Synthesis and in vitro bioactivity evaluation of new glucose and xylitol ester derivatives of 5-aminosalicylic acid

Article abstract of DOI:10.1039/c5ra19623j

New glucose and xylitol esters of 5-amino salicylic acid (5-ASA) were synthesized followed by evaluation of their in vitro antimicrobial, anti-cancer and anti-inflammatory activities. The results of the antimicrobial activity assessment revealed that the new final esters were more effective against Gram-negative as well as Gram-positive bacteria than the original drug. Furthermore, the new final products were confirmed by a cytotoxicity assay over HT-29 and 3T3 cell lines to be less toxic for normal cells compared to the initial drug. On the other hand, however, their suppressive effect against cancerous cells was somewhat lower. Meanwhile, the anti-inflammatory activity assay over a RAW264.7 macrophage cell line demonstrated that the NO inhibition activity of the conjugated drug to the previously mentioned saccharides, especially to glucose, has slightly improved compared to the non-conjugated drug. Finally, in silico screening was also performed in order to predict the potential interactions and binding energy of the novel products against cyclooxygenase (COX-1/COX-2) and lipoxygenase (5-LOX) proteins. Findings indicated that the new products had greater hydrogen bonds and binding affinities with the active sites of proteins towards 5-ASA.

Full text of DOI:10.1039/c5ra19623j

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PAPER
Synthesis and in vitro bioactivity evaluation of new
glucose and xylitol ester derivatives of 5-
aminosalicylic acid†
Cite this: RSC Adv., 2015, 5, 97295
a
a
abc
Samira Yousefi,* Saadi Bayat, Mohd Basyaruddin Abdul Rahman,
ad
e
ab
Intan Safinar Ismail, Elnaz Saki and Emilia Abdulmalek*
New glucose and xylitol esters of 5-amino salicylic acid (5-ASA) were synthesized followed by evaluation of
their in vitro antimicrobial, anti-cancer and anti-inflammatory activities. The results of the antimicrobial activity
assessment revealed that the new final esters were more effective against Gram-negative as well as Gram-
positive bacteria than the original drug. Furthermore, the new final products were confirmed by
a cytotoxicity assay over HT-29 and 3T3 cell lines to be less toxic for normal cells compared to the initial
drug. On the other hand, however, their suppressive effect against cancerous cells was somewhat lower.
Meanwhile, the anti-inflammatory activity assay over a RAW264.7 macrophage cell line demonstrated that
the NO inhibition activity of the conjugated drug to the previously mentioned saccharides, especially to
glucose, has slightly improved compared to the non-conjugated drug. Finally, in silico screening was also
performed in order to predict the potential interactions and binding energy of the novel products against
cyclooxygenase (COX-1/COX-2) and lipoxygenase (5-LOX) proteins. Findings indicated that the new
products had greater hydrogen bonds and binding affinities with the active sites of proteins towards 5-ASA.
Received 23rd September 2015
Accepted 6th November 2015
DOI: 10.1039/c5ra19623j
www.rsc.org/advances
6
and nitrogen species, make it a multivalent drug. Hence, the
1
. Introduction
9,10
11
derivatives of 5-ASA with polymers,
nanoparticles and
12
Inammatory bowel disease (IBD) is a high-incidence disease amino acids have been prepared in order to expand its
which is induced by a complex interplay among environmental, applications. Its complexes with metals have shown greater
1
,2
13
genetic and immunoregulatory factors. IBD includes two antimicrobial activity than 5-ASA alone. For optimizing clin-
14
15
major constituents, namely, ulcerative colitis and Crohn's ical use, chitosan and azo-bond derivatives of 5-ASA have
disease, and if not cured properly, can lead to outbreak of been produced to deliver the drug to targeted sites.
3,4
collateral associated cancers. Therefore, high priority should
Despite the development of numerous benecial 5-ASA
be accorded to the achievement of better therapeutic effects for derivatives, patient life quality has so far not been improved.
the treatment of IBD. For more than half a century, 5-amino- This is due to the fact that, when the drug is taken orally or used
salicylic acid (5-ASA) has been the most widely prescribed anti- in the form of a suppository, the main portion of the drug is
2
inammatory drug for the treatment of IBD. It reduces gastro- absorbed in the small intestine and hardly reaches the colon.
5
16
intestinal toxicity and is tolerable for the majority of patients Its low stability in the gastrointestinal tract and poor water
4,6,7
15,17,18
because it has limited side-effects.
In addition, its chemo- solubility
are other problems which result in the necessary
preventive activity, particularly against colitis-associated consumption of a higher dosage of the drug in order to be
8
cancers, and its antioxidant property against reactive oxygen effective, the requirement of long-term treatment and a greater
number of side-effects. In some cases, the disease relapses and
4,19,20
surgery is required.
a
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM
On the other hand, the use of carbohydrate drug derivatives
has been increasingly favoured in recent times. The versatile
applications of carbohydrates in a variety of elds have attracted
Serdang, Selangor, Malaysia. E-mail: syouse12@yahoo.com; emilia@upm.edu.my
b
Enzyme and Microbial Technology Centre, Faculty of Biotechnology and Bimolecular
Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
c
Structural and Synthetic Biology Research Centre, Malaysia Genome Institute, 43600 the attention of many researchers due to their biodegradability,
21
Bangi, Selangor, Malaysia
nontoxicity, as well as amphipathic and emulsifying charac-
d
22,23
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia,
teristics.
A wide range of anticancer, antibiotic, antiviral or
4
3400 Serdang, Selangor, Malaysia
fungicidal active compounds relied on conjugating with carbo-
e
Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular
Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
22,24
hydrates for development of diverse medicinal applications.
Furthermore, it has been found that saccharides increase pene-
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c5ra19623j
22
tration into cell membranes and decrease toxicity. In addition,
1
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the anti-inammatory properties of some synthetic compounds Furthermore, 3-O-peruoroalkylated D-glucose and O-per-
25
possessing glycosidic linkages have been recognized. By uoroalkylated D-xylitol amphiphiles have been prepared and
comparison to ibuprofen, ibuprofen fructose ester exhibited biological assessment displayed stabilisation effects of sugars on
3
6
fewer side-effects, longer stability in aqueous solution and better peruorocarbon emulsions.
uptake of glycosylated drugs into tissue. Meanwhile, compared
26
Thus, considering that conjugation of drugs with saccha-
to the parent drug, glucose-conjugated aspirin improved its rides enriches the bioavailability and pharmaceutical properties
27
28,29
solubility and anti-cancer activities. By comparison with non- of drug,
glucose and xylitol esters of 5-ASA were prepared as
saccharide derivatives, porphyrin–saccharide incapacitated models of sugar and sugar alcohol derivatives of 5-ASA, followed
human breast cancer due to greater binding to cancerous by the evaluation of the in vitro bioactivity of new products for
28,29
cells.
