An investigation of phenylthiazole antiflaviviral agents

Article abstract of DOI:10.1016/j.bmc.2011.04.041

Flaviviruses are one of the most clinically important pathogens and their infection rates are increasing steadily. The phenylthiazole ring system has provided a template for the design and synthesis of antiviral agents that inhibit the flaviviruses by tar

Full text of DOI:10.1016/j.bmc.2011.04.041

Bioorganic & Medicinal Chemistry 19 (2011) 3845–3854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
An investigation of phenylthiazole antiflaviviral agents
Abdelrahman S. Mayhoub a, Mansoora Khaliq b, Carolyn Botting b, Ze Li a, Richard J. Kuhn b,
Mark Cushman a,
⇑
a Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and The Purdue Center for Cancer Research, Purdue University, West Lafayette, IN
47907, United States
b Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Flaviviruses are one of the most clinically important pathogens and their infection rates are increasing
steadily. The phenylthiazole ring system has provided a template for the design and synthesis of antiviral
agents that inhibit the flaviviruses by targeting their E-protein. Unfortunately, there is a correlation
between phenylthiazole antiflaviviral activity and the presence of the reactive and therefore potentially
toxic mono- or dibromomethyl moieties at thiazole-C4. Adding a linear hydrophobic tail para to the phe-
nyl ring led to a new class of phenylthiazole antiflaviviral compounds that lack the toxic dibromomethyl
moiety. This led to development of a drug-like phenylthiazole 12 that had high antiflaviviral selectivity
(TI = 147).
Received 10 February 2011
Revised 13 April 2011
Accepted 21 April 2011
Available online 3 May 2011
Keywords:
Envelope-protein
Flaviviruses
Phenylthiazoles
Dengue virus
Yellow fever virus
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
but there are no human vaccines for the vast majority of flavivi-
ruses including dengue viruses, nor effective therapy for treatment
Flavivirus is a genus of the positive-sense ssRNA family Flavivi-
ridae, which includes many clinically important species such as
dengue, Japanese encephalitis and West Nile viruses.
of the clinical cases.5
There are many flaviviral proteins that have been targeted for
drug discovery such as helicase,6,7 methyl transferase,8,9 and serine
protease.10,11 In addition, the viral RNA is also reported to be a tar-
get for some antiviral agents.12 Among the flaviviral targets, E-pro-
tein plays a crucial role at the first step in viral infection, since it
contains the fusogenic loop.13 Structural comparisons of E-protein
in the immature and mature virus stages suggest that the E-protein
undergoes substantial conformational and translational changes
through the virus replication cycle, thereby causing the native
homodimer to change into a fusogenic homotrimer.13 Moreover,
crystallization of a dengue virus type 2 E-protein (Fig. 1) in the
presence and the absence of b-OG14 showed an orientation change
between domains I and II and paved the road for structure-based
design of antiviral agents that could occupy the b-OG pocket. Since
the b-OG-containing crystal structure revealed conformational
changes relative to the unoccupied protein, it is believed that the
b-OG pocket is an ideal target for designing new antiflaviviral
agents.
More than 50 million cases of dengue viral infection are re-
ported per year in more than 80 countries in which the mosquito
Aedes aegypti is endemic.1 Approximately 909,000 clinical cases
of dengue viral infection were reported in 2008 in North, Central,
and South America. Of these cases, there were 306 reported deaths
as the consequence of the more severe illnesses dengue hemor-
rhagic fever and dengue shock syndrome, and the number is
increasing steadily.2 In the United States, after decades of absence,
the dengue virus is again emerging, causing an epidemic in Hawaii
in 2001.3 The features of flavivirus infection range from an asymp-
tomatic state to the severe hemorrhagic disorders that include the
classical typical clinical manifestations (fever) and atypical symp-
toms that involve encephalitis, myocarditis, hepatitis and chole-
cystitis.4 Currently there are limited licensed flaviviral vaccines,
Abbreviations: BHK, Baby hamster kidney cells; b-OG, n-Octyl-b-D-glucoside;
Starting from the hit phenylthiazole 1 that was obtained by vir-
tual screening,15 followed by extensive structural optimization,
compound 2 was developed with a notably more selective and po-
tent antiflaviviral activity (Fig. 2).16 These results have encouraged
further structural optimization studies to search for more potent
antiviral agents based on the phenylthiazole scaffold.17 Compound
EMCV, Encephalomyocarditis virus; E-protein, Envelope-protein; FBS, Fetal bovine
serum; Luc, Luciferase; MEM, Minimal essential medium; NBS, N-bromosuccini-
mide; NCS, N-chlorosuccinimide; PCR, Polymerase chain reaction; SAR, Structure–
activity relationship; ssRNS, Single-stranded RNA; TI, Therapeutic index; YFV,
Yellow fever virus; IRES, Internal ribosome entry site.
⇑
Corresponding author. Tel.: +1 765 494 1465; fax: +1 765 494 6790.
E-mail address: cushman@purdue.edu (M. Cushman).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.041

3846
A. S. Mayhoub et al. / Bioorg. Med. Chem. 19 (2011) 3845–3854
O
S
O
O
OCH3
S
a
OCH3
Br
+
N
Ar
NH2
4a-p
Cl
3
Ar
5a-p
Br
O
OCH3
Figure 1. Dengue viral 2 E-protein, domain I: red; domain II: yellow; domain III:
blue. The b-OG binding pocket is located between domains I and II.18
b
N
S
2 has a high TI, but it had two main drawbacks. First, it is a simple
methyl ester derivative with a short plasma half-life and its corre-
sponding free acid was shown to lack any antiflaviviral activity.18
Second, it contains the dibromomethyl moiety that is expected to
be vulnerable to endogenous nucleophiles and consequently a high
in vivo toxicity could be expected. In the next step in this project,18
the focus was shifted to finding metabolically stable bioisosteres of
the methyl ester that retained antiviral potency, combined with
possibly less toxic dibromomethyl replacements. In those studies
the SARs of the thiazole-C4 and -C5 substituents were defined
and the pharmacophore model shown in Figure 2 was built.18 So
far, several metabolically stable 4-chlorophenylthiazoles have
been derived from this model with TI’s up to 256. In this article,
the structure–activity relationships (SARs) at the thiazole-C2 posi-
tion have been investigated in order to enable the rational design
of more effective and less cytotoxic antiviral compounds.
Ar
6a-k
4a, 5a, 6a Ar = Ph
4b, 5b, 6b Ar = 2-ClC6H4
4c, 5c, 6c Ar = 3-BrC6H4
4d, 5d, 6d Ar = 4-BrC6H4
4e, 5e, 6e Ar = 2-BrC6H4
4f, 5f, 6f Ar = 4-FC6H4
4g, 5g, 6g Ar = 2-FC6H4
4h, 5h, 6h Ar = 4-MeOC6H4
4i, 5i, 6i Ar = 4-CF3C6H4
4j, 5j, 6j Ar = 3,4-Cl2C6H3
4k, 5k, 6k Ar = 2-naphthyl
4l, 5l Ar = 4-tertBut-C6H4
4m, 5m Ar = 4-nButylC6H4
4n, 5n Ar = 4-IC6H4
4o, 5o Ar = CH2C6H5
4p, 5p Ar = CH2pClC6H4
2. Result and discussion
2.1. Chemistry
Scheme 1. Reagents and conditions: (a) ethanol, heat to reflux, 12–24 h, 49–92%;
(b) NBS, UV irradiation, heat to reflux for 24 h, CCl4, 27–93%.
