Synthesis, antimicrobial and anticancer activities of some new N-methylsulphonyl and N-benzenesulphonyl-3-indolyl heterocycles

2.1 Chemistry

Melting points were determined in open capillary tubes on an Electrothermal 9100 digital melting point apparatus (Büchi, Switzerland) and were uncorrected. Elemental analyses were performed on a Perkin–Elmer 2400 analyzer (USA) and were found within ±0.4% of the theoretical values. IR spectra were recorded on a Perkin–Elmer 1600 FTIR (USA) in KBr disks. The ¹H NMR spectra were measured with a Bruker Avance digital spectrophotometer 300 MHz in DMSO-d₆ and chemical shifts were recorded in δ ppm relative to TMS as internal stander solvent. Mass spectra (EI) were run at SECTOR-FILD MS and GC–MS (single-phase) 200 V (50/60 Hz) 30 A (Germany). N-methylsulphonyl (1a) and N-benzenesulphonyl-3-acetylindoles (1b) have been prepared as reported (Mndzhoyan et al., 1969).

2.2 Antimicrobial evaluation

The antimicrobial activity of the newly synthesized compounds was determined in vitro using the disk diffusion method (Bauer et al., 1966) against a variety of pathogenic microorganisms: Salmonella typhimurium (ATCC 14028), Pseudomonas fluorescens (S 97) (Gram-positive bacteria), Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6635) (Gram-negative bacteria) and two strains of fungi Candida albicans (ATCC 10231) and Aspergillus fumigatus (isolated from clinical samples and identified to the species level according to different API 20E system (Analytab Products) (bioMerieux, Australia). The antimicrobial activities of the tested compounds were estimated by placing presterilized filter paper disks (6 mm in diameter) impregnated with 20 and 10 μg disk⁻¹ in Nutrient and MacConky agar media for bacteria and on Sabouraud dextrose agar for fungus. Dimethyl formamide (DMF) which showed no inhibition zone was used as a solvent for impregnation. The inhibition zones (IZ) of the tested compounds were measured after 24–48 h incubation at 37 °C for bacteria and after 5 days incubation at 28 °C for fungi. Chloramphenicol (C1863, Sigma–Aldrich) and cephalothin (C3050, Sigma–Aldrich) were used as reference drugs for bacteria. Whereas, cycloheximide (C7698, Sigma–Aldrich) was used as reference drug for fungi.

2.3 Anticancer evaluation

3.1 Chemistry

The reaction routes for the synthesis of the title compounds were described, as shown in Schemes 1 and 2. Claisen–Schmidt reaction of N-methylsulphonyl-3-acetylindole (1a) and N-benzenesulphonyl-3-acetylindole (1b) with some aromatic aldehydes namely, p-methoxy, p-chloro or m-hydroxybezaldehydes in ethanol in the presence of aqueous potassium hydroxide (25%) led to the formation of 1-(N-methylsulphonyl-1H-indol-3-yl)-3-aryl-prop-2-ene-1-ones (2a–c) and 1-(N-benzenesulphonyl-1H-indol-3-yl)-3-aryl-prop-2-ene-1-ones (3a–c), respectively (Scheme 1). Compounds 2a,b and 3a,b are previously reported (El-Sawy et al., 2009), and the newly compounds 2c and 3c were confirmed by their correct elemental analyses and spectral data (c.f. Experimental section).

Cyclocondensation of chalcones 2a–c or 3a–c with urea in dry ethanol in the presence of glacial acetic acid as a catalyst gave 4-(N-substituted-1H-indol-3-yl)-6-aryl-pyrimidin-2(1H)-ones (4a–c) and (5a–c), respectively (Scheme 1). Similarly, chalcones 2a–c or 3a–c reacted with thiourea and gave the corresponding 4-(N-substituted-1H-indol-3-yl)-6-aryl-pyrimidin-2(1H)-thiones (6a–c) and (7a–c), respectively (Scheme 1). Moreover, reaction of chalcones 2a–c or 3a–c with guanidine hydrochloride in dry ethanol in the presence of anhydrous sodium acetate yielded 4-(N-substituted-1H-indol-3-yl)-6-aryl-pyrimidin-2-amines (8a–c) and (9a–c), respectively (Scheme 1).

