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Dengue Fever
Organic Compounds to Combat Neglected Tropical Diseases 41
2.2. Viral Entry Inhibitors
Any virus needs to gain access to the cellular machinery of its host cell in order to
replicate. The inhibition of viral access to the host cell cytoplasm is a well
explored strategy for treating viral infections [21] and some efforts have been
made to develop novel drugs against DENV that could use this mechanism of
action.
Among the structural proteins encoded by the DENV genome, 3 are considered
structural proteins: capsid (C), premembrane (prM), and the envelope (E)
proteins. The DENV E-protein plays an important role in viral entry, mediating
the fusion between the viral membrane and the host membrane; consequently, it
has emerged as a potential therapeutic target.
Molecular docking of the E-protein has been utilized as a strategy to design small
organic compounds with the ability to preclude viral access to the host cell's
cytoplasm. The β-OG binding pocket of this protein is a hydrophobic pocket
described by Modis et al. to be involved in the pH-dependency of the fusion of
DENV to the host cell [22] and has been employed in theoretical studies aimed at
the rational design of anti-dengue agents.
A high-throughput docking virtual screening experiment using the β-OG binding
pocket enabled the selection of 111 compounds as possible therapeutic agents
from a library of almost 600,000 compounds. Two compounds that had positive
results in the biological assays were selected for detailed SAR analysis to increase
the potency of this class of compounds against DENV (Scheme 10) [23]. The
findings suggested that the amine group R1 and aryl group R2 were crucial for the
activity; additionally it was observed that several substitutions were tolerated on
the aryl ring R2 and that a change of the pyrimidine ring to a quinazoline ring
improved the potency. Based on this information from the SAR analysis, a
compound bearing high efficacy against all serotypes of laboratory strains and
clinical isolates of DENV was synthesised. Further characterization showed that
this compound interfered early in the DENV life cycle, blocking its entry to the
host cell cytoplasm after its internalization into endosomes, probably by targeting
E-protein [23].
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42 Organic Compounds to Combat Neglected Tropical Diseases Rezende et al.
S
N
O
O
NH
S
N
O
O
NH
O
S
N
O
O
OH
H
2
N
H
2
N
O
S
N
O
O
NH
N
N
N
R
1
S
N
O
O
NH
O
N
N
N
R
2
R
1
N
3
R
2
N
3
O
NH
O
NH
O
R2
ONH
O
O
NH
O
CDI/THF
CDI/THF
sodium ascorbate/
CuSO
4
/BuOH/H
2
O
sodium ascorbate/
CuSO
4
/BuOH/H
2
O
R
1
/R
2
Scheme 9: Top: Synthetic scheme of benz[d]isothiazol-3(2H)-one derivatives. Bottom: The
substituents present in the most active compounds.
Another virtual screening of the same pocket of E-protein, applying a different
search protocol to a different compound library, resulted in 3 hits and these
contained the thiazole moiety (Scheme 11) [24]. Several analogues bearing this
central thiazole moiety were synthesised, tested for their activity against another
kind of flavivirus—yellow fever virus — [25], and a highly promising derivative
containing 2 bromine atoms was synthesised (Scheme 11). This brominated
compound was later applied as a lead compound to develop novel agents against
DENV. Non-brominated derivatives were also synthesised and tested for their
activity against flavivirus. Novel non-brominated phenylthiazole compounds with
a similar capacity to inhibit DENV were obtained and a novel SAR model of
phenylthiazoles with anti-flaviviral activity was proposed (Scheme 11) [26, 27].
Dengue Fever
Organic Compounds to Combat Neglected Tropical Diseases 43
N
N
S
H
N
NH
N
NH
2
N
N
S
H
N
NH
O
R
1
R
1
R
2
R
2
N
N
S
H
N
N
Cl
A
B
C
EC
50
: 0.9EC
50
: 1.69
EC
50
: 0.07
Scheme 10: A and B: The most active compounds discovered from virtual screening of the E-
protein pocket and the testing of 111 hits. C: The most active compound discovered from SAR
analysis of the compounds derived from A and B.
S
H
N
N
HN
N
S
O
H
N
O
H
N
S
N
N
N
O
Cl
Cl
S
N
S
Cl
NH
2
S
N
Cl
O
O
Br
Br
N
S
OMe
O
nBu
N
S
R
1
R
2
R
3
N
S
OH
O
nBu
N
S
SMe
O
nBu
N
S
nBu
N
HN
NH
NH
2
A
B
C
D
99.6%
Polar Group
Linear lipophilic
Lenght: 4units
Br atom not
necessary
91,9%
94.1%
99.8%
99.2%
Scheme 11: A: Hits containing a central thiazole ring obtained from the virtual screening of the β-
OG pocket of the DENV E-protein [24]. B: The highly anti-flaviviral phenylthiazole derivative
and its ability to inhibit DENV (shown as % of DENV inhibition) [25, 27]. C: The SAR mod el of
phenylthiazoles as anti-flaviviral agents [27]. D: Highly anti-flaviviral non-brominated
phenylthiazole derivatives and their ability to inhibit DENV (shown as % o f DENV inhibition)
[27].
