209 g (72%): m.p. 263e265 ꢀC; IR (KBr):
n
2896,
1H NMR
4.32 (s, 2H, SCH2), 6.00 (s, 2H, OCH2O),
2182, 1579, 1529, 1503, 1489, 1361, 1282, 1142 cmꢂ1
(500 MHz, DMSO-d6):
;
(0.130 g). Yield: 0.279g (89%): m.p.173e175 ꢀC; IR (KBr):
n
2938, 2176,
d
1635, 1590, 1458, 1334, 1283, 1240, 1123 cmꢂ1
DMSO-d6):
;
1H NMR (200 MHz,
6.88e6.89(m,1H, Ar), 6.96e6.97 (m,1H, Ar), 7.04 (s,1H, Ar), 7.37e7.40
(m, 1H, Ar), 7.48e7.51 (m, 2H, Ar), 7.60 (s, 1H, Ar), 8.04e8.06 (m, 2H,
Ar), 8.35 (s, 1H, Ar), 8.79 (s, 1H, Ar); Anal. C24H15ClKN3O4S3 (C, H, N).
d
3.64 (s, 3H, OCH3), 3.78 (s, 6H, 2ꢃ OCH3), 4.37 (s, 2H,
SCH2), 6.85(s, 2H,Ar), 7.58e7.62(m, 3H,Ar), 7.74(s,1H,Ar), 8.08e8.13
(m, 2H, Ar), 8.69 (s, 1H, Ar); Anal. C25H20ClKN4O5S3 (C, H, N).
4.5.10. N-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-(4-(4-
chlorophenyl)thiazol-2-yl)benzenesulfonyl}cyanamide potassium
salt (5n)
4.5.16. N-{4-Chloro-2-(3,4,5-trimethoxybenzylthio)-5-[5-(4-
chlorophenyl)-1,3,4-thiadiazol-2-yl]benzenesulfonyl}cyanamide
potassium salt (5t)
Starting from 4f (0.221 g) and 1,3-benzodioxol-5-ylmethyl
Starting from 4d (0.222 g) and 3,4,5-trimethoxybenzyl chloride
chloride (0.102 g). Yield: 0.234 g (76%): m.p. 260e262 ꢀC; IR
(0.130 g). Yield: 0.175 g (53%): m.p. 148e150 ꢀC; IR (KBr):
n
2923,
2851, 2175, 1654, 1637, 1587, 1561, 1542, 1512, 1459, 1334,1277, 1179,
1144, 1125 cmꢂ1 1H NMR (500 MHz, DMSO-d6):
3.63 (s, 3H,
(KBr):
n
2895, 2185, 1635, 1578, 1503, 1488, 1472, 1361, 1285,
4.32 (s, 2H, SCH2), 6.00
1142 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
;
d
(s, 2H, OCH2O), 6.87e6.89 (m, 1H, Ar), 6.96e6.97 (m, 1H, Ar), 7.04 (s,
1H, Ar), 7.55e7.57 (m, 2H, Ar), 7.60 (s,1H, Ar), 8.06e8.08 (m, 2H, Ar),
8.41 (s, 1H, Ar), 8.77 (s, 1H, Ar); Anal. C24H14Cl2KN3O4S3 (C, H, N).
OCH3), 3.77 (s, 6H, 2ꢃ OCH3), 4.36 (s, 2H, SCH2), 6.84 (s, 2H, Ar),
7.65e7.66 (m, 2H, Ar), 7.72 (s, 1H, Ar), 8.09e8.11 (m, 2H, Ar), 8.68 (s,
1H, Ar); Anal. C25H19Cl2KN4O5S3 (C, H, N).
4.5.11. N-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-(3-
phenyl-1,2,4-oxadiazol-5-yl)benzenesulfonyl}cyanamide potassium
salt (5o)
4.5.17. N-[4-Chloro-2-(3,4,5-trimethoxybenzylthio)-5-(4-
phenylthiazol-2-yl)benzenesulfonyl]cyanamide potassium salt (5u)
Starting from 4e (0.204 g) and 3,4,5-trimethoxybenzyl chloride
Starting from 4g (0.196 g) and 1,3-benzodioxol-5-ylmethyl chlo-
(0.130 g). Yield: 0.310 g (99%): m.p. >320 ꢀC; IR (KBr):
n
3078, 2935,
2176, 1593, 1529, 1506, 1452, 1329, 1289, 1142, 1123 cmꢂ1; 1H NMR
(200 MHz, DMSO-d6):
ride (0.102 g). Yield: 0.215 g (88%): m.p. 274e276 ꢀC; IR (KBr):
n
2923,
2179, 1625, 1585, 1560, 1500, 1488, 1362, 1349, 1253, 1226,
1143 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
4.38 (s, 2H, SCH2), 6,01
d
3.63 (s, 3H, OCH3), 3.77 (s, 6H, 2ꢃ OCH3),
d
4.34 (s, 2H, SCH2), 6.04 (s, 2H, Ar), 7.37e7.40 (m, 1H, Ar), 7.48e7.51
(m, 2H, Ar), 7.65 (s, 1H, Ar), 8.04e8.05 (m, 2H, Ar), 8.35 (s, 1H, Ar),
8.79 (s, 1H, Ar); Anal. C26H21ClKN3O5S3 (C, H, N).
(s, 2H, OCH2O), 6.90 (d, J ¼ 7.81 Hz, 1H, Ar), 6.98 (d, J ¼ 7.81 Hz, 1H,
Ar), 7.05 (s, 1H, Ar), 7.59e7.63 (m, 3H, Ar), 7.70 (s, 1H, Ar), 8.10e8.11
(m, 2H, Ar), 8.59 (s, 1H, Ar); Anal. C23H14ClKN4O5S2 (C, H, N).
