Design, synthesis, and biological evaluation of antiviral agents targeting flavivirus envelope proteins

Article abstract of DOI:10.1021/jm800412d

Flavivirus envelope proteins (E proteins) have been shown to play a pivotal role in virus assembly, morphogenesis, and infection of host cells. Inhibition of flavivirus infection of a host cell by means of a small molecule envelope protein antagonist is an attractive strategy for the development of antiviral agents. Virtual screening of the NCI chemical database using the dengue virus envelope protein structure revealed several hypothetical hit compounds. Bioassay results identified a class of thiazole compounds with antiviral potency in cell-based assays. Modification of these lead compounds led to a series of analogues with improved antiviral activity and decreased cytotoxicity. The most active compounds 11 and 36 were effective in the low micromolar concentration range in a cellular assay system.

Full text of DOI:10.1021/jm800412d

4660
J. Med. Chem. 2008, 51, 4660–4671
Design, Synthesis, and Biological Evaluation of Antiviral Agents Targeting Flavivirus Envelope
Proteins
Ze Li,† Mansoora Khaliq,‡ Zhigang Zhou,† Carol Beth Post,† Richard J. Kuhn,‡ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences and the Purdue
Cancer Center, Purdue UniVersity, West Lafayette, Indiana 47907, Department of Biological Sciences, Purdue UniVersity, West Lafayette,
Indiana 47907
ReceiVed April 10, 2008
Flavivirus envelope proteins (E proteins) have been shown to play a pivotal role in virus assembly,
morphogenesis, and infection of host cells. Inhibition of flavivirus infection of a host cell by means of a
small molecule envelope protein antagonist is an attractive strategy for the development of antiviral agents.
Virtual screening of the NCI chemical database using the dengue virus envelope protein structure revealed
several hypothetical hit compounds. Bioassay results identified a class of thiazole compounds with antiviral
potency in cell-based assays. Modification of these lead compounds led to a series of analogues with improved
antiviral activity and decreased cytotoxicity. The most active compounds 11 and 36 were effective in the
low micromolar concentration range in a cellular assay system.
Introduction
Many flaviviruses are arthropod-borne, human pathogens that
can cause encephalitis, hemorrhagic fever, shock syndrome, and
jaundice. Examples of pathogenic flaviviruses include dengue
virus, yellow fever virus, West Nile virus, tick-borne encephalitis
virus, and Japanese encephalitis virus. A number of antiflaviviral
agents with a wide variety of putative targets have been reported,
including those that act on viral membranes,1 translation,2 c-Src
Figure 1. Structure of dengue 2 E protein. Domain I: red; domain II:
protein kinase,3 RNAa polymerase,4 R-glucosidase,5,6 mRNA,7
yellow; domain III: blue. The ꢀ-OG binding pocket is located between
domains I and II.
cellular IMP dehydrogenase,8 2′-O-methyltransferase,9 and
RNA-dependent RNA polymerase.10 In addition, peptide anti-
viral agents targeting West Nile virus envelope protein (E
cell membrane after the E protein undergoes a series of
protein) have been reported to be active against both dengue
conformational changes.
virus and West Nile virus.11 However, at the present time, there
Cryo-electron microscopy and X-ray crystallography studies
are no clinically useful antiflaviviral agents.
of various flavivirus E protein fragments have revealed that
The flaviviruses infect host cells through receptor-mediated
domains I and II are connected by a mobile hinge that allows
endocytosis to form a virus-containing endosome, followed by
rotation of DII relative to DI-DIII during the processes of viral
fusion of the viral envelope with the host cell membrane.12 The
maturation and fusion of the viral envelope with the host cell
membrane.14–17 Mutations that affect the pH threshold for either
fusion process is orchestrated by the viral E proteins, which
fusion or virulence are clustered in this hinge region.18–23
are made up of three domains: domain I (DI), the middle
domain, is attached at one end to domain II (DII), the fusion
Furthermore, crystallization of a dengue virus type 2 E protein
domain, and at the other end contacts domain III (DIII), the
fragment in the presence of the detergent n-octyl-ꢀ-D-glucoside
receptor binding domain (Figure 1).13 Domain II contains the
(ꢀ-OG) has led to structures with and without occupation of
the hinge pocket by ꢀ-OG.14 Occupation of the pocket by ꢀ-OG
fusion peptide loop near its tip, which is inserted into the host
requires an altered conformation of the kl loop toward an “open”
position, and the angle that DII makes with DI-DIII also differs
between two crystal structures with and without ꢀ-OG.14 The
ꢀ-OG pocket may therefore represent an ideal target for
structure-based design of potential antiviral agents because
ligands that bind there could alter the conformational equilibrium
associated with the hinge angle and inhibit virus maturation.14,15
Moreover, the effect on hinge equilibrium could interfere with
the fusion of the viral envelope with the host cell membrane.
Computational screening of an NCI library using the ꢀ-OG
binding pocket recently identified a small set of compounds that
were predicted to bind in the pocket. An array of 23 compounds
were selected for antiviral testing vs yellow fever virus
replication. Of these, nine compounds inhibited viral growth
and several of them had EC50 values in the micromolar range
and were potent enough to warrant further consideration. These
* To whom correspondence should be addressed. Phone: +1 765 494
1465. Fax: +1 765 494 6790. E-mail: cushman@pharmacy.purdue.edu.
†
Department of Medicinal Chemistry and Molecular Pharmacology,
School of Pharmacy and Pharmaceutical Sciences and the Purdue Cancer
Center, Purdue University.
‡
Department of Biological Sciences, Purdue University.
a Abbreviations: RNA, ribonucleic acid; mRNA, messenger ribonucleic
acid; IMP, inosine monophosphate; NCI, National Cancer Institute; SAR,
structure-activity relationship; DMSO-d6, hexadeuterodimethyl sulfoxide;
NMR, nuclear magnetic resonance; TMS, tetramethylsilane; HRMS, high
resolution mass spectrum; ES+, positive ion electrospray; EDCI, 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide; DMF, dimethylformamide; NBS,
N-bromosuccinimide; YFV, yellow fever virus; IRES, internal ribosome
entry site; Luc, luciferase; 3′ NTR, 3′ nontranslated region; PCR, polymerase
chain reaction; EMCV, encephalomyocarditis virus; MEM, minimum
essential medium; FBS, fetal bovine serum; BHK, baby hamster kidney,
PBS, phosphate-buffered saline; NIH, National Institutes of Health; NIAID,
National Institute of Allergy and Infectious Diseases; RCE, Regional Centers
of Excellence.
10.1021/jm800412d CCC: $40.75
2008 American Chemical Society
Published on Web 07/09/2008

AntiViral Agents Targeting FlaViVirus EnVelope Proteins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4661
inhibitors, this moiety was replaced with a bioisosteric amide
linkage. The Figure 5 depicts compounds 1, 2, and their
analogues 3-8, which have been synthesized and bioassayed.
According to the docked models of the hit M02, one of the
two phenyl rings attached to the 3,5-diphenyl-1H-pyrazole was
calculated to point to an independent hydrophobic region. This
phenyl ring has been thought to be redundant and was deleted
in our first generation drug design because no comparable partial
structures in the other hits can overlay with this group in the
docked models. However, considering the fact that this phenyl
ring may nevertheless play an important role in the hit’s antiviral
activity, a class of compounds based on the structure of the hit
M02 has been designed, synthesized, and bioassayed (Figure
6). The only modification in structure 11 is that the R,ꢀ-
unsaturated ketone in M02 has been replaced by a bioisosteric
amide, which was based on the same considerations with regard
to toxicity as described above. To clarify the role of the 3,5-
diphenyl-1H-pyrazole moiety of M02 in its antiviral activity,
3,5-diphenyl-1H-pyrazole (17) itself was tested for antiviral
activity.
By comparing the docked models of P02 and M02, a class
of branched compounds (18, 19, and 20, Figure 7) was designed,
synthesized, and bioassyed. These compounds can be considered
as analogues in which a derivative of the substituted thiazole
moiety of M02 has been attached to a fragmented analogue of
P02.
Another class of compounds was designed to mimick the hit
D03, which provides a convenient template for diversification.
An array of analogues of D03 shown in Figure 8 has therefore
been synthesized and bioassayed. It was expected that these
structurally related compounds could provide useful SAR
information.
Figure 2. Chemical structures of the “hits” identified through in silico
screening and biological evaluation. The antiviral activities were
determined using yellow fever virus.
three hit compounds, M02, P02, and D03 (Figure 2), provided
a starting point for lead optimization in the present study.
A final set of compounds was derived from fragments of M02
and D03 (Figure 9). The phenylthiazole moiety of D03 was
attached to an analogue of the 3,4-dichlorostyryl moiety of M02,
resulting in 34. Introduction of bromine into the methyl group
of 32 led to the dibromosubstituted compound 36. This
compound, showing unexpected stability, presented an interest-
ing chemical probe to test the influence of electronic and steric
effects on antiviral activity.