Moreover, glycotargeting has demonstrated that carbo- antibacterial, anti-inammatory and anticancer activities and
hydrate ligands can be feasibly used to target protein receptors at their comparison with the parent drug (5-ASA).
30
sites of localization. Additionally, carbohydrate fatty acid
derivatives showed signicant potential for improving antibac-
2
. Results and discussion
23
terial agents. Sugar alcohols also have been proven to possess
biological activities. Sugar alcohols selectively reduced the acute
lethal toxicity of 1-(2-chloroethyl)-3-(methyl alpha-D-glucopyranos-
2.1. Synthesis
In this work, esterication was purposed for preparing 5-ASA
derivatives, since the carbohydrate esters have been shown to be
31
6-yl)-1-nitrosourea without reducing its anti-tumour activity.
Anti-tumoural and antimicrobial specications of xylitol esters
involved in varied biological processes such as antineoplastic,
21,32
have been documented as well.
Xylitol has been advocated as
37,38
antidiabetic, anti-inammatory, etc.
Furthermore, it has been
an optimal choice for linkage to drugs, due to the fact that its anti-
biolm activity has been proven to have efficiency in suppressing
severe yeast condition and other harmful bacteria, as well as ulcers
found that ester bond is fairly xed in the acidic medium and
readily ruptured in basic environment which is compatible
39
with gut nature and may enable the drug glycoside on colonic
33,34
and stomach cancers.
Moreover, in vitro pharmacological
tract releases the active drug through enzymatic action. More-
over, protection of hydroxyl functions by O-isopropylidenation
has been performed as it is a well-known method due to its
investigations using xylitol butyric derivatives have shown poten-
35
tial in the treatment of sickle cell anaemia and cancer.
ꢀ
Scheme 1 Reagents and conditions: (i) H
under N gas, 5 h.
3
PO
4
, 80 C; (ii) acetone, ZnCl
2
, H
3
PO
4
, 5 h; (iii) DCC, DMAP, DCM, 24 h, r.t; (iv) acetic acid 85%, reflux
2
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40
convenient application in synthetic studies. The di-and/or mono- involved in the reaction. A highly efficient, one step and regio-
O-isopropylidene derivatives have been utilized widely in total selective method for the direct O-isopropylidenation of
syntheses of some bioactive macromolecules and various natural D-glucose and D-xylitol is using anhydrous acetone in the pres-
41
products since their low toxicity and biological activities ence of an acid catalyst. Subsequently, esterication was
such as anti-inammatory and antipyretic activities have been carried out through a facile and one-step method which eludes
4
0,41
0
documented.
introduced be to useful for biochemical studies as a key starting (DCC) and 4-dimethylaminopyridine (DMAP) as catalysts in
In addition, C-3 derivatives of D-glucose were harsh reaction conditions by using N,N -dicyclohexylcarbodiimide
42
44–46
material. For instance 1,2:5,6-di-O-isopropylidene-D-glucofur- dichloromethane (DCM) solvent.
anose has been used as a starting material for synthesis of glucose the isopropylidene group occurs under mildly acidic conditions,
aspirin. A series of 3-O-alkyl and 3-O-haloalkyl-D-glucoses and hence, nally, deprotection of compounds was achieved by
As regards, the hydrolysis of
47
27
likewise in another work a series of fatty acid D-glucose derivatives reuxing under nitrogen gas in presence of acetic acid. The
1
were prepared from 1,2:5,6-di-O-isopropylidene-D-glucofuranose structures of the synthesized products were conrmed by
H
13
and several of them showed antimicrobial efficacy comparable NMR, C NMR, FT-IR and mass spectroscopies.
21,43
with commercially available antimicrobials.
A chemical route designed to obtain 5-ASA monosaccharide
esters is shown in Scheme 1. The synthesis was commenced
with the selective protection of the hydroxyl groups of sugars
and protection of amine group of 5-ASA to avoid becoming
2
.2. Antibacterial evaluation
The antibacterial activity evaluation of the new monosaccharide
esters of 5-ASA (f and g) against both Gram-positive bacteria and
a
Table 1 In vitro antibacterial activity of samples, the results were duplicated and averages were recorded
Microbes
Diameter of inhibition zone in millimeter (ꢁ1 mm)
Entry
Sample name
Staphylococcus aureus (+)
Bacillus subtilis (+)
Escherichia coli (ꢂ)
Salmonella choleraesuis (ꢂ)
1
2
3
4
5
5-ASA
Streptomycin
Ethanol
f
NA
35
NA
14
NA
25
NA
NA
NA
NA
27
NA
10
NA
25
NA
NA
NA
g
14
12
a
NA: no activity found at the tested concentration. (+) Gram-positive and (ꢂ) Gram-negative bacteria.
a
Table 2 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of samples
ꢂ1
MIC/MBC (mg mL
)
Entry
Microbes
5-ASA
f
g
Streptomycin*
1
2
3
4
Escherichia coli (ꢂ)
NA/NA
NA/NA
3.33/>3.33
NA/NA
1.92/>3.33
NT/NT
1.60/3.33
NT/NT
1.60/3.33
NT/NT
1.33/2.77
NT/NT
24.11/28.93
NT/NT
16.74/24.11
NT/NT
Salmonella choleraesuis (ꢂ)
Staphylococcus aureus (+)
Bacillus subtilis (+)
a
NA: no activity found at the tested concentration. (+) Gram-positive and (ꢂ) Gram-negative bacteria. NT: not tested. Streptomycin*: concentration
ꢂ1
unit mg mL
.
a
Table 3 In vitro nitric oxide (NO) suppression activity and cytotoxicity of samples on RAW 264.7 cells at 50 mM, S.E.M: standard error of the mean
NO inhibition
NO inhibition
Cytotoxicity
Entry
Sample name
(%) ꢁ S.E.M
IC50 (mM) ꢁ S.E.M
IC50 (mM)
1
2
3
4
Curcumin
5-ASA
f
g
99.3 ꢁ 0.2
9.0 ꢁ 1.1
13.2 ꢁ 1.6
9.2 ꢁ 3.0
14.7 ꢁ 0.2
ND
ND
>100
>100
>100
>100
ND
a
ND: not detected.
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Gram-negative bacteria exhibited, both new compounds were mediators. Nitric oxide (NO) is a key inammatory mediator
more effective against Escherichia coli and Staphylococcus aureus which is secreted by inducible nitric oxide synthase (iNOS) from
bacteria than the initial drug which presented insignicant activated macrophages during the inammatory response.
activity against all tested organisms. However, the antibacterial When it occurs in excess, this factor contributes to a variety of
properties of the new products were not comparable with conditions and diseases, including swelling, pain, asthma,
48–50
antimicrobial agents (streptomycin). Based on Table 1, there cardiovascular disorders, cancer and other diseases.