During the process of cellular entry, DENV and other enveloped viruses bind to
heparan sulphate in the cellular membrane. Sulphated polysaccharides are known
inhibit enveloped viruses, hampering their access to the cytoplasm [28]. Among
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44 Organic Compounds to Combat Neglected Tropical Diseases Rezende et al.
the examples of sulphated polysaccharides are the over-sulphated algal mannans
and xylomannans [29], obtained using SO3-pyridine, and glucans and
galactomannans from Gastrodia elata [30], Leucaena leucocephala, and Mimosa
scabrella [31] sulphated using chlorosulphonic acid. A similar concept was
observed in the in vitro inhibition of DENV by various carbohydrate-binding
agents [32] and zosteric [33] acid, which hampered virus-cell adhesion.
2.3. Inhibitors of RNA Replication
Among the non-structural proteins, NS5 protein has 2 enzymatic functions
essential for viral propagation: methyltransferase (MTase) and the RNA-
dependent RNA polymerase (RdRp). RdRp is responsible for the synthesis of
viral RNAs, a particular function that is not provided by the host cell’s machinery;
consequently, the viral RdRp presents as an attractive anti-viral target [34].
Viral polymerase activity can be targeted using either nucleoside analogs, which
are phosphorylated to a nucleotide before they compete with natural nucleotides
for the enzymatic active site and terminate replication, or non-nucleoside
compounds, which target allosteric sites in the protein. Nucleoside inhibitors of
viral polymerases are the prevailing commercially available class of drugs for
treating viral infections.
Scheme 12: Top: Acetylene-substituted deaza-adenosine compounds with po tent and specific anti-
flaviviral activity. Bottom: The strategy used for the functionalisation of the ribose with the
acetylene group using Grignard reagent.
Dengue Fever
Organic Compounds to Combat Neglected Tropical Diseases 45
To develop inhibitors of DENV RdRp, over 90 adenosine analogs were analysed,
resulting in the identification of an acetylene-substituted deaza-adenosine
compound with potent and specific anti-flaviviral activity (NITD008), synthesised
through the oxidation of the hydroxyl group of ribose to a carbonyl followed by
acetylene functionalization using Grignard reagent (Scheme 12, above) [35]. The
anti-viral actions of this compound were thoroughly characterised and several
lines of evidence support the proposition that its anti-viral activity resulted from
RNA chain termination. Later, a similar compound (NITD449) was shown to
have important anti-flaviviral activity but poor oral availability. To overcome this
problem, its diisobutyryl analogue (NITD203) was synthesised resulting in an
enhancement of its bioavailability (Scheme 12) [36].
O
HO
OH
OH
O
O
O
OH
O
HO
OH
OH
O
O
O
OH
O
O
HO
OH
OH
O
O
O
O
HO
OH
O
O
O
OH
O
O
HO
OH
OH
O
O
O
OH
O
O
OH
O
HO
HO
OH
O
HO
O
OH
O
O
O
OH
O
O
OH
O
Bz
OH
HO
O
HO
OH
OH
O
O
O
OH
O
O
OH
O
O
HO
Bz
Flacourtoside A
Flacourtoside B
Flacourtoside C
Flacourtoside D
Flacou
r
toside E
Flacourtoside F
Scheme 13: The structure of glycosides extracted from Flacourtia ramontchi that inhibited DENV
polymerase.
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46 Organic Compounds to Combat Neglected Tropical Diseases Rezende et al.
Molecular hybridization, the synthesis of novel compounds through the fusion of
2 active chemical groups, has also been applied as an approach to developing
novel compounds for fighting DENV infection. The search for novel adenosine
analogs of DENV reflects the fact that purine is widely known to be a potentially
anti-viral chemical group. It was proposed the chemical linkage of purine scaffold
with a β-lactam ring that, besides its primary uses as an antibacterial moiety;
possess other pharmacological activities, including antiviral. Although promising
lead compounds were obtained for other viral infections, none of the hybrids
showed activity against DENV [37].
In the field of non-nucleoside polymerase inhibitors, natural products have shown
some promise. From extracts obtained from different pa rts of 400 plants randomly
collected in Madagascar, the tree Flacourtia ramontchi was selected for a bioassay-
guided purification project. Six phenolic glycosides, named flacourtosides A–F
(Scheme 13), were isolated from F. r amontchi and their inhibitory activity against
DENV was described to be via polymerase inhibition [38].
