Synthetic Approaches for Sulfur Derivatives Containing 1,2,4-Triazine Moiety: Their Activity for in Vitro Screening towards Two Human Cancer Cell Lines

Abstract

A series of sulfur 1,2,4-triazine derivatives were prepared and evaluated as anticancer compounds for two human breast cancer cell lines (MCF-7, MDA-MB-231) with some of them acting as low micromolar inhibitors. Evaluation of the cytotoxicity using a 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) assay, the inhibition of [³H]thymidine incorporation into DNA, and collagen synthesis inhibition demonstrated that these products exhibit cytotoxic effects on these breast cancer cell lines in vitro. The most effective were disulfide and sulfenamide compounds with two valence sulfur atoms. A structure–activity relationship study was performed using X-ray analysis and theoretical calculations at an ab initio density functional theory (DFT) level.

Heteroaromatic compounds possess a wide spectrum of biological activities. Indeed the class of those containing 1,2,4-triazines has been attracting attention for a long time and have received considerable interest due to their medicinal value.¹⁾ 1,2,4-Triazine moiety has been found to be present in the skeleton of various natural products and a large number of compounds containing this moiety exhibits antimicrobial, antitubercular, analgesic, anti-inflamantory, anticonvulsant, antiviral and antidepressant activities.^2,3) Recognizing that a 1,2,4-triazine could be considered as an azapyrimidine, early work focused on the development of 6-azapyrimidine nucleotides and nucleosides.^4,5) This moiety has attracted much attention as a novel anticonvulsant for the treatment of epilepsy,^6–8) and in recent years, the 1,2,4-triazine ring system has been the object of much effort directed at identifying new anticancer drugs such as the hydrazide, or anti-human immunodeficiency virus (HIV) agents, exemplified by bromide.⁹⁾ Additionally, 1,2,4-triazines are potential blood platelet aggregation inhibitors, kinase inhibitors, and agonists/antagonists for other central nervous system (CNS) targets.¹⁰⁾

Heteroaromatic compounds containing sulfur atom as organosulfur compounds are also important classes of organic compounds which exhibit numerous pharmaceutical activities.¹¹⁾ In contrast to those, the number of heterocycles including azole scaffold seems to be less known in the literature, especially 1,2,4-triazines.

In this report, we have designed and synthesized a small library of novel sulfur triazine compounds bearing a sulfur atom whose valency is different depending on the compound e.g. two valence typical for sulfide in (–S–R) or (–S–N), four valence in (–SO–N) sulfinylamide, six valence in (–SO₂–N) sulfonamide substituent. These compounds were selected for in vitro screening towards two human cancer cell lines. It is obvious that potentially biological activities are closely correlated to molecular structures. Therefore the structural investigations using X-ray analysis and theoretical calculations at ab initio density functional theory (DFT) level were performed in order to find these structural and electronic parameters which can be responsible for biological activity of the analyzed sulfur derivatives.

Results and Discussion

In this project we have explored new substitution patterns at the 1,2,4-triazine ring containing different valence of sulfur atom. The procedure started from 5,6-diphenyl-1,2,4-triazine-3-thiol (1).¹²⁾ During our study we found that the reaction of 1 with halogenating agent led to the formation of very unstable sulfenyl bromide 1a which, in the presence of amine undergoes further transformation. The proposed mechanism leading to the formation of the sulfenamides and disulfide 2¹³⁾ is outlined in Chart 1.

Chart 1. Synthesis of Derivatives 1–6

Reagents and conditions: (a) NBS, DMF, 2 h, rt; (b) Amine, THF, 24 h, rt; (c) m-CPBA, CH₂Cl₂, 24 h, 0°C; (d) 5,6-Diphenyl-1,2,4-triazine-3-thiol 1.

When the non-isolable sulfenyl bromide 1a reacts with morpholine and N-pyrrolidine we afforded the corresponding products 3 and 4. In contrast to that when the reaction mixture with N-bromosuccinimide (NBS) was performed disulfide delivered as the consequence of the reaction with thiol. The final products 5 and 6 were obtained by treating sulfenamide 3 and 4 with m-chloroperbenzoic acid in dichloromethane. The structures of newly obtained compounds were confirmed by high resolution (HR)-MS, spectroscopic data and X-ray analysis (2 and 7) and then characterized by physicochemical techniques in Experimental. Furthermore, for the biological screening we have proposed to check activity of sulfur compounds (sulfide) obtained in our earlier study.¹⁴⁾ Synthesis of sulfides 7, 8, 9 was prepared using 3,3′-bis-chloro-5,5′-bi-1,2,4-triazine as starting compound, which in the nucleophilic substitution reaction with appropriate thiol delivered the expected compounds (Chart 2).

Chart 2. Synthesis of Derivatives 7–9

Reagents and conditions: K₂CO₃, thiol, DMSO, 24 h.