In order to synthesize methyl 4-pentylphenylthiazole-5-car-
boxylate 5q and its propyl analogue 5r, the thioamides 4q, r were
prepared from the corresponding amides 7a, b using Lawesson’s
reagent in dry THF (Scheme 2). These two thioamides 4q, r were
Treatment of methyl
a-chloroacetoacetate (3) with the appro-
priate thioamide derivatives 4a–p in absolute ethanol afforded,
in each case, the corresponding methyl ester derivatives 5a–p
(Scheme 1). Bromination of intermediates 5a–k, utilizing NBS
and UV light as a free radical initiator, gave the desired dibromom-
ethyl derivatives 6a–k, usually in moderate to good yields
(Scheme 1). The presence of the methine proton was confirmed
by 1H NMR spectra which revealed, in each case, a singlet at about
d 7.8 ppm. In addition, the dibromomethyl carbons of the products
were responsible for signals in the 13C NMR spectra that appeared
between 30.9–31.7 ppm.
treated with methyl a-chloroacetoacetate (3) as described for the
synthesis of the other methyl thiazole esters 5a–p in Scheme 1.
Hydrolysis of methyl ester 5m afforded the corresponding free
acid 8, which was converted into the acid chloride 9 by heating
with thionyl chloride (Scheme 3). Treatment of acid chloride 9 with
sodium methanethiolate afforded the corresponding thioester 10
as depicted in Scheme 3.
Virtual Screening
Hit Compound
Lead Compound
Development of SAR Model
H-bond acceptor or donor:
Metabolically
Toxic
Br
disfavored
unstable
Br
O
S
Hydrophilic:
Hydrophobic: disfavored
favored
S
OMe
H2N
N
Structural
optimization
N
S
O
S
Br
N
Hydrophobic:
X
Br
disfavored
Cl
x = O, CH2, N, S
Ar= 4-Cl-C6H4
Cl
Only one Br
atom is
necessary
Ar
2
1
EC50 = 2.8 µM
GI50 = 222 µM
TI = 78
EC50 = 31 µM
GI50 = 61µM
TI = 2
Rat plasma t1/2 = 1.4 h
Figure 2. Chemical structure of the hit compound 1 and lead compound 2, and SAR model of phenylthiazoles as antiflaviviral agents.

A. S. Mayhoub et al. / Bioorg. Med. Chem. 19 (2011) 3845–3854
3847
Thiazole methylketone derivative 11 was prepared in moderate
yield by heating thioamide 4m with 3-chloropentane-2,4-dione in
absolute ethanol (Scheme 4). The methyl ketone 11 was gently
heated with aminoguanidine hydrochloride in the presence lith-
ium chloride as a catalyst to afford hydrazinecarboximidamide
derivative 12 (Scheme 4).
O
S
NH
NH2
2 a
n
n
4q,r
7a,b
O
S
2.2. Biological results
OMe
N
All of the thiazole derivatives have been evaluated in a yellow
fever virus luciferase cellular assay. Compounds that showed suffi-
cient inhibitory activity (over 50%) on viral replication at a concen-
b
tration of 50 lM were considered to be active and were tested to
determine their antiviral EC50 values for inhibition of viral replica-
tion, as well as their GI50 values for inhibition of growth of unin-
fected cells.
n
5q,r
Initially, the para chlorine atom present on the C2-thiazole phe-
nyl ring was removed to investigate its biological effect (compound
6a). Interestingly, the resulting unsubstituted phenyl analogue 6a
revealed very weak antiflaviviral inhibitory activity (Table 1). This
initial result emphasized the biological importance of the phenyl
ring substitution. Next, the chlorine atom was replaced with fluo-
rine, bromine, and a methoxy group. The bromine-containing
derivative 6d showed a similar EC50 value to the lead compound
7a, 4q, 5q n = 4
7b, 4r, 5r n= 2
Scheme 2. Reagents and conditions: (a) Lawesson’s reagent, dry THF, 23 °C, 1 h,
55–57%; (b) methyl
51–55%.
a
-chloroacetoacetate, absolute ethanol, heat to reflux for 24 h,
O
Table 1
COOH
Cl
Antiviral activities and cytotoxicities of compounds versus yellow fever virusa
N
S
N
S
b
c
b
Compd
Inhibitiona (%)
GI50
(lM)
EC50 (lM)
TI
a
5m
2
99.6
<50
<50
<50
<50
<50
<50
<50
<50
À1672.6
À8.2
78.4
<50
91.9
À222.5
À23.8
À6.5
98.6
83.2
<50
<50
93
81.9
<50
<50
222.5 35.0
NDd
2.83 1.0
NDd
78
5a
5b
5c
5d
5e
5f
5g
5h
5i
NDd
NDd
NDd
NDd
NDd
NDd
n-Bu
n-Bu
NDd
NDd
8
9
NDd
NDd
NDd
NDd
O
S
NDd
NDd
S
NDd
NDd
c
5j
5k
5l
NDd
NDd
N
433.5 23.3
35.7 22.7
12
22
NDd
NDd
5m
5n
5o
5p
5q
5r
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
8
63.1 6.4
NDd
2.8 0.8
NDd
NDd
NDd
NDd
NDd
n-Bu
41.5 7.6
499.0 0.9
NDd
38.9 8.2
34.3 3.9
NDd
1.1
12
10
NDd
NDd
26
Scheme 3. Reagents and conditions: (a) (i) NaOH, methanol/H2O (3:5), heat to
reflux for 2 h, (ii) HCl, 100%; (b) SOCl2, heat to reflux for 2 h, 95%; (c) sodium
methanethiolate, dry CH2Cl2, 23 °C, 30 min, 81%.
26.5 1.5
407.5 78.3
NDd
1
121
3.3 2.1
NDd
NDd
NDd
<50
<50
NDd
NDd
NDd
NDd
91.0
42.2
26.0
83.2
94.1
99.8
99.2
261.7 27.9
10.6 3.1
26
NH2
NH
O
S
NDd
NDd
HN
N
NDd
NDd
365.7 63.2
46.5 5.6
59.4 4.2
352.8 28.8
202.9 14.7
45.6 5.6
10.1 3.0
2.4 0.3
N
10
11
12
1
6
147
b
N
S
a
4m
a
Measured as a reduction in luciferase activity of BHK cells infected with YF-
IRES-Luc at 50 M in comparison to the control.
l
b
n-Bu
The GI50 is the concentration of the compound causing a 50% growth inhibition
of uninfected BHK cells.
The EC50 is the concentration of the compound resulting in a 50% inhibition in
virus production.
ND indicates that the value was not determined; compounds that produced
inhibitory activity of less than 50% in the preliminary screening test were consid-
ered to be inactive and their exact EC50 and GI50 values were not determined.
n-Bu
c
11
12
d
Scheme 4. Reagents and conditions: (a) ethanol, 3-chloro-2,4-pentanedione, heat
to reflux, 24 h, 68%; (b) aminoguanidine hydrochloride, absolute ethanol, LiCl, heat
to reflux for 24 h, 46%.

3848
A. S. Mayhoub et al. / Bioorg. Med. Chem. 19 (2011) 3845–3854
2 (Table 1), while its fluorinated 6f and its methoxy-containing
derivative 6h revealed much weaker antiflaviviral activity (Table 1).