Here-
was no great difference between the antibacterial activities of f upon, the NO inhibition activity of the nal esters (f and g) in
and g. Both new products demonstrated no outstanding inhi- IFN-g/LPS-stimulated RAW264.7 macrophages (at 50 mM
bitions against Salmonella choleraesuis (Gram-negative) and concentration) was screened and compared with the parent
Bacillus subtilis (Gram-positive) bacteria. On the other hand, drug. Despite the NO inhibition activity of the new synthesized
both f and g showed an average inhibition zone of 14 ꢁ 1 mm compounds and 5-ASA was not as effective as positive control
against Staphylococcus aureus. Furthermore, the average inhi- (curcumin), with less than 50% NO inhibition being achieved,
bition zones of 10 ꢁ 1 mm and 12 ꢁ 1 mm were conjugation of 5-ASA to glucose increased the NO inhibition (%)
observed against Escherichia coli for f and g, respectively. activity of 5-ASA by almost 1.5 fold, from 9.0 mM to 13.2 mM.
Accordingly, the inhibition activities of both f and g were Meanwhile, conjugating to xylitol was revealed to be less effec-
found to be slightly greater against Staphylococcus aureus tive with the NO inhibition (%) activity of 9.2 mM. These results
bacteria than against Escherichia coli ones (Table 1). Hence, the may contribute to the improvement of anti-inammatory
MIC and MBC tests were proceeded for the bacteria which properties in the future. Additionally, the cell viability test
showed sensitivity against samples in disc diffusion assay. results conrmed that NO inhibition at 50 mM was not due to
Based on Table 2, minimum inhibition concentration of g the toxicity of the samples (Table 3).
against both E. coli and S. aureus was slightly lower than f,
ꢂ1
moreover, 3.33 mg mL MIC was found for 5-ASA against
2.4. Anti-cancer evaluation
Staphylococcus aureus (Table 2).
One of the predominant problems during the chemotherapy
period is the side effects of drugs over normal cells, therefore,
diminishing the toxicity of drugs against normal cells with
maintaining anti-cancer properties of them is the priority.
According to the evaluation of cytotoxicity against cancerous
cells (HT-29 cell line), the inhibition activity of the new prod-
2
.3. Anti-inammatory evaluation
Inammation is a defensive response of the immune system
against infections, tissue damage or harmful foreign stimuli
and is accompanied by the production of pro-inammatory
ꢂ1
ꢂ1
ucts, f (IC50: 8.1 mg mL ) and g (IC50: 9.9 mg mL ), was slightly
ꢂ1
reduced in comparison with 5-ASA (IC50: 5.1 mg mL ) (Table 4
Table 4 In vitro anticancer activity of samples against HT-29 and 3T3
cell lines ꢁ SD (standard deviation)
and Fig. 1). On the other hand, the cytotoxicity test displayed
that both f and g were much less harmful than the parent drug
ꢂ1
ꢂ1
Sample
name
IC50 (mg mL ) for
IC50 (mg mL ) for for normal cells (3T3 cell lines), since 50% of 3T3 cells were
ꢂ1
Entry
HT-29 cell line ꢁ SD
3T3 cell line ꢁ SD inhibited by approximately 2918.5 mg mL of f and 9254.0 mg
ꢂ1
ꢂ1
mL of g, while only 6.1 mg mL of 5-ASA inhibited 50% of
normal cells (Table 4 and Fig. 1). Consequently, although the
anticancer activity of the new compounds was slightly less
potent than that of 5-ASA, their signicant reduced toxicity to
1
2
3
5-ASA
f
g
5.1 ꢁ 0.01
8.1 ꢁ 0.01
9.9 ꢁ 0.03
6.1 ꢁ 0.008
2918.5 ꢁ 0.008
9254.0 ꢁ 0.01
Fig. 1 Graphs were plotted for HT-29 and 3T3 cell lines with the percentage of cell viability against their respective concentration. HT-29 and
T3 cell lines after treatment by: Series1: 5-ASA, Series2: g, Series3: f.
3
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normal cells makes them promising options for future drug and is a prerequisite for successful structure-based drug design
development. because it is used to screen virtual libraries of drug-like mole-
54
cules to obtain leads for further drug development, and
55
anticipation of the biological activity of molecules before
experimental work which can be time-consuming and costly.
2
.5. Molecular docking analysis
56
Recently, the computer-based analysis are utilizing as a key tool
to drug discovery in the eld of molecular modelling. Indeed,
molecular docking is a computer-assisted drug design with the
aim of characterizing the predominant mode(s) of the small
molecules (ligand) into the binding pocket of target proteins
Despite many evidence that proved the inhibition effect of 5-
ASA against cyclooxygenase protein (COX) and lipoxygenase, its
2,57,58
mechanism of action remains controversial.
Cyclo-
oxygenase has two main isoforms: COX-1 and COX-2. COX-1
causes cytoprotection in the gastrointestinal tract, whereas
COX-2 mediates inammation. On the other hand, lipoxygenase
(three-dimensional structure) as well as estimating the binding
51–53
affinity.