Design
The binding models derived from docking results indicate
that compounds M02 and P02 might bind to the E protein ꢀ-OG
binding site in a similar way. An overlay of their docked models
suggested that these molecules occupy a pocket that is located
in the hinge region between domains I and II (Figure 1).
Computational analysis revealed that the middle part of the
pocket is the most hydrophobic and the entrance part is the most
hydrophilic (Figure 3). The bottom region of the pocket contains
some hydrophilic amino acid residues that could interact with
ligands through electrostatically favorable interactions as in the
case of P02. One end of the ligand can reach into the pocket,
and electrostatically favorable interactions could be formed
between the aminoguanidine groups with Thr280 in domain I
and Asp203, Gln200, Gln271, and Ser274 in domain II (Figure
4B). Topologically, the entrance of the pocket shows a rather
wide space in which the ends of the hit compounds assume
various positions, whereas the middle part of the pocket has
the most limited space.
An analysis of the protein-ligand interaction suggests that
tight-binding inhibitors might be obtained by grafting half of
the structure of P02 onto half of the structure of M02. Driven
by the hypothesis that the aminoguanidine group in P02 can
potentially form electrostatically favorable interactions with the
residues in the receptor pocket, one-half of the structure of P02
was incorporated by connecting it to M02’s thiazole ring, which
is a common structural feature that many of the hits contain.
This contributed to the design of the first two target compounds
1 and 2 (Figure 5). The double bond in the R,ꢀ-unsaturated
ketone present in inhibitor M02 is electrophilic and could react
nondiscriminately with biological nucleophiles, thus resulting
in toxicity. Accordingly, in the design of the first two potential
Synthesis
The synthetic route to compounds 1-8 is outlined in Scheme
1. Radical bromination of methyl 3-oxobutanoate (37) was used
to construct methyl 2-bromo-3-oxobutanoate (38),24 which
reacted with thiourea at room temperature to give rise to the
thiazole intermediate, 2-amino-4-methylthiazole-5-carboxylic
acid methyl ester (39).25 Reaction of this intermediate with 1-(4-
isocyanatophenyl)ethanone in pyridine afforded compound 3.
Hydrolysis of 3 with sodium hydroxide to the corresponding
acid 4 and then amidation with 2,4-dichloro- or 3,4-dichlo-
robenzylamine mediated by 1-ethyl-3-(3′-dimethylaminopropy-
l)carbodiimide (EDCI) readily provided the desired compounds
7 and 8. Target hydrazones 1 and 2 were synthesized by reacting
7 with semicarbazide hydrochloride or aminoguanidine hydro-
chloride, respectively.26 Compounds 5 and 6 were prepared from
3 similarly.
Compounds 9-16 were synthesized through the route shown
in Scheme 2, in which a copper-catalyzed N-arylation reaction
of the pyrazole 17 is the key step.27 The reaction is highly
sensitive to air. In our experiments, the reaction was conducted
in a sealed pressure vessel filled with argon gas. Conversions
of 10 to 11-16 were performed smoothly.
The branched potential inhibitors 18–20 were made as
outlined in Scheme 3. Bisalkylation of 2-amino-4-methylthiaz-

4662 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Li et al.
Figure 3. Lipophilic potential analysis of the E protein ꢀ-OG binding site showing the entrance region is relatively more hydrophilic and the
middle region is the most lipophilic region. The lipophilic potential ranges from -0.241 (blue, hydrophilic) to 0.147 (brown, hydrophobic). The left
picture views the pocket from entrance side, and the right picture views the pocket from the bottom side. The red stick model is P02 and the stick
model colored by atom type is M02.
Figure 4. (A) Two active compounds docked in the ꢀ-OG pocket of dengue E protein. The structure colored magenta is P02, and the structure
colored blue is M02. (B) Potential electrostatically favorable interactions formed between residues Asp203, Gln200, Gln271, Ser274, Thr48, and
Thr280 with P02. Part of the protein is shown in cartoon representation with red color for domain I and yellow color for domain II.
ole-5-carboxylic acid methyl ester (39) with 1-(4-(bromomethyl)-
phenyl)ethanone (41) was performed with sodium hydride as
the base to afford 18. In the presence of ethyl acetate, the major
product of the reaction was 20. Compound 18 was further
converted to 19 through the acid 42 via a hydrolysis-amidation
sequence similar to the synthetic route to compounds 10 and 11.
Analogues of D03 were prepared following the routes
depicted in Scheme 4. Heterocyclic ring closure of 4-chlo-
rothiobenzamide (43) with 1-chloroacetone (44) afforded com-
pound 21. Reaction of 1,3-dichloroacetone (45) with aromatic
thioamides, followed by nucleophilic substitution of the chloride
intermediates with thiophenols, readily provided 23-30 in good
yield.28,29
Potential inhibitors 31-36 were obtained by the route detailed
in Scheme 5. Heterocyclic ring closure of arylthioamides with
2-chloro-3-oxo-butyric acid methyl ester (46)30,31 furnished
compounds 31-33. Hydrolysis of 32 to the corresponding acid
and then amidation or esterification afforded compounds 34 and
35. Bromination of 32 with NBS provided the dibromosubsti-
tuted product 36.
that showed over 50% inhibitory activity on viral replication at
a concentration of 50 µM were considered to be active and were
tested to determine their EC50 and CC50 values. The biological
data are summarized in Table 1.
Bioassay results showed that compounds 2, 3, and 8 exhibited
better antiviral activity against yellow fever virus replication
in a cellular bioassay system than the parent compounds M02,
but they were not more potent than P02. The results suggest
that a core structure consisting of a thiazole ring and an aromatic
ring linked by a urea moiety could be a promising structural
template for potential antiviral agents. The activity of 3 appears
to be structurally specific. For instance, compound 3 lost its
activity upon being hydrolyzed to the corresponding acid 4. In
addition, compounds 1 and 7 are inactive vs the active
compounds 2 and 8, even though the structures are very similar.
Although the bioassay results revealed several active compounds
in this class, their potencies are still moderate.
Although most of the compounds in the 9-16 array did not
show over 50% inhibition of virus production at a concentration
of 50 µM, the target molecule 11 showed enhanced antiviral
activity and decreased cytotoxicity compared with the parent
compound M02. The antiviral activity of 11 (EC50 5 ( 1.7 µM)
is 10 times that of parent compound and cytotoxicity is 2.7 times
less, resulting in a therapeutic index of 37. These data validate
Biological Results and Discussion
All of the compounds were evaluated in a yellow fever virus
inhibition assay (see Experimental Section). The compounds

AntiViral Agents Targeting FlaViVirus EnVelope Proteins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4663
Figure 6. Analogues of M02.
Figure 5. The first generation of designed molecules 1 and 2 and
analogues.
Bioassay results showed that the branched compound 18 is
active against yellow fever virus, with an EC50 value of 25 µM.
The results suggest that replacement of the diphenylpyrazole
moiety of structure M02 with tertiary amines could be a possible
strategy to develop more potent flavivirius inhibitors.
the hypothesis that replacing the Michael acceptor in M02 with
a bioisosteric amide would reduce the undesired cytotoxicity.
The weak antiviral activities of the other analogues may indicate
that an aromatic substituent attached to the thiazole ring through
a stable linker such as an amide is necessary for optimal antiviral
activity. The hydrolytic instability of the ester linker could
explain why the ester analogues, such as compounds 12-16,
did not display antiviral activity. Obviously, the amide 11 should
be more stable than the esters to enzymatic hydrolysis. Testing
the contribution of the 3,5-diphenyl-1H-pyrazole moiety to
antiviral activity of M02 revealed that the 3,5-diphenyl-1H-
pyrazole (17) fragment is at least as active as the parent M02
(EC50 44 vs 51 µM).
Compounds 23-27 mimicking D03 exhibited antiviral
activity at 30-50 µM range, which is comparable to the
activity of D03 itself (EC50 31 µM). It seems that the overall
physical-chemical properties of the compounds are the
determining factor for their biological activity. Slight modi-
fications in structure do not cause a significant change in
antiviral activity. A preliminary SAR analysis suggested that
an electron-rich aromatic ring attached to a thiazole ring that
is linked to a 2- or 4-substituted aniline group may represent
a template for antiviral agents. The presence of a pyridine

4664 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Li et al.
Figure 9. Analogues of M02 and D03.