As a preliminary conclusion, both the size and the electronegativity
of the substituent may be considered to be important factors for
the antiviral properties. To test this tentative conclusion, a more
bulky trifluoromethyl-containing analogue 6i was synthesized
and tested. Compound 6i showed a significantly higher EC50 value
(Table 1). The effect of changing the halogen position was investi-
gated next. Ortho and meta halogen-containing analogues 6b, c, e,
and g were prepared. None of the ortho-substituted analogues
showed any antiflaviviral activity, and only the meta-substituted
derivative 6c revealed weak activity characterized by inhibition
of viral replication occurring at the cytotoxic concentration
(Table 1). Therefore, it was hypothesized that para substitution
confers biological activity while meta substitution is less effective.
Next, the disubstituted compounds 6j and k were synthesized.
Both compounds produced less than 50% inhibition of viral replica-
Since the branched compound 5l and the more bulky deriva-
tives 5o, p were found to be inactive (Table 1), it was hypothesized
that the hydrophobic pocket of the flaviviral E-protein might prefer
linear hydrophobic residues. Therefore, the optimal length of the
alkyl chain was studied. One carbon longer and shorter homo-
logues 5q and r of compound 5m were synthesized. The pentyl
and propyl derivatives 5q, r showed 12–14 times higher EC50 val-
ues than the butyl derivative 5m (Table 1), which means that a
length of four carbons is optimal.
Compound 5m has an EC50 value in the low micromolar range
and it lacks the dibromomethyl moiety. Its TI is lower than the lead
compound 2 and it contains a metabolically vulnerable methyl es-
ter that is expected to be hydrolyzed in human plasma to the cor-
responding inactive free acid 8 (Table 1). To improve the metabolic
stability as well as to increase the TI of 5m, the SAR analysis of sub-
stituents at the thiazole-5 position that was obtained from the pre-
vious study18 was applied to the methyl ester moiety. That
investigation improved the metabolic stability of the lead com-
pound 2 from 1.4 h to more than a day as measured in rat plasma
and it also improved the TI up to four times.18 Accordingly, the cor-
responding thioester and methyl ketone analogues of 5m were
synthesized. Although the corresponding methyl thioester and
methyl ketone congeners of the lead compound 2 were previously
found to be more active than the lead compound 2,18 the same
chemical modifications on 5m produced two less active com-
pounds 10 and 11 (Table 1).
The previously determined SAR model contains one more fea-
ture, a hydrophilically favorable region of the receptor, that was as-
sumed to exist around the ester moiety of the ligand (Fig. 2).18 Also,
active compounds that carried a polar amide moiety were calcu-
lated to form hydrogen bonds with E-protein polar residues
Ser274 and Gln271 that are close to the solvent-accessible region.
In addition, the hydrazinecarboximidamide moiety at the methyl
ester position was previously proposed to be tightly bound to three
polar residues in the solvent-accessible region (Gln200, Asp203,
and Gln271).15 A hydrazinecarboximidamide moiety was chosen
as a replacement for the methyl ester of 5m because it could pos-
sibly interact favorably with the polar residues on the surface of
the E-protein. The hydrazinecarboximidamide derivative 12 was
synthesized and it displayed a TI of 147 (Table 1). The TI of com-
pound 12 is improved in comparison with its corresponding
methyl ester 5m (Table 1) due to a decrease in cytotoxicity. That
may be because introducing a hydrazinecarboximidamido moiety
increases water solubility and that may hinder the ability of the
cation of 12 to penetrate biological membranes. Compound 12
was subjected to hydrolytic stability analysis utilizing lyophilized
rat plasma. The half-life of compound 12 was found to be 8.12 h.
This value is around 6 times higher than the lead compound 2,
which had a half-life of 1.41 h18 under the same experimental con-
ditions. Therefore, compound 12 provided the desired increased
metabolic stability, along with improved antiviral potency, lower
cytotoxicity, and an improved TI. In order to confirm that the im-
proved TI of compound 12 resulted from inhibition of viral replica-
tion and was not an artifact resulting from inhibition of luciferase,
an inhibition assay was performed on cells transfected with lucif-
erase-pcDNA3 (an optimized mammalian expression vector
expressing the fire-fly luciferase gene). No reduction of luciferase
activity was observed (data not shown).
tion at 50 lM in the preliminary screening test (Table 1). The non-
brominated naphthyl derivative 5k was the only compound that
does not contain a dibromomethyl moiety that had antiflaviviral
activity (Table 1). This observation is an advancement because
one of the original aims for optimizing the thiazole-C4 position
was to find a more chemically stable alternative to the dibromom-
ethyl moiety, since the presence of reactive bromides increases the
risk of toxicity.18–21 Although a great deal of effort was expended
to meet this goal, none of the dibromo replacements led to better
antiviral activity than the brominated lead compound 2, with the
exception of the monobromo analogue, which is expected to be
more susceptible to endogenous nucleophilic substitution because
of less steric hindrance and consequently more toxicity may be ex-
pected in vivo.18
The moderate activity of compound 5k can be rationalized from
the observation of the calculated position of the phenyl moiety of
the phenylthiazoles. The phenyl moiety was calculated to be
imbedded among hydrophobic residues (Val130, Phe193, Leu207,
Leu191, Phe279, and Ile270) regardless of the position and the ori-
entation of the top parts of the molecule (Fig. 3). Therefore, a set of
phenylthiazoles that carries different hydrophobic substitutions on
the para position of the phenyl ring with different spatial charac-
teristics was built (compounds 5l–p). Among this new set of com-
pounds 5l–p, compound 5m showed a very interesting result
(Table 1). It was the first phenylthiazole derivative that lacks the
mono- or dibromomethyl moiety and has an equivalent EC50 value
to the lead compound 2; however, its therapeutic index (TI) was
still lower than the lead compound 2.
The antiflaviviral activity of compound 12 can be rationalized
from molecular modeling (Fig. 4). The hydrazinecarboximidamido
moiety of the ligand is calculated to hydrogen bond to the Glu49
carboxylate. High flexibility was observed for the hydrazinecarbox-
imidamido moiety in lower ranked binding poses where different
hydrogen bonding possibilities have been observed between the
hydrazinecarboximidamide and other polar amino acid residues
such as Lys47 and Glu126. The hydrophobic n-butylphenyl tail is
Figure 3. The phenyl moiety of the highest-ranked binding poses of the lead
compound 2 surrounded by hydrophobic residues (bottom view) (PDB ID: 1OKE).
The stereoview is programmed for wall-eyed (relaxed) viewing.

A. S. Mayhoub et al. / Bioorg. Med. Chem. 19 (2011) 3845–3854
3849
Figure 4. Hypothetical model of compound 12 in the dengue viral 2 E-protein b-OG binding pocket (PDB ID: 1OKE). The stereoview is programmed for wall-eyed (relaxed)
viewing.
modeled within the hydrophobic core of the b-OG pocket as shown
in Figure 4.
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 5i,22 5l,23 5o, and 5p24 were previously reported.
3. Conclusion
4.2. Preparation of thioamides
Optimizing the thiazole-C2 position of the phenylthiazole scaf-
fold led to the first non-brominated phenylthiazole 5k to show
antiflaviviral activity. Further structural optimization documented
a significant impact of substituents present on the para position of
the phenyl ring on antiflaviviral activity. With this in mind, the 2-
naphthyl moiety of 5k was optimized to the n-butylphenyl
analogue 5m. Compound 5m was the first non-brominated phe-
nylthiazole derivative that had an EC50 value that was equivalent
to the lead compound 2. To improve the metabolic stability and
antiviral selectivity of 5m, the SAR analysis of the thiazole-5 posi-
tion that was obtained from the previous study18 was applied to
the methyl ester moiety of 5m and that furnished a drug-like com-
pound 12 with an excellent TI value and higher plasma stability
character. Lastly, a new comprehensive SAR model has been estab-
lished that includes the thiazole-C2, -C4, and -C5 positions (Fig. 5).
