Design, synthesis, and biological evaluation of thiazoles targeting flavivirus envelope proteins

Article abstract of DOI:10.1021/jm1013538

A series of third-generation analogues of methyl 4-(dibromomethyl)-2-(4- chlorophenyl)thiazole-5-carboxylate (1), which had the most potent antiviral activity among the first- and second-generation compounds, have been synthesized and tested against yellow fever virus using a cell-based assay. The compounds were designed with the objectives of improving metabolic stability, therapeutic index, and antiviral potency. The biological effects of C4 and C5 substitution were examined. The methylthio ester and the dihydroxypropylamide analogues had the best antiviral potencies and improved therapeutic indices and metabolic stabilities relative to the parent compound 1.

Full text of DOI:10.1021/jm1013538

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Thiazoles Targeting
Flavivirus Envelope Proteins
Abdelrahman S. Mayhoub,†,§ Mansoora Khaliq,‡ Richard J. Kuhn,‡ and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and the Purdue Center for Cancer Research,
Purdue University, West Lafayette, Indiana 47907, United States
‡Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, United States
ABSTRACT: A series of third-generation analogues of methyl
4-(dibromomethyl)-2-(4-chlorophenyl)thiazole-5-carboxylate
(1), which had the most potent antiviral activity among the first-
and second-generation compounds, have been synthesized and
tested against yellow fever virus using a cell-based assay. The
compounds were designed with the objectives of improving
metabolic stability, therapeutic index, and antiviral potency.
The biological effects of C4 and C5 substitution were examined.
The methylthio ester and the dihydroxypropylamide analogues
had the best antiviral potencies and improved therapeutic indices and metabolic stabilities relative to the parent compound 1.
’ INTRODUCTION
Among the 39 molecules previously synthesized and tested
for antiviral activity against yellow fever virus, compound 1
(Figure 1b) displayed the most potent antiviral activity.13 This
compound also exhibited potent inhibitory activity against
dengue virus. These results have encouraged further structural
optimization studies to search for more potent antiviral agents
based on the phenylthiazole scaffold.14
The structural details of the binding of the lead compound 1 to
its receptor are not known with certainty. It adopted two distinct
docking poses having a very small calculated energy difference
(Figure 2). A focused library of phenylthiazole structural analo-
gues has therefore been synthesized so that SARs can be
rigorously defined in order to enable the rational design of more
effective antiviral agents. This article will focus on examination of
the effects of C4 and C5 substitution (Figure 1c) on the antiviral
activity.
Flavivirus is a genus of the positive-sense single-stranded RNA
family Flaviviridae, which includes many clinically important
species such as dengue, Japanese encephalitis, and West Nile
viruses. More than 50 million cases of dengue viral infections are
reported per year in more than 80 countries in which the
mosquito Aedes aegypti is endemic.1 Of these cases, approxi-
mately 500 000 patients suffer the more severe and often lethal
illnesses known as dengue hemorrhagic fever and dengue shock
syndrome.1 These diseases remain a significant problem in
Africa, Europe, the Middle East, and West and Central Asia,
where the infection is endemic, as well as in North America.2
Although there are limited licensed vaccines against some
flaviviruses such as yellow fever and Japanese encephalitis, there
are no vaccines for other types such as dengue viruses nor
effective therapy for treatment of the clinical cases.3
In order to probe steric and electronic effects, the dibro-
momethyl and methyl ester moieties were replaced by a
variety of substituents. Since the methyl ester moiety is
expected to be metabolically labile to nonspecific esterases
in blood plasma and the corresponding free acid analogue has
already been established to be an inactive compound, C5
structural optimization included replacement of the methyl
ester by bioisosteres that are more metabolically stable but
still retain the antiviral potency. Fortunately, ester instability
is a common problem in drug development that has been
successfully dealt with multiple times before.15 In the present
case, the methyl ester is replaced by other more metabolically
stable bioisosters such as amides, thioesters, ketones, and
more bulky esters.
There are many flaviviral proteins that could be considered as
targets for drug discovery such as helicase,4,5 methyl trans-
ferase,6,7 and serine protease.8,9 In addition, the viral RNA is
also reported to be a target for some antimicrobial agents.10 The
flaviviral E-protein plays a crucial role at the first step in viral
infection, since it contains a receptor-binding site and also plays a
role in fusion. It undergoes substantial conformational and
translational changes through the virus replication cycle, thereby
causing the native homodimer to change into a fusogenic
homotrimer.11 Moreover, crystallization of a dengue virus type
2 E protein (Figure 1a) in the presence and the absence of n-
octyl-β-D-glucoside (β-OG)12 paved the road for structure-based
design of antiviral agents that could occupy the β-OG pocket.
Since the β-OG-containing crystal structure revealed conforma-
tional changes relative to the unoccupied protein, it is believed
that the β-OG pocket is an ideal target for designing new
antiflaviviral agents.
Received: October 18, 2010
Published: February 28, 2011
r
2011 American Chemical Society
1704
dx.doi.org/10.1021/jm1013538 J. Med. Chem. 2011, 54, 1704–1714
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. (a) Dengue viral 2 E protein: domain I, red; domain II, yellow; domain III, blue. The β-OG binding pocket is located between domains
I and II.13 (b) Structure of lead compound 1. (c) Targeted lead modifications. R1, R2, and R3 stand for bulky groups or aromatic systems, and R4
represents ester bioisosteres.
was prepared by utilizing thioamide 18 and the appropriate
diketo derivative 17 (Scheme 2). The only reaction that was
found to brominate the desired methyl group of 19 efficiently
without detectable bromoketone was one involving exactly
2 equiv NBS in CCl4 with UV irradiation. Using other
chemical-free radical initiators afforded mixtures of different
brominated compounds. The bromination was assigned to be
on the Ar-methyl carbon and not on the ketone-methyl car-
bon, based on the spectral data. For instance, the Ar-methyl
carbon signal of 19 at 18.34 ppm was not present in the 13C
NMR spectrum of the product 20, which revealed only two
signals corresponding to dibromomethyl and acylmethyl
groups at 32.00 and 31.28 ppm in the aliphatic region. In
addition, the mass spectrum showed a base peak at m/z 395
Figure 2. Hypothetical models of the lead compound 1 in the dengue
viral 2 E protein β-OG binding pocket. Two different poses opposite
corresponding to the acylium cation (Mþ - CH3).
The amide derivatives with hydroxyalkyl or carbohydrate
moieties were prepared by treatment of the acid chloride 4 with
the appropriate amines in DMF (Scheme 3). To synthesize the
desired thiazole-C4 derivatives, chlorination of the commercially
available dicarbonyl compounds 26a-c was performed using
sulfuryl chloride to afford the corresponding R-chloro derivatives
27a-d in high yields (Scheme 4). The 1H NMR spectra of these
compounds exhibited singlets at approximately δ 5 ppm due to
the methine proton. Treatment of the R-chloro derivatives with
4-chlorobenzothioamide (18) in ethanol afforded, in each case,
the corresponding 2-p-chlorophenylthiazole derivatives 28-31
(Scheme 4).
To prepare phenylthiazoles with hydrogen bond acceptor
moieties at position 4, two reactions were adopted. The first
one included treatment of the ether 33 with 1 equiv of NBS
under free radical conditions to afford the corresponding
aldehyde 34 in reasonable yield as reported by Markees
(Scheme 5).16 To prepare the methyl esters 40, 43, and 45
(Chart 1), a modified version of the Markees procedure was used,
as previously reported.17
The aldehyde derivative 35 was treated with acetyl bromide
and a catalytic amount of AlCl3 to afford the desired dichlor-
omethyl derivative 37, which could not be obtained by treatment
of 2 with NCS (Scheme 6). The nitrile derivative 36 was ob-
tained from the reaction of the corresponding aldehyde 35 and
ammonia solution. Oxidation of the imine intermediate in situ by
elemental iodine provided the required nitrile in 99% yield
(Scheme 6). The sulfone derivative 39 was obtained from the
corresponding monobromo derivative 38 (Scheme 7).
each other are shown. Only protein residues within a 6 Å radius around
the ligand are represented, and waters of crystallization were removed
for the sake of clarity. Compound 1 parallel binding poses to β-OG is
represented as red stick. Antiparallel is represented as blue stick. β-OG
is represented as green stick, and the protein residues are represented as
cyan blue lines (PDB code 1OKE).
’ RESULTS AND DISCUSSION
Chemistry. The acid chloride 4 served as a key intermediate
for the replacement of the metabolically labile ester with more
stable bioisosteres. As shown in Scheme 1, two pathways were
used to synthesize it. Hydrolysis of ester 1 gave a very low yield of
the corresponding free acid because, under the reaction condi-
tions, the carboxylate salt formed in situ underwent SN2 reaction
with the adjacent alkyl bromide, forming a cyclic lactone
byproduct. On the other hand, bromination of the acid chloride
3, utilizing NBS and UV light as a free radical initiator, gave the
desired dibromo acid chloride derivative 4 in a good yield with no
detectable mono or tribromo byproduct. The 1H NMR spectrum
of compound 4 revealed three signals, one singlet and two
doublets, at δ 7.30, 7.49, and 8.01 ppm, corresponding to the
methine and 1,4-disubstituted phenyl moieties, respectively.
To synthesize the desired amide derivatives 5-12, the acid
chloride 4 was allowed to react with primary or secondary amines
in dichloromethane or with ammonia solution at room tempera-
ture for 5-10 min (Scheme 1). The esters were prepared by
dissolving the acid chloride 4 in the required alcohols
(Scheme 1). Similarly, treatment of acid chloride 4 with alka-
nethiols or their sodium salts afforded the corresponding thioe-
sters as depicted in Scheme 1.
Biological Results. All of the thiazole derivatives have been
evaluated in a yellow fever virus luciferase cellular assay.