Scheme 1a
Figure 7. The branched potential flavivirus inhibitors.
a Reagents and conditions: (a) NBS, benzene, AIBN, RT for 2 h, 100%;
(b) thiourea, ethanol, RT for 3 h, 81%; (c) 1-(4-isocyanatophenyl)ethanone,
pyridine, RT for 12 h, 83%; (d) semicarbazide hydrochloride for 5 or
aminoguanidine hydrochloride for 6, ethanol,triethylamine for 5, AcOH,
heated to reflux for 6 h, 70% for 5, and 65% for 6; (e) NaOH, EtOH,MeOH,
heated to reflux for 3 h, 73%; (f) 2,4-dichlorobenzylamine for 7 or 3,4-
dichlorobenzylamine for 8, EDCI, pyridine, RT for 12 h, 60-70%; (g)
semicarbazide hydrochloride for 1 or aminoguanidine hydrochloride for 2,
ethanol, triethylamine for 1, AcOH, heated to reflux for 6 h, 73% for 1,
and 67% for 2.
The most active compound was 36, which showed 99%
inhibition of yellow fever virus and dengue virus replication at
the initial 50 µM concentration tested. The EC50 value of the
compound is 0.9 µM vs yellow fever virus replication. Encour-
agingly, although this compound contains a dibromomethyl
substituent on the thiazole ring that was considered to be likely
to cause toxicity by alkylating biological nucleophiles, it was
in fact not very cytotoxic (CC50 153 µM), resulting in a good
therapeutic index of 170. Obviously, this compound provides
an intriguing lead that deserves further study. Comparing the
structure of 36 with the others in this class, it seems that the
dibromomethyl substituent is important for antiviral activity.
Compound 32, the synthetic precursor of 36, is inactive against
yellow fever virus and it does not contain the dibromomethyl
substituent.
Figure 8. The analogues of D03.
ring, such as in compounds 28 and 29, or the absence of the
aniline group, such as in compound 30, causes these
analogues to lose antiviral activity.
The most promising compounds 11 and 36 were docked in
the dengue virus E protein ꢀ-OG binding pocket in order to

AntiViral Agents Targeting FlaViVirus EnVelope Proteins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4665
Scheme 4a
Scheme 2a
a Reagents and conditions: (a) Cu2O, 2-hydroxybenzaldehyde oxime,
CsCO3, CH3CN, argon, 80-90 °C for 8 h, 65%; (b) LiOH, methanol,
ethanol, H2O, heated to reflux for 3 h, 80%; (c) benzylamines or alkyl
alcohols, EDCI, pyridine, RT for 12 h, 60-70%.
Scheme 3a
a Reagents and conditions: (a) ethanol, heated to reflux for 2 h, 80%;
(b) ethanol, heated to reflux for 2 h, 65%; (c) R2SH, CsCO3, DMF, 80 °C
for 2 h, 70-80%.
Scheme 5a
a Reagents and conditions: (a) NaH, DMF, 0 °C-RT for 4 h, 74% (b)
NaH, DMF, ethyl acetate, 0 °C-RT for 4 h, 70%; (c) NaOH, methanol,
ethanol, heated to reflux for 1 h, 70%; (d) 2,4-dichlorobenzylamine, EDCI,
pyridine, RT for 12 h, 72%.
a Reagents and conditions: (a) ethanol, heat to reflux for 2 h, 65-85%;
(b) LiOH, methanol, ethanol, water, heated to reflux for 1 h, 80%; (c) 3,4-
dichlorobenzylamine for 34 or adamantan-1-yl-methanol for 35, EDCI,
pyridine, RT for 12 h, 60%; (d) NBS, AIBN, CCl4, heated to reflux for
12 h, 60%.
gain insight into their probable binding modes (Figure 10). These
two compounds may bind in the ꢀ-OG binding pocket in an
area surrounded by amino acid residues including Thr280,
Val130, Leu198, Gln200, Ala205, Ile270, Ser274, Gln271,
Leu277, Lys47, Thr48, Glu49, Leu135, Ala50, and Thr137. The
entire binding site forms a channel. Compound 11 places its
3,5-diphenyl-1H-pyrazole group in the middle region, leaving
its amide tail positioned in the entrance region. It is still unclear
what role the second phenyl ring of the 3,5-diphenyl-1H-
pyrazole in compound 11 plays in its antiviral activity. The
docked model suggests that this phenyl ring, with its bulky size,
could block the compound from docking deeply into the channel.
The smaller compound 36 has been suggested by docking
simulation to bind deeply into the channel. The binding could
be tighter than other compounds binding to the entrance and
middle regions, thus resulting in stronger potency. The dibro-
momethyl substituent of compound 36, as the biological data
indicate, is likely to be a key structural element for the antiviral
activity. One bromine atom may hinder nucleophilic displace-
ment of the other bromine atom by biological nucleophiles, thus
lowering the cytotoxicity. On the other hand, the docked model
suggests that one of the bromine atoms may fit a small region
between the entrance region and the middle region. The
nonbonded interactions obtained from this extra binding may
result in stronger biological activity.
Conclusion
Although the active compounds have variable structures,
many of them share the common feature of a central thiazole
ring. SAR analysis suggests that the hits M02, P02, and D03
indeed provide promising structural templates for potential
antiflaviviral agents. Replacing the Michael acceptor in M02
with an amide in compound 11 significantly enhanced antiviral
activity and diminished the undesired cytotoxicity. Alteration
of the chloro and amino substitution pattern of the lead
compound D03 provided equally potent derivatives 23-27.
The biological testing results indicate that compounds 11 and
36 inhibit yellow fever virus replication with EC50 values in
the low micromolar range. Clearly, the preliminary antiviral
potency displayed by these thiazole derivatives warrants further
investigation. Future work will focus on modification of two

4666 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Li et al.
Table 1. Antiviral Activities vs Yellow Fever Virus and Cytotoxicities
of the Original Hits and Second Generation Compounds
N-(2,4-dichlorobenzyl)-4-methylthiazole-5-carboxamide (7) (100
mg, 0.21 mmol) in ethanol (5 mL) was added to the reaction
solution followed by acetic acid (0.1 mL). The reaction mixture
was heated to reflux for 6 h. The reaction mixture was allowed to
cool to room temperature. After removal of solvent under reduced
pressure, the resulting residue was washed with cold ethyl acetate
(1 mL × 3) and then recrystallized from ethanol to provide the
desired compound as a white solid (80 mg, 73%): mp 184-186
°C. 1H NMR (500 MHz, DMSO-d6): δ 9.51 (s, 1 H), 9.24 (s, 1 H),
8.50 (s, 1 H), 7.77 (d, J ) 7.5 Hz, 2 H), 7.57 (s, 1 H), 7.46 (d, J
) 7.5 Hz, 1 H), 7.40 (d, J ) 7.5 Hz, 1 H), 7.32 (d, J ) 7.5 Hz, 1
H), 6.46 (s, 2 H), 4.39 (d, J ) 3.0 Hz, 2 H), 2.44 (s, 3 H), 2.12 (s,
3 H). 13C NMR (125 MHz, DMSO-d6): δ 162.3, 159.0, 157.7,
143.9, 139.2, 136.0, 133.1, 133.0, 132.4, 130.4, 128.7, 127.7, 127.0
× 2, 118.2 × 2, 40.5, 17.1, 13.4. IR (KBr) 3271, 2922, 2851, 1667,
1531, 1454, 1373, 1309, 1248, 1187 cm-1. MS (ES-) calcd for
C22H20Cl2N7O3S 532.08, found 532.06. Anal. (C22H21Cl2N7O3S·
0.5H2O) C, H, N.
compound
EC50 (µM)a
CC50 (µM)b
TIc
M02
P02
D03
1
2
3
4
5
6
7
51 ( 7.2
13 ( 3
31 ( 0.3
>50
70 ( 12
398 ( 0.1
61 ( 3
1.37
30.6
2.0
37.6 ( 2.8
13.9 ( 1.5
>50
154 ( 27
155 ( 50
4.1
11.2
>50
>50
>50
8
9
37.0 ( 7.0
>50
138 ( 25
186 ( 6
3.7
37
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
>50
5 ( 1.7
>50
>50
>50
(E)-2-(3-(4-(1-(2-Carbamimidoylhydrazono)-ethyl)-phenyl)-
ureido)-N-(2,4-dichlorobenzyl)-4-methylthiazole-5-carboxam-
ide (2). Aminoguanidine hydrochloride salt (20 mg, 0.22 mmol),
2-(3-(4-acetylphenyl)-ureido)-N-(2,4-dichlorobenzyl)-4-methylthi-
azole-5-carboxamide (7) (100 mg, 0.21 mmol), and acetic acid (0.1
mL) were added to ethanol (5 mL). The reaction mixture was heated
to reflux for 6 h and allowed to cool to room temperature. After
removal of solvent under reduced pressure, the resulting residue
was washed with cold ethyl acetate (1 mL × 3) and then
recrystallized from ethanol to provide the desired compound as a
white solid (69 mg, 67%): mp 132-134 °C. 1H NMR (500 MHz,
DMSO-d6): δ 11.4 (s, 1 H), 10.72 (s, 1 H), 8.52 (s, 1 H), 7.90 (d,
J ) 9.0 Hz, 2 H), 7.89 (s, 4 H), 7.56 (d, J ) 2.0 Hz, 1 H), 7.49 (d,
J ) 9.0 Hz, 2 H), 7.39 (d, J ) 8.5 Hz, 1 H), 7.33 (d, J ) 8.5 Hz,
1 H), 4.38 (d, J ) 5.5 Hz, 2 H), 2.46 (s, 3 H), 2.30 (s, 3 H). 13C
NMR (125 MHz, DMSO-d6): δ 162.4, 158.5, 156.5, 152.1, 151.4,
140.5, 136.0, 133.1, 132.4, 130.4, 128.8, 127.9 × 2, 127.6, 117.7
>50
>50
44 ( 5
25 ( 4
>50
123 ( 48
200 ( 26
2.8
8
>50
>50
>50
29 ( 16
46 ( 0.5
37 ( 13
40 ( 0.3
46 ( 6
>50
151 ( 5
119 ( 14
155 ( 15
131 ( 15
133 ( 2
5.2
2.6
4.2
3.2
2.9
>50
>50
>50
>50
× 2, 117.4, 40.5, 17.2, 14.9. IR (KBr) 1329, 1270, 913, 700 cm-1
.