General procedure: Amides 7 (1 mmol) and Lawesson’s reagent
(490 mg, 1.2 mmol) were added to dry THF (15 mL). The reaction
mixture was stirred at room temperature for 1 h. The solvent
was evaporated under reduced pressure and the residue was
partitioned between aq NaHCO3 (25 mL) and ethyl acetate
(25 mL). The organic solvent was separated and dried over anhy-
drous MgSO4. The crude product was further purified by silica
gel flash chromatography, using hexane–ethyl acetate (4:1), to
yield the corresponding thioamides as yellow solids (42–60%).
Non-commercially available amides 4-n-pentylbenzamide (7a),25
and n-propylbenzamide (7b)26 were prepared as previously
reported.
4.2.1. 4-Pentylbenzothioamide (4q)
Yellow solid (57%): mp 56 °C. 1H NMR (CDCl3) d 8.24 (br s, 1H),
7.75 (d, J = 8.1 Hz, 2H), 7.50 (br s, 1H), 7.15 (d, J = 8.1 Hz, 1H), 2.59
(t, J = 7.5 Hz, 2H), 1.58 (m, J = 7.2 Hz, 2H), 1.29 (m, 4H), 0.88 (t,
J = 7.2 Hz, 3H); 13C NMR (CDCl3) d 202.05, 147.80, 136.18, 128.42,
127.11, 35.71, 31.36, 30.75, 22.46, 14.00; CIMS m/z (rel intensity)
208 (MH+, 100); HR-MS (CI), m/z 208.1158 MH+, calcd for
4. Experimental section
4.1. General
Melting points were determined in capillary tubes using a Mel-
Temp apparatus and are not corrected. 1H NMR spectra were run at
300 MHz and 13C spectra were determined at 75.46 MHz in deuter-
ated chloroform (CDCl3) or dimethyl sulfoxide (DMSO-d6). Chemi-
cal shifts are given in parts per million (ppm) on the delta (d) scale.
C12H18NS 208.1160.
4.2.2. 4-Propylbenzothioamide (4r)
Yellow solid (55%): mp 57 °C. 1H NMR (CDCl3) d 7.88 (br s, 1H),
7.77 (d, J = 8.4 Hz, 2H), 7.24 (br s, 1 H), 7.23 (d, J = 8.4 Hz, 1H), 2.54
(t, J = 7.5 Hz, 2H), 1.57 (m, J = 7.5 Hz, 2H), 0.86 (t, J = 7.5 Hz, 3H);
13C NMR (CDCl3) d 202.49, 147.51, 136.35, 128.52, 126.93, 37.77,
24.18, 13.68; ESI-MS m/z (rel intensity) 180 (MH+, 100); HR-MS
(ESI), m/z 180.0841 MH+, calcd for C10H14NS 180.0841.
H-bond acceptor or donor:
disfavored
Hydrophilic:
Hydrophobic: disfavored
favored
4.3. Preparation of methyl thiazole-5-carboxylates 5a–r
Hydrophobic:
General procedure: Appropriate thiobenzamides or thioaceta-
X
disfavored
mides (1 mmol) and
a-chloroacetoacetate 3 (150 mg, 1.2 mmol)
N
S
were added to absolute ethanol (15 mL). The reaction mixture
was heated at reflux for 24 h. After removal of solvent under re-
duced pressure, the residue was purified by silica gel chromatogra-
phy using hexanes–ethyl acetate (7:3) to provide the desired
compounds.
X is a polar moiety
No Br atom is
necessary
Linear lipophilic: favored
Optimal length: 4 units
Y
Bulkness:
disfavoured
4.3.1. Methyl 4-methyl-2-phenylthiazole-5-carboxylate (5a)
White solid (120 mg, 70%): mp 110–112 °C. 1H NMR (CDCl3) d
7.94 (m, 2H), 7.44 (m, 3H), 3.87 (s, 3H), 2.77 (s, 3H); 13C NMR
Figure 5. New SAR model of phenylthiazoles as antiflaviviral agents.

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A. S. Mayhoub et al. / Bioorg. Med. Chem. 19 (2011) 3845–3854
(CDCl3) d 169.9, 162.5, 161.2, 132.7, 130.9, 128.9 Â 2, 126.7 Â 2,
121.2, 52.0, 17.4; IR (KBr) 2925, 2851, 1717, 1266, 1095,
764 cmÀ1; ESI-MS m/z (rel intensity) 233.97 (MH+, 100). Anal.
Calcd for C12H9Br2NO2S: C, 36.85; H, 2.32; N, 3.58. Found: C,
37.23; H, 2.14; N, 3.51.
4.3.8. Methyl 2-(4-methoxyphenyl)-4-methylthiazole-5-carb-
oxylate (5h)
White solid (62 mg, 79%): mp 118–120 °C. 1H NMR (CDCl3) d
7.90 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 3.87 (s, 3H), 3.85
(s, 3H), 2.75 (s, 3H); 13C NMR (CDCl3) d 169.8, 162.7, 161.8,
161.1, 128.3 Â 2, 125.6, 120.2, 114.2 Â 2, 55.3, 51.9, 17.4; IR (KBr)
2994, 2949, 2840, 1715, 1521, 1437, 1271, 1258, 1096, 823,
758 cmÀ1; ESI-MS m/z (rel intensity) 263.94 (MH+, 100). Anal.
Calcd for C13H13NO3S: C, 59.30; H, 4.98; N, 5.32. Found: C, 59.12;
H, 4.92; N, 5.21
4.3.2. Methyl 2-(2-chlorophenyl)-4-methylthiazole-5-carboxy-
late (5b)
White solid (60 mg, 73%): mp 146–148 °C. 1H NMR (CDCl3) d
8.32 (m, 1H), 7.49 (m, 1H), 7.38 (m, 2H), 3.90 (s, 3H), 2.80 (s,
3H); 13C NMR (CDCl3) d 131.0, 130.8, 130.6, 127.0, 52.0, 17.3; IR
(KBr) 1714, 1266, 1100, 763 cmÀ1; ESI-MS m/z (rel intensity)
267.87 (MH+, 100). Anal. Calcd for C12H10ClNO2S: C, 53.83; H,
3.76; N, 5.23. Found: C, 53.48; H, 3.59; N, 5.08.
4.3.9. Methyl 2-(3,4-dichlorophenyl)-4-methylthiazole-5-carb-
oxylate (5j)
White solid (88%): mp 68 °C. 1H NMR (CDCl3) d 8.05 (d,
J = 1.2 Hz, 1H), 7.73 (dd, J = 1.5, 8.1 Hz, 1H), 7.47 (dd, J = 1.5,
8.0 Hz, 1H), 3.88 (s, 3H), 2.75 (s, 3H); 13C NMR (CDCl3) d 166.88,
162.28, 161.36, 135.05, 133.47, 132.60, 130.95, 128.27, 125.69,
122.14, 52.25, 17.41; ESI-MS m/z (rel intensity) 304/302 (MH+,
40/56); HR-MS (ESI), m/z 301.9806 MH+, calcd for C12H10Cl2NO2S
301.9804; HPLC purity 95.05%.