The viability of MCF-7 and MDA-MB-231 breast cancer cells was measured by the method of Carmichael using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium¹⁵⁾ (Table 1).

Table 1. Cytotoxic and Cytostatic Activities of New Sulfur 1,2,4-Triazine Derivatives 1–9 after 24 h Incubation

Compd	MTT assay, IC50a) (µM)			[3H]Thymidine incorporation, IC50a) (µM)
	MCF-7	MDA-MB-231	Fibroblasts	MCF-7	MDA-MB-231	Fibroblasts
1	ND	ND	ND	100±2	ND	ND
2	25±3	25±2	ND	35±2	39±1	ND
3	91±2	88±1	ND	87±1	84±2	ND
4	30±2	25±2	ND	15±2	40±2	ND
5	ND	ND	ND	100±1	ND	ND
6	ND	ND	ND	ND	ND	ND
7	ND	ND	ND	ND	ND	ND
8	ND	ND	ND	ND	ND	ND
9	ND	ND	ND	ND	ND	ND
Cisplatin	82±2	93±1	ND	98±2	86±2	98±2

a) Data presented the mean±standard deviation (S.D.) of each compound from four independent experiments.

Although growth inhibition was concentration dependent in either cell line, it was more pronounced at shorter times, in MCF-7 than MDA-MB-231 (Table 1). In terms of reduction in cell viability, the compounds rank in both MCF-7 and MDA-MB-231 cells in the order 2>4>3, the remaining compounds were inactive. We have studied the effect of compounds 1–9 on DNA synthesis in both MDA-MB-231 and MCF-7 breast cancer cells (Table 1).

All of the tested compounds showed concentration dependent activity, yet with different potency. The concentrations of 1, 5–9 needed to inhibit [³H]thymidine incorporation into DNA by 50% (IC₅₀) in MDA-MB-231 and MCF-7 cells was above 100 µM suggesting low cytotoxic potency. The concentrations of 4, 2, 3 needed 50% reduction in [³H]thymidine incorporation into DNA in breast cancer MCF-7 (IC₅₀) obtained in the range 15; 35 and 87 µM and in MDA-MB-231 40, 39 and 84 µM, respectively.

Collagen biosynthesis was measured in MCF-7 and MDA-MB-231 breast cancer cells treated with various concentrations of compounds 1–9 (Table 2) for 24 h. In both cell lines compounds 4 and 2 were found to be more effective inhibitors of collagen biosynthesis. IC₅₀ for 4 and 2 (in MDA-MB-231; 40 µM and 40 µM, in MCF-7; 17 µM and 38 µM, respectively) showed specific inhibitory effect of compounds 4 and 2 on collagen biosynthesis.

Table 2. Collagen Synthesis, Measured by 5-[³H]Proline Incorporation into Proteins Susceptible to the Action of Callogenase, in MCF-7 and MDA-MB-231 Breast Cancer Cells in the Presence of Compounds 1–9

Compd	IC50a) (µM)
	MCF-7	MDA-MB-231
1	100±2	110±1
2	38±2	40±1
3	75±3	90±2
4	17±2	40±2
5	100±1	ND
6	ND	ND
7	ND	ND
8	ND	ND
9	ND	ND
Cisplatin	30±2	64±1

a) Mean values from three independent experiments done in duplicates ±S.D. are presented.

The X-ray analysis was performed for compounds 2 and 7. The crystal structure of 5,5′,6,6′-tetraphenyl-bis-(1,2,4-triazine)-3,3′-disulfide (2) is known¹³⁾ and deposited in the Cambridge Structural Database CSD (ID: KEJRAT).^16,17) The basic crystal data of 2 are presented in Experimental part and its molecular structure observed in crystal is shown in Fig. 1. The crystal and structural data of 2 received by us are in good agreement with the data from CSD and they are not discussed in detail in this paper. It should be emphasized, that molecule 2 has a gauche conformation with respect to S–S bond with the torsion angle φ=C3A–S1A–S1B–C3B of 74.19(19)°.

Fig. 1. A View of the X-Ray Molecular Structure of 2 and 7 with the Atomic Labeling

Non-H atoms are represented by displacement ellipsoids of 50 and 30% probability in 2 and 7, respectively.

The X-ray investigation confirmed the synthesis pathway and assumed molecular structures of 3,3′-bis-p-chlorophenylsulfanyl-5,5′-bi-1,2,4-triazine 7. This compound crystallized with four molecules in an asymmetric part of the unit cell (see: Experimental). Because these four independent molecules exhibit almost the same metric parameters, only the molecular structure of molecule AB (Fig. 1) is discussed.

