Novel 5-substituted 2-(aylmethylthio)-4-chloro-N-(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamides: Synthesis, molecular structure, anticancer activity, apoptosis-inducing activity and metabolic stability

Article abstract of DOI:10.3390/molecules21060808

A series of novel 5-substituted 2-(arylmethylthio)-4-chloro-N-(5-aryl-1,2,4-triazin-3-yl) benzenesulfonamide derivatives 27-60 have been synthesized by the reaction of aminoguanidines with an appropriate phenylglyoxal hydrate in glacial acetic acid. A maj

Full text of DOI:10.3390/molecules21060808

molecules
Article
Novel 5-Substituted 2-(Aylmethylthio)-4-chloro-
N-(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamides:
Synthesis, Molecular Structure, Anticancer Activity,
Apoptosis-Inducing Activity and Metabolic Stability
Beata Zołnowska 1,*, Jarosław Sławin´ski 1,*, Aneta Pogorzelska 1, Krzysztof Szafran´ski 1,
˙
4
Anna Kawiak 2,3, Grzegorz Stasiłojc´ , Mariusz Belka 5, Szymon Ulenberg 5, Tomasz Ba˛czek 5
and Jarosław Chojnacki 6
1
Department of Organic Chemistry, Medical University of Gdan´sk, Al. Gen. J. Hallera 107,
Gdan´sk 80-416, Poland; anetapogorzelska@gumed.edu.pl (A.P.); k.szafranski@gumed.edu.pl (K.S.)
Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdan´sk and Medical
2
University of Gdan´sk, ul. Abrahama 58, Gdan´sk 80-307, Poland; kawiak@biotech.ug.edu.pl
Laboratory of Human Physiology, Medical University of Gdan´sk, ul. Tuwima 15, Gdan´sk 80-210, Poland
Laboratory of Cell Biology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology
3
4
UG-MUG, Medical University of Gdan´sk, ul. De˛binki 1, Gdan´sk 80-211, Poland; gstasilojc@gumed.edu.pl
Department of Pharmaceutical Chemistry, Medical University of Gdan´sk, Al. Gen. J. Hallera 107,
5
Gdan´sk 80-416, Poland; mariusz.belka@gumed.edu.pl (M.B.); szymon.ulenberg@gumed.edu.pl (S.U.);
tbaczek@gumed.edu.pl (T.B.)
Department of Inorganic Chemistry, Gdan´sk University of Technology, Narutowicza 11/12,
6
Gdan´sk 80-233, Poland; jarekch@pg.gda.pl
˙
*
Correspondence: zolnowska@gumed.edu.pl (B.Z.); jaroslaw@gumed.edu.pl (J.S.);
˙
˙
Tel.: +48-58-349-1098 (B.Z.); +48-58-349-1229 (J.S.); Fax: +48-58-349-1277 (B.Z. & J.S.)
Academic Editors: Jean Jacques Vanden Eynde and Annie Mayence
Received: 13 May 2016; Accepted: 17 June 2016; Published: 22 June 2016
Abstract: A series of novel 5-substituted 2-(arylmethylthio)-4-chloro-N-(5-aryl-1,2,4-triazin-3-yl)
benzenesulfonamide derivatives 27–60 have been synthesized by the reaction of aminoguanidines
with an appropriate phenylglyoxal hydrate in glacial acetic acid. A majority of the compounds
showed cytotoxic activity toward the human cancer cell lines HCT-116, HeLa and MCF-7, with IC50
values below 100 µM. It was found that for the analogues 36–38 the naphthyl moiety contributed
significantly to the anticancer activity. Cytometric analysis of translocation of phosphatidylserine as
well as mitochondrial membrane potential and cell cycle revealed that the most active compounds 37
(HCT-116 and HeLa) and 46 (MCF-7) inhibited the proliferation of cells by increasing the number
of apoptotic cells. Apoptotic-like, dose dependent changes in morphology of cell lines were also
noticed after treatment with 37 and 46. Moreover, triazines 37 and 46 induced caspase activity in
the HCT-116, HeLa and MCF-7 cell lines. Selected compounds were tested for metabolic stability in
the presence of pooled human liver microsomes and NADPH, both R2 and Ar = 4-CF3-C6H4 moiety
in 2-(R2-methylthio)-N-(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamides simultaneously increased
metabolic stability. The results pointed to 37 as a hit compound with a good cytotoxicity against
HCT-116 (IC50 = 36
metabolic stability.
µM), HeLa (IC50 = 34 µM) cell lines, apoptosis-inducing activity and moderate
Keywords: sulfonamide; synthesis; metabolic stability; anticancer; apoptosis; mitochondrial
membrane potential
Molecules 2016, 21, 808; doi:10.3390/molecules21060808
www.mdpi.com/journal/molecules

Molecules 2016, 21, 808
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1. Introduction
For decades chemotherapeutics have played the most important role in the fight against cancer.
Unfortunately, the serious toxicities of conventional cytotoxic medicines have impelled and are still
impelling researchers to focus on the development of new potent and selective anticancer drugs.
Heterocyclic scaffolds, especially nitrogen-containing heterocyclic compounds, play an important
role in the design of novel drugs because of their utility for various biological receptors with a
high degree of binding affinity. Among the heterocycles, the triazines, with their wide biological
profile, occupy a prominent position [
heterocycles in Nature, and it has been reported to possess a broad spectrum of biological properties,
including anticonvulsant [ ], neuroprotective [ ], sedative [ ], anxiolytic [ ], benzodiazepine receptor
inhibitory activity [ ], antiparkinson [ ], antidepressant [ ], anti-inflammatory [ ], antimicrobial [10],
1]. The 1,2,4-triazine ring as one of the most ubiquitous
2
3
4
5
6
7
8
9
antiparasitic [11] activities. Some 1,2,4-triazines have become well-known drugs, such as the antiviral
azaribine [12], the anticonvulsant lamotrigine [13], the anti-inflammatory and analgesic apazone [14],
the antibacterial ceftriaxone [15], the insecticide pymetrozine [16] or vardenafil, useful in the treatment
of male erectile dysfunction [17]. In addition, there are also many reports indicating significant
anticancer properties for the 1,2,4-triazine fragment. For example, tirapazamine (I, Figure 1) is
currently in various clinical trial phases for the treatment of human non-small cell, cervical, ovarian,
head and neck cancers. Tirapazamine works by inducing DNA damage in poorly oxygenated tumor
cells [18]. The 6-azauridine II, known as an inhibitor of de novo pyrimidine biosynthesis, is an
effective agent for remission induction of acute myelocytic leukemia in children [19]. Close analogs of
6-azauridine, S-alkyl derivatives of 1,2,4-triazinone III, show cytotoxic activities against human breast
cancer (MCF-7), colon carcinoma (HCT-116) and hepatocellular carcinoma (Hep-G2) cell lines [20].
Some 1,2,4-triazine analogs IV have distinct antiptoliferative activities against non-small cell lung
cancer (NCI-H460), breast cancer (MCF-7), CNS cancer (SF-268), with a low cytotoxicity [21].
Figure 1. Structures of known anticancer 1,2,4-triazines (I–V) and novel 2-(arylmethylthio)-4-chloro
-N-(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamide derivatives (VI).
Our systematic studies on arylsulfonamides has resulted in promising anticancer agents [22–29],
and among them we have reported that some trisubstituted 1,2,4-triazines
V (Figure 1),
exhibited anticancer activity against colon, CNS, melanoma, ovarian, breast, renal, leukemia
cell lines [29]. Searching for innovative low-molecular chemotherapeutics, a structure-based
molecular hybridization strategy has been applied as a common approach to develop multi-targeted

Molecules 2016, 21, 808
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compounds [30]. Thus, we designed and synthesized a series of new 4-chloro-2-mercapto-N-
(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamides VI (Figure 1), bearing a disubstituted 1,2,4-triazine
and a 2-mercaptobenzenesulfonamide moiety in the molecular skeleton, (Figure 1) as potential
anticancer agents. Various substituent groups were introduced to the triazine ring as well as the
benzenesulfonamide fragment in positions 2 and 5 for structure-activity relationship (SAR) analysis.
The prepared compounds were evaluated for their antiproliferative activities against three human
cancer cell lines, namely, HCT-116 (colon cancer), HeLa (cervical cancer) and MCF-7 (breast cancer).
For the most active compounds apoptosis- inducing activities on HCT-116, HeLa and MCF-7 cell lines
were further investigated.
In vitro tests for metabolic stability, which is one of the important parameters characterizing
pharmacokinetic properties of a molecule, were performed on selected compounds. The experiments
were run in the presence of human liver microsomes and NADPH.
2. Results and Discussion
2.1. Chemistry
The starting substrates, 6-chloro-3-methylthio-7-R1-1,1-dioxo-1,4,2-benzodithiazines
3-amino-6-chloro-7-R1-1,1-dioxo-1,4,2-benzodithiazines
10 33 34], N-(benzenesulfonyl)cyanamide
potassium salts 11 18 22 25 33 35] as well as aminoguanidines 19 20 and 23 25 29 33 35 36] were
prepared according to the respective known methods. Novel substrates 21 22 and 26 were synthesized
analogously by the reaction of hydrazine monohydrochloride with the potassium salts 13 14
as was shown in Scheme 1. Finally, 4-chloro-2-(R2-methylthio)-5-R1-N-(5-aryl-1,2,4-triazin-3-yl)
benzenesulfonamides 27 60 were readily obtained by treatment of aminoguanidines 19 26 with the
appropriate phenylglyoxal hydrate in glacial acetic acid at reflux for 24–45 h as outlined in Scheme 2.
1–5 [31,32],
6
–
[ ,
–
[
,
,
,
–
–
[ , , ,
–
–
–
–
Scheme 1. Synthesis of 1-amino-2-[4-chloro-5-R1-2-(R2-methylthio)benzenesulfonyl]guanidine (19
–
26).
Reagents and Conditions: (a) 25% NH3 aq/EtOH, r.t., 144 h; (b) Excess K2CO3, THF, reflux, 24 h;
(c) R1CH2Cl, ethanol or water, r.t. or 0 ˝C, 1–4 h; (d) NH2-NH2 ˆ HCl, dry toluene, reflux 3–9 h.

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Scheme 2. Synthesis of 4-chloro-2-(R2-methylthio)-5-R1-N-(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamides
27–60.
1
The structures of the final compounds 27
–
60 were confirmed by IR, H-NMR and 13C-NMR
1
spectroscopy. H-NMR spectra showed the absence of characteristic of aminoguanidine NH signals at
4 and 6 ppm, confirming the success of the condensation reactions between the amine and carbonyl
groups of the reagents leading to the heterocyclic ring formation. In addition, the presence in the
1H-NMR spectra of a singlet at about 9 ppm corresponding to the H-6 proton in the 1,2,4-triazine
fragment was observed. Moreover, X-ray analysis was done to confirm the proposed structures using
the representative compound 59.
Compound 59 crystallizes in the triclinic system in the space group P1. The asymmetric
unit contains two sulfonamide molecules and four solvating dimethylformamide (DMF) molecules.
The unit cell of the crystal contains two asymmetric parts (Z = 2). The two independent sulfonamide
molecules have the same topological atom connectivity (constitution) but cannot be superimposed on
each another (they are almost related by a mirror plane passing through SO2 group and bisecting the
S–N–C and S–C–C planes). The solvent DMF molecules are not only filling the voids in the structure,
but also participate in hydrogen bonding—two of carbonyl groups are acceptors of hydrogen from
N-H of 1,2,4-triazine residue (O14-N4, O16-N9, see Table 1). The other two DMF molecules use their
CO groups as acceptors for amidic NH groups (O13-N6, O15-N1, Table 1). Other intermolecular
interactions responsible for crystal packing are π-π stacking operating between R1 (N3, C22, N4, N5,
C23, C24) and R5 (C25–C30) with the distance between ring geometry centers (centroids) of 3.8252(3)Å
and R6 (N8, C54, N9, N10, C55, C56) and R10 (C57–C52) 3.8952(3)Å. All other rings are separated by
more than 4.4Å and interactions among them were neglected.