4.5.18. N-{4-Chloro-5-[4-(4-chlorophenyl)thiazol-2-yl]-2-(3,4,5-
trimethoxybenzylthio)benzenesulfonyl}cyanamide potassium salt(5v)
Starting from 4f (0.221 g) and 3,4,5-trimethoxybenzyl chloride
4.5.12. N-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-[3-(4-
chlorophenyl)-1,2,4-oxadiazol-5-yl]benzenesulfonyl}cyanamide
potassium salt (5p)
(0.130 g). Yield: 0.231 g (70%): m.p. >320 ꢀC; IR (KBr):
n
2934, 2173,
1H NMR (500 MHz,
3.63 (s, 3H, OCH3), 3.76 (s, 6H, 2ꢃ OCH3), 4.34 (s, 2H,
SCH2), 6.83 (s, 2H, Ar), 7.55e7.57 (m, 2H, Ar), 7.65 (s, 1H, Ar),
Starting from 4h (0.214 g) and 1,3-benzodioxol-5-ylmethyl
1596, 1507, 1458, 1331, 1287, 1141, 1123 cmꢂ1
DMSO-d6):
;
chloride (0.102 g). Yield: 0.255 g (85%): m.p. 283e285 ꢀC; IR
d
(KBr):
n 2184, 1589, 1560, 1503, 1491, 1474, 1345, 1255,

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390
8.06e8.08 (m, 2H, Ar), 8.41 (s, 1H, Ar), 8.76 (s, 1H, Ar); Anal.
(50 MHz, DMSO-d6): d 36.08, 123.78, 127.77, 128.01, 128.91, 129.50,
C26H20Cl2KN3O5S3 (C, H, N).
129.62, 129.65, 129.78, 129.86, 131.91, 134.22, 135.82, 140.09, 142.61,
158.97, 162.29, 169.29 ppm; Anal. C22H18ClN5O3S3 (C, H, N).
4.5.19. N-[4-Chloro-2-(3,4,5-trimethoxybenzylthio)-5-(3-phenyl-
1,2,4-oxadiazol-5-yl)benzenesulfonyl]cyanamidepotassium salt(5w)
Starting from 4g (0.196 g) and 3,4,5-trimethoxybenzyl chloride
4.6.4. 1-{2-Benzylthio-4-chloro-5-[5-(4-chlorophenyl)-1,3,4-
thiadiazol-2-yl]benzenesulfonyl}-3-hydroxyguanidine (6d)
(0.130 g). Yield: 0.266 g (87%): m.p. 283e285 ꢀC; IR (KBr):
n
2930,
2176, 1594, 1507, 1333, 1144, 1124 cmꢂ1; 1H NMR (500 MHz, DMSO-
d6):
Starting from 5d (0.143 g). Yield: 0.095
245e246 ꢀC; IR (KBr):
3454, 3378, 3334, 3254, 2924, 2854,
1625, 1576, 1531, 1495, 1456, 1248, 1151, 1130 cmꢂ1 1H NMR
(500 MHz, DMSO-d6): 4.47 (s, 2H, SCH2), 7.29e7.51 (m, 7H, Ar
g (67%): m.p.
n
d
3.64 (s, 3H, OCH3), 3.78 (s, 6H, 2ꢃ OCH3), 4.40 (s, 2H, SCH2),
;
6.85 (s, 2H, Ar), 7.59e7.63 (m, 3H, Ar), 7.76 (s, 1H, Ar), 8.09e8.14 (m,
d
2H, Ar), 8.56 (s, 1H, Ar); Anal. C25H20ClKN4O6S2 (C, H, N).
and 2ꢃ NH), 7.65e7.69 (m, 2H, Ar), 7.72 (s, 1H, H-3), 8.08e8.12
(m, 2H, Ar), 8.75 (s, 1H, H-6), 9.47 (s, 1H, NH), 10.07 (s, 1H,
4.5.20. N-{4-Chloro-5-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]-
2-(3,4,5-trimethoxybenzylthio)benzenesulfonyl}cyanamide
potassium salt (5x)
OH); 13C NMR (50 MHz, DMSO-d6):
d 36.08, 107.38, 123.64,
127.78, 128.02, 128.32, 128.91, 129.50, 129.68, 129.90, 134.22,
135.79, 136.53, 140.07, 142.75, 158.97, 162.59, 168.08 ppm; Anal.
C22H17Cl2N5O3S3 (C, H, N).
Starting from 4h (0.214 g) and 3,4,5-trimethoxybenzyl chloride
(0.130 g). Yield: 0.316 g (98%): m.p. >320 ꢀC; IR (KBr):
n
2937, 2838,
2180, 1591, 1561, 1506, 1460, 1334, 1301, 1148, 1121 cmꢂ1 1H NMR
(500 MHz, DMSO-d6):
;
4.6.5. 1-[2-Benzylthio-4-chloro-5-(4-phenylthiazol-2-yl)
benzenesulfonyl]-3-hydroxyguanidine (6e)
d
3.63 (s, 3H, OCH3), 3.76 (s, 6H, 2ꢃ OCH3), 4.39
(s, 2H, SCH2), 6.84 (s, 2H, Ar), 7.66e7.68 (m, 2H, Ar), 7.75 (s, 1H, Ar),
8.11e8.12(m, 2H, Ar),8.55(s,1H, Ar);Anal. C25H19Cl2KN4O6S2 (C, H, N).
Starting from 5e (0.134 g). Yield: 0.116 g (87%): m.p. 225e227 ꢀC;
IR (KBr):
n
3481, 3367, 3276, 2927, 1615, 1584, 1528, 1496, 1475,
4.43 (s, 2H,
1393, 1258, 1149 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
4.6. General procedure for the synthesis of 1-(2-
mercaptobenzenesulfonyl)-3-hydroxyguanidines 6aex
SCH2), 7.12e7.40 (m, 6H, Ar and NH), 7.47e7.51 (m, 4H, Ar), 7.66 (s,
1H, Ar), 8.05e8.06 (m, 2H, Ar), 8.35 (s, 1H, Ar), 8.82 (s, 1H, Ar), 9.44
(s, 1H, NH), 10.05 (s, 1H, OH); 13C NMR (50 MHz, DMSO-d6):
d 36.14,
A suspension of the appropriate N-(benzenesulfonyl)cyanamide
potassium salt 5aex (0.25 mmol) and hydroxylamine hydrochlo-
ride (0.052 g, 0.75 mmol) in dry MeCN (3 ml) was refluxed for
20e24 h. After cooling to room temperature, stirring was
continued overnight. The precipitate was filtered off, washed with
MeOH, 50% MeOH/H2O, water, MeOH and cold MeCN giving pure
product.