>50
MS (ES+) calcd for C22H23Cl2N8O2S 533.10, found 532.88. HRMS
(ES+) calcd for C22H20Cl2N7O3S 533.1042, found 533.1040.
Methyl 2-(3-(4-Acetylphenyl)ureido)-4-methylthiazole-5-car-
boxylate (3). 1-(4-Isocyanatophenyl)ethanone (500 mg, 3.1 mmol)
was added to a solution of methyl 2-amino-4-methylthiazole-5-
carboxylate (39) (550 mg, 3.1 mmol) in pyridine (15 mL). The
reaction mixture was stirred at room temperature for 12 h. The
solid was filtered and washed with cold methanol (5 mL × 3) to
provide the desired compound as a white solid (876 mg, 83%): mp
>50
>50
0.9 ( 0.7
153 ( 35
170
a The EC50 is the concentration of the compound resulting in a 50%
inhibition in virus production. b The CC50 is the concentration of the
compound causing a 50% growth inhibition of uninfected BHK cells. c TI
is the therapeutic index and is equal to CC50/EC50.
of these structural templates, as well as NMR and crystal-
lographic studies of drug-receptor complexes, testing vs drug-
resistant mutant viruses, and evaluation of efficacy in animal
models. On the basis of the data in hand, there is every reason
to hope that derivatives of 11 or 36 could be developed into
therapeutically useful antiviral agents.
1
270-272 °C. H NMR (300 MHz, DMSO-d6): δ 9.45 (s, 1 H),
7.90 (d, J ) 9.0 Hz, 2 H), 7.60 (d, J ) 9.0 Hz, 2 H), 3.76 (s, 3 H),
2.52 (s, 3 H), 2.50 (s, 3 H). 13C NMR (125 MHz, pyridine-d5): δ
197.5, 164.7, 164.4, 155.9, 155.7, 145.7, 133.3, 131.3 × 2, 119.7
× 2, 114.5, 52.8, 27.5, 17.8. IR (KBr) 3330, 1714, 1684, 1659,
1540, 1317, 1255, 1186, 762, 667 cm-1. MS (ES+) calcd for
C15H16N3O4S 334.36, found 334.93. Anal. (C15H15N3O4S) C, H,
N.
Experimental Section
Experimental procedures for the syntheses of compounds 12-16,
23-31, 33, and 34, including analytical data, are reported in
Supporting Information. Proton (1H NMR) and carbon (13C NMR)
spectra were recorded in CDCl3 or DMSO-d6 on a Bruker ARX-
300 or on a Bruker AVANCE 500 spectrometer. The NMR solvents
are specified individually for each compound. Chemical shifts are
given in parts per million (ppm) on the delta (δ) scale. The solvent
peak or the internal standard tetramethylsilane were used as
reference values. For 1H NMR: CDCl3 ) 7.27 ppm, TMS ) 0.00
ppm. Multiplicities were recorded as s (singlet), d (doublet), t
(triplet), and m (multiplet). All reagents and dry solvents were
purchased from Aldrich. All reactions were conducted under argon
or nitrogen atmosphere, unless otherwise specified.
2-[3-(4-Acetylphenyl)-ureido]-4-methylthiazole-5-carboxylic
Acid (4). Sodium hydroxide (200 mg, 5 mmol) was added to a
solution of methyl 2-(3-(4-acetylphenyl)ureido)-4-methylthiazole-
5-carboxylate (3) (100 mg, 0.3 mmol) in methanol (5 mL) and
ethanol (5 mL). The reaction mixture was heated to reflux for 3 h
and allowed to cool to room temperature. After removal of solvent
under reduced pressure, water (10 mL) was added to the residue.
The solution was filtered and acidified to pH 2-3 with hydrochloric
acid. The generated solid was filtered and washed with cold hexanes
(1 mL × 3) to provided the desired compound as a white solid (60
1
mg, 62%): mp 232-234 °C. H NMR (500 MHz, DMSO-d6): δ
9.43 (s, 2 H), 7.90 (d, J ) 8.5 Hz, 2 H), 7.60 (d, J ) 8.5 Hz, 2 H),
2.49 (s, 3 H), 2.46 (s, 3 H). 13C NMR (125 MHz, pyridine-d5): δ
197.5, 166.6, 164.7, 156.1, 153.9, 145.9, 134.6, 131.3 × 2, 119.7
× 2, 116.9, 27.5, 17.6. IR (KBr) 3367, 1716, 1598, 1535, 1486,
1410, 1313, 1272, 1176, 752, 680 cm-1. MS (ES-) calcd for
C14H12N3O4S 318.34, found 318.23. Anal. (C14H13N3O4S·1.5H2O)
C, H, N.
(E)-2-(3-(4-(1-(2-Carbamoylhydrazono)-ethyl)-phenyl)-ureido)-
N-(2,4-dichlorobenzyl)-4-methylthiazole-5-carboxamide (1). Semi-
carbazide hydrochloride salt (20 mg, 0.22 mmol) was added to
ethanol (5 mL). Et3N was added dropwise to this mixture until it
became a clear solution. A solution of 2-(3-(4-acetylphenyl)-ureido)-

AntiViral Agents Targeting FlaViVirus EnVelope Proteins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4667
Figure 10. The docked models of compounds 11 (left) and 36 (right). The compounds are colored by atom type and the dengue ꢀ-OG binding
pocket is displayed as a Connolly surface model.
(E)-Methyl 2-(3-(4-(1-(2-Carbamoylhydrazono)-ethyl)-phe-
nyl)-ureido)-4-methylthiazole-5-carboxylate (5). Semicarbazide
hydrochloride salt (111 mg, 1 mmol) was added to ethanol (5 mL).
Et3N was added dropwise to this mixture until it became a clear
solution. A solution of methyl 2-(3-(4-acetylphenyl)ureido)-4-
methylthiazole-5-carboxylate (3) (333 mg, 1 mmol) in ethanol (10
mL) was added to the reaction mixture, followed by acetic acid
(0.5 mL). The reaction mixture was heated to reflux for 3 h and
allowed to cool to room temperature. After removal of solvent under
reduced pressure, the resulting residue was washed with cold ethyl
acetate (1 mL × 3) and then recrystallized from ethanol to provide
the desired compound as a white solid (323 mg, 83%): mp 265-267
°C. 1H NMR (500 MHz, DMSO-d6): δ 9.82 (s, 1 H), 9.21 (s, 1 H),
7.77 (d, J ) 8.5 Hz, 2 H), 7.44 (d, J ) 8.5 Hz, 2 H), 6.45 (s, 2 H),
3.72 (s, 3 H), 2.48 (s, 3 H), 2.11 (s, 3 H). 13C NMR (125 MHz,
DMSO-d6): δ 162.9, 161.0, 157.7, 152.0, 144.3, 139.0, 133.1, 130.4,
127.0 × 2, 118.3 × 2, 117.9, 52.0, 17.2, 13.4. IR (KBr) 2382,
2300, 1698, 1535, 1493, 1376, 1328, 1259, 1219, 1070 cm-1. MS
(ES-) calcd for C16H17N6O4S 389.11, found 388.98. Anal. calcd
for C16H18N6O4S·0.4H2O: C, 48.33; H, 4.77; N, 21.14. Found: C,
48.80; H, 4.70; N, 20.65.
