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

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 μM), HeLa (IC₅₀ = 34 μM) cell lines, apoptosis-inducing activity and moderate metabolic stability.

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 [1]. The 1,2,4-triazine ring as one of the most ubiquitous heterocycles in Nature, and it has been reported to possess a broad spectrum of biological properties, including anticonvulsant [2], neuroprotective [3], sedative [4], anxiolytic [5], benzodiazepine receptor inhibitory activity [6], antiparkinson [7], antidepressant [8], anti-inflammatory [9], antimicrobial [10], 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].

Our systematic studies on arylsulfonamides has resulted in promising anticancer agents [22,23,24,25,26,27,28,29], 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 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 1–5 [31,32], 3-amino-6-chloro-7-R¹-1,1-dioxo-1,4,2-benzodithiazines 6–10 [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.

The structures of the final compounds 27–60 were confirmed by IR, ¹H-NMR and ¹³C-NMR spectroscopy. ¹H-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 $P\overline{1}$. 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. Crystal data and structure refinement of 59.

Empirical Formula	C38H42ClN7O8S2
Formula weight	824.35
Temperature	120(2) K
Wavelength	1.54186 Å
Crystal system	Triclinic
Space group	$P\overline{1}$
Unit cell dimensions	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)°
Volume	3879.4(5) Å3
Z	4
Density (calculated)	1.411 Mg/m3
Absorption coefficient	2.399 mm−1
F(000)	1728
Crystal size	0.03 × 0.03 × 0.25 mm3
Theta range for data collection	1.984 to 64.981°
Index ranges	−8 ≤ h ≤ 8, −23 ≤ k ≤ 26, −25 ≤ l ≤ 23
Reflections collected	19645
Independent reflections	11676 [R(int) = 0.1757]
Completeness to θ = 67.686°	83.1%
Refinement method	Full-matrix least-squares on F2
Data/restraints/parameters	11,676/0/1014
Goodness-of-fit on F2	1.253
Final R indices [I > 2σ(I)]	R1 = 0.1476, wR2 = 0.3359
R indices (all data)	R1 = 0.2313, wR2 = 0.4260
Extinction coefficient	0.0114(11)

). 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.

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.

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. Cytotoxicity of compounds 27–60 toward human cancer cell lines ^a.

Compound	IC50 (μM)
	HCT-116	HeLa	MCF-7
27	75 ± 1	84 ± 1	80 ± 2
28	200 ± 4	100 ± 6	120 ± 5
29	720 ± 36	190 ± 7	110 ± 2
30	85 ± 2	90 ± 4	67 ± 1
31	140 ± 7	150 ± 6	77 ± 2
32	88 ± 3	87 ± 4	87 ± 4
33	67 ± 1	76 ± 3	88 ± 1
34	51 ± 1	73 ± 1	68 ± 3
35	68 ± 3	69 ± 1	78 ± 2
36	49 ± 2	55 ± 2	96 ± 4
37	36 ± 1	34 ± 2	70 ± 3
38	38 ± 2	42 ± 1	69 ± 1
39	83 ± 2	93 ± 1	95 ± 3
40	77 ± 1	85 ± 2	98 ± 3
41	97 ± 2	85 ± 1	99 ± 1
42	77 ± 2	82 ± 1	83 ± 2
43	92 ± 3	130 ± 10	87 ± 2
44	160 ± 6	160 ± 8	90 ± 5
45	97 ± 2	82 ± 1	90 ± 1
46	64 ± 1	80 ± 2	59 ± 2
47	75 ± 5	79 ± 1	78 ± 3
48	80 ± 3	80 ± 2	87 ± 3
49	110 ± 2	120 ± 6	81 ± 5
50	115 ± 3	130 ± 8	120 ± 4
51	190 ± 13	*	95 ± 5
52	70 ± 2	*	69 ± 1
53	76 ± 2	74 ± 2	88 ± 2
54	84 ± 5	82 ± 1	96 ± 5
55	150 ± 3	105 ± 4	150 ± 6
56	75 ± 2	80 ± 2	89 ± 3
57	73 ± 1	80 ± 1	90 ± 1
58	98 ± 2	88 ± 6	92 ± 6
59	140 ± 5	150 ± 2	100 ± 5
60	230 ± 18	230 ± 5	270 ± 13
Cisplatin	3.8 ± 0.2	2.2 ± 0.2	3 ± 0.1

^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 μM of tested compounds were approximately 100%.

