Synthesis, molecular structure, and in vitro antitumor activity of new 4-chloro-2-mercaptobenzenesulfonamide derivatives

Article abstract of DOI:10.1016/j.ejmech.2004.11.014

The reaction of 3-amino-2-(2-alkylthio-4-chlorobenzenesulfonyl)guanidines 2a-j with 1,2-dicarbonyl compounds are described. Depending on structure of 1,2-dicarbonyl reagent novel 2-alkylthio-5-chloro-N-(1,2,4-triazin-3-yl) benzenesulfonamides 3-15, 1-(2-a

Full text of DOI:10.1016/j.ejmech.2004.11.014

European Journal of Medicinal Chemistry 40 (2005) 377–389
www.elsevier.com/locate/ejmech
Original article
Synthesis, molecular structure, and in vitro antitumor activity of new
4-chloro-2-mercaptobenzenesulfonamide derivatives
Jarosław Sławin´ski a,*, Maria Gdaniec b
a Department of Chemical Technology of Drugs, Medical University of Gdan´sk, Al. Gen. J. Hallera 107, 80-416 Gdan´sk, Poland
b Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan´, Poland
Received 17 September 2004; received in revised form 15 November 2004; accepted 17 November 2004
Available online 29 January 2005
Abstract
The reaction of 3-amino-2-(2-alkylthio-4-chlorobenzenesulfonyl)guanidines 2a–j with 1,2-dicarbonyl compounds are described. Depend-
ing on structure of 1,2-dicarbonyl reagent novel 2-alkylthio-5-chloro-N-(1,2,4-triazin-3-yl)benzenesulfonamides 3–15, 1-(2-alkylthio-4-
chlorobenzenesulfonyl)-3-(2-oxobutane-3-ylidenoimino)guanidines 16–18 and 2-alkylthio-4-chloro-N-(1,2-dihydroxycyclobuta[e]1,2,4-
triazin-3-yl)benzenesulfonamides 19–21 are obtained. The structures of these compounds were confirmed on the basis of elemental analysis,
spectral data and X-ray analysis. The compounds 4, 5, 7, 9, 10, 12–15, 17, 18 and 20 were screened at the National Cancer Institute (NCI) for
their in vitro activities against a panel of 56 tumor cell lines and relationship between structure and antitumor activity are discussed. The
compounds 10, 12, 17 and 20 were inactive, whereas the other compounds exhibited reasonable activity against one or more human tumor cell
lines. The prominent compound 18 showed significant activity against cell lines of colon cancer (HCT-116), renal cancer (786-0) and mela-
noma (M14) (GI50 in the range 0.33–1.08 µM) as well as good selectivity toward non-small cell lung cancer (HOP-62) cells (GI50 = 0.05 µM,
TGI = 0.38 µM and LC50 = 4.83 µM).
© 2005 Elsevier SAS. All rights reserved.
Keywords: 4-Chloro-2-mercapto-N-(1,2,4-triazin-3-yl)benzenesulfonamide; Synthesis; Crystal structures; Antitumor effect
1. Introduction
studies we synthesized a new series of arylsulfonylami-
noguanidine derivatives possessing electron-withdrawing sub-
stituents, either at the N-terminal nitrogen atom of the hydra-
zine moiety of type I [24] or at the opposite nitrogen atom of
guanidine moiety of type II and III [25,26] (Fig. 1), and found
many of them exhibited a pronounced anticancer activity
[24–26].
This prompted us to develop a method for the synthesis of
their cyclic analogues. Hence, in the present study we elabo-
rated an efficient method for syntheses of novel 2-alkylthio-
4-chloro-N-(1,2,4-triazin-3-yl)benzenesulfonamides of type
IV and related 2-alkylthio-4-chloro-5-methyl-N-(1,2-dihy-
droxycyclobuta[e]1,2,4-triazin-3-yl)benzenesulfonamides of
type V (Fig. 1).
Numerous of structurally novel sulfonamide derivatives
have been reported to possess anticancer or/and anti-HIV
[1–5] properties. We have also described the syntheses of a
number of 4-chloro-2-mercaptobenzenesulfonamide possess-
ing bulky aromatic/heterocyclic moieties substituting the sul-
fonamide functionality. These compounds, depending on their
structure, exhibited either anticancer [6–17] or anti-HIV-1
[6,7,15,18–20] activities and have been described by Neamati
et al. [21,22] as a novel class of potent HIV-1 integrase inhibi-
tors. We further found that S-substituted 4-chloro-2-
mercaptobenzenesulfonamide derivatives possessing pri-
mary (unsubstituted) sulfonamide moiety or cyano group
attached to the nitrogen atom of the sulfonamide moiety also
showed substantial anticancer properties [23].
2. Results and discussion
Recently, in search for even more potent 2-mercapto-
benzenesulfonamides and for structure–activity relationship
2.1. Chemistry
The previously described methods were employed for the
* Corresponding author. Tel.: +48-58-349-3255; fax: +48-58-349-3257.
E-mail address: jaroslaw@amg.gda.pl (J. Sławin´ski).
synthesis of compounds 1a–j [27] and 2a–c [24]. Analo-
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.11.014

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Fig. 1
.
gously were prepared novel 3-amino-2-(2-alkylthio-4-chloro-
5-R1-benzenesulfonyl)guanidines 2d–j as shown in Scheme 1.
The synthesis of the target cyclocondensation products
3–14 were achieved by reacting the corresponding ami-
noguanidines 2a–c and 2e–j with suitable 1,2-diarylethane-
1,2-diones in refluxed ethanol (3–6) or a mixture of ethanol
and dioxane (9–12), as well as dry DMSO (7–8, 13–14) at
80–85 °C for 8–30 h (Scheme 1). Subsequent reaction of 9
with POCl3 carried out at elevated temperature led to the for-
mation of the expected 2-benzylthio-4-chloro-5-cyano-N-
(1,2,4-triazin-3-yl)benzenesulfonamide (15) in 85% yield
(Scheme 1).
Scheme 1
(2-oxobutane-3-ylideneimino)guanidines 16–18.
.
Syntheses of the 2-alkylthio-4-chloro-5-R1-N-(1,2,4-triazin-3-yl)benzenesulfonamides 3–15 and 1-(2-alkylthio-4-chloro-5-R1-benzenesulfonyl)-3-

J. Sławin´ski, M. Gdaniec / European Journal of Medicinal Chemistry 40 (2005) 377–389
379
When the aminoguanidines 2b, d and 2h, in turn, were
subjected to the reaction with diacetyl the corresponding 1-[2-
alkylthio-4-chloro-5-(methyl or phenylcarbamoyl)ben-
zenesulfonyl]-3-(2-oxobutane-3-ylideneimino)guanidines
16–18 were obtained as final products in 65–89% of sepa-
rated yields (Scheme 1). It is worthy noting, that prolonga-
tion of the reaction time up to 24 h does not provide either the
cyclocondensation product or higher yield. The first step of
this reaction i.e. condensation of the terminal amino group of
the hydrazine moiety with one carbonyl group, is fast. How-
ever, the intermediate product of type 16–18 adopts the stable
sulfonylamino tautomeric form as shown in Scheme 1. This
may results in relatively low reactivity of the opposite imino
group to the reaction with second carbonyl group [25].
It is well known that the reactions of squaric acid (3,4-
dihydroxy-3-cyclobutene-1,2-dione) and its esters with ali-
phatic or aromatic amines lead to the formation of the corre-
sponding amide or bisamide derivatives [28–30].As depicted
in Scheme 2, treatment of aminoguanidine 2a, b, d with
1 molar equivalent of squaric acid in refluxing ethanol
afforded the 2-alkylthio-4-chloro-5-methyl-N-(1,2-dihy-
droxycyclobuta[e]1,2,4-triazin-3-yl)benzenesulfonamide
19–21 in moderate (34–42%) yields. As expected, the same
product 20 was formed after treatment of aminoguanidine 2b
with 1 molar equivalent of 3,4-diethoxy-3-cyclobutene-1,2-
dione in ethanol, but pure product was isolated in poor yield.
The formation of these N-(1,2-dihydroxycyclobuta[e]1,2,4-
triazin-3-yl) derivatives is believed to arise from the follow-
ing two-step process: nucleophilic addition of amino groups
to the double C=C bond of squaric acid with simultaneous
elimination of water molecules resulting in the intermediate
A, and finally tautomerization.A similar mechanism was pro-
posed for the reaction with diethyl squarate (Scheme 2).
All the final products were characterized by IR and NMR
spectroscopy as shown in experimental protocols. Elemental
analyses were in accordance with the proposed structure.
X-ray crystallography was undertaken in order to investi-
gate in detail tautomeric structures on representative com-
pounds 4, 10 and 11. Molecular structures of compounds 4,
10 and 11 are shown in Figs. 2–4, respectively.
Compounds 4 and 10 crystallize as sulfonamide tau-
tomers, i.e. the hydrogen atom is bound to the sulfonamide
nitrogen atom. This form was confirmed by localization of H
atoms from electron-density difference maps and by geometri-
cal parameters characteristic of the sulfonamide form. For
example, the exocyclic C7–N1 bonds in 4 and 10 (1.378 and
1.381 Å, respectively), are ca. 0.06 Å longer than in 11.
Contrary to 4 and 10, compound 11 crystallizes in the sul-
fonimide form with hydrogen atom joined to N3 of the het-
erocycle (Fig. 4). This tautomer was confirmed by localiza-
tion of the H atom in electron-density maps, by molecular
geometry of 11 and by observed assembly mode of the mol-
ecules in the solid state. For example, the exocyclic C7–
N1 and endocyclic N2–C9 and N4–C8 bonds of the 1,2,4-
triazine fragment (1.318, 1.311 and 1.318 Å, respectively)
show a substantial double-bond character pointing to a sul-
fonimide tautomer of 11. Change of the tautomeric form is
also reflected in the geometry of the 1,2,4-triazine cycle which
Scheme 2
yl)benzenesulfonamides 19–21.
. Synthesis and proposed mechanism of the formation of 2-alkylthio-4-chloro-5-methyl-N-(1,2-dihydroxycyclobuta[e]1,2,4-triazin-3-