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Table 1. Crystal data and structure refinement of 59.
Empirical Formula
C38H42ClN7O8S2
Formula weight
Temperature
Wavelength
824.35
120(2) K
1.54186 Å
Crystal system
Space group
Triclinic
P1
a = 7.8336(6) Å, α = 84.84(3)˝
b = 22.4284(8) Å, β = 86.95(4)˝
c = 22.2739(9) Å, γ = 85.13(5)˝
3879.4(5) Å3
Unit cell dimensions
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
4
1.411 Mg/m3
2.399 mm´1
1728
0.03 ˆ 0.03 ˆ 0.25 mm3
1.984 to 64.981˝
´8 ď h ď 8, ´23 ď k ď 26, ´25 ď l ď 23
19645
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to θ = 67.686˝
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
11676 [R(int) = 0.1757]
83.1%
Full-matrix least-squares on F2
11,676/0/1014
1.253
R1 = 0.1476, wR2 = 0.3359
R1 = 0.2313, wR2 = 0.4260
0.0114(11)
Extinction coefficient
The sulfonamide group seems to be deprotonated in the solid state, as there is a C–H bond from
a DMF molecule directed to the sulfonamidic N (Figure 2) which would not be beneficial without
assumed ionization of the –SO2NH– fragment. Additionally, protonation of the nitrogen atom in
position 2 of the 1,2,4-triazine is required by electrical neutrality and by formation of hydrogen bond
to the carbonyl group from the neighbor DMF molecule. Thus, two charge-assisted hydrogen bonds
are created: C–H¨ ¨ ¨ N(´)–S and (+)N–H¨ ¨ ¨ O between the sulfonamide and the solvating DMF.
Figure 2. View of half of the independent unit (one sulfonamide and two DMF molecules) showing the
general arrangement of atoms in 59 and N–H¨ ¨ ¨ O type hydrogen bonds.

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Bond lengths within the triazine ring are not helpful to attribute double bonds in the substructure.
The 1,2,4-triazine residue is flat and almost coplanar with 3,4-dimethoxyphenyl which may be a
consequence of beneficial π-π interactions with the neighbor molecule in crystal. The second solvent
DMF molecule forms hydrogen bond with the amide N–H proton donor in the other branch of the
main molecule. The other functional groups are not unusual and their geometry will therefore not be
further analyzed.
2.2. Biological Evaluations
2.2.1. Cytotoxic Activity
Compounds 27–60 were evaluated in vitro for their effects on the viability of three human cancer
cell lines: HCT-116 (colon cancer), HeLa (cervical cancer) and MCF-7 (breast cancer). The concentration
required for 50% inhibition of cell viability IC50 was calculated and compared with the reference drug
cisplatin, the results were shown in Table 2. To describe of cytotoxic potency, the following scale
was applied: IC50 < 25
75 ď IC50 ď 100 µM—weak, IC50 >100 µM—inactive compounds.
µ
M—very strong, 25
ď
IC50
ď
50 µM—strong, 50 < IC50 < 75 µM—moderate,
Table 2. Cytotoxicity of compounds 27–60 toward human cancer cell lines a.
IC50 (µM)
Compound
HCT-116
HeLa
MCF-7
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
75 ˘ 1
200 ˘ 4
720 ˘ 36
85 ˘ 2
140 ˘ 7
88 ˘ 3
67 ˘ 1
51 ˘ 1
68 ˘ 3
49 ˘ 2
36 ˘ 1
38 ˘ 2
83 ˘ 2
77 ˘ 1
97 ˘ 2
77 ˘ 2
92 ˘ 3
160 ˘ 6
97 ˘ 2
64 ˘ 1
75 ˘ 5
80 ˘ 3
110 ˘ 2
115 ˘ 3
190 ˘ 13
70 ˘ 2
76 ˘ 2
84 ˘ 5
150 ˘ 3
75 ˘ 2
73 ˘ 1
98 ˘ 2
140 ˘ 5
230 ˘ 18
3.8 ˘ 0.2
84 ˘ 1
100 ˘ 6
190 ˘ 7
90 ˘ 4
150 ˘ 6
87 ˘ 4
76 ˘ 3
73 ˘ 1
69 ˘ 1
55 ˘ 2
34 ˘ 2
42 ˘ 1
93 ˘ 1
85 ˘ 2
85 ˘ 1
82 ˘ 1
130 ˘ 10
160 ˘ 8
82 ˘ 1
80 ˘ 2
79 ˘ 1
80 ˘ 2
120 ˘ 6
130 ˘ 8
*
80 ˘ 2
120 ˘ 5
110 ˘ 2
67 ˘ 1
77 ˘ 2
87 ˘ 4
88 ˘ 1
68 ˘ 3
78 ˘ 2
96 ˘ 4
70 ˘ 3
69 ˘ 1
95 ˘ 3
98 ˘ 3
99 ˘ 1
83 ˘ 2
87 ˘ 2
90 ˘ 5
90 ˘ 1
59 ˘ 2
78 ˘ 3
87 ˘ 3
81 ˘ 5
120 ˘ 4
95 ˘ 5
69 ˘ 1
88 ˘ 2
96 ˘ 5
150 ˘ 6
89 ˘ 3
90 ˘ 1
92 ˘ 6
100 ˘ 5
270 ˘ 13
3 ˘ 0.1
*
74 ˘ 2
82 ˘ 1
105 ˘ 4
80 ˘ 2
80 ˘ 1
88 ˘ 6
150 ˘ 2
230 ˘ 5
2.2 ˘ 0.2
60
Cisplatin
a
Analysis was performed using the MTT assay after 72 h of incubation. Values are expressed as the mean
˘
SD
of at least three independent experiments; * Viability of cell lines at 100
approximately 100%.
µM of tested compounds were

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The most active compounds 37 and 38 belonged to the 1-naphthyl series (R2 = 1-naphthyl) and
showed outstanding average cytotoxic activity (47
cell lines, HCT-116, HeLa and MCF-7. Moreover, compounds 34
cell lines viability, exhibiting average IC50 values below 71
µ
M and 50
µ
M, respectively) against all tested
36 and 46 moderately inhibited all
–
µM. Among tested analogues only six
compounds (17.6%) were completely inactive with average IC50 > 100 µM.
As shown in Table 2, the HCT-116 cell line presented the relatively highest susceptibility and
was affected by eleven compounds (33
Meanwhile, the HeLa cell line was susceptible toward six compounds (34
M, and the MCF-7 by six compounds (30 34 37 38 46 and 52) with IC50 values ranging between 59
–38
,
46
,
47
,
52,
56 and 57) in the range of IC50 values 36–75
µM.
–38 and 53) with IC50; 34–74
µ
,
,
,
,
and 70 µM.
We found that, among 4-chloro-2-(R2-methylthio)-5-R1-N-(5-aryl-1,2,4-triazin-3-yl)benzene
-sulfonamides with R2 = 1-naphthyl, presence of Ar = 4-CF3-C6H4 group provided strong cytotoxicity
of 37 toward the HeLa (IC50 = 34
µM) as well as HCT-116 (IC50 = 36 µM) cells and replacement of
1-naphthyl substituent in this compound by the smaller aromatic group, i.e., 34 (R2 = 4-CF3-C6H4),
40 (R2 = 3,5-dimethylisoxazol-4-yl) caused decrease in activity to IC50; 51–85
µM or the loss of activity
when R2 = Ph (compd 28, see Table 2). It should be noted, that replacement of Ar = 4-CF3-C6H4
group (37) by Ar = 4-MeO-C6H4 (38) or 3-F-C6H4 (36) did not contribute to improving the anticancer
activity. The presence of Ar = 4-CF3-C6H4 also influenced the strongest cytotoxic activity of 46
(R1 = 4-Cl-C6H4NHCO, R2 = Ph, IC50 = 59
µM) toward the MCF-7 cell line. Nevertheless, the exchange
of R1 = 4-Cl-C6H4NHCO in compd 46 to small methyl group strongly inactivate the compound 28
(R1 = Me, R2 = Ph) but introduction of 4-Me-C6H4NHCO as the R1 substituent in compd 52 decreased
its cytotoxic activity to IC50 = 69 µM.
2.2.2. Investigation of Apoptotic Activity
The ability to induce apoptosis in cancer cells is a desired feature of a potential chemotherapeutic
agent. Thus, the apoptosis-inducing activity was studied through biochemical markers, such as: DNA
fragmentation, loss of mitochondrial membrane potential (∆ψm), phosphatidylserine translocation
and caspase activation. Apoptotic-like changes in morphology of tested cell lines were also evaluated.
The experiments were performed with the most active compounds (37 and 46) exhibited the highest
activity in MTT tests.
Cell Morphology
To evaluate the changes in morphology of the treated cells, they were incubated with increasing
concentrations of 37 (HCT-116, HeLa) or 46 (MCF-7) for 24 h and observed using light microscope.
The most characteristic morphological changes (shrinkage of the cells, detachment from the surface)
occurred in the HCT-116 and HeLa cells treated with 37 (Figure 3).
Cell Cycle Analysis
One of the most common mechanisms in anticancer drug treatment is changes in cell cycle,
which can be measured by DNA content [37]. Cell cycle distribution in HCT-116, HeLa and MCF-7 cells
was examined to determine cycle arrest and/or presence of sub-G1 after treatment with compounds
37 and 46. Cells were coincubated with trazines and a reference drug (cisplatin) for 24 h, respectively.
Cell populations data are expressed as the mean ˘ SD of at least three independent experiments.

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Figure 3. Morphology of cell lines: HCT-116 (
(100 M); MCF-7 ( ) treated with 46 (0–100 M) and cisplatin (100
with 10ˆ objective, of cells treated for 24 h.
a
); HeLa (
b
) treated with 37 (0–100
µM) and cisplatin
µ
c
µ
µM). Light microscopy photographs,
Sub-G1 cell population increased in dose dependent manner (HCT-116 from 3.6%
˘
1.66% (control)
to 48.6% 13.5%; HeLa from 5.7% 3.4% (control) to 21% 3.4%) after treatment with compound 37
˘
˘
˘
for 24 h. Figure 4 shows one representative experiment. Similar results were obtained for MCF-7 cells
coincubated with compound 46 for 24 h (from 5.0% ˘ 3.8% (control) to 20% ˘ 5.6%) (Figure 4).
Coincubation of HCT-116, HeLa and MCF-7 with appropriate triazines (37 and 46) caused
appearance of sub-G1 stage and high concentrations led to fragmentation of DNA, resulting in a
sub-G0/G1 peak of cell cycle. A significant accumulation of the HCT-116, HeLa and MCF-7 population
in the sub-G1 phase indicated that the cells had undergone programmed cell death (apoptosis) for all
of the analyzed compounds. Triazine coincubation did not cause significant differences in the cell cycle
phases of the analyzed cell lines.

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Figure 4. HCT-116, HeLa and MCF-7 cell cycle 24 h induction with 1,2,4-triazine derivatives
(representative results). HCT-116 ( ); HeLa ( ) treated with 37 (0–100 M) and cisplatin (100 M);
MCF-7 (c) treated with 46 (0–100 µM) and cisplatin (100 µM).
a
b
µ
µ
Mitochondrial Membrane Potential (∆ψ ) Analysis
m
Loss of the mitochondrial membrane potential (∆ψm) is one of the earliest indicators of
an apoptosis [38]. It can be detected using fluorescent dye JC-1 (5,51,6,61-tetrachloro-1,11,3,31
-tetraethylbenzimidazolylcarbocyanine iodide) exited with blue laser. JC-1 spontaneously forms
dimer complexes (J aggregates) with red fluorescent or remain as a monomers with green fluorescence
in cells with high and low ∆ψ , respectively.
m
As shown in Figure 5 HCT-116, HeLa and MCF-7 cells in the control group, high red fluorescence
(J aggregates) were observed. However, exposure of cells even to low and moderate concentrations
of compounds 37 and 46 remarkably decreased ∆ψm which is the earliest indicators of an apoptotic
cell death.