4.3.3. Methyl 2-(3-bromophenyl)-4-methylthiazole-5-carboxy-
late (5c)
White solid (65 mg, 70%): mp 96–98 °C. 1H NMR (CDCl3) d 8.14
(s, 1H), 7.86 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 7.5, 1H), 7.32 (dd, J = 7.5,
7.5 Hz, 1H), 3.90 (s, 3H), 2.78 (s, 3H); 13C NMR (CDCl3) d 133.7,
130.4, 129.4, 125.3, 52.2, 17.3; IR (KBr) 2990, 2848, 1713, 1518,
1424, 1257, 1098, 779 cmÀ1; ESI-MS m/z (rel intensity) 311.88
(MH+, 100). Anal. Calcd for C12H10BrNO2S: C, 46.17; H, 3.23; N,
4.49. Found: C, 46.07; H, 3.10; N, 4.39.
4.3.10. Methyl 4-methyl-2-(naphthalen-2-yl)thiazole-5-carb-
oxylate (5k)
White solid (49%): mp 58 °C. 1H NMR (CDCl3) d 8.43 (s, 1H),
7.97–7.80 (m, 4H), 7.52 (dt, J = 1.5, 4.8, 7.5 Hz, 2H), 3.88 (s, 3H),
2.81 (s, 3H); 13C NMR (CDCl3) d 169.97, 162.60, 161.34, 134.47,
133.03, 130.10, 128.80, 127.80, 127.43, 126.88, 126.56, 123.73,
121.31, 52.12, 17.55; ESI-MS m/z (rel intensity) 284 (MH+, 100);
HR-MS (ESI), m/z 284.0748 MH+, calcd for C16H14NO2S 284.0745;
HPLC purity 95.30%.
4.3.4. Methyl 2-(4-bromophenyl)-4-methylthiazole-5-carboxy-
late (5d)
White solid (70 mg, 75%): mp 136–138 °C. 1H NMR (CDCl3) d
7.82 (s, J = 8.5 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 3.89 (s, 3H), 2.77
(s, 3H); 13C NMR (CDCl3) d 133.0, 130.4, 128.0, 52.2, 17.3; IR
(KBr) 2918, 2848, 1716, 1519, 1431, 1277, 1096, 821 cmÀ1; ESI-
MS m/z (rel intensity) 312.05 (MH+, 100). Anal. Calcd for
4.3.11. Methyl 2-(4-butylphenyl)-4-methylthiazole-5-carb-
oxylate (5m)
Colorless viscous oil (92%). 1H NMR (CDCl3) d 7.85 (d, J = 8.0 Hz,
2H), 7.22 (d, J = 8.4 Hz, 2H), 3.85 (s, 3H), 2.75 (s, 3H), 2.64 (t,
J = 7.8 Hz, 3H), 1.58 (m, J = 7.4 Hz, 2H), 1.34 (m, J = 7.4 Hz, 2H),
0.91 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3) d 170.21, 162.67, 161.21,
146.43, 130.40, 129.02, 126.71, 120.70, 52.01, 35.52, 33.26, 22.26,
17.48, 13.87; ESI-MS m/z (rel intensity) 290 (MH+, 98); HR-MS
(ESI), m/z 290.1215 MH+, calcd for C16H20NO2S 290.1209; HPLC
purity 98.05%.
C12H10BrNO2S: C, 46.17; H, 3.23; N, 4.49. Found: C, 45.78; H,
3.10; N, 4.50.
4.3.5. Methyl 2-(2-bromophenyl)-4-methylthiazole-5-
carboxylate (5e)
White solid (70 mg, 75%): mp 126–128 °C. 1H NMR (CDCl3) d
8.14 (d, J = 6.6 Hz, 1H), 7.70 (d, J = 6.6 Hz, 1H), 7.40 (dd, J = 6.6,
6.6 Hz, 1H), 7.29 (dd, J = 6.6, 6.6 Hz, 1H). 3.90 (s, 3H), 2.80 (s,
3H); 13C NMR (CDCl3) d 166.5, 162.6, 159.7, 134.1, 133.3, 131.5,
131.1, 127.5, 122.8, 121.6, 52.1, 17.2; IR (KBr) 2920, 2850, 1716,
1527, 1265, 1101, 761 cmÀ1; ESI-MS m/z (rel intensity) 312.01
(MH+, 100). Anal. Calcd for C12H10BrNO2S: C, 46.17; H, 3.23; N,
4.49. Found: C, 46.07; H, 3.11; N, 4.40.
4.3.12. Methyl 2-(4-iodophenyl)-4-methylthiazole-5-carb-
oxylate (5n)
White solid (86%): mp 148–149 °C. 1H NMR (CDCl3) d 7.74 (d,
J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H), 3.87 (s, 3H), 2.75 (s, 3H);
13C NMR (CDCl3)
d 168.67, 162.43, 161.32, 138.14, 132.25,
128.10, 121.57, 97.52, 52.21, 17.48; ESI-MS m/z (rel intensity)
360 (MH+, 100); HR-MS (ESI), m/z 359.9555 MH+, calcd for C12H11I-
NO2S 359.9550; HPLC purity 97.35%.
4.3.6. Methyl 2-(4-fluorophenyl)-4-methylthiazole-5-carboxy-
late (5f)
White solid (52 mg, 70%): mp 90–92 °C. 1H NMR (CDCl3) d 7.95
(m, 2H), 7.14 (m, 2H), 3.89 (s, 3H), 2.77 (s, 3H); 13C NMR (CDCl3) d
169.8, 162.7, 161.8, 161.1, 128.7 Â 2, 125.6, 120.2, 116.2, 115.9,
52.1, 17.4; IR (KBr) 2958, 2924, 2850, 1726, 1521, 1439, 1264,
1091, 834, 756 cmÀ1; ESI-MS m/z (rel intensity) 252 (MH+, 100).
Anal. Calcd for C12H10FNO2S: C, 57.36; H, 4.01; N, 5.57. Found: C,
56.64; H, 4.10; N, 5.70.
4.3.13. Methyl 4-methyl-2-(4-pentylphenyl)thiazole-5-carb-
oxylate (5q)
Colorless oil (166 mg, 55%). 1H NMR (CDCl3) d 7.82 (d, J = 8.4 Hz,
2H), 7.20 (d, J = 8.4 Hz, 2H), 3.83 (s, 3H), 2.74 (s, 3H), 2.59 (t,
J = 7.5 Hz, 2H), 1.59 (m, J = 7.2 Hz, 2H), 1.30 (m, 4 H), 0.87 (t,
J = 7.5 Hz, 3H); 13C NMR (CDCl3) d 170.16, 162.61, 146.41, 130.37,
128.99, 126.68, 120.67, 51.96, 35.78, 31.38, 30.79, 22.46, 17.44,
13.95; ESI-MS m/z (rel intensity) 304 (MH+, 100); HR-MS (ESI),
m/z 304.1368 MH+, calcd for C17H22NO2S 304.1366; HPLC purity
95.10%.
4.3.7. Methyl 2-(2-fluorophenyl)-4-methylthiazole-5-carboxy-
late (5g)
White solid (60 mg, 80%): mp 100–102 °C. 1H NMR (CDCl3) d
8.34 (t, J = 9.0 Hz, 1H), 7.43 (m, 1H), 7.19 (m, 2H); 13C NMR (CDCl3)
d 165.13, 160.99, 161.05, 159.15, 132.02, 131.91, 128.88, 124.57,
116.26, 52.04, 17.29; ESI-MS m/z (rel intensity) 252 (MH+, 100);
HR-MS (ESI), m/z 252.0492 MH+, calcd for C12H11FNO2S
252.0489; HPLC purity 95.00%.