Scheme 14: The synthetic procedure used to obtain sulfon ylanthranilic acid derivatives and the
IC
50
values for the most active compounds. The triaryl pyrazoline scaffold is boxed.
Another effort to develop DENV inhibitors was published by the company
Novartis. Applying a scintillation assay developed by the company, high-
O
B
O
NH
N
SO
O
Br
Cl
Br
R
1
O
B
O
N
N
R
1
MeOOC
R
2
HN
S
O
O
Br
MeOO
C
R
2
NH
2
S
N
N
O
Cl
R1
H
H
F
R2
Benzyl
HOOC
R
2
HN
S
O
O
N
N
R
1
CsCO
3
,
CH
3
CN;
+
pyridine
1
. Pd(P h
3
)
4
, K
2
CO
3
, DMF;
2
. LiOH, Me O H
Tria ryl pyra z o line
IC50(uM)
Methyl-2-naphtalene
Methyl-2-naphtalene
0.7
0.26
0.37
Dengue Fever
Organic Compounds to Combat Neglected Tropical Diseases 47
throughput screening of the corporate compound archive resulted in the
identification of N-sulfonylanthranilic acid as a potentially active compound.
Employing a synthetic approach based on the Suzuki coupling of modified
pyrazole boronic esters with 3-bromobenzenesulfonamides, several derivatives
were synthesised [39]. The assaying of the obtained compounds for viral and
human polymerases identified 3 derivatives that were highly specific to, and
active against, DENV (Scheme 14) [39]. In a similar approach, a small library of
200 small molecular compounds with diverse structures was screened for activity
against flavivirus. Triaryl pyrazoline was identified as an inhibitor of DENV
through its actions on viral replication.
2.4. Other Relevant Compounds
Several studies that aimed to treat DENV infection by designing and screening
drugs with alternative or unknown mechanisms of action have been reported in
the literature. Inosine monophosphated (IMPDH) is an enzyme that acts during a
rate-limiting step in the synthesis of GTP and has been used as a target for several
diseases, including viral diseases. Some long-known compounds, such as
mycophenolic acid, a non-nucleoside inhibitor of IMPDH [40] isolated from the
fermentation of Penicilium stoloniferum [41] and clinically useful in organ
transplantation [42], and Vx-497, a phenyloxazole derivative that is a potent
reversible inhibitor of IMPDH, have been valued as anti-dengue compounds with
promising results in vitro [43].
Among the novel synthetic compounds with the capacity to inhibit IMPDH, a
uracil-based compound synthesised primarily via the Gould–Jacobs reaction has
been obtained and shown to be a potent in vitro inhibitor of DENV (Scheme 15)
[44]. Another nucleotide-derived core, 6-methylmercaptopurine, seemed to inhibit
DENV in a cell culture by interacting with IMPDH, but, due to pharmacokinetic
issues, in vivo experiments do not support the use of this drug, even though it may
be suitable for modification [45].
N
N
NO
OOH
OH
O
N
H
N
NO
O
CO
2
C
2
H
5
CO
2
C
2
H
5
NH
N
NO
O
O
CO
2
C
2
H
5
O
CO
2
C
2
H
5
1.Dowtherm A
2. HCl1N
NaOEt, EtOH
Scheme 15: The synthesis of compounds active against IMPDH via the Goul d-Jacobs reaction.
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48 Organic Compounds to Combat Neglected Tropical Diseases Rezende et al.
High-throughput screening of the Novartis corporate chemical library gave rise to
2 potential lead compounds for treating dengue: NITD-982 and NITD2636, which
inhibited in vitro DENV production with EC
90
s of 5.2 nM [46] and 1.7 µM [47],
respectively. Both compounds seemed to act by inhibiting viral translation. NITD-
982 was shown to inhibit dihydroorotate dehydrogenase, an enzyme of the
pyrimidine synthesis pathway, but no in vivo activity was reported, probably due
to pyrimidine intake. Even though NITD-2636 inhibited both host and viral
translation, its mechanism of action is not yet fully understood. Interestingly,
however, it was observed to have significant in vivo activity (Scheme 16). An
NITD-2636 derivative bearing a cyano group, NITD-451, was shown to have
enhanced activity against DENV.
Scheme 16: Top: The synthetic approach used to convert NITD-2636 to NITD-451. Bottom: The
molecular structure of NITD-982.
The compound 5-methoxyquinazoline-2,4-diamine was identified by the
screening of a compound library, using a DENV -replicon cell line, as a moderate
inhibitor of DENV with a low cytotoxicity [48]. The synthesis of a library of
similar compounds enabled the development of derivatives with better activity.