This prediction is of particular practical importance
ꢂ1
Table 5 The docking results (AutoDock 4.2), regarding the binding free energy: [DG (kcal mol )], distances or lengths of hydrogen bonds: [D ( A˚ )]
and hydrogen bonds between compounds and amino acids involved in COX-1, COX-2 and 5-LOX
Hydrogen bonds between atoms of compounds and amino acids into related protein
COX-1
COX-2
5-LOX
Compound name
structure
Atom of
compound Amino acid D
Atom of
compound Amino acid
Atom of
compound acid
Amino
&
DG
DG
D
DG
D
Ty355
COO
2.59
2.61,
(
p-OH)
ꢂ8.42 COO
Arg120
—
—
—
—
—
—
—
—
—
—
(
NH
2
, NH) 2.69
F
Arg120 (NH
His90 (N)
Leu352 (COO) 2.76
Gln192(COO) 2.89
2
)
3.20
2.79
S]O
NH
NH
2
2
—
—
—
ꢂ10.85
—
—
Ty355
p-OH)
Arg120
NH
COO
2.59
NH
2
Ala527 (COO) 2.95
(
COO
2.61,
, NH) 3.03
COO
Tyr355 (p-OH) 2.93
Ala606
ꢂ4.33
ꢂ3.94
ꢂ3.68 NH
2
2.48
(
2
(COO)
Ser530
COO)
Arg120
NH)
Val349
COO)
Ser353
NH
Ile673
(COO)
Gln363
(COO)
His367
(N)
Tyr181
(p-OH)
Tyr181
p-OH
2.62
3.12
p-OH
Ty385 (p-OH) 2.80
p-OH
2.71
2.72
(
C
6
-OH
-OH
-OH
NH
2
Met522 (COO) 2.93
C
1
-OH
-OH
-OH
-OH
(
ꢂ5.78 C4
3.02
2.90
ꢂ5.78 C1
-OH
-OH
Tyr355 (p-OH) 2.62
Arg120 (NH) 3.11
Arg120 (NH) 3.01
ꢂ5.40 C2
2.83
2.97
2.63
(
C
4
C
6
C
5
(
2
)
C–O–C
C
6
(
p-OH)
Met522
COO)
Arg120
NH)
Ser530
COO)
Ile406
(COO)
Asn407
(H N–C]O)
2
Gln363
(COO)
NH
2
2.71
2.94
p-OH
Val523 (COO) 2.58
NH
2
2
3.00
3.03
(
C
5
-OH
NH
2
Ty385 (p-OH) 2.77
NH
(
ꢂ4.22 p-OH
2.90
ꢂ4.07 C4
-OH
-OH
-OH
Arg120 (NH) 2.96
Arg120 (NH) 2.71
Ty355 (p-OH) 2.93
ꢂ4.55 C3
-OH
-OH
-OH
2.53
2.74
2.64
(
C
3
C
4
Gln363
(
COO)
Thr364
HC-OH)
C
3
C
5
(
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Fig. 2 Showing the examples of samples docked into the binding site of related protein in the best of their conformation. Images were rendered
from Pymol (capital letter) and Ligplot softwares (small letter): images (A) and (a) illustrating 5-ASA (blue) into COX-2, images (B) and (b) illustrating
the best mode of f (purple) into COX-1 and images (C) and (c) illustrating the best pose of g (green) into 5-LOX. The meaning of the items for
images taken by ligplot are as follows:
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LOX) is known for its essential role in leading to pro-
RSC Advances
(
In terms of the binding energy, the binding affinities of the
59
inammatory mediators especially in human body. In the new products into COX-1 observed to be moderate in compar-
ꢂ1
family of lipoxygenase, 5-lipoxygenase (5-LOX) has received ison with ibuprofen (DG: ꢂ8.42 kcal mol ), however,
ꢂ1
attention due to its therapeutic potential. For treatment of compounds f (DG: ꢂ5.78 kcal mol ) revealed greater binding
ꢂ1
inammation, some dual COX/5-LOX non-steroidal anti- energy toward both g (DG: ꢂ4.22 kcal mol ) and 5-ASA (DG:
ꢂ1
inammatory drugs (NSAIDs) have been designed and ach- ꢂ4.33 kcal mol ) which exhibited almost the equal binding
59
ieved great successes. Therefore, to understand possible energies towards each other. Although the binding energies of
interactions and predict binding ability of new synthesized the new derivatives into COX-2 were not comparable with the co-
ꢂ1
compounds with the relevant amino acids in active site of the crystallized S58 (DG: ꢂ10.85 kcal mol ), but based on binding
ꢂ1
protein docking study has been performed into COX-1, COX-2 affinities f (DG: ꢂ5.78 kcal mol ) showed almost one and half
and 5-LOX enzymes and compared with parent drug.
fold greater binding energy than the parent drug (DG: ꢂ3.49
ꢂ1
In docking study toward COX-1 protein the co-crystallized kcal mol ) and seems to be a more reasonable candidate than
ꢂ1
ibuprofen was utilized as a positive control ligand. The key g (DG: ꢂ4.12 kcal mol ). Also, studying of binding energy of the
amino acids of COX-1 consist: His90, Arg120, Val349, Tyr355, new derivatives against 5-LOX predicted both f (DG: ꢂ5.40 kcal
ꢂ1
ꢂ1
Arg53, Met522 and Glu524 (ref. 57 and 59) where carboxylic mol ) and g (DG: ꢂ4.55 kcal mol ) have stronger binding
ꢂ1
group of ibuprofen formed two hydrogen bonds between NH affinities than 5-ASA (DG: ꢂ3.68 kcal mol ), (Table 5 and
2
and NH groups of Arg120 and one hydrogen bond between Fig. 2).
phenolic OH of Ty355. The co-crystallized S58, was used to
parameterize molecular docking study against COX-2. The main
amino acids in this interaction include: Val349, Ser530, Leu352,
3
. Conclusion
Tyr385, Tyr348, Trp387, Gly526, Ala527, Met522, Leu384, His90, Glucose and xylitol esters of 5-amino salicylic acid were
Arg120, Tyr355, Arg53, Phe518 and Gly526, where S58 formed synthesized successfully in average yields with the approxi-
four hydrogen bonds with His90, Arg120, Leu352 and Gln192 mately moderate to superior bioactivities toward 5-ASA which
(ref. 57 and 59). It has been found that the active site of 5-LOX is offering a larger therapeutic window for preparation of a library
around catalyst non-hem iron atom. The iron is coordinated by of different 5-ASA's monosaccharide derivatives. Comparing
three conserved histidines (histidines 367, 372, and 550), as well bioactivities between sugar and sugar alcohol showed there was
as the main-chain carboxylate of the C terminus (Ile673). The no signicant difference between antibacterial activity of them
other predominantly involved amino acids contain: Tyr181, also both derivatives exhibited slightly greater inhibition
60
Ala603, Ala606, His600 and Thr364. As regards, there is no activity against Staphylococcus aureus (Gram-positive) than
reference co-crystallized ligand into 5-LOX, the same parame- against Escherichia coli (Gram-negative) ones which is in
ters applied in docking against COX-1 and COX-2 were utilized. accordance with previous studies on carbohydrate deriva-
2
1,62
Additionally, hydrogen bond interactions between the samples tives.
and the amino acids in active site conrmed that docking better candidate for suppressing nitroxide over macrophage
carried out in the targeted site. As cited in the literatures,
Also it can be noted that 5-ASA's glucose ester was
59,61
if cells than xyilitol ester. Moreover, cytotoxicity assay for inhib-
the RMSD (root mean square deviation) of the best docked iting HT-29 cells, revealed that f had greater potential than g,
˚
conformation of co-crystal ligand is #2 A from the experimental whereas g was less harmful than f against 3T3 cell lines. On the
one the used scoring function is reliable. The RMSD value of other hand, in silico docking study anticipated that although
˚
˚
0
.98 A for ibuprofen and 1.57 A for docked S58 validated the against all proteins f showed better binding energies, by
accuracy of the AutoDock4.2 performance. comparison to the parent drug, both new ester derivatives of 5-
In docking against COX-1, 5-ASA exhibited two hydrogen ASA (f and g) formed more hydrogen bonds with active site
bonds with NH and NH groups of Arg120 and one hydrogen residues of the aforementioned proteins as well as had greater
2
bond with phenolic OH of Ty355 from its carboxyl group. More- binding energy which is encouraging to further experimental
over, four hydrogen bonds were observed between functional investigation over chosen proteins and in vivo medicinal
groups of f with Val349, Ser353, Arg120, and Ser530. g conserved chemistry exploration.