. To describe of cytotoxic potency, the following scale was applied: IC₅₀ < 25 μM—very strong, 25 ≤ IC₅₀ ≤ 50 μM—strong, 50 < IC₅₀ < 75 μM—moderate, 75 ≤ IC₅₀ ≤ 100 μM—weak, IC₅₀ >100 μM—inactive compounds.

The most active compounds 37 and 38 belonged to the 1-naphthyl series (R² = 1-naphthyl) and showed outstanding average cytotoxic activity (47 μM and 50 μM, respectively) against all tested cell lines, HCT-116, HeLa and MCF-7. Moreover, compounds 34–36 and 46 moderately inhibited all cell lines viability, exhibiting average IC₅₀ values below 71 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 (33–38, 46, 47, 52, 56 and 57) in the range of IC₅₀ values 36–75 μM. Meanwhile, the HeLa cell line was susceptible toward six compounds (34–38 and 53) with IC₅₀; 34–74 μM, and the MCF-7 by six compounds (30, 34, 37, 38, 46 and 52) with IC₅₀ values ranging between 59 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 (Δψ_m), 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.

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.

Mitochondrial Membrane Potential (Δψ_m) Analysis

Loss of the mitochondrial membrane potential (Δψ_m) is one of the earliest indicators of an apoptosis [38]. It can be detected using fluorescent dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-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 Δψ_m, respectively.

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 Δψ_m which is the earliest indicators of an apoptotic cell death.

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.

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 [39,40,41].

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. Results of metabolic stability study.

Compound	In Vitro Metabolic Half-Life t1/2 (min)
30	24.9
31	17.6
34	>60 (154)
35	42.4
36	13.9
37	32.8
38	<5
46	>60 (78.1)
47	17.5
52	26.5

).

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 t_1/2 value from >60 to 42.4 min, while an analogous change between 46 and 47 caused a significant decrease of t_1/2 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.

Comparison of t_1/2 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).

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 400–4000 cm⁻¹. ¹H-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 [31,32], 3-amino-6-chloro-7-R¹-1,1-dioxo-1,4,2-benzodithiazines 6–10 [33,34], N-(2-alkylthio-4-chloro-5-R¹-benzenesulfonyl)cyanamide potassium salts 11–18 [22,25,33,35], 1-amino-2-(4-chloro-5-R¹-2-alkylthiobenzenesulfonyl)guanidines 19–20 and 23–25 [25,29,33,35,36]. 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_max 3479, 3345, 3341 (NH); 2926, 2851 (C–H); 1268, 1131 (SO₂) cm⁻¹; ¹H-NMR (500 MHz, DMSO-d₆) 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, 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) δ 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%.