Initially, compounds with different substituted positions were obtained and better
results were obtained when position 5 was substituted for an electron-donating
group (Scheme 17). The activity was optimized by varying the substituents at
position 5 and the compound substituted with a tert-butoxy group showed a
higher activity in the DENV-replicon cell line with a selectivity index (SI) of over
N
HO
F
N
OTf
F
Zn(CN)
2
Pd(PPh
3
)
4
Cl
Cl
O
N
N
N
Cl
CF
3
O
O
NITD-2636
NITD-982
NITD-451
N
R
1
N
Tf
2
O,
Dengue Fever
Organic Compounds to Combat Neglected Tropical Diseases 49
1000 (Scheme 17). Even though the mechanism of action of this compound has
not been elucidated it seems to be a promising compound for the future
development of anti-dengue drugs [48].
D-glucose was used as a starting material for the synthesis of azole derivatives.
Among several compounds obtained by the synthetic procedures used, only 2
showed a moderate inhibition of DENV and are potential lead compounds
obtained from renewable sources (Scheme 18) [49]. No mechanistic hypothesis
for the observed results was made by the authors.
Scheme 17: Left: The method used for the synthesis of 5-methoxyquinazoline-2,4-diamine
derivatives. Right: The structure of the most active compound (boxed).
Scheme 18: The synthetic approach used to obtain 2 active derivatives from glucose.
The DENV proteins prM/M, E, and NS1 are known to possess sites for N-linked
glycosylation and, during translation, the oligosaccharide (Glc)
3
(Man)
9
(GlcNAc)
2
is transferred to specific asparagine (Asn) residues in a cotranslational process
Br
CN
R
F
CN
R
N
NNH
2
NH
2
R
N
NNH
2
NH
2
O
Guanidine carbonate,
or
Position 5: Best results - electron-donating
EC
50
= 2.8 nM
SI > 1000
O
MeO
O
OHO
HO
Me
Me
O
MeO
O
OBr
HO
Me
Me
O
MeO
O
OBr
O
Me
Me
N
S
N
N
O
Br
O
MeO
O
O
O
Me
Me
N
N
S
O
Br
O
HO
O
O
O
Me
Me
O
HO
O
O
N
Me
Me
N
H
O
Br
O
HO
O
O
Me
Me
O
N
N
Br
NBS, PPh3 IBX/DMSO
Halo benzoic hydrazide PIDA/MeOH
EC
50
: 29.9 uM
EC
50
: 64.6 uM
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50 Organic Compounds to Combat Neglected Tropical Diseases Rezende et al.
catalysed by oligosaccharyltransferase. In the endoplasmic reticulum (ER), this
glycoprotein is processed by α-glucosidases I and II before it is associated with
calnexin and calreticulin, which are crucial proteins for glycoprotein folding. This
makes α-glucosidase a suitable target for anti-viral treatment [50].
Castanospermine (CP) is a polyhydroxylated indolizidine alkaloid isolated from
Castanospermum australae [51] and also obtained by total synthesis using
different approaches (Scheme 19) [52]. It is structurally an azasugar related to D-
glucose, known as a ER glucosidase I inhibitor, consequently reducing viral
secretion and infection. In vivo studies have shown its ability to improve the
survival of mice [53].
N
OH
HO
OH
HO
H
b
a
NO
OO
H
O
O
OH
Bn
Bn
GP
PG
O
N
O
R
H
O
OGP
BnO
OBn
H
N
O
O
O
O
GP
GP
GP
H
N
O
O
OGP
BnO
OBn
R
H
N
O
O
O
GP
GP
GP
OO
O
O
GP
PG
PG
RCM
H
N
O
O
O
GP
GP
GP
O
O
O
O
O
I
PG
GP
PG
zinc-mediated
fragmentation
transannular
cyclization
nitrenium ion
cyclization
concerted ring opening
a
b
Scheme 19: Synthetic approaches to the synthesis of castanospermine.
Some iminosugars, such as deoxynorijimycin, are glucose mimetics that act as
competitive inhibitors for α-glucosidase. Deoxynorijimycin, an N-containing
sugar isolated from Morus sp. [54], has shown limited in vivo DV inhibition [55,
56]. However, continuous N-alkylation towards higher and more lipophilic
compounds led to the discovery of CM-10-18 [55, 57], an active compound in the
more lethal dengue haemorrhagic fever (DHF), but with a high toxicity. The
evolution towards an aromatic ether showed an in vivo active compound with a
good pharmacokinetic profile and low cytotoxicity (Scheme 20) [58].
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