Initially, optimization of R4 at thiazole-C5 (Figure 1c) was
In order to synthesize a derivative 20 containing dibromo-
methyl and methylketo functionalities, the methyl ketone 19
1705
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714

Journal of Medicinal Chemistry
ARTICLE
Scheme 1a
a Reagents and conditions: (a, a0) NBS, UV irradiation, heat to reflux for 24 h, CCl4, 87% for step a and 63% for step a0; (b, b0) (i) 80% methanol, NaOH,
heat to reflux for 2 h, (ii) SOCl2, heat to reflux for 2 h, 95% for step b and 53% for step b0. (c) CH2Cl2, alkylamine, 23 °C, 2-10 min, 10-86%, or NH3
solution, 40 °C, 30 min, 84%; (d) absolute ethanol or 2-propanol, Et3N, heat to reflux for 6 h, 49-81%; (e) ethanethiol heat to reflux for 4 h, or MeONa/
CH2Cl2, 23 °C, 0.5 h, 73-91%.
performed in order to increase both the metabolic stability of
the lead compound and its potency. Therefore, the ester
group was replaced by more metabolically stable groups such
as amides, thioesters, or ketones. Among the newly synthe-
sized amides (Scheme 1), the unsubstituted amide 6 and the
secondary methylamide derivative 8 revealed slightly better
EC50 values than the lead compound 1 (Table 1). Increasing
the alkyl chain length, from methyl to ethyl, retained the
potency, but the toxicity was increased (Table 1, compound
11). On the other hand, increasing the side chain polarity by
replacement of the ethyl group in compound 11 by a methoxy
group improved the antiviral activity (compound 5). How-
ever, tertiary amides, such as 7 and 9, showed 5- to 10-fold
lower activity. Replacement of the methyl group of 8 with an
allyl group (compound 10) did not improve the potency.
Further increasing the unsaturation led to an increase in the
toxicity (compound 12).
of the amide and increasing its hydrophobicity and steric bulk
decrease the antiviral activity. In order to explore this trend
further, the ethyl ester 13 (EC50 = 8.7 μM) and the isopropyl
ester 14 (EC50 = 19.9 μM) were synthesized and compared
with the methyl ester 1 (EC50 = 2.8 μM). The results obtained
with the esters also document a correlation of decreased
antiviral activity with an increase in hydrophobicity. This
would be consistent with a hydrophobically and sterically
unfavorable region of the envelope protein surrounding the
C-5 substituent of the ligand.
With this information in hand, the metabolically unstable
methyl ester moiety was further replaced by a methyl ketone,
as well as methyl and ethyl thioester analogues (structures 20,
16, and 15). Bulkier derivatives were excluded because they
were expected to have lower biological activities. Both the
ketone 20 (EC50 = 1.3 μM) and the ethyl thioester 15 (EC50
=
1.6 μM) derivatives displayed approximately double the
antiviral potency compared with the lead compound 1
(EC50 = 2.8 μM). More significantly, the methyl thioester
16 (EC50 = 1.4 μM, GI50 368.7 μM) had higher antiviral
potency and was also less cytotoxic in uninfected cells than 1
(GI50 = 222.1 μM), resulting in a therapeutic index (TI) for
Comparison of the EC50 values for the secondary amide 8
(EC50 = 1.8 μM) vs the corresponding tertiary amide 7 (EC50
= 20 μM), as well as the secondary amide 11 (EC50 = 2.7 μM)
vs the corresponding tertiary amide 9 (EC50 = 13.7 μM),
suggests that abrogating the hydrogen-bond-donating ability
1706
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714

Journal of Medicinal Chemistry
ARTICLE
Scheme 2a
Scheme 4a
a Reagents and conditions: (a) absolute ethanol, heat to reflux for 24 h,
73%; (b) NBS, UV irradiation, heat to reflux for 12 h, CCl4, 50%; (c)
DMF-DMA, dry toluene, heat to reflux to 24 h, 66%.
a Reagents and conditions: (a) CH2Cl2, SO2Cl2, 23 °C, 2 h, 90-96%;
(b) absolute ethanol, heat to reflux for 24 h, 63-96%.
Scheme 3a
Scheme 5a
a Reagents and conditions: (a) (i) 80% methanol, NaOH, heat to reflux
for 2 h, 84%, (ii) SOCl2, heat to reflux for 2 h, 96%, (iii) MeNH2 HCl,
3
K2CO3, CH2Cl2, 23 °C, 1 h, 91%; (b) NBS (1 equiv), UV irradiation,
heat to reflux for 2 h, CCl4, 56%.
In a summary, simple amide, ketone, and thioester derivative
exhibited similar to better antiviral activity relative to the lead
compound 1 with higher metabolic stability in rat blood plasma.
Since the N- and O-hydrophobic substitutions decreased the
antiviral activity, it may be hypothesized that this part either lies
in a hydrophilic region within the binding pocket or faces the
solvent-accessible surface. Therefore, the next structural optimi-
zation efforts included adding hydrophilic moieties to the alkyl
side chain. Among the ester isosteres that showed significant
antiviral activity, the amide was chosen because of the commer-
cial availability of hydroxy- and sugar-containing primary amines.
To systematically examine this hypothesis, amides with a variable
number of hydroxy groups were synthesized (Scheme 3). Com-
pound 23, which contains two hydroxyl groups, revealed an EC50
value 3-fold better than the lead compound. Removal of the
terminal hydroxylmethyl group afforded compound 22, which
displayed a higher EC50 value. Compounds 24 and 25 revealed
much lower biological activity than 23.
a Reagents and conditions: (a) DMF, 23 °C, 0.5-1 h, 12-86%.
16 of 263, which compares favorably with that of the lead
compound 1 (TI 79). The metabolic stability of a methyl
thioester in the rat plasma in some cases has been found to be
much higher than the corresponding ester.18 Compound 16
was subjected to hydrolytic stability analysis utilizing lyoph-
ilized rat plasma. The half-life of compound 16 was found to
be 24.725 h. This value is around 17 times higher than the lead
compound 1, which had a half-life of 1.415 h under the same
experimental conditions. Therefore, compound 16 provided
the desired increased metabolic stability, along with im-
proved antiviral potency, lower cytotoxicity, and an impr-
oved TI.
The higher activity of compound 23 could be rationalized
from the molecular modeling (Figure 3). The modeling was
1707
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714

Journal of Medicinal Chemistry
ARTICLE
Chart 1. Polar Dibromomethyl Replacement
Scheme 7a
a Reagents and conditions: (a) NBS (1.1 equiv), Bz2O2, CCl4, heat to
gentle reflux for 24 h, 50%; (b) MeSO2Na, absolute ethanol, heat to
reflux for 12 h, 83%.
Although the central hydroxyl group is not calculated to interact
directly with any residues in the binding pocket, it points out
toward the solvent-accessible surface so that it might be hydrated
and could also stabilize the molecule within the active site.
The second point of optimization of the lead compound 1 is at
thiazole-C4. Since the dibromomethyl derivative 1 revealed
potent antiviral activity and since its nonhalogenated and di-
chloromethyl analogues showed no or substantially reduced
antiviral properties, it was concluded that the dibromomethyl
moiety is an important region for the antiviral activity. Therefore,
a series of C4-optimized derivatives were designed to test the
steric and the electronic properties at the dibromomethyl posi-
tion. First, the dibromomethyl moiety was replaced by different
alkyl and aryl substituents (compounds 28-31). All of the
derivatives showed much lower activity than the lead compound.
Next, the dibromomethyl was replaced by more polar moieties
(Chart 1). High EC50 values were observed for all of the
compounds with the exception of compound 33 (Table 1,
compounds 32-36, 40-45).
Scheme 6a
Although both polar and nonpolar dibromomethyl replace-
ments failed to improve the antiviral activity of the lead com-
pound 1, one more attempt was made to further investigate the
possibility of increasing the antiviral activity of the lead com-
pound 1 by adding a hydrogen bond acceptor moiety at the
thiazole-C4 position. Starting from the molecular modeling
study of the lead compound 1 and its amide analogues, two
glutamine residues have been observed to be possible targets for
C4-thiazole structural modification. In the case of the lead
compound 1, the binding pose that is parallel to the β-OG
molecule indicated the possibility of hydrogen bond formation
between the dibromomethyl moiety and both Gln200 and
Gln271 residues (Figure 4). The same observation was found
in the case of amide analogue 24. Therefore, replacement of the
dibromomethyl by a sulfinyl (SO2) moiety was considered in
which the two oxygen atoms are proposed to make two distinct
hydrogen bonds with the glutamine residues. However, when
this hypothetical compound was docked in the β-OG site, it was
far away from the desired residues. In contrast, adding one CH2
unit between the sulfinyl moiety and thiazole ring (compound
39) was calculated to make the two oxygen atoms closer to the
desired residues. Surprisingly, compound 39 stimulated the viral
replication instead of inhibiting it (Table 1).
a Reagents and conditions: (a) anhydrous AlCl3, THF, NaN3, heat to
gentle reflux for 24 h, 21%; (b) I2, NH3 solution, THF, 23 °C, 1 h, 99%;
(c) AcCl (2 equiv), anhydrous AlCl3, dichlorobenzene, heat to reflux for
24 h, 99%.
performed using the published structure of the dengue virus
envelope protein, which is valid is this case because the sequence
alignment of the dengue virus protein vs the yellow fever virus
protein reveals that they have 45% identity and 61% similarity.19
Among flaviviruses, the envelope proteins are predicted to be
very similar in their overall fold and domain arrangement,
including the hinge region, which is highly conserved. The
terminal hydroxy group of the dihydroxypropyl moiety of 23 is
calculated to have a binding pose that is very close to that of the
sugar moiety of β-OG (Figure 3). It is calculated to form two
hydrogen bonds with Ser274 and Gln271, which might improve
the binding energy of 23 and consequently its inhibitory activity.