130.2, 128.3, 119.7 × 2, 117.6, 42.1, 27.1, 17.4. IR (KBr) 3246,
1537, 1176 cm-1. MS (ES-) calcd for C21H17Cl2N4O3S 475.05,
found 474.95. Anal. (C21H18Cl2N4O3S·2/3H2O) C, H, N.
2-(3-(4-Acetylphenyl)-ureido)-N-(3,4-dichlorobenzyl)-4-meth-
ylthiazole-5-carboxamide (8). 3,4-Dichlorobenzylamine (90 mg,
0.5 mmol) and EDCI (100 mg, 0.52 mmol) were added to a solution
of 2-[3-(4-acetyl-phenyl)-ureido]-4-methyl-thiazole-5-carboxylic
acid (4) (150 mg, 0.46 mmol) in pyridine (5 mL). The reaction
mixture was stirred at room temperature overnight. After removal
of solvent under reduced pressure, the resulting residue was purified
by chromatography (ethyl acetate-hexanes ) 2:3) to provide the
desired compound as a white solid (153 mg, 70%): mp 262-264
1
°C. H NMR (300 MHz, DMSO-d6): δ 9.53 (s, 1 H), 8.57 (dd, J
) 5.7, 5.7 Hz, 1 H), 7.93 (d, J ) 8.7 Hz, 2 H), 7.63 (d, J ) 8.7
Hz, 2 H), 7.60 (d, J ) 8.4 Hz, 1 H), 7.55 (d, J ) 1.8 Hz, 1 H),
7.30 (dd, J ) 8.4, 1.8 Hz, 1 H), 4.36 (d, J ) 6.0 Hz, 2 H), 2.52 (s,
3 H), 2.50 (s, 3 H). 13C NMR (75 MHz, DMSO-d6): δ 197.3, 162.7,
141.9, 133.1, 132.4, 131.7, 131.4, 130.6 × 2, 133.0, 130.2, 130.1,
128.6, 118.6 × 2, 42.6, 27.3, 17.4. IR (KBr) 3252, 1534, 1177
cm-1. MS (ES+) calcd for C21H19Cl2N4O3S 477.05, found 477.0.
Anal. (C21H18Cl2N4O3S·1.5 H2O) C, H, N.
(E)-Methyl 2-(3-(4-(1-(2-Carbamimidoylhydrazono)-ethyl)-
phenyl)-ureido)-4-methylthiazole-5-carboxylate (6). Aminoguani-
dine hydrochloride salt (111 mg, 1 mmol) and methyl 2-(3-(4-
acetylphenyl)ureido)-4-methylthiazole-5-carboxylate (3) (333 mg,
1 mmol) were added to ethanol (10 mL), followed by acetic acid
(0.5 mL). The reaction solution was heated to reflux for 3 h and
allowed to cool to room temperature. After removal of solvent under
reduced pressure, the resulting residue was washed with cold ethyl
acetate (1 mL × 3) and then recrystallized from ethanol to provide
the desired compound as a white solid (291 mg, 75%): mp 212-214
°C. 1H NMR (500 MHz, DMSO-d6): δ 11.0 (s, 2 H), 10.4 (s, 1 H),
7.90 (d, J ) 8.5 Hz, 2 H), 7.50 (d, J ) 8.5 Hz, 2 H), 3.73 (s, 3 H),
2.49 (s, 3 H), 2.26 (3 H). IR (KBr) 2382, 1319 cm-1. MS (ES+)
calcd for C16H20N7O3S 390.13, found 389.76. HRMS (ES+) calcd
for C16H20N7O3S 390.1348, found 390.1340. Anal. (C16H19N7O3S·4/
3HCl) C, H, N.
Ethyl 2-(3,5-Diphenyl-1H-pyrazol-1-yl)-4-methylthiazole-5-
carboxylate (9). 3,5-Diphenyl-1H-pyrazole (17) (80 mg, 0.4 mmol),
2-bromo-4-methyl-thiazole-5-carboxylic acid ethyl ester (40) (125
mg, 0.5 mmol), Cu2O (10 mg, 0.07 mmol), and 2-hydroxybenzal-
dehyde oxime (10 mg, 0.07 mmol) were added to a solution of
cesium carbonate (325 mg, 1 mmol) in predegassed anhydrous
CH3CN (5 mL) in a pressure reaction vessel. Argon gas was induced
into the reaction mixture for 20 min. Then the vessel was sealed
with a cap. The reaction mixture was heated to 80-90 °C for 8 h.
After removal of solvent under reduced pressure, the residue was
dissolved in methanol and purified by chromatography (ethyl
acetate-hexanes ) 1:9) to provide the desired compound as a white
solid (100 mg, 65%): mp 114-116 °C. 1H NMR (300 MHz,
CDCl3): δ 7.92 (d, J ) 6.9 Hz, 2 H), 7.55 (m, 2 H), 7.44 (m, 6 H),
6.79 (s, 1 H), 4.31 (ddd, J ) 6.9, 6.9, 6.9 Hz, 2 H), 2.51 (s, 3 H),
1.34 (dd, J ) 7.2, 7.2 Hz, 3 H). 13C NMR (75 MHz, CDCl3): δ
162.1, 162.0, 158.2, 153.8, 145.9, 131.4, 129.6 × 2, 129.5, 129.0,
128.9, 128.6 × 2, 127.7 × 2, 126.0 × 2, 119.2, 108.4, 61.0, 17.4,
14.2. IR (KBr) 1712, 1499, 1256, 1095, 759 cm-1. MS (ES+) calcd
for C22H20N3O2S 390.12, found 390.12. Anal. (C22H19N3O2S) C,
H, N.
2-(3,5-Diphenyl-1H-pyrazol-1-yl)-4-methylthiazole-5-carboxy-
lic Acid (10). Lithium hydroxide (120 mg, 5 mmol) was added to
a solution of ethyl 2-(3,5-diphenyl-1H-pyrazol-1-yl)-4-methylthi-
azole-5-carboxylate (9) (70 mg, 0.18 mmol) in methanol (5 mL),
ethanol (10 mL), and water (5 mL). The reaction mixture was heated
to reflux for 3 h. After removal of solvent under reduced pressure,
the residue was dissolved in water (10 mL) and the resulting
solution was filtered. The solution was acidified to pH 2-3 with
hydrochloric acid and the resulting solid was filtered. The crude
product was recrystallized from methanol (10 mL) to provide the
desired compound as a white solid (51 mg, 80%): mp 232-234
2-(3-(4-Acetylphenyl)-ureido)-N-(2,4-dichlorobenzyl)-4-meth-
ylthiazole-5-carboxamide (7). 2,4-Dichlorobenzylamine (90 mg,
0.5 mmol) and EDCI (100 mg, 0.52 mmol) were added to a solution
of 2-[3-(4-acetyl-phenyl)-ureido]-4-methyl-thiazole-5-carboxylic
acid (4) (150 mg, 0.46 mmol) in pyridine (5 mL). The reaction
mixture was stirred at room temperature overnight. After removal
of solvent under reduced pressure, water (1 mL) was added to the
residue. The resulting mixture was stirred for 1 h at room
temperature. The solid was filtered and washed with cold methanol
(1 mL × 3) to provide the desired compound as a white solid (138
1
mg, 63%): mp 214-216 °C. H NMR (300 MHz, DMSO-d6): δ
9.49 (s, 1 H), 8.54 (dd, J ) 5.7, 5.7 Hz, 1 H), 7.92 (d, J ) 8.4 Hz,
2 H), 7.63 (d, J ) 8.4 Hz, 2 H), 7.43 (d, J ) 8.4 Hz, 1 H), 7.35 (d,
J ) 8.4 Hz, 1 H), 4.41 (d, J ) 5.7 Hz, 2 H), 2.52 (s, 3 H), 2.47 (s,
3 H). 13C NMR (125 MHz, pyridine-d5): δ 197.5, 164.3, 163.1,
156.1, 145.6, 136.9, 135.9, 134.7, 134.0, 132.8, 131.3, 130.9 × 2,

4668 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Li et al.
°C. 1H NMR (500 MHz, DMSO-d6): δ 7.93 (d, J ) 7.5 Hz, 2 H),
7.57 (br, s, 2 H), 7.48 (m, 6 H), 7.23 (s, 1 H), 2.35 (s, 3 H). 13C
NMR (75 MHz, CDCl3): δ 163.7, 161.9, 157.4, 154.2, 146.6, 131.8,
130.4 × 2, 130.2, 130.1, 129.8 × 2, 128.7 × 2, 126.8 × 2, 126.0,
pressure, the reaction residue was poured into water (5 mL). The
generated solids were filtered, washed with cold hexanes (1 mL ×
3), and recrystallized from methanol (10 mL) to provide the desired
compound as yellow solid (2.4 g, 70%): mp 182-184 °C. 1H NMR
(300 MHz, CDCl3): δ 7.92 (d, J ) 8.1 Hz, 2 H), 7.26 (d, J ) 8.0
Hz, 2 H), 5.55 (s, 2 H), 3.85 (s, 3 H), 2.58 (s, 6 H), 2.03 (s, 3 H).
