Synthesis and characterization of compounds

AYNBDO = 5-Nitro-1,3-benzodioxine (C₈H₇NO₄) ( 2): 5 g (36 mmol) of 3-Nitrophenol (1) was melted in a round bottom flask at 120°C with constant stirring followed by the addition of 6 mL 1:1 (v/v) mixture of 37% solution of commercial formaldehyde and 12.6 M concentrated HCl (Scheme 1). The reaction mixture was refluxed for 2 h yielding yellow precipitates, while the progress of reaction was monitored with thin layer chromatography (TLC). After completion of reaction, 50 mL of 5 M sodium hydroxide (NaOH) was added in the reaction mixture for quenching. The crude product was extracted with EtOAc (50 mL thrice) and dried at reduced pressure to give pure yellow powdered product 2. The yield of the product was 3.6 g (55%). Electron ionization–mass spectrometry (EI-MS) m/z 181.04. 1H nuclear magnetic resonance (NMR) in CDCl₃, 7.81 (d, 1H, Ar), 7.29 (t, 1H, Ar), 7.17 (d, 1H, Ar), 5.27 (s, 2H, Ar–CH₂–O), 5.22 (s, 2H, O–CH₂–O).

Synthesis of 5-Nitro-1,3-benzodioxine (2).

AYNH₂ = 5-Amino-1,3-benzodioxine (C₈H₉NO₂) ( 3): Product 2 (1.67 g, 10 mmol) and SnCl₂ dihydrate (11.275 g, 50 mmol) were dissolved in EtOH in a round bottom flask. The reaction mixture was heated at 70°C with continuous stirring for half hour and the progress of the reaction was monitored with TLC (Scheme 2). The reaction was brought to room temperature and then was basified with 5% NaOH aqueous solution to attain neutral pH. Product 3 was achieved as yellow powder after extracting the reaction mixture with EtOAc (50 mL × 3) followed by removal of solvents in vacuum and purification by silica gel column chromatography with gradient elution of 1:1 mixture of Hexane:Dichloromethane (DCM) to pure DCM. The yield of the product was 0.97 g (70%). EI-MS m/z 151.0. 1H NMR in CDCl₃, 6.94 (t, 1H, Ar), 6.33 (d, 1H, Ar), 6.28 (d, 1H, Ar), 5.18 (s, 2H, Ar–CH₂–O), 4.71 (s, 2H, O–CH₂–O), 3.41 (s, 2H, –NH₂).

Synthesis of 5-Amino-1,3-benzodioxine (3).

AY80 = N-(4-((dimethylamino)methyl)-3-hydroxyphenyl)acetamide (C₁₁H₁₆N₂O₂) ( 5): To a solution of 3-Acetaminophenol (4) (5 g, 33 mmol) in 40% Dimethyl amine (4.5 g, 40 mmol) and MeOH (4 mL) was added 37% formaldehyde (2.68 g, 33 mmol), and the reaction mixture was placed in an ice bath (Scheme 3). After 15 min, the reaction mixture turned into solid precipitates, which were filtered and washed with ice cold water (100 mL). These precipitates were oven dried to obtain pure product 5 as white powder. The yield of the product was 6.2 g (90%). EI-MS m/z 208.26. 1H NMR in dimethyl sulfoxide (DMSO), 7.17 (s, 1 H, –NH–), 7.00 (d, 1H, Ar), 6.84 (d, 2H, Ar), 3.57 (s, 2H, Ar–CH₂–N), 2.28 (s, 6H, N–CH₃), 2.12 (s, 3H, –CO–CH₃).

Synthesis of N-(4-((dimethylamino)methyl)-3-hydroxyphenyl)acetamide (5).

AYZHC = 4-amino-2-(hydroxymethyl)phenol (C₇H₉NO₂) ( 7): 109 mg (1 mmol) of 4-Aminophenol (6) was treated with 0.1 mL Formaldehyde and 0.1 mL concentrated HCl with same procedure as compound 2 to afford product 7 as light yellow powder (Scheme 4). The yield of the product was 56 mg (40%). EI-MS m/z 139.15. 1H NMR in DMSO, 11.09 (s, 1H, OH), 8.19 (appeared as d, 1H, Ar), 7.99 (dd, 1H, Ar), 6.91 (d, 1H, Ar), 4.49 (s, 2H, Ar–CH₂–OH).

Synthesis of 4-amino-2-(hydroxymethyl)phenol (C₇H₉NO₂) (7).