three hydrogen bonds between its functional groups with Arg120,
Met522 and Ser530. In case of molecular docking into COX-2, 5-
ASA conserved two hydrogen bonds between carboxyl of Ala527
and phenolic OH of Ty355 with its amine and carboxylic acid
4
. Experimental
4.1. Materials
groups, respectively. Additionally, both new compounds became All materials in this project were purchased in analytical grade
involved in three more hydrogen bonds with amino acids in and used without further purication, otherwise stated. Merck
active site of COX-2 protein than 5-ASA. f exhibited ve hydrogen silica gel 60 (70–230 mesh) was used for ash chromatography.
bonds with Met522, Try385, Arg120 and Try355. Similarly, g Analytical thin layer chromatography (TLC) was implemented
formed ve hydrogen bonds with Tyr385, Val523, Arg120, and by using Merck 60 F254 precoated silica gel plate (0.2 mm
Try355. In docking study toward 5-LOX, 5-ASA formed one thickness). Functional groups were detected by Fourier trans-
hydrogen bond from its amine group with carboxyl group of form infrared spectroscopy (FT-IR); (Perkin Elmer Spectrum
1
Ala606. Whiles, both new compounds formed ve hydrogen 100). NMR data were obtained by 500 MHz for H NMR and
1
3
bonds with amino acids in active site of 5-LOX protein.
125.8 MHz for C NMR (JEOL JNM ECA) spectrometer. Direct
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infuse mass spectrometry (DIMS) was applied for mass char- through ash chromatography with the same mobile phase
acterization using ACQUITY® SQD with Single Quadrupole applied for TLC to obtain (b) and (c), Scheme 1.
Detector (Waters Corporation, Milford, MA USA). Mass of
compound (a) has been recorded by GC-MS QP2010 Plus SHI- 4.4. General procedure for esterication and preparation of
MADZU. Optical rotations of products were measured on (d) and (e)
a JASCO P-2000 Polarimeter.
Over an ice bath, a (0.1 g, 0.5 mmol), b or c (0.2 g, b: 0.7 mmol, c:
All solvents prepared from J.T. Baker. 5-ASA, DCC, DMAP,
0.8 mmol) and DMAP (0.09 g, 0.7 mmol) were dissolved in DCM.
˚
Molecular sieves 3 A (8 to 12 mesh) were from ACROS Company.
Aer that, DCC (0.15 g, 0.7 mmol) was added batch-wise (over
D-Glucose and D-xylitol were bought from Fisher Scientic
Company. Nutrient agar and nutrient broth have been supplied
from Sigma Aldrich. Bacterial strain subcultures were prepared
in department of Microbiology, Faculty of Medicine and Health
Sciences, UPM University Malaysia. Melting points were
measured by Barnstead Electrothermal instrument. RAW 264.7
murine macrophages cells obtained from American Type
Culture Collection (ATCC, Rockville, MD, USA).
1
2
5–20 minutes) to the mixture. Subsequently, it was stirred for
4 h at room temperature. Aer initial identication by TLC
(eluent solvent 8 : 2 ethyl acetate/hexane) dicyclohexylurea was
separated by ltration and the solvent was removed under high
vacuum. Next, the mixture was dissolved in the cold hexane (5 ꢃ
50 mL) to detach unreacted DCC. Later, precipitations were
collected by decanting hexane. The surplus DMAP was extracted
by NH Cl (20%) and DCM. Subsequently, the organic layer was
dried with MgSO and evaporated. Further purication was
4
4
fullled through column chromatography (mobile phase 8 : 2,
ethyl acetate/hexane) to obtain (d) or (e), Scheme 1.
4
.2. Protection of 5-ASA and preparing (a)
The synthesis of 5-acetamido-2-hydroxy benzoic acid was con-
ducted in accordance with previously described procedures.
Under the fume hood, 5-ASA (3 g, 19.6 mmol) in an Erlenmeyer
placed over warm water bath (80 C) was dissolved in acetic
63,64
4.5. General procedure for deprotection and preparation of
(f) and (g)
ꢀ
In a round-bottomed ask, d or e (0.1 g, 0.2 mmol) and acetic
acid (85%, 10 mL) were combined together and reuxed under
nitrogen gas for 5–7 h to form a clear solution. The mixture was
then concentrated under the reduced pressure and neutralized
by NaHCO3 (5%). Purication was performed with column
chromatography (9 : 1 ethyl acetate/methanol) to acquire (f) or
anhydride (20 mL) and 5 to 10 drops of phosphoric acid (85%)
were added to the mixture, which was then stirred for about 15
to 30 min. When the mixture was dissolved completely, it was
removed from the warm water bath and 10 to 20 drops of
distilled water were cautiously added to the warm mixture.
Subsequently, over an ice bath 50 mL of cool distilled water was
added and crystallization was induced by scratching the
container's walls. Aerwards, the obtained crystals were ltered
using the Buchner funnel and were washed several times with
the cold distilled water to remove the rest of the acid. Ethanol
(g), Scheme 1.
4.6. Characterization
4.6.1. N-Acetyl-5-aminosalicylate or 5-acetamido-2-hydroxy
1
(
95%) was used to recrystallize the dried crystals and thus benzoic acid (a). H NMR (500 MHz, DMSO-d
6
): d (ppm) ¼
obtain pure (a). The product was identied initially with TLC 2.08 (3H, CH3), 6.83 (1H, d, J ¼ 7.7 Hz, Ar-CH), 7.55 (1H, d, J ¼
1
3
(
mobile phase 8 : 2, ethyl acetate/methanol) and visualized 7.9 Hz, Ar-CH), 8.05 (1H, s, Ar-CH), 10.41 (1H, s, COOH).
C
under the UV lamp, Scheme 1.