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) δ 3.75 (s, 3H, OCH₃), 4.41 (s, 2H, SCH₂), 4.54 (s, 2H, NH₂), 6.92–7.03 (m, 4H, 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 × 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_max 3430, 3196 (NH), 3074, 2921 (C–H), 1585, 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) ν_max 3457, 3182 (NH), 3089, 2929 (C–H), 1585, 1560 (C=C, C=N), 1320, 1123 (SO₂) cm⁻¹; ¹H-NMR (200 MHz, DMSO-d₆) δ 2.37 (s, 3H, CH₃), 4.32 (s, 2H, SCH₂), 7.10–7.13 (m, 3H, arom), 7.28–7.30 (m, 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) δ 19.1, 36.2, 121.3, 126.1, 126.2, 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_max 3188 (NH), 2918, 2831 (C–H), 1606, 1575 (C=C, C=N), 1533 (NH_def.), 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) ν_max 3199 (NH), 2934 (C–H), 1631, 1582 (C=C, C=N), 1561 (NH_def.), 1283, 1132 (SO₂) cm⁻¹; ¹H-NMR (200 MHz, DMSO-d₆) δ 2.28 (s, 3H, CH₃), 3.81 (s, 3H, OCH₃), 4.29 (s, 2H, SCH₂), 7.10–7.13 (m, 3H, 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_max 3447, 3125 (NH), 3000, 2936 (C–H), 1615, 1554, 1519 (C=C, C=N), 1316, 1133 (SO₂) cm⁻¹; 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_max 3436 (NH), 3073, 2936 (C–H), 1594, 1550 (C=N, C=C), 1332, 1129 (SO₂) cm⁻¹; ¹H-NMR (200 MHz, DMSO-d₆) δ 2.28 (s, 3H_, CH₃), 3.79 (s, 3H, OCH₃), 3.85 (s, 6H, 2×OCH₃), 4.32 (s, 2H, SCH₂), 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) δ: 19.1, 36.3, 56.7, 60.7, 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) ν_max 3436, 3186 (NH), 3092, 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]-benzenesulfonamide (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 (KBr) ν_max 3444 (NH), 3078, 2925 (C–H), 1581, 1556 (C=C, C=N), 1325, 1131 (SO₂) cm⁻¹; ¹H-NMR (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-sulfonamide (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_max 3435 (NH), 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%.

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_max 3432 (NH), 3052, 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]-benzenesulfonamide (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 3438 (NH), 3080, 2927 (C–H), 1580, 1554 (C=C, C=N), 1323, 1162 (SO₂) cm⁻¹; ¹H-NMR (500 MHz, DMSO-d₆) 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, 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) δ 19.6, 34.9, 124.8, 125.9, 126.6, 126.9, 128.6, 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-sulfonamide (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_max 3450 (NH), 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-benzenesulfonamide (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_max 3435 (NH), 3071, 2963, 2925, 2854, 2737 (C–H), 1551, 1514 (C=C, C=N), 1326, 1156 (SO₂) cm⁻¹; ¹H-NMR (500 MHz, DMSO-d₆) δ 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) δ 10.3, 11.2, 19.6, 25.6, 110.3, 115.3, 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) ν_max 3434 (NH), 3072, 2754 (C-H), 1550, 1519 (C=C,C=N), 1325, 1156 (SO₂) cm⁻¹; ¹H-NMR (500 MHz, 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) 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) δ 2.08 (s, 3H, CH₃), 2.13 (s, 3H, CH₃), 2.43 (s, 3H, CH₃), 3.87 (s, 3H, OCH₃), 4.08 (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 (NH_def.), 1315, 1174 (SO₂) cm⁻¹; ¹H-NMR (200 MHz, DMSO-d₆) δ 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), 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_max 3436, 3326 (NH), 2935 (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) ν_max 3419, 3328 (NH), 2933 (C–H), 1651 (C=O), 1600, 1540 (C=N, C=C), 1318, 1133 (SO₂) cm⁻¹; ¹H-NMR (200 MHz, DMSO-d₆) δ 3.72 (s, 3H, OCH₃), 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) ν_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) δ 4.42 (s, 2H, SCH₂), 7.17–7.36 (m, 3H, arom), 7.43–7.63 (m, 6H, 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%.

