Anti-dengue, cytotoxicity, antifungal, and in silico study of the newly synthesized 3-O-Phospo-α-D-glucopyranuronic acid compound

Article abstract of DOI:10.1155/2018/8648956

The aim of the current study was to synthesize new bioactive compounds and evaluate their therapeutic relevance.The chemical structure of compound 7 (methyl 3-O-phospo-D-glucopyranuronic acid was elucidated by physical and advance spectral technique. Also, this compound was assessed for various in vitro biological screening. The results showed that compound 7 has promising antifungal activity against selected fungal strains. Computational study was also carried out to find antimalarial efficacy of the synthesized compounds. Compounds (2-7) were tested for cytotoxicity by MTT assay, and no considerable cytotoxicity was observed.Molecular docking studywas performed to predict the bindingmodes of newcompound (7).Thedocking results revealed that the compound has strong attraction towards the target protein, as characterized by good bonding networks. On the basis of the acquired results, it can be predicted that compound (7) might show good inhibitory activity against dengue envelope protein.

Full text of DOI:10.1155/2018/8648956

Hindawi
BioMed Research International
Volume 2018, Article ID 8648956, 5 pages
https://doi.org/10.1155/2018/8648956
Research Article
Anti-Dengue, Cytotoxicity, Antifungal, and In Silico Study of
the Newly Synthesized 3-O-Phospo-ꢀ-D-Glucopyranuronic
Acid Compound
1
,2
3
1
Tareq Abu-Izneid , Abdur Rauf , Saud Bawazeer,
4
5
Abdul Wadood , and Seema Patel
ꢀ
Faculty of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia
Department of Pharmaceutical Sciences, College of Pharmacy, Al Ain University of Science and Technology, Al Ain Campus, UAE
Department of Chemistry, University of Swabi, Khyber Pakhtunkhwa, Pakistan
Department of Pharmacy, Abdul Wali Khan University, Mardan ꢁꢂꢁꢄꢄ, Pakistan
Bioinformatics and Medical Informatics Research Center, San Diego State University, San Diego ꢆꢁꢀꢇꢁ, USA
ꢁ
ꢂ
ꢃ
ꢅ
Correspondence should be addressed to Tareq Abu-Izneid; tizneid@gmail.com and Abdur Rauf; mashaljcs@yahoo.com
Received 23 June 2018; Revised 21 October 2018; Accepted 8 November 2018; Published 3 December 2018
Academic Editor: Marcelo A. Soares
Copyright © 2018 Tareq Abu-Izneid et al. ꢀis is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
ꢀ
e aim of the current study was to synthesize new bioactive compounds and evaluate their therapeutic relevance. ꢀe chemical
structure of compound 7 (methyl 3-O-phospo-ꢀ-D-glucopyranuronic acid was elucidated by physical and advance spectral
technique. Also, this compound was assessed for various in vitro biological screening. ꢀe results showed that compound 7 has
promising antifungal activity against selected fungal strains. Computational study was also carried out to ꢁnd antimalarial eꢂcacy
of the synthesized compounds. Compounds (2-7) were tested for cytotoxicity by MTT assay, and no considerable cytotoxicity was
observed. Molecular docking study was performed to predict the binding modes of new compound (7). ꢀe docking results revealed
that the compound has strong attraction towards the target protein, as characterized by good bonding networks. On the basis of
the acquired results, it can be predicted that compound (7) might show good inhibitory activity against dengue envelope protein.
1
. Introduction
immune defense. But excess or perpetual activation of the
complement system creates an inꢃammatory milieu and can
cause blood coagulation.
Viruses are a major cause of diseases worldwide. Hepatitis C
virus (HCV), human papillomavirus (HPV), inꢃuenza virus,
herpes simplex virus (HSV), Japanese encephalitis virus,
rotavirus, human immunodeꢁciency virus (HIV), respiratory
syncytial virus (RSV), and Ebola virus are some of the most
common pathogenic viruses. While HIV is largely contained
in recent years, the emergence of dengue virus, Ebola virus,
and Zika virus has been very alarming. Viruses bind to cell
surface heparan sulfate proteoglycans and enter into the host
cells. Viral proteases bind to host mannan binding lectins
Numerous phytochemicals have shown diꢄerent degrees
of inhibitory responses towards various pathogenic viruses.
Polyphenols, alkaloids, sulfated polysaccharides, and glyco-
sides among others have shown in vitro antiviral responses.