380
J. Sławin´ski, M. Gdaniec / European Journal of Medicinal Chemistry 40 (2005) 377–389
Fig. 2
.
ORTEP drawing [37] of 4 with labeling scheme and displacement ellipsoids at 40% probability level (dashed lines show minor conformation of the
thiomethylene fragment).
has significantly spread C–N bond-lengths for the sulfonim-
ide form of 11 (1.311–1.369 Å) whereas all C–N bonds are
similar in length for the sulfonamide tautomers of 4 and 10
(1.326–1.346 and 1.329–1.334 Å, respectively).
Compound 4 which has more limited hydrogen-bonding
potential, when compared with 10 and 11, gives in crystals
solely self-complementary hydrogen bond interaction shown
in Fig. 5a. Compound 10, which analogously to 11 crystal-
lizes as the DMSO solvate and whose molecule is equipped
with primary amide functionality, forms in crystals one-
dimensional hydrogen-bonded assemblies via complemen-
tary interaction between primary amide group and sulfona-
Fig. 3
molecule is shown).
. ORTEP drawing [37] of 10 with labeling scheme and displacement ellipsoids at 40% probability level (only one of the two orientations of the DMSO

J. Sławin´ski, M. Gdaniec / European Journal of Medicinal Chemistry 40 (2005) 377–389
381
Fig. 4. ORTEP drawing [37] of 11 with labeling scheme and displacement ellipsoids at 40% probability level (only one of the two orientations of the DMSO
molecule is shown).
mide moiety (Fig. 5b) and this interaction points
unambiguously to sulfonamide character of 10. On the other
hand, hydrogen bond interaction observed in crystals of 11
between the primary amide group and (3E)-1,2,4-triazin-
3(2H)-imine moiety, as shown in Fig. 5c, provides definitive
tautomer confirmation. Hydrogen bonds between these two
complementary functionalities assemble molecules of 11 into
one-dimensional network, with DMSO molecules connected
to the amide groups via hydrogen bonds.
Fig. 5. Hydrogen-bonding motifs in crystals structures of (a) compound 4, (b) compound 10 and (c) compound 11.

382
J. Sławin´ski, M. Gdaniec / European Journal of Medicinal Chemistry 40 (2005) 377–389
Despite the fact that all three studied molecules are to a
concentration tested. Selectivity of the compound with respect
to one or more cell lines of the screen is characterized by a
high deviation (D) of the particular cell line parameter com-
pared to the MG_MID value.
large extend flexible, the conformations which they adopt in
crystals show certain common features. The aromatic sub-
stituents of 1,2,4-triazine form a propeller, i.e. they are twisted
in the same direction relative to the planar heterocyclic ring.
The torsion angles N2–C7–N1–S1 (–7.8, –20.8 and –1.5° for
4, 10 and 11, respectively) C7–N1–S1–C1 (–58.2, –66.7 and
–68.1° for 4, 10 and 11, respectively) and N1–S1–C1–C2
(–72.0, –57.8 and –60.2° for 4, 10 and 11, respectively),
describing orientation of the sulfonamide or sulfonimide
group relative to the heterocyclic moiety and conformation
of the arylsulfonamide or -sulfonimide moiety, reveal similar
shape of the large molecular fragment. As could be expected,
the most flexible constituent of these molecules is the ben-
zylthio group attached to the benzene ring showing different
conformations in the three studied structures.
The following is to be noted regarding the tumor cell
growth inhibition data with the tested compounds: (a) the
compounds 10, 12, 17 and 20 were inactive (log GI50 [M] >
–4), whereas the other compounds 4, 5, 7, 9, 13–15 and 18
exhibited reasonable activity against one or more human can-
cer cell lines (Table 1); (b) relatively broad spectrum of tumor
cell growth inhibition was found for the compounds 4, 5 and
14–15 (Table 1), while the compounds 7, 9, 13 and 18 dem-
onstrated a moderate selectivity toward one or more tumor
cell lines (D log GI50 ranged from 1.11 to 2.77; Table 2).
The following conclusion may be drawn from the struc-
ture–activity relationship study. In a series of 5,6-diphenyl-
1,2,4-triazin-3-yl derivatives electronic character of substitu-
ent on benzene ring at position 5 is important factor
influencing cytotoxicity. Thus, the presence of electron-
withdrawing carbamoyl or 4-chlorophenylcarbamoyl groups
at this position significantly enhances selectivity (9,
R1 = CONH2 and 13, 4-Cl-PhNHCO). For example, replace-
ment of the methyl group in 4 (R1 = Me) for carbamoyl (9,
R1 = CONH2) caused a reasonable increase of potency against
the cell lines of colon cancer (HT29), CNS (SNB-75), mela-
noma (SK-MEL-28) and ovarian cancer (OVCAR-4) (log
GI50 in the range from –6.99 to –5.56), respectively (Table 2).
Similarly, the presence of 4-chlorophenylcarbamoyl moiety
at this position (13) enhances selectivity toward renal cancer
RXF 393 cells (13, D log GI50 = 2.77) (Table 2). In contrast,
the presence of unsubstituted phenylcarbamoyl group at this
position (12, R1 = PhNHCO) leads to total loss of cytotoxic
activity. The loss in potency was also observed when the phe-
nyl groups at five and six positions on 1,2,4-triazine ring in 9
(R3 = Ph) was replaced for 2-furyl moieties (10, R3 = 2-
furyl).
2.2. Biology
The compounds 4, 5, 7, 9, 10, 12–15, 17, 18 and 20 were
submitted to the US National Cancer Institute (NCI; Bethesda,
MD), for in vitro testing against a panel approximately
56 tumor cell lines, derived from nine different cancer types:
leukemia, lung, colon, CNS, melanoma, ovarian, renal, pros-
tate and breast. The compounds were at five concentrations
at 10-fold dilution. A 48 h continuous drug exposure proto-
col was used and sulforhodamine B (SRB) protein assay was
used to estimate cell growth. Details of this system and the
information which is encoded by the activity pattern over all
cell lines, have been published [31–33]. The antitumor activ-
ity of tested compounds is given by three parameters for each
cell line: log GI50 value (GI50 = molar concentration of the
compound that inhibits 50% net cell growth), log TGI value
(TGI = molar concentration of the compound leading to total
inhibition) and log LC50 value (LC50 = molar concentration
of the compound leading to 50% net cell death). Further-
more, a mean graph midpoint (MG_MID) is calculated for
each of the mentioned parameters, giving an averaged activ-
ity parameters over all cell lines. For the calculation of the
MG_MID, insensitive cell lines are included with the highest
Substitution of five position with electron-withdrawing
cyano group (15, R1 = CN) enhances activity against cell lines
of leukemia HL-60(TB) and RPMI-8226, whereas the sub-
stitution of this position with 4-methylphenylcarbamoyl moi-
Table 1
Overview of the results of the anticancer screening for compounds 4, 5, 7, 9, 10, 12–15, 17, 18 and 20a
b
Compound
Number of the cell
lines investigated
Number of the cell lines giving positive log GI50, log TGI and log LC50
Log GI50 (M)
Range
Log TGI (M)
Range
Log LC50 (M)
Range
Number
54
Number
53
Number
4
5
7
9
13
14
15
18
55
56
56
56
55
52
53
55
–5.25 to –4.43
–4.60 to –4.04
–4.49 to –4.08
–4.54 to –4.06
–4.43 to –4.05
–4.44 to –4.02
–4.54 to –4.03
–4.65 to –4.04
–6.41 to –4.10
31
28
6
–4.23 to –4.01
–4.22 to –4.01
–4.16 to –4.03
–4.14 to –4.07
–4.13 to –4.05
–4.24 to –4.01
–4.21 to –4.02
–5.32
55
–4.77 to –4.41
–6.17 to –4.10
–6.99 to –4.11
–7.29 to –4.10
–4.93 to –4.31
–5.20 to –4.35
–7.26 to –4.01
49
41
16
36
20
3
54
22
3
51
40
22
18
1
51
39
42
7
Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen (see Table 2 or Refs. [31–33] for details).
a Compounds 10, 12, 17 and 20 were inactive.
b The response parameters: log GI50, log TGI and log LC50 are interpolated values representing the molar concentrations at which percentage growth is +50,
0 and –50, respectively.