4.3.14. Methyl 4-methyl-2-(4-propylphenyl)thiazole-5-carboxy-
late (5r)
White solid (140 mg, 51%): mp 41 °C. 1H NMR (CDCl3) d 7.84
(d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 3.85 (s, 3 H), 2.75 (s,

A. S. Mayhoub et al. / Bioorg. Med. Chem. 19 (2011) 3845–3854
3851
3H), 2.60 (t, J = 7.5 Hz, 2H), 1.64 (m, J = 7.5 Hz, 2H), 0.93 (t,
J = 7.2 Hz, 3H); 13C NMR (CDCl3)
170.22, 162.68, 161.20,
146.20, 130.43, 129.08, 126.70, 120.72, 52.01, 37.86, 24.23,
17.47, 13.69; ESI-MS m/z (rel intensity) 276 (MH+, 100); HR-MS
(ESI), m/z 276.1058 MH+, calcd for C15H18NO2S 276.1053; HPLC
purity 95.20%.
4.4.6. Methyl 4-(dibromomethyl)-2-(4-fluorophenyl)thiazole-5-
carboxylate (6f)
d
White solid (128 mg, 63%): mp 160–162 °C. 1H NMR (CDCl3) d
8.05 (m, 2H), 7.69 (s, 1H), 7.17 (m, 2H), 3.94 (s, 3H); 13C NMR
(CDCl3) d 170.27, 166.53, 160.99, 158.89, 129.34, 128.58, 119.84,
116.49, 52.94, 31.16; IR (KBr) 3063, 2960, 1717, 1287, 842,
631 cmÀ1; ESI-MS m/z (rel intensity) 407.73 (MH+, 100); HPLC pur-
ity 96.87%.
4.4. Preparation of 6a–k
4.4.7. Methyl 4-(dibromomethyl)-2-(2-fluorophenyl)thiazole-5-
carboxylate (6g)
General procedure: The ester 5 (0.5 mmol) and NBS (531 mg,
3 mmol) were added to CCl4 (10 mL). The reaction mixture was
heated at reflux for 6 h, during which time it was irradiated by
an ultraviolet sunlamp (GE, 215 W). After removal of solvent under
reduced pressure, the residue was purified by silica gel column
chromatography (ethyl acetate–hexanes 1:4) to provide the de-
sired compounds.
White solid (142 mg, 70%): mp 132–134 °C. 1H NMR (CDCl3) d
8.49 (ddd, J = 7.5, 7.5, 1.8 Hz, 1H), 7.73 (s, 1H), 7.49 (m, 1H), 7.32
(m, 1H), 7.23 (m, 1H), 3.95 (s, 3H); 13C NMR (CDCl3) d 163.6,
161.4, 161.1, 159.4, 157.7, 132.8, 129.5, 124.8, 120.7, 116.1, 52.8,
31.3; IR (KBr) 1707, 1587, 1516, 1455, 1291, 1100, 633 cmÀ1
;
ESI-MS m/z (rel intensity) 407.72 (MH+, 100); HPLC purity 96.81%.
4.4.1. Methyl 4-(dibromomethyl)-2-phenylthiazole-5-carboxy-
late (6a)
4.4.8. Methyl 4-(dibromomethyl)-2-(4-methoxyphenyl)-
thiazole-5-carboxylate (6h)
White solid (117 mg, 60%): mp 176–178 °C. 1H NMR (CDCl3) d
8.03 (m, 2H), 7.71 (s, 1H), 7.50 (m, 3H), 3.94 (s, 3H); 13C NMR
(CDCl3) d 171.5, 161.0, 158.8, 132.1, 131.7, 129.0 Â 2, 127.0 Â 2,
116.9, 52.8, 31.2; IR (KBr) 3057, 2947, 2918, 2849, 1716, 1521,
1434, 1283, 1092, 719, 629 cmÀ1; ESI-MS m/z (rel intensity)
389.69 (MH+, 100). Anal. Calcd for C12H9Br2NO2S: C, 36.85; H,
2.32; N, 3.58. Found, C, 37.23; H, 2.14; N, 3.51.
White solid (127 mg, 60%): mp 104–106 °C. 1H NMR (CDCl3) d
7.97 (m, 2H), 7.69 (s, 1H), 6.96 (m, 2H), 3.92 (s, 3H), 3.87 (s, 3H);
13C NMR (CDCl3) d 171.4, 162.4, 161.2, 158.7, 128.7 Â 2, 128.6,
125.0, 114.3 Â 2, 55.4, 52.7, 31.4; IR (KBr) 2919, 2850, 1701,
1422, 1321, 1167, 814, 640 cmÀ1; ESI-MS m/z (rel intensity)
263.94 (MH+, 100). Anal. Calcd for C13H11Br2NO3S): C, 37.08; H,
2.63; N, 3.33. Found: C, 37.05; H, 2.59; N, 3.24.
4.4.2. Methyl 2-(2-chlorophenyl)-4-(dibromomethyl)thiazole-5-
carboxylate (6b)
4.4.9. Methyl 4-(dibromomethyl)-2-(4-(trifluoromethyl)-
phenyl)thiazole-5-carboxylate (6i)
White solid (133 mg, 63%): mp 154–156 °C. 1H NMR (CDCl3) d
8.53 (m, 1H), 7.72 (s, 1H), 7.50 (m, 1H), 7.43 (m, 2H), 3.96 (s,
3H); 13C NMR (CDCl3) d 166.5, 161.2, 157.3, 132.3, 131.7, 131.4,
130.6, 130.5, 127.3, 121.0, 52.8, 31.3; IR (KBr) 3045, 2956, 2848,
1706, 1520, 1433, 1283, 1099, 756, 632 cmÀ1; ESI-MS m/z (rel
intensity) 423.49 (MH+, 100). Anal. Calcd for C12H9Br2NO2S: C,
36.85; H, 2.32; N, 3.58. Found: C, 37.23; H, 2.14; N, 3.51.
White solid (67 mg, 87%): mp 81–82 °C. 1H NMR (CDCl3) d 8.16
(d, J = 8.4 Hz, 2H), 8.75 (d, J = 8.4 Hz, 2H), 7,70 (s, 1H), 3.96 (s, 3H);
13C NMR (CDCl3) d 169.48, 160.81, 159.13, 135.19, 131.01, 127.59,
127.24, 126.37, 125.94, 53.25, 31.12; CIMS m/z (rel intensity) 462/
460/458 (MH+, 57/100/50); HR-MS (ESI), m/z 457.8670 MH+, calcd
for C13H9Br2F3NO2S 456.8667.
4.4.10. Methyl 4-(dibromomethyl)-2-(3,4-dichlorophenyl)-
thiazole-5-carboxylate (6j)
4.4.3. Methyl 2-(3-bromophenyl)-4-(dibromomethyl)thiazole-
5-carboxylate (6c)
White solid (849 mg, 93%): mp 144–145 °C. 1H NMR (CDCl3) d
8.16 (d, J = 2.1 Hz, 1H), 7.83 (dd, J = 2.1, 8.4 Hz, 1H), 7.68 (s, 1H),
7.55 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H); 13C NMR (CDCl3) d 168.63,
161.53, 160.76, 159.05, 136.00, 133.72, 131.90, 131.12, 128.66,
126.04, 120.58, 53.08, 30.83; APCIMS m/z (rel intensity) 464/462/
460/458 (MH+, 21/80/100/36); HR-MS (CI), m/z 457.8019 MH+,
calcd for C12H8Br2Cl2NO2S 457.9014; HPLC purity 97.09%.