13C NMR (75 MHz, CDCl3): δ 197.3, 170.5, 163.6, 160.4, 155.9,
141.4, 136.5, 128.9 × 2, 126.2 × 2, 116.8, 51.6, 50.4, 26.5, 22.9,
17.3. IR (KBr) 3375, 1682, 1643, 1507, 1437, 1100 cm-1. MS
(ES+) calcd for C17H19N2O4S 347.10, found 347.01. Anal.
(C17H19N2O4S·0.5H2O) C, H, N.
121.3, 110.0, 17.4. IR (KBr) 2925, 1675, 1493, 1316, 759 cm-1
.
MS (ES-) calcd for C20H14N3O2S 360.09, found 359.87. Anal.
(C22H15N3O2S·2.17H2O) C, H, N.
N-(3,4-Dichlorobenzyl)-2-(3,5-diphenyl-1H-pyrazol-1-yl)-4-
methylthiazole-5-carboxamide (11). 3,4-Dichlorobenzylamine (25
mg, 0.13 mmol) was added to a solution of 2-(3,5-diphenyl-1H-
pyrazol-1-yl)-4-methylthiazole-5-carboxylic acid (10) (45 mg, 0.125
mmol) in anhydrous pyridine (3 mL). EDCI (20 mg, 0.13 mmol)
was added to the resulting solution. The reaction mixture was stirred
at room temperature overnight. After removal of solvent under
reduced pressure, the resulting residue was purified by silica gel
chromatography (ethyl acetate-hexanes ) 1:4) to provide the
desired compound as a white solid (47 mg, 70%): mp 144-146
2-(4-Chlorophenyl)-4-methylthiazole (21). 4-Chlorobenzothioa-
mide (43) (170 mg, 1 mmol) was added to a solution of
1-chloroacetone (44, 360 mg, 4 mmol) in absolute ethanol (5 mL).
The reaction mixture was heated to reflux for 2 h. After removal
of solvent under reduced pressure, the residue was purified by silica
gel chromatography (ethyl acetate-hexanes ) 1:4) to provide the
desired compound as a white solid (167 mg, 80%): mp 128-130
1
°C. H NMR (500 MHz, CDCl3): δ 7.90 (d, J ) 7.5 Hz, 2 H),
1
°C. H NMR (500 MHz, CDCl3): δ 8.30 (d, J ) 8.7 Hz, 2 H),
7.53 (d, J ) 7.5 Hz, 2 H), 7.43 (m, 8 H), 7.18 (dd, J ) 8.5, 2.0 Hz,
1 H), 6.79 (s, 1 H), 6.04 (dd, J ) 6.0, 6.0 Hz, 1 H), 4.55 (d, J )
7.53 (d, J ) 8.7 Hz, 2 H), 7.22 (s, 1 H), 2.79 (s, 3 H). 13C NMR
(125 MHz, CDCl3): δ 169.4, 148.4, 140.3, 130.1 × 2, 129.5 × 2,
6.0 Hz, 2 H), 2.48 (s, 3 H). IR (KBr) 2994, 1770, 1056, 744 cm-1
.
124.8, 115.2, 14.4. IR (KBr) 1598, 1093, 1003, 831, 750, 697 cm-1
.
MS (ES+) calcd for C27H21Cl2N4OS 519.07, found 518.95. HRMS
(ES+) calcd for C27H20Cl2N4OSNa 541.0633, found 541.0636.
Anal. (C27H20Cl2N4OS) C, H, N.
MS (ES+) calcd for C10H9ClNS 210.01, found 210.03. Anal. calcd
for C10H8ClNS·2H2O: C, 48.88; H, 4.92; N, 5.70. Found: C, 48.46;
H, 4.38; N, 5.25.
Methyl 2-(Bis(4-acetylbenzyl)amino)-4-methylthiazole-5-car-
boxylate (18). Sodium hydride (95%, 30 mg, 1 mmol) was added
to a solution of methyl 2-amino-4-methylthiazole-5-carboxylate (39)
(86 mg, 0.5 mmol) in anhydrous DMF (5 mL) at 0 °C under argon
gas. The reaction mixture was stirred for 1 h until it became a clear
solution. 1-(4-(Bromomethyl)phenyl)ethanone (41) (211 mg, 1
mmol) was added to this solution and the resulting mixture was
stirred at 0 °C for 1 h and at room temperature for additional 3 h.
Ice water (10 mL) was added to the reaction mixture. The resulting
solution was extracted with ethyl acetate (10 mL × 3). The organic
layer was concentrated under reduced pressure. The residue was
purified by silica gel chromatography (ethyl acetate-hexanes )
1:4) to provide the desired compound as a white solid (260 mg,
4-(Chloromethyl)-2-(4-chlorophenyl)thiazole (22). 4-Chlo-
robenzothioamide (350 mg, 2.0 mmol) was added to a solution of
1,3-dichloroacetone (45) (500 mg, 4.0 mmol) in absolute ethanol
(10 mL). The reaction mixture was heated to reflux for 2 h. After
removal of solvent under reduced pressure, the residue was
recrystallized from hexanes to provide the desired compound as a
1
white solid (315 mg, 65%): mp 74-76 °C. H NMR (500 MHz,
CDCl3): δ 7.88 (d, J ) 8.5 Hz, 2 H), 7.42 (d, J ) 8.5 Hz, 2 H),
7.31 (s, 1 H), 4.74 (s, 2 H). 13C NMR (125 MHz, CDCl3): δ 167.8,
153.3, 136.3, 131.6, 129.1 × 2, 127.7 × 2, 117.4, 40.8 (d). IR
(KBr) 3374, 3107, 2917, 1656, 1497, 1453, 1398, 1262, 1244, 1104,
1091, 1038, 838, 722, 652 cm-1. MS (ES+) calcd for C10H8Cl2NS
243.97, found 243.89. Anal. (C10H7Cl2NS·1/3H2O) C, H, N.
2-((2-(4-Chlorophenyl)thiazol-4-yl)methylthio)aniline (D03).
CsCO3 (160 mg, 0.50 mmol) was added to a solution of 2-ami-
nobenzenethiol (40 mg, 0.33 mmol) in anhydrous DMF (5 mL) at
0 °C under argon. Argon gas was bubbled through the reaction
mixture for 30 min. Then the reaction mixture was allowed to warm
to room temperature. 4-(Chloromethyl)-2-(4-chlorophenyl)thiazole
(22) (80 mg, 0.33 mmol) was added to the reaction mixture, and
the resulting mixture was stirred at room temperature for 0.5 h.
The reaction mixture was heated to reflux for additional 2 h and
allowed to cool to room temperature. After removal of solvent under
reduced pressure, the resulting residue was purified by silica gel
chromatography (ethyl acetate-hexanes ) 1:9) to provide the
desired compound as a colorless oil (76 mg, 70%). 1H NMR (500
MHz, CDCl3): δ 7.86 (d, J ) 8.5 Hz, 2 H), 7.40 (d, J ) 8.5 Hz,
2 H), 7.25 (m, 1 H), 7.11 (ddd, J ) 7.5, 7.5, 0.5 Hz, 1 H), 6.75 (s,
1 H), 6.69 (dd, J ) 7.5, 0.5 Hz, 1 H), 6.65 (ddd, J ) 7.5, 7.5, 0.5
Hz, 1 H), 4.38 (br, s, 2 H), 4.06 (s, 2 H). 13C NMR (125 MHz,
CDCl3): δ 166.8, 154.3, 148.6, 136.7, 136.0, 131.7, 130.2, 129.0
× 2, 127.6 × 2, 118.2, 116.9, 115.7, 114.7, 34.8. IR (KBr) 2922,
1606, 1478, 1219, 1091, 831 cm-1. MS (ES+) calcd for
C16H14ClN2S2 333.02, found 332.95. Anal. (C16H13ClN2S2) C, H,
N.
