Synthesis and cytotoxicity of aminomethylselenopheno[3,2-b]thiophene sulfonamides

The reaction of 5-[(aminomethyl)ethynyl]thiophene-2-sulfonamides with in situ generated selenium(IV) chloride was used to synthesize 5-aminomethyl-substituted 6-chloroselenopheno[3,2-b]thiophene-2-sulfonamides. The cytotoxicity of these compounds was studied against HT-1080 (human fibrosarcoma), MH-22A (mouse hepatoma), CCL-8 (mouse sarcoma), MES-SA (human uterine sarcoma), MCF-7 (estrogen receptor-positive human breast adenocarcinoma) cell lines, as well as the normal NIH 3T3 cell line (mouse fibroblasts).

Selenium, its organic and inorganic derivatives are generally known to exhibit a broad range of biological activity. It has been established that selenium as nutritional supplement strengthens the immune system and reduces the incidence of oncological diseases.1 ^, 2 Certain organic selenium derivatives show antitumor,3 ^– 8 antiviral,9 and antimicrobial10 ^, 11 activity. It has been shown that seleniumcontaining compounds have a remarkable ability to affect the activity of redox enzymes, such as glutathione peroxidase and reductase, by modulation of thioredoxin system activity.12 ^– 14 It should be noted that selenium halides are widely used as reagents in organic synthesis that add at double and triple bonds.15 ^– 18

In general the interest toward chemistry of seleniumcontaining heterocyclic systems is largely motivated by the diverse biological activity of such compounds19 ^– 21 and by the possible use as synthons in the construction of π-systems.22 ^– 24 For example, selenopheno[3,2-b]thiophenes have attracted attention because their structural analogs, 5-substituted thieno[3,2-b]thiophene-2-sulfonamides, are active as drugs for the treatment of glaucoma; the best results were observed with bis(2-methoxyethyl)aminomethylsubstituted derivatives.25 ^, 26 However, the methods of synthesis and biological properties of the structurally related selenophenothiophenes have not yet been described.

This work was devoted to the synthesis and cytotoxicity studies of new heterocyclic compounds, derivatives of 6-chloroselenopheno[3,2-b]thiophene sulfonamides. We developed a new two-step synthetic route for the preparation of these compounds. The first step was a reaction of 5-bromo-2-thiophene sulfonamide (1) with terminal alkynes, catalyzed by bis(triphenylphosphino)-palladium dichloride and copper(I) iodide, which gave substituted 5-[(aminomethyl)ethynyl]thiophene-2-sulfonamides 2–6 in 69–99% yields (Scheme 1).27 The sulfonamide group did not require additional protection when the reaction was performed in a mixture of anhydrous ethyl acetate and triethylamine. The avoidance of more toxic solvents, such as dimethylformamide, dimethyl acetamide or secondary amines, provided environmental and energy-saving advantages to this method. The selected reaction conditions allowed to decrease the reaction time for the formation of ethynylthiophenes 2–6 to 1 h.

Scheme 1

In the second step, dioxane solutions of ethynylthiophenes 2–6 were added dropwise to a solution of selenium tetrachloride, which was prepared in situ from selenium dioxide and concentrated hydrochloric acid. The addition of SeCl₄ to the multiple bonds of compounds 2–6 probably produced intermediates A, which underwent intramolecular cyclization to selenophenes 7–9. It should be noted that the in situ prepared selenium(IV) chloride was less reactive than SeBr4.27 ^, 28 However, that should be considered as an advantage in this case, providing for fewer side reactions. As a result, the isolation of chlorosubstituted selenophenothiophenes 7–9 did not present any major difficulties, but the yields (31–69%) were somewhat lower, compared to the bromo-substituted analog of compound 8 (84%).27 The yield of compound 7 was lower than in the case of compounds 8 and 9, as there was a possibility of selenium tetrachloride reaction with the hydroxyethyl group.

Prior to biological testing, the synthesized compounds 2–6 were treated with HCl solution in ether, giving the respective salts 2·HCl–6·HCl, well soluble under physiological conditions.