The bond lengths and angles of the triazine and phenyl rings and their sulfanyl spacers are in normal ranges.¹⁸⁾ The X-ray analysis showed, that two 3-p-chlorophenylsulfanyl-1,2,4-triazinyl parts of molecule 7 adopt the trans conformation with the torsion angle ψ=N4A–C5A–C5B–N4B of −179.5(4)°. In this conformation short intramolecular hydrogen bond C6A–H6A…N4B is observed (Table 3). The 1,2,4-triazine rings are planar within 0.018(4) Å (ring A) and 0.032(4) Å (ring B) with the length of the central C5A–C5B bond of 1.480(4) Å being in good agreement with the corresponding values of 1.485(6) Å in related structure of 3,3′-bis(2-pyridylthio)-5,5-bi-1,2,4-triazine.¹⁴⁾ The 3-p-chlorophenylsulfanyl substituents have trans-gauche conformation relative to the respective 1,2,4-triazine rings with the torsion angles N2–C3–S7–C11 of −171.8(3) and 166.8(3)° and C3–S7–C11–C12 of 62.1(4) and −64.6(4)° in part A and B, respectively. In the crystal of 7, the screw-related molecules AB and translation-related molecules CD with AB and EF with CD are linked to form chains along the Y axis by C–H…N intermolecular hydrogen bonds (Table 3).

Table 3. Hydrogen-Bond Geometry (Å, ^o) in 7

D–H…A	D–H	H…A	D…A	D–H…A
C6A–H6A…N4B	0.93	2.48	2.801(6)	100
C6D–H6D…N4C	0.93	2.47	2.784(6)	100
C6E–H6E…N4F	0.93	2.47	2.797(5)	101
C6H–H6H…N4G	0.93	2.44	2.768(6)	101
C12A–H12A…N1Ai	0.93	2.62	3.423(6)	145
C12C–H12C…N1Bii	0.93	2.57	3.422(5)	153
C12E–H12E…N1Diii	0.93	2.56	3.397(6)	150

(i)=1−x, −1/2+y, 1−z; (ii)=x, 1+y, z; (iii)=x, −1+y, z.

Theoretical calculations at the ab initio DFT/B3LYP/6-311++G(d, p) level for all the investigated compounds 1–9 were performed in order to characterize those structural, energetic and electronic parameters which can be connected with biological activity of these compounds. The total energies, dipole moments, energies of the frontier orbitals highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) and HOMO–LUMO energy gap are summarized in Table 4.

Table 4. The Molecular Energies, E (kcal/mol), Energies of HOMO and LUMO Orbitals, E_HOMO (kcal/mol) and E_LUMO (kcal/mol), HOMO–LUMO Energy Gap, E_LUMO–E_HOMO (kcal/mol) and Dipole Moments, D_m (D), Calculated at DFT/B3LYP/6-311++G(d, p) Level for 1–9

Compd	E	EHOMO	ELUMO	ELUMO–EHOMO	Dm
1	−715889.74	−152.15	−57.07	95.09	3.477
2	−1431020.55	−149.30	−55.72	93.58	2.521
3	−895773.10	−149.36	−56.15	93.21	3.792
4	−848563.26	−142.73	−54.19	88.54	2.493
5	−895751.93	−149.35	−59.47	89.88	5.079
6	−942961.23	−160.82	−63.64	97.19	5.938
7	−1717800.76	−163.64	−82.95	80.69	0.003
8	−1781413.19	−157.47	−81.76	75.71	0.001
9	−1756546.56	−162.81	−83.94	78.87	0.000

View of the molecules 1–9 in conformation obtained after energy minimization and geometry optimization with the vector of dipole moment is presented in Fig. 2.

Fig. 2. The Molecular Structures and Dipole Moments of 1–9 Obtained from DFT/B3LYP/6-311++G(d, p) Calculations

The theoretical calculations showed that the conformations of the molecules 2 and 7 do not differ significantly from those observed in the crystalline state giving φ=80.30° and ψ=179.91° in 2 and 7, respectively. The sulfenylmorpholine, sulfenylpyrrolidine and sulfonylpyrrolidine substituents adopted a cis conformation with respect to the 1,2,4-triazine ring in 3, 4 and 6, respectively, with the torsion angle N2(triazine)–C3(triazine)–S–N of 4.52° in 3, 1.47° in 4 and 3.50° in 6, while this torsion angle being −93.57° indicates a gauche conformation of sulfinylpyrrolidine substituent in 5. The central 1,2,4-triazine rings in 8 and 9, similarly as in 7, have a trans conformation to each other as shown by the torsion angle N4–C5–C5′–N4′ of 180.0° in both molecules. In a conformation corresponding to the energy minimum the molecules 1–6 are polar with the dipole moment varying from 2.511 D in 2 to 5.938D in 6 and directed towards the phenyl substituents of the triazine ring, while the molecules 7–9 are non-polar due to inversion symmetry in conformation trans on the C5–C5′ central bond (Table 4).