In a study, cardiac glycosides inhibited HIV-1 gene expres-
sion, preventing the viral assembly, by manipulating Na-
K-ATPase pump action [4]. Lanatoside C, a cardiac gly-
coside, has showed inhibitory eꢄect on all four serotypes
of dengue virus [5]. Polyphenolic compounds, ꢃavonoids,
chalcones, and other phenolics were the common docking
ligands for dengue virus protein targets such as protease
(NS2B-NS3pro), helicase (NS3 helicase), methyltransferase
(MTase), RNA-dependent RNA polymerase (RdRp), and the
(
MBL), the pattern recognition molecules, which leads to
the activation of complement system [1–3]. ꢀe complement
system includes serum proteins, receptors, and proteases.
ꢀ
is system assists the antibodies and phagocytic cells in

2
BioMed Research International
dengue virus envelope protein [6]. Deacetyl-3-cinnamoyl-
azadirachtin from Azadirachta indica inhibited NS3/4A pro-
tease of HCV [7].
ꢁ.ꢂ. Methyl ꢂ-O-Phospo-ꢀ-D-Glucopyranuronic Acid (ꢈ).
White needle solid; yield: 90 mg, 65%, IR; 3443.50 for OH
stretching, 2919.21 CH stretching, 2875.07 C=O stretching
1
Glucuronide or glucuronoside is glucuronic acid bound
to other moieties by glycosidic bond. In fact, glucuronides are
glycosides. Glucuronide derivatives constitute an important
class of pharmaceutical active compounds which possess
antiviral activity. Amantadine glucuronide and its derivative
when combined with rimantadine inhibited the reproduction
of inꢃuenza virus strains A/H1N1 and A/H3N2 [8]. Inꢃuenza
virus inhibitory activities of spacer-linked 1-thioglucuronide
analogues of glycyrrhizin have been observed [9].
and 1682. 24, COOH stretching. EI-MS; 287, H-NMR (600
MHz, CDCl ): ꢁ 5.01 (d, H-1, J=3.00), 3.58 (d, H-2. J=9.6),
3
H
4.36 (d, H-3, J=9.6), 3.56 (d, H-4, J=9.6), 4.56 (d, H-5, J=9.6)
and 3.29 (s, OCH ).
3
3
. Biological Activity
ꢂ.ꢀ. Cytotoxicity Study by MTT Methods. In vitro cyto-
toxicity study of the compounds (2-7) was performed
by using mice hepatocytes and LCMK-2 monkey kid-
ney epithelial cells [13]. Incubation of the compounds (2-
In a study glucuronide 3-O-sulfated GlcA interfered with
the binding of dengue virus to host cell receptors [10].
Further, this glucuronide had low cytotoxicity and it was
eꢄective at EC value lower than that of sucrose octasulfate,
7) was achieved in 24h and subsequently the cell abil-
50
ity was recognized by MTT (3-[4,5-dimethlthiazol-2yl]-2,5-
diphenyltetrazolium bromide) method. In this procedure, the
cells were kept in RPMI-1640 medium which was acquired
from Gibco BRL. ꢀis medium comprises 110 ꢂg/ml penicillin
sodium salt, 2 mg/ml sodium bicarbonate (Na CO ), 10%
an inhibitor of dengue virus infection [10].
Hence, the aim of current ꢁnding was to synthesize
methyl 3-O-phospo-ꢀ-D-glucopyranuronic acid, to conduct
in silico analysis, and to predict pharmaceutical relevance.
2
3
fetal bovine serum (FBS), and 100 ꢂg/ml streptomycin sulfate.
3
3
Initial seeding of the 7.1×10 LCMK-2 cells and 8.6× 10 mice
hepatocytes was performed in 96-well plates. ꢀe cells were
treated with the synthesized compounds (2-7) at diꢄerent
concentrations and also with a vehicle containing 0.2 %
DMSO. Subsequently, they were subjected to incubation for
48h followed by conducting MTT assays.
2
. Experimental
ꢁ
.ꢀ. Material and Methods. ꢀe chemical and reagents used
in this study were of analytical grade. ꢀe methylglucoside,
1
-O-methyl glucose (1), was purchased from Sigma Aldrich.
IR spectra were recorded on a FT-IR Nicolet 380 spec-
1
trophotometer (UK), as KBr disks, from 400-4000. H-NMR
spectra were measured on AVANCE AV600 Crycroprob
ꢂ.ꢁ. Antifungal Activity. ꢀe antifungal properties of com-
pounds (2-7) were assayed according to standard procedure.
spectrometer in CDCl . Mass spectra were recorded by using
3
JEOL-600H-2 mass spectrometer; EI source 70 eV.