J. Sławin´ski, M. Gdaniec / European Journal of Medicinal Chemistry 40 (2005) 377–389
383
Table 2
The in vitro activity and selectivity toward most sensitive tumor cell lines for compounds 4, 5, 7, 9, 13–15 and 18
Compound
Most sensitive tumor cell lines
Log GI50 (M)a
Selectivity toward tumor cell Mean value for all tested cell
lines (D)b for log GI50 (M).
The value is shown if D > 1
lines (MG_MID)c for log
GI50 (M)
4
Non-small cell lung: EKVX
Non-small cell lung: NCI-H322M
Leukemia: RPMI-8226
Prostate : DU-145
Melanoma: SK
–5.25
–4.85
–4.94
–4.77
–4.76
–4.75
–6.17
–4.71
–4.68
–6.99
–6.30
–6.30
–5.56
–4.74
–7.29
–4.78
–4.82
–4.93
–4.88
–4.80
–5.20
–5.15
–5.01
–7.26
–5.18
–6.47
–6.12
–5.10
–5.97
–5.32
–5.63
–5.43
–5.26
–5.21
–5.15
–5.55
–5.46
–5.23
–5.07
–4.91
–4.68
5
7
9
–4.61
–4.34
–4.45
Leukemia: MOLT
CNS: SF-539
1.83
Ovarian: OVCAR-3
Colon: HCT-116
Colon: HT29
2.53
1.85
1.85
1.11
CNS: SNB-75
Melanoma: SK-MEL-28
Ovarian: OVCAR-4
Breast: MCF7
13
14
15
18
Renal: RXF 393
2.77
–4.52
–4.64
–4.61
–4.65
Renal: SN12C
Leukemia: MOLT-4
Leukemia: RPMI-8226
Melanoma: LOX IMV1
Breast: MDA-MB-435
Renal: RXF 393
Leukemia: HL-60(TB)
Leukemia: RPMI-8226
Non-small cell lung: HOP-62
Non-small cell lung: EKVX
Colon: HCT-116
2.60
1.82
1.47
Renal: 786-0
Renal: ACHN
Melanoma: M14
1.32
Melanoma: LOX IMV1
Leukemia: HL-60(TB)
Leukemia: MOLT-4
Leukemia: K-562
Leukemia: SR
Leukemia: RPMI-8226
CNS: SF-539
Prostate : DU-145
Breast: MDA-MB-231/ATCC
Ovarian: OVCAR-8
Ovarian: OVCAR-4
Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen (see Refs [31–33] for details).
a The response parameter: log GI50 is interpolated value representing the molar concentration at which percentage growth is + 50.
b The reported data represent the logarithmic difference between the parametric value referred to the most sensitive cell line and the same mean parameter, D
is considered low if < 1, moderate > 1 and < 3, high if > 3.
c MG_MID = mean graph midpoint = arithmetical mean value for all tested cancer cell lines. If the indicated effect was not attainable within the used
concentration interval, the highest concentration was used for the calculation.
ety (14) results in almost equal potency (compare 14 with 4
in Tables 1 and 2). It is pertinent to note, however, that replace-
ment of the 1,2,4-triazine ring for a fused non-aromatic 1,2-
dihydroxycyclobuta[e]1,2,4-triazine ring system leads to inac-
tive compound 20.
On the other hand, replacement of the benzylthio group
(4) at position 2 of benzene ring for carbamoylmethylthio
moiety resulted in compound 7 (R1 = Me, R2 = CONH2)
(D log GI50 = 1.83, Table 2) exhibited a moderate level of
selectivity toward CNS (SF-539) cells (log GI50 = –6.17, log
TGI = –4.52 and log LC50 = –4.03).
The most active compound 18 (R1 = PhNHCO, R2 = Ph)
indicating that combination of phenylcarbamoyl group at posi-
tion 5 and 3-substituted aminoguanidine moiety attached to
sulfonyl group results in compound with suitable properties,
while its analogue 17 (R1 = Me, R2 = H) was inactive. The
compound 18 exhibited advantageous activity against leuke-
mia subpanel among others (Table 2). Relatively highest sen-