The biological activity of compounds 2 and 7–9 seems to be strictly connected with conformation at the central bond S–S in 2 and C5–C5′ in 7–9. Therefore, for the molecules 2 and 7 the energy effects of the free rotation between the triazine rings, taking into account the one degree of freedom described by torsion angle φ in 2 and ψ in 7, were calculated using the AM1 method. The differences in heat of formation, ΔHF, of the conformations were calculated after energy minimization and optimization of all geometrical parameters for each rotation, with a 10° increment from −180° to 180° of φ and ψ (Fig. 3). The calculations showed, that the calculated conformations with minima of energy (gauche in 2 and trans in 7) are in good agreement with those observed in the crystalline state for these molecules. The energy barriers for the conformation interconversion of about 8.5 kcal/mol for 2 and 4.0 kcal/mol for 7 show, that both molecules can practically rotate freely about the central bond under physiological conditions.

Fig. 3. The Energy Effect upon S–S (φ; ■ for 2) and C5–C5′ (ψ; ▲ for 7) Rotation as Calculated Using the AM1 Semi-empirical Method

The HOMO–LUMO energy gap and ionization potential (IP=−E_HOMO) being both in the range of about 20 kcal/mol and increasing according to the relation 8<9<7<4<5<3<2<1<6 and 4<5<3<2<1<8<6<9<7 for E_LUMO–E_HOMO and IP, respectively, indicate similar stability and reactivity of all the investigated compounds. The HOMO orbital is concentrated on the whole molecule in 2 and 3, in the triazine ring and sulfur atom substituent in 4–6, on the triazine ring in 7 and on the triazine (in 8) and benzothiazole substituents (in 9) of the sulfanyl group. In all the investigated molecules the LUMO orbital were distributed on triazine ring and additionally on phenyl substituents in 2–6. The drawings of the HOMO and LUMO orbitals for 2, 5 and 7 are presented in Fig. 4.

Fig. 4. Schematic Drawings of the HOMO and LUMO Orbitals of 2, 5 and 7 as Calculated Using DFT/B3LYP/6-311++G(d, p) Method

The analysis of the molecular structure of compounds 2, 3 and 4 with anticancer activity shows that the presence of the S–S spacer between triazine rings (2) and S–N bond to morpholine (3) and pyrrolidine (4) rings can be responsible for biological activity. The calculated Natural Bond Orbital (NBO) charges on S atoms of the disulfide chain and S atom and N atom of the morpholine and pyrrolidine rings are respectively 0.208e and 0.207e for 2, 0.514e and −0.691e for 3, 0.516e and −0.703e for 4, 0.355e and −0.737e for 5 and 2.226e and −0.773e for 6. It can be observed, that the positive net charge on S atom depends on the valence of sulfur atom and increases in direction (–S–)<(–SO–)<(–SO₂–), while the negative net charge on N atom is very similar in morpholine and pyrrolidine ring.

In order to predict a lipophilic character of the molecules 1–9 their partition coefficients P were used. The calculated values of logP are as follows: 4.26 for 1, 8.96 for 2, 3.92 for 3, 4.59 for 4, 5.56 for 5, 3.64 for 6, 6.23 for 7, 9.72 for 8 and 5.80 for 9. It can be noticed, that anticancer inactive molecules with the bi-1,2,4-triazine system (with exception of 2) have a much higher affinity for a lipid phase as compared with the other investigated compounds.

Conclusion

Our experimental study identified only two structures, 2 and 4, as the most cytotoxic compounds among a series of sulfur substituted triazine scaffolds 1–9, which we have synthesized to date. The structure–activity relationship (SAR) analysis revealed that the presence of the S–S spacer between triazines scaffold and S–N bond and between triazine moiety and pyrrolidine ring is responsible for biological activity. Moreover, the polarity of molecules and moderate affinity to the lipid phase favour anticancer activity of 2–4. However, these conditions are not sufficient to achieve the desired biological activity, because they are determined by inactive compounds 5 and 6. Further investigations on the mechanisms of the cytotoxicity and preparation of new triazine derivatives are now in progress.

Experimental

Cell Culture

Human breast cancer MDA-MB-231, MCF-7 and human skin fibroblasts cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin, 50 µg/mL streptomycin at 37°C. Cells were cultured in Costar flasks and subconfluent cells were detached with 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) in calcium-free phosphate buffered saline, counted in hemocytometers and plated at 5105 cells per well of 6-well plates (Nunc) in 2 mL of growth medium (DMEM without phenol red with 10% CPSR1). Cells reached about 80% of confluency at day 3 and in most cases such cells were used for the assays.

Cell Viability Assay

The assay was performed according to the method of Carmichael¹⁵⁾ using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confluent cells, cultured for 24 h with various concentrations of studied compounds in 6-well plates were washed three times with phosphate buffered saline (PBS) and then incubated for 4 h in 1 mL of MTT solution (0.5 mg/mL of PBS) at 37°C in atmosphere of 5% CO₂ in an incubator. The medium was removed and 1 mL of 0.1 mol/L HCl in absolute isopropanol was added to the cells attached. Absorbance of converted dye in the living cells was measured at a wavelength of 570 nm. Cell viability of breast cancer cells cultured in the presence of ligands was calculated as percentage of controlled cells.