ꢀ
e tube dilution assay was performed to determine the
fungal inhibitory eꢄect of the compounds. Each compound
was dissolved in DMSO at 2ꢂg/ml to prepare stock solution.
SDA (Sabouraud dextrose agar) (4 ml) were poured into
ꢁ
.ꢁ. Synthesis of Methyl ꢂ-O-Phospo-ꢀ-D-Glucopyranuronic
Acid (ꢈ). Methyl 3-O-phospo-ꢀ-D-glucopyranuronic acid
∘
each tube and autoclaved for 15 min at 120 C and then
(
7) (Scheme 1) was synthesized by using the following
∘
allowed to cool at 15 C. To the SDA medium, stock solution
procedure. 1-O-Methyl glucose 1 (1.00 mmol) was reacted
(
66.6 ꢂL) was added, which gave the ꢁnal concentration
with benzaldehyde (1.00 mmol) in tetrahydrofuran (THF;
2
mg/mL. All tubes were allowed to solidify in the slanted
1
.00 mmol) as per standard procedure [10] and then reacted
position at room temperature. Each tube was incubated with
inoculum removed from a 7-day-old culture of fungal strain.
with benzyl chloride (1.00 mmol), in dimethylformamide
(
DMF; 1.00 mmol) in basic media (NaOH; 1.00 mmol) to
ꢀ
e antifungal activity of each sample and standard drug was
obtain intermediate [11, 12]; then reaction mixture was
reꢃuxed for 48-72 h to obtain compound 2. To synthesize
compound 3, compound 2 (1.00 mmol) was reacted with
di-Fm-phosphoramidite (1.00 mmol) in presence of 1H-
tetrazole (1.00 mmol) and then oxidized with hydrogen
peroxide [10]. Compound 3 (1.00 mmol) is then reacted
with pieridine to disconnect the Fm group, which presented
compound 4. Compound 4 aꢅer acidic work-up resulted in
compound 5. Compound 5 (1.00 mmol) was reacted with
recorded aꢅer 7 days of incubation. ꢀe antifungal activity
was performed in triplicate. ꢀen the results were analyzed
for the visible growth of fungi and % activity was calculated.
ꢂ.ꢂ. Molecular Docking. Molecular docking was carried out
to predict the binding modes of newly synthesized 3-O-
phosphated glucuronide derivative in the binding pockets
of dengue virus envelope protein and antifungal enzymes.
ꢀe 3D structures of dengue virus entry protein (PDB
ID: 1OKE) and antifungal enzymes, dihydrofolate reduc-
tase (PDB ID: 4HOF), aspartic protease (PDB ID: 3Q70),
and N-myristoyl transferase (PDB ID: 1IYL), were down-
loaded from protein databank (http://www.rcsb.org/) [14].
ꢀe 3D structures were then subjected to protonation and
energy minimization, using default parameters of MOE
(http://www.chemcomp.com/) [15]. ꢀe three-dimensional
structure of 3-O-phosphated glucuronide derivative (7) was
∘
triethylsilane and 10% Pd/C in methanol at 25 C followed by
oxidation of primary alcohol [10], which generated methyl
3
-O-phospo-ꢀ-D- glucopyranuronic acid (7) Yield; 700 mg
(
37%). ꢀe residue obtained in each step was puriꢁed by
normal phase chromatography over silica gel by using chlo-
roform: methanol as a solvent system. ꢀe purity of the
compounds was conꢁrmed by TLC. ꢀe structure of identi-
ꢁ
ed compounds was conꢁrmed by comparing their spectral
data with reported one [10].

BioMed Research International
3
OH
Ph
O
Ph
O
Ph
N
O
O
O
O
O
a.
b.
OMe
O
c.
O
d.
O
HO
HO
O
HO
PO
O PO
3
FmO
FmO
HO
BnO
BnO
BnO
2
OMe
OMe
OMe
1
3
2
4
OH
O
6
Ph
O
COOH
e.
O
f.
g.