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Figure 5. Mitochondrial membrane potential (∆ψm). Cytometric analysis of ∆ψ with 2.5
µ
g/mL JC-1
M) 24 h, carbonyl cyanide m-chlorophenylhydrazone
M) 24 h; MCF-7 ( ) treated with 46 (0–100 M) 24 h,
M) 24 h. Results represent the mean of number of cells
SD (n = 8). Statistically significant differences between
m
in: HCT-116 (
(CCCP) (100
a
µ
), HeLa (
M) for 15 min or cisplatin (100
M) for 15 min or cisplatin (100
with high ∆ψ (high JC-1 fluorescence)
b) treated with 37 (0–100 µ
µ
c
µ
CCCP (100
µ
µ
˘
m
treated and control cells are indicated (* p < 0.05; *** p < 0.005).
Translocation of Phosphatidylserine to Outer Leaflet of Cell Membrane
The quantification of cell death was evaluated by measuring the exposure of phosphatidylserine
on the outer leaflet of plasma membrane. Four subpopulations were identified according to their
fluorescence: PI-low/FITC-low (live cells), PI-high/FITC-low (necrotic cells), PI-low/FITC-high
(early apoptotic cells), PI-high/FITC-high (late apoptotic cells).
Appearance of increased population of PI-low/FITC-high (early apoptotic cells), PI-high/FITC-high
(late apoptotic cells) gates was noticed for HCT-116 and HeLa cells coincubated with compound 37
(Figure 6). The increased levels of apoptotic cells were concentration dependent. For MCF-7 cells
treated with compound 46 the results were not conclusive, as the differences between treated cells and
control cells were statistically significant only for low 25 µM of 46 (Figure 6).
Caspase Activation
Caspase activation plays a central role in the execution of apoptosis and is required for the
occurrence of its biochemical and morphological hallmarks, such as DNA fragmentation, formation
of apoptotic bodies and chromatin condensation. The ability of the examined compounds to
induce caspase activity was determined with the use of a fluorescent labeled caspase inhibitor,
a carboxyfluorescein (FAM) derivative of valylalanylaspartic acid (VAD) fluoromethyl ketone (FMK).
FAM-VAD-FMK contains a target sequence recognized by active caspases (caspases 1 through 9).
Binding to this sequence inhibits the enzymatic activity of caspases and allows for the determination of
their activity through the direct measurement of fluorescent intensity of the bound inhibitor. The results
of the research showed that the tested compounds 37 and 46 induced caspase activity in the examined
cells lines, as shown by an increase in FAM-VAD-FMK fluorescence in the cell population with activated
caspases (Figure 7). The strongest influence on caspase activation was noticed for the HeLa cell line,
where that cell population increased by 31% in the presence of 37 at a concentration of 100 µM.

Molecules 2016, 21, 808
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Figure 6. Features of dying process of HCT-116 (
results). Dotblots show cells stained with Annexin V-FITC Apoptosis Kit. HCT-116, HeLa cells treated
with 37 (0–100 M) and cisplatin (100 M); MCF-7 cells treated with 46 (0–100 M) and cisplatin
(100 M) for 24 h. Results show the mean of number of cells in each quadrant Q1, Q2, Q3, Q4 (necrotic
cells, late apoptotic cells, alive cells, apoptotic cells, respectively).
a); HeLa (b) and MCF-7 (c) cell lines (representative
µ
µ
µ
µ

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Figure 7. Induction of caspase activity in HCT-116, HeLa and MCF-7 cells by 1,2,4-triazine derivatives.
HCT-116 ( ), HeLa ( ) and MCF-7 ( ) cells were treated with the indicated concentrations of compounds
37 and 46 for 24 h ( ), 72 h ( ) and 72 h ( ), respectively. Enzyme activity was determined by flow
a
b
c
a
b
c
cytometry with the use of a caspase inhibitor, FAM-VAD-FMK. Values represent mean
independent experiments.
˘
SD of three
2.2.3. Metabolic Stability
Metabolism as one of the drugs’ pharmacokinetic characteristics can be evaluated during the
early preclinical stage to assessment of degree of drug candidate conversion into a set of metabolites.
Metabolic stability can be assessed by incubation of a potent drug in a presence of liver microsomes
and NADPH to give an insight to metabolic properties [39–41].
For further evaluation of metabolic stability the compounds with outstanding activities and
varied structural features including R1, R2 and Ar substituents (30
, 31, 34–38, 46, 47, 52) were selected.
Human liver microsomes were chosen as a model enzymatic system and in vitro metabolic half-life
was assessed (Table 3).
Based on the enzymatic test results some structure-metabolic stability relationships can be noted.
The most stable compound 34 bears a 4-CF3-C6H4 moiety in both the R1 and Ar positions. Replacement
of Ar = 4-CF3-C6H4 (34) by 4-MeO-C6H4 (compound 35) resulted in decrease of the t1/2 value from
>60 to 42.4 min, while an analogous change between 46 and 47 caused a significant decrease of t1/2
from >60 to 17.5 min. On the other hand, taking into account the results for 30 (Ar = 3-MeO-C6H4)
and 31 (Ar = 3,4-diMeO-C6H3), it was found that an additional methoxy (MeO) group in the Ar
substituent slightly decreased the metabolic stability. These facts suggest that the methoxy substituent
is a major soft spot in the described series of compounds and moreover, its undesirable influence can
be diminished by incorporation of CF3 instead of a MeO substituent.

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Table 3. Results of metabolic stability study.
Compound
In Vitro Metabolic Half-Life t1/2 (min)
30
31
34
35
36
37
38
46
47
52
24.9
17.6
>60 (154)
42.4
13.9
32.8
<5
>60 (78.1)
17.5
26.5
Comparison of t1/2 for 34 (R1 = 4-CF3-C6H4) and 37 (R1 = 1-naphthyl) indicated that 1-naphthyl
(R1) also seems to decrease metabolic stability. We assumed that the undesirable influence of the
1-naphthyl (R1) and 4-MeO-C6H4 (Ar) substituents accelerated in the case of compound 38, which was
characterized as the least stable derivative. Further synthesis in this group of compounds should avoid
incorporation of the two abovementioned substituents. Although a 4-CF3-C6H4 (R1) substituent is
preferable for metabolic stability in comparison with a 1-naphthyl moiety, the high biological response
observed for 37 predisposes it to be a lead compound, as it shows a good balance between stability
and cytotoxic properties.
In order to explain the differences between the metabolic stability of distinctive compounds
(34–35, 37–38) we applied a tool accessible on-line for accurate prediction of xenobiotic metabolism
sites, called XenoSite Cytochrome P450 Prediction Models [42]. Among the available models, one is
able to predict which atoms on a molecule are likely to be oxidized by human liver microsomes.
In silico results showed that the least stable derivative 38 has three more sites vulnerable for metabolic
biotransformation than 34 (see Figure 8). Moreover, a slight decrease of metabolic stability of 35 and 37
compared to 34 may resulted from presence of additional sites of oxidation on methoxy group and
methylene linker (35, Figure 8) as well as on methylene linker and naphthalene ring (37, Figure 8).
Figure 8. Sites of metabolism predicted for 34, 35, 37 and 38 by XenoSite software [42]. Yellow
color indicates more vulnerability to biotransformation than blue. Some significant differences are
additionally pointed out by red arrows.

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3. Materials and Methods
3.1. General Information
The melting points were uncorrected and measured using Boethius PHMK apparatus
(Veb Analytic, Dresden, Germany). IR spectra were measured on Thermo Mattson Satellite FTIR
spectrometer (Thermo Mattson, Madison, WI, USA) in KBr pellets; an absorption range was
1
400–4000 cm´1. H-NMR and 13C-NMR spectra were recorded on a Varian Gemini 200 apparatus
(Varian, Palo Alto, CA, USA) or a Varian Unity Plus 500 apparatus. Chemical shifts are expressed
at
δ values relative to Me4Si (TMS) as an internal standard. The apparent resonance multiplicity
is described as: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), and br
(broad) signal. Elemental analyses were performed on 2400 Series II CHN Elemental Analyzer
(PerkinElmer, Shelton, CT, USA) and the results were within
˘
0.4% of the theoretical values. Thin-layer
chromatography (TLC) was performed on Kieselgel 60 F254 plates (Merck, Darmstadt, Germany)
and visualized by UV. The commercially unavailable substrates were obtained according to the
following methods described previously: 6-chloro-3-methylthio-7-R1-1,1-dioxo-1,4,2-benzo-dithiazines
1
–
5
[
31
-4-chloro-5-R1-benzenesulfonyl)cyanamide potassium salts 11
-5-R1-2-alkylthiobenzenesulfonyl)guanidines 19
20 and 23 25
,
32], 3-amino-6-chloro-7-R1-1,1-dioxo-1,4,2-benzodithiazines
18 22
25 29 33
6
25
,
–
,
10
33
35
[33,34], N-(2-alkylthio
35], 1-amino-2-(4-chloro
36]. The NMR spectra of
–
[
,
,
–
–
[
,
,
,
newly synthesized compounds 21–22, 26–60 are given in the Supplementary Materials.
3.2. Synthesis
3.2.1. Procedures for the Preparation of Aminoguanidines 21–22 and 26
A mixture of the appropriate cyanamide potassium salt 13, 14 or 18 (7 mmol) and hydrazine
monohydrochloride (0.48 g, 7 mmol) in anhydrous toluene (14 mL) was refluxed with stirring for
3–9 h. After cooling to room temperature, the reaction mixture was left in the freezer overnight.
The precipitate was collected by filtration, dried and suspended in water (9 mL). The solid was
separated and crystallized from ethanol (21–22) or p-dioxane (26). In this manner, the following
aminoguanidines were obtained.
1-Amino-2-{4-chloro-5-methyl-2-[(naphthalen-1-ylmethyl)thio]benzenesulfonyl}guanidine (21). Starting from
N-{4-chloro-5-methyl-2-[(naphthalen-1-ylmethyl)thio]benzenesulfonyl}cyanamide monopotassium
salt 13 (3.09 g), the title compound 21 was obtained (2.55 g, 80%) mp 217–219 ˝C; IR (KBr)
ν
max 3453,
3348, 3217 (NH), 2925, 2853 (C-H), 1385, 1133 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ 2.31 (s, 3H,
CH3), 4.44 (s, 2H, C-NH2), 4.74 (s, 2H, SCH2), 6.93 (s, 2H, NNH2), 7.46 (t, 1H, arom), 7.51 (s, 1H, H-3,
arom), 7.57–7.59 (m, 3H, arom), 7.84 (s, 1H, H-6, arom), 7.87 (d, J = 8.3 Hz, 1H, arom), 7.95 (d, J = 7.8 Hz,
1H, arom), 8.23 (d, J = 8.3 Hz, 1H, arom), 8.38 (s, 1H, NH) ppm; 13C-NMR (125 MHz, DMSO-d6)
δ
19.7, 35.2, 124.9, 126.1, 126.6, 126.9, 128.6, 128.8, 129.2, 131.1, 132.1, 132.3, 132.8, 134.1, 136.6, 137.3,
140.4, 155.9, 157.6 ppm; anal. C 52.20, H 4.30, N 12.61% calcd. for C19H19ClN4O2S2, C 52.47, H 4.40,
N 12.88%.
1-Amino-2-{4-chloro-5-methyl-2-[(3,5-dimethylisoxazol-4-ylmethyl)thio]benzenesulfonyl}guanidine (22). Starting
from N-{4-chloro-5-methyl-2-[(3,5-dimethylisoxazol-4-ylmethyl)thio]benzenesulfonyl}cyanamide
monopotassium salt 14 (2.87 g), the title compound 22 was obtained (2.34 g, 84%) mp 204–206 ˝C;
IR (KBr)
ν
3479, 3345, 3341 (NH); 2926, 2851 (C–H); 1268, 1131 (SO ) cm´1; 1H-NMR (500 MHz,
max
2
DMSO-d6)
2H, NNH2), 7.43 (s, 1H, H-3, arom), 7.82 (s,1H, H-6, arom), 8.39 (s, 1H, NH) ppm; 13C-NMR (125 MHz,
DMSO-d6) 10.4, 11.3, 19.7, 25.3, 109.7, 129.7, 131.0, 133.0, 135.1, 136.6, 141.8, 159.4, 160.3, 167.3 ppm;
anal. C 41.88, H 4.56, N 17.62% calcd. for C14H18ClN5O3S2, C 41.63, H 4.49, N 17.34%.
δ
2.21 (s, 3H, CH3), 2.30–2.31 (m, 6H, 2 CH3), 4.08 (s, 2H, SCH2), 4.49 (s, 2H, CNH2), 6.97 (s,
ˆ
δ