DNA Synthesis Assay

To examine the effect of the study compounds on cells proliferation, MCF-7 and MDA-MB-231 cells were seeded in 6-well plates and grown as described above. Cells cultures were incubated with varying concentrations of studied compounds and 0.5 µCi of [³H]thymidine for 24 h at 37°C.¹⁹⁾ The cells were harvested by trypsinization and washed several times in the cold PBS (10 min/1500 g) until the radioactivity (dpm) in the washes were similar to the reagent control. Radioactivity was determined by liquid scintillation counting. [³H]Thymidine uptake was expressed as dpm/well.

Collagen Synthesis Assay

Incorporation of a radioactive precursor into proteins was measured after labeling the cells with 5-[³H]proline (5 µCi/mL) for 24 h in growth medium with varying concentrations of studied compounds. Incorporation of a tracer into collagen was determined by digesting proteins with purified Clostridium histolyticum collagenase, according to the method of Peterkofsky et al.²⁰⁾

General Methods

Melting points were determined on Boethius melting point apparatus and are uncorrected. The ¹H-NMR spectra were recorded on a Varian Gemini 400 MHz spectrometr with tetramethylsilane, or trimethylsilyl (TMS) as internal standard in deuterated solvents. The chemical shifts are given in δ (ppm). Mass spectra were measured on AMD 604 spectrometer. Compound 1 was prepared according to the literature procedure.¹²⁾

Synthesis of 5,5ʹ,6,6ʹ-Tetraphenyl-bis-(1,2,4-triazine)-3,3ʹ-disulfide 2

To a mixture of 2.5 g (9.40 mmol) of 5,6-diphenyl-1,2,4-triazine-3-thiol in 15 mL of N,N-dimethylformamide (DMF), 1.6 g (9.3 mmol) N-bromosuccinimide was added. The mixture was stirred for 2 h (monitoring by TLC). After the end of reaction, the mixture was poured into ice/H₂O, then was filtered and concentrated in vacuo. The precipitate was filtered off and the crude product was purified by column chromatography on silica gel, using CH₂Cl₂ as eluent, to give pure compound 2. Yield 80%; mp 184°C; ¹H-NMR (CDCl₃, 400 MHz) δ: 7.24–7.40 (m, 5H), 7.47–7.50 (m, 5H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 128.50, 128.64, 129.49, 129.76, 129.81, 131.17, 134.72, 144.88, 155.21, 155.90, 167.73; HR-MS electron ionization (EI, m/z) Calcd for C₃₀H₂₁N₆S₂ [M+H]⁺ 529.12636. Found 529.12713.

General Procedure for the Synthesis of 3 and 4

To a mixture of 0.14 mmol 5,5ʹ-6,6ʹ-tetraphenyl-bis-(1,2,4-triazine)-3,3ʹ-disulfide in 2 mL of THF, 0.24 mmol amine was added. The mixture was stirred at room temperature for 24 h (monitoring by TLC). The precipitate was concentrated in vacuo and the crude product was purified by column chromatography on silica gel, using CH₂Cl₂ : hexane (3 : 1) as eluent, to give pure compounds 3 and 4.

5,6-Diphenyl-3-sulfenylmorpholinamide-1,2,4-triazine 3

Yield 20%; mp 115°C; ¹H-NMR (CDCl₃, 400 MHz) δ: 3.85 (t, J=4.8 Hz, 4H), 4.03 (t, J=4.8 Hz, 4H), 7.31–7.62 (m, 10H), ¹³C-NMR (CDCl₃, 100 MHz) δ: 44.0, 66.8, 128.3, 128.7, 129.1, 129.6, 130.2, 136.2, 136.5, 149.0, 155.9, 159.6; HR-MS electron spray ionization (ESI, m/z) Calcd for C₁₉H₁₈N₄SONa [M+Na]⁺ 373.1103. Found 373.1094.

5,6-Diphenyl-3-sulfenylpyrrolidinamide-1,2,4-triazine 4

Yield 57%; mp 178°C; ¹H-NMR (CDCl₃, 400 MHz) δ: 2.04–2.08 (m, 4H), 3.78 (br s, 4H), 7.26–7.32 (m, 5H), 7.76–7.45 (m, 3H), 7.50–7.53 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 25.5, 46.6, 155.9, 158.3, 127.9, 128.2, 129.1, 129.7, 129.9, 136.7, 147.8; HR-MS electron spray ionization (ESI, m/z) Calcd for C₁₉H₁₉N₄S [M+H]⁺ 335.13249. Found 335.13070.