As all of the above-mentioned C4-optimization attempts failed
to replace the dibromomethyl with a more potent moiety, other
halogens were tried. The dichloromethyl analogue 37 had very
weak antiviral activity, but the monobromo derivative 38 showed
approximately 1-fold lower EC50 value than the lead compound.
1708
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714

Journal of Medicinal Chemistry
ARTICLE
Table 1. Antiviral Activities and Cytotoxicities of Com-
pounds vs Yellow Fever Virus
compd
% inhibitiona
GI50 b (μM)
EC50 c (μM)
1
99.6
93.6
99.8
93.2
99.7
99.8
99.9
99.9
99.7
98.1
84.4
99.7
99.6
99.9
99.8
99.8
99.9
53.2
43.4
-56.7
14.7
20.9
50.3
38.1
99.7
37.5
99.9
7.8
222.1 ( 51.4
81.4 ( 39.1
99.7 ( 37.0
162.9 ( 23.3
74.6 ( 5.1
63.5 ( 14.9
40.8 ( 0.9
33.3 ( 5.4
23.9 ( 3.9
94.6 ( 28.9
372.3 ( 22.3
81.4 ( 39.1
368.7 ( 7.5
83.5 ( 10.9
38.2 ( 9.8
40.8 ( 0.9
158.0 ( 20.4
NAd
2.8 ( 1.0
1.7 ( 0.6
1.7 ( 0.5
20.0 ( 2.5
1.8 ( 0.7
13.7 ( 0.6
1.6 ( 0.4
2.7 ( 0.8
1.7 ( 0.2
8.7 ( 2.9
19.9 ( 2.5
1.6 ( 0.1
1.4 ( 0.1
1.3 ( 0.5
11.4 ( 6.4
2.9 ( 0.5
1.1 ( 0.1
NAd
5
6
7
8
9
10
11
12
13
14
15
16
20
21
22
23
24
25
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
Figure 3. Hypothetical model of compound 23 in the dengue viral 2 E
protein β-OG binding pocket (PDB code 1OKE).
NAd
NAd
NAd
NAd
49.6 ( 3.6
NAd
58.7 ( 17.0
NAd
465 ( 40
387.9 ( 25.0
50.8 ( 4.2
NAd
311.0 ( 25.0
158.3 ( 157.6
2.9 ( 0.5
NAd
36.4 ( 2.1
NAd
42.8 ( 17.4
NAd
Figure 4. Hypothetical model of the lead compound 1 in the dengue
viral 2 E protein β-OG binding pocket.
99.9
99.9
-74.7
30.1
11.3
5.5
34.1 ( 1.4
74.6 ( 5.1
NAd
NAd
NAd
52.8 ( 18.1
1.6 ( 0.5
NAd
NAd
NAd
on the metabolically unstable methyl ester. The SARs at C5 were
defined to a great extent, and the SARs at C4 were investigated.
In addition, more metabolically stable methyl ester analogues
were successfully prepared that had no decrease in the antiviral
potency, which will increase the potential utility of this series of
compounds as antiflaviviral agents.
NAd
NAd
74.0
99.6
1.5
99.7 ( 37.0
162.9 ( 23.3
NAd
51.0 ( 18.0
26.5 ( 1.8
NAd
Developing selective antiflaviviral agents is still a challenging
field. Of the vast number of articles on the discovery and
development of antiviral agents, relatively few deal with dengue,
West Nile, or other flaviviruses. The current report of an
antiflaviviral agent having a therapeutic index of 256 with
enhanced metabolic stability is a significant achievement that
will hopefully stimulate further research in this field.
a Measured as a reduction in luciferase activity of BHK cells infected with
YF-IRES-Luc at 50 μM in comparison to the control. b The GI50 is the
concentration of the compound causing a 50% growth inhibition of
uninfected BHK cells. c The EC50 is the concentration of the compound
resulting in a 50% inhibition in virus production. d NA indicates that the
value was not determined.
Interestingly, the cytotoxicity of compound 38 was in the
acceptable range (GI50 > 40 μM). Although the monobromo
derivative 38 was the only C4-optimized structure that exceeds
the antiviral activity of the lead compound, it is expected to be
more vulnerable to attack by endogenous nucleophiles than the
dibromo analogue because of steric factors.
’ EXPERIMENTAL SECTION
All biologically tested compounds had purity of g95% as established
by HPLC. 1H NMR spectra were run at 300 MHz, and 13C spectra were
determined at 75.46 MHz in deuterated chloroform (CDCl3), dimethyl
sulfoxide (DMSO-d6), methanol (CD3OD), or acetone (CD3COCD3).
Chemical shifts are given in parts per million (ppm) on the δ scale.
Chemical shifts were calibrated relative to those of the solvents. Mass
spectra were recorded at 70 eV. All reactions were conducted under
argon or nitrogen atmosphere, unless otherwise specified. Compounds
2,13 3,20 27a,21 27b,22 22, 32, 35, and 40-4517 were prepared according
to the reported procedures.
’ CONCLUSION
The main aims of this study were to define the SARs at C4 and
C5 of the 4-chlorophenylthiazole scaffold as well as to improve
1709
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714

Journal of Medicinal Chemistry
ARTICLE
4-(Dibromomethyl)-5-chlorocarbonyl-2-(4-chlorophenyl)th-
iazole (4). Method A. 5-Chlorocarbonyl-(4-chlorophenyl)-4-methyl-
thiazole (3) (3.538 g, 13 mmol) and NBS (20.7 g, 78 mmol) were added
to CCl4 (75 mL). The reaction mixture was heated at reflux for 48 h and
irradiated with UV light (produced from a sun-lamp) 10 times (5 min
every 30 min). The yellowish-white precipitate was purified by silica gel
chromatography (ethyl acetate-hexanes 1:1) to provide the desired
compound as a white solid (3.55 g, 63.7%).
Method B. NaOH (80 mg, 2 mmol) was added to a solution of methyl
ester 1 (423 mg, 1 mmol) in methanol (20 mL) and water (5 mL). The
reaction mixture was heated at reflux for 6 h and then allowed to cool to
room temperature. The reaction mixture was filtered, and the pH of the
liquid phase was adjusted to 2 with hydrochloride acid. The solid was
filtered and dried and purified by silica gel chromatography (hexanes-
ethyl acetate-glacial acetic acid 50:49:1) to provide the carboxylic acid.
The free acid (411 mg, 1 mmol) was heated at reflux with thionyl
chloride (7 mL) for 2 h. The solvent was evaporated under pressure. The
pale yellow residue was collected and recrystallized from chloroform to
yield a white solid product (231.7 mg, 53%): mp 131-132 °C. 1H NMR
(CDCl3) δ 8.01 (d, J = 8.7 Hz, 2 H), 7.49 (d, J = 8.7 Hz, 2 H), 7.30
(s, 1 H); 13C NMR (CDCl3) δ 167.43, 155.31, 136.57, 131.21, 128.83,
127.67, 122.44; CIMS m/z (rel intensity) 432/430/428 (MHþ, 40/45/
41), 352/350/348 (Mþ - Br, 26/100/38); HRMS (CI), m/z 347.8652
(M - HBr)þ, calcd for C11H5BrCl2NOS 347.8647.
Preparation of Amide Derivatives 5 and 7-12. The acid
chloride 4 (430 mg, 1 mmol) was suspended in dry CH2Cl2 (5 mL)
under argon. Amine (2 mmol) was added, and the mixture was stirred at
room temperature for 2-10 min. Aqueous HCl (0.5 M, 15 mL) was
added, and the solution was extracted with ethyl acetate (30 mL). The
crude residue obtained after evaporation of solvent was purified by silica
gel flash chromatography using hexane-ethyl acetate (1:1) to afford the
desired product.
4-(Dibromomethyl)-2-(4-chlorophenyl)-N-methoxythia-
zole-5-carboxamide (5). Off-white solid (7.1 mg, 11%), mp 85 °C.
IR (film) 3584, 3047, 2960, 1715, 1593 cm-1; 1H NMR (CDCl3) δ 7.98
(d, J = 7.5 Hz, 2 H), 7.69 (s, 1 H), 7.45 (d, J = 8.7 Hz, 2 H), 3.94 (s, 3 H);
13C NMR (CDCl3) δ 170.09, 161.35, 158.90, 137.85, 130.60, 129.36,
128.27, 52.93, 31.01; MS m/z (rel intensity) 443/441/439 (MHþ, 51/
100/31), 394 (36); HRMS (ESI) m/z 438.8514 MHþ, calcd for
C12H10Br2ClN2O2S 438.8513; HPLC purity 98.36%.
(ESI), m/z 486.8860 MNaþ, calcd for C15H15Br2ClN2OSNa 486.8852;
HPLC purity 99.42%.
N-Allyl-4-(dibromomethyl)-2-(4-chlorophenyl)thiazole-5-
carboxamide (10). Colorless solid (8 mg, 18%), mp 135-137 °C. 1H
NMR (CDCl3) δ 7.95 (d, J = 8.4 Hz, 2 H), 7.73 (s, 1 H), 7.45 (d, J = 8.4
Hz, 2 H), 5.95 (m, 2 H), 5.32 (brs, 1 H), 5.26 (m, 1 H), 4.07 (d, J = 1.2
Hz, 2 H); 13C NMR (CDCl3) δ 166.63, 159.55, 157.03, 137.70, 132.89,
130.45, 129.36, 128.20, 123.21, 117.66, 42.67, 31.79; MS m/z (rel
intensity) 452/450/448 (Mþ, 70/100/26); HRMS (ESI), m/z
447.8652 Mþ, calcd for C14H11Br2ClN2OS 447.8647; HPLC purity
98.17%.
4-(Dibromomethyl)-2-(4-chlorophenyl)-N-ethylthiazole-
5-carboxamide (11). Colorless solid (24 mg, 48%), mp 140-141 °C.