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1
sitivity to 18 was also found for cell lines of colon (HCT-
116), renal (786-0) and melanoma (M14), whereas a
reasonable level of selectivity (D log GI50 = 2.60, D log TGI
= 2.30 and D log LC50 = 1.29) indicated that the lung cancer
HOP-62 cells were the most susceptible to inhibition by com-
pound 18 (log GI50 = –7.26, log TGI = –6.41 and log LC50
= –5.32).
NH), 1342, 1130 (SO2) cm–1; H NMR (DMSO-d6) d 2.31
(s, 3H, CH3), 2.44 (s, 3H, SCH3), 4.51 (s, 2H, NH2), 6.97 (s,
2H, NNH2), 7.28 (s, 1H, H-3), 7.81 (s, 1H, H-6), 8.40 (s, 1H,
NH) ppm. Anal. (C9H13ClN4O2S2) C, H, N.
4.1.1.2. 3-Amino-2-(2-carbamoylmethylthio-4-chloro-5-
methylbenzenesulfonyl)guanidine (2e). Yield: 1.37 g, 78%,
m.p. 183–185 °C dec.; IR (KBr) 3447, 3355, 3216, 3162
(NH2, NH), 1683 (C=O), 1345, 1135 (SO2) cm–1; 1H NMR
(DMSO-d6) d 2.31 (s, 3H, CH3), 3.68 (s, 2H, SCH2), 4.52 (s,
2H, NH2), 6.99 (s, 2H, NNH2), 7.23 (s, 1H, HaNC=O), 7.53
(s, 1H, H-3), 7.55 (s, 1H, HbNC=O), 7.82 (s, 1H, H-6), 8.44
(s, 1H, NH) ppm. Anal. (C10H14ClN5O3S2) C, H, N.
3. Conclusions
We have developed a method for the synthesis of new series
of 4-chloro-2-mercaptobenzenesulfonamide derivatives with
the nitrogen atom of the sulfonamide moiety attached either
to 1,2,4-triazin-3-yl or 1,2-dihydroxycyclobuta[e]1,2,4-
triazin-3-yl ring systems as well as their ‘open chain’ ana-
logues. These compounds depending on their structure exhib-
ited anticancer activity. Compounds 9, 13 and 18 were the
most potent of all derivatives tested. Moreover, the com-
pound 18 acted as potent inhibitor against HOP-62 non-
small cell lung cancer line. The results we have obtained hith-
erto indicated that anticancer activity of the newly synthesized
compounds depends on the electronic nature of substituents.
4.1.1.3. 3-Amino-2-(5-carbamoyl-4-chloro-2-ethoxycarbon-
ylmethylthiobenzenesulfonyl)guanidine (2f). Yield: 1.78 g,
87%, m.p. 216–218 °C dec.; IR (KBr) 3448, 3406, 3354, 3336,
3218 (NH2, NH), 1716 (C=O), 1663 (C=O), 1310, 1131 (SO2)
cm–1; 1H NMR (DMSO-d6) d 1.20 (t, J = 6.8 Hz, 3H, CH3),
4.03 (s, 2H, SCH2), 4.13 (q, J = 6.8 Hz, 2H, CH2), 4.51 (s,
2H, NH2), 7.01 (s, 2H, NNH2), 7.41 (s, 1H, H-3), 7.67 (s, 1H,
HaNC=O), 7.87 (s, 1H, H-6), 7.97 (s, 1H, HbNC=O), 8.48 (s,
1H, HN) ppm. Anal. (C12H16ClN5O5S2) C, H, N.
4.1.1.4. 3-Amino-2-(2-benzylthio-5-carbamoyl-4-chloroben-
zenesulfonyl)guanidine (2g). Yield: 2.18 g, 95%, m.p. 219–
221 °C dec.; IR (KBr) 3454, 3342 (NH2, NH), 1684 (C=O),
1320, 1135 (SO2) cm–1; 1H NMR (DMSO-d6) d 3.57 (s, 4H,
dioxane), 4.35 (s, 2H, SCH2), 4.52 (s, 2H, NH2), 6.99 (s, 2H,
NNH2), 7.26–7.28 (m, 1H, aromatic), 7.33–7.36 (m, 2H, aro-
matic); 7.45–7.46 (m, 3H, aromatic, H-3), 7.65 (s, 1H,
HaNC=O), 7.87 (s, 1H, H-6), 7.94 (s, 1H, HbNC=O), 8.48 (s,
1H, HN) ppm.Anal. (C15H16ClN5O3S2·1/2C4H8O2) C, H, N.
4. Experimental protocols
4.1. Synthesis
Melting points were taken on a Bu˝chi SMP 20 apparatus
and are reported uncorrected. IR spectra in KBr were recorded
on a Perkin-Elmer FT IR 1600 spectrophotometer. H and
1
13C NMR spectra were recorded on a Varian Gemini
(200 MHz) and Varian Unity Plus (500 MHz) spectrometer
using TMS as internal standard (d values in ppm). Mass spec-
tra were recorded on a Finnigan MAT 95 spectrometer at
70 eV. The results of elemental analyses for C, H and N were
within 0.4% of the theoretical values.
4.1.1.5. 3-Amino-2-(2-benzylthio-4-chloro-5-phenylcarbam-
oylbenzenesulfonyl)guanidine (2h). Yield: 2.40 g, 98%, m.p.
237–239 °C dec.; IR (KBr) 3448, 3348, 3213, 3101 (NH2,
NH), 1684 (C=O), 1316, 1131 (SO2) cm–1. Anal.
(C21H20ClN5O3S2) C, H, N.
4.1.1. General procedure for the preparation of 3-amino-
2-(2-alkylthio-4-chloro-5-R1- benzenesulfonyl)guanidines
(2d–j)
4.1.1.6. 3-Amino-2-[2-benzylthio-4-chloro-5-(4-chlorophe-
nylcarbamoyl)benzenesulfonyl]guanidine (2i). Yield: 1.70 g,
65%, m.p. 252–254 °C dec.; IR (KBr) 3471, 3356, 3314, 3214
(NH2, NH), 1687 (C=O), 1333, 1133 (SO2) cm–1; 1H NMR
(DMSO-d6) d 3.51 (s, 2H, SCH2), 4.52 (s, 2H, NH2), 7.02 (s.
2H, NNH2), 7.26–7.29 (m, 1H, aromatic), 7.34–7.37 (m, 2H,
aromatic), 7.41 (d, J = 8.5 Hz, 2H, aromatic), 7.47–7.49 (m,
2H, aromatic), 7.56 (s, 1H, H-3), 7.71 (d, J = 8.5 Hz, 2H,
aromatic), 7.98 (s, 1H, H-6), 8.51 (s, 1H, NH), 10.70 (s, 1H,
NHC=O) ppm. Anal. (C21H19Cl2N5O3S2) C, H, N.
To a suspension of the corresponding N-(benzenesulfonyl)-
cyanamide potassium salt 1d–j (5 mmol) in dry dioxane
(25 ml) hydrazine hydrochloride (0.68 g, 10 mmol) was added.
The reaction was stirred under reflux for 4–5 h and left to
stand at room temperature overnight. The precipitate was fil-
tered off, washed with dioxane (3 × 3 ml), dried and treated
with water (50 ml). After vigorously stirring for 10 min the
precipitate was collected by filtration, dried and crystallized
from a mixture of ethanol and dioxane (1:1).
In this manner, the following aminoguanidines were
obtained.
4.1.1.7. 3-Amino-2-[2-benzylthio-4-chloro-5-(4-methylphe-
nylcarbamoyl)benzenesulfonyl]guanidine (2j). Yield: 1.89 g,
75%, m.p. 229–231 °C dec.; IR (KBr) 3466, 3313 (NH2, NH),
1654 (C=O), 1314, 1135 (SO2) cm–1; 1H NMR (DMSO-d6)
d 2.26 (s, 3H, CH3), 4.38 (s, 2H, SCH2), 4.51 (s, 2H, NH2),
4.1.1.1. 3-Amino-2-(4-chloro-5-methyl-2-methylthiobenzene-
sulfonyl)guanidine (2d). Yield: 1.17 g, 76%, m.p. 249–
251 °C dec.; IR (KBr) 3464, 3354, 3340, 3265, 3223 (NH2,