NMR (125.8 MHz, DMSO-d ) d ¼ 24.2 (C-1), 113.8, 114.4, 120.9,
6
1
1
2
29.7; (C-2, C-3, C-4, C-5), 130.4 (C-6), 156.7 (C-7), 165.9 (C-8),
69.8 (C-9). FT-IR (n): 3561, 3239, 3064, 2818, 2699, 2573,
4.3. General procedure for protection of monosaccharides
490, 1796, 1674, 1618, 1537, 1486, 1208, 1018. Ms: GC-mass,
and preparation of (b) and (c)
+
(
M) m/z 195.0, [calcd; 195.053, formula (C
9
H
9
NO
4
)]. Yield:
ꢀ
Glucose and xylitol were selectively protected based on previous 67.5%. M.p: 195 C. White, needle-shape crystals.
35,65,66
works
presence of 5 to 10 drops of H
w), monosaccharide (1 g, D-glucose: 5.5 mmol, D-xylitol: 6.5 s, CH ), 1.29 (3H, s, CH ), 1.33 (3H, s, CH ), 1.45 (3H, s, CH ),
with a slightly different method. In brief, in the
4.6.2. 1,2:5,6-Di-O-isopropylidene-D-glucofuranose or diacetone-
1
3
PO and molecular sieves (2% w/ glucose (b). H NMR (500 MHz, acetone-d
4
6
): d (ppm) ¼ 1.22 (3H,
3
3
3
3
mmol), freshly fused anhydrous ZnCl (0.77 g, 5.7 mmol) and 2.22 (1H, s, OH), 3.95 (2H, dd, J ¼ 6.3 Hz, J ¼ 1.8 Hz, H-a, H-b),
2
acetone (100 mL) were immediately mixed together in a ask 4.12 (1H, t, J ¼ 5.3 Hz, H-c), 4.28 (1H, t, J ¼ 4.8 Hz, H-d), 4.41–

tted with an anhydrous calcium chloride guard tube. The 4.50 (1H, m, H-e), 5.42 (1H, t, J ¼ 5.1 Hz, H-f), 5.78 (1H, d, J ¼ 3.6
13
mixture was stirred vigorously for 5 h at room temperature. The Hz, H-g). C NMR (125.8 MHz, acetone-d
primary detection was performed by TLC (mobile phase 9 : 1 26.2; (C-1, C-2, C-3, C-4), 66.4 (C-5), 73.8 (C-6), 75.9 (C-7), 80.1 (C-
6
) d ¼ 23.8, 24.3, 25.7,
ethyl acetate/hexane). The mixture was ltered and neutralized 8), 84.9 (C-9), 105.2 (C-10), 110.0 (C-11), 113.2 (C-12). FT-IR (n):
gradually with NaHCO
3 4
(10%), then dried with MgSO and freed 3421, 2984, 1457, 1373, 1321, 1218, 1162, 1061, 1003. MS: HRMS
+
from acetone under the reduced pressure. Aerwards, the (M + Na) , m/z 283.282, [calcd; 260.126, formula (C H O )].
1
2
20 6
ꢀ
product was extracted by hexane (5 ꢃ 50 mL) and hexane was Yield: 61%. M.p: 109–110 C. White crystals. [a] 24/D ꢂ0.12, (c ¼
collected and separated from precipitations by decanting. Once 0.4 in acetone).
hexane evaporated under the diminished pressure, the precip-
itations were collected. Further purication was carried out xylitol (c). H NMR (500 MHz, chloroform-d
4.6.3. 2,3,4,5-Di-isopropylidene-D-xylitol or diacetone-
1
1
): d (ppm) ¼ 1.29
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(
6H, s, 3 each, 2 ꢃ CH
3
), 1.34 (6H, s, 3 each, 2 ꢃ CH
3
), 2.14 (1H, OH), 6.97 (1H, d, J ¼ 8.8 Hz, Ar-CH), 7.19 (1H, d, J ¼ 9.0 Hz,
1
3
s, OH), 3.49 (2H, dd, J ¼ 6.3 Hz, J ¼ 2.5 Hz, H-a, H-b), 3.72–3.78 Ar-CH), 7.58 (1H, s, Ar-CH). C NMR (125.8 MHz, methanol-d )
4
(
3H, m, H-c, H-d, H-e), 3.90–3.99 (1H, m, H-f), 4.12–4.15 (1H, m, d ¼ 63.9 (C-1), 64.0 (C-2), 69.9 (C-3), 71.9 (C-4), 73.8 (C-5), 114.4
13
H-g). C NMR (125.8 MHz, chloroform-d ) d ¼ 25.5, 26.2, 27.0, (C-6), 116.4 (C-7), 116.6 (C-8), 120.1 (C-9), 149.6 (C-10), 156.4 (C-
1
2
7
2
7.1; (C-1, C-2, C-3, C-4), 62.2 (C-5), 65.6 (C-6), 75.1 (C-7, C-8), 11), 168.0 (C-12). FT-IR (n): 3601, 3536, 3446, 3121, 2986, 2977,
ꢂ
7.8 (C-9), 109.7 (C-10), 110.05 (C-11). FT-IR (n): 3445, 2962, 1719, 1601, 1549, 1475, 1310, 1282, 1000. MS: HRMS (M ꢂ H) ,
7
858, 1465, 1460, 1383, 1375, 1215, 1125, 1003. MS: HRMS (M + m/z 286.259 [calcd; 287.101, formula (C12H17NO )]. Yield: 38%.
+
ꢀ
Na) , m/z 255.267 [calcd; 232.131, formula, (C11
5
H
20
O
5
)]. Yield: M.p: 164–166 C. White crystals.
ꢀ
7%. M.p: 30–33 C. White crystals. [a] 24/D ꢂ0.02, (c ¼ 0.4 in
acetone).
.6.4. 3-O-N-Acetyl-5-aminosalicylate-1,2:5,6-di-O-isopropylidene-
4
.7. Anti-bacterial assay
4.7.1. Disc diffusion method. In vitro antibacterial test was
), 2.08 (3H, s, carried out in keeping with the recommended standards of the
), 3.59 (2H, dd, J ¼ 7.2 Hz, J ¼ 3.3 Hz, H-a, H-b), 3.73 (2H, National Committee for Clinical Laboratory Standards based on
dd, J ¼ 10.5 Hz, J ¼ 5.7 Hz, H-c, H-d), 3.90 (1H, t, J ¼ 6.3 Hz, H-e), 4.28 determination of inhibition zone in millimetres (mm) in
1H, t, J ¼ 7.6 Hz, H-f), 5.47 (1H, s, Ar-OH), 5.95 (1H, d, J ¼ 3.4 Hz, H-g), nutrient agar (NA). In addition, the microbe cultures were
.46 (1H, d, J ¼ 7.8 Hz, Ar-CH), 7.63 (1H, d, J ¼ 7.9 Hz, 1H, Ar-CH), standardized to 0.5McFarland standard which is approximately
4
1
D-glucofuranose (d). H NMR (500 MHz, methanol-d ): d (ppm) ¼ 1.28
4
(
6H, bs, 3 each, 2 ꢃ CH
3
), 1.49 (6H, s, 3 each, 2 ꢃ CH
3
O]C–CH
3
(
7
13
8
7
.91 (1H, s, NH), 8.05 (s,1H, Ar-CH). C NMR (125.8 MHz, meth- 10 cells.