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1-Amino-2-(2-benzylthio-4-chloro-5-methoxyphenylcarbamoylbenzenesulfonyl)guanidine (26). Starting from
N-(2-benzylthio-4-chloro-5-methoxyphenylcarbamoylbenzenesulfonyl)cyanamide monopotassium
salt 18 (3.68 g), the title compound 26 was obtained (2.52 g, 70%) mp 238–240 ˝C; IR (KBr)
ν
max 3312,
3212 (NH), 2921, 2851 (C–H), 1678 (C=O), 1541, 1510 (C=C, C=N), 1312, 1134 (SO2) cm´1; 1H-NMR
(500 MHz, DMSO-d6) 3.75 (s, 3H, OCH3), 4.41 (s, 2H, SCH2), 4.54 (s, 2H, NH2), 6.92–7.03 (m, 4H,
δ
arom), 7.29–7.41 (m, 3H, arom), 7.48–7.64 (m, 5H, arom), 7.97 (m, 1H, arom), 8.52 (s, 1H, NH), 10.43 (s,
1H, NHCO) ppm; anal. C 50.56, H 4.15, N 13.15 calcd. for C22H22ClN5O4S2, C 50.81, H 4.26, N 13.47%.
3.2.2. Procedures for the Preparation of 4-Chloro-2-(R2-methylthio)-5-R1-N-(5-aryl-1,2,4-triazin-3-yl)
benzenesulfonamides 27–60
A mixture of the appropriate aminoguanidine 19–26 (1 mmol) and phenylglyoxal hydrate (1 mmol)
in glacial acetic acid (2.5 mL) was refluxed with stirring for 24–45 h. The solid of the compound was
obtained either after stirring overnight at room temperature, or after additional stirring on an ice bath
for 12 h (compds. 30–31, 42–44, 47). The formed precipitate was collected by filtration, washed with
glacial acetic acid (2 ˆ 1 mL) and dried. The final products 27–60 were purified as described below.
2-Benzylthio-4-chloro-N-[5-(3-fluorophenyl)-1,2,4-triazin-3-yl]-5-methylbenzenesulfonamide (27). Starting
from 19 (0.385 g) and 3-fluorophenylglyoxal hydrate (0.170 g) after stirring for 33 h, the crude 27
(0.431 g) was obtained. The crude 27 was crystallized twice from EtOH and the title compound 27
(0.290 g, 58%) was isolated mp 216–218 ˝C; IR (KBr)
ν
3430, 3196 (NH), 3074, 2921 (C–H), 1585,
max
1556 (C=C, C=N), 1302, 1159 (SO2) cm´1; 1H-NMR (200 MHz, DMSO-d6)
δ 2.29 (s, 3H, CH3), 4.31 (s,
2H, SCH2), 7.09–7.10 (m, 3H, arom), 7.26–7.37 (m, 2H, arom), 7.50–7.67 (m, 4H, arom), 7.82–7.86 (m, 1H,
arom), 8.13 (s, 1H, H-6, arom), 9.31 (s, 1H, triazine), 14.25 (brs, 1 H, SO2NH) ppm; 13C-NMR (50 MHz,
DMSO-d6) δ 19.1, 36.2, 114.8, 115.3, 121.1, 125.1, 127.3, 128.3, 128.4, 129.1, 131.6, 131.7, 132.5, 132.7, 134.9,
135.1, 135.4, 136.5, 137.6, 160.1, 165.0 ppm; anal. C 55.32, H 3.36, N 11.36% calcd. for C23H18ClFN4O2S2,
C 55.14, H 3.29, N 11.18%.
2-Benzylthio-4-chloro-5-methyl-N-[5-(4-trifluoromethylphenyl)-1,2,4-triazin-3-yl]benzenesulfonamide (28).
Starting from 19 (0.385 g) and 4-trifluoromethylphenylglyoxal hydrate (0.220 g) after stirring for 33
h, the title compound 28 (0.298 g, 72%) was obtained without further purification mp 287–289 ˝C; IR
(KBr)
(200 MHz, DMSO-d6)
2H, arom), 7.57 (s, 1H, H-3, arom), 7.92–7.96 (m, 2H, H-6, arom), 8.15–8.17 (m, 3H, arom), 9.42 (s, 1H,
triazine), 14.10 (brs, 1 H, SO2NH) ppm; 13C-NMR (50 MHz, DMSO-d6)
19.1, 36.2, 121.3, 126.1, 126.2,
ν
max 3457, 3182 (NH), 3089, 2929 (C–H), 1585, 1560 (C=C, C=N), 1320, 1123 (SO2) cm´1; 1H-NMR
δ
2.37 (s, 3H, CH3), 4.32 (s, 2H, SCH2), 7.10–7.13 (m, 3H, arom), 7.28–7.30 (m,
δ
126.3, 127.3, 128.2, 128.5, 129.2, 129.4, 129.5, 132.3, 133.2, 135.7, 136.4, 136.7, 137.7, 137.8, 137.9 ppm;
anal. C 52.45, H 3.41, N 10.28% calcd. for C23H18ClFN4O2S2, C 52.32, H 3.29, N 10.17%.
2-Benzylthio-4-chloro-N-[5-(4-methoxyphenyl)-1,2,4-triazin-3-yl]-5-methylbenzenesulfonamide (29). Starting
from 19 (0.385 g) and 4-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 31 h, the crude 29
(0.381 g) was obtained. The crude 29 was crystallized from MeCN and the title compound 29 (0.324 g,
˝
63%) was isolated mp 232–234 C; IR (KBr)
ν
3188 (NH), 2918, 2831 (C–H), 1606, 1575 (C=C, C=N),
max
1533 (NHdef.), 1298, 1180 (SO2) cm´1; 1H-NMR (200 MHz, DMSO-d6)
δ 2.40 (s, 3H, CH3), 3.88 (s, 3H,
OCH3), 4.27 (s, 2H, SCH2), 7.05–7.17 (m, 4H, arom), 7.24–7.31 (m, 3H, arom), 7.48 (s, 1H, H-3, arom),
7.95 (d, 2H, J = 8.93 Hz, arom), 8.12 (s, 1H, H-6), 9.08 (s, 1H, triazine) ppm; anal. C 56.02, H 3.96, N
10.91% calcd. for C24H21ClN4O3S2, C 56.19, H 4.13, N 10.92%.
2-Benzylthio-4-chloro-N-[5-(3-methoxyphenyl)-1,2,4-triazin-3-yl]5-methylbenzenesulfonamide (30). Starting
from 19 (0.385 g) and 3-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 33 h, the title
˝
compound 30 (0.400 g, 78%) was obtained without further purification mp 198–200 C; IR (KBr)
max 3199 (NH), 2934 (C–H), 1631, 1582 (C=C, C=N), 1561 (NHdef.), 1283, 1132 (SO2) cm´1; 1H-NMR
(200 MHz, DMSO-d6) 2.28 (s, 3H, CH3), 3.81 (s, 3H, OCH3), 4.29 (s, 2H, SCH2), 7.10–7.13 (m, 3H,
ν
δ

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arom), 7.26–7.32 (m, 3H, arom), 7.42–7.52 (m, 4H, H-3, arom.), 8.08 (s, 1H, H-6, arom), 9.27 (s, 1H,
triazine) ppm; anal. C 56.13, H 4.01, N 10.96% calcd. for C24H21ClN4O3S2, C 56.19, H 4.13, N 10.92%.
2-Benzylthio-4-chloro-N-[5-(3,4-dimethoxyphenyl)-1,2,4-triazin-3-yl]-5-methylbenzenesulfonamide
(31).
Starting from 19 (0.385 g) and 3,4-dimethoxyphenylglyoxal hydrate (0.212 g) after stirring for 32 h,
the title compound 31 (0.443 g, 82%) was obtained without further purification mp 198–200 ˝C; IR
(KBr)
ν
3447, 3125 (NH), 3000, 2936 (C–H), 1615, 1554, 1519 (C=C, C=N), 1316, 1133 (SO ) cm´1
;
max
2
1H-NMR (200 MHz, DMSO-d6)
δ 2.35 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.28 (s, 2H,
SCH2), 7.08–7.19 (m, 4H, arom), 7.28–7.32 (m, 2H, arom), 7.47 (s, 1H, H-3, arom), 7.56 (d, J = 1.91 Hz,
1H, arom), 7.67 (dd, J1 = 8.50 Hz, J2 = 2.00 Hz, 1H, arom), 8.08 (s, 1H, H-6, arom), 9.20 (s, 1H, triazine)
ppm; anal. C 55.08, H 4.13, N 10.11% calcd. for C25H23ClN4O4S2, C 55.29, H 4.27, N 10.32%.
2-Benzylthio-4-chloro-5-methyl-N-[5-(3,4,5-trimethoxyphenyl)-1,2,4-triazin-3-yl]benzenesulfonamide (32).
Starting from 19 (0.385 g) and 3,4,5-trimethoxyphenylglyoxal hydrate (0.242 g) after stirring for 33 h,
the title compound 32 (0.525 g, 92%) was obtained without further purification mp 163–164 ˝C; IR
(KBr)
ν
3436 (NH), 3073, 2936 (C–H), 1594, 1550 (C=N, C=C), 1332, 1129 (SO ) cm´1; 1H-NMR (200
max
2
MHz, DMSO-d6)
7.17–7.19 (m, 3H, arom), 7.31–7.35 (m, 4H, arom), 7.52 (s, 1H, H-3, arom), 8.04 (s, 1H, H-6, arom), 9.34
(brs, 1H, triazine), 14.25 (brs, 1H, SO2NH) ppm; 13C-NMR (50 MHz, DMSO-d6)
: 19.1, 36.3, 56.7, 60.7,
δ
2.28 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 3.85 (s, 6H, 2 OCH3), 4.32 (s, 2H, SCH2),
ˆ
δ
107.1, 127.4, 127.7, 127.9, 128.5, 129.2, 132.0, 132.1, 135.4, 136.4, 137.3, 146.2, 153.6 ppm; anal. C 54.82, H
4.56, N 10.07% calcd. for C26H25ClN4O5S2, C 54.49, H 4.39, N 9.78%.
4-Chloro-N-[5-(3-fluorophenyl)-1,2,4-triazin-3-yl]-5-methyl-2-[4-(trifluoromethyl)benzylthio]benzenesulfonamide
(
33). Starting from 20 (0.451 g) and 3-fluorophenylglyoxal hydrate (0.170 g) after stirring for 33 h, the
crude 33 (0.446 g) was obtained. The crude 33 was purified by heating under reflux with EtOH and the
title compound 33 (0.330 g, 58%) was isolated mp 212–214 ˝C; IR (KBr)
3436, 3186 (NH), 3092,
ν
max
2923, 2851 (C–H), 1589, 1554 (C=C, C=N), 1326, 1126 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ
2.35 (s, 3H, CH3), 4.42 (s, 2H, SCH2), 7.39 (d, J = 8.30 Hz, 2H, arom), 7.43–7.53 (m, 4H, arom), 7.54–7.65
(m, 2H, arom), 7.76 (d, J = 7.80 Hz, 1H, arom), 8.08 (s, 1H, H-6, arom), 9.28 (brs, 1H, triazine) ppm; anal.
C 50.71, H 3.11, N 9.89% calcd. for C24H17ClF4N4O2S2, C 50.66, H 3.01, N 9.85%.
4-Chloro-5-methyl-2-[4-(trifluoromethyl)benzylthio]-N-[5-(4-trifluoromethylphenyl)-1,2,4-triazin-3-yl]-benze
nesulfonamide (34). Starting from 20 (0.451 g) and 4-trifluoromethylphenylglyoxal hydrate (0.220 g)
after stirring for 33 h, the crude 34 (0.447 g) was obtained. The crude 34 was purified by heating
under reflux with EtOH and the title compound 34 (0.331 g, 53%) was isolated mp 256–259 ˝C; IR
1
(KBr)
ν
3444 (NH), 3078, 2925 (C–H), 1581, 1556 (C=C, C=N), 1325, 1131 (SO ) cm´1; H-NMR
max
2
(500 MHz, DMSO-d6)
δ
2.33 (s, 3H, CH3), 4.42 (s, 2H, SCH2), 7.40 (d, 2H, J = 7.80 Hz, arom), 7.49 (d,
2H, J = 8.30 Hz, arom), 7.60 (s, 1H, H-3, arom), 7.89 (d, 2H, J = 8.30 Hz, arom), 8.08 (d, J = 8.30 Hz, 2H,
arom), 8.13 (s, 1H, H-6, arom), 9.39 (brs, 1H, triazine) ppm; anal. C 48.66, H 3.00, N 9.12% calcd. for
C25H17ClF6N4O2S2, C 48.51, H 2.77, N 9.05%.
4-Chloro-N-[5-(4-methoxyphenyl)-1,2,4-triazin-3-yl]-5-methyl-2-[4-(trifluoromethyl)benzylthio]benzene-su
lfonamide (35). Starting from 20 (0.451 g) and 4-methoxyphenylglyoxal hydrate (0.182 g) after stirring
for 29 h, the crude 35 (0.462 g) was obtained. The crude 35 was purified by heating under reflux with
˝
EtOH and the title compound 35 (0.387 g, 66%) was isolated mp 255–258 C; IR (KBr)
ν
3435 (NH),
max
3084, 2931 (C–H), 1579, 1536 (C=C, C=N), 1299, 1125 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ
2.37 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 4.39 (s, 2H, SCH2), 7.04 (d, 2H, J = 8.8 Hz, arom), 7.44 (d, 2H,
arom), 7.48-7.58 (m, 3H, arom), 7.89 (d, 2H, J = 7.8 Hz, arom), 8.10 (s, 1H, H-6, arom), 9.04 (brs, 1H,
triazine), 14.51 (brs, 1H, SO2NH) ppm; 13C-NMR (125 MHz, DMSO-d6)
δ 19.7, 35.8, 56.5, 88.9, 115.4,
123.6, 124.9, 125.6, 128.1, 128.3, 130.3, 132.9, 134.8, 142.4 ppm; anal. C 51.75, H 3.49, N 9.68% calcd. for
C25H20ClF3N4O3S2, C 51.68, H 3.47, N 9.64%.