General Procedure for the Synthesis of 5 and 6

To a mixture of 0.19 mmol of 5,6-diphenyl-3-sulfenylpyridin-1,2,4-triazine (4) in 10 mL of CH₂Cl₂, 0.39 mmol of m-chloroperbenzoic acid (m-CPBA) was added. The mixture was stirred at 0°C for 24 h (monitoring by TLC). The precipitate was concentrated in vacuo and the crude product was purified by column chromatography on silica gel, using CH₂Cl₂ : CH₃COCH₃ (100 : 1) as eluent, to give pure compound 5 and 6.

5,6-Diphenyl-3-sulfinylpyrrolidinamide-1,2,4-triazine 5

Yield 39%; mp 191°C; ¹H-NMR (CDCl₃, 400 MHz) δ: 2.04 (br s, 4H), 4.05 (br s, 4H), 7.26–7.41 (m, 10H), ¹³C-NMR (CDCl₃, 100 MHz) δ: 25.2, 25.5, 46.7, 46.9, 128.5, 128.6, 129.3, 129.4, 131.0, 135.3, 136.2, 159.4, 162.9; HR-MS (ESI, m/z) Calcd for C₁₉H₁₈N₄SO [M+H]⁺ 350.12043. Found 350.12093.

5,6-Diphenyl-3-sulfonylpyrrolidinamide-1,2,4-triazine 6

Yield 40%; mp 167°C; ¹H-NMR (CDCl₃, 400 MHz) δ: 2.04 (br s, 4H), 4.05 (br s, 4H), 7.26–7.41 (m, 10H), ¹³C-NMR (CDCl₃, 100 MHz) δ: 25.2, 25.5, 46.7, 46.9, 128.5, 128.6, 129.3, 129.4, 131.0, 135.3, 136.2, 159.4, 162.9; HR-MS (ESI, m/z) Calcd for C₁₉H₁₈N₄SO₂ [M+H]⁺ 366.16046. Found 366.16833.

General Procedure for the Synthesis of 7–9

To a mixture of 0.66 g (4.79 mmol) of K₂CO₃ in 7 mL of DMSO, 4.39 mmol of thiol was added. The mixture was stirred at room temperature for 15 min. After that 2.18 mmol of 3,3ʹ-bis-chloro-5,5ʹ-bi-1,2,4-triazine 2 was added. The mixture was stirred for 24 h (monitoring by TLC). After the end of reaction, the mixture was poured into ice/H₂O and was extracted with ether (5×10 mL). The combined extracts were dried over MgSO₄, then filtered and concentrated in vacuo. The precipitate was filtered off and the crude product was purified by column chromatography on silica gel, using CH₂Cl₂ as eluent, to give pure compounds 7, 8 and 9.

3,3ʹ-Bis-p-chlorophenylsulfanyl-5,5ʹ-bi-1,2,4-triazine 7

Yield 36%; mp 195–200°C; ¹H-NMR (CDCl₃, 400 MHz) δ: 7.48 (d, J=8.4 Hz, 2H), 7.59 (d, J=8.8 Hz, 2H), 9.55 (s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 122.24, 126.69, 126.78, 130.36, 135.61, 142.27, 150.12, 174.88; HR-MS (ESI, m/z) Calcd for C₁₈H₁₂N₆S₂Cl₂ [M+H] 447.98516. Found 447.98622.

3,3ʹ-Bis-5,5ʹ6,6ʹ-tetraphenylsulfanyl-1,2,4-triazin-5,5ʹ-bi-1,2,4-triazine 8

Yield 62%; mp 250°C; ¹H-NMR (CDCl₃, 400 MHz) δ: 7.32 (t, J=8 Hz, 2H), 7.39–7.46 (m, 4H), 7.5–7.61 (m, 4H), 9.85 (s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 128.71, 128.80, 128.54, 129.54, 129.82, 130.16, 131.44, 134.46, 134.51, 144.01; HR-MS (EI, m/z) Calcd for C₃₆H₂₃N₁₂S₂ [M+H] 687.16046. Found 687.15833.

3,3ʹ-Bis-benzothiazolesulfanyl-5,5ʹ-bi-1,2,4-triazine 9

Yield 87%; mp 246–250°C; ¹H-NMR (CDCl₃, 400 MHz) δ: 7.52 (dt, J=1.2, J=8.2 Hz, 1H), 7.58 (dt, J=1.2, J=8.2 Hz, 1H), 7.97 (dd, J=0.8 Hz, J=8.0 Hz 1H), 8.12 (dd, J=0.8, J=8.0, 1H), 9.87 (s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 112.2, 121.4, 124.7, 127.2, 129.3, 129.9, 140.2, 155.8; HR-MS (ESI, m/z) Calcd for C₂₀H₁₀N₈S₄ [M⁺] 490.99840. Found 490.99530.