116.92, 126.52, 126.85, 127.73, 128.34, 128.71, 128.89, 129.22, 129.50,
129.67, 133.40, 133.88, 135.97, 140.12, 140.70, 154.37, 159.06,
161.28 ppm; Anal. C23H19ClN4O3S3 (C, H, N).
4.6.6. 1-{2-Benzylthio-4-chloro-5-[4-(4-chlorophenyl)thiazol-2-yl]
benzenesulfonyl}-3-hydroxyguanidine (6f)
Starting from 5f (0.143 g). Yield: 0.109
225e226 ꢀC; IR (KBr):
3488, 3370, 3336, 3248, 3028, 2924, 1621,
1577, 1531, 1495, 1474, 1454 cmꢂ1 1H NMR (500 MHz, DMSO-d6):
4.43 (s, 2H, SCH2), 7.12e7.44 (m, 5H, Ar and NH), 7.47e7.48 (m,
g (77%): m.p.
n
4.6.1. 1-[2-Benzylthio-4-chloro-5-(5-phenyl-1,3,4-oxadiazol-2-yl)
benzenesulfonyl]-3-hydroxyguanidine (6a)
;
d
Starting from 5a (0.130 g). Yield: 0.128 g (99%): m.p. 214e216 ꢀC;
2H, Ar), 7.54e7.56 (m, 2H, Ar), 7.66 (s, 1H, Ar), 8.07e8.09 (m, 2H,
IR (KBr):
n
3444, 3336, 3206, 2923, 1622, 1586, 1553, 1492, 1450,
4.48
Ar), 8.42 (s, 1H, Ar), 8.81 (s, 1H, Ar), 9.45 (s, 1H, NH), 10.06 (s, 1H,
1326, 1280, 1162, 1117 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
OH); 13C NMR (50 MHz, DMSO-d6):
d 36.13, 117.57, 126.70, 127.73,
(s, 2H, SCH2), 7.19 (br s, 1H, NH), 7.30e7.33 (m, 1H, Ar), 7.37e7.40
(m, 3H, Ar), 7.64e7.71 (m, 3H, Ar), 7.74 (s, 1H, H-3), 8.11e8.12 (m,
2H, Ar), 8.56 (s, 1H, H-6), 9.47 (s, 1H, NH), 10.08 (s, 1H, OH); 13C NMR
128.25, 128.89, 129.24, 129.50, 129.63, 132.73, 133.19, 133.42,
135.95, 140.13, 140.83, 153.07, 159.07, 161.52 ppm; Anal.
C23H18Cl2N4O3S3 (C, H, N).
(50 MHz, DMSO-d6):
d 35.99, 117.73, 123.32, 127.07, 127.79, 128.58,
128.92, 129.51, 129.82, 130.17, 132.59, 134.51, 135.73, 139.85, 143.62,
158.90, 161.83, 164.57 ppm; Anal. C22H18ClN5O4S2 (C, H, N).
4.6.7. 1-[2-Benzylthio-4-chloro-5-(3-phenyl-1,2,4-oxadiazol-5-yl)
benzenesulfonyl]-3-hydroxyguanidine (6g)
Starting from 5g (0.130 g). Yield: 0.107
224e225 ꢀC; IR (KBr):
3483, 3370, 3329, 2923, 2854, 1613, 1579,
1561, 1538, 1351, 1150 cmꢂ1 1H NMR (200 MHz, DMSO-d6):
4.51
g (83%): m.p.
4.6.2. 1-{2-Benzylthio-4-chloro-5-[5-(4-chlorophenyl)-1,3,4-
oxadiazol-2-yl]benzenesulfonyl}-3-hydroxyguanidine (6b)
n
;
d
Starting from 5b (0.139 g). Yield: 0.132
238e240 ꢀC; IR (KBr):
3444, 3342, 3203, 3067, 2900, 1620, 1580,
1548, 1482, 1328, 1290, 1153, 1123 cmꢂ1; 1H NMR (500 MHz, DMSO-
d6): 4.48 (s, 2H, SCH2), 7,20 (br s, 1H, NH), 7.30e7.32 (m, 1H, Ar),
g
(96%): m.p.
(s, 2H, SCH2), 7.28e7.60 (m, 7H, Ar and NH), 7.63e7.65 (m, 3H, Ar),
7.77 (s, 1H, Ar), 8.11e8.15 (m, 2H, Ar), 8.67 (s, 1H, Ar), 9.49 (s, 1H,
n
NH),10.09 (s,1H, OH); 13C NMR (50 MHz, DMSO-d6):
d 36.00,117.79,
d
126.14, 127.47, 127.83, 128.57, 128.94, 129.52, 129.63, 131.21, 132.11,
135.19, 135.62, 139.77, 145.16, 158.84, 168.29, 173.47 ppm; Anal.
C22H18ClN5O4S2 (C, H, N).
7.37e7.40 (m, 3H, Ar and NH), 7.50e7.51 (m, 2H, Ar), 7.72e7.73 (m,
3H, Ar), 7.73 (s,1H, H-3), 8.11e8.13 (m, 2H, Ar), 8.56 (s,1H, H-6), 9.47
(s, 1H, NH), 10.07 (s, 1H, OH); 13C NMR (50 MHz, DMSO-d6):
d 36.01,
117.57, 122.22, 127.79, 128.57, 128.86, 128.92, 129.51, 129.98, 130.18,
134.52, 135.71, 137.29, 139.84, 143.73, 158.90, 161.96, 163.79 ppm;
Anal. C22H17Cl2N5O4S2 (C, H, N).