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4-Chloro-N-[5-(3-fluorophenyl)-1,2,4-triazin-3-yl]-5-methyl-2-[(naphthalene-1-yl)methylthio]benzene-sulfonamide
36). Starting from 21 (0.435 g) and 3-fluorophenylglyoxal hydrate (0.170 g) after stirring for 43 h, the
crude 36 (0.417 g) was obtained. The crude 36 was purified by heating under reflux with EtOH and
(
˝
the title compound 36 (0.277 g, 50%) was isolated mp 234–237 C; IR (KBr)
ν
3432 (NH), 3052,
max
2922 (C–H), 1552, 1524 (C=C, C=N), 1442, 1168 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ
2.40 (s,
3H, CH3), 4.75 (s, 2H, SCH2), 7.26 (t, 1H, arom), 7.42–7.57 (m, 5H, arom), 7.58–7.64 (m, 1H, arom),
7.68 (s, 1H, H-3, arom), 7.74 (d, J = 8.30 Hz, 1H, arom), 7.81–7.82 (m, 1H, arom), 7.86–7.89 (m, 1H,
arom), 8.08–8.12 (m, 2H, arom), 9.20 (brs, 1H, triazine) ppm; anal. C 58.53, H 3.45, N 9.95% calcd. for
C27H20ClFN4O2S2, C 58.85, H 3.66, N 10.17%.
4-Chloro-5-methyl-2-[(naphthalene-1-yl)methylthio]-N-[5-(4-trifluoromethylphenyl)-1,2,4-triazin-3-yl]-benze
nesulfonamide (37). Starting from 21 (0.435 g) and 4-trifluoromethylphenylglyoxal hydrate (0.220 g)
after stirring for 30 h, the crude 37 (0.526 g) was obtained. The crude 37 was purified by heating under
˝
reflux with EtOH and the title compound 37 (0.417 g, 69%) was isolated: mp 247–251 C; IR (KBr)
ν
max
1
3438 (NH), 3080, 2927 (C–H), 1580, 1554 (C=C, C=N), 1323, 1162 (SO2) cm´1; H-NMR (500 MHz,
DMSO-d6)
1H, H-3, arom), 7.76 (d, J = 7.8 Hz,1H, arom), 7.82–8.02 (m, 3H, arom), 8.02–8.28 (m, 4H, arom), 9.30
(brs, 1H, triazine) ppm; 13C-NMR (125 MHz, DMSO-d6)
19.6, 34.9, 124.8, 125.9, 126.6, 126.9, 128.6,
δ 2.38 (s, 3H, CH3), 4.76 (s, 2H, SCH2), 7.28 (t, 1H, arom), 7.39–7.57 (m, 3H, arom), 7.67 (s,
δ
128.8, 129.1, 129.1, 130.0, 131.9, 132.2, 132.7, 133.5, 133.9, 137.0 ppm; anal. C 55.63, H 3.20, N 8.99%
calcd. for C28H20ClF3N4O2S2, C 55.95, H 3.35, N 9.32%.
4-Chloro-N-[5-(4-methoxyphenyl)-1,2,4-triazin-3-yl]-5-methyl-2-[(naphthalene-1-yl)methylthio]benzene-su
lfonamide (38). Starting from 21 (0.435 g) and 4-methoxyphenylglyoxal hydrate (0.182 g) after stirring
for 40 h, the crude 38 (0.461 g) was obtained. The crude 38 was purified by heating under reflux with
˝
EtOH and the title compound 38 (0.389 g, 75%) was isolated mp 262–265 C; IR (KBr)
ν
3450 (NH),
max
3060, 2925 (C–H), 1607, 1541 (C=C, C=N), 1264, 1176 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ
2.42 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 4.73 (s, 2H, SCH2), 7.06 (d, J = 8.8 Hz, 2H, arom), 7.30 (t, 1H,
arom), 7.44–7.56 (m, 3H, arom), 7.60 (s, 1H, H-3, arom), 7.78 (d, J = 8.3 Hz, 1H, arom), 7.84–8.02 (m, 3H,
arom), 8.09 (d, J = 7.8 Hz, 1H, arom), 8.13 (s, 1H, H-6 arom), 9.00 (brs, 1H, triazine), 14.39 (brs, 1H,
SO2NH) ppm; 13C-NMR (125 MHz, DMSO-d6):
δ 18.2, 37.1, 55.2, 110.9, 113.7, 115.4, 115.8, 117.7, 122.0,
122.7, 124.9, 125.9, 126.6, 128.4, 128.7, 130.3, 131.5, 132.7, 133.1, 133.4, 136.3, 136.5, 137.8, 142.7, 150.9,
168.8 ppm; anal. C 59.56, H 4.09, N 9.89% calcd. for C28H23ClN4O3S2, C 59.72, H 4.12, N 9.95%.
4-Chloro-2-[(3,5-dimethylizoxazol-4-yl)methylthio]-N-[5-(3-fluorophenyl)-1,2,4-triazin-3-yl]-5-methyl-benz
enesulfonamide (39). Starting from 22 (0.404 g) and 3-fluorophenylglyoxal hydrate (0.170 g) after stirring
for 25 h, the crude 39 (0.380 g) was obtained. The crude 39 was purified by heating under reflux with
˝
EtOH and the title compound 39 (0.178 g, 33%) was isolated mp 208–211 C; IR (KBr)
ν
3435 (NH),
max
3071, 2963, 2925, 2854, 2737 (C–H), 1551, 1514 (C=C, C=N), 1326, 1156 (SO2) cm´1; 1H-NMR (500 MHz,
DMSO-d6) 2.06 (s, 3H, CH3), 2.11 (s, 3H, CH3), 2.41 (s, 3H, CH3), 4.10 (s, 2H, SCH2), 7.34–7.46 (s, 1H,
arom), 7.48–7.66 (m, 2H, arom), 7.72 (s, 1H, H-3, arom), 7.77 (d, J = 7.8 Hz, 1H, arom), 8.09 (s, 1H, H-6,
arom), 9.24 (bs, 1H, triazine) ppm; 13C-NMR (125 MHz, DMSO-d6)
10.3, 11.2, 19.6, 25.6, 110.3, 115.3,
δ
δ
115.5, 125.6, 130.7, 131.9, 132.0, 133.9, 135.3, 135.3, 135.4, 159.9, 161.9, 163.9, 166.7 ppm; anal. C 51.05, H
3.75, N 13.70% calcd. for C22H19ClFN5O3S2, C 50.81, H 3.68, N 13.47%.
4-Chloro-2-[(3,5-dimethylizoxazol-4-yl)methylthio]-5-methyl-N-[5-(4-trifluoromethylphenyl)-1,2,4-triazin-3-yl]
benzenesulfonamide (40). Starting from 22 (0.404 g) and 4-trifluoromethylphenylglyoxal hydrate (0.220 g)
after stirring for 45 h, the crude 40 (0.516 g) was obtained. The crude 40 was purified by heating under
˝
reflux with EtOH and the title compound 40 (0.250 g, 43%) was isolated mp 218–221 C; IR (KBr)
3434 (NH), 3072, 2754 (C-H), 1550, 1519 (C=C,C=N), 1325, 1156 (SO ) cm´1; 1H-NMR (500 MHz,
ν
max
2
DMSO-d6)
δ 2.08 (s, 3H, CH3), 2.14 (s, 3H, CH3), 2.39 (s, 3H, CH3), 4.11 (s, 2H, SCH2), 7.67 (s, 1H, H-6,
arom), 7.90 (d, J = 8.3 Hz, 2H, arom), 8.12 (d, 2H, arom), 8.15 (s, 1H, H-6, arom), 9.36 (brs, 1H, triazine)

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ppm; 13C-NMR (125 MHz, DMSO-d6)
δ
10.3, 11.2, 19.6, 25.6, 110.0, 121.1, 123.3, 125.4, 126.5, 126.6,
127.6, 129.9, 130.4, 133.7, 135.2, 137.0, 160.0, 166.9 ppm; anal. C 51.15, H 3.55, N 10.10% calcd. for
C23H19ClF3N5O3S2 , C 50.84, H 3.20, N 9.88%.
4-Chloro-2-[(3,5-dimethylizoxazol-4-yl)methylthio]-N-[5-(4-methoxyphenyl)-1,2,4-triazin-3-yl]-5-methyl-
benzenesulfonamide (41). Starting from 22 (0.404 g) and 4-methoxyphenylglyoxal hydrate (0.182 g) after
stirring for 24 h, the crude 41 (0.435 g) was obtained. The crude 41 was purified by heating under
˝
reflux with EtOH and the title compound 41 (0.364 g, 68%) was isolated mp 224–227 C; IR (KBr)
ν
max
3418, 3218 (NH), 3071, 2925, 2847, (C–H), 1577, 1541 (C=C, C=N), 1267, 1157 (SO2) cm´1; 1H-NMR
(500 MHz, DMSO-d6) 2.08 (s, 3H, CH3), 2.13 (s, 3H, CH3), 2.43 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 4.08
δ
(s, 2H, SCH2), 7.03 (d, J = 8.8 Hz, 2H, arom), 7.62 (s, 1H, H-3, arom), 7.86 (d, J = 8.30 Hz, 2H, arom),
8.10 (s, 1H, H-6, arom), 9.03 (brs, 1H, triazine), 14.40 (brs, 1H, SO2NH) ppm; 13C-NMR (125 MHz,
DMSO-d6) δ 10.3, 11.2, 19.7, 25.5, 56.5, 110.2, 115.4, 125.0, 130.1, 133.5, 160.0, 166.8 ppm; anal. C 52.03,
H 4.20, N 13.39% calcd. for C23H22ClN5O4S2, C 51.92, H 4.17, N 13.16%.
4-Benzylthio-2-chloro-5-{N-[5-(4-methoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-N-phenylbenzamide
(42).
Starting from 23 (0.451 g) and 4-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 31 h,
the crude 42 (0.486 g) was obtained. The crude 42 was crystallized from MeCN and the title compound
42 (0.383 g, 67%) was isolated mp 283–286 ˝C; IR (KBr)
ν
max 3409, 3323 (NH), 2923, 2843 (C–H), 1664
(C=O), 1604, 1576 (C=C, C=N), 1535 (NHdef.), 1315, 1174 (SO2) cm´1; 1H-NMR (200 MHz, DMSO-d6)
3.77 (s, 3H, OCH3), 4.38 (s, 2H, SCH2), 7.04 (d, J = 9.09 Hz, 2H, arom), 7.14–7.22 (m, 4H, arom),
δ
7.32–7.42 (m, 4H, arom), 7.63 (s, 1H, H-3, arom), 7.73 (d, J = 7.67 Hz, 2H, arom), 7.94 (d, J = 8.92 Hz, 2H,
arom), 8.26 (s, 1H, H-6, arom), 9.09 (s, 1H, triazine), 10.63 (s, 1H, NHCO), 14.50 (s, 1H, SO2NH) ppm;
anal. C 58.03, H 2.99, N 11.18% calcd. for C30H24ClN5O4S2, C 58.29, H 3.19, N 11.33%.
4-Benzylthio-2-chloro-5-{N-[5-(3-methoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-N-phenylbenzamide
(43).
Starting from 23 (0.451 g) and 3-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 33 h, the
crude 43 (0.453 g) was obtained. The crude 43 was purified by heating under reflux with EtOH and the
title compound 43 (0.399 g, 71%) was isolated mp 270–272 ˝C; IR (KBr)
ν
3436, 3326 (NH), 2935
max
(C–H), 1681 (C=O), 1603, 1554 (C=N, C=C), 1314, 1137 (SO2) cm´1; 1H-NMR (500 MHz, DMSO-d6)
δ
3.73 (s, 3H, OCH3), 4.39 (s, 2H, SCH2), 7.11–7.20 (m, 5H, arom), 7.34–7.42 (m, 6H, arom), 7.63–7.68 (m,
4H, H-3, arom), 8.21 (s, 1H, H-6, arom), 9.23 (s, 1H, triazine), 10.54 (s, 1H, NHCO) ppm; anal. C 58.49,
H 3.30, N 11.42% calcd. for C30H24ClN5O4S2, C 58.29, H 3.19, N 11.33%.
4-Benzylthio-2-chloro-5-{N-[5-(3,4-dimethoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-N-phenylbenzamide (44).
Starting from 23 (0.451 g) and 3,4-dimethoxyphenylglyoxal hydrate (0.212 g) after stirring for 32 h, the
crude 44 (0.500 g) was obtained. The crude 44 was crystallized from EtOH and the title compound 44
(0.407 g, 68%) was isolated mp 252–253 ˝C; IR (KBr)
1600, 1540 (C=N, C=C), 1318, 1133 (SO2) cm´1; 1H-NMR (200 MHz, DMSO-d6)
ν
max 3419, 3328 (NH), 2933 (C–H), 1651 (C=O),
3.72 (s, 3H, OCH3),
δ
3.78 (s, 3H, OCH3), 4.40 (s, 2H, SCH2), 6.99–7.24 (m, 6H, arom), 7.32–7,39 (m, 3H, arom), 7.61–7.71 (m,
5H, arom), 8.25 (s, 1H, arom), 9.17 (s, 1H, triazine), 10.57 (s, 1H, NHCO) ppm; anal. C 57.50, H 4.03, N
10.81% calcd. for C31H26ClN5O5S2, C 57.45, H 4.04, N 10.81%.
4-Benzylthio-2-chloro-N-(4-chlorophenyl)-5-{N-[5-(3-fluorophenyl)-1,2,4-triazin-3-yl]sulfamoyl}benzamide
(
45). Starting from 24 (0.524 g) and 3-fluorophenylglyoxal hydrate (0.170 g) after stirring for 33 h,
the title compound 45 (0.470 g, 73%) was obtained without further purification mp 280–282 ˝C; IR
(KBr) max 3429, 3276 (NH), 3065, 2923 (C–H), 1657 (C=O), 1558, 1527 (C=N, C=C), 1310, 1166 (SO2)
cm´1; 1H-NMR (200 MHz, DMSO-d6)
4.42 (s, 2H, SCH2), 7.17–7.36 (m, 3H, arom), 7.43–7.63 (m, 6H,
ν
δ
arom), 7.66–7.75 (m, 4H, arom), 7.93 (d, J = 9.61 Hz, 1H, arom), 8.25 (s, 1H, H-6, arom), 9.28 (s, 1H,
triazine), 10.72 (s, 1H, CONH), 14.45 (brs, 1H, SO2NH) ppm; anal. C 54.58, H 3.22, N 11.01% calcd. for
C29H20Cl2FN5O3S2, C 54.36, H 3.15, N 10.93%.