X-Ray Analysis

X-Ray data of 7 were collected on the Bruker SMART APEX II CCD diffractometer; crystal sizes 0.69×0.66×0.15 mm, CuKα (λ=0.1.54178 Å) radiation, φ and ω scans, multi-scan absorption correction (T_min/T_max=0.1086/0.5086.²¹⁾ The structure was solved by direct methods using SHELXS97²²⁾ and refined by full-matrix least-squares with SHELXL97.¹⁸⁾ The H atoms were positioned geometrically and treated as riding on their parent C atoms with C–H distances of 0.93 Å (aromatic). All H atoms were refined with isotropic displacement parameters taken as 1.5 times those of the respective parent atoms. Compound 7 crystallized in non-centrosymmetric space group P2₁ with four molecules in asymmetric part of the unit cell. These four independent molecules are almost perfectly related by two pseudo-inversion centers located at (0.066, 0.088, 0.433) and (0.438, 0.078, 0.060), which confirms the occurrence of non-crystallographic symmetry (NCS)²³⁾ in crystal of 7. The refinement of the structure gave Flack parameter of 0.45(2) for 5214 Friedel pairs, suggesting that the crystal is possibly inversion twined. Therefore, in last cycles of refinement the appropriate TWIN/BASF instructions of SHELXL97 were used giving the fractional contributions of the twin components of 0.48/0.52. All calculations were performed using WINGX version 1.64.05 package.²⁴⁾ CCDC-1030065 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; Fax: [phone]; e-mail: [e-mail]].

Crystal Data of 7

C₁₈H₁₀N₆S₂Cl₂, M=445.34, monoclinic, space group P2₁, a=18.9516(9), b=7.2403(3), c=27.8497(13) Å, β=96.201(2)°, V=3799.0(3) ų, Z=8, d_calc=1.557 mg m⁻³, F(000)=1808, µ(CuKα)=5.282 mm⁻¹, T=150 K, 30715 measured reflections (θ range 2.34–67.52°), 12531 unique reflections (R_int=0.047), final R=0.048, wR=0.128, S=1.032 for 11295 reflections with I>2σ(I).

Crystal Data of 2

C₃₀H₂₀N₆S₂, M=528.64, triclinic, space group P1ˉ, a=5.8912(3), b=11.6794(6), c=19.6911(10) Å, α=95.026(4), β=92.640(4), γ=102.670(4)°, V=1313.81(12) ų, Z=2, d_calc=1.336 Mg m⁻³, F(000)=548, µ(CuKα)=2.084 mm⁻¹, T=296 K, 10331 measured reflections (θ range 4.31–66.55°), 4284 unique reflections (R_int=0.053), final R=0.090, wR=0.243, S=1.074 for 3271 reflections with I>2σ(I).

Theoretical Calculations

The theoretical calculations for all investigated compounds were performed with GAUSSIAN 03²⁵⁾ at the DFT/B3LYP level with 6–311++G(d, p) basis set. The structures were fully optimized without any symmetry constraints and the initial geometries were built from the crystallographic data of 2 and 7. The AM1 semi-empirical SCF-MO method²⁶⁾ implemented in the program package GAUSSIAN 03 was undertaken to investigate the conformational preferences of molecules 2 and 7. The net atomic charges for 1–9 were calculated by Natural Bond Order (NBO) method.^27–29) The values of logP (P—partition coefficient) were obtained using HyperChem 8.0 package.³⁰⁾ Calculations were carried out at the Academic Computer Centre in Siedlce.

Acknowledgment

The authors thank D. Sc. Danuta Branowska for helpful and stimulating discussions.