1H NMR (CDCl3) δ 7.94 (d, J = 8.4 Hz, 2 H), 7.43 (d, J = 8.4 Hz, 2 H),
6.84 (s, 1 H), 3.48 (q, J = 7.2 Hz, 4 H), 1.24 (t, J = 7.2 Hz, 6 H); 13C
NMR (CDCl3) δ 166.72, 160.65, 153.39, 137.18, 130.66, 129.27,
128.06, 124.45, 43.20, 31.18, 15.81; APCIMS m/z (rel intensity) 441/
439/437 (MHþ, 42/100/48); HRMS (CI) m/z 436.8723 MHþ, calcd
for C13H12Br2ClN2OS 436.8720; HPLC purity 98.27%.
4-(Dibromomethyl)-2-(4-chlorophenyl)-N-(prop-2-ynyl)thi-
azole-5-carboxamide (12). White solid (9.6 mg, 20%), mp 177-
178 °C. 1H NMR (CDCl3) δ 7.94 (d, J = 8.7 Hz, 2 H), 7.70 (s, 1 H), 7.46
(d, J = 8.7 Hz, 2 H), 6.01 (brs, 1 H), 4.23 (s, 2 H), 2.3 (s, 1 H); 13C NMR
(CDCl3) δ 167.03, 159.38, 157.52, 137.83, 130.37, 129.39, 128.23,
122.37, 72.68, 31.60, 30.00, 29.59; MS m/z (rel intensity) 453/451/
499/477 (MHþ, 92/100/47/41.1); HRMS (ESI), m/z 446.8567 MHþ,
calcd for C14H10Br2ClN2OS 446.8569; HPLC purity 100%.
4-(Dibromomethyl)-2-(4-chlorophenyl)thiazole-5-carboxa-
mide (6). Excess saturated ammonium hydroxide solution (30 mL) was
added gradually to acid chloride 4 (85 mg, 2 mmol) with continuous
stirring at 40-45 °C. The suspension was heated at 90 °C for 30 min,
and the solid was filtered, washed with water several times, and purified
by crystallization from EtOH to afford the product as a white solid (78.3
mg, 84%): mp 219-220 °C. 1H NMR (DMSO) δ 8.00 (d, J = 8.4 Hz, 2
H), 7.80 (s, 1 H), 7.64 (d, J = 8.4 Hz, 2 H); 13C NMR (DMSO) δ 166.35,
158.20, 157.11, 137.24, 131.44, 130.63, 129.21, 125.00, 33.61; CIMS
m/z (rel intensity) 412/410/408 (MHþ, 13/100/38); HRMS (ESI)
m/z 430.8238 MNaþ, calcd for C11H7Br2ClN2OSNa 430.8227; HPLC
purity 95.51%.
Alkyl 4-(Dibromomethyl)-2-(4-chlorophenyl)thiazole-5-
carboxylates 13 and 14. The acid chloride 4 (30 mg, 0.07 mmol)
was heated under reflux with the alcohol (2 mL) for 6 h. The reaction
mixture was allowed to cool, the solvent was evaporated under reduced
pressure, and the solid residue was purified by silica gel flash chroma-
tography using hexane-ethyl acetate (9:1) to afford the products. The
physical properties and spectral data are listed below.
4-(Dibromomethyl)-2-(4-chlorophenyl)-N,N-dimethylthia-
zole-5-carboxamide (7). White solid (51.1 mg, 83%), mp 63-64 °C.
1H NMR (CDCl3) δ 7.93 (d, J = 8.4 Hz, 2 H), 7.43 (d, J = 8.4 Hz, 2 H),
6.87 (s, 1 H), 3.11 (s, 6 H); 13C NMR (CDCl3) δ 166.50, 161.42, 153.57,
137.42, 130.60, 129.28. 128.06, 123.76, 39.72, 31.14, 31.27; MS m/z (rel
intensity) 441/439/437 (MHþ, 11.2/15.4/9.6), 394 (100); HRMS
(ESI), m/z 436.8728 Mþ, calcd for C13H12Br2ClN2OS 436.8720; HPLC
purity 95.24%.
Ethyl 4-(Dibromomethyl)-2-(4-chlorophenyl)thiazole-5-
1
carboxylate (13). White solid (20 mg, 81%), mp 185-187 °C. H
4-(Dibromomethyl)-2-(4-chlorophenyl)-N-methylthiazole-
5-carboxamide (8). Colorless solid (9.5 mg, 20%), mp 165-167 °C.
1H NMR (CDCl3) δ 7.93 (d, J = 8.4 Hz, 2 H), 7.73 (s, 1 H), 7.43 (d, J =
8.4 Hz, 2 H), 5.98 (brs, 1 H), 3.02 (s, 3 H); 13C NMR (CDCl3) δ 166.56,
160.35, 156.80, 137.65, 130.46, 129.34, 128.18, 123.35, 31.85, 29.59;
CIMS m/z (rel intensity) 426/424/422 (MHþ, 15/71/22); HRMS
(ESI), m/z 422.8568 MHþ, calcd for C12H10Br2ClN2OS 422.8564;
NMR (CDCl3) δ 7.98 (d, J = 8.4 Hz, 2 H), 7.69 (s, 1 H), 7.45 (d, J = 8.4
Hz, 2 H), 4.40 (q, J = 7.2 Hz, 2 H), 1.38 (t, J = 7.2 Hz, 3 H); 13C NMR
(CDCl3) δ 169.88, 160.44, 158.68, 137.77, 130.63, 129.33, 128.23,
120.01, 62.29, 31.15, 14.11; CIMS m/z (rel intensity) 441/439/437
(MHþ, 20/67/55), 393 (100); HRMS (EI), m/z 437.8562 MHþ, calcd
for C13H11Br2ClNO2S 437.8560; HPLC purity 96.98%.
Isopropyl 4-(Dibromomethyl)-2-(4-chlorophenyl)thiazole-
5-carboxylate (14). White solid (15.3 mg, 49%), mp 176-178 °C.
1H NMR (CDCl3) δ 7.99 (d, J = 8.4 Hz, 2 H), 7.70 (s, 1 H), 7.45 (d, J =
8.4 Hz, 2 H), 5.25 (m, J = 7.2 Hz, 1 H), 1.39 (d, J = 7.2 Hz, 6 H); 13C
NMR (CDCl3) δ 169.02, 159.98, 158.47, 139.64, 137.74, 130.69,
129.33, 128.22, 70.43, 46.87, 31.21, 21.75; CIMS m/z (rel intensity)
456/454/452 (MHþ, 10/47/23), 393 (100); HRMS (EI), m/z
450.8647 Mþ, calcd for C14H12Br2ClNO2S 450.8644; HPLC purity 100%.
S-Ethyl 4-(Dibromomethyl)-2-(4-chlorophenyl)thiazole-
5-carbothioate (15). The acid chloride 4 (38 mg, 0.09 mmol) was
HPLC purity 95.13%.
4-(Dibromomethyl)-2-(4-chlorophenyl)-N,N-diethylthia-
zole-5-carboxamide (9). Colorless solid (37.2 mg, 86%), mp 63 °C.
1H NMR (CDCl3) δ 7.92 (d, J = 8.4 Hz, 2 H), 7.72 (s, 1 H), 7.44 (d, J =
8.4 Hz, 2 H), 5.94 (brs, 1 H), 3.47 (q, J = 7.2 Hz, 2 H), 1.26 (t, J = 7.2 Hz,
3 H); 13C NMR (CDCl3) δ 166.47, 159.63, 156.67, 137.62, 130.46,
129.33, 128.15, 123.71, 44.21, 41.02, 31.93, 13.55; MS m/z (rel
intensity) 491/489/487 (MNaþ, 8.1/3.3/6.0), 394 (100); HRMS
1710
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714

Journal of Medicinal Chemistry
ARTICLE
heated under reflux with ethanethiol (0.1 mL, 1.9 mmol) under solvent-
free conditions for 4 h. The reaction mixture was allowed to cool. The
solvent was taken off under reduced pressure, and the solid residue was
purified by silica gel flash chromatography using hexane-ethyl acetate
(7:3) to afford the product together with a disulfide byproduct, which
was removed by another silica gel flash chromatography column using
hexane-ethyl acetate (97.5:2.5) to yield a white solid (5.5 mg, 10%): mp
90-91 °C. 1H NMR (CDCl3) δ 8.00 (d, J = 8.7 Hz, 2 H), 7.56 (s, 1 H),
7.46 (d, J = 8.7 Hz, 2 H), 3.13 (q, J = 7.5 Hz, 2 H), 1.38 (t, J = 7.5 Hz, 3
H); 13C NMR (CDCl3) δ 181.01, 156.11, 149.58, 144.87, 138.00,
130.89, 129.39, 128.36; APCIMS m/z (rel intensity) 458/456/454
(MHþ, 7/29/24), 393 (100); HRMS (ESI), m/z 453.8337 MHþ, calcd
for C13H11Br2ClNOS2 453.8332; HPLC purity 99.04%.
7.93, (s, 1 H), 7.76 (d, J = 12 Hz, 2 H), 7.40 (d, J = 8.3 Hz, 2 H), 5.34 (d,
J = 12 Hz, 2 H), 3.17 (s, 3 H), 2.93 (s, 3 H); 13C NMR (CDCl3) δ
178.70, 166.27, 154.92, 137.04, 131.26, 131.07, 130.40, 129.16, 128.09,
127.63, 94.83, 45.39, 37.52, 33.65; ESIMS m/z (rel intensity) 467/465/
563 (MHþ, 65/100/42); HRMS (ESI), m/z 462.8880 MHþ, calcd for
C15H14Br2ClN2OS 462.8877; HPLC purity 100%.
Preparation of Hydroxyalkyl and Carbohydrate Amide
Derivatives. General Procedure. Primary amine (1 mmol) was
added to acid chloride 4 (41 mg, 1 mmol) in dry dichloromethane
(2 mL). The reaction mixture was stirred at room temperature for
0.5-1 h. The solvent was evaporated under reduced pressure. The solid
residue was purified by crystallization from methanol. The physical and
spectral data of the obtained compounds are listed below.