It was reported that, reaction of chalcone with ethyl acetoacetate in the presence of aqueous potassium hydroxide (10%) gives rise to cyclohexanone (Sreevidya et al., 2010). In the present work and under the above mentioned conditions, chalcones 2a–c or 3a–c reacted with ethyl acetoacetate (1:1) and gave the newly compounds namely, ethyl-4-(N-substituted-1H-indol-3-yl)-6-aryl-2-oxo-cyclohexa-3-enecarboxylates (10a–c) and (11a–c), respectively (Scheme 1).

Reaction of compound 10a with hydrazine hydrate under reflux in absolute ethanol in the presence of glacial acetic acid as a catalyst afforded 12a. Its IR spectrum showed absorption band at 3218, 1665, 1620 for NH, C⚌O and C⚌N groups successively. Its ¹H NMR (DMSO-d₆) lacks the signals at δ 4.48 and 1.69 ppm of COOCH₂CH₃ and showed 1H, singlet signal (D₂O exchangeable) at δ 9.50 ppm which can be attributed to the proton of NH group besides the signals at δ 8.12 (s, 1H, H-2 indole), 7.67–7.01 (m, 8H, Ar—H), 6.66 (s, 1H, CH⚌C), 3.98 (s, 3H, OCH₃), 3.88 (s, 3H, CH₃), 2.81 and 3.35 (2d, 2H, CH—CH) 2.33 and 2.35 ppm (dd, 2H, CH—CH₂). Its mass spectrum showed a molecular ion peak at m/z 453 (3%) (c.f. Experimental section). The structure of compound 12a was actually identified by the IR, ¹H NMR and MS spectra as, 4,5-dihydro-4-(p-methoxyphenyl)-6-(N-methylsulphonyl-1H-indol-3-yl)-2H-indazol-3(H)one (Scheme S1). The reaction may be proceded through condensation between C⚌O of cyclohexanone and NH₂ of hydrazine, followed with cyclization by loosing a molecule of ethanol. Similarly, reaction of 10b,c or 11a–c with hydrazine hydrate under the above mention condition led to the formation of 4,5-dihydro-4-aryl-6-(N-substituted sulphonyl-1H-indol-3-yl)-2H-indazol-3(H)ones (12b,c) and (13a–c), respectively (Scheme 1) (See Scheme S2).

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Scheme 1 Synthetic route for the preparation of N-substituted sulphonyl-3-indolyl chalcone, pyrimidine and cyclohexanone derivatives.

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Scheme 2 Synthetic route of the preparation of N-substituted sulphonyl-3-indolyl pyrazole and isoxazole derivatives; compounds 14–18, R, a = CH3; b = Ph.

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On the other hand, condensation of chalcones 2c or 3c with hydrazine hydrate in absolute ethanol in the presence of few drops of glacial acetic acid gave 4,5-dihydro-3-(N-substituted-1H-indol-3-yl)-5-(m-hydroxy-phenyl)pyrazoles (14a) and (14b), respectively (Scheme 2). whereas, reaction of 2c or 3c with hydrazine hydrate under reflux in a mixture of acetic anhydride and glacial acetic acid (2:1) afforded the corresponding N-acetylpyrazole derivatives 15a and 15b (Scheme 2). Incidentally, compounds 15a and 15b were obtained via the reaction of 2c and 3c with acetic acid hydrazide in refluxing ethanol which showed no depression in a dmixed m.p.s with that previously obtained (Scheme 2).

Additionally, reaction of 2c or 3c with phenylhydrazine gave N-phenylpyrazole derivatives 16a and 16b. While, reaction of 2c or 3c with benzylhydrazine hydrochloride in the presence of anhydrous sodium acetate gave N-benzylpyrazole derivatives 17a and 17b (Scheme 2).