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Neunhoeffer H., “Comprehensive Heterocyclic Chemistry II,” Vol. 6, ed. by Katrizky A. R., Rees C. W., Pergamon, Oxford, 1996, p. 571.
• 2) Arshad M., Khan T. A., Khan M. A., Int. J. Pharm. Sci. Res., 5, 149 (2014).
• 3) Kumar R., Sirohi T. S., Singh H., Yadav R., Roy R. K., Chaudhary A., Pandeya S. N., Mini Rev. Med. Chem., 14, 168–207 (2014).
• 4) Neunhoeffer H., Wiley F. P., “Chemistry of 1,2,3-Triazines and 1,2,4-Triazines, Tetrazines, and Pentazines,” ed. by Weissberger A., Taylor C. E., Wiley, New York, 1978.
• 5) Ziemlicka J., “Nucleic Acid Chemistry,” Part I, ed. by Townsend L. B., Tipson R. S., Wiley, New York, 1978, p. 451.
• 6) Leach M. J., Marden C. M., Miller A. A., Epilepsia, 27, 490–497 (1986).
• 7) Miller A. A., Sawyer D. A., Roth B., Peck A. W., Leach M. J., Wheatley P. L., Parsons D. N., Morgan R. J. I., Curr. Probl. Epilepsy, 4, 165 (1986).
• 8) Wootton R., Soul-Lawton J., Rolan P. E., Sheung C. T. C. F., Cooper J. D. H., Posner J., Br. J. Clin. Pharmacol., 43, 23 (1997).
• 9) Abdel-Rahman R. M., Pharmazie, 56, 1 (2001).
• 10) Lindsley C. W., Layton M. E., “Science of Synthesis: Houben-Weyl Methods of Molecular Transformations,” Vol. 17, ed. by Weinreb, S. M., Thieme, New York, 2004, p. 357.
• 11) Corbet J.-P., Mignani G., Chem. Rev., 106, 2651–2710 (2006).
• 12) Sabena S., Verma M., Saxena A. K., Shanker K., Arzneim-Forsch. Drug Res., 44, 766 (1994).
• 13) López-Torres E., Mendiola M. A., Pastor C. J., Inorg. Chem., 45, 3103–3112 (2006).
• 14) Ławecka J., Olender E., Karczmarzyk Z., Wysocki W., Branowska D., Urbańczyk-Lipkowska Z., Kalicki P., Heterocycl. Commun., 20, 5 (2014).
• 15) Carmichael J., DeGraff W. G., Gazdar A. F., Mina J. D., Mitchell J. B., Cancer Res., 47, 936–942 (1987).
• 16) Allen F. H., Acta Crystallogr., 58, 380–388 (2002).
• 17) Bruno I. J., Cole J. C., Edgington P. R., Kessler M., Macrae C. F., McCabe P., Pearson J., Taylor R., Acta Crystallogr., 58, 389–397 (2002).
• 18) Allen F. H., Kennard O., Watson D. G., Brammer L., Orpen A. G., Taylor R., J. Chem. Soc., Perkin Trans. 2, 1987, S1 (1987).
• 19) Coward L., Smith M., Kirk M., Barnes S., Am. J. Clin. Nutr., 68, 14865 (1998).
• 20) Peterkofsky B., Pałka J., Wilson S., Takeda K., Shah V., Endocrinology, 128, 1769–1779 (1991).
• 21) Bruker (2005). SADABS (Version 2004/1). Bruker AXS Inc., Madison, Wisconsin, U.S.A.
• 22) Sheldrick G. M., Acta Crystallogr., 64, 112–122 (2008).
• 23) Müller P., Herbst-Irmer R., Spek A. L., Schneider T. R., Sawaya M. R., “Crystal Structure Refinement, A Crystallographer’s Guide to SHELXL,” ed. by Müller P., International Union of Crystallography, Oxford University Press, 2006.
• 24) Farrugia L. J., J. Appl. Cryst., 45, 849–854 (2012).
• 25) Frisch M. J., Trucks G. W., Schlegel H. B., Scuseria G. E., Robb M. A., Cheeseman J. R., Montgomery J. A. Jr., Vreven T., Kudin K. N., Burant J. C., Millam J. M., Iyengar S. S., Tomasi J., Barone V., Mennucci B., Cossi M., Scalmani G., Rega N., Petersson G. A., Nakatsuji H., Hada M., Ehara M., Toyota K., Fukuda R., Hasegawa J., Ishida M., Nakajima T., Honda Y., Kitao O., Nakai H., Klene M., Li X., Knox J. E., Hratchian H. P., Cross J. B., Bakken V., Adamo C., Jaramillo J., Gomperts R., Stratmann R. E., Yazyev O., Austin A. J., Cammi R., Pomelli C., Ochterski J. W., Ayala P. Y., Morokuma K., Voth G. A., Salvador P., Dannenberg J. J., Zakrzewski V. G., Dapprich S., Daniels A. D., Strain M. C., Farkas O., Malick D. K., Rabuck A. D., Raghavachari K., Foresman J. B., Ortiz J. V., Cui Q., Baboul A. G., Clifford S., Cioslowski J., Stefanov B. B., Liu G., Liashenko A., Piskorz P., Komaromi I., Martin R. L., Fox D. J., Keith T., Al-Laham M. A., Peng C. Y., Nanayakkara A., Challacombe M., Gill P. M. W., Johnson B., Chen W., Wong M. W., Gonzalez C., Pople J. A., Gaussian 03, Revision E.01, Gaussian, Inc., Wallingford, CT, 2004.
• 26) Dewar M. J. S., Zoebisch E. G., Healy E. F., Stewart J. J. P., J. Am. Chem. Soc., 107, 3902–3909 (1985).
• 27) Carpenter J. E., Weinhold F., J. Mol. Struct. THEOCHEM, 169, 41–62 (1988).
• 28) Foster J., Weinhold F., J. Am. Chem. Soc., 102, 7211–7218 (1980).
• 29) Reed A. E., Weinhold F., J. Chem. Phys., 78, 4066–4073 (1983).
• 30) Hypercube Inc., “Reference Manual. HyperChem® Release 8.0 for Windows® Molecular Modeling System,” Hypercube Inc., Waterloo, ON, Canada, 2008.