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4-Benzylthio-2-chloro-N-(4-chlorophenyl)-5-{N-[5-(4-trifluoromethylphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-
benzamide (46). Starting from 24 (0.524 g) and 4-trifluoromethylphenylglyoxal hydrate (0.220 g) after
stirring for 33 h, the crude 46 (0.561 g) was obtained. The crude 46 was purified by heating under
˝
reflux with MeCN and the title compound 46 (0.553 g, 80%) was isolated mp 273–275 C; IR (KBr)
ν
max
3290 (NH), 3062, 2922 (C–H), 1658 (C=O), 1560, 1529 (C=C, C=N), 1325, 1165 (SO2) cm´1; 1H-NMR
(200 MHz, DMSO-d6) 4.43 (s, 2H, SCH2), 7.16–7.28 (m, 3H, arom), 7.34–7.46 (m, 6H, H-3, arom),
7.73–7.77 (m, 3H, arom), 7.85–7.89 (m, 2H, arom), 8.09–8.13 (m, 2H, arom), 8.30 (s, 1H, H-6, arom), 9.36
(s, 1H, triazine), 10.80 (s, 1H, NHCO) ppm; 13C-NMR (50 MHz, DMSO-d6)
35.9, 121.4, 126.3, 126.4,
δ
δ
126.6, 127.5, 128.0, 128.3, 128.6, 129.0, 129.2, 129.3, 129.6, 130.9, 132.2, 132.8, 133.4, 134.3, 136.2, 136.4,
137.9, 140.8, 155.1, 163.9 ppm; anal. C 52.36, H 3.15, N 10.23% calcd. for C30H20Cl2F3N5O3S2, C 52.18,
H 2.92, N 10.14%.
4-Benzylthio-2-chloro-N-(4-chlorophenyl)-5-{N-[5-(4-methoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}benzamide
(47). Starting from 24 (0.524 g) and 4-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 30 h,
the crude 47 (0.502 g) was obtained. The crude 47 was purified by˝heating under reflux with MeCN
3411 (NH), 2925,
max
and the title compound 47 (0.363 g, 56%) was isolated mp 282–284 C; IR (KBr)
ν
2843 (C–H), 1663 (C=O), 1604, 1577 (C=N, C=C), 1537 (NHdef.), 1314, 1174 (SO2) cm´1; 1H-NMR (200
MHz, DMSO-d6)
δ 3.79 (s, 3H, OCH3), 4.39 (s, 2H, SCH2), 7.03 (d, J = 8.97 Hz, 2H, arom), 7.17–7.23 (m,
3H, arom), 7.32–7.37 (m, 2H, arom), 7.44 (d, J = 8.88 Hz, 2H, arom), 7.64 (s, 1H, H-3, arom), 7.75 (d,
J = 8.92 Hz, 2H, arom), 7.94 (d, 2H, arom), 8.27 (s, 1H, H-6, arom), 9.09 (s, 1H, triazine), 10.74 (s, 1H,
NHCO) ppm; anal. C 55.01, H 3.46, N 10.58% calcd. for C30H23Cl2N5O4S2, C 55.22, H 3.55, N 10.87%.
4-Benzylthio-2-chloro-N-(4-chlorophenyl)-5-{N-[5-(3-methoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-benzamide
(48). Starting from 24 (0.524 g) and 3-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 33 h,
the crude 48 (0.358 g) was obtained. The crude 48 was purified by hea˝ting under reflux with MeCN
3318 (NH),
max
and the title compound 48 (0.356 g, 56%) was isolated mp 256–258 C; IR (KBr)
ν
3088, 2921 (C–H), 1684 (C=O), 1597, 1553 (C=C, C=N), 1311, 1162 (SO2) cm´1; 1H-NMR (200 MHz,
DMSO-d6) δ 3.76 (s, 3H, OCH3), 4.42 (s, 2H, SCH2), 7.20–7.38 (m, 4H, arom), 7.43–7.46 (m, 6H, arom),
7.67–7.75 (m, 4H, arom), 8.26 (s, 1H, H-6, arom), 9.27 (s, 1H, triazine), 10.70 (s, 1H, NHCO) ppm; anal.
C 55.36, H 3.60, N 10.80% calcd. for C30H23Cl2N5O4S2, C 55.22, H 3.55, N 10.87%.
4-Benzylthio-2-chloro-N-(4-chlorophenyl)-5-{N-[5-(3,4-dimethoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-benzamide
(49). Starting from 24 (0.524 g) and 3,4-dimethoxyphenylglyoxal hydrate (0.212 g) after stirring for 33
h, the crude 49 (0.570 g) was obtained. The crude 49 was crystallized from dry p-dioxane and the title
˝
compound 49 (0.264 g, 39%) was isolated: mp: 257–259 C; IR (KBr)
ν
3435, 3327 (NH), 2921 (C–H),
max
1656 (C=O), 1600, 1544 (C=C, C=N), 1318, 1132 (SO2) cm´1; 1H-NMR (200 MHz, DMSO-d6)
δ 3.76 (s,
3H, OCH3), 3.78 (s, 3H, OCH3), 4.41 (s, 2H, SCH2), 6.99–7.04 (m, 1H, arom), 7.23–7.26 (m, 3H, arom),
7.39–7.45 (m, 4H, arom), 7.63–7.72 (m, 3H, arom), 8.27 (s, 1H, H-6, arom), 9.19 (s, 1H, triazine), 10.69
(s, 1H, CONH) ppm; anal. C 54.60, H 3.75, N 10.27% calcd. for C31H25Cl2N5O5S2, C 54.55, H 3.69,
N 10.26%.
4-Benzylthio-2-chloro-N-(4-chlorophenyl)-5-{N-[5-(3,4,5-trimethoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-
benzamide (50). Starting from 24 (0.524 g) and 3,4,5-trimethoxyphenylglyoxal hydrate (0.242 g) after
stirring for 33 h, the crude 50 (0.385 g) was obtained. The crude 50 was crystallized from dry p-dioxane
˝
and the title compound 50 (0.182 g, 47%) was isolated mp 260–262 C; IR (KBr)
ν
3301, 3102 (NH),
max
2925, 2853 (C–H), 1682 (C=O), 1596, 1552 (C=C, C=N), 1333, 1131 (SO2) cm´1; 1H-NMR (200 MHz,
DMSO-d6) 3.69 (s, 3H, OCH3), 3.79 (s, 6H, 2 OCH3), 4.43 (s, 2H, SCH2), 7.24–7.46 (m, 8H, arom),
δ
ˆ
7.64–7.73 (m, 4H, arom), 8.22 (s, 1H, H-6, arom), 9.31 (s, 1H, triazine), 10.66 (s, 1H, CONH), 14.5
(brs, 1H, SO2NH) ppm; anal. C 53.58, H 3.56, N 9.49% calcd. for C32H27Cl2N5O6S2, C 53.93, H 3.82,
N 9.83%.