The cytotoxic properties of compounds 2–6, 8, 9 were studied in vitro with the following tumor cell lines: HT-1080 (human fibrosarcoma), MH-22A (mouse hepatoma), CCL-8 (mouse sarcoma), MES-SA (human uterine sarcoma), MCF-7 (estrogen receptor-positive human breast adenocarcinoma), as well as the normal NIH 3T3 cell line (mouse fibroblasts). The concentrations of study compounds that were lethal to 50% of the cells in vitro (IC₅₀) (Table 1) were determined according to the standard method by the intensity of cell wall staining in the presence of Crystal violet and mitochondrial enzyme staining in the presence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.29

Table 1 The in vitro cytotoxic activity of sulfonamides 2-9

According to the obtained data, 5-[(aminomethyl)-ethynyl]thiophene-2-sulfonamides 2–6 did not show pronounced cytotoxic activity against tumor cells. It should be taken into account that the studied derivatives had lower acute toxicity in vitro (LD₅₀ 1684–2195 mg/kg). At the same time, the introduction of selenophene ring noticeably increased the cytotoxic effect. Thus, the mouse hepatoma cell line MH-22A and human uterine sarcoma cell line MES-SA were sensitive to bis(2-methoxyethyl)aminomethyl and morpholine derivatives of 6-chloroselenopheno[3,2-b]thiophene-2-sulfonamide (compounds 8 and 9, IC₅₀ 68–79 μM). In addition, the derivative 8 showed inhibitory activity against human fibrosarcoma cell line HT-1080 (IC50 65 μM) and mouse sarcoma cell line CCL-8 (IC₅₀ 92 μM). The investigated selenophenothiophene sulfonamides were not toxic to normal cells (LD₅₀ 1159–1559 mg/kg).

Thus, we have developed a convenient method for the synthesis of new selenophene-containing fused heterocycles, 3-chloroselenopheno[3,2-c]thiophene sulfonamides. The method for the preparation of selenophenothiophenes is simple, does not require unusual reaction conditions and can be applied to the synthesis of a wider range of compounds. The cytotoxic activity of the synthesized 3-chloroselenopheno[3,2-c]thiophenes was studied in vitro. The obtained results indicate that this type of compounds is nontoxic to normal cells in vitro and therefore may serve as a promising lead for further studies.

Experimental

¹H and ¹³C NMR spectra were acquired on a Varian Mercury 400 instrument (400 and 100 MHz, respectively), with residual proton signals (δ 2.50 ppm for DMSO-d ₆, δ 7.26 ppm for CDCl3) or signals of ¹³C nuclei of the deuterated solvent (δ 39.4 ppm for DMSO-d ₆, δ 77.2 ppm for CDCl₃) as internal standard. Mass spectra were recorded with a Waters Micromass 3100 Mass Detector coupled to a Waters Acquity UPLC system, using an Acquity UPLC BEH C18 column (1.7 μm, 2.1×50 mm, 0.4 ml/min), MeCN (from 5 to 100%, 8 min) – HCOOH (0.5% in H₂O). Elemental analysis was performed on a Carlo Erba EA 1108 elemental analyzer. The reaction progress and purity of the obtained compounds were controlled by TLC on Merck Kieselgel plates, visualization under UV light. Propargylamines were obtained by reactions of propargyl bromide with secondary amines. The tumor cell lines were obtained from the collection of ATCC (American Type Culture Collection).

Synthesis of 5-[(aminomethyl)ethynyl]thiophene-2-sulfonamides 2–6 and their hydrochlorides (General method). Pd(PPh₃)₂Cl₂ (26.2 mg, 0.1 mmol) and CuI (9.5 mg, 0.05 mmol) were added to triethylamine (1 ml) under argon atmosphere and stirred for 5 min. Then a solution of 5-bromo-2-thiophene sulfonamide (1) (242 mg, 1 mmol) in EtOAc (5 ml) and the appropriate tertiary propargylamine (2 mmol) were added. The reaction mixture was stirred for 1 h at 50°C under inert atmosphere. The reaction mixture was then cooled, diluted with EtOAc, and treated with a few drops of aqueous ammonia, stirred for 5–10 min and filtered through a thin layer of silica gel. The solution was evaporated to dryness and the product was isolated by silica gel column chromatography (eluent CH₂Cl₂–EtOAc–MeOH). In order to obtain hydrochlorides, the isolated product was dissolved in 1:1 EtOAc–Et₂O mixture and treated with anhydrous ethereal HCl solution to pH 1. The mixture was stirred until the reaction was complete (TLC control), evaporated to dryness, and diluted with anhydrous ether. The precipitate was collected by filtration and dried.

5-[3-(Piperidin-1-yl)prop-1-yn-1-yl]thiophene-2-sulfonamide (2). Yield 98%, beige powder, mp 118–120°C. ¹H NMR spectrum (DMSO-d ₆), δ, ppm (J, Hz): 1.31–1.34 (2H, m, CH₂); 1.52–1.59 (4H, m, 2CH₂); 2.49–2.58 (4H, m, CH₂NCH₂); 3.60 (2H, s, CH₂N); 7.30 (1H, d, J = 3.7, H-4), 7.46 (1H, d, J = 3.7, H-3); 7.80 (2H, s, NH₂). ¹³C NMR spectrum (DMSO-d ₆), δ, ppm: 23.4; 25.4; 47.5; 52.6; 76.4, 93.0, 126.3; 129.9; 135.9; 145.5. Mass spectrum, m/z (I _rel, %): 285 [M+H]⁺ (100). 5-[3-(Piperidin-1-yl)prop-1-yn-1-yl]thiophene-2-sulfonamide hydrochloride (2·HCl). Yield 99%, white powder, mp 175–178°C. Found, %: C 44.81; H 5.50; N 8.26. C₁₂H₁₇ClN₂O₃S₂. Calculated, %: C 44.92; H 5.34; N 8.73.