1
74%): mp 162-164 °C. H NMR (500 MHz, CDCl3): δ 7.92 (d,
J ) 8.1 Hz, 4 H), 7.31 (d, J ) 8.1 Hz, 4 H), 4.73 (s, 4 H), 3.78 (s,
3 H), 2.60 (s, 9 H). 13C NMR (125 MHz, CDCl3): δ 197.4 × 2,
171.1, 162.8, 160.2, 140.7 × 2, 136.7 × 2, 128.9 × 4, 127.7 × 4,
109.9, 53.4 × 2, 51.4, 26.5 × 2, 17.5. IR (KBr) 2949, 1682, 1525,
1265, 1094 cm-1. MS (ES+) calcd for C24H25N2O4S 437.15, found
437.00. Anal. (C24H25N2O4S) C, H, N.
2-(Bis(4-acetylbenzyl)amino)-N-(2,4-dichlorobenzyl)-4-meth-
ylthiazole-5-carboxamide (19). 2,4-Dichlorobenzylamine (86 mg,
0.5 mmol) was added to a solution of 2-(bis(4-acetylbenzyl)amino)-
4-methylthiazole-5-carboxylic acid (42) (210 mg, 0.5 mmol) in
anhydrous pyridine (10 mL). EDCI (100 mg, 0.6 mmol) was added
to the resulting solution. The reaction mixture was stirred at room
temperature overnight. After removal of solvent under reduced
pressure, the resulting residue was purified by silica gel chroma-
tography (ethyl acetate-hexanes ) 1:4) to provide the desired
compound as a colorless oil (210 mg, 72%). 1H NMR (300 MHz,
CDCl3): δ 7.90 (d, J ) 8.1 Hz, 4 H), 7.37 (d, J ) 8.5 Hz, 1 H),
7.36 (s, 1 H), 7.30 (d, J ) 8.1 Hz, 4 H), 7.20 (m, 1 H), 6.01 (dd,
J ) 6.3, 6.3 Hz, 1 H), 4.69 (s, 4 H), 4.57 (d, J ) 6.3 Hz, 2 H),
2.59 (s, 6 H), 2.54 (s, 3 H). 13C NMR (75 MHz, CDCl3): δ 197.4
× 2, 169.2, 162.8, 160.0, 154.0, 140.9 × 2, 136.5 × 2, 131.2, 129.2,
128.5, 127.4, 128.8 × 4, 127.6 × 4, 127.3, 10.9, 53.3 × 2, 41.2,
26.5 × 2, 17.8. IR (KBr) 3375, 1682, 1521, 1267 cm-1. MS (ES+)
calcd for C30H28Cl2N3O3S 580.12, found 580.07. Anal.
(C30H27Cl2N3O3S) C, H, N.
Methyl 2-(N-(4-Acetylbenzyl)acetamido)-4-methylthiazole-5-
carboxylate (20). Sodium hydride (500 mg, 22 mmol) was added
to a solution of methyl 2-amino-4-methylthiazole-5-carboxylate (39)
(1.7 g, 10 mmol) in DMF (20 mL). The reaction mixture was stirred
at 0 °C for 30 min. A solution of 1-(4-(bromomethyl)phenyl)etha-
none (41) (4.2 g, 20 mmol) in ethyl acetate (5 mL) was slowly
added to the reaction mixture. The reaction mixture was stirred at
room temperature for 4 h. After removal of solvent under reduced
2-(4-Chlorophenyl)-4-methyl-thiazole-5-carboxylic Acid Meth-
yl Ester (32). 4-Chloro-thiobenzamide (51 mg, 0.3 mmol) and
2-chloro-3-oxo-butyric acid methyl ester (46, 45 mg, 0.3 mmol)
were added to absolute ethanol (5 mL). The reaction mixture was
heated to reflux for 2 h. After removal of solvent under reduced
pressure, the residue was purified by silica gel chromatography
(ethyl acetate-hexanes ) 1:9) to provide the desired compound
as a white solid (60 g, 75%): mp 130-132 °C. 1H NMR (500 MHz,
CDCl3): δ 7.89 (d, J ) 8.5 Hz, 2 H), 7.41 (d, J ) 8.5 Hz, 2 H),
3.89 (s, 3 H), 2.77 (s, 3 H). 13C NMR (125 MHz, CDCl3): δ 168.4,
162.4, 161.2, 136.9, 131.2, 129.1 × 2, 127.9 × 2, 121.5, 52.1, 17.4.

AntiViral Agents Targeting FlaViVirus EnVelope Proteins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4669
IR (KBr) 1717, 1519, 1435, 1398, 1328, 1277, 1090, 830, 758
cm-1. MS (ES+) calcd for C12H11ClNO2S 268.01, found 267.92.
Anal. (C12H10ClNO2S) C, H, N.
reduced pressure, water (5 mL) was added to the residue. The pH
was adjusted to 2-3 with hydrochloric acid. The generated solid
was filtered and washed with cold hexanes (1 mL × 3) to provide
the desired compound as an off-white solid (67 mg, 70%): mp
75-77 °C. H NMR (300 MHz, DMSO-d6): δ 7.92 (d, J ) 8.0
Hz, 4 H), 7.30 (d, J ) 8.0 Hz, 4 H), 4.72 (br, s, 4 H), 2.60 (s, 9 H);
MS (ES+) calcd for C23H22N2O4S 422.13, found 422.86.
2-(4-Chloro-phenyl)-4-methyl-thiazole-5-carboxylic Acid Ad-
amantan-1-ylmethyl Ester (35). 2-(4-Chloro-phenyl)-4-methyl-
thiazole-5-carboxylic acid methyl ester (32, 80 mg, 0.3 mmol) and
LiOH (45 mg, 1.8 mmol) were added to ethanol-methanol-H2O
(3 mL, 1:1:1). The reaction mixture was heated at reflux for 1 h
and allowed to cool to room temperature. The reaction mixture was
filtered. After removal of solvent under reduced pressure, water (5
mL) was added. After adjusting pH to 3-4 with HCl, the solid
was filtered and washed with water and a small amount of ethanol.
The solid was dried to provide 2-(4-chloro-phenyl)-4-methyl-
thiazole-5-carboxylic acid as a white solid (47, 60 mg, 80%) that
was used directly in the next step without further purification. 2-(4-
Chlorophenyl)-4-methyl-thiazole-5-carboxylic acid (47, 60 mg, 0.25
mmol) and EDCI (50 mg, 0.25 mmol) and adamantan-1-yl-methanol
(40 mg, 0.25 mmol) were added to pyridine (5 mL). The reaction
mixture was stirred at room temperature for 12 h. After removal
of solvent under reduced pressure, water (5 mL) was added. The
obtained solution was extracted by ethyl acetate (2 mL × 3). The
organic layer was washed by brine (1 mL × 3). After removal of
solvent under reduced pressure, the residue was purified by silica
gel chromatography (ethyl acetate-hexanes ) 1:9) to provide the
desired compound as a colorless oil (60 mg, 60%). 1H NMR (300
MHz, CDCl3): δ 7.93 (d, J ) 8.7 Hz, 2 H), 7.43 (d, J ) 8.7 Hz,
2 H), 3.89 (s, 2 H), 2.79 (s, 3 H), 2.02 (s, 3 H), 1.70 (m, 12 H).
13C NMR (75 MHz, CDCl3): δ 168.4, 162.2, 160.8, 136.9, 131.3,
129.2 × 2, 127.9 × 2, 122.2, 74.7, 39.2, 36.8, 33.2, 29.6, 27.9,
1
2-Chloro-3-oxo-butyric Acid Methyl Ester (46). Sufuryl
chloride (18 g, 110 mmol) and a catalytic amount of AIBN were
added to a solution of methyl 3-oxobutanoate (12 g, 100 mmol) in
anhydrous benzene (50 mL). The reaction mixture was stirred at
room temperature for 2 h. After removal of solvent under reduced
pressure, the residue was purified by silica gel chromatography
(ethyl acetate-hexanes ) 2:3) to provide the desired compound
1
as a colorless oil (20 g, 100%). H NMR (300 MHz, CDCl3): δ
4.73 (s, 1 H), 3.37 (s, 3 H), 2.38 (s, 3 H).
Molecular Modeling. Compounds 1-36 were built within Sybyl
7.1 and minimized to 0.01 kcal/mol by the Powell method, using
Gasteiger-Huckel charges and the Tripos force field. To save
calculation time, solvent was not explicitly included and instead
the dielectric constant was set to a value of 4. The minimized
molecules underwent 10 rounds of simulated annealing to further
relax the conformations of these manually drawn molecules.
Conformations resulting from each simulation were selected and
the energies were minimized using the same parameter set as the
one used during molecule construction. The conformer with the
lowest energy was used as the initial structure for each molecule
to be docked into the ꢀ-OG binding pocket of the dengue virus E
protein.
Docking Simulation. The energy-optimized compounds were
docked into the ꢀ-OG binding domain in E protein of the dengue
virus that was obtained by removal of the n-octyl-ꢀ-D-glucoside
(BOG). The parameters were set as the default value that the GOLD
software suggested.
Bioassay Methods. BHK cells. BHK-15 cells (baby hamster
kidney cells) obtained from the American Type Culture Collection
(ATCC, Rockville, MD) were maintained in minimal essential
medium (MEM) (Invitrogen, Carlsbad, CA) containing 7.5% fetal
bovine serum (FBS). Cells were grown in incubators at 37 °C in
the presence of 5% CO2.
17.5, 0.9. IR (KBr) 2903, 2848, 1714, 1437, 1260, 1092 cm-1
.