Moreover, reaction of 2c or 3c with hydroxyl amine hydrochloride in the presence of anhydrous sodium acetate led to the formation of 4,5-dihydro-3-(N-substituted-1H-indol-3-yl)-5-(m-hydroxyphenyl)isoxazoles (19a) and (19b) (Scheme 2).

The structures of the newly synthesized compounds were confirmed on the bases of elemental analyses as well as spectral data (IR, ¹H NMR, and MS spectra) (c.f. Experimental section).

3.2 Antimicrobial activity

All the newly synthesized compounds were tested for their antimicrobial activities against a variety of pathogenic microorganisms: S. typhimurium (ATCC 14028), P. fluorescens (S 97) (Gram-positive bacteria), S. aureus (ATCC 25923), B. subtilis (ATCC 6635) (Gram-negative bacteria) and two strains of fungi C. albicans (ATCC 10231) and A. fumigatus (isolated from clinical samples and identified to the species level according to different API 20E system (Analytab Products) (bioMerieux, Australia) using the disk diffusion method at concentrations of 10 and 20 μg disk⁻¹ (Table 1). The results showed that, compounds 14a, 14b, 15a and 15b were found to be the most active of all the tested compounds, with inhibition zones of 30, 35, 31 and 30 mm, respectively, against S. typhimurium (ATCC 14028) compared to the reference drug chloramphenicol (42 mm) at 20 μg disk⁻¹, and showed inhibition zones of 20 mm against S. typhimurium (ATCC 14028) compared to the reference drug chloramphenicol (28 mm) at 10 μg disk⁻¹. The rest of the tested compounds were non-active against all microorganisms (Table 1).

3.3 Anticancer activity

Eighteen new compounds namely, pyrimidin-2(1H)-ones 4a–c and 5a–c, pyrimidin-2(1H)-thiones 6a–c and 7a–c and pyrimidin-2-amines 8a–c and 9a–c were primary screened for their in vitro cytotoxicity against (HEPG2), (MCF7) and (HCT-116) cell lines at a concentration of 10 μg mL⁻¹ (Table 2). Compounds 4c, 6c and 8c were found to be the highly active compounds with cytotoxicity of 99.6, 94.2 and 95.1%, respectively, against HEPG2, and showed activity of 100.7, 99.0 and 100.3%, respectively, against MCF7 and also, showed activity of 100.0, 100.3 and 99.3%, respectively, against HCT-116. While, compounds 5c and 9c showed activity of 94.3 and 96.3%, respectively, against HEPG2 and showed activity of 97.4 and 90.1%, respectively, against HCT-116 (Table 2).

The compounds that gave cytotoxicity more than 90% at 10 μg mL⁻¹ were used to calculate their IC₅₀ value, which corresponds to the concentration required for 50% inhibition of all cell viability. Doxorubicin is one of the most effective anticancer agents used as reference drug (Table 3). In case of HEPG2 cancer cell line, compounds 4c, 5c, 6c, 8c and 9c showed more potent activity with IC₅₀ = 1.6, 2.9, 3.1, 2.4 and 1.7 μg mL⁻¹, respectively, than the reference drug doxorubicin (IC₅₀ = 24.47 μg mL⁻¹). In case of MCF7 cancer cell line, compounds 4c, 6c and 8c showed more potent activity with IC₅₀ = 201, 2.6 and 1.8 μg mL⁻¹, respectively, than the reference drug doxorubicin(IC₅₀ = 38.2 μg mL⁻¹) (Table 3). In case of HCT-116 cancer cell line, compounds 4c, 5c, 6c, 8c and 9c showed more potent activity with IC₅₀ = 3.7, 5.1, 4.7, 3.5 and 2.8 μg mL⁻¹, respectively, than the reference drug doxorubicin (IC₅₀ = 37.6 μg mL⁻¹) (Table 3).

Interpretation of the obtained results and considering the structure activity relationship (SAR) of the tested compounds showed that, compounds 4c, 5c, 6c, 8c and 9c that contain hydroxyl group at p-position of phenyl moiety at pyrimidine ring are associated with remarkable cytotoxicity against all cancer cell lines under investigation HEPG2, MCF7 and HCT-116.