6
Z. NGAINI AND N. A. MORTADZA
3.2. General procedure to the synthesis of aspirinate-azo derivatives (2a-d)
Halogenated azo derivative 1a-d (3 mmol) in dichloromethane (20 mL) was added into a
solution containing acetylsalicylic acid (0.54 g, 3 mmol) in dichloromethane (20 mL). The
mixture was stirred for 15 min. Dicyclohexylcarbodiimide (0.62, 3 mmol) was added into the
mixture, followed by N,N-dimethyl-4-aminopyridine (0.37 g, 3 mmol). The reaction mixture
was stirred for 4 h at 0–10 °C. The white precipitate of dicyclohexylurea was filtered and the
filtrate was evaporated under vacuo. The crude was purified by flash column chromatography
on silica gel using hexane. The solid product was recrystallized from hot ethanol to give
2a-d. The characterization of 2a-d is available in the supplementary materials (S6).
3.3. General procedure to the synthesis of azo derivatives (3a-d)
Sodium nitrite solution (1 M, 2 mL) was added dropwise to a mixture of halogenated aniline
(0.46 g, 5 mmol) in HCl (2 M, 6 mL) in an ice bath (0–5 °C). The mixture was then added into
a solution of aspirin (0.90 g, 5 mmol) and sodium hydroxide (0.4 g, 10 mmol) in distilled water
(10 mL). The solution was cooled to 0–5 °C and stirred for 1 h. The mixture was acidified with
HCl (2 M) to form precipitate. The solid was filtered, washed and recrystallized from ethanol
to give 3a-d. The characterization of 3a-d is available in the supplementary materials (S6).
3.4. Antibacterial studies
The antibacterial activities of the synthesised compounds were studied against E. coli and
S. aureus. E. coli was cultured on Luria–Bertani (LB) broth and incubated at 37 °C for 24 h at
200 rpm. Transmittances (T) were recorded in UV-Visible spectrophotometers. Erlenmeyer
flasks containing 100 mL of culture medium were added with 50, 80 and 100 ppm concen-
trations of compounds, inoculated with 0.99 mL of inoculums and stirred in a culture cham-
ber at 37 °C with 180 rpm. Inoculums with DMSO were used as negative control. Aliquots
were extracted every 1 h interval for 6 h and transmittances (T) were recorded in a UV-Visible
spectrophotometer at 560 nm wavelength. T values were extrapolated to the number of
cfu/mL (colony forming units/mL) for E. coli expressed in ln Nt. The antibacterial screening
was also performed using S. aureus (Abd Halim and Ngaini 2016).
3.5. Molecular docking
Molecular docking studied were done using AutoDock Vina 1.1.2 program and AutoDock
Tools 1.5.6 were used prior to docking with AutoDock Vina program (Morris et al. 2009; Trott
and Olson 2010). The cubic grid box of 40 Å size (x, y, and z) with a spacing of 0.375 Å were
centred on the active site of the protein. The X-ray structure of E. coli phospholipid binding
protein MIaC (PDB entry: 5UWA) and phosphatidylinositol-Specific Phospholipase C
Membrane Binding of S. aureus (PDB entry: 4F2B) was retrieved from Protein Data Bank
4. Conclusion
Three series of azo derivatives 1a-d, aspirinate-halogenated azo derivatives 2a-d and halo-
genated azo-aspirin derivatives 3a-d synthesised from natural product derived compound