4.6.8. 1-{2-Benzylthio-4-chloro-5-[3-(4-chlorophenyl)-1,2,4-
oxadiazol-5-yl]benzenesulfonyl}-3-hydroxyguanidine (6h)
Starting from 5h (0.139 g). Yield: 0.111
253e255 ꢀC; IR (KBr):
3411, 3326, 3281, 3029, 2924, 1644, 1589,
1563, 1543, 1504, 1472, 1456, 1344, 1164 cmꢂ1; 1H NMR (500 MHz,
DMSO-d6): 4.48 (s, 2H, SCH2), 7.29e7.49 (m, 7H, Ar and NH),
g (81%): m.p.
n
4.6.3. 1-[2-Benzylthio-4-chloro-5-(5-phenyl-1,3,4-thiadiazol-2-yl)
benzenesulfonyl]-3-hydroxyguanidine (6c)
d
Starting from 5c (0.134 g). Yield: 0.110 g (83%): m.p. 234e236 ꢀC;
7.67e7.69 (m, 2H, Ar), 7.73 (s, 1H, Ar), 8.10e8.12 (m, 2H, Ar), 8.63 (s,
IR (KBr):
n
3451, 3332, 3275, 2923,1622,1576,1521,1495,1456,1349,
1H, Ar), 9.45 (s, 1H, NH), 10.06 (s, 1H, OH); 13C NMR (50 MHz,
1150 cmꢂ1
;
1H NMR (500 MHz, DMSO-d6):
d
4.46 (s, 2H, SCH2),
DMSO-d6): d 36.02, 117.62, 125.00, 127.83, 128.55, 128.94, 129.26,
7.29e7.61 (m, 10H, Ar and 2ꢃ NH), 7.73 (s, 1H, H-3), 8.05e8.08 (m,
129.52, 129.79, 131.22, 135.21, 135.59, 136.83, 139.76, 145.30, 158.84,
167.49, 173.62 ppm; Anal. C22H17Cl2N5O4S2 (C, H, N).
2H, Ph), 8.74 (s, 1H, H-6), 9.47 (s,1H, NH),10.06 (s, 1H, OH); 13C NMR

_
ꢀ
K. Brozewicz, J. Sławinski / European Journal of Medicinal Chemistry 55 (2012) 384e394
391
4.6.9. 1-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-(5-phenyl-
1501, 1489, 1473, 1254, 1156, 1130 cmꢂ1
;
1H NMR (200 MHz,
1,3,4-oxadiazol-2-yl)benzenesulfonyl}-3-hydroxyguanidine (6i)
DMSO-d6): d 4.35 (s, 2H, SCH2), 6.01 (s, 2H, OCH2O), 6.87e7.03
Starting from 5i (0.141 g). Yield: 0.077 g (55%): m.p. 230e232 ꢀC;
(m, 3H, Ar), 7.12e7.42 (br s, 2H, NH), 7.54e7.58 (m, 2H, Ar),
7.65 (s, 1H, Ar), 8.07e8.12 (m, 2H, Ar), 8.43 (s, 1H, Ar), 8.82 (s, 1H,
Ar), 9.45 (s, 1H, NH), 10.06 (s, 1H, OH); MALDI-TOF m/z obsd:
IR (KBr):
n
3436, 3141, 2926, 1704, 1591, 1530, 1496, 1450, 1351,
4.32 (s, 2H, SCH2), 6.00
1172 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
(s, 2H, OCH2O), 6.88e6.89 (m, 1H, Ar), 6.96e6.97 (m, 1H, Ar), 7.02 (s,
1H, Ar), 7.18 (br s, 1H, NH), 7.38 (br s, 1H, NH), 7.63e7.70 (m, 4H, Ar),
8.09e8.10 (m, 2H, Ar), 8.54 (s,1H, H-6), 9.44 (s,1H, NH),10.04 (s,1H,
OH); MALDI-TOF m/z obsd: 560.2, [M þ H]þ, 582.2, [M þ Na]þ calcd:
559.0; Anal. C23H18ClN5O6S2 (C, H, N).
609.1, [M
C24H18Cl2N4O5S3 (C, H, N).
þ
H]þ, 631.1, [M
þ
Na]þ calcd: 608.0; Anal.
4.6.15. 1-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-(3-phenyl-
1,2,4-oxadiazol-5-yl)benzenesulfonyl}-3-hydroxyguanidine (6o)
Starting from 5o (0.141 g). Yield: 0.116 g (83%): m.p. 235e237 ꢀC;
4.6.10. 1-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-[5-(4-
chlorophenyl)-1,3,4-oxadiazol-2-yl]benzenesulfonyl}-3-
hydroxyguanidine (6j)
IR (KBr):
1550, 1502, 1491, 1475, 1353, 1255, 1154 cmꢂ1
DMSO-d6): 4.40 (s, 2H, SCH2), 6.01 (s, 2H, OCH2O), 6.89 (d,
J ¼ 7.81 Hz, 1H, Ar), 6.98 (d, J ¼ 7.81 Hz, 1H, Ar), 7.03 (s, 1H, Ar), 7.17
(br s, 1H, NH), 7.39 (br s, 1H, NH), 7.60e7.64 (m, 3H, Ar), 7.72 (s, 1H,
Ar), 8.10e8.11 (m, 2H, Ar), 8.64 (s, 1H, Ar), 9.45 (s, 1H, NH), 10.05 (s,
1H, OH); Anal. C23H18ClN5O6S2 (C, H, N).
n
3486, 3370, 3302, 3204, 2923, 2854, 1612, 1586, 1560,
;
1H NMR (500 MHz,
d
Starting from 5j (0.150 g). Yield: 0.125 g (84%): m.p. 240e242 ꢀC;
IR (KBr):
n
3450, 3348, 3249, 2907, 1619,1581, 1544, 1504, 1487, 1368,
4.38
1328, 1288, 1249, 1156 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
(s, 2H, SCH2), 6.01 (s, 2H, OCH2O), 6.89 (d, J ¼ 7.81 Hz,1H, Ar), 6.96 (d,
J ¼ 7.81 Hz, 1H, Ar), 7.02 (s, 1H, Ar), 7.17 (br s, 1H, NH), 7.39 (br s, 1H,
NH), 7.71e7.73 (m, 3H, Ar), 8.10e8.12 (m, 2H, Ar), 8.54 (s,1H, Ar), 9.45
(s, 1H, NH), 10.05 (s, 1H, OH); MALDI-TOF m/z obsd: 594.0, [M þ H]þ,
616.0, [M þ Na]þ calcd: 593.0; Anal. C23H17Cl2N5O6S2 (C, H, N).
