Supporting information
Synthesis, bioactivity evaluation and docking study of 5-amino salicylic
acid’s fatty acid derivatives
Samira Yousefia*, Saadi Bayata, Mohd Basyaruddin Abdul Rahmana,b, Intan Safinar Ismaila,c, Elnaz Sakid, Sze
Wei Leongc and Emilia Abdulmaleka*
a
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
b
Structural and Synthetic Biology Research Centre, Malaysia Genome Institute, 43600 Bangi, Selangor, Malaysia
c
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
d
Department of Pathology, Faculty of Medicine and Health sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Experimental method
Materials and instruments
All chemicals were purchased and used without further purification. Analytical thin layer chromatography (TLC)
was performed using Merck 60 F254 precoated silica gel plate (0.2 mm thickness). Flash chromatography was
performed using Merck silica gel 60 (70-230 mesh). Fourier transforms infrared spectroscopy (FTIR); Perkin
Elmer Spectrum 100 was used for identification of functional groups. NMR data were recorded on 500 MHz for
1
HNMR and 125.8 MHz for 13C NMR (JEOL JNM ECA). High resolution mass of new compounds have detected
by Waters Single Quadrupole Detector (SQD).
Synthesis of 5-ASA fatty acid amide derivatives
5-ASA (0.5 g, 3.2 mmol) along with fatty acid (0.9 g lauric acid, 1.5 cm3 linoleic acid and 1.5 cm3 oleic acid; 4.9
mmol) and (0.6 g, 4.9 mmol) DMAP in dichloromethane solvent (DCM) were placed in flask fitted to calcium
chloride guard tube, molecular sieve (5% w/w) was added to the mixture and was stirred. In continue, DCC (1.0
g, 4.9 mmol) was added batch wise to the mixture at 0°C over 30 minutes. After that triethyl amine was added to
reach pH ≈ 7. The mixture was stirred for 3 to 5h in room temperature, (Fig. 1).
Initial characterizations of all products were performed by TLC (mobile phase 9:1 ethyl acetate/methanol) and
were visualized under UV lamp. Surplus materials were isolated by filtration. Solvent was removed under high
vacuum at 40°C. The rest of DCC was extracted by cold hexane (3×50 cm3) and precipitations were separated by
decanting hexane. Excess acid was neutralized by NaHCO3 (5%) and the rest of materials were extracted by DCM
(3×100 cm3). Following that, organic layer was dried with MgSO4 and evaporated under high vaccume. Further
purification has been done by flash chromatography (8:2 ethyl acetate/methanol).
In conclusion, three new 5-ASA’s oleic acid, linoleic acid and lauric acid amide derivatives were synthesized
successfully in moderate yields.
Fig 1. (a): 5-ASA, (b): fatty acids (oleic acid, linoleic acid, lauric acid); R: C 17H33, C17H31, C11H23;, (c): fatty acid derivatives of 5-ASA (products). (1c): 5-ASA
oleic acid amide, (2c): 5-ASA linoleic acid amide, (3c): 5-ASA lauric acid amide
Spectroscopic data of new compounds
5-ASA oleic amide (1c): 1H NMR (500 MHz, methanol-d4): δ (ppm) = 0.85 (t, J=6.6 Hz, 3H), 1.18 (br s, 20H),
1.38 (qnt, J=7.1 Hz, 2H), 1.49 (m, 4H), 2.29 (t, J=7.4 Hz, 2H), 5.02 (s, 1H), 5.31 (m, 2H), 6.78 (d, J= 8.2 Hz,
1H), 6.98 (d, J= 7.6, 1H), 7.39 (s, 1H), 8.26 (s, 1H), 10.20 (s,1H), 13C NMR (125.8 MHz, methanol-d4) δ = 14.1,
23.4, 23.8, 29.1, 29.4, 29.5, 29.8, 30.0, 31.3, 31.9, 32.0, 33.5, 38.7, 112.1, 112.5, 115.9, 122.2, 130.7, 143.3, 156.5,
167.9, 168.2, FT-IR (cm-1): 3283, 2920, 2855, 1657, 1540, 1450, 1193, 1081. Mass: [M] +, m/z 417.507,
[calculated mass 417.58, formula (C25H39NO4)]. Yield: 73%. Melting point: 138-140°C, white crystals.