5-[3-(4-Hydroxypiperidin-1-yl)prop-1-yn-1-yl]thiophene-2-sulfonamide (3). Yield 75%, beige powder, mp 138–140°C. 1H NMR spectrum (DMSO-d ₆), δ, ppm (J, Hz): 1.18–1.40 (2H, m, CH₂); 1.51–1.71 (2H, m, CH₂); 2.28–2.48 (2H, m) and 2.75–2.88 (2H, m, CH₂NCH₂); 3.50 (1H, br. s, CHOH); 3.68 (2H, s, CH₂N); 4.64 (1H, br. s, OH); 7.31 (1H, d, J = 3.9, H-4); 7.46 (1H, d, J = 3.9, H-3); 7.80 (2H, s, NH₂). ¹³C NMR spectrum (DMSO-d ₆), δ, ppm: 33.6; 45.6; 47.9; 49.5; 65.4; 109.9; 125.9; 129.9; 132.4; 145.9. Mass spectrum, m/z (I _rel, %): 301 [M+H]⁺ (100). 5-[3-(4-Hydroxypiperidin-1-yl)prop-1-yn-1-yl]thiophene-2-sulfonamide hydrochloride hydrate (3·HCl·H ₂ O). Yield 99%, white powder, mp 80–82°C. Found, %: C 40.70; H 5.09; N 7.42. C₁₂H₁₇ClN₂O₃S₂. Calculated, %: C 40.61; H 5.40; N 7.89.

5-{3-[2-Hydroxyethyl(methyl)amino]prop-1-yn-1-yl}thiophene-2-sulfonamide (4). Yield 69%, beige powder, mp 128–130°C. ¹H NMR spectrum (DMSO-d ₆), δ, ppm (J, Hz): 2.27 (3H, s, NCH₃), 2.47–2.48 (2H, m, CH₂OH); 3.47–3.51 (2H, m, NCH ₂CH₂); 3.61 (2H, s, CH₂N); 4.44–4.47 (1H, m, OH); 7.28 (1H, d, J = 3.8, H-4); 7.45 (1H, d, J = 3.8, H-3); 7.79 (2H, s, NH₂). ¹³C NMR spectrum (DMSO-d ₆), δ, ppm: 41.9; 46.3, 57.7; 58.9; 76.6; 92.8; 126.3, 129.9; 132.0; 145.5. Mass spectrum, m/z (I _rel, %): 275 [M+H]⁺ (100). 5-{3-[2-Hydroxyethyl(methyl)amino]-prop-1-yn-1-yl}thiophene-2-sulfonamide hydrochloride (4·HCl). Yield 99%, white powder, mp 188–190°C.

5-{3-[Bis(2-methoxyethyl)amino]prop-1-yn-1-yl}thiophene-2-sulfonamide (5). Yield 99%, yellow oil. ¹H NMR spectrum (DMSO-d ₆), δ, ppm (J, Hz): 2.67 (4H, t, J = 5.7, 2NCH₂CH ₂O); 3.24 (6H, s, 2OCH₃); 3.42 (4H, t, J = 5.7, 2NCH ₂CH₂O); 3.71 (2H, s, CH₂N); 7.27 (1H, d, J = 3.8, H-4); 7.45 (1H, d, J = 3.8, H-3); 7.79 (2H, s, NH₂). ¹³C NMR spectrum (DMSO-d ₆), δ, ppm: 43.7; 52.8; 57.9; 70.5; 76.4; 93.1; 126.4; 129.9; 132.0; 145.5. Mass spectrum, m/z (I _rel, %): 333 [M+H]⁺ (100). 5-{3-[Bis-(2-methoxyethyl)-amino]prop-1-yn-1-yl}thiophene-2-sulfonamide hydrochloride (5·HCl). Yield 99%, white powder, mp 140–145°C. Found, %: C 42.23; H 5.83; N 7.28. C₁₃H₂₁ClN₂O₄S₂. Calculated, %: C 42.33; H 5.74; N 7.59.