S-Methyl 4-(Dibromomethyl)-2-(4-chlorophenyl)thiazole-
5-carbothioate (16). The acid chloride 4 (50 mg, 0.12 mmol) was
stirred with sodium methanthiol (12 mg, 1.7 mmol) in dry dichlor-
omethane (10 mL) for 30 min. The solvent was taken off under reduced
pressure, and the solid residue was partitioned between EtOAc (10 mL)
and water (10 mL). The organic layer was separated, dried, and
evaporated. The yellow precipitate was further purified by crystallization
from MeOH to afford the product as pale yellow needles (36 mg, 91%):
mp 180-182 °C. 1H NMR (CDCl3) δ 7.99 (d, J = 8.1 Hz, 2 H), 7.56 (s,
1 H), 7.45 (d, J = 8.1 Hz, 2 H), 2.55 (s, 1 H); 13C NMR (CDCl3) δ
183.20, 169.11, 156.20, 138.11, 130.46, 129.45, 128.44, 127.34, 31.59,
12.97; APCIMS m/z (rel intensity) 444/442/440 (MHþ, 19/41/33),
395 (MHþ - CH3, 100); HRMS (ESI), m/z 439.8185 MHþ, calcd for
C12H9Br2ClNOS2 439.8175; HPLC purity 100%.
4-(Dibromomethyl)-2-(4-chlorophenyl)-N-(2-hydroxyeth-
yl)thiazole-5-carboxamide (22). White solid (11 mg, 27%), mp
132-133 °C. 1H NMR (CDCl3) δ 7.93 (d, J = 8.4 Hz, 2 H), 7.72 (s,
1 H), 7.45 (d, J = 8.4 Hz, 2 H), 6.45 (brs, 1 H), 3.87 (t, J = 7 Hz, 2 H),
3.64 (t, J = 7 Hz, 2 H), 2.2 (brs, 1 H); 13C NMR (CDCl3) δ 166.89,
165.66, 157.10, 137.77, 130.48, 129.42, 128.26, 123.27, 61.51, 42.58,
31.88; CIMS m/z (rel intensity) 457/455/453 (MHþ, 65/79/17);
HRMS (ESI), m/z 451.8594 Mþ, calcd for C13H11Br2ClN2OS
451.8596; HPLC purity 96.21%.
(S)-4-(Dibromomethyl)-2-(4-chlorophenyl)-N-(2,3-dihydrox-
ypropyl)thiazole-5-carboxamide (23). White solid (13 mg, 12%),
mp 144-146 °C. 1H NMR (CD3OD) δ 8.03 (d, J = 8.7 Hz, 2 H), 7.80
(s, 1 H), 7.53 (d, J = 7.8 Hz, 2 H), 3.54 (m, J = 4.9 Hz, 1 H), 3.29 (dd, J =
1.5 and 4.9 Hz, 2 H), 3.21 (dd, J = 1.5 and 4.9 Hz, 2 H); 13C NMR
(CD3OD) δ 166.28, 159.07, 158.18, 143.36, 138.65, 132.21, 130.59,
129.39, 71.70, 65.23, 44.28, 32.71; ESIMS m/z (rel intensity) 485/483/
481 (MHþ, 17/46/41); HPLC purity 99.56%.
4-(Dibromomethyl)-2-(4-chlorophenyl)-N-(ribityl)thiazole-
5-carboxamide (24). White solid (11.9 mg, 86%), mp 217-219 °C.
1H NMR (CD3OD) δ 8.03 (d, J = 7.5 Hz, 2 H), 7.77 (s, 1 H), 7.52 (d, J =
7.8 Hz, 2 H), 2.98 (s, 3 H), 2.85 (s, 3 H), 2.80 (s, 1 H); 13C NMR
(CD3OD) δ 170.96, 164.87, 159.65, 138.83, 132.26, 130.59, 129.47,
123.25, 74.10, 74.04, 69.97, 64.58, 36.98, 31.90; ESIMS m/z (rel
intensity) 548/546/544/542 (MHþ, 17/81/31/53); HRMS (ESI),
m/z 542.8996 MHþ, calcd for C16H18Br2ClN2O5S 542.8986; HPLC
purity 98.98%.
4-(Dibromomethyl)-2-(4-chlorophenyl)-N-(D-glucosyl)thia-
zole-5-carboxamide (25). White solid (10 mg, 18%), mp 190-
191 °C. 1H NMR (CD3OD) δ 8.24 (d, J = 7.8 Hz, 2 H), 7.82 (s, 1 H),
7.75 (d, J = 7.8 Hz, 2 H), 5.24 (s, J = 3.4 Hz, 1 H), 4.04 (dd, J = 3.5 and 10
Hz, 1 H), 3.89-3.70 (m, 5 H); 13C NMR (CD3OD) δ 169.07, 163.20,
154.42, 143.82, 136.25, 132.24, 130.56, 129.35, 91.10, 73.34, 72.19,
62.69, 62.19, 56.76. 31.93; ESIMS m/z (rel intensity) 597/595/593
(MNaþ, 87/100/55); HRMS (ESI), m/z 592.8757 MNaþ, calcd for
C17H17Br2ClN2O6SNa 592.8755; HPLC purity 98.59%.
1-(2-(4-Chlorophenyl)-4-methylthiazol-5-yl)ethanone
(19). 4-Chlorothiobenzamide (18, 1.71 g, 10 mmol) and 3-chloropen-
tane-2,4-dione (1.604 g, 12 mmol) were added to absolute ethanol
(50 mL). The reaction mixture was heated at reflux for 24 h. After
removal of solvent under reduced pressure, the residue was crystallized
from ethanol-methanol-ethyl acetate (10:85:5) to provide the desired
1
compound as a white needles (1.852 g, 73%): mp 114-115 °C. H
NMR (CDCl3) δ 7.91 (d, J = 8.7 Hz, 2 H), 7.44 (d, J = 8.7 Hz, 2 H), 2.77
(s, 3 H), 2.57 (s, 3 H); 13C NMR (CDCl3) δ 190.33, 167.86, 159.44,
137.16, 131.17, 129.24, 127.97, 30.67, 18.34; EIMS m/z (rel intensity)
253/251 (MHþ, 26/70), 236 (Mþ - CH3, 100); HRMS (EI), m/z
251.0172 Mþ, calcd for C12H10ClNOS 251.0173; HPLC purity 98.98%.
1-(4-(Dibromomethyl)-2-(4-chlorophenyl)thiazol-5-yl)eth-
anone (20). Compound 19 (251 mg, 1 mmol) and NBS (358 mg, 2
mmol) were added to CCl4 (5 mL). The reaction mixture was irradiated
for 5 min frequently (15 min intervals) by an ultraviolet sunlamp (GE,
215 W) and heated at reflux for 12 h. After removal of solvent under
reduced pressure, the residual NBS was removed by adding saturated
aqueous NaOH (3 mL), and the mixture was filtered and washed with
distilled water. The collected brown solid was purified by silica gel
chromatography using ethyl acetate-hexanes (1:1) to provide the
desired compound as a white solid (204 mg, 50%): mp 193-195 °C.
1H NMR (CDCl3) δ 8.00 (d, J = 8.7 Hz, 2 H), 7.67 (s, 1 H), 7.47 (d, J =
8.7 Hz, 2 H), 2.59 (s, 3 H); 13C NMR (CDCl3) δ 189.56, 168.67, 160.61,
158.02, 138.03, 130.40, 129.41, 128.34, 32.00, 31.28; CIMS m/z (rel
intensity) 412/410/408 (MHþ, 30/78/32); HRMS (ESI), m/z
406.8378 MHþ, calcd for C12H8Br2ClNOS 406.8382; HPLC purity
95.13%.
Ethyl 2-Chloro-3-(furan-2-yl)-3-oxopropanoate (27c). Sul-
furyl chloride (3.3 mmol) was added dropwise to a stirred solution of
diketo compounds 26c (3 mmol) in dry methylene chloride (10 mL).
The mixture was vigorously stirred for 1 h. The solvent was
evaporated.20 The product was collected as a colorless to faint-yellow
oil and purified by column chromatography, using 6:4 hexane-ethyl
acetate. Yellow oil (621 mg, 96%). 1H NMR (CDCl3) δ 8.22 (s, 1 H),
7.47 (s, 1 H), 6.82 (s, 1 H), 5.20 (s, 1 H), 4.85 (q, J = 7.2 Hz, 2 H), 1.26
(t, J = 7.2 Hz, 3 H); 13C NMR (CDCl3) δ 182.80, 164.86, 149.09,
144.38, 109.02, 63.25, 59.75, 13.97; ESIMS m/z (rel intensity) 218/216
(MHþ, 23/100); HRMS (ESI), m/z 216.0184 MHþ, calcd for
C9H9ClO4 216.0189; HPLC purity 96.81%.
(E)-1-(4-(Dibromomethyl)-2-(4-chlorophenyl)thiazol-5-yl)-3-
(dimethylamino)prop-2-en-1-one (21). A mixture of thiazole
derivative 20 (1 mmol) and DMF-DMA (0.357 mL, 3 mmol) was taken
in dry toluene (20 mL), and the mixture was heated at reflux for 24 h and
then left to cool at room temperature. The solvent was evaporated under
reduced pressure. The reddish-yellow precipitated product was washed
with petroleum ether (60/80 °C) and dried. Recrystallization from
benzene afforded the desired compound as an orange solid (303 mg,
66%): mp 148-149 °C. 1H NMR (CDCl3) δ 7.96 (d, J = 8.3 Hz, 2 H),
Preparation of Alkyl 2-(4-Chlorophenyl)-4-alkylthiazole-
5-carboxylates (28-31). General Procedure. 4-Chlorothioben-
zamide (171 mg, 1 mmol) and R-chlorodiketo derivatives 27a-d (1.2
mmol) were added to absolute ethanol (15 mL). The reaction mixture
1711
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714

Journal of Medicinal Chemistry
ARTICLE
was heated to reflux for 24 h. After removal of solvent under reduced
pressure, the residue was purified by silica gel chromatography
(hexanes-ethyl acetate 7:3) to provide the desired compounds. The
physical and spectral data of products are listed below.