White solid (140 mg, 60%): mp 111–113 °C. 1H NMR (CDCl3) d
8.21 (s, 1H), 7.92 (dd, J = 10.8, 0.6 Hz, 1H), 7.69 (s, 1H), 7.63 (dd,
J = 10.8, 0.6 Hz, 1H), 7.35 (m, 1H), 3.95 (s, 3H); 13C NMR (CDCl3) d
169.5, 160.8, 158.8, 134.5, 133.8, 130.5, 129.7, 125.6, 123.2,
120.3, 52.9, 30.9; IR (KBr) 3221, 1705, 1427, 1316, 1176, 814,
640 cmÀ1; ESI-MS m/z (rel intensity) 467.65 (MH+, 100); HPLC pur-
ity 100%.
4.4.11. Methyl 4-(dibromomethyl)-2-(naphthalen-2-yl)thiazole-
5-carboxylate (6k)
4.4.4. Methyl 2-(4-bromophenyl)-4-(dibromomethyl)thiazole-
5-carboxylate (6d)
Off-white solid (224 mg, 27%): mp >360 °C. 1H NMR (CDCl3) d
8.57 (s, 1H), 8.08 (d, J = 1.8 Hz, 1H), 7.94–7.80 (m, 3H), 7.75 (s,
1H), 7.59–7.56 (m, 2H), 3.96 (s, 3H); 13C NMR (CDCl3) d 171.65,
161.12, 159.01, 134.82, 132.96, 129.50, 129.01, 128.96, 127.87,
127.26, 127.08, 123.79, 119.84, 52.93, 31.36; CIMS m/z (rel inten-
sity) 443/441/439 (MH+, 50/100/64); HR-MS (CI), m/z 439.8958
MH+, calcd for C16H12Br2NO2S 439.8955; HPLC purity 95.09%.
White solid (140 mg, 60%): mp 120–122 °C. 1H NMR (CDCl3) d
7.93 (d, J = 8.4 Hz, 2H), 7.70 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 3.95
(s, 3H); 13C NMR (CDCl3) d 170.1, 160.8, 158.9, 132.3 Â 2, 131.0,
128.4, 126.3, 120.0, 52.9, 31.0; IR (KBr) 3046, 1712, 1447, 1289,
1093, 829, 622 cmÀ1; ESI-MS m/z (rel intensity) 467.48 (MH+,
100); HPLC purity 96.00%.
4.4.5. Methyl 2-(2-bromophenyl)-4-(dibromomethyl)thiazole-
5-carboxylate (6e)
4.5. 2-(4-Butylphenyl)-4-methylthiazole-5-carboxylic acid (8)
White solid (128 mg, 63%): mp 159–161 °C. 1H NMR (CDCl3) d
8.25 (d, J = 8.1 Hz, 1H), 7.63 (s, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.52
(m, 2H), 3.82 (s, 3H); 13C NMR (CDCl3) d 164.74, 162.74, 159.80,
133.03, 132.10, 131.87, 131.56, 131.04, 128.75, 123.26, 53.29,
31.4; ESI-MS m/z (rel intensity) 468/470/472/474 (MH+, 23/91/
100/26); HR-MS (ESI), m/z 467.7902 MH+, calcd for C12H9Br3NO2S
467.7899; HPLC purity 95.86%.
NaOH (200 mg, 5 mmol) was added to a solution of methyl ester
5m (290 mg, 1 mmol) in methanol (6 mL) and water (10 mL). The
reaction mixture was heated under reflux for 2 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 to provide the corresponding
carboxylic acid as a white solid (100%): mp 147–148 °C. 1H NMR

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A. S. Mayhoub et al. / Bioorg. Med. Chem. 19 (2011) 3845–3854
(DMSO-d6) d 7.87 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 2.65 (s,
3H), 2.62 (t, J = 7.2 Hz, 3H), 1.60 (m, J = 7.2 Hz, 2H), 1.36 (m,
J = 7.2 Hz, 2H), 0.86 (t, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) d
170.21, 163.89, 160.50, 148.44, 130.17, 127.40, 35.57, 33.69,
22.64, 18.04, 14.69; ESI-MS m/z (rel intensity) 276 (MH+, 36), 178
(100); HR-MS (ESI), m/z 276.1063 MH+, calcd for C15H8NO2S
276.1058.
added. The reaction mixture was heated at reflux for 24 h. The
solvent was evaporated under reduced pressure. The crude prod-
uct was purified by crystallization from 70% methanol, then
recrystallized from methanol to afford the desired compound as
a
off-white solid (78 mg, 46%): mp 230–231 °C. 1H NMR
(DMSO-d6) d 11.49 (br s, 1H), 7.80 (d, J = 7.8 Hz, 2H) 7.76 (br s,
3H), 7.31 (d, J = 7.8 Hz, 2H), 2.61 (t, J = 7.2 Hz, 2H), 2.59 (s, 3H),
2.42 (s, 3H), 1.56 (m, J = 6.9 Hz, 2H), 1.30 (m, J = 6.9 Hz, 2H),
0.89 (t, J = 7.5 Hz, 3H); 13C NMR (DMSO-d6) d 170.23, 161.05,
157.50, 152.36, 150.52, 135.40, 135.33, 134.34, 131.14, 39.80,
37.97, 26.92, 23.36, 23.30, 18.93; ESI-MS m/z (rel intensity) 330
(MH+, 100); HR-MS (ESI), m/z 330.1751 MH+, calcd for
4.6. 2-(4-Butylphenyl)-4-methylthiazole-5-carbonyl chloride
(9)
The carboxylic acid 8 (275 mg, 1 mmol) was heated under re-
flux with thionyl chloride (6 mL) for 2 h. The solvent was evapo-
rated under reduced pressure. The brown residue was collected
and purified by silica gel flash chromatography, using hexane–
ethyl acetate (7:3), to yield the corresponding acid chloride as vis-
cous colorless oil (95%). 1H NMR (CDCl3) d 7.88 (d, J = 8.4 Hz, 2H),
7.26 (d, J = 8.4 Hz, 2H), 2.72 (s, 3H), 2.66 (t, J = 7.8 Hz, 3H), 1.61
(m, J = 7.5 Hz, 2H), 1.36 (m, J = 7.5 Hz, 2H), 0.94 (t, J = 7.5 Hz, 3H);
C17H24N5S; HPLC purity 95.43%.
4.10. Bioassay methods
4.10.1. BHK cells
BHK-15 cells obtained from the American Type Culture Collec-
tion (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.
13C NMR (CDCl3)
d 174.11, 164.12, 158.19, 147.79, 129.74,
129.25, 126.97, 126.82, 35.63, 33.21, 22.29, 18.51, 13.89; CIMS
m/z (rel intensity) 295/293 (MH+, 36/100); HR-MS (CI), m/z
294.0725 MH+, calcd for C15H17ClNOS 294.0719.
4.10.2. 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 over-
lapping 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.27
4.7. S-Methyl 2-(4-butylphenyl)-4-methylthiazole-5-
carbothioate (10)
The acid chloride 9 (58 mg, 0.2 mmol) was stirred with sodium
methanthiol (12 mg, 1.7 mmol) in dry dichloromethane for
30 min. The solvent was evaporated 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 crys-
tallization from MeOH to afford the title compound as a yellow
solid (81%): mp 35 °C. 1H NMR (CDCl3) d 7.85 (d, J = 8.1 Hz, 2H),
7.24 (d, J = 8.1 Hz, 2H), 2.76 (s, 3H), 2.63 (t, J = 7.6 Hz, 3H), 1.60
(m, J = 7.5 Hz, 2H), 1.35 (m, J = 7.5 Hz, 2H), 0.92 (t, J = 7.5 Hz,
3H); 13C NMR (CDCl3) d 183.58, 169.68, 158.13, 146.71, 130.20,
129.43, 129.08, 126.85, 35.56, 33.27, 22.29, 18.34, 13.89, 12.58;
ESI-MS m/z (rel intensity) 306 (MH+, 100); HR-MS (ESI), m/z
306.0990 MH+, calcd for C16H20NOS2 306.0986; HPLC purity
95.01%
4.10.3. 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 har-
vested 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.