OMe
O
HO
H O PO
O
HO
H O PO
H O PO
2
3
2
3
2
3
BnO
OH
3
OMe
6
OH
5
7
OMe
Sꢆꢇꢈꢉꢈ 1: Synthesis of methyl 3-O-phospo-ꢀ-D-glucopyranuronic acid (7). Reagents: (a) ZnCl, rt (b) BnBr, NaH, THF, 0C (c) iPr2NP-
(
OFm)2, ꢀH-tetrazole, H O ,THF, (d) piperidine, CH Cl , (e) work-up with buꢄer, pH 6.0, (f) Et3SiH, 10% Pd/C, MeOH (g) NaOCl, NaOClO,
2
2
2
2
piperidoxyl, acetonitrile, rt, pH 6.9.
built by using Molecular Builder Module program imple-
mented in MOE and saved as a (.mdb) ꢁle for molecular
docking. Subsequently, the energy of the built compound was
minimized up to 0.05 gradient using MMFF94s force ꢁeld
implemented in MOE. ꢀe modelled compound was docked
into the active site of proteins using the Triangular Matching
docking method (default) and 10 diꢄerent conformations
were generated. To obtain minimum energy structures, the
ligand was allowed to be ꢃexible during docking. At the
end of docking, the predicted ligand-protein complexes were
analyzed for molecular interactions.
the hydrophobic pocket of envelope protein showed that
this compound established ꢁve polar interactions and three
hydrophobic interactions (Figure 1(a)). ꢀe polar interactions
were established between the oxygen atoms of compound
7 and the active site residues Glu49, ꢀr48, Ala50, Gln200,
and Gln271 of the envelope protein. Compound 7 showed
hydrophobic interactions with the nonpolar residues Ala50,
Leu198, and Leu277 (Figure 1(a)). On comparison of the bind-
ing mode of the synthesized compound with the reference
compound (cocrystallized compound), it was observed that
both compounds showed about similar binding modes with
dengue envelope protein (Figure 1(b)). ꢀe docking results
showed that the synthesized compound has strong aꢂnity
with the target protein, as represented by good bonding
networks with the target protein. On the basis of the obtained
results, we can predict that this compound might show good
inhibitory activity against dengue envelope protein.
4. Results and Discussion
ꢃ.ꢀ. Interaction of Compound with Dengue Envelope Protein.
Previous research had showed that the crystal structure of sol-
uble ectodomain of dengue virus 2 envelope has a hydropho-
bic pocket present in the hinge region between domains I
and II [16]. ꢀis hydrophobic pocket binds to n-octyl-ꢃ-D-
glucoside or ꢃ-OG (a small detergent molecule); therefore,
it is known as ꢃ-OG binding site and it is proposed as an
appropriate target for developing small molecule inhibitors
of viral host fusion process [17]. ꢀis pocket is important for
the low-pH-triggered conformational rearrangement during
fusion [16]. So, compounds blocking the ꢃ-OG binding
site interfere with conformational changes in the envelope
protein [18]. ꢀe sequence similarity among four diꢄerent
dengue serotypes was carried out using NCBI BLAST. Result
showed that the sequence of dengue 2 virus envelope protein
ꢃ.ꢁ. Antifungal Activity. Antifungal activity of the synthe-
sized compounds (2-7) was measured as per standard pro-
cedure. ꢀe results indicated that compounds 2-7 exhibited
excellent reduction in the growth of selected fungal strains.
Among all tested compounds, compound 7 exhibited maxi-
mum eꢄect (35-45 mm) against Aspergillus flavus, Candida
albicans, and Fusarium solani. Compounds 2-6 showed
moderate activity (10-35 mm) against selected fungal strains
(
Table 1). Hence, the results suggest environmentally friendly
treatment avenue for fungal infections.
1
OKE is almost identical with other DENV-2 sequences
ꢃ.ꢂ. Docking against Antifungal Enzymes. To validate and
specify the drug target for antifungal activity of these com-
pounds, three diꢄerent target proteins from C. albicans were
selected as antifungal targets. Among them, only one tar-
get protein, i.e., dihydrofolate reductase (DHFR) from C.
albicans, was preferred as it showed good docking score (-
5.8605) and interactions with compound 7, as compared to
secreted aspartic protease (4.9656) and N-myristoyl trans-
ferase (4.7592) in docking simulation.
ꢀe docking conformation of compound 7 in the binding
pocket of DHFR showed that this compound establishes
seven hydrogen bonds with the active site residues Val10,
Met25, Trp27, Glu32, Ile33, and Tyr118 of DHFR (Figure 2).
ꢀis compound also formed hydrophobic interaction with
found in the NCBI website, with identity score about 99%.