Methyl 2-(4-Chlorophenyl)-4-ethylthiazole-5-carboxylate
(28). White solid (277.6 mg, 98%), mp 86-87 °C. 1H NMR (CDCl3) δ
7.93 (d, J = 8.4 Hz, 2 H), 7.20 (d, J = 8.4 Hz, 2 H), 3.96 (q, J = 7.5 Hz,
2 H), 3.89 (s, 3 H), 1.36 (t, J = 7.5 Hz, 3 H); 13C NMR (CDCl3) δ
172.21, 166.97, 160.32, 136.90, 131.44, 129.17, 127.94, 120.00, 52.10,
24.31, 13.52; ESIMS m/z (rel intensity) 284/282 (MHþ, 35/100);
HRMS (ESI), m/z 282.0360 MHþ, calcd for C13H13ClNO2S 282.0350;
HPLC purity 97.80%.
Methyl 2-(4-Chlorophenyl)-4-isopropylthiazole-5-carboxy-
late (29). White solid (186 mg, 63%), mp 70-71 °C. 1H NMR
(CDCl3) δ 7.92 (d, J = 8.4 Hz, 2 H), 7.39 (d, J = 8.4 Hz, 2 H), 3.98 (m,
1 H), 3.88 (s, 1 H), 1.345 (d, J = 6.9 Hz, 6 H); 13C NMR (CDCl3) δ
171.00, 168.55, 162.20, 138.09, 133.67, 129.09, 127.95, 120.10, 52.04,
29.10, 21.99; ESIMS m/z (rel intensity) 298/296 (MHþ, 34/100);
HRMS (ESI), m/z 296.0509 MHþ, calcd for C14H15ClNO2S 296.0506;
HPLC purity 97.78%.
298/297 (MHþ, 38/100); HRMS (ESI), m/z 297.0464 MHþ, calcd for
C13H14ClN2O2S 297.0459; HPLC purity 95.00%.
2-(4-Chlorophenyl)-4-formyl-N-methylthiazole-5-carbox-
amide (34). Methyl ether 33 (300 mg, 1 mmol) and NBS (178 mg, 1
mmol) were added to CCl4 (15 mL). The reaction mixture was heated at
reflux for 1 h and was illuminated 4 times by a 60 W bulb for 5 min with
time intervals of 15 min. The solvent was removed under reduced
pressure. The residue was partitioned between EtOAc (10 mL) and
NaOH (0.1 M NaOH, 5 mL). The organic layer was separated, dried
over anhydrous MgSO4, and removed under reduced pressure. The
semisolid residue was purified by silica gel flash chromatography using
hexane-ethyl acetate (3:1) to afford the product as a white solid (56%):
mp 231-232 °C. 1H NMR (CDCl3) δ 10.15 (s, 1 H), 9.78 (brs, 1 H),
7.93 (d, J = 8.4 Hz, 2 H), 7.46 (d, J = 8.4 Hz, 2 H), 3.04 (d, J = 4.8 Hz, 3
H); 13C NMR (CDCl3) δ 188.92, 168.13, 159.29, 147.60, 145.36,
137.68, 130.49, 129.53, 127.96, 29.65; MS m/z (rel intensity) 283/281
(MHþ, 51/100); HRMS (ESI), m/z 281.0157 MHþ, calcd for
C12H10ClN2O2S 281.0146; HPLC purity 95.13%.
Methyl 2-(4-Chlorophenyl)-4-cyanothiazole-5-carbox-
ylate (36). Method A.23 To a stirred solution of AlCl3 (40 mg. 0.3
mmol) in THF (3 mL) was added NaN3 (61 mg, 0.94 mmol) followed
by aldehyde 35 (44 mg, 0.15 mmol). The mixture was heated to gentle
reflux. After 12 h the reaction mixture was diluted with 10% HCl (3 mL)
and THF was removed under reduced pressure. The organic layer was
extracted with ethyl acetate (3 ꢀ 5 mL), dried, and evaporated. The
residue was purified by column chromatography on silica gel using a
mixture of hexane-ethyl acetate (4:1) to obtain the product (9 mg,
21%).
Methyl 4-tert-Butyl-2-(4-chlorophenyl)thiazole-5-carbox-
1
ylate (30). White solid (270 mg, 87%), mp 105-106 °C. H NMR
(CDCl3) δ 7.91 (d, J = 8.4 Hz, 2 H), 7.41 (d, J = 8.4 Hz, 2 H), 3.87 (s, 3
H), 1.53 (s, 9 H); 13C NMR (CDCl3) δ 171.62, 165.84, 162.00, 136.61,
131.63, 129.10, 127.82, 121.34, 52.27, 36.61, 29.36; ESIMS m/z (rel
intensity) 312/310 (MHþ, 27/100); HRMS (ESI), m/z 310.0672
MHþ, calcd for C15H17ClNO2S 310.0663; HPLC purity 95.63%.
Ethyl 2-(4-Chlorophenyl)-4-(furan-2-yl)thiazole-5-carbox-
ylate (31). 4-Chlorothiobenzamide (256.5 mg, 1.5 mmol) and R-
chlorodiketo derivative 27c (4.33 mg, 2 mmol) were added to absolute
ethanol (15 mL). The reaction mixture was heated at reflux for 24 h.
After removal of solvent under reduced pressure, the residue was
purified by silica gel chromatography (hexanes-ethyl acetate 7:3, then
9:1) to provide the desired compound as a white solid (481 mg, 96%):
mp 82-83 °C. 1H NMR (CDCl3) δ 8.59 (s, 1 H), 7.98 (d, J = 8.4 Hz, 2
H), 7.83 (d, J = 8.4 Hz, 2 H), 7.44 (s, 1 H), 7.23 (s, 1 H), 2.47 (q, J = 7.2
Hz, 2 H), 1.40 (t, J = 7.2 Hz, 3 H); ESIMS m/z (rel intensity) 336/334
(MHþ, 19/100); HRMS (ESI), m/z 334.0309 MHþ, calcd for
C16H13ClNO3S 334.0229; HPLC purity 95.33%.
Method B.24 A solution of aldehyde 35 (20 mg, 0.07 mmol) and iodine
(12.7 mg, 0.1 mmol) in ammonia solution (3 mL of 28% solution) and
THF (0.5 mL) was stirred at room temperature for 1 h (until the
solution became colorless). Then the reaction mixture was charged with
aqueous Na2S2O3 (5% solution) followed by extraction with ethyl
acetate (2 ꢀ 5 mL) to give the crude nitrile 36, which was purified by
column chromatography on silica gel using a mixture of hexane-ethyl
acetate (7:3) to yield a white solid (18.5 mg, 99%): mp 142-143 °C. 1H
NMR (CDCl3) δ 7.92 (d, J = 8.7 Hz, 2 H), 7.48 (d, J = 8.7 Hz, 2 H), 4.02
(s, 3 H); 13C NMR (CDCl3) δ 171.24, 159.01, 138.63, 133.28, 129.65,
129.27, 128.23, 112.49, 53.43; CIMS m/z (rel intensity) 280/278 (Mþ,
30/100); HRMS (CI), m/z 278.9992 MHþ, calcd for C12H9ClN2O2S
278.9989; HPLC purity 100%.
Methyl 4-(Dichloromethyl)-2-(4-chlorophenyl)thiazole-5-
carboxylate (37)25. The aldehyde derivative 35 (25 mg, 0.09 mmol),
acetyl chloride (13 μL, 0.178 mmol), and anhydrous aluminum chloride
(3.8 mg, 0.028 mmol) were added to dichlorobenzene (3 mL). The
reaction mixture was heated at reflux for 24 h. The solvent was taken off
under reduced pressure and the orange oily residue was purified by silica
gel flash chromatography using hexane-ethyl acetate (7:3) to yield 37
as an off-white solid (29 mg, 99%): mp 133 °C. 1H NMR (CDCl3) δ
8.27 (s, 3 H), 7.98 (d, J = 8.7 Hz, 2 H), 7.46 (d, J = 8.7 Hz, 2 H), 3.95 (s, 3
H); 13C NMR (CDCl3) δ 168.07, 160.64, 155.78, 137.79, 130.66,
129.33, 128.30, 124.30, 75.49, 52.96; ESIMS m/z (rel intensity) 304/
302/300 (MHþ - HCl, 25/62.8/100); HRMS (ESI), m/z 299.9650
(Mþ - HCl), calcd for C12H7Cl2NO2S 299.9647; HPLC purity
99.78%.