4.6.16. 1-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-[3-(4-
chlorophenyl)-1,2,4-oxadiazol-5-yl]benzenesulfonyl}-3-
(hydroxyguanidine) (6p)
Starting from 5p (0.150 g). Yield: 0.117 g (79%): m.p. 258e260 ꢀC;
4.6.11. 1-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-(5-phenyl-
1,3,4-thiadiazol-2-yl)benzenesulfonyl}-3-hydroxyguanidine (6k)
IR (KBr):
n
3436, 3321, 2923, 1624, 1586, 1558, 1502, 1491, 1472,
4.39 (s, 2H,
1342, 1253, 1158 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
Starting from 5k (0.145 g). Yield: 0.118
231e233 ꢀC; IR (KBr):
3486, 3441, 3352, 3322, 3098, 2923, 2854,
1626, 1577, 1558, 1540, 1500, 1468, 1457, 1373, 1247, 1160 cmꢂ1; 1H
NMR (500 MHz, DMSO-d6): 4.37 (s, 2H, SCH2), 6.01 (s, 2H,
g
(82%): m.p.
SCH2), 6.01 (s, 2H, OCH2O), 6.88 (d, J ¼ 7.81 Hz, 1H, Ar), 6.96 (d,
J ¼ 7.81 Hz, 1H, Ar), 7.03 (s, 1H, Ar), 7.18 (br s, 1H, NH), 7.39 (br s, 1H,
NH), 7.67e7.69 (m, 2H, Ar), 7.72 (s, 1H, Ar), 8.10e8.12 (m, 2H, Ar),
8.63 (s, 1H, Ar), 9.45 (s, 1H, NH), 10.05 (s, 1H, OH); Anal.
C23H17Cl2N5O6S2 (C, H, N).
n
d
OCH2O), 6.89 (d, J ¼ 7.81 Hz, 1H, Ar), 6.97 (d, J ¼ 7.81 Hz, 1H, Ar),
7.03 (s, 1H, Ar), 7.16 (br s, 1H, NH), 7.40 (br s, 1H, NH), 7.59e7.60
(m, 3H, Ar), 7.71 (s, 1H, Ar), 8.06e8.07 (m, 2H, Ar), 8.74 (s, 1H,
Ar), 9.46 (s, 1H, NH), 10.05 (s, 1H, OH); MALDI-TOF m/z obsd:
4.6.17. 1-[4-Chloro-2-(3,4,5-trimethoxybenzylthio)-5-(5-phenyl-
1,3,4-oxadiazol-2-yl)benzenesulfonyl]-3-hydroxyguanidine (6q)
Starting from 5q (0.153 g). Yield: 0.133 g (88%): m.p. 235e237 ꢀC;
576.0, [M
C23H18ClN5O5S3(C, H, N).
þ
H]þ, 598.0, [M
þ
Na]þ calcd: 575.0; Anal.
IR (KBr):
n
3434, 3344, 2999, 2929,1626,1589,1555,1507,1460,1338,
3.63 (s, 3H, OCH3), 3.77
1126 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
4.6.12. 1-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-[5-(4-
chlorophenyl)-1,3,4-thiadiazol-2-yl]benzenesulfonyl}-3-
hydroxyguanidine (6l)
(s, 6H, 2ꢃ OCH3), 4.39 (s, 2H, SCH2), 6.82 (s, 2H, Ar), 7.19 (br s, 1H,
NH), 7.39 (br s, 1H, NH), 7.63e7.64 (m, 3H, Ar), 7.75 (s, 1H, H-3),
8.08e8.09 (m, 2H, Ar), 8.54 (s, 1H, H-6), 9.45 (s, 1H, NH), 10.05 (s, 1H,
OH); MALDI-TOF m/z obsd: 606.1, [M þ H]þ, 628.1, [M þ Na]þ calcd:
605.0; Anal. C25H24ClN5O7S2 (C, H, N).
Starting from 5l (0.154 g). Yield: 0.122
234e236 ꢀC; IR (KBr):
3458, 3354, 3297, 2922, 2853, 1626, 1577,
1535, 1503, 1489, 1363, 1253, 1150, 1128 cmꢂ1 1H NMR
(500 MHz, DMSO-d6): 4.36 (s, 2H, SCH2), 6.00 (s, 2H, OCH2O),
g (80%): m.p.
n
;
d
4.6.18. 1-{4-Chloro-5-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-2-
(3,4,5-trimethoxybenzylthio)benzenesulfonyl}-3-hydroxyguanidine
(6r)
6.89e7.02 (m, 3H, Ar), 7.15 (br s, 2H, NH), 7.38 (br s, 1H, NH),
7.65e7.67 (m, 2H, Ar), 7.71 (s, 1H, Ar), 8.08e8.10 (m, 2H, Ar), 8.73
(s, 1H, Ar), 9.44 (s, 1H, NH), 10.05 (s, 1H, OH); MALDI-TOF m/z
obsd: 610.0, [M þ H]þ, 632.0, [M þ Na]þ calcd: 609.0; Anal.
C23H17Cl2N5O5S3 (C, H, N).