AYTHPP = 5,10,15,20-Tetrakis(4-hydroxyphenyl)-21 H,23 H-porphine (C₄₄H₃₀N₄O₄) ( 10): Compound 10 was synthesized as follows: 50 mL Propionic acid was heated at 90°C in a round bottom flask fitted with water condensor, and a solution of 4-Hydroxybenzaldehyde (8) (2 g, 16.37 mmol) dissolved in 2.5 mL propionic acid was added very slowly in hot propionic acid. After continuous heating and constant stirring of the solution at reflux temperature for half an hour, pyrrole (9) (1.2 mL, 16.37 mmol) solution in 2.5 mL propionic acid was added in the above-mentioned reaction mixture dropwise for half an hour. The reaction was refluxed and monitored with TLC (Scheme 5). After completion (about 2 h), the reaction mixture was cooled to room temperature followed by addition of 50 mL of 2.5 M NaOH for quenching. Crude products containing a mixture of different oligomers and tar were obtained by solvent extraction with EtOAc (50 mL × 3) and evaporating the EtOAc at reduced pressure. Compound 10 was purified from the crude product through silica gel column chromatography by using EtOAc and Hexane as mobile phase (3:1 to pure EtOAc). The yield of pure product was 1.1 g (10%). EI-MS m/z 678.1. 1H NMR in DMSO, 9.94 (s, 4H, OH), 8.85 (s, 8H, pyrrole), 7.97 (d, 8H, Ar), 7.18 (d, 8H, Ar).

Synthesis of 5,10,15,20-Tetrakis(4-hydroxyphenyl)-21H,23H-porphine (10).

AYTPyP = 5,10,15,20-Tetra(4-pyridyl)-21H,23H-porphine (C₄₀H₂₆N₈) ( 12): Compound 12 was synthesized with same procedure as compound 10 at 18.6 mmol amount level, with minor variation in the workup of reaction mixture (Scheme 6). After completion of reaction (45 min), the reaction mixture was poured into a beaker containing 75 mL 10% sodium acetate aqueous solution to approximately attain the pH 3. The reaction mixture was precipitated at cooling to give crude product 12, which was filtered and washed with ice cold water (100 mL) and oven dried. Finally, it was passed through silica gel column chromatography with DCM as mobile phase for purification. The yield of the product was 0.9 g (8%). ESI-MS 619.22. 1H NMR in CDCl₃, 9.03 (d, 8H, Ar), 8.85 (s, 8H, pyrrole), 8.13 (d, 8H, Ar).

Synthesis of 5,10,15,20-Tetra(4-pyridyl)-21 H,23 H-porphine (12).

AY102, 103 and 127 all were synthesized with similar procedure as given in the following.

102 = N,N″-Di-(3′-methylthio phenyl)-1,4,5,8-naphthalene diimide (C₂₈H₁₈N₂O₄S₂) ( 15): Naphthalene dianhydride (13) (268 mg, 1 mmol) is suspended in 13 mL acetic acid at 140°C for 1 h in a round bottom flask. 3-Mehtylthioaniline (14) (1.2 mL, 10 mmol) is added in above suspension, and the reaction mixture is refluxed for 5 h (Scheme 7). Progress of the reaction is checked with TLC, and after completion, the reaction mixture is concentrated to 3 mL solution. This solution is poured into a beaker containing 13 mL MeOH to give precipitates of crude product 15. The precipitates are filtered, dried and recrystallized with 50 mL acetic acid to give pure compound 15. The yield of the product was 505 mg (99%). EI-MS 509.9. 1H NMR in CDCl₃, 8.829 (s, 4H, Ar-naphth.), 7.45 (t, 2H, Ar), 7.37 (d, 2H, Ar), 7.18 (s, 2H, Ar), 7.07 (d, 2H, Ar).

Synthesis of N,N″-Di-(3′-methylthio phenyl)-1,4,5,8-naphthalene diimide (15).

103 = N,N″-Di-(4,4′-phenyl disulfide)-1,4,5,8-naphthalene diimide (C₂₆H₁₂N₂O₄S₂) ( 17): The yield of the product was 60%. EI-MS 480.5. 1H NMR in CDCl₃, 7.66 (d, 4H, Ar), 7.54 (d, 4H, Ar), 7.41 (s, 4H, Ar-naphth.).

Synthesis of N,N″-Di-(4,4′-phenyl disulfide)-1,4,5,8-naphthalene diimide (17).

127 = N,N″-Di-(propargyl)-1,4,5,8-naphthalene diimide (C₂₀H₁₀N₂O₄) ( 19): The yield of the product was 99%. EI-MS 342.0. 1H NMR in DMSO, 8.72 (s, 4H, Ar-naphth.), 4.80 (s, 4H, -N-CH₂), 3.20 (s, 2H, alkyne).

Synthesis of N,N″-Di-(propargyl)-1,4,5,8-naphthalenediimide (19).

HeLa (Henrietta Lacks) cell lines and culture conditions. HeLa cervical cancer cells were obtained from ATCC (ATCC^® CCL-2™, Singapore) and cultured in RPMI-1640 supplemented with 10% foetal bovine serum, 1% l-glutamine, 1% minimal essential media non-essential amino acid (MEM NEAA) and 1% Penicillin–Streptomycin (Life Technologies, USA). The cells were maintained in a 5% CO₂ incubator at 37°C. For cytotoxicity assays, HeLa cells were cultured in 24-well plates by inoculating 5 × 105 cells per well per millilitre and incubating at 37°C in a 5% CO₂ incubator, which resulted in the formation of complete monolayers within 48 h.