MS (ES+) calcd for C22H25ClNO2S 402.12, found 402.02. Anal.
(C22H24ClNO2S) C, H, N.
2-(4-Chloro-phenyl)-4-dibromomethyl-thiazole-5-carboxylic
Acid Methyl Ester (36). 2-(4-Chlorophenyl)-4-methyl-thiazole-5-
carboxylic acid methyl ester (32, 133 mg, 0.5 mmol) and NBS (531
mg, 3 mmol) were added to CCl4 (10 mL). The reaction mixture
was heated at reflux for 12 h. After removal of solvent under
reduced pressure, the residue was purified by silica gel chroma-
tography (ethyl acetate-hexanes ) 1:4) to provide the desired
1
compound as a white solid (127 mg, 60%): mp 154-156 °C. H
NMR (300 MHz, CDCl3)L δ 7.96 (d, J ) 8.5 Hz, 2 H), 7.69 (s, 1
H), 7.44 (d, J ) 8.5 Hz, 2 H), 3.94 (s, 3 H). 13C NMR (75 MHz,
CDCl3): δ 170.0, 160.8, 158.8, 137.8, 130.5, 129.3 × 2, 128.2 ×
2, 120.0, 52.9, 31.0. IR (KBr) 3044, 1714, 1514, 1447, 1287, 1091,
833, 719, 623 cm-1. Anal. (C12H8Br2ClNO2S) C, H, N.
YFV-IRES-Luc. A fire-fly luciferase reporter gene was inserted
into pYF23, a derivative of pACNR, which is the full-length cDNA
clone of YFV 17D, to construct YFV-IRES-Luc, a luciferase-
reporting full-length virus. To facilitate this construction, an NsiI
restriction site was introduced at the beginning of the 3′ NTR
immediately following the UGA termination codon of NS5 in
pYF23 using standard overlapping PCR mutagenesis. To construct
YFV-IRES-Luc, an IRES-FF-Luc (EMCV IRES-fire fly luciferase)
cassette was amplified by PCR from YFRP-IRES-Luc, a YFV
replicon, and inserted into the NsiI restriction site.
Generation of YFV-IRES-Luc Virus. In vitro transcribed YFV-
IRES-Luc RNA was transfected into BHK-15 cells via electropo-
ration. Subconfluent monolayers of cells were grown in T-75 culture
flasks and were harvested by trypsinization, then subsequently
washed twice with phosphate-buffered saline (PBS) before being
resuspended in 400 µL PBS. These cells were then combined with
40 µL of in vitro transcribed RNA, and then placed in a 2-mm gap
cuvette (BioRad, Hercules, CA). The cells were subjected to
electroporation by being pulsed two times at settings of 1.5 kV, 25
µF, and 200 Ω using a GenePulser II apparatus (BioRad). After
the two pulses, the cells were given a 5 min recovery time at room
temperature. Following recovery, cells were resuspended in MEM
supplemented with 10% FBS. At 4 days post-transfection, the
resulting YFV-IRES-Luc virus was harvested and the titer of the
virus determined by a standard plaque assay. The infectivity of
the virus could be assayed directly as a measure of the luciferase
amounts produced in infected cells over a period of time.
Inhibition of YFV-IRES-Luc Virus Growth. BHK cells were
plated in a 96-well plate and grown at 37 °C. At confluency, cells
were infected with YF-IRES-Luc virus32 at a multiplicity of
infection (MOI) of 0.1. A low MOI was utilized to ensure that
Methyl 2-Bromo-3-oxobutanoate (38). NBS (18 g, 110 mmol)
and a catalytic amount of AIBN were added to a solution of methyl
3-oxobutanoate (37) (12 g, 100 mmol) in anhydrous benzene (50
mL). The reaction mixture was stirred at room temperature for 2 h.
After removal of solvent under reduced pressure, the residue was
purified by silica gel chromatography (ethyl acetate-hexanes )
2:3) to provide the desired compound as a colorless oil (20 g,
1
100%). H NMR (300 MHz, CDCl3): δ 4.73 (s, 1 H), 3.37 (s, 3
H), 2.38 (s, 3 H).
Methyl 2-Amino-4-methylthiazole-5-carboxylate (39). Thio-
urea (4 g, 50 mmol) was added to a solution of methyl 2-bromo-
3-oxobutanoate (38, 10 g, 50 mmol) in ethanol (100 mL). The
reaction mixture was stirred at room temperature for 3 h. After
removal of the solvent under reduced pressure, the crude product
was recrystallized from fresh ethyl acetate (30 mL) and methanol
(15 mL). The solid was filtered and washed with diethyl ether to
provide the desired compound as a white solid (7 g, 81%): mp
1
132-134 °C. H NMR (300 MHz, DMSO-d6): δ 3.57 (s, 3 H),
2.26 (s, 3 H); MS (ES+) calcd for C6H9N2O2S 173.03, found
172.95.
2-(Bis(4-acetylbenzyl)amino)-4-methylthiazole-5-carboxylic Acid
(42). Sodium hydroxide (200 mg, 5 mmol) was added to a solution
of methyl 2-(N-(4-acetylbenzyl)acetamido)-4-methylthiazole-5-car-
boxylate (18, 100 mg, 0.23 mmol) in methanol (5 mL) and ethanol
(5 mL). The reaction mixture was heated to reflux for 1 h and
allowed to cool to room temperature. After removal of solvent under

4670 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Li et al.
fewer cells were infected so that the spread of released virus could
be monitored. Cells were then overlaid with culture media contain-
ing serial dilutions of compounds at concentrations below the CC50
values. Controls included uninfected cells, infected cells, and
DMSO-treated infected cells. Cells were incubated at 37 °C, 5%
CO2 for ∼36 h, lysed using 50 µL of cell culture lysis buffer
(Promega Inc., Madison, WI), and 20 µL of cell extracts were placed
into a 96-well opaque plate. Luciferase activity was determined
from the luminescence generated with fire-fly luciferase substrate
(Promega Inc., Madison, WI). Luminescence was measured in a
96-well plate luminometer, LMax II (Molecular Devices, Sunnyvale,
CA). A reduction in luciferase activity indicates inhibition of YFV-
IRES-Luc virus growth. The luciferase luminescence as a function
of compound concentration was analyzed by nonlinear regression
analysis using GraphPad Prizm to estimate the EC50 of each
compound. The EC50 was defined as the concentration of the
compound to cause 50% reduction of luciferase activity in infected
cells as compared to the DMSO treated cells.
Dengue Immunofluorescence Assay. In brief, Den-2 (strain
16681) was added to monolayers of BHK cells in a 24-well plate
at an moi of 0.025 for 1 h at room temperature. Following infection,
the excess/unbound virus was removed by washing with PBS. Cells
were then overlaid with MEM + 2.5% FBS containing the
compound at 50 µM concentration. The cells were then incubated
at 34 °C for 48 h before performing immunofluorescence assay.
Cell monolayers were fixed and incubated with the antidengue
capsid antibody for 2 h, followed by incubation with the secondary
antibody conjugated with TRITC. Three randomly selected fields
per well were captured in both the TRITC (tetramethylrhodamine-5
(and 6)-isothiocyanate) and DAPI (4′,6-diamidino-2-phenylindole
dihydrochloride) channels. All infected cells were compared to total
cells in all three fields and percent inhibition was determined.
Cell Viability Assay. Cellular viability was measured spectro-
photometrically by monitoring the reduction of a tetrazolium salt
to a formazan dye by mitochondrial dehydrogenase in living cells.
BHK cells were plated in a 96-well plate and grown at 37 °C. At
confluency, cells were overlaid with culture media containing serial
dilutions of compounds (compound stocks were generated by
dissolving compounds in DMSO). Untreated and DMSO treated
cells served as positive controls. Cells were then incubated at 37
°C, 5% CO2 for ∼36 h. At ∼36 h post-treatment, media on cells
was replaced with fresh media to remove the compounds. Then
100 µL of XTT-substrate from the Quick Cell Proliferation Kit
(Biovision Inc., CA) was added to each well. Cells were incubated
at 37 °C for a further 2 h. Plates were then removed and OD450
measured using a 96-well plate reader (Molecular Devices, Sunny-
vale, CA). The OD450 values for cells treated with a compound
were compared to those obtained for cells treated with the control
(1% DMSO) to determine the CC50 of each compound. The 1%
DMSO in the control was necessary to solubilize some of the
compounds, and it did not affect cell viability.
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Acknowledgment. This work was sponsored by the NIH/
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Emerging Infectious Diseases Research (RCE) Program. We
wish to acknowledge membership within and support from the
Region V “Great Lakes” RCE (NIH award 1-U54-AI-057153).
This research was conducted in a facility constructed with
support from Research Facilities Improvement Program grant
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