Starting from 5r (0.161 g). Yield: 0.125
227e229 ꢀC; IR (KBr):
3457, 3355, 3271, 2927, 1631, 1588, 1556,
1506, 1481, 1334, 1158 cmꢂ1 1H NMR (500 MHz, DMSO-d6):
g (78%): m.p.
n
;
d
3.63 (s, 3H, OCH3), 3.77 (s, 6H, 2ꢃ OCH3), 4.40 (s, 2H, SCH2),
4.6.13. 1-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-(4-
phenylthiazol-2-yl)benzenesulfonyl}-3-hydroxyguanidine (6m)
6.82 (s, 2H, Ar), 7.19 (br s, 1H, NH), 7.42 (br s, 1H, NH), 7.71e7.73
(m, 2H, Ar), 7.76 (s, 1H, Ar), 8.10e8.11 (m, 2H, Ar), 8.54 (s, 1H, Ar),
9.46 (s, 1H, NH), 10.06 (s, 1H, OH); MALDI-TOF m/z obsd: 640.2,
[M þ H]þ, 662.2, [M þ Na]þ calcd: 639.0; Anal. C25H23Cl2N5O7S2
(C, H, N).
Starting from 5m (0.145 g). Yield: 0.115
234e236 ꢀC; IR (KBr):
3438, 3336, 2921, 1637, 1579, 1531, 1501,
1488, 1362, 1278, 1254, 1151, 1125 cmꢂ1; 1H NMR (500 MHz, DMSO-
d6):
g (80%): m.p.
n
d
4.34 (s, 2H, SCH2), 6.00 (s, 2H, OCH2O), 6.88 (d, J ¼ 7.81 Hz,1H,
Ar), 6.95 (d, J ¼ 7.81 Hz, 1H, Ar), 7.03 (s, 1H, Ar), 7.12e7.50 (m, 4H, Ar
and NH), 7.64 (s, 1H, Ar), 8.04e8.06 (m, 2H, Ar), 8.36 (s, 1H, Ar), 8.82
(s, 1H, Ar), 9.44 (s, 1H, NH), 10.05 (s, 1H, OH); MALDI-TOF m/z obsd:
575.2, [M
C24H19ClN4O5S3 (C, H, N).
4.6.19. 1-[4-Chloro-2-(3,4,5-trimethoxybenzylthio)-5-(5-phenyl-
1,3,4-thiadiazol-2-yl)benzenesulfonyl]-3-hydroxyguanidine (6s)
Starting from 5s (0.156 g). Yield: 0.107 g (69%): m.p. 187e189 ꢀC;
þ
H]þ, 597.2, [M
þ
Na]þ calcd: 574.0; Anal.
IR (KBr):
n
3475, 3363, 3280, 2933, 1614, 1592, 1557, 1506, 1458,
3.64
1334, 1246, 1143, 1123 cmꢂ1; 1H NMR (500 MHz, DMSO-d6):
d
(s, 3H, OCH3), 3.78 (s, 6H, 2ꢃ OCH3), 4.39 (s, 2H, SCH2), 6.82 (s, 2H,
Ar), 7.15e7.45 (m, 2H, 2ꢃ NH), 7.59e7.62 (m, 3H, Ar), 7.76 (s, 1H, Ar),
8.05e8.10 (m, 2H, Ar), 8.74 (s, 1H, Ar), 9.47 (s, 1H, NH), 10.06 (s, 1H,
OH); MALDI-TOF m/z obsd: 622.0, [M þ H]þ, 644.1, [M þ Na]þ calcd:
621.0; Anal. C25H24ClN5O6S3 (C, H, N).
4.6.14. 1-{2-[(1,3-Benzodioxol-5-yl)methylthio]-4-chloro-5-[4-(4-
chlorophenyl)thiazol-2-yl]benzenesulfonyl}-3-hydroxyguanidine (6n)
Starting from 5n (0.154 g). Yield: 0.125
g (82%): m.p.
261e263 ꢀC; IR (KBr):
n
3438, 3306, 3230, 2923, 1629, 1575, 1544,

_
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392
4.6.20. 1-{4-Chloro-5-[5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-
(3,4,5-trimethoxybenzylthio)benzenesulfonyl}-3-hydroxyguanidine
(6t)
minimized to the putative global low-energy conformation using
the standard Tripos molecular mechanics force field [28,29] with
a distance-dependent (1/r) dielectric function and the Powell
conjugate-gradient algorithm with convergence criterion of
0.05 kcal/mol. Atomic partial charges were computed using the
GeisteigereHückel method [30,31]. In CoMSIA studies, a common
probe atom with 1.0 Ǻ radius, þ1.0 formal charge and þ1.0 hydro-
phobicity was used to evaluate three interaction fields: steric, elec-
trostatic and hydrophobic. Because our data set is structurally
consistent all molecules in the created database were aligned to the
common core of 1-(4-chloro-2-mercaptobenzenesulfonyl)-3-hydro-
xyguanidine and 6k as the most active compound was chosen as
a template molecule (Fig. 2a). CoMSIA field descriptors used in the
modeling gave some insight into 3D structureeantiproliferative
activity relationship (Fig. 3).
Starting from 5t (0.165 g). Yield: 0.131
228e230 ꢀC; IR (KBr):
3433, 3339, 3274, 2927, 2839, 1620, 1594,
1542, 1508, 1461, 1334, 1240, 1153, 1126 cmꢂ1 1H NMR (200 MHz,
DMSO-d6):
g (80%): m.p.
n
;
d
3.63 (s, 3H, OCH3), 3.77 (s, 6H, 2ꢃ OCH3), 4.38 (s, 2H,
SCH2), 6.81 (s, 2H, Ar), 7.13 (br s, 1H, NH), 7.40 (br s, 1H, NH),
7.66e7.67 (m, 2H, Ar), 7.75 (s, 1H, Ar), 8.08e8.10 (m, 2H, Ar), 8.74
(s, 1H, Ar), 9.45 (s, 1H, NH), 10.05 (s, 1H, OH); MALDI-TOF m/z obsd:
656.0, [M
þ
H]þ, 678.1, [M
þ
Na]þ calcd: 655.0; Anal.