2-(4-Chlorophenyl)-4-(methoxymethyl)-N-methylthia-
zole-5-carboxamide (33). NaOH (4.00 g, 100 mmol) was added to
a solution of methyl carboxylate 32 (6.237 g, 21 mmol) in ethanol
(40 mL) and water (5 mL). The reaction mixture was heated under
reflux for 5 h and then allowed to cool to room temperature. The
reaction mixture was filtered, and the pH value of the liquid phase was
adjusted to 2 with hydrochloride acid. The solid was filtered and dried to
provide an off-white solid (4.980 g, 84%). The crude obtained solid
material (4.88 g, 17.2 mmol) was heated under reflux with thionyl
chloride (25 mL) for 6 h. The solvent was evaporated under reduced
pressure. The brown residue was collected and purified by silica gel flash
chromatography, using hexane-ethyl acetate (7:3), to yield the corre-
sponding acid chloride as a white solid (2.507 g, 96%). Methylamine
hydrochloride (88 mg, 1.3 mmol) and potassium carbonate (27 mg, 0.5
mmol) were dissolved in water (10 mL), and then the corresponding
acid chloride (380 mg, 1.26 mmol) was added after 5 min. The reaction
mixture was stirred at room temperature for 1 h. The white flocculant
solid was collected by filtration and washed with HCl (0.1 M, 5 mL) and
then water (3 ꢀ 10 mL). The white solid was further purified by
crystallization from EtOAc to yield a white solid (339.4 mg, 91%): mp
113-114 °C. 1H NMR (CDCl3) δ 8.07 (brs, 1 H), 7.86 (d, J = 8.7 Hz, 2
H), 7.41 (d, J = 8.7 Hz, 2 H), 4.78 (s, 2 H), 3.46 (s, 3 H), 2.99 (d, J = 4.8
Hz, 3 H); 13C NMR (CDCl3) δ 166.05, 161.56, 151.46, 136.78, 135.11,
131.33, 129.25, 127.80, 69.32, 58.04, 26.90; ESIMS m/z (rel intensity)
Methyl 4-(Bromomethyl)-2-(4-chlorophenyl)thiazole-5-car-
boxylate (38). Methyl ester 2 (1.045 g, 3.9 mmol), NBS (767 mg, 4.3
mmol), and benzoyl peroxide (10 mg) were added to CCl4 (25 mL).
The reaction mixture was heated at reflux for 24 h. After removal of
solvent under reduced pressure, the residual NBS was removed by
adding saturated aqueous NaOH (20 mL), filtering, and washing with
distilled water. The collected yellowish-white precipitate was purified by
silica gel chromatography (ethyl acetate-hexanes 1:4) to provide the
desired compound as a white solid (676 mg, 50%): mp 151-152 °C.1H
1712
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714

Journal of Medicinal Chemistry
ARTICLE
NMR (CDCl3) δ 7.92 (d, J = 8.7 Hz, 2 H), 7.43 (d, J = 8.7 Hz, 2 H), 4.97
(s, 2 H), 3.93 (s, 3 H); 13C NMR (CDCl3) δ 169.45, 161.37, 158.71,
137.55, 130.86, 129.36, 128.12, 124.05, 52.68, 24.80; ESIMS m/z (rel
intensity) 349/347/345 (MHþ, 11/38/29); HRMS (ESI), m/z 344.9229
Mþ, calcd for C12H9BrClNO2S 344.9226; HPLC purity 100%.
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,
medium on cells was replaced with fresh medium to remove the
compounds. Then 10 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 was measured using a 96-well plate reader (Molecular Devices,
Sunnyvale, CA). The OD450 value for cells treated with a compound was
compared to that obtained from cells treated with 1% DMSO, and the
GI50 for each compound was calculated.
Molecular Modeling. Molecule Construction and Energy Opti-
mization. Compounds 1, 23-25, and 39 were built with Sybyl 7.1
software and minimized to 0.01 kcal/mol by the Powell method, using
Gasteiger-H€uckel charges and the Tripos force field. To save calcula-
tion time, solvents were not taken into account, and instead the dielectric
constant was set to a value of 4 to mimic the aqueous environment. The
minimized molecules underwent 10 rounds of simulated annealing to
search for the optimized conformation. During the simulation process,
the starting conformation in each round was heated to 700 K within
1000 fs and then cooled to 200 K in the same period. Conformations
were recorded at each temperature level (700 and 200 K). The
conformers located at the starting point at the each round of simulation
were selected for further energy refinement using the same parameter set
as the ones in molecular construction. The minimized conformer with
the lowest energy was selected as the optimized conformation of the
molecule which was docked into the β-OG binding pocket of the yellow
fever virus E-protein (PDB code 1OKE).
Docking Simulation. The energy-optimized compounds were
docked into the β-OG binding domain in the E-protein of the dengue
virus after removal of the n-octyl-β-D-glucoside (β-OG). The parameters
were set as the default values for GOLD. The maximum distance
between hydrogen bond donors and acceptors for hydrogen bonding
was set to 3.5 Å. After docking, the first pose conformation of
compounds 1, 23-25, and 39 were merged into the ligand-free protein.
The new ligand-protein complex was subsequently subjected to energy
minimization using the Amber force field with Amber charges. During
the energy minimization, the structure of the compounds of interest and
only chain A of the viral E protein were allowed to move. Chain B was
kept frozen. The energy minimization was performed using the Powell
method with a 0.05 kcal/(mol Å) energy gradient convergence criterion
and a distance dependent dielectric function.
In Vitro Hydrolytic Stability Assay Utilizing Rat Plasma. Compounds
1 and 16 were tested for their hydrolytic stability in solutions of
reconstituted rat plasma. Compounds 1 and 16 (15-5 μmol) and
7 μmol of 4-bromopyrazole as an internal standard were dissolved in
DMSO (1.0 mL). This solution was filtered through a 0.45 μM filter
(Millex-HN). Lyophilized rat plasma (1.0 mL) (lot no. 048K7420,
Sigma Chemical Co., St. Louis, MO) was reconstituted with water of
HPLC (1.0 mL). The plasma solution was incubated at 37 °C for 15 min
and was then diluted with 0.01 M saline (0.250 mL) to afford an 80%
plasma solution. The plasma solution was incubated again at 37 °C for an
additional 5 min. An aliquot of the compounds 1 and 16 in DMSO (100 μL)
was added to the rat plasma (0.75 mL), and the mixture was incubated at
37 °C throughout the course of the experiment. Aliquots (10 μL) of the
compound-plasma mixture were collected at various time intervals and
diluted with methanol (90 μL) to precipitate any proteins present. The
aliquots were mixed and centrifuged at 10 000 rpm for 5-10 min to
pellet the precipitated proteins. After centrifugation, the supernatants
(20 μL) of the aliquots were analyzed by HPLC to determine the
residual amount of tested compounds present in the sample. The aliquot
supernatants were analyzed using a Waters binary HPLC system (model
1525, 10 μL injection loop) and a Waters dual wavelength absorbance
UV detector (model 2487) set for 254 nm. Data were collected and
Methyl 2-(4-Chlorophenyl)-4-[(methylsulfonyl)methyl]th-
iazole-5-carboxylate (39). Compound 38 (46 mg, 0.133 mmol)
and sodium methylsulfinate (24 mg, 0.26 mmol) were added to absolute
ethanol (3 mL). The reaction mixture was heated at reflux for 12 h. After
removal of solvent under reduced pressure, the off-white solid was
partitioned between EtOAc (5 mL) and water (10 mL). The organic
layer was separated, dried over MgSO4, and evaporated under reduced
pressure. The collected solid material was purified by crystallization from
MeOH-EtOAc to provide the product as a white solid (38.1 mg, 83%):
mp 178-179 °C. 1H NMR (CDCl3) δ 7.89 (d, J = 8.4 Hz, 2 H), 7.44 (d,
J = 8.4 Hz, 2 H), 5.01 (s, 2 H), 3.93 (s, 3 H), 3.09 (s, 3 H); 13C NMR
(CDCl3) δ 169.80, 161.41, 150.42, 137.83, 130.62, 129.48, 128.06,
126.68, 55.24, 52.88, 41.33; CIMS m/z (rel intensity) 347/345 (Mþ, 4/
19), 268/266 (Mþ - CH3SO2, 39/100); HRMS (CI), m/z 344.9902
Mþ, calcd for C13H12ClNO4S 344.9896; HPLC purity 96.02%.
Bioassay Methods. BHK Cells. BHK-15 cells obtained from the
American Type Culture Collection (ATCC, Rockville, MD) were
maintained in MEM (Invitrogen, Carlsbad, CA) containing 10% FBS.
Cells were grown in incubators at 37 °C in the presence of 5% CO2.
YFV-IRES-Luc. A firefly luciferase reporter gene was inserted into
pYF23, a derivative of pACNR that 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 30NTR immediately following the UGA termina-
tion codon of NS5 in pYF23 using standard overlapping PCR mutagen-
esis. 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.26
Generation of YFV-IRES-Luc Virus. In vitro transcribed YFV-IRES-
Luc RNA was transfected into BHK-15 cells using Lipofectamine
(Invitrogen, Carlsbad,CA). 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 virus at a multiplicity of infection (MOI) of 0.1. A low
MOI was utilized to ensure that fewer cells were infected so that the
spread of released virus could be monitored. Cells were then overlaid
with culture medium containing serial dilutions of compounds at
concentrations below the GI50 values. Controls included uninfected
cells, infected cells and DMSO-treated infected cells. Cells were
incubated at 37 °C, 5% CO2 for ∼36 h and lysed using 50 μL of cell
culture lysis buffer (Promega Inc., Madison, WI), and 10 μL of cell
extracts was placed into a 96-well opaque plate. Luciferase activity was
determined from the luminescence generated with firefly luciferase
substrate (Promega Inc., Madison, WI). Luminescense 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 com-
pound concentration was analyzed by nonlinear regression analysis
using GraphPadPrizm to estimate the IC50 of each compound. The IC50
was defined as the concentration of the compound that causes 50%
reduction of luciferase activity in infected cells compared to the DMSO-
treated cells.
Cell Viability Assay. BHK cells were plated in a 96-well plate and
grown at 37 °C. At confluency, cells were overlaid with culture medium
containing serial dilutions of compounds (compound stocks were
1713
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714

Journal of Medicinal Chemistry
ARTICLE
processed using the Breeze software (version 3.3) on a Dell Optiplex
GX280 personal computer. The mobile phase consisted of 85:15 (v/v)
methanol/water, and the Sunrise HPLC column (4.6 mm ꢀ 150 mm)
was packed with C18 silica from Waters. The column was maintained at
room temperature during the analyses. The half-lives of 1 and 16 were
calculated from regression curves fitted to plots of the compound
concentration versus time.