anol-d ) d ¼ 23.8 (C-1), 26.4, 26.7; (C-2, C-3, C-4, C-5), 72.1 (C-6), 75.9
The antibacterial activity of (f) and (g) (2 mg mL concen-
C-7), 80.0 (C-8), 83.4 (C-9), 85.1 (C-10), 109.4 (C-11), 110.3 (C-12), 115.9 tration) was evaluated against pathogenic Gram-negative
C-13), 116.0 (C-14), 121.1 (C-15), 121.9 (C-16), 127.1 (C-17), 130.3 (C- bacteria, (Escherichia coli (E. coli) and Salmonella choleraesuis)
8), 157.8 (C-19), 167.9 (C-20), 168.0 (C-21). FT-IR (n): 3477, 2987, and Gram-positive bacteria, (Staphylococcus aureus (S. aureus)
928, 1734.20, 1455, 1378, 1214, 1165, 1070, 1008. MS: HRMS (M + and Bacillus subtilis) via the disc diffusion method and
ꢂ1
4
(
(
1
2
+
ꢂ1
Na) , m/z 460.439 [calcd; 437.169, formula (C21
M.p: 182–185 C. Light gray crystals.
9
H27NO )]. Yield: 57%. compared with the initial drug (2 mg mL concentration). Pure
ꢀ
ethanol (which was employed to dissolve samples) and strep-
ꢂ1
4
.6.5. 1-O-N-Acetyl-5-aminosalicylate-2,3,4,5-di-isopropylidene- tomycin (100 mg mL concentration) were applied as a negative
1
D-xylitol (e). H NMR (500 MHz, methanol-d ): d (ppm) ¼ 1.15 (3H, control and positive control, respectively.
4
brs, CH ), 1.35 (9H, s, 3 each, 3 ꢃ CH ), 1.76 (3H, s, O]C–CH ),
In brief, the samples, which had been previously sterilised
.40 (1H, t, J ¼ 5.4 Hz, H-a), 3.56–3.61 (2H, m, H-b, H-c), 3.85–3.93 under UV, were inoculated onto 5 mm diameter paper discs
1H, m, H-d), 3.99–4.18 (3H, m, H-e, H-f, H-g), 5.21 (1H, s, OH), 6.83 which were then positioned on the NA surface of the plates on
1H, d, J ¼ 8.5 Hz), 7.43 (1H, d, J ¼ 7.8 Hz), 8.24 (1H, s, NH), 8.52 which microbial growth took place. The plates were inverted
3
3
3
3
(
(
13
ꢀ
(
2
(
1
(
1
1H, s). C NMR (125.8 MHz, methanol-d
6.1, 26.5; (C-2, C-3, C-4, C-5) 61.7 (C-6), 63.8 (C-7), 72.1 (C-8), 72.5 growth occurred. Aer incubation, by ruler from the back of
C-9), 78.3 (C-10), 110.5 (C-11), 111.1 (C-12), 117.9 (C-13), 118.0 (C- inverted Petri plates, the diameters of the zones around the
4), 122.0 (C-15), 127.8 (C-16), 130.8 (C-17), 153.6 (C-18), 169.8 samples which indicated amount of inhibition were measured
C-19), 170.0 (C-20). FT-IR (n): 3496, 3224, 3145, 2987, 2810, 1778, to millimetre (Table 1).
688, 1648, 1570, 1465, 1437, 1384, 1377, 1268, 1195, 1010, 1000.
4.7.2. Determination of minimum inhibitory concentra-
4
) d ¼ 22.7 (C-1), 24.3, and incubated at 30–37 C for 18–24 hours until sufficient
+
MS: HRMS (M + Na) , m/z 432.423 [calcd; 409.174, formula tion (MIC) and minimum bactericidal concentration (MBC).
ꢀ
(C
20 8
H27NO )]. Yield: 47%. M.p: 151–153 C. White crystals.
The MIC/MBC assay was performed through the macro broth
1
4
.6.6. 3-O-5-Aminosalicylate-D-glucopyranoside (f). H NMR serial dilution technique as previously described methods by
500 MHz, methanol-d
): d(ppm) ¼ 2.08 (4H, s, 4 ꢃ OH), 3.61 (1H, (Nakamura et al., 1999, Karaman et al., 2003, Singariya et al.,
dd, J ¼ 5.8 Hz, J ¼ 2.2 Hz, H-a), 3.75–3.77 (2H, m, H-b, H-c), 3.89 2011, Fabry, et al., 1998) with a slight modication.
2H, s, NH ), 3.99 (1H, t, J ¼ 4.2 Hz, H-d), 4.03 (1H, t, J ¼ 4.9 Hz,
MICs were also investigated for the microbial strains which
H-e), 4.20 (1H, t, J ¼ 5.2 Hz, H-f), 5.47 (1H, s, Ar-OH), 5.95 (1H, d, J were determined to be sensitive to the compounds in the disc
1.8 Hz, H-g), 7.45 (1H, d, J ¼ 8.8 Hz, Ar-CH), 7.64 (1H, d, J ¼ 7.9 diffusion assay.
(
4
67–70
(
2
¼
1
3
ꢂ1
Hz, Ar-CH), 8.05 (1H, s, Ar-CH). C NMR (125.8 MHz, methanol-
Samples initially prepared at the concentration of 4 mg mL
d
4
) d (ppm) ¼ 62.2 (C-1), 69.9 (C-2), 70.2 (C-3), 74.2 (C-4), 76.9 (C- in ethanol, aer that, they were diluted serially into eight
5), 94.6 (C-6), 116.0 (C-7), 116.2 (C-8), 118.6 (C-9), 122.0 (C-10), consecutive tubes to obtain concentration range, 0.93–3.33 mg
ꢂ1
129.9 (C-11), 151.9 (C-12), 165.9 (C-13). FT-IR (n): 3575, 3475, mL (0.2 mL of samples into 1 mL of nutrient broth which pre-
3
327, 3154, 2971, 2833, 2689, 1768, 1768, 1662, 1624, 1556, 1475, inoculated with bacteria adjusted to the 0.5 Mcfarland standard
+
8
ꢂ1
1269, 1220, 1122. MS: HRMS (M + Na) , m/z 338.263 [calcd; concentration, 10 cfu mL ). The nal volume in each tube was
ꢀ
3
15.095, formula (C H NO )]. Yield: 41%. M.p: 191–193 C. 1.2 mL. Similar preparation was carried out for streptomycin
1
3
17
8
ꢂ1
White crystals.