4.8. 1-(2-(4-Butylphenyl)-4-methylthiazol-5-yl)ethanone (11)
4.10.4. 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 re-
leased virus could be monitored. Cells were then overlaid with
culture media containing serial dilutions of compounds at con-
centrations below the GI50 values. Controls included uninfected
cells, infected cells, and DMSO-treated infected cells. Cells were
4-n-Butylthiobenzamide (4m, 450 mg, 2.3 mmol) and 3-chloro-
pentane-2,4-dione (0.32 mL, 2.8 mmol) were added to absolute
ethanol (10 mL). The reaction mixture was heated at reflux for
24 h. After evaporation of solvent under reduced pressure, the
brown residue was collected and purified by silica gel flash chro-
matography, using hexane–ethyl acetate (9:1), to yield the desired
compound as a yellowish oil (432 mg, 68%). 1H NMR (CDCl3) d 7.75
(d, J = 8.7 Hz, 2H), 7.13 (d, J = 8.7 Hz, 2H), 2.65 (s, 3H), 2.53 (t,
J = 5.2 Hz, 2H), 2.40 (s, 3H), 1.52 (m, J = 5.2 Hz, 2H), 1.29 (m,
J = 5.2 Hz, 2H), 0.85 (t, J = 5.0 Hz, 3H); 13C NMR (CDCl3) d 190.10,
169.35, 159.26, 146.47, 130.62, 130.18, 128.95, 126.68, 35.47,
33.17, 30.53, 22.26, 18.33, 13.85; ESI-MS m/z (rel intensity) 274
(MH+, 100); HR-MS (ESI), m/z 274.1262 MH+, calcd for C16H20NOS
274.1260; HPLC purity 99.38%.
incubated at 37 °C, 5% CO2 for ꢀ36 h, lysed using 50
l
L of cell
L of cell
culture lysis buffer (Promega Inc., Madison, WI), and 10
l
extracts placed into a 96-well opaque plate. Luciferase activity
was determined from the luminescence generated with fire-fly
luciferase substrate (Promega Inc., Madison, WI). Luminescense
was measured in a 96-well-plate luminometer, LMax II (Molecu-
lar Devices, Sunnyvale, CA). A reduction in luciferase activity indi-
cates inhibition of YFV-IRES-Luc virus growth. The luciferase
luminescence as a function of compound concentration was ana-
lyzed by non-linear regression analysis using GraphPadPrizm 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.
4.9. 2-(1-(2-(4-Butylphenyl)-4-methylthiazol-5-yl)ethylidene)-
hydrazinecarboximidamide (12)
The thiazole derivative 11 (230 mg, 0.83 mmol) was dissolved
in absolute ethanol (10 mL), and aminoguanidine hydrochloride
(110 mg, 1 mmol) and a catalytic amount of LiCl (5 mg) were

A. S. Mayhoub et al. / Bioorg. Med. Chem. 19 (2011) 3845–3854
3853
4.10.5. Cell viability assay
of 4-bromopyrazole as an internal standard were dissolved in
DMSO (1.0 mL). This solution was filtered through a 0.45 M filter
BHK cells were plated in a 96-well plate and grown at 37 °C. At
confluency, cells were overlaid with culture media containing seri-
al 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
l
(Millex-HN). Lyophilized rat plasma (1.0 mL) (LOT# 048K7420, Sig-
ma Chemical Co., St. Louis, Mo) was reconstituted with water
(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 compound 12 in
10
l
L of XTT-substrate from the Quick Cell Proliferation Kit (Biovi-
DMSO (100
mixture was incubated at 37 °C throughout the course of the
experiment. Aliquots (10 L) of the compound-plasma mixture
were collected at various time intervals and diluted with methanol
(90 L) to precipitate any proteins present. The aliquots were
lL) was added to the rat plasma (0.75 mL) and the
sion 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, 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.
l
l
mixed and centrifuged at 10,000 rpm for 5–10 min to pellet the
precipitated proteins. After centrifugation, the supernatants
(20 lL) of the aliquots were analyzed by HPLC to determine the
4.10.6. Luciferase-pcDNA3
residual amount of tested compounds present in the sample. The
A fire-fly luciferase gene was inserted into a pcDNA3 backbone
to construct a mammalian expression vector of fire-fly luciferase
(Addgene plasmid 18964). Briefly, the fire-fly luciferase gene was
excised from pGL3-Basic Vector (Promega Inc., Madison, WI), and
inserted in pcDNA3 (Invitrogen, Carlsbad, CA) by using theHindIII
and XbaI restriction sites.28
aliquot supernatants were analyzed using a Waters binary HPLC
system (Model 1525, 10 lL injection loop) and a Waters dual
wavelength absorbance UV detector (Model 2487) set for
254 nM. Data were collected and processed using the Breeze soft-
ware (version 3.3) on a Dell Optiplex GX280 personal computer.
The mobile phase consisted of 85:15 (v/v) methanol/water and
the SunriseÒ HPLC column (4.6 mm  150 mm) was packed with
C18 Silica from Waters. The column was maintained at room tem-
perature during the analyses. The half-life of 12 was calculated
from regression curves fitted to plots of the compound concentra-
tion versus time.
4.10.7. Direct luciferase inhibition assay
BHK cells were plated in a 96-well plate and grown at 37 °C. At
confluency, the cells were visually inspected for uniform growth
and the Luciferase-pcDNA3 plasmid was introduced into the cells
using Lipofectamine (Invitrogen, Carlsbad, CA) transfection reagent
according to the manufacturer’s instructions. At 3 h post infection,
the complexes were removed and replaced with culture media
containing serial dilutions of compound 12 (diluted in DMSO),
above and below the EC50 value. Controls included uninfected cells,
infected cells and DMSO-treated infected cells. Cells were then
Acknowledgments
This work was sponsored by the NIH/NIAID Regional Center of
Excellence for Biodefense and Emerging Infectious Diseases Re-
search (RCE) Program. Support is gratefully acknowledged from
the Region V Great Lakes RCE (NIH award 1-U54-AI-057153) and
NIAID (P01AI055672). This research was also supported by a fel-
lowship to A.S.M. from the Egyptian government. Figure 1 and part
of Figure 2 are reproduced from reference 18 and from Mayboub, A.
S. et al. J. Med. Chem. 2011, 54, 1704 and appear here with permis-
sion from the American Chemical Society.
incubated at 37 °C, 5% CO2 for 20 h, lysed using 50
lysis buffer (Promega, Inc., Madison, WI), and 10
were placed into a 96-well opaque plate. Luciferase activity was
determined from the luminescence generated with 50 L fire-fly
lL of cell culture
l
L of cell extracts
l
luciferase substrate (Promega Inc., Madison, WI). An LMax II 96-
well-plate luminometer was used to measure luminescence
(Molecular Devices, Sunnyvale, CA). The luciferase readings for
cells overlaid with Compound 12 were compared to those obtained
from cells overlaid with 1% DMSO using GraphPadPrizm. A cell via-
bility assay was also performed as detailed in Section 4.10.5.
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