C25H23Cl2N5O6S3 (C, H, N).
4.6.21. 1-[4-Chloro-2-(3,4,5-trimethoxybenzylthio)-5-(4-
phenylthiazol-2-yl)benzenesulfonyl]-3-hydroxyguanidine (6u)
Starting from 5u (0.157 g). Yield: 0.124
233e235 ꢀC; IR (KBr):
3423, 3317, 2933, 1617, 1593, 1540, 1507,
1458, 1333, 1277, 1144, 1127 cmꢂ1 1H NMR (200 MHz, DMSO-d6):
g
(80%): m.p.
The aligned molecules were set in a Cartesian coordinate box,
a 3D regular lattice with 2 Ǻ grid spacing, and similarity indices
were calculated at each grid point using following equation:
n
;
d
3.63 (s, 3H, OCH3), 3.77 (s, 6H, 2ꢃ OCH3), 4.36 (s, 2H, SCH2),
X
2
AqF;kðjÞ ¼
wprobe;kwikeꢂ
a
riq
7.17e7.40 (m, 3H, Ar and NH), 7.47e7.51 (m, 2H, Ar), 7.69 (s, 1H, Ar),
8.05e8.07 (m, 2H, Ar), 8.36 (s, 1H, Ar), 8.82 (s, 1H, Ar), 9.46 (s, 1H,
NH),10.06 (s, 1H, OH); MALDI-TOF m/z obsd: 621.3, [M þ H]þ, 643.3,
[M þ Na]þ calcd: 620.1; Anal. C26H25ClN4O6S3 (C, H, N).
i
A is the similarity index at grid point q, summed overall atoms (i)
of the molecule j; wprobe,k is the probe atom, wik is the actual value of
the physicochemical property (k) of atom i; riq is the mutual
distance between the probe atom at grid point q and atom i of the
4.6.22. 1-{4-Chloro-5-[4-(4-chlorophenyl)thiazol-2-yl]-2-(3,4,5-
trimethoxybenzylthio)benzenesulfonyl}-3-hydroxyguanidine (6v)
Starting from 5v (0.165 g). Yield: 0.134 g (82%): m.p. 241e243 ꢀC;
test molecule;
a is the attenuation factor.
Because of used in this calculation Gaussian-type distance
dependence, the similarity indices can be calculated at all grid
points (both inside and outside the molecular surface); therefore,
no cut-off values need to be defined. The only field parameter
IR (KBr):
n
3429, 3314, 2931, 1617, 1597, 1540, 1508, 1461, 1334, 1274,
3.64 (s, 3H, OCH3), 3.77
1129 cmꢂ1; 1H NMR (200 MHz, DMSO-d6):
d
(s, 6H, 2ꢃ OCH3), 4.37 (s, 2H, SCH2), 6.81 (s, 2H, Ar), 7.26e7.34 (br s,
2H, NH), 7.54e7.58 (m, 2H, Ar), 7.70 (s, 1H, Ar), 8.07e8.11 (m, 2H,
Ar), 8.43 (s, 1H, Ar), 8.82 (s, 1H, Ar), 9.46 (s, 1H, NH), 10.07 (s, 1H,
OH); MALDI-TOF m/z obsd: 655.1, [M þ H]þ, 677.1, [M þ Na]þ calcd:
654.0; Anal. C26H24Cl2N4O6S3 (C, H, N).
available that can be modified at this step is a, which determines
the steepness of the Gaussian function, and was set to the default,
yet optimal value (0.3). Here, steric indices are related to the third
power of the atomic radii, electrostatic descriptors are derived from
atomic partial charges, hydrophobic fields are derived from atom-
4.6.23. 1-[4-Chloro-2-(3,4,5-trimethoxybenzylthio)-5-(3-phenyl-
1,2,4-oxadiazol-5-yl)benzenesulfonyl]-3-hydroxyguanidine (6w)
Starting from 5w (0.153 g). Yield: 0.077
228e230 ꢀC; IR (KBr):
3440, 3334, 2933, 2837, 1622, 1592, 1545,
1506, 1461, 1380, 1351, 1334, 1286, 1247, 1159, 1128 cmꢂ1 1H NMR
(200 MHz, DMSO-d6):
g (51%): m.p.
n
;
d
3.64 (s, 3H, OCH3), 3.78 (s, 6H, 2ꢃ OCH3),
4.42 (s, 2H, SCH2), 6.83 (s, 2H, Ar), 7.20e7.40 (m, 2H, 2ꢃ NH),
7.60e7.64 (m, 3H, Ar), 7.78 (s, 1H, Ar), 8.09e8.13 (m, 2H, Ar), 8.64 (s,
1H, Ar), 9.46 (s, 1H, NH), 10.06 (s, 1H, OH); Anal. C25H24ClN5O7S2 (C,
H, N).
4.6.24. 1-{4-Chloro-5-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]-2-
(3,4,5-trimethoxybenzylthio)benzenesulfonyl}-3-hydroxyguanidine
(6x)
Starting from 5x (0.161 g). Yield: 0.122 g (76%): m.p. 243e245 ꢀC;
IR (KBr):
n
3457, 3353, 3268, 2936, 1631, 1589, 1558, 1505, 1459,
1335, 1157 cmꢂ1
;
1H NMR (500 MHz, DMSO-d6):
d
3.63 (s, 3H,
OCH3), 3.77 (s, 6H, 2ꢃ OCH3), 4.40 (s, 2H, SCH2), 6.81 (s, 2H, Ar), 7.19
(br s, 1H, NH), 7.40 (br s, 1H, NH), 7.67e7.69 (m, 2H, Ar), 7.77 (s, 1H,
Ar), 8.10e8.12 (m, 2H, Ar), 8.63 (s, 1H, Ar), 9.45 (s, 1H, NH), 10.05 (s,
1H, OH); Anal. C25H23Cl2N5O7S2 (C, H, N).
4.7. Molecular modeling
QSAR studies were conducted to better understand the biolog-
ical activity of this series of compounds.