5-ASA linoleic amide (2c): 1H NMR (500 MHz, methanol-d4): δ (ppm) = 0.96 (t, J=7.8 Hz, 3H), 1.39 (br s, 14H),
1.82 (qnt, J= 6.9 Hz, 2H), 2.14 (dd, J= 7.9, 3.1 Hz, 4H), 2.23(dd, J=8.1, 3.3 Hz, 2H), 2.65 (t, J=7.1Hz , 2H), 5.40
(s, 1H), 5.81 (m, 4H), 6.65 (s, 1H), 7.42 (d, J=8.2 Hz, 1H), 7.62 (d, J=7.2 Hz, 1H), 8.91 (s, 1H), 10.37 (s, 1H),
13
C NMR (125.8 MHz, methanol-d4) δ = 14.1, 23.7, 24.5, 29.2, 29.5, 29.8, 30.0, 30.1, 30.1, 30.1, 33.8, 37.6, 112.9,
113.1, 116.0, 122.3, 127.8, 131.0, 143.3, 156.5, 167.9, 168.4. FT-IR (cm-1): 3540, 3476, 3078, 2986, 2946, 2789,
2650, 1681, 1570, 1475, 1455, 1346, 1237, 1167, 1115. Mass: [M+H] +, m/z 416.488, [calculated mass 415.565,
formula (C25H37NO4)]. Yield: 65%. Melting point: 203-205°C, white crystals.
5-ASA lauric amide (3c): 1H NMR (500 MHz, methanol-d4): δ (ppm) = 0.91(t, J=7.2 Hz, 3H), 1.25 (s, 16H), 1.79
(m, 2H), 2.22 (t, J= 7.7 Hz, 2H), 5.14 (s, 1H), 5.67 (s, 1H), 6.61 (d, J= 8.1 Hz, 1H), 6.80 (d, J= 7.8 Hz, 1H), 7.8
(s, 1H), 11.01 (s, 1H), 13C NMR (125.8 MHz, Methanol-d4) δ =14.1, 23.5, 23.8, 28.3, 28.5, 29.6, 29.8, 30.9, 31.1,
31.1, 36.1, 112.8, 113.1, 116.0, 122.2, 143.3, 156.2, 167.7, 168.4. FT-IR (cm-1): 3489, 3355, 3120, 2991, 2861,
1742, 1679, 1557, 1485, 1442, 1337, 1239, 1104. Mass: [M+H] +, m/z 336.423, [calculated mass 335.452, formula
(C19H29NO4)]. Yield: 78%. Melting point: 261-263 °C, light pink crystals.
Spectra of compound 1c
Spectra of compound 2c
Spectra of compound 3c
Anti-bacterial method and results
Shortly, 5 mm diameter paper discs were impregnated with samples (previously were sterilized under UV) and
were placed onto the NA surface of plates that microbes were grown. The microbe culture was standardized to
0.5McFarland standard which is approximately 10 8cells.
The plates were inverted and incubated at 30-37°C for 18-24 hours until sufficient growth occurred. After
incubation, by ruler, the diameters of the zones around samples which indicate amount of inhibition were
measured to millimeter from the back of inverted petri plates.
Antibacterial activity screening of all new products against both Staphylococcus aureus (Gram-positive) and
Escherichia coli (Gram-negative) bacteria, manifested notable activity toward initial drug which presented
insignificant activity. Also all new products exhibited subtle more antibacterial specification against Grampositive than Gram-negative bacteria, (Table 1, Fig 2).
Table 1. Antibacterial activity of some new fatty acid derivatives of 5-ASA, all results have been duplicated and averages were recorded.
Microbes
Diameter of inhibition zone/(±1mm)
Entry
Sample name
Staphylococcus aureus
(Gram-positive)
Escherichia coli
(Gram-negative)
1
5-ASA oleic amide
15
10
2
5-ASA linoleic amide
13
10
3
5-ASA lauric amide
18
12
4
5-ASA
NA
NA
5
streptomycin
35
27
6
ethanol
NA
NA
NA: no activity found
Fig. 2. Showing antibacterial results against (A): Staphylococcus aureus and (B) Escherichia coli bacteria. No 1: 5-ASA oleic amide, No 2:
5-ASA linoleic amide, No 3: 5-ASA lauric amide, No 4: 5-ASA, No 5: positive control (streptomycin), No 6: negative control (ethanol)
Docking study and figures
(B)
(A)
To understand possible interactions and predict binding ability of new synthesized compounds with relevant
amino acids in active site of the protein docking study has been performed into COX-1, COX-2 and 5-LOX
enzymes and compared with parent drug (5-ASA).