5-[3-(Morpholin-4-yl)prop-1-yn-1-yl]thiophene-2-sulfonamide (6). Yield 60%, beige powder, mp 120–122°C. 1H NMR spectrum (CDCl₃), δ, ppm (J, Hz): 2.62 (4H, t, J = 4.7, CH₂OCH₂); 3.54 (2H, s, CH₂N); 3.76 (4H, t, J = 4.7, CH₂NCH₂); 4.98 (2H, br. s, NH₂); 7.09 (1H, d, J = 3.8, H-4); 7.49 (1H, d, J = 3.8, H-3). ¹³C NMR spectrum (CDCl3), δ, ppm: 47.0; 51.6; 65.9; 76.8; 92.3; 126.1; 129.9; 132.2; 145.7. Mass spectrum, m/z (I _rel, %): 287 [M+H]⁺. 5-[3-(Morpholin-4-yl)prop-1-yn-1-yl]thiophene-2-sulfonamide hydrochloride (6·HCl). Yield 99%, white powder, mp 175–178°C. Found, %: C 40.62; H 4.71; N 8.26. C₁₁H₁₅ClN₂O₃S₂. Calculated, %: C 40.92; H 4.68; N 8.68.

Synthesis of selenophenothiophenes 7–9 (General method). A solution of the appropriate 5-[(aminomethyl)-ethynyl]thiophene-2-sulfonamide 4–6 (0.7 mmol) in dioxane (10 ml) was added dropwise to a solution of SeCl4 that was prepared in situ from SeO₂ (46 mg, 0.42 mmol) and 10 drops of concd. HCl. The reaction progress was controlled by TLC. After the cyclization reaction was complete, the reaction mixture was treated with EtOAc and solid Na₂CO₃. The mixture was filtered through a thin layer of silica gel. The organic phase was dried over anhydrous Na₂SO₄ and evaporated to dryness.

6-Chloro-5-{[2-hydroxyethyl(methyl)amino]methyl}-selenopheno[3,2- b ]thiophene-2-sulfonamide (7). Yield 31%, yellow powder, mp 84–86°C. ¹H NMR spectrum (DMSO-d ₆), δ, ppm (J, Hz): 2.46 (3H, s, CH₃); 2.58–2.68 (2H, m, CH ₂OH); 3.52–3.61 (2H, m, NCH ₂CH₂); 3.85–3.40 (2H, m, CH₂N); 4.51–4.60 (1H, m, OH); 7.84 (2H, s, NH₂); 7.97 (1H, s, H Ar). ¹³C NMR spectrum (DMSO-d ₆), δ, ppm: 52.7; 56.3; 58.9; 67.3; 102.4; 127.5; 133.8; 140.3; 144.5; 146.9. Mass spectrum, m/z (Irel, %): 385 [M+H]⁺ (20), 387 [M+H]⁺ (40), 389 [M+H]⁺ (100), 391 [M+H]⁺ (30).

6-Chloro-5-{[bis(2-methoxyethyl)amino]methyl}selenopheno[3,2- b ]thiophene-2-sulfonamide (8). Yield 69%, yellow powder, mp 86–88°C. ¹H NMR spectrum (CDCl₃), δ, ppm (J, Hz): 2.86 (4H, t, J = 5.7, 2NCH₂CH₂O); 3.33 (6H, s, 2OCH₃); 3.52 (4H, t, J = 5.7, 2NCH₂CH₂O); 4.02 (2H, s, CH₂N); 5.21 (2H, br. s, NH₂); 7.81 (1H, s, H Ar). ¹³C NMR spectrum (CDCl₃) δ, ppm: 54.3; 54.5; 58.7; 74.1; 113.5; 127.6; 133.3; 142.3; 143.4; 150.8. Mass spectrum, m/z (I _rel, %): 443 [M+H]⁺ (20), 445 [M+H]⁺ (50), 447 [M+H]⁺ (100), 449 [M+H]⁺ (30).

6-Chloro-5-(morpholin-4-ylmethyl)selenopheno[3,2- b ]-thiophene-2-sulfonamide (9). Yield 59%, yellow powder, mp 170–172°C. ¹H NMR spectrum (DMSO-d ₆), δ, ppm (J, Hz): 2.76–3.30 (2H, m, CH₂N); 3.59–3.84 (4H, m, CH₂OCH₂); 4.06–4.71 (4H, m, CH₂NCH₂); 7.91 (2H, s, NH₂); 8.02 (1H, s, H Ar). ¹³C NMR spectrum (DMSO-d ₆), δ, ppm: 54.2; 58.1; 70.8; 111.5; 127.5; 133.8; 140.6; 144.1; 151.3. Mass spectrum, m/z (I _rel, %): 397 [M+H]⁺ (20), 399 [M+H]⁺ (50), 401 [M+H]⁺ (100), 403 [M+H]⁺ (30).