A heterocyclic molecule with significant activity against dengue virus

Article abstract of DOI:10.1016/j.bmcl.2009.01.031

There are no specific approved drugs or vaccines for the treatment or prevention of infectious dengue virus and there are very few compounds known that inhibit the replication of this virus. This letter describes the concise synthesis of two uracil-based

Full text of DOI:10.1016/j.bmcl.2009.01.031

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1425–1427
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A heterocyclic molecule with significant activity against dengue virus
a
a
b
b
Vasu Nair a, , Guochen Chi , Qingning Shu , Justin Julander , Donald F. Smee
*
a Department of Pharmaceutical and Biomedical Sciences and the Center for Drug Discovery, University of Georgia, Athens, GA 30602, USA
b Institute for Antiviral Research, Utah State University, Logan, UT 84322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
There are no specific approved drugs or vaccines for the treatment or prevention of infectious dengue
virus and there are very few compounds known that inhibit the replication of this virus. This letter
describes the concise synthesis of two uracil-based multifunctional compounds. One of these compounds
(1) has strong activity against dengue virus. It also exhibits low activity against a few other RNA viruses,
but is highly active against yellow fever virus, a related flavivirus. It is likely that the mechanism of action
of the antiviral activity of this compound is through its inhibition of the enzyme, inosine monophosphate
dehydrogenase (IMPDH). Molecular modeling studies reveal that the compound can have specific hydro-
gen bonding interactions with a number of amino acids in the active site of IMPDH, a stacking interaction
with the bound natural substrate, IMP, and the ability to interfere with the binding of NAD+ with IMPDH,
prior to the hydration step.
Received 10 December 2008
Revised 6 January 2009
Accepted 12 January 2009
Available online 15 January 2009
Keywords:
Dengue
Inhibitor
Antiviral
Synthesis
Heterocycle
Mechanism
Ó 2009 Elsevier Ltd. All rights reserved.
The etiological agents of dengue fever (DF) and dengue hemor-
rhagic fever (DHF) are four closely related dengue viruses, which
are antigenically similar but immunologically distinct serotypes
of the family called Flavivirus.1–3 The major vector for these viruses
is the mosquito, Aedes aegypti. Estimates make the number of cases
of dengue fever as high as 100 million annually, which makes this
arthropod-borne viral infection a serious global health problem.4
Dengue virus genome is a single, positive-stranded RNA of about
11 kDa with a 50-cap but without a polyadenylated terminus. After
fusion and entry, translation of genomic RNA occurs in infected
cells.1,3 Processing of the viral polyprotein is apparently catalyzed
by both viral and cellular enzymes. NS proteins are involved in
the replication cycle. For example, the NS3 viral protease protein
is essential for viral replication.5 The NS5 conserved protein has a
methyltransferase motif in the N-terminal domain and an RNA-
dependent RNA polymerase in the C-terminal domain. The trans-
ferase and polymerase are possible viral targets in antiviral strate-
gies.5–7 However, there are no specific approved drugs or vaccines
for the treatment or prevention of DF and DHF. This letter reports
on the synthesis and antiviral activity of a novel, uracil-based mul-
tifunctional compound, 1, which is a potent inhibitor of the repli-
cation of dengue virus. In contrast, its deaza analog, 2, was
inactive against this flavivirus (Fig. 1).
prepared through the coupling of 3 and 4,10 and diethyl ethoxym-
ethylenemalonate under basic conditions, provided the aminom-
ethylenemalonate
6 (42%). Intermediate 6 was heated in
Dowtherm A (heat transfer fluid), which resulted in intramolecular
cyclization to produce the pyridopyrimidine ester 7 (74%). Hydro-
lysis of ester 7 under acidic conditions afforded the target com-
pound 1 as a crystalline solid in 67% yield. The structure of 1 was
established by UV, 1H and 13C NMR, and HRMS data.11 Compound
2 was synthesized by a related methodology.12
Antiviral activity and mechanism: Compound 1 exhibited rela-
tively strong activity against dengue virus type 2 (New Guinea C)
with an EC50 of 5.7
(CPE) inhibition] and 6.3
CC50 of >100 g/ml producing a TI (therapeutic index) of >18 and
>16, respectively, in Vero cells.13 Confirmatory virus yield reduc-
tion (VYR) assay gave an EC90 value of 3.8 g/ml and a CC50 of
>100 g/ml, resulting in a TI of >26. The activity appears to be spe-
l
g/ml [visual inspection of cytopathic effect
l
g/ml (neutral red uptake assay) and
l
l
l
cific for this compound as the related synthesized compound, 2,
that is without the endocyclic nitrogen in the second ring, does
not exhibit significant activity against this virus family. Compound
1 represents one of the few compounds that have strong antiviral
activity against dengue virus. Interestingly, this compound is mod-
erately active by neutral red assay and highly active (TI > 63) in the
virus yield reduction (VYR) assay against yellow fever virus (17D
strain), another virus within the Flavivirus genus.
The mechanism of the antiviral activity of compound 1 is likely
associated with its ability to inhibit the enzyme, inosine mono-
phosphate dehydrogenase (IMPDH; EC 1.1.1.205). IMPDH catalyses
the oxidative conversion of inosine 50-monophosphate (IMP) to
Synthesis: Synthesis of the target compound was achieved using
the Gould–Jacobs reaction8,9 as the key step (Scheme 1, see Note 11
for details). Thus, condensation of 6-amino-1,3-dibenzyluracil (5),
* Corresponding author. Tel.: +1 706 542 6293; fax: +1 706 583 8283.
E-mail address: vnair@rx.uga.edu (V. Nair).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.031