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) δ 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) δ 35.9, 121.4, 126.3, 126.4, 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 and the title compound 47 (0.363 g, 56%) was isolated mp 282–284 °C; IR (KBr) ν_max 3411 (NH), 2925, 2843 (C–H), 1663 (C=O), 1604, 1577 (C=N, C=C), 1537 (NH_def.), 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 heating under reflux with MeCN and the title compound 48 (0.356 g, 56%) was isolated mp 256–258 °C; IR (KBr) ν_max 3318 (NH), 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_max 3435, 3327 (NH), 2921 (C–H), 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_max 3301, 3102 (NH), 2925, 2853 (C–H), 1682 (C=O), 1596, 1552 (C=C, C=N), 1333, 1131 (SO₂) cm⁻¹; ¹H-NMR (200 MHz, DMSO-d₆) δ 3.69 (s, 3H, OCH₃), 3.79 (s, 6H, 2×OCH₃), 4.43 (s, 2H, SCH₂), 7.24–7.46 (m, 8H, arom), 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%.

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) ν_max 3427, 3278 (NH), 3064, 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) ν_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) δ 2.31 (s, 3H, CH₃), 4.43 (s, 2H, SCH₂), 7.16–7.21 (m, 5H, arom), 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) ν_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) δ 2.31 (s, 3H, CH₃), 3.78 (s, 3H, OCH₃), 4.40 (s, 2H, SCH₂), 7.05 (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) ν_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) δ 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) δ 20.8, 35.9, 55.7, 114.2, 119.9, 120.1, 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)-benzamide (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_max 3329, 3093 (NH), 2921, 2852 (C–H), 1640 (C=O), 154, 1518 (C=N, C=C), 1318, 1132 (SO₂) cm⁻¹; ¹H-NMR (200 MHz, DMSO-d₆) δ 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), 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) ν_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) δ 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) δ 20.8, 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_max 3331 (NH), 3092, 2932, 2835 (CH₂, CH₃), 1676 (C=O), 1608, 1543 (C=N, C=C), 1313, 1162 (SO₂) cm⁻¹; ¹H-NMR (200 MHz, DMSO-d₆) δ 3.77 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 4.39 (s, 2H, 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_max 3333 (NH), 3083, 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) δ 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) δ 35.9, 55.5, 55.7, 114.2, 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_max 3393, 3262 (NH), 2924, 2851 (C–H), 1679 (C=O), 1543, 1514 (C=C, C=N), 1312, 1126 (SO₂) cm⁻¹; ¹H-NMR (200 MHz, DMSO-d₆) δ 3.76-3.78 (m, 9H, 3×OCH₃), 4.41 (s, 2H, 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 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) δ 3.71-3.79 (m, 12H, 4×OCH₃), 4.42 (s, 2H, SCH₂), 6.92 (d, J = 8.9 Hz, 2H, arom), 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 R_int 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: [email protected] 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 × 10³ cells/well and treated for 72 h with the examined compounds in the concentration range 1–100 μM (1, 10, 25, 50 and 100 μM). Following treatment, MTT (0.5 mg/mL) was 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.

3.4.1. Cell Morphology

The HCT-116, HeLa and MCF-7cells were seeded on 24-well plates (5 × 10⁴/per well) in 300 μL of medium and incubated with 37 (HCT-116, HeLa) or 46 (MCF-7) for 24 h. Cells incubated with increasing concentrations of compounds (25–100 μM) were observed after 24 h using light microscope Axiovert 200 (Zeiss, Oberkochen, Germany).

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 ± 40 nm with a 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).

3.4.3. Mitochondrial Membrane Potential (Δψ_m) Analysis

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 μg/mL of JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) was added to the cell medium. As a negative control 100 μM CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was used 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 ± 30 nm) and FL-2 (585 ± 42 nm) channels, respectively. Percentage of cell population in red or green fluorescence gate indicated the level of Δψ_m. Decreasing red fluorescence cell population indicated lower Δψ_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.

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 μL 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 μM of alprazolam (Sigma-Aldrich) serving as internal standard (IS). Control 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 (t_1/2) 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 × 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 Δψ_m, 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 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 μM) and HeLa (IC₅₀ = 34 μM) cell lines with apoptosis-inducing activity, and satisfactory metabolic stability.