Fig 3. Showing images visualized by Pymol program for the samples docked into COX-1 in the best of their conformation into the binding site
(B)
(A)
(C)
(D)
(E)
(A): 5-ASA (stick), (B): 5-ASA lauric amide (green), (C): 5-ASA linoleic amide (yellow), (D): 5-ASA oleic amide (green), (E): Ibuprofen
(orange)
Fig 4. Showing images visualized by Ligplot for the samples docked into COX-1 in the best of their conformation into the binding site
(A)
(C)
(E)
(A): 5-ASA, (B): 5-ASA lauric amide, (C): 5-ASA linoleic amide, (D): 5-ASA oleic amide, (E): Ibuprofen
(B)
(D)
Table 2. The docking results (AutoDock 4.2), regarding the binding free energy, distances and hydrogen bonds between compounds and amino acids involved in
COX-1
Compound name
Ibuprofen
5-ASA
5-ASA lauric amide
5-ASA linoleic amide
5-ASA oleic amide
∆G (kJ/mol): Binding free energy
∆G (kJ/mol)
Hydrogen bonds between atoms of compounds and amino acids into COX-1
Atom of
compound
Amino acid
Distance (Å)
COO
Ty355 (p-OH)
2.59
COO
Arg120 (NH2, NH)
2.61, 2.69
COO
Ty355 (p-OH)
2.57
COO
Arg120 (NH2, NH)
2.63, 2.65
COO
Arg120(NH2)
2.67
COO
Ty355 (p-OH)
2.84
p-OH
Met522(COO)
2.84
COO
Ser530 (OH)
3.04
COO
Ty385 (p-OH)
2.45
-35.22
-18.11
-34.93
-29.32
-28.36
Fig 5. Showing images visualized by Pymol for the samples docked into COX-2 in the best of their conformation into the binding site
(A)
(C)
(B)
(D)
(E)
(A): 5-ASA (pink), (B): 5-ASA lauric amide (purple), (C): 5-ASA linoleic amide (pink), (D): 5-ASA oleic amide (yellow), (E): S58 (light
pink)
Fig 6. Showing images visualized by Ligplot for the samples docked into COX-2 in the best of their conformation into the binding site
(A)
(C)
(E)
(A): 5-ASA, (B): 5-ASA lauric amide, (C): 5-ASA linoleic amide, (D): 5-ASA oleic amide, (E): S58
(B)
(D)
Table 3. The docking results (AutoDock 4.2), regarding the binding free energy, distances and hydrogen bonds between compounds and amino acids involved in
COX-2
Compound name
S58
5-ASA
5-ASA lauric amide
5-ASA linoleic amide
5ASA oleic amide
∆G (KJ/mol): Binding free energy
∆G (kJ/mol)
Hydrogen bonds between atoms of compounds and amino acids into COX-2
Atom of compound
Amino acid
Distance (Å)
F
Arg120 (NH2)
3.20
S=O
His90 (N)
2.79
NH2
Leu352 (COO)
2.76
NH2
Gln192(COO)
2.89
NH2
Ala527 (COO)
2.95
COO
Tyr355 (p-OH)
2.93
COO
Arg120 (NH2)
2.54
COO
Ty355 (OH)
2.86
N-C=O
Arg12 (NH2)
3.08
p-OH
Ty355 (p-OH)
3.07
COO
Gln192(NH2)
2.87
COO
Ser353(COO)
2.80
-45.39
-16.48
-30.54
-31.38
-29.66
Fig 7. Showing images visualized by Pymol for the samples docked into 5-LOX in the best of their conformation into the binding site
(a)
(A)
Fe
(B)
(b)
Fe
(A) and (a): 5-ASA (yellow), (B) and (b): 5-ASA lauric amide (light green); (a), (b): showing mode of compounds around Iron.
Fig 8. Showing images visualized by Pymol for the samples docked into 5-LOX in the best of their conformation into the binding site
(C)
(c)
Fe
(D)
(d)
Fe
(C) and (c): 5-ASA linoleic amide (light green), (D) and (d): 5-ASA oleic amide (yellow); (c), (d): showing mode of compounds around Iron.
Fig 9. Showing images visualized by ligplot for the samples docked into 5-LOX in the best of their conformation into the binding site
(A)
(C)
(A): 5-ASA, (B): 5-ASA lauric amide, (C): 5-ASA linoleic amide, (D): 5-ASA oleic amide
The meaning of the items for images were taken by ligplot are as follows:
(B)
(D)
Tale 4. The docking results (AutoDock 4.2), regarding the binding free energy, distances and hydrogen bonds between compounds and amino acids involved in
5-LOX
Compound name
∆G (kJ/mol)
Hydrogen bonds between atoms of compounds and amino acids into 5-LOX
Atom of
compound
Amino acid
Distance (Å)
5-ASA
-15.39
NH2
Ala606 (COO)
2.48
5-ASA lauric amide
-19.24
COO
Ile673 (COO)
2.99
NH
Glu614 (COO)
2.49
5-ASA linoleic amide
-14.56
COO
Arg401 (NH2)
2.83
COO
Gln363 (N-C=O)
3.03
NH
Tyr181 (p-OH)
2.32
5-ASA oleic amide
-21.92
∆G (kJ/mol): Binding free energy
Fig 10. (A): illustrating position of 5-ASA lauric amide as a representative of other new compounds docked into COX-1 in contrast with co-crystalized ligand
Ibuprofen (B): illustrating position 5-ASA oleic amide as a representative of other new compounds docked into COX-2 in contrast with co-crystalized ligand S58.
As it is seems in both images the docked ligands exactly superimposed on the native ones.
(A)
(B)