1426
V. Nair et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1425–1427
cantly lower RNA antiviral activity. The carboxylate moiety of 1
is positioned to form hydrogen bonds with Asn303 and Gly326.
In addition, the ‘phenolic’ OH and the adjacent amide carbonyl
may form a hydrogen bonding network with Asp 274 via a water
molecule. The two phenyl rings may have stacking or other inter-
actions, one with His 93 and the other with the carbonyl of Gln
441. Our molecular modeling studies confirm the ability of inhibi-
tor 1 to bind in the active site region of the inhibitor. This binding
would interfere with the interaction of the cofactor, NAD+, and
IMPDH in the formation of the ternary complex with the substrate,
which is required for hydration of the hypoxanthine ring.15 Sup-
port for our molecular modeling data and interpretation comes
from the X-ray crystal structure of the complex of IMPDH, IMP,
and mycophenolic acid (MPA) together with the mutagenesis and
kinetic data for the mechanism of the non-competitive inhibition
of IMPDH by MPA.24 It is also of relevance to note that MPA exhib-
its antiviral activity against dengue,25 yellow fever virus26 and
West Nile virus.27 Further validation that the mechanism of the
antiviral activity of compound 1 is connected with its ability to in-
hibit IMPDH came from the observation that addition of guanosine
to the cell culture assay medium reversed the antiviral activity,
presumably by replenishing the levels of the GTP pool.
In conclusion, we have synthesized a new heterocyclic com-
pound, 1, that shows relatively strong in vitro antiviral activity
against dengue virus. This compound is also highly active against
another virus of the genus, Flavivirus, the yellow fever virus. In
addition, the compound exhibits antiviral activity, albeit low,
against other RNA viruses. The mechanism of the antiviral activity
is likely associated with inhibition of the enzyme, IMPDH and this
is supported by the observation that addition of guanosine to the
cell culture medium reverses the antiviral activity. Molecular dock-
ing data point to specific hydrogen bonding interactions with a
number of amino acids in the active site of IMPDH and to a stacking
interaction with the bound natural substrate, IMP. Inhibitor 1 rep-
resents one of the few compounds that exhibit significant activity
against dengue virus.
Figure 1. Structures of target compounds.
xanthosine 50-monophosphate (XMP) with the involvement of the
coenzyme, nicotinamide adenine dinucleotide (NAD+).14–16 IMPDH
is an important target enzyme in drug discovery. Consistent with
this suggestion is the observation that some nucleosides, which
are inhibitors of IMPDH as their monophosphates, have been found
to have anticancer, antiviral and immunosuppressive activity.17–22
Inhibition of IMPDH results in the reduction of the GTP pool and
linear correlations do exist between GTP inhibition and antiviral
activity against RNA viruses.23 The mechanism of the antiviral
activity of compound 1 may be associated with its inherent ability
to inhibit IMPDH through interference of the formation of ternary
complex normally formed between the enzyme, the substrate IMP
and the cofactor, NAD+.
In order to investigate the binding of inhibitor 1 with IMPDH in
the presence of IMP, extensive molecular modeling studies were
carried out using SYBYL 7.2 and GOLD 3.2. Figure 2 shows the pre-
ferred docking pose of inhibitor 1 in the active site region of
IMPDH.
The binding allows for favorable stacking interactions between
the hypoxanthine base of substrate IMP and the fused heterocyclic
ring of the inhibitor. Additionally, the pyridine ring nitrogen of the
inhibitor is appropriately situated to form hydrogen bonds with
Thr333 and Gln441. This is a key interaction with the enzyme as
the absence of this nitrogen (as in compound 2) results in signifi-
Scheme 1. Synthesis of antiviral compound 1. Reagents and conditions: (a) NaOEt, EtOH, reflux; (b) EtOCH@C(CO2Et)2, NaOEt, EtOH, reflux; (c) Dowtherm A, reflux; (d)
dioxane, 1 N HCl, reflux.