Flavivirus RNA Replication. Antimicrob. Agents Chemther. 2006,
50, 1320–1329.
(11) Perera, R. k.; Kuhn, R. J. Structural Proteomics of Dengue
Virus. Curr. Opin. Microbiol. 2008, 11, 369–377.
(12) Modis, Y.; Ogata, S.; Clements, D.; Harrison, S. C. A Ligand-
Binding Pocket in the Dengue Virus Envelope Glycoprotein. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 6986–6991.
(13) Ze, L.; Khaliq, M.; Zhou, Z.; Post, C. B.; Kuhn, R. J.; Cushman, M.
Design, Synthesis, and Biological Evaluation of Antiviral Agents Targeting
Flavivirus Envelope Proteins. J. Med. Chem. 2008, 51, 4660–4671.
(14) (a) Wang, Q.; Patel, S. J.; Vangrevelinghe, E.; Xu, H. Y.; Rao, R.;
Jaber, D.; Schul, W.; Gu, F.; Heudi, O.; Ma, N. L.; Poh, M. K.; Phong,
W. Y.; Keller, T. H.; Jacoby, E.; Vasudevan, S. G. A Small Molecule
Dengue Virus Entry Inhibitor. Antimicrob. Agents Chemother. 2009,
53, 1823–1831. (b) Poh, M. K.; Yip, A.; Zhang, S.; Priestle, J. P.; Ma,
N. L.; Smit, J. M.; Wilschut, J.; Shi, P.; Wenk, M. R.; Schul, W. A Small
Molecule Fusion Inhibitor of Dengue Virus. Antiviral Res. 2008,
84, 260–266.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@
purdue.edu.
Present Addresses
§On leave from Faculty of Pharmacy, AL-Azhar University,
Cairo 1884, Egypt.
(15) (a) Saunders, J.; Cassidy, M.; Freedman, S. B.; Harley, E. A.;
Iversen, L. L.; Kneen, C.; MacLeod, A. M.; Merchant, K. J.; Snow, R. J.;
Baker, R. Novel Quinuclidine-based Ligands for the Muscarinic Choli-
nergic Receptor. J. Med. Chem. 1990, 33, 1128–1138. (b) Orlek, B. S.;
Blaney, F. E.; Brown, F.; Clark, M. S. G.; Hadley, M. S.; Hatcher, J.; Riley,
G. J.; Rosenberg, H. E.; Wadsworth, H. J.; Wyman, P. Comparison of
Azabicyclic Esters and Oxadiazoles as Ligands for the Muscarinic
Receptor. J. Med. Chem. 1991, 34, 2726–2735. (c) Sakamoto, T.; Cullen,
M. D.; Hartman, T. L.; Watson, K. M.; Buckheit, R. W.; Pannecouque,
C.; De Clercq, E.; Cushman, M. Synthesis and Anti-HIV Activity of New
Metabolically Stable Alkenyldiarylmethane Non-Nucleoside Reverse
Transcriptase Inhibitors Incorporating N-Methoxy Imidoyl Halide and
1,2,4-Oxadiazole Systems. J. Med. Chem. 2007, 3314–3321.
(16) Markees, D. J. Reaction of Benzyl Methyl Ethers with Bromine
and N-Bromosuccinimide. J. Org. Chem. 1958, 23, 1490–1492.
(17) Mayhoub, A. S.; Talukdar, A.; Cushman, M. Oxidation of Benzyl
Methyl Ethers with NBS Selectively Affords EitherAromatic Aldehydes or
Aromatic Methyl Esters. J. Org. Chem. 2010, 75, 3507–3510.
(18) Cullen, M. D.; Deng, B.; Hartman, T. L.; Watson, K. M.;
Buckheit, R. W.; Pannecouque, C., Jr.; De Clercq, E.; Cushman, M.
Synthesis and Biological Evaluation of Alkenyldiarylmethane HIV-1
Non-Nucleoside Reverse Transcriptase Inhibitors That Possess In-
creased Hydrolytic Stability. J. Med. Chem. 2007, 50, 4854–4867.
(19) (a) Burke, D. S.; Monath, T. Flaviviruses; Lippincott Williams
and Wilkins: Philadelphia, PA, 2001. (b) Bressanelli, S.; Stiasny, K.;
Allison, S. L.; Stura, E. A.; Duquerroy, S.; Lescar, J.; Heinz, F. X.; Rey,
F. A. Structure of a Flavivirus Envelope Glycoprotein in Its Low-pH-
Induced Membrane Fusion Conformation. EMBO J. 2004, 23, 728–738.
(20) Csavassy, G.; Gyorfi, Z. A. Thiazole Compounds. I. Synthesis
and Reactions of 2-Aryl-5-(diazoacetyl)-4-methylthiazoles. Justus Liebigs
Ann. Chem. 1974, 8, 1195–1205.
’ ABBREVIATIONS USED
BHK, baby hamster kidney cells; β-OG, n-octyl-β-D-glucoside;
DMF-DMA, N,N-dimethylformoxide dimethyl acetal; EMCV,
encephalomyocarditis virus; E-protein, Envelop-protein; FBS,
fetal bovine serum; Luc, luciferase; MEM, minimal essential
medium; NBS, N-bromosuccinimide; NCSe, N-chlorosuccini-
mide; PCR, polymerase chain reaction; SAR, structure-activity
relationship;TI, therapeutic index; YFV, yellow fever virus; IRES,
internal ribosome entry site
’ REFERENCES
(1) Munoz-Jordan, J. L.; Sanchez-Burgos, G. G; Laurent-Rolle, M.;
Garcia-Sastre, A. Inhibition of Interferon Signaling by Dengue Virus.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14333–14338.
(2) http://en.wikipedia.org/wiki/West_Nile_virus.
(3) Sampath, A.; Padmanabhan, R. Molecular Targets for Flavivirus
Drug Discovery. Antiviral Res. 2009, 81, 6–15 and references therein.
(4) Borowski, P.; Lang, M.; Haag, A.; Schmitz, H.; Choe, J.; Chen,
H.-M.; Hosmane, R. S. Characterization of Imidazo[4,5-d]pyridazine
Nucleosides as Modulators of Unwinding Reaction Mediated by West
Nile Virus Nucleoside Triphosphatase/Helicase: Evidence for Activity
on the Level of Substrate and/or Enzyme. Antimicrob. Agents Chemother.
2002, 46, 1231–1239.
(5) Zhang, N.; Chen, H.-M.; Koch, V.; Schmitz, H.; Minczuk, M.;
Stepien, P.; Fattom, A. I.; Naso, R. B.; Kalicharran, K.; Borowski, P.;
Hosmane, R. S. Potent Inhibition of NTPase/Helicase of the West Nile
Virus by Ring-Expanded (“Fat”) Nucleoside Analogues. J. Med. Chem.
2003, 46, 4776–4789.
(21) De Kimpe, N.; De Cock, W.; Schamp, N. A Convenient
Synthesis of 1-Chloro-2-alkanones. Synthesis 1987, 2, 188–190.
(22) Sreedhar, B.; Reddy, P. S.; Madhavi, M. Rapid and Catalyst-
Free R-Halogenation of Ketones Using N-Halosuccinimides in DMSO.
Synth. Commun. 2007, 37, 4149–4156.
(6) Luzhkov, V. B.; Selisko, B.; Nordqvist, A.; Peyrane, F.; Decroly,
E.; Alvarez, K.; Karlen, A.; Canard, B.; Qvist, J. A. Virtual Screening and
Bioassay Study of Novel Inhibitors for Dengue Virus mRNA Cap
(Nucleoside-20O)-methyltransferase. Bioorg. Med. Chem. 2007,
15, 7795–7802.
(23) Suzuki, H.; Nakaya, C. A Convenient One-Step Method of
Converting Electron-Rich Aromatic Aldehydes into Nitriles. Synthesis
1992, 641–642.
(7) Fabrega, C.; Hausmann, S.; Shen, V.; Shuman, S.; Lima, C. D.
Structure and Mechanism of mRNA Cap (Guanine-N7) Methyltrans-
ferase. Mol. Cell 2004, 13, 77–89.
(24) Jiun-Jie, S.; Jim-Min, F. Direct Conversion of Aldehydes to
Amides, Tetrazoles, and Triazines in Aqueous Media by One-Pot
Tandem Reactions. J. Org. Chem. 2003, 68, 1158–1160.
(25) Wolfson, A.; Shokin, O.; Tavor, D. Acid Catalyzes the Synthesis
of Aromatic Gem-Dihalides from Their Corresponding Aromatic Alde-
hydes. J. Mol. Catal. A: Chem. 2005, 226, 69–76.
(8) Mueller, N. H.; Pattabiraman, N.; Ansarah-Sobrinho, C.;
Viswanathan, P.; Pierson, T. C.; Padmanabhan, R. Identification and
Biochemical Characterization of Small-Molecule Inhibitors of West Nile
Virus Serine Protease by a High-Throughput Screen. Antimicrob. Agents
Chemther. 2008, 52, 3385–3393.
(9) Mueller, N. H.; Yon, C.; Ganesh, V. K.; Padmanabhan, R.
Characterization of the West Nile Virus Protease Substrate Specificity
and Inhibitors. Int. J. Biochem. Cell Biol. 2007, 39, 606–614.
(10) Puig-Basagoiti, F.; Tilgner, M.; Forshey, B. M.; Philpott, S. M.;
Espina, N. G.; Wentworth, D. E.; Goebel, S. J.; Masters, P. S.; Falgout, B.;
Ren, P.; Ferguson, D. M.; Shi, P. Triaryl Pyrazoline Compound Inhibits
(26) Jones, C. T.; Patkar, C. G.; Kuhn, R. J. Construction and
Application of Yellow Fever Virus Replicons. Virology 2005, 331, 247–259.
1714
dx.doi.org/10.1021/jm1013538 |J. Med. Chem. 2011, 54, 1704–1714