from initial concentration of 50 mg mL to obtain concentra-
1
ꢂ1
4.6.7. 1-O-5-Aminosalicylate-D-xylitol (g). H NMR (500 tion range, 11.62–41.66 mg mL as a positive control. Two
MHz, methanol-d
H-a, H-b), 3.57–3.59 (1H, m, H-c), 3.65–3.70 (2H, m, H-d, H-e), tube containing sample without inoculum and the tube con-
.08 (2H, s, NH ), 4.33–4.41(2H, m, H-f, H-g), 5.28 (1H, s, Ar- taining the growth medium and inoculum. The tubes were
4
): d ¼ 2.09 (4H, s, 4 ꢃ OH), 3.40–3.46 (2H, m, control tubes also were set-up for each test batch. These include
4
2
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ꢂ1
covered with a sterile sealer. Contents of each tube were mixed 3.125, 6.25, 12.5, 25, 50, and 100 mg mL for HT-29 cell lines.
ꢀ
and incubated at 37 C. Aer 24 h, MIC was recorded as the For 3T3 cell lines, the concentrations used were 0, 0.1, 1, 100,
ꢂ1
lowest concentration of the test compound that demonstrated 1000 and 10 000 mg mL . For both cell lines, a concentration of
no turbidity or visible growth of organism. Each sample tested zero was used for untreated control cells and 5-ASA was applied
in this study was screened two times against each organism.
as a reference drug as well. The incubation was carried out over
The MBC was determined by sub-culturing the test dilution 24 h and 48 h periods. When reduction of yellow MTT to purple
on nutrient agar and further incubated for 18–24 h. The MBC formazan crystals was completed, the optical density (OD) of
is dened as the minimum level of compound concentration samples was calculated by ELISA reader at a wavelength of
produces no growth of microbial colonies on NA medium (Table 2). 570 nm (Table 4). IC50 value (the concentration of a drug that is
required for 50% inhibition) was recorded using the following
equation:
4.8. Anti-inammatory assay
The NO suppression of f and g in IFN-g/LPS-stimulated
RAW264.7 macrophages at 50 mM test concentration was
examined, and curcumin was used as a positive control.
Subsequently, the results were compared with the parent drug.
Furthermore, an evaluation of cytotoxicity was carried out to
support that the NO inhibition was not caused by sample
toxicity.
Cell viability (%) ¼ (OD of sample/OD of control) ꢃ 100. (1)
Aer triplication and validation, graphs were plotted with
the percentage of cell viability against their respective concen-
trations (Fig. 1).
4
.10. Docking study
.10.1. Preparation of proteins and ligands. The crystal
structures of proteins were retrieved from Protein Data Bank
4
.8.1. NO investigation. According to Leong et al.'s
4
48
method, NO suppression was investigated. Briey, into a 96-
well culture plate with 50 mL of DMEM (Dulbecco's Modied
Eagle's Medium) including 10% fetal bovine serum (FBS) and
5
9,61
(
1
http://www.rscb.org/pdb).
Both COX-1, COX-2 (PDB code:
˚
˚
57,59
EQG, 1CX2, X-ray resolutions 2.61 A, 3 A, respectively)
exist
1% penicillin/streptomycin in a 95% air and 5% CO2 atmo-
as a homodimeric assembly, none of binding sites of them
come in the interface of two domains, thus, only a subunit of
the homodimeric COX-1 and COX-2 were utilized. 5-LOX (PDB
sphere RAW 264.7 murine macrophages cells were seeded
ꢀ
(
50 000 cells per well) and grown for 24 h at 37 C. Thereaer,
ꢂ1
cells in 5 mg mL of LPS (Escherichia coli, serotype 0111:B4)
and 1 ng mL of interferon-gamma (IFN-g) were provoked in
˚
61
ꢂ1
code: 3O8Y, X-ray resolution 2.39 A) exists as a monomer. The
water was removed from crystal structures to avoid masking the
the presence or absence of test compounds for 17 h. Nitrite
concentration was measured by reacting 50 mL of cell culture
supernatant with 50 mL of Griess reagent (1% sulfanilamide and
61
surface of protein from ligand. AutoDockTools-1.5.6 program
was utilized to add hydrogen and Kollman charges to protein.
3
1
D structures of ligands were constructed using Pymol version
.3 (ref. 57 and 74) and were converted to PDB.
0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5%
phosphoric acid) at room temperature. Aer 5 min of incuba-
tion at room temperature, the optical density was determined
with a microplate reader, (Table 3).
4.10.2. Preparing grid box. Rigid protein and exible ligand
docking was fullled with 0.375 grid spacing. Grid of 50 ꢃ 50 ꢃ
˚
5
6
0 A size was set for COX-1 and COX-2 which centered at (x y z)
4.8.2. Cell cytotoxicity determination for macrophage cell
8.328, 22.231 and 189.725 for COX-1, 23.947, 21.582, 15.436
line. 100 mL DMEM was added in each well to remove super-
natant, following that 20 mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
˚
for COX-2. Grid size for 5-LOX was created 60 ꢃ 60 ꢃ 60 A and
(x y z) centered at 4.41, 20.412 and ꢂ0.648 for 5-LOX where
surrounded the active side residues.
ꢂ1
diphenyltetrazolium bromide (MTT, 5 mg mL ) was added,
then the plate was incubated in a 95% air and 5% CO atmo-
2
4
.10.3. Preparing AutoDock. 100 runs of Lamarckian
ꢀ
sphere at 37 C for 4 h. Subsequently, the mixture of culture
61
Genetics Algorithm were carried out using AutoDock4.0 (ref.
1) (http://autodock.scripps.edu). All docking parameters were
media and MTT in all wells were detached and the purplish
formazan crystals formed were dissolved in dimethyl sulfoxide
6
set to their default values. Eventually, the lowest binding energy
was obtained for highest cluster members. All images were
rendered using Pymol version 1.3 and binding interactions were
analyzed using Ligplot soware version v.1.4.5.
(DMSO) and incubation was carried out at room temperature
for 15 min. The color intensity was then calculated at 570 nm at
room temperature, (Table 3).
4
.9. Anti-cancer assay
Acknowledgements
In vitro MTT test was carried out for cell viability assessment
against HT-29 and 3T3 cell lines. The HT-29 cell line which We are grateful for nancial support of this work by ERGS (KPM)
obtained from cultured human colon cancer cells was used in with grant No. 5527054 from University Putra Malaysia (UPM).
the study of intestinal cells. The 3T3 cell line which is the
standard broblast cell line and was originally acquired from
mouse embryo tissue was applied as a model for normal cells.
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