J. Sławin´ski, M. Gdaniec / European Journal of Medicinal Chemistry 40 (2005) 377–389
385
7.02 (s, 2H, NNH2), 7.14 (d, J = 7.5 Hz, 2H, aromatic), 7.25–
7.28 (m, 1H, aromatic), 7.33–7.36 (m, 2H, aromatic), 7.47
(d, J = 7.5 Hz, 2H, aromatic), 7.54–7.57 (m, 3H, aromatic),
7.95 (s, 1H, H-6), 8.50 (s, 1H, HN), 10.45 (s, 1H, HNC=O)
ppm. Anal. (C22H22ClN5O3S2) C, H, N.
8.06 (s, 1H, aromatic), 8.17 (s, 1H, H-6) ppm; 13C NMR
(DMSO-d6) d 20.89 (CH3), 37.86 (SCH2), 110.75, 113.89,
114.16, 115.40, 121.65, 121.70, 126.67, 128.83, 129.73,
129.97, 130.74, 133.71, 135.77, 137.33, 137.96, 139.59,
146.45, 148.54, 149.06, 151.06, 152.75 (21C, aromatic) ppm.
Anal. (C25H19ClN4O4S2) C, H, N.
4.1.2. Preparation of 2-alkylthio-4-chloro-5-methyl-N-(5,6-
diaryl-1,2,4-triazin-3-yl)benzenesulfonamides (3–6)
A stirred suspension of the corresponding 3-amino-2-(2-
alkylthio-4-chloro-5-methylbenzenesulfonyl)guanidine 2a–c
(2 mmol) and benzil (0.43 g, 2 mmol) or 2,2′-furil (0.38 g,
2 mmol) (5) in ethanol (10 ml) was refluxed for 24 h and left
to stand in a refrigerator overnight. The precipitate that depos-
ited was filtered off, washed with ethanol (2 × 1 ml), and
dried. The product was purified by crystallization either from
ethanol or from a mixture of ethanol and dioxane (4:1).
In this manner, the following benzenesulfonamides were
obtained.
4.1.2.4. 4-Chloro-2-ethylthio-5-methyl-N-(5,6-diphenyl-1,2,4-
triazin-3-yl)benzenesulfonamide (6). Yield: 0.65 g, 67%, m.p.
105–108 °C dec.; IR (KBr) 3401, 3189 (NH), 2966, 2925,
2860 (CH3, CH2), 1305, 1163 (SO2) cm–1; 1H NMR (DMSO-
d6) d 1.15 (t, J = 7.3 Hz, 3H, CH3), 2.26 (s, 3H, CH3-Ar),
3.02 (q, J = 7.3 Hz, 2H, SCH2), 7.16–7.20 (m, 2H, aromatic),
7.29–7.51 (m, 10H, aromatic, and NH), 8.08 (s, 1H, H-6)
ppm. Anal. (C23H21ClN4O2S2) C, H, N.
4.1.3. 2-Carbamolymethylthio-4-chloro-5-methyl-N-(5,6-
diphenyl-1,2,4-triazin-3-yl)benzenesulfonamide (7)
A stirred solution of 3-amino-2-(2-carbamoylmethylthio-
4-chloro-5-methylbenzenesulfonyl)guanidine 2e (0.70 g,
2 mmol) and benzil (0.43 g, 2 mmol) in dry DMSO (10 ml)
was heated at 80–85 °C for 30 h. The reaction mixture was
diluted with methanol (80 ml) then quenched with water
(75 ml) and kept overnight in a refrigerator. The precipitate
that deposited was filtered off, washed with methanol (3 ×
1 ml) and dried. The product was purified by crystallization
from ethanol to give 7 as white crystals (0.37 g, 35%); m.p.
206–208 °C. IR (KBr) 3447, 3343 (NH2, NH), 2974, 2921,
2855 (CH3, CH2), 1669 (C=O), 1334, 1160 (SO2) cm–1; 1H
NMR (DMSO-d6) d 2.25 (s, 3H, CH3), 3.74 (s, 2H, SCH2),
7.17–7.19 (m, 2H, aromatic), 7.21 (s, 1H, HaN–C=O), 7.31–
7.35 (m, 7H, aromatic and NH), 7.38–7.41 (m, 1H, aro-
matic), 7.45–7.48 (m, 1H, aromatic), 7.55 (s, 1H, H-3), 7.59
(s, 1H, HbN–C=O), 8.06 (s, 1H, H-6) ppm. Anal.
(C24H20ClN5O3S2) C, H, N.
4.1.2.1. 4-Chloro-2-ethoxycarbonylmethylthio-5-methyl-N-
(5,6-diphenyl-1,2,4-triazin-3-yl)benzenesulfonamide
(3). Yield: 0.64 g, 58%, m.p. 170–172 °C dec.; IR (KBr) 3198
(NH), 2976, 2925, 2848 (CH3, CH2), 1726 (C=O), 1343, 1147
1
(SO2) cm–1; H NMR (CDCl3) d 1.14 (t, J = 7.2 Hz, 3H,
CH3), 2.30 (s, 3H, CH3-Ar), 3.74 (s, 2H, SCH2), 4.06 (m,
J = 7.2 Hz, 2H, OCH2), 7.26–7.48 (m, 11H, aromatic and
NH), 7.53 (s, 1H, H-3), 8.24 (s, 1H, H-6) ppm; 13C NMR
(CDCl3) d 14.54 (CH3), 20.0 (CH3-Ar), 37.53 (SCH2), 62.39
(OCH2), 128.75, 128.89, 129.16, 129.74, 130.06, 130.58,
131.95, 132.39, 134.12, 135.18, 135.25, 135.56, 137.72,
140.29, 153.00, 155.52, 159.00 (17C, aromatic), 169.32
(C=O) ppm; EIMS: m/z (%) 555 (M+, 1), 554 (M – 1, 3), 435
(13), 405 (12), 403 (30), 391 (18), 371 (23), 179 (15), 178
(100), 165 (17). Anal. (C26H23ClN4O4S2) C, H, N.
4.1.2.2. 2-Benzylthio-4-chloro-5-methyl-N-(5,6-diphenyl-
1,2,4-triazin-3-yl)benzenesulfonamide (4). Yield: 0.69 g, 62%,
m.p. 204–205 °C dec.; IR (KBr) 3448 (NH), 2913, 2855 (CH3,
CH2), 1349, 1167 (SO2) cm–1; 1H NMR (CDCl3) d 2.28 (s,
2H, CH3), 4.16 (s, 2H, SCH2), 7.05–7.11 (m, 3H, aromatic),
7.24–7.28 (m, 2H, aromatic), 7.29–7.36 (m, 6H, aromatic and
NH), 7.38–7.41 (m, 5H, aromatic), 7.42–7.47 (m, 1H, aro-
matic) 8.21 (s, 1H, H-6) ppm; EIMS: m/z (%) 560 (M + 1, 7),
559 (M+, 5), 558 (M – 1, 14), 494 (11), 467 (12), 435 (10),
405 (22), 404 (15), 403 (59), 337 (37), 249 (28), 245 (29),
179 (17), 178 (100), 165 (23), 91 (65). Anal.
(C29H23ClN4O2S2) C, H, N.
4.1.4. 5-Carbamoyl-4-chloro-2-ethoxycarbonylmethylthio-
N-(5,6-diphenyl-1,2,4-triazin-3-yl)benzenesulfonamide (8)
A
solution of 3-amino-2-(5-carbamoyl-4-chloro-2-
ethoxycarbonylmethylthiobenzenesulfonyl)guanidine 2f
(0.92 g, 2 mmol) and benzil (0.43 g, 2 mmol) in dry DMSO
(10 ml) was stirred at 80–85 °C for 8 h. The resulting mixture
was diluted with methanol (70 ml) then quenched with water
(30 ml) and left overnight in a refrigerator. The solid that pre-
cipitated was filtered off, washed with methanol (3 × 1 ml),
dried and crystallized from ethanol to give 8 as white crystals
(0.68 g, 58%); m.p. 266–269 °C dec. IR (KBr) 3466, 3331
(NH2, NH), 2978, 2925, 2855 (CH3, CH2), 1728 (C=O, ester),
4.1.2.3. 2-Benzylthio-4-chloro-5-methyl-N-[5,6-di(2-furyl)-
1,2,4-triazin-3-yl]benzenesulfonamide (5). Yield: 0.54 g,
50%, m.p. 200–203 °C dec.; IR (KBr) 3448 (NH), 2913, 2855
(CH3, CH2), 1331, 1152 (SO2) cm–1; 1H NMR (DMSO-d6)
d 2.38 (s, 3H, CH3), 4.32 (s, 2H, SCH2), 6.50–6.52 (d, 1H,
aromatic), 6.72–6.74 (m, 2H, aromatic), 6.92–6.94 (d, 1H,
aromatic), 7.09–7.17 (m, 3H, aromatic), 7.28–7.33 (m, 3H,
aromatic and NH), 7.56 (s, 1H, H-3), 7.91 (s, 1H, aromatic),
1
1699 (C=O, amide), 1331, 1152 (SO2) cm–1; H NMR
(DMSO-d6) d 1.02 (t, J = 6.8 Hz, 3H, CH3), 3.94–3.96 (q,
J = 6.8 Hz, 2H, CH2), 4.13 (s, 2H, SCH2), 7.06–7.08 (m, 2H,
aromatic), 7.30–7.45 (m, 9H, aromatic and NH), 7.55 (s, 1H,
H-3), 7.72 (s, 1H, HaN–C=O), 8.02 (s, 1H, HbN–C=O), 8.14
(s, 1H, H-6) ppm. Anal. (C26H22ClN5O5S2) C, H, N.