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4-Benzylthio-2-chloro-5-{N-[5-(3-fluorophenyl)-1,2,4-triazin-3-yl]sulfamoyl}-N-(4-methylphenyl)benzamide
51). Starting from 25 (0.504 g) and 3-fluorophenylglyoxal hydrate (0.170 g) after stirring for 33 h, the
crude 51 (0.470 g) was obtained. The crude 51 was purified by heating under reflux with EtOH and the
title compound 51 (0.322 g, 52%) was isolated mp 266–268 ˝C; IR (KBr)
3427, 3278 (NH), 3064,
(
ν
max
2921 (C–H), 1654, 1528 (C=C, C=N), 1316, 1165 (SO2) cm´1; 1H-NMR (200 MHz, DMSO-d6)
δ 2.30 (s,
3H, CH3), 4.41 (s, 2H, SCH2), 7.17–7.20 (m, 4H, arom), 7.33–7.35 (m, 2H, H-3, arom), 7.45–7.68 (m, 7H,
arom), 7.95 (d, J = 9.9 Hz, 1H, arom), 8.23 (s, 1H, H-6, arom), 9.28 (s, 1H, triazine), 10.49 (s, 1H, NHCO),
14.5 (brs, 1H, SO2NH) ppm; anal. C 58.22, H 3.80, N 11.31% calcd. for C30H23ClFN5O3S2, C 58.09, H
3.74, N 11.29%.
4-Benzylthio-2-chloro-N-(4-methylphenyl)-5-{N-[5-(4-trifluoromethylphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-
benzamide (52). Starting from 25 (0.504 g) and 4-trifluoromethylphenylglyoxal hydrate (0.220 g) after
stirring for 33 h, the crude 52 (0.560 g) was obtained. The crude 52 was purified by heating under
˝
reflux with EtOH and the title compound 52 (0.370 g, 55%) was isolated mp 277–279 C; IR (KBr)
max 3343, 3347 (NH), 3091, 2920 (C–H), 1668 (C=O), 1558, 1517 (C=C, C=N), 1316, 1120 (SO2) cm´1
1H-NMR (200 MHz, DMSO-d6)
2.31 (s, 3H, CH3), 4.43 (s, 2H, SCH2), 7.16–7.21 (m, 5H, arom),
ν
;
δ
7.34–7.36 (m, 2H, arom), 7.62 (d, J = 8.0 Hz, 2H, arom), 7.72 (s, 1H, H-3, arom), 7.89 (d, J = 8.2 Hz, 2H,
arom), 8.11 (d, 2H, arom), 8.28 (s, 1H, H-6, arom), 9.36 (s, 1H, triazine), 10.61 (s, 1H, NHCO), 14.13 (brs,
1H, SO2NH) ppm; 13C-NMR (50 MHz, DMSO-d6)
δ 20.8, 35.9, 119.8, 126.3, 126.4, 127.5, 128.3, 128.5,
128.6, 129.2, 129.5, 129.6, 130.9, 132.6, 133.4, 134.4, 136.2, 136.4, 136.5, 136.9, 140.5, 155.0, 163.6 ppm;
anal. C 55.28, H 3.10, N 10.09% calcd. for C31H23ClF3N5O3S2, C 55.56, H 3.46, N 10.45%.
4-Benzylthio-2-chloro-5-{N-[5-(4-methoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-N-(4-methylphenyl)-benzamide
(
53). Starting from 25 (0.504 g) and 4-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 33 h,
the title compound 53 (0.490 g, 77%) was obtained without further purification mp 255–258 ˝C; IR
(KBr) max 3315 (NH), 3087, 2921, 2851 (C–H), 1675 (C=O), 1577, 1540 (C=C, C=N), 1315, 1162 (SO2)
cm´1; 1H-NMR (200 MHz, DMSO-d6)
2.31 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 4.40 (s, 2H, SCH2), 7.05
ν
δ
(d, J = 8.3 Hz, 2H, arom), 7.18–7.21 (m, 5H, arom), 7.34–7.36 (m, 2H, arom), 7.62–7.65 (m, 3H, H-3,
arom), 7.95 (d, J = 8.3 Hz, 2H, arom), 8.27 (s, 1H, H-6, arom), 9.10 (s, 1H, triazine), 10.57 (s, 1H, NHCO),
14.5 (brs, 1H, SO2NH ) ppm; 13C-NMR (50 MHz, DMSO-d6)
δ 20.8, 35.9, 55.7, 115.2, 119.9, 124.5, 127.5,
127.9, 128.6, 129.1, 129.5, 130.5, 131.7, 132.4, 133.3, 133.7, 136.3, 133.7, 136.6, 140.2, 154.2, 163.7, 164.6
ppm; anal. C 58.72, H 4.03, N 10.98% calcd. for C31H26ClN5O4S2, C 58.90, H 4.15, N 11.08%.
4-Benzylthio-2-chloro-5-{N-[5-(3-methoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-N-(4-methylphenyl)benzamide
(
54). Starting from 25 (0.504 g) and 3-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 33 h,
the title compound 54 (0.480 g, 76%) was obtained without further purification mp 237–239 ˝C; IR
(KBr)
max 3328 (NH), 3029, 2918 (C–H), 1678 (C=O), 1554, 1519 (C=C, C=N), 1314, 1162 (SO2) cm´1
1H-NMR (200 MHz, DMSO-d6)
2.31 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 4.42 (s, 2H, SCH2), 7.19–7.21
(m, 6H, arom), 7.36–7.46 (m, 4H, arom), 7.58–7.69 (m, 4H, H-3, arom), 8.24 (s, 1H, H-6), 9.28 (s, 1H,
triazine), 10.50 (s, 1H, NHCO) ppm; 13C-NMR (50 MHz, DMSO-d6)
20.8, 35.9, 55.7, 114.2, 119.9, 120.1,
ν
;
δ
δ
121.3, 127.5, 127.9, 128.6, 129.2, 129.4, 130.5, 130.9, 132.5, 133.2, 133.7, 136.2, 136.5, 140.3, 154.2, 159.9,
163.5 ppm; anal. C 59.06, H 4.25, N 11.12% calcd. for C31H26ClN5O4S2, C 58.90, H 4.15, N 11.08%.
4-Benzylthio-2-chloro-5-{N-[5-(3,4-dimethoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-N-(4-methylphenyl)-benz
amide (55). Starting from 25 (0.504 g) and 3,4-dimethoxyphenylglyoxal hydrate (0.212 g) after stirring
for 33 h, the crude 55 (0.570 g) was obtained. The crude 55 was purified by heating under reflux with
˝
EtOH and the title compound 55 (0.480 g, 72%) was isolated mp 250–252 C; IR (KBr)
ν
1
3329,
max
3093 (NH), 2921, 2852 (C–H), 1640 (C=O), 154, 1518 (C=N, C=C), 1318, 1132 (SO2) cm´1; H-NMR
(200 MHz, DMSO-d6) 2.30 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 4.41 (s, 2H, SCH2),
7.00–7.04 (m, 1H, arom), 7.15–7.25 (m, 5H, arom), 7.37–7.42 (m, 2H, arom), 7.56–7.68 (m, 5H, arom),
δ

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8.25 (s, 1H, H-6, arom), 9.19 (s, 1H, triazine), 10.49 (s, 1H, NHCO) ppm; anal. C 56.03, H 4.19, N 10.36%
calcd. for C32H28ClN5O5S2, C 56.23, H 4.26, N 10.58%.
4-Benzylthio-2-chloro-N-(4-methylphenyl)-5-{N-[5-(3,4,5-trimethoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-
benzamide (56). Starting from 24 (0.504 g) and 3,4,5-trimethoxyphenylglyoxal hydrate (0.242 g) after
stirring for 33 h, the crude 56 (0.516 g) was obtained. The crude 56 was purified by heating under
˝
reflux with EtOH and the title compound 56 (0.464 g, 67%) was isolated mp 259–261 C; IR (KBr)
max 3435, 3308 (NH), 3033, 2924 (C–H), 1640 (C=O), 1558, 1522 (C=C, C=N), 1342, 1129 (SO2) cm´1
1H-NMR (200 MHz, DMSO-d6)
2.29 (s, 3H, CH3), 3.71 (s, 3H, OCH3), 3.80 (s, 6H, 2 OCH3), 4.43 (s,
2H, SCH2), 7.14–7.26 (m, 5H, arom), 7.40–7.63 (m, 7H, H-3, arom), 8.20 (s, 1H, H-6, arom), 9.32 (s, 1H,
triazine), 10.45 (s, 1H, NHCO), 14.20 (brs, 1H, SO2NH) ppm; 13C-NMR (50 MHz, DMSO-d6)
20.8,
ν
;
δ
ˆ
δ
35.9, 56.2, 60.5, 106.6, 119.8, 127.4, 127.6, 127.7, 128.7, 129.3, 129.4, 130.0, 132.3, 133.2, 133.9, 136.2, 136.4,
140.4, 143.5, 153.5, 154.5, 163.4 ppm; anal. C 56.89, H 4.02, N 9.95% calcd. for C33H30ClN5O6S2, C 57.26,
H 4.37, N 10.12%.
4-Benzylthio-2-chloro-N-(4-methoxyphenyl)-5-{N-[5-(4-methoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}benzamide
(
57). Starting from 26 (0.520 g) and 4-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 33 h,
the title compound 57 (0.571 g, 88%) was obtained without further purification mp 270–272 ˝C; IR
(KBr)
ν
3331 (NH), 3092, 2932, 2835 (CH , CH ), 1676 (C=O), 1608, 1543 (C=N, C=C), 1313, 1162
max
2
3
(SO2) cm´1; H-NMR (200 MHz, DMSO-d6)
δ
3.77 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 4.39 (s, 2H,
1
SCH2), 6.96 (d, J = 9.0 Hz, 2H, arom), 7.05 (d, J = 9.00 Hz, 2H, arom), 7.17–7.20 (m, 3H, arom), 7.33–7.35
(m, 2H, arom), 7.63–7.68 (m, 3H, arom), 7.95 (d, J = 8.8 Hz, 2H, arom), 8.26 (s, 1H, H-6, arom), 9.10 (s,
1H, triazine), 10.49 (s, 1H, NHCO) ppm; 13C-NMR (50 MHz, DMSO-d6)
δ 35.8, 55.5, 55.9, 114.3, 115.3,
121.4, 124.5, 127.5, 127.9, 128.6, 129.1, 130.5, 131.7, 132.2, 132.5, 133.8, 136.3, 140.1, 156.0, 163.5, 164.6
ppm; anal. C 57.50, H 4.09, N 10.85% calcd. for C31H26ClN5O5S2, C 57.44, H 4.04, N 10.81%.
4-Benzylthio-2-chloro-N-(4-methoxyphenyl)-5-{N-[5-(3-methoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}benzamide
(58). Starting from 26 (0.520 g) and 3-methoxyphenylglyoxal hydrate (0.182 g) after stirring for 33 h,
the crude 58 (0.550 g) was obtained. The crude 58 was purified by heating under reflux with EtOH
˝
and the title compound 58 (0.470 g, 72%) was isolated mp 242–244 C; IR (KBr)
ν
3333 (NH), 3083,
max
2922 (CH2, CH3), 2360 (OCH3), 1679 (C=O), 1600, 1552, 1510 (C=N, C=C), 1307, 1162 (SO2) cm´1
1H-NMR (200 MHz, DMSO-d6)
3.77 (s, 6H, 2 OCH3), 4.41 (m, 2H, SCH2), 6.96 (d, J = 8.8 Hz, 2H,
arom), 7.19–7.21 (m, 4H, arom), 7.35–7.45 (m, 4H, arom), 7.59–7.68 (m, 4H, arom), 8.23 (s, 1H, arom),
9.27 (s, 1H, triazine), 10.43 (s, 1H, NHCO) ppm; 13C-NMR (50 MHz, DMSO-d6)
35.9, 55.5, 55.7, 114.2,
;
δ
ˆ
δ
120.0, 121.3, 121.4, 127.5, 128.0, 128.6, 129.2, 130.5, 130.9, 132.2, 132.5, 133.7, 136.2, 140.2, 156.0, 160.0,
163.2 ppm; anal. C 57.32, H 3.88, N 10.70% calcd. for C31H26ClN5O5S2, C 57.44, H 4.04, N 10.81%.
4-Benzylthio-2-chloro-5-{N-[5-(3,4-dimethoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}-N-(4-methoxyphenyl)-
benzamide (59). Starting from 26 (0.520 g) and 3,4-dimethoxyphenylglyoxal hydrate (0.212 g) after
stirring for 33 h, the title compound 59 (0.625 g, 92%) was obtained without further purification mp
269–271 ˝C; IR (KBr)
ν
3393, 3262 (NH), 2924, 2851 (C–H), 1679 (C=O), 1543, 1514 (C=C, C=N),
max
1
1312, 1126 (SO2) cm´1; H-NMR (200 MHz, DMSO-d6)
δ
3.76-3.78 (m, 9H, 3 OCH3), 4.41 (s, 2H,
ˆ
SCH2), 6.92–7.06 (m, 3H, arom), 7.22–7.25 (m, 3H, H-3, arom), 7.38–7.49 (m, 2H, arom), 7.59–7.67 (m,
5H, arom), 8.26 (s, 1H, H-6, arom), 9.19 (s, 1H, triazine), 10.43 (s, 1H, NHCO) ppm; 13C-NMR (50 MHz.
DMSO-d6)
δ 35.9, 55.5, 55.9, 111.5, 112.3, 114.2, 121.3, 124.5, 127.6, 127.7, 128.7, 129.2, 132.2, 132.3,
133.8, 136.1, 136.2, 140.4, 149.3, 154.5, 156.0, 163.3 ppm; anal. C 56.52, H 4.05, N 10.22% calcd. for
C32H28ClN5O6S2, C 56.68, H 4.17, N 10.33%.
4-Benzylthio-2-chloro-N-(4-methoxyphenyl)-5-{N-[5-(3,4,5-trimethoxyphenyl)-1,2,4-triazin-3-yl]sulfamoyl}
-benzamide (60). Starting from 26 (0.520 g) and 3,4,5-trimethoxyphenylglyoxal hydrate (0.242 g) after
stirring for 33 h, the crude 60 (0.610 g) was obtained. The crude 60 was purified by heating under