V. Nair et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1425–1427
1427
chromatography (hexanes:ethyl acetate, 4:1) gave 400 mg (41.8%) of 6 as a
waxy solid. 1H NMR (500 MHz, CDCl3): 11.1 (d, 1H, J = 12 Hz), 8.04 (d, 1H,
J = 12 Hz), 7.28–7.50 (m, 10H), 5.55 (s, 1H), 5.25 (s, 2H), 5.20 (s, 2H), 4.28 (q,
2H, J = 7.0 Hz), 4.22 (q, 2H, J = 7.0 Hz), 1.33 (t, 3H, J = 7.0 Hz), 1.30 (t, 3H,
J = 7.0 Hz). ESI MS calcd for C26H28N3O6 (M+H)+ 478, found 478. Compound 6
(380 mg) in 3 ml of Dowtherm A was heated under reflux for 1 h. After cooling,
the mixture was purified by flash chromatography (hexanes:ethyl acetate, 4:1)
to afford 255 mg of 7 as a solid (74.2%). 1H NMR (500 MHz, CDCl3): 13.42 (br s,
1H), 9.02 (s, 1H), 7.47–7.52 (m, 4H), 7.28–7.35 (m, 6H), 5.55 (s, 2H), 5.23 (s,
2H), 4.41 (q, 2H, J = 7.0 Hz), 1.41 (t, 3H, J = 7.0 Hz). ESI HRMS calcd for
C24H22N3O5 (M+H)+ 432.1559, found 432.1555. Compound
7 (86.3 mg,
0.2 mmol) in 6 ml of dioxane and 5 ml of 1 N HCl was heated under reflux
for 48 h and then cooled. The solid that crystallized out was collected and
washed with a mixture of chloroform and hexanes (1:1, 8 ml) to give 54 mg
(66.9%) of the target molecule 1. Mp 248–250 °C. 1H NMR (500 MHz, DMSO-
d6): 8.84 (s, 1H), 7.21–7.36 (m, 10H), 5.45 (s, 2H), 5.11 (s, 2H). 13C NMR
(125 MHz, DMSO-d6): 169.3, 166.5, 164.2, 157.0, 154.2, 150.6, 137.1, 136.7,
128.7, 128.6, 127.9, 127.7, 127.5, 127.4, 110.2, 98.1, 45.9, 44.5. UV (MeOH)
240 nm (
404.1246, found 404.1244.
e 40,800), 311 nm (e
7,600). ESI HRMS calcd for C22H18N3O5 (M+H)+
12. Data for compound 2: Mp 211–213 °C. 1H NMR (CDCl3, 500 MHz): 14.5 (s, 1H),
10.54 (br s, 1H), 8.27 (d, 1H, J = 9.0), 7.55 (d, 2H, J = 7.0), 7.31–7.39(m, 6H), 7.22
(d, 2H, J = 7.0), 6.75 (d, 1H, J = 9.0), 5.38 (s, 2H), 5.33 (s, 2H). 13C NMR (CDCl3,
125 MHz): 166.8, 164.3, 161.4, 150.4, 144.6, 140.7, 135.6, 134.6, 129.4, 129.39,
Figure 2. Molecular modeling (SYBYL 7.2, GOLD 3.3) of IMPDH complexed with
inhibitor 1 (shown in green) and substrate IMP. The inhibitor interferes with the
binding of NAD+ with the enzyme.
128.9, 128.5, 128.4, 126.6, 110.7, 106.3, 101.0, 48.4, 45.4. UV (MeOH) 236 nm (
41,400); 330 nm (
found 403.1297.
e
e
9,500). ESI HRMS calcd for C23H19N2O5 (M+H)+ 403.1294,
Acknowledgment
13. Antiviral assays were determined as follows: inhibitor 1 was prepared as 8
half-log dilutions with a top concentration of 100 lg/ml. DENV-2 (New Guinea
This research was supported by Research Awards U19 AI
056540 and NO1 AI30048 from the National Institute of Allergy
and Infectious Diseases, NIH.
C) was prepared at a dose of 103 pfu/ml and added to Vero cells shortly after
the addition of compound. Visual CPE was recorded, after which cells were
stained with neutral red for determination of viability. To verify activity, a virus
yield reduction assay was run to determine the concentration of compound
necessary to reduce the virus titer by 1log10 (EC90).
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