386
J. Sławin´ski, M. Gdaniec / European Journal of Medicinal Chemistry 40 (2005) 377–389
4.1.5. Preparation of 2-benzylthio-5-(carbamoyl or
phenylcarbamoyl)-4-chloro-N-(5,6-diaryl-1,2,4-triazin-3-
yl)benzenesulfonamides (9–12)
4.1.6. Preparation of 2-benzylthio-4-chloro-5-[(4-chloro
or 4-methyl)phenylcarbamoyl]-N-(5,6-diphenyl-1,2,4-tri-
azin-3-yl)benzenesulfonamides (13–14)
A stirred suspension of the corresponding 3-amino-2-(2-
benzylthio-5-carbamoyl-4-chlorobenzenesulfonyl)gua-
nidine 2g or 2h (2 mmol) and the appropriate 1,2-
diarylethane-1,2-dione (2 mmol) in a mixture prepared from
ethanol (20 ml) and dioxane (10 ml) was refluxed for 12–15 h
and kept overnight at room temperature. The precipitate that
deposited was filtered off, washed with ethanol (2 × 1 ml)
and dried. The product was purified by crystallization from a
mixture of ethanol and dioxane (2:1).
A stirred solution of the corresponding 3-amino-2-(2-
benzylthio-4-chloro-5-[(4-chloro or 4-methyl)phenyl-
carbamoyl]benzenesulfonyl)guanidine 2i or 2j (2 mmol) and
benzil (0.43 g, 2 mmol) in dry DMSO (10 ml) was heated at
80–85 °C for 20 h. The reaction mixture was diluted with
methanol (100 ml) and kept overnight in a refrigerator. The
product that precipitated was collected by filtration, washed
with methanol (3 × 2 ml) and dried. The contaminations were
extracted with boiling ethanol (40 ml per 1 g of crude reac-
tion product) to afford pure product.
In this manner, the following benzenesulfonamides were
obtained.
In this manner, the following compounds were obtained.
4.1.5.1. 2-Benzylthio-5-carbamoyl-4-chloro-N-(5,6-diphenyl-
1,2,4-triazin-3-yl)benzenesulfonamide (9). Yield: 0.87 g, 74%,
m.p. 293–294 °C dec.; IR (KBr) 3436, 3360 (NH2, NH), 2855
4.1.6.1. 2-Benzylthio-4-chloro-5-(4-chlorophenylcarbamoyl)-
N-(5,6-diphenyl-1,2,4-triazin-3-yl)benzenesulfonamide
(13). Yield: 0.71 g, 51%, m.p. 260–262 °C dec.; IR (KBr)
3388, 3324 (NH), 2966, 2919, 2854 (CH2), 1669 (C=O), 1305,
1163 (SO2) cm–1; 1H NMR (DMSO-d6) d 4.44 (s, 2H, SCH2),
7.13–7.26 (m, 7H, aromatic), 7.32–7.45 (m, 11H, aromatic
and NH), 7.73–7.75 (m, 3H, aromatic), 8.18 (s, 1H, H-6),
10.72 (s, 1H, HNC=O) ppm.Anal. (C35H25Cl2N5O3S2) C, H,
N.
1
(CH2), 1646 (C=O), 1342, 1163 (SO2) cm–1; H NMR
(DMSO-d6) d 4.41 (s, 2H, SCH2), 7.11–7.20 (m, 5H, aro-
matic), 7.31–7.45 (m, 10H, aromatic and NH), 7.65 (s, 1H,
H-3), 7.72 (s, 1H, HaNC=O), 7.99 (s, 1H, HbNC=O), 8.12 (s,
1H, H-6) ppm. Anal. (C29H22ClN5O3S2) C, H, N.
4.1.5.2. 2-Benzylthio-5-carbamoyl-4-chloro-N-[5,6-di(2-
furyl)-1,2,4-triazin-3-yl]benzenesulfonamide (10). Yield:
0.59 g, 52%, m.p. 297–299 °C dec.; IR (KBr) 3442, 3354
(NH2, NH), 2925, 2855 (CH2), 1646 (C=O), 1361, 1161 (SO2)
cm–1; 1H NMR (DMSO-d6) d 4.37 (s, 2H, SCH2), 6.42–6.43
(d, 1H, aromatic), 6.67–6.67 (m, 1H, aromatic), 6.73–6.74
(m, 1H, aromatic), 6.91–6.92 (d, 2H, aromatic), 7.11–7.15
(m, 4H, aromatic and NH), 7.31–7.33 (d, 2H, aromatic), 7.59
(s, 1H, H-3), 7.72 (s, 1H, HaNC=O), 7.91 (s, 1H, aromatic),
7.97 (s, 1H, aromatic), 8.04 (s, 1H, HbNC=O), 8.24 (s, 1H,
H-6) ppm. Anal. (C25H18ClN5O5S2) C, H, N.
4.1.6.2. 2-Benzylthio-4-chloro-5-(4-methylphenylcarbamoyl)-
N-(5,6-diphenyl-1,2,4-triazin-3-yl)benzenesulfonamide
(14). Yield: 0.77 g, 57%, m.p. 240–241 °C dec.; IR (KBr)
3389, 3354 (NH), 2919, 2855 (CH3, CH2), 1646 (C=O), 1314,
1164 (SO2) cm–1; 1H NMR (DMSO-d6) d 2.28 (s, 3H, CH3),
4.42 (s, 2H, SCH2), 7.12–7.26 (m, 9H, aromatic), 7.31–7.42
(m, 9H, aromatic and NH), 7.57–7.59 (d, 2H, aromatic), 7.70
(s, 1H, H-3), 8.15 (s, 1H, H-6), 10.53 (s, 1H, HNC=O) ppm.
Anal. (C36H28ClN5O3S2) C, H, N.
4.1.5.3. 2-Benzylthio-5-carbamoyl-4-chloro-N-[5,6-di(4-
methoxyphenyl)-1,2,4-triazin-3-yl]benzenesulfonamide
(11). Yield: 0.60 g, 46%, m.p. 275–277 °C dec.; IR (KBr)
3366, 3319 (NH2, NH), 2966, 2931, 2873 (CH3, CH2), 1661
(C=O), 1361, 1176 (SO2) cm–1; 1H NMR (DMSO-d6) d 3.77
(s, 3H, OCH3), 3.79 (s, 3H, OCH3), 4.37 (s, 2H, SCH2), 6.89
(d, J = 8.8 Hz, 2H, aromatic), 6.95 (d, J = 8.8 Hz, 2H, aro-
matic), 7.09 (d, J = 8.8 Hz, 2H, aromatic), 7.11–7.18 (m, 4H,
aromatic and NH), 7.27 (d, J = 8.8 Hz, 2H, aromatic), 7.33–
7.35 (m, 2H, aromatic), 7.61 (s, 1H, H-3), 7.74 (s, 1H,
HaNC=O), 8.01 (s, 1H, HbC=O), 8.12 (s, 1H, H-6) ppm.Anal.
(C31H26ClN5O5S2) C, H, N.
4.1.7. 2-Benzylthio-4-chloro-5-cyano-N-(5,6-diphenyl-
1,2,4-triazin-3-yl)benzenesulfonamide (15)
A suspension of 9 (0.74 g, 1.25 mmol) in phosphorus oxy-
chloride (12 ml) was stirred at first at room temperature for
15 min, and then the temperature was raised to 60 °C for
2.5 h. The suspension was further stirred at 65–70 °C for 8 h
and allowed to stand at room temperature overnight. The
resulting solution was poured onto crushed ice (130 g) with
vigorously stirring for at least 1 h. The solid that precipitated
was collected by filtration, washed thoroughly with several
portions of cold water (pH 7), dried and crystallized from a
mixture of ethanol and dioxane (4:1) to give 15 as white crys-
tals (0.65 g, 85%); m.p. 232–234 °C. IR (KBr) 3441 (NH),
2966, 2927, 2856, 2771 (CH2), 2234 (C≡N), 1345, 1163 (SO2)
4.1.5.4. 2-Benzylthio-4-chloro-5-phenylcarbamoyl-N-(5,6-
diphenyl-1,2,4-triazin-3-yl)benzenesulfonamide (12). Yield:
0.64 g, 48%, m.p. 218–221 °C dec.; IR (KBr) 3342,
3311(NH), 2966, 2923, 2857 (CH2), 1662 (C=O), 1334, 1165
(SO2) cm–1; 1H NMR (DMSO-d6) d 4.44 (s, 2H, SCH2), 7.13–
7.26 (m, 8H, aromatic), 7.32–7.42 (m, 11H, aromatic and
NH), 7.71–7.70 (m, 3H, aromatic), 8.18 (s, 1H, H-6), 10.58
(s, 1H, HNC=O) ppm. Anal. (C35H26ClN5O3S2) C, H, N.
1
cm–1; H NMR (DMSO-d6) d 3.55 (s, 4H, CH2, dioxane),
4.45 (s, 2H, SCH2), 7.06–7.08 (m, 2H, aromatic), 7.17–7.24
(m, 3H, aromatic), 7.29–7.36 (m, 9H, aromatic and NH),
7.39–7.43 (m, 1H, aromatic), 7.48–7.51 (m, 1H, aromatic),
7.84 (s, 1H, H-3), 8.34 (s, 1H, H-6) ppm. Anal.
(C29H20ClN5O2S2·1/2 C4H8O2) C, H, N.