Molecules 2016, 21, 808
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˝
reflux with EtOH and the title compound 60 (0.519 g, 73%) was isolated mp 263–265 C; IR (KBr)
ν
max
3309 (NH), 2924, 2834 (C–H), 1639 (C=O), 1558, 1513 (C=N, C=C), 1334, 1132 (SO2) cm´1; 1H-NMR
(200 MHz, DMSO-d6) 3.71-3.79 (m, 12H, 4 OCH3), 4.42 (s, 2H, SCH2), 6.92 (d, J = 8.9 Hz, 2H, arom),
δ
ˆ
7.23–7.26 (m, 3H, arom), 7.40–7.46 (m, 4H, arom), 7.53–7.62 (m, 3H, arom), 8.19 (s, 1H, H-6, arom), 9.32
(s, 1H, triazine), 10.38 (s, 1H, NHCO) ppm; anal. C 55.84, H 3.88, N 9.55% calcd. for C33H30ClN5O7S2,
C 55.97, H 4.27, N 9.89%.
3.3. X-ray Structure Determination
Crystals of 59 were grown from DMF solution. All specimens obtained were in form of very thin
needles, having poor diffraction power. The copper radiation (almost no diffraction was observed with
the Mo lamp) and long exposure times (4 min) were applied. The obtained signal to noise ratio was still
smaller than usually, so final Rint and R1 indices are above the regular standards for small molecules.
Nevertheless, structure solution is with no doubts generally correct. Diffraction intensity data were
collected on an IPDS 2T dual-beam diffractometer (STOE & Cie GmbH, Darmstadt, Germany) at
120.(2) K with Cu-Kα radiation of a microfocus X-ray source (50 kV, 0.6 mA, λ = 154.186 pm, GeniX 3D
Cu High Flux, Xenocs, Sassenage, France), The crystal was thermostated in nitrogen stream at 120 K
using CryoStream-800 device (Oxford CryoSystem, Oxford, UK) during the entire experiment. Data
collection and data reduction were controlled by X-Area 1.75 program [43]. An absorption correction
was performed on the integrated reflections by a combination of frame scaling, reflection scaling and a
spherical absorption correction. Outliers have been rejected according to Blessing’s method [44].
The structure was solved using direct methods with SHELXS-13 program and refined by
SHELXL-2013 [45] program run under control of WinGx [46]. All C-H type hydrogen atoms were
attached at their geometrically expected positions and refined as riding on heavier atoms with the
usual constraints. The N-H hydrogen atoms were found in the differential Fourier electron density
map and were refined without constraints.
Crystallographic data for the analysis have been deposited with the Cambridge Crystallographic
data Centre, CCDC reference number is 1471508. Copies of this information may be obtained free
of charge from CCDC, 12 Union Road, Cambridge, CB21EZ, UK (Fax: +44-1223-336033; E-mail:
deposit@ccdc.cam.ac.uk or www: http://www.ccdc.cam.ac.uk).
3.4. Cell Culture and Cell Viability Assay
All chemicals, if not stated otherwise, were obtained from Sigma-Aldrich (St. Louis, MO, USA).
The MCF-7 cell line was purchased from Cell Lines Services (Eppelheim, Germany), the HeLa and
HCT-116 cell lines were obtained from the Department of Microbiology, Tumor and Cell Biology,
Karolinska Institute (Stockholm, Sweden). Cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and
˝
100 µg/mL streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37 C
in an incubator (HeraCell, Heraeus, Langenselbold, Germany).
Cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
-tetrazoliumbromide) assay. Stock solutions of the studied compounds were prepared in 100% DMSO.
Working solutions were prepared by diluting the stock solutions with DMEM medium, the final
concentration of DMSO did not exceed 0.5% in the treated samples. Cells were seeded in 96-well
plates at a density of 5
concentration range 1–100
ˆ
103 cells/well and treated for 72 h with the examined compounds in the
M (1, 10, 25, 50 and 100 M). Following treatment, MTT (0.5 mg/mL) was
µ
µ
˝
added to the medium and cells were further incubated for 2 h at 37 C. Cells were lysed with DMSO and
the absorbance of the formazan solution was measured at 550 nm with a plate reader (1420 multilabel
counter, Victor, Jügesheim, Germany). The optical density of the formazan solution was measured
at 550 nm with a plate reader (Victor 1420 multilabel counter). The experiment was performed in
triplicate. Values are expressed as the mean ˘ SD of at least three independent experiments.

Molecules 2016, 21, 808
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3.4.1. Cell Morphology
The HCT-116, HeLa and MCF-7cells were seeded on 24-well plates (5
of medium and incubated with 37 (HCT-116, HeLa) or 46 (MCF-7) for 24 h. Cells incubated with
ˆ
104/per well) in 300
µL
increasing concentrations of compounds (25–100
Axiovert 200 (Zeiss, Oberkochen, Germany).
µM) were observed after 24 h using light microscope
3.4.2. Cell Cycle Analysis
Cells were coincubated with compounds and a reference drug (cisplatin) for 24 h, respectively.
Following treatment, cells were fixed in cold 70% ethanol for 30 min. Fixed cells were stained for
15 min in buffer containing 500
µg/mL RNAse A (EURx, Gdansk, Poland) and 2.5
µg/mL of DAPI
(Sigma-Aldrich, Munich, Germany). The fluorescence of DAPI was measured at 440
flow cytometer (LSR II, BD Biosciences, Mountain View, CA, USA). Each experiment was performed at
least in triplicate. Data were analyzed off-line using the BD FACSDiva (BD Biosciences).
˘
40 nm with a
3.4.3. Mitochondrial Membrane Potential (∆ψ ) Analysis
m
The HCT-116, HeLa nad MCF-7 cells were coincubated with trazines and a reference drug
(cisplatin) for 24 h, respectively. Half an hour before the end of 24 h-treatment 2.5
(5,51,6,61-tetrachloro-1,11,3,31-tetraethylbenzimidazolylcarbocyanine iodide) was added to the cell
medium. As a negative control 100 M CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was used
µg/mL of JC-1
µ
15 min before JC-1 treatment. Following treatment cells were twice washed with PBS and analyzed
with flow cytometer (LSR II, BD Biosciences).
Green and red fluorescence were observed using FL-1 (530
channels, respectively. Percentage of cell population in red or green fluorescence gate indicated the
˘
30 nm) and FL-2 (585
˘
42 nm)
level of ∆ψ . Decreasing red fluorescence cell population indicated lower ∆ψ .
m
m
3.4.4. Translocation of Phosphatidylserine to Outer Leaflet of Cell Membrane
Analysis was performed using FITC-conjugated annexin-V (Annexin V-FITC Apoptosis Kit I, BD
Biosciences) according to the manufacturer’s instructions (BD Biosciences) as described previously [47].
HCT-116, HeLa and MCF-7 cells were coincubated for 24 h with traizines 37 and 46, respectively.
Following treatment cells were washed in PBS, centrifuged and then resuspended in binding buffer.
˝
Afterwards, the cells were incubated for 15 min at 37 C with FITC-conjugated annexin-V and
propidium iodide. The samples were then analyzed using a LSR II flow cytometer (BD Biosciences)
using 525
˘
20 nm (Annexin V-FITC) and 570
˘
26 nm (PI). The subpopulations were identified
according to their fluorescence: PI-low/FITC-low (live cells), PI-high/FITC-low (necrotic cells),
PI-low/FITC-high (early apoptotic cells), PI-high/FITC-high (late apoptotic cells).
3.4.5. Caspase Activity Determination
Caspase activity was determined with the FLICA Apoptosis Detection Kit (Immunochemistry
Technologies, Bloomington, MN, USA) according to the manufacturer’s instructions. Cells were treated
with the examined compounds for the indicated periods of time after which cells were collected and
suspended in a buffer containing the caspase inhibitor—a carboxyfluorescein-labeled fluoromethyl
ketone peptide. Cells were subsequently incubated for 1 h 37 ˝C under 5% CO2 cells and next were
washed with washing buffer. The fluorescence intensity of fluorescein was determined with flow
cytometry (BD FACSCalibur) and caspase activity was determined as the amount of fluorescence
emitted from caspase inhibitors bound to the caspases.

Molecules 2016, 21, 808
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3.4.6. Statistical Analysis
Statistical differences between control and triazine coincubated cells were determined using the
t-student test, after checking the normal distribution of data. The analyses were performed using 6 to
15 replicates run in at least three independent experiments.
3.5. Metabolic Stability
Stock solutions of studied compounds were prepared at concentration of 10 mM in DMSO.
Working solutions were prepared by dilution of stock with reaction buffer or acetonitrile, final
concentration of organic solvent did not exceed 1%. Incubation mixture contained 10 µM of a studied
derivative, 1 mM of NADPH (Sigma-Aldrich) and 0.5 mg/mL of human liver microsomes (HLM,
Sigma-Aldrich) in potassium phosphate buffer (0.1 M, pH 7.4). Incubation was carried out in thermostat
at 37 ˝C and started by addition of studied compound. 50
µ
L samples were taken after 5, 15, 30, 45
and 60 min. Enzymatic reaction was terminated by the addition of the equal volume of ice-cold
acetonitrile containing 10 M of alprazolam (Sigma-Aldrich) serving as internal standard (IS). Control
µ
incubations were performed without NADPH to assess chemical instability. After collection, samples
were immediately centrifuged (10 min, 10,000 rpm) and resulted supernatant was directly analyzed or
kept in
´
80 ˝C until LC-MS analysis. Natural logarithm of a compound over IS peak area ratio was
plotted versus incubation time. Metabolic half-time (t1/2) was calculated from the slope of the linear
regression, as reported in [48].
LC-MS analysis was performed on an Agilent 1260 system coupled to SingleQuad
6120 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Ascentis Express ES-CN
(2.1 mm
ˆ
100 mm, 2.7 µm, Supelco Analytical, Bellefonte, PA, USA) was used in reversed-phase
mode with gradient elution starting with 50% of phase A (10 mM ammonium formate in water) and
50% of phase B (10 mM ammonium formate in acetonitrile-water mixture, 95:5 v/v). The amount of
phase B was linearly increased to 100% in 5 min. Total analysis time was 10 min at 25 ˝C, flow rate
was 0.4 mL/min and the injection volume was 20 µL. The mass spectrometer was equipped with
electrospray ion source and operated in positive ionization. Mass analyzer was set individually to
each compound to detect [M + H]+ pseudomolecular ions. MSD parameters of the ESI source were as
˝
follows: nebulizer pressure 50 psig (N2), drying gas 13 mL/min (N2), drying gas temperature 300 C,
capillary voltage 3.5 kV, fragmentor voltage 100 V.
4. Conclusions
We have synthesized a series of novel 4-chloro-2-(R2-methylthio)-5-R1-N-(5-aryl-1,2,4-triazin-3-yl)
benzenesulfonamide derivatives using aminoguanidines and appropriate phenylglyoxal hydrates
in glacial acetic acid. The molecular structures of novel compounds were confirmed by
NMR, IR, and also by single crystal X-ray structure analysis for representative compound 59
.
All N-(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamides were tested for their in vitro cytotoxic activity
against the HCT-116, HeLa and MCF-7 cell lines. The most active compounds 37 and 38 belonged to
the 1-naphthyl series (R2 = 1-naphthyl) and showed outstanding average cytotoxic activity against all
tested cell lines with special regard to the HCT-116 (IC50: 36–38 µM) and HeLa (IC50: 34–42 µM) cell
lines. The MCF-7 cell line occurred less sensitive to 1,2,4-triazine derivatives, the strongest cytotoxic
potency showed 46. The ability to induce apoptosis in cancer cells was studied through biochemical
markers, such as: DNA fragmentation, loss of mitochondrial membrane potential, phosphatidylserine
translocation and caspase activation. Apoptotic-like changes in morphology of HCT-116, HeLa,
MCF-7 cell lines, significant accumulation of the HCT-116, HeLa and MCF-7 population in the
sub-G1 phase and DNA fragmentation, loss of ∆ψm, appearance of increased population of early
and late apoptotic cells, induction of caspase activity indicated apoptosis of the cells in presence of
37 and 46
. In vitro tests for metabolic stability in the presence of human liver microsomes showed
that both R2 and Ar substituents (4-CF3-C6H4) substantially increased the metabolic stability of

Molecules 2016, 21, 808
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4-chloro-2-(R2-methylthio)-5-R1-N-(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamides. The results pointed
to 37 as a hit compound with a good biological response against HCT-116 (IC50 = 36 M) and HeLa
µ
(IC50 = 34 µM) cell lines with apoptosis-inducing activity, and satisfactory metabolic stability.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
6/808/s1.
Acknowledgments: The project was financed by National Science Centre based on the decision number
DEC-2013/09/B/NZ7/00048.
˙
Author Contributions: B.Z. and J.S. created the concept, and designed the study. A.P. and K.S. performed
˙
chemical research. B.Z. and J.S. analyzed both chemical and biological data and wrote the manuscript together.
A.K. and G.S. tested the biological activity and made cytometric analysis of the compounds and interpreted the
results. J.C. performed crystallographic analysis. M.B, S.U. and T.B. carried out all experiments concerning the
metabolic stability. All Authors read and approved the final version of the article.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 21–22, 26–60 are available from the authors.
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