SYBYL (Tripos Inc., St. Louis, USA, Ver. 8.0) molecular modeling
software package running on Linux workstation was used to perform
QSAR analysis. 3D structures of all 5-dose assayed 19 compounds
were constructed using Sketch Molecule module and were energy-
Fig. 2. (a) Template molecule (6k) used for alignment (common substructure shown in
blue). (b) Training set compounds aligned on minimum energy conformation of
compound 6k. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

_
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K. Brozewicz, J. Sławinski / European Journal of Medicinal Chemistry 55 (2012) 384e394
393
Fig. 3. Contour maps of CoMSIA fields contributing to ligand binding generated by PLS analysis in Model_B. Compound 6p (stick model) is shown as a reference to depict the field
regions. The contours of the steric map are shown in yellow and green (a), those of the electrostatic map are shown in red and blue (b) and contour map of hydrophobic field in cyan
(c). Greater values of activity are correlated with: more bulk near green; less bulk near yellow; more positive charge near blue, and more negative charge near red; hydrophobic
moieties near cyan. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Plot of observed vs. predicted activities (MGeMID pGI50) for the training set compounds based on the best QSAR model (A) and CoMSIA region-focusing model (B).
based parameters [32]. Biological activities for this set of
compounds fall within the range 10ꢂ5 to 10ꢂ6 M (data presented in
supporting materials in Table 6) and this led to challenges to obtain
good model of high cross-validated r2 (q2) value.
molecular structure, represented in the model by 0.541 (Model_A)
or 0.550 (Model_B) fraction (descriptors: CMR, CLogP, PSA and PV)
of explanatory variables (data presented in supporting materials in
Table 7).
ꢀ
ꢁ
2
P
Ypred ꢂ Yactual
Y
q2 ¼ 1 ꢂ
4.8. COMPARE analysis
P
ðYactual ꢂ YmeanÞ2
Y
The biological activity results (NCI-60 panel inhibition profiles)
of 11 representatives were subjected to COMPARE analysis [33,34].
Some of them showed good correlation (PCC ¼ 0.510e0.806) with
nucleic acids interfering agents like dactinomycin, bruceantin,
chromomycin A3 and echinomycin (Table 4). The highest correla-
tion coefficients were identified when compounds 6d, 6g and 6r
Ypred is a predicted value; Yactual is an actual (experimental)
value; Ymean is the best estimate of the mean of all values that might
be predicted.
In order to get an optimal QSAR model, different descriptors
were used, namely, log of the n-octanol/water partition coefficient
(CLogP), molar refractivity (CMR), polar volume (PV) and polar
surface area (PSA). The constructed best QSAR model (Model_A)
was further optimized. The best results were obtained at a column
filtering (Cf) of 0.5 kcal/mol. CoMSIA Hydrophobic explanatory
variable contributed mostly in the obtained model (data presented
in supporting materials in Table 7) and CoMSIA region-focusing
method was employed to derive Model_B (Fig. 4).
The model confirms that the bulky substituent, especially (1,3-
benzodioxol-5-yl)methylthio (6iep), at 2 position of 1-benzen-
esulfonyl-3-hydroxyguanidine core accompanied with positive
electrostatic charge projecting above and electronegative below the
heterocyclic residue (1,3,4-thiadiazole ring) in 5 position of the core
structure correlate with better activity (6c, 6d, 6k, 6l, 6s, 6t). The
model does not judge weather phenyl or 4-chlorophenyl is
preferred at heterocycle moiety.
Table 4
COMPARE
analysis
of
eleven
5-dose
assayed
1-benzenesulfonyl-3-
hydroxyguanidines (6c, 6d, 6f, 6g, 6i, 6j, 6m, 6p, 6r, 6s, and 6v). Compounds with
the highest correlation coefficient were bolded out.
Target vectora
No.b PCC rangec
Correlated compoundsc
Dactinomycin
(NSC 3053)
Bruceantin
(NSC 165563)
Chromomycin A3
(NSC 58514)
Echinomycin
(NSC 526417)
Didemnin B
11
10
8
0.510e0.806 6c, 6d, 6f, 6g, 6i, 6j, 6m, 6p, 6r, 6s, 6v
0.539e0.791 6c, 6d, 6g, 6i, 6j, 6l, 6p, 6r, 6s, 6v
0.596e0.799 6c, 6d, 6g, 6i, 6j, 6r, 6s, 6v
0.555e0.760 6c, 6d, 6g, 6i, 6j, 6p, 6s, 6v
0.522e0.712 6c, 6d, 6g, 6p, 6s, 6v
8
6
(NSC 325319)
a
For definitions and methods of calculation of the correlation coefficient (PCC)
The main outcome of this complex model is that the potency of
this series of compounds seems to accompany not only local steric,
electrostatic or hydrophobic properties but overall structural
features associated with the whole molecule attributes like molar
refractivity, hydrophobicity and polar surface properties of the
from the COMPARE analysis, see Ref. [33,34].
b
Number of correlated compounds giving PCC >0.5.
Pearson Correlation coefficient (PCC) range for the described compounds.
Compounds in NIH-DTP STANDARD_AGENTS database, giving correlation
c
d
coefficient >0.5.

_
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K. Brozewicz, J. Sławinski / European Journal of Medicinal Chemistry 55 (2012) 384e394
394
[11] J.-W. Chern, Y.-C. Liaw, C.-S. Chen, J.-G. Rong, C.-L. Huang,C.-H. Chan, A.H.-J. Wang,
were used as Seed [34]. This could indicate DNA as a potential
molecular target for the synthesized 1-benzenesulfonyl-3-
hydroxyguanidines and further biological studies are needed to
verify the suggested by COMPARE algorithm hypothesis.
Heterocycles 36 (1993) 1091e1103.
ꢀ
[12] J. S1awinski, Eur. J. Med. Chem. 39 (2004) 179e188.
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The authors are very grateful to Dr. Joel Morris, Chief of Drug
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