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387
4.1.8. General procedure for the preparation of 1-(2-alky-
lthio-4-chloro-5-R1-benzenesulfonyl)-3-(2-oxobutane-3-
ylideneimino)guanidines (16–18)
128.11, 128.73, 128.85, 129.09, 129.28, 132.59, 133.88,
136.11, 138.78, 138.93, 140.78, 149.18, 155.39, 163.74
(NHC=O), 197.33 (C=O) ppm. Anal. (C25H23ClN5O4S2) C,
H, N.
To a stirred suspension of the corresponding 3-amino-2-
(2-alkylthio-4-chloro-5-R1-benzenesulfonyl)guanidine 2b, d
or 2h (2 mmol) and ethanol (10 ml) diacetyl (2 mmol) was
added. The resulting mixture was refluxed for 4–5 h and left
overnight at room temperature. The solid that precipitated was
collected by filtration, washed with ethanol (2 × 1 ml), dried
and purified either by crystallization from ethanol or from a
mixture of ethanol and chloroform (2:1) for 18.
4.1.9. General procedure for the preparation of 2-alkyl-
thio-4-chloro-5-methyl-N-(1,2-dihydroxycyclobuta[e]1,2,4-
triazin-3-yl)benzenesulfonamides (19–21)
To a suspension of the corresponding 3-amino-2-(2-
alkylthio-4-chloro-5-methylbenzenesulfonyl)guanidine 2a, b
or 2d (2 mmol) in ethanol (10 ml) 3,4-dihydroxy-3-
cyclobutene-1,2-dione (2 mmol) or 3,4-diethoxy-3-cyclo-
butene-1,2-dione (2 mmol) was added. The reaction mixture
was stirred at reflux for 1.5–2 h, and then left overnight at
room temperature. The product thus obtained was collected
by filtration, washed with ethanol (2 × 1 ml) and dried. The
contaminations were extracted with boiling ethanol (15 ml
per 1 g of crude reaction product).
According to the above procedure, the following ami-
noguanidines were obtained.
4.1.8.1. 1-(2-Benzylthio-4-chloro-5-methybenzenesulfonyl)-
3-(2-oxobutane-3-ylideneimino)guanidine (16). Starting from
3-amino-2-(2-benzylthio-4-chloro-5-methylbenzenesul-
fonyl)guanidine 2b (0.77 g) and diacetyl (0.17 g), the title
compound 16 was obtained (0.59 g, 65%); m.p. 160–162 °C.
IR (KBr) 3455, 3341, 3297 (NH), 2948, 2921, 2854 (CH3,
CH2), 1691 (C=O), 1627 (C=N), 1300, 1135 (SO2) cm–1; 1H
NMR (CDCl3) d 1.93 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.39
(s, 3H, CH3), 4.15 (s, 2H, SCH2), 6.45 (br.s, 1H, C=NH),
7.05 (br.s, 1H, NH–N=C), 7.21–7.29 (m, 5H, aromatic), 7.31
(s, 1H, H-3), 7.98 (s, 1H, H-6), 9.02 (br.s, 1H, SO2NH) ppm.
Anal. (C19H21ClN4O3S2) C, H, N.
According to the above procedure, the following com-
pounds were obtained.
4.1.9.1. 4-Chloro-2-ethoxycarbonylmethylthio-5-methyl-N-
(1,2-dihydroxycyclobuta[e]1,2,4-triazin-3-yl)benzenesulfona-
mide (19). Starting from 3-amino-2-(4-chloro-2-ethoxy-
carbonylmethylthio-5-methylbenzenesulfonyl)guanidine 2a
(0.76 g) and 3.4-dihydroxy-3-cyclobutene-1,2-dione (0.23 g),
the title compound 19 was obtained (0.31 g, 34%); m.p. 201–
202 °C dec. IR (KBr) 3385, 3312, 3189, 3114 (OH, NH),
2981, 2931 (CH3, CH2), 1718 (C=O), 1618, 1558 (C=N),
4.1.8.2. 1-(4-Chloro-5-methyl-2-methylthiobenzenesulfonyl)-
3-(2-oxobutane-3-ylideneimino)guanidine (17). Starting from
3-amino-2-(4-chloro-5-methyl-2-methylthiobenzenesul-
fonyl)guanidine 2d (0.62 g) and diacetyl (0.17 g), the title
compound 17 was obtained (0.67 g, 89%); m.p. 190–191 °C
dec. IR (KBr) 3454, 3342, 3306 (NH), 2923, 2854 (CH3,
CH2), 1685 (C=O), 1643, 1610 (C=N), 1300, 1135 (SO2)
1
1343, 1134 (SO2) cm–1; H NMR (200 MHz, DMSO-d6) d
1.19 (t, J = 7.0 Hz, 3H, CH3), 2.34 (s, 3H, CH3-Ar), 3.95 (s,
2H, SCH2), 4.14 (q, J = 7.0 Hz, 2H, OCH2), 7.20–7.60 (br.s,
2H, 2 × OH), 7.45 (s, 1H, H-3), 7.89 (s, 1H, H-6), 9.83 (s,
1H, SO2NH) ppm; 13C NMR (50 MHz, DMSO-d6) d 14.26
(CH3), 19.12 (CH3-Ar), 34.99 (SCH2), 61.38 (OCH2), 128.43,
128.65, 130.59, 132.98, 133.20, 134.55, 136.69, 140.16,
157.59, 169.49 (C=O) ppm. Anal. (C16H15ClN4O6S2) C, H,
N.
1
cm–1; H NMR (CDCl3) d 2.05 (s, 3H, CH3), 2.39 (s, 3H,
CH3), 2.45 (s, 3H, CH3), 2.55 (s, 3H, CH3), 6.58 (br.s, 1H,
C=NH), 7.22–7.45 (s, 2H, aromatic), 7.99 (s, 1H, NH), 9.33
(br.s, 1H, SO2NH) ppm; 13C NMR (DMSO-d6) d 10.39
(CH3C=N), 15.51 (SCH3), 19.12 (CH3-Ar), 24.98 (CH3C=O),
126.18, 130.77, 131.54, 137.51, 137.92, 138.37, 149.02,
155.29, 197.32 (C=O) ppm. Anal. (C13H17ClN4O3S2) C, H,
N.
4.1.9.2. 2-Benzylthio-4-chloro-5-methyl-N-(1,2-dihydroxy-
cyclobuta[e]1,2,4-triazin-3-yl)benzenesulfonamide
(20).
Starting from 3-amino-2-(2-benzylthio-4-chloro-5-methyl-
benzenesulfonyl)guanidine 2b (0.77 g) and 3.4-dihydroxy-3-
cyclobutene-1,2-dione (0.23 g), the title compound 20 was
obtained (0.39 g, 42%); m.p. 203–204 °C dec. IR (KBr) 3376,
3307, 3189, 3110 (OH, NH), 2966, 2921, 2860 (CH3, CH2),
1618, 1553 (C=N), 1343, 1133 (SO2) cm–1; 1H NMR
(200 MHz, DMSO-d6) d 2.32 (s, 3H, CH3), 4.31 (s, 2H,
SCH2), 7.25–7.75 (m, 8H, aromatic and 2 × OH), 7.87 (s,
1H, H-6), 9.85 (s, 1H, SO2NH) ppm; 13C NMR (50 MHz,
DMSO-d6) d 20.68 (CH3), 38.17 (CH2), 129.03, 129.45,
129.78, 129.94, 130.28, 130.63, 130.98, 132.08, 133.75,
137.40, 138.06, 138.20, 141.23, 159.16, 166.96 ppm. Anal.
(C19H15ClN4O4S2) C, H, N.
4.1.8.3. 1-(2-Benzylthio-4-chloro-5-phenylcarbamoylbenze-
nesulfonyl)-3-(2-oxobutane-3-ylideneimino)guanidine (18).
Starting from 3-amino-2-(2-benzylthio-4-chloro-5-phenylcar-
bamoylbenzenesulfonyl)guanidine 2h (0.98 g) and diacetyl
(0.17 g), the title compound 18 was obtained (0.84 g, 75%);
m.p. 137–139 °C. IR (KBr) 3481, 3348, 3280 (NH), 2923,
2854 (CH3,CH2), 1676, 1657 (C=O), 1629, 1598 (C=N),
1
1316, 1129 (SO2) cm–1; H NMR (CDCl3) d 1.93 (s, 3H,
CH3), 2.35 (s, 3H, CH3), 4.18 (s, 2H, SCH2), 6.55 (s, 1H,
C=NH), 7.15–7.32 (m, 10H, aromatic), 7.62 (s, 2H, aro-
matic), 8.25 (s, 1H, NH), 8.33 (s, 1H, NHC=O), 9.16 (s, 1H,
SO2NH) ppm; 13C NMR (DMSO-d6) d 10.37 (CH3C=N),
26.01 (CH3C=O), 36.05 (SCH2), 119.92, 124.29, 127.66,
Starting from 2b (0.77 g) and 3.4-diethoxy-3-cyclobutene-
1,2-dione (0.34 g), the title compound 20 was obtained (0.19 g,