However, the identity of 1OKE sequence and other types
of DENV sequences falls between 69% and 54%, with the
least identity obtained when 1OKE was compared with the
envelope protein of DENV-4. ꢀe higher the identity of two
or more sequences, the more similar their protein structure is.
If the target proteins share the identity of 50%, their protein
structure is suꢂciently reliable for drug design purpose [19].
ꢀe docking results showed that the newly synthesized
compound 7 was well-accommodated in the binding pocket
of ꢃ-OG. ꢀe docking scores for reference compound
(
octyl- ꢃ-OG) and compound 7 were -7.0046 and -5.6691,
respectively. ꢀe docking conformation of compound 7 in

4
BioMed Research International
Tꢊꢋꢌꢈ 1: Antifungal activity of compounds 2-7.
Zone of inhibition (mm)
%
Name of the fungus
A. flavus
C. albicans
C. glabrata
F. solani
2
3
4
5
6
7
STD
Amphotericin B
Miconazole
Miconazole
Miconazole
MIC (ꢂg/ml)
30 ± 1.44
30±1.14
25±1.48
28±1.88
35±2.06
44±1.16
20.20
110.8
110.8
110.2
-
-
-
-
-
-
45±1.17
18±1.20
15±1.46
18±1.28
20±2.27
-
-
-
20±1.20
15±1.88
10±2.00
35±1.49
M. canis
T. logifusus
20±2.00
18±1.01
-
-
-
-
-
-
-
-
Miconazole
Miconazole
98.4
100.5
15±1.24
30±2.02
(a)
(b)
Fꢍꢎꢏꢐꢈ 1: Binding mode of newly synthesized compound 7 (a) and reference compound (b) in the active site of dengue envelope protein.
active site residues Ala11, Leu29, Phe36, and Ile112. ꢀis
strong bonding network might be one of the reasons for this
compound’s good antifungal activity.
ꢃ.ꢃ. Cytotoxic Effect. Compounds (2-7) were assessed for
their cytotoxicity by using MTT assay. No considerable
cytotoxicity was observed.
ꢃ.ꢅ. Antifungal Effect. ꢀe results of the antifungal activity
of compounds (2-7) are illustrated in Table 1. Among all
the tested compounds, compound 7 showed good antifungal
activity against A. flavus, C. albicans, and F. solani with % zone
of inhibition which ranges in 35-45 mm, followed by com-
pounds 2-6 (Table 1). ꢀe current ꢁnding demonstrated wide
antifungal potency of compound 7 followed by compounds
2-6 against selected pathogenic fungi.
5
. Conclusion
ꢀ
e current ꢁnding describes the synthesis, characterization,
in vitro antifungal, cytotoxicity, and in silico anti-dengue
study of methyl 3-O-phospo-ꢀ-D-glucopyranuronic acid (7).
ꢀ
e results showed that compound 7 has promising antifun-
gal activity against selected fungal strains. Cytotoxicity was
negligible. ꢀe docking showed that compound (7) has strong
attraction towards the target protein, characterized by good
Fꢍꢎꢏꢐꢈ 2: Binding mode of newly synthesized compound 7 in the
active site of DHFR.

BioMed Research International
5
bonding networks. On the basis of the developed results, we
can predict that compound (7) might show good inhibitory
activity against dengue envelope protein.
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Data Availability
ꢀ
e data used to support the ꢁndings of this study are
available from the corresponding author upon request.
Conflicts of Interest
[
ꢀ
e authors declare that they have no conꢃict of interest
regarding the publication of this paper.
[
11] D. M. Clode and D. M. Clode, “Carbohydrate Cyclic Acetal
Authors’ Contributions
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491–513, 1979.
Tareq Abu-Izneid and Abdur Rauf supervised the project and
conducted the experimental part. Abdul Wadood performed
the docking analysis. Seema Patel and Saud Bawazeer were
involved in editing and they gave the ꢁnal shape to this
manuscript. All authors read the paper and approved it for
submission.
[
12] C. Herbivo, Z. Omran, J. Revol, H. Javot, and A. Specht, “Syn-
thesis and characterization of cell-permeable caged phosphates
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Acknowledgments
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Hoboken, NJ, USA, 2003.
ꢀ
e authors would like to thank the Deanship of Scientiꢁc
Research at Umm Al-Qura University (Bahth Program, Grant
Code: 15- MED-3-1-0026, Investigators: Dr Tareq Abu-Izneid
and Dr Saud Bawazeer) for the ꢁnancial support.
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