388
J. Sławin´ski, M. Gdaniec / European Journal of Medicinal Chemistry 40 (2005) 377–389
21%); m.p. 202–204 °C dec. IR and 1H NMR were identical
with authentic sample 20.
I > 2r(I) and 473 refined parameters are: R1 = 0.0461,
wR2 = 0.1056 (R1 = 0.0933, wR2 = 0.1260 for all 6148 data).
Atom labeling is shown in Fig. 4. The DMSO molecule is
disordered over two positions with the ratio of occupancy fac-
tors being 82:18.
4.1.9.3. 4-Chloro-5-methyl-2-methylthio-N-(1,2-dihydroxy-
cyclobuta[e]1,2,4-triazin-3-yl)benzenesulfonamide
(21).
Crystallographic data for compounds 4, 10 and 11 have
been deposited with Cambridge Crystallographic Data Cen-
ter (CCDC deposition numbers CCDC 244778–244780).
Copies of the data can be obtained upon request from CCDC,
12 Union Road, Cambridge CB2 1EZ, UK, quoting the depo-
sition numbers.
Starting from 3-amino-2-(4-chloro-5-methyl-2-methylthio-
benzenesulfonyl)guanidine 2d (0.62 g) and 3.4-dihydroxy-3-
cyclobutene-1,2-dione (0.23 g), the title compound 21 was
obtained (0.29 g, 37%); m.p. 215–217 °C dec. IR (KBr) 3375,
3312, 3190, 3115 (OH, NH), 2978, 2922 (CH3), 1618, 1559
(C=N), 1343, 1134 (SO2) cm–1; 1H NMR (200 MHz, DMSO-
d6) d 2.33 (s, 3H, CH3), 2.46 (s, 3H, SCH3), 7.25–7.55 (br.s,
2H, 2 × OH), 7.31 (s, 1H, H-3), 7.86 (s, 1H, H-6), 9.76 (s,
1H, SO2NH) ppm. Anal. (C13H11ClN4O4S2) C, H, N.
Acknowledgements
The authors are very grateful to Dr. V.L. Narayanan, Chief
of Drug Synthesis, Chemistry Branch, National Cancer Insti-
tute (Bethesda, MD) for the in vitro screening.
4.2. X-ray structure analyses
The diffraction data were collected at room temperature
with a KumaCCD diffractometer using graphite monochro-
mated Mo Ka radiation. The intensity data were collected and
processed using Oxford Diffraction CrysAlis Software [34].
The structures were solved by direct methods with the pro-
gram SHELXS-97 [35] and refined by full-matrix least-
squares method on F2 with SHELXL-97 [36].
Crystal data for 4: C29H23ClN4O2S2, triclinic, space group
P1, a = 9.6195(8), b = 11.7985(9), c = 12.5981(10) Å,
␣ = 92.024(6), b = 94.633(6), c = 108.828(7)°,
V = 1346.04(19) Å3, Z = 2, dx = 1.379 g cm–3, T = 293 K.
10258 data were collected up to 2hmax = 50° for a crystal
with dimensions 0.6 × 0.2 × 0.02 mm3 (Rint = 0.043,
Rr = 0.062). Final R indices for 2944 reflections with
I > 2r(I) and 389 refined parameters are: R1 = 0.0534,
wR2 = 0.1398 (R1 = 0.0848, wR2 = 0.1530 for all 4725 data).
Atom labeling is shown in Fig. 2. The thiomethylene frag-
ment of the molecule is disordered and adopts two different
conformations with the occupancy ratio 78:22. The hydro-
gen atoms of the methyl group C29 are also disordered.
Crystal data for 10: C25H18ClN5O5S2·C2H6OS, triclinic,
References
[1] A. Casini, A. Scozzafava, A. Mastrolorenzo, C.T. Supuran, Curr.
Cancer Drug Targets 2 (2002) 55–75.
[2] A. Scozzafava, T. Owa, A. Mastrolorenzo, C.T. Supuran, Curr. Med.
Chem. 10 (2003) 925–953.
[3] A. Scozzafava, A. Casini, C.T. Supuran, Curr. Med. Chem. 9 (2002)
1167–1185.
[4] C.T. Supuran, A. Casini, A. Scozzafava, Med. Res. Rev. 23 (2003)
535–558.
[5] Z. Brzozowski, F. STczewski, J. Med. Chem. 45 (2002) 430–437.
[6] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 52 (1995) 91–101.
[7] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 53 (1996) 269–276.
[8] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 52 (1995) 287–292.
[9] E. Pomarnacka, Acta Polon. Pharm. Drug Res. 53 (1996) 373–378.
[10] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 54 (1997) 49–53.
[11] E. Pomarnacka, Z. Brzozowski, Acta Polon. Pharm. Drug Res. 54
(1997) 215–221.
[12] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 54 (1997) 293–298.
[13] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 55 (1998) 233–238.
[14] E. Pomarnacka,A. Kornicka,Acta Polon. Pharm. Drug Res. 55 (1998)
297–304.
space group P1,
a = 9.3236(5), b = 10.5164(5),
[15] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 55 (1998) 375–379.
[16] E. Pomarnacka, Acta Polon. Pharm. Drug Res. 55 (1998) 481–486.
[17] Z. Brzozowski, A. Kornicka, Acta Polon. Pharm. Drug Res. 56 (1999)
135–142.
c = 15.6016(8) Å, ␣ = 72.448(4), b = 88.733(4),
c = 81.883(4)°, V = 1443.58(13) Å3, Z = 2, dx = 1.487 g cm–3,
µ(Mo Ka) = 0.401 mm–1 T = 293 K. 13017 data were col-
lected up to 2hmax = 50° for a crystal with dimensions 0.4 ×
0.4 × 0.05 mm3 (Rint = 0.034, Rr = 0.027). Final R indices for
4562 reflections with I > 2r(I) and 406 refined parameters
are: R1 = 0.0462, wR2 = 0.1173 (R1 = 0.0516, wR2 = 0.1216 for
all 5028 data). Atom labeling is shown in Fig. 3. The DMSO
molecule is disordered over two positions with the ratio of
occupancy factors being 58:42.
Crystal data for 11: C31H26ClN5O5S2·C2H6OS, mono-
clinic, space group P21/n, a = 17.7172(8), b = 9.4902(5),
c = 20.7410(10) Å, b = 90.498(4)°, V = 3487.3(3) Å3, Z = 4,
dx = 1.383 g cm–3, µ(Mo Ka) = 0.266 mm–1, T = 293 K.
17140 data were collected up to 2hmax = 50° for a crystal
with dimensions 0.7 × 0.3 × 0.02 mm3 (Rint = 0.051,
Rr = 0.064). Final R indices for 3579 reflections with
[18] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 55 (1998) 49–56.
[19] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 55 (1998) 473–480.
[20] E. Pomarnacka, A. Kornicka, Farmaco 56 (2001) 571–577.
[21] N. Neamati, A. Mazumder, S. Sunder, J.M. Owen, R.J. Schultz,
Y. Pommier, Antiviral Chem. Chemother. 8 (1997) 485–495.
[22] L.C. Kuo, H. Assefa, S. Kamath, Z. Brzozowski, J. Sławin´ski,
F. STczewski, et al., J. Med. Chem. 47 (2004) 385–399.
[23] J. Sławin´ski, Eur. J. Med. Chem. 39 (2004) 179–188.
[24] J. Sławin´ski, P. Bednarski, R. Grünert, P. Reszka, Pol. J. Chem. 77
(2003) 53–64.
[25] J. Sławin´ski, P. Bednarski, P. Reszka, Pol. J. Chem. 78 (2004) 369–
379.
[26] Z. Brzozowski, F. STczewski, M. Gdaniec, Eur. J. Med. Chem. 37
(2002) 285–293.
[27] J. Sławin´ski, Pol. J. Chem. 75 (2001) 1309–1316.
[28] G. Maahs, P. Hegenberg, Angew. Chem. 78 (1966) 927–931.

J. Sławin´ski, M. Gdaniec / European Journal of Medicinal Chemistry 40 (2005) 377–389
389
[29] W.A. Kinney, N.E. Lee, D.T. Garrison, E.J. Podlesny Jr., J.T. Sim-
monds, D. Bramlett, et al., J. Med. Chem. 35 (1992) 4720–4726.
[34] Oxford Diffraction, Crysalis CCD and RED, ver. 1.169, Oxford Dif-
fraction, Oxfordshire, UK, 2002.
[35] G.M. Sheldrick, SHELXS-97: Program for a Crystal Structure Solu-
tion, University of Göttingen, Germany, 1997.
[30] J.A. Butera, M.M. Antane, S.A. Antane, T.M. Argentieri, C. Freeden,
R.F. Graceffa, et al., J. Med. Chem. 43 (2000) 1187–1202.
[36] G.M. Sheldrick, SHELXL-97: Program for the Refinement of a Crys-
tal Structure from Diffraction Data, University of Göttingen, Ger-
many, 1997.
[37] Siemens, Stereochemical Workstation Operation Manual, Release
3.4, SiemensAnalytical X-Ray Instruments, Inc., Madison, WI, USA,
1989.
[31] M.R. Boyd, Am. Assoc. Cancer Res. 30 (1989) 652–663.
[32] A.P. Monks, D.A. Scudiero, P. Skehan, K.D. Shoemaker, D. Poull,
C. Vistica, et al., J. Natl. Cancer Inst. 83 (1991) 757–766.
[33] J.N. Weinstein, T.G. Myers, P.M. O’Connor, S.H. Friend, A.J. For-
nance Jr., K.W. Kohn, et al., Science 275 (1997) 343–349.