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was irradiated under microwave at 1308C (with the heating pro-
gram starting at 150 W) for 8 min. Thereafter, the reaction mixture
was concentrated and extracted with EtOAc/H2O. The combined
organic layer was dried over Na2SO4, concentrated, and purified by
column chromatography using EtOAc/hexane (starting from 3:7 to
3:2) as the eluent. The resulting product was dried overnight in
a vacuum oven at 508C to provide compound 8 in 76–86% yield.
was heated at 508C and filtered under suction. The filtrate was
cooled to 08C and adjusted to pH 2 with 6m HCl. The precipitate
that formed was filtered and purified by flash column chromatog-
raphy using EtOAc/hexane (from 1:2 to 1:1) as eluent. The product
was dried overnight under vacuum at 508C to obtain compound
11 in 48–65% yield.
General procedure for the synthesis of 3: The synthesis for com-
pound 3 is similar to that used for the synthesis of 2a12. However,
the resulting precipitate was purified by flash column chromatog-
raphy using EtOAc/hexane (from 2:3 to 2:1) as eluent. The product
was dried overnight under vacuum at 508C to obtain compound 3
in 67–74% yield.
General procedure for the synthesis of 8 (R2 =H): Commercially
available acid chloride (9.0 mmol) or crude product 6b (L=Cl,
9.0 mmol) was added slowly to a mixture of thiosemicarbazide
(19.6 mmol) in THF (20 mL) at 08C. The reaction mixture was stirred
at room temperature for 1.5 h. The reaction mixture was then con-
centrated, and the residue was triturated with water. The product
that precipitated from the reaction mixture was filtered, washed
with water, and dried overnight under vacuum at 508C to yield
product 8 in 60–87% yield. This product was used directly in the
next reaction.
General procedure for the synthesis of 12: The corresponding
amine or aniline 4 (4.4 mmol) was added dropwise to a mixture of
bromoacetyl bromide (5.3 mmol) in CH3CN (50 mL) at 08C. After
addition, the mixture was stirred at room temperature for 1 h.
CH3CN was removed by rotary evaporation, and the residue was
extracted with an EtOAc/H2O solvent system. The combined organ-
ic layer was concentrated and purified by flash column chromatog-
raphy using EtOAc/hexane (from 1:10 to 3:10) as eluent. The prod-
uct was dried overnight under vacuum at 508C to obtain com-
pound 12 in 68–87% yield.
General procedure for the synthesis of 2a: NaOH (2m, 15 mL)
was added to compound 8 (3.4 mmol), and the reaction mixture
was irradiated under microwave at 1508C (with the heating pro-
gram starting at 150 W) for 3 min. Thereafter, the solution was neu-
tralized with acetic acid and extracted with EtOAc (3ꢁ15 mL). The
combined organic layers were dried over Na2SO4, concentrated,
and purified by flash column chromatography using EtOAc/hexane
(from 1:5 to 1:2) as the eluent. The product was dried overnight
under vacuum at 508C to provide compound 2a in 71–94% yield.
General procedure for the synthesis of 13: Compound
3
(0.3 mmol) was dissolved in a mixture of KOH (0.6 mmol) in 60%
aqueous EtOH (20 mL). The corresponding intermediate 12
(0.3 mmol) was added, and the reaction mixture was stirred for 1 h
at room temperature. After completion of the reaction (monitored
by TLC), EtOH was removed by rotary evaporation, and the residual
mixture was neutralized with dilute acetic acid followed by extrac-
tion with EtOAc (3ꢁ15 mL). The combined organic layers were
concentrated and purified using a short silica column with EtOAc/
hexane (from 3:10 to 1:4) as eluent. The resulting semi-pure prod-
uct was dried under high vacuum at room temperature and used
for the next step of the reaction.
Synthesis of 3-(4-aminophenyl)-1H-1,2,4-triazole-5(4H)-thione
(2a12): Concentrated HCl (1 mmol) was added to a mixture of iron
powder (10 mmol) in 80% aqueous EtOH (5 mL), and the reaction
mixture was stirred in a warm (408C) water bath. Thereafter, 3-(4-
nitrophenyl)-1H-1,2,4-triazole-5(4H)-thione 2a6 (R1 =NO2, 2 mmol)
was added portionwise, and the resulting reaction mixture was
stirred and heated at reflux for 45 min. The EtOH was then re-
moved by rotary evaporator, and the residual reaction mixture was
adjusted to ~pH 13 with NaOH. The reaction mixture was then fil-
tered by suction filtration, and the filtrate was adjusted to pH 5
with glacial acetic acid. The resulting precipitate was filtered and
purified by flash column chromatography using EtOAc/hexane
(from 1:1 to 2:1) as eluent. The resulting product was dried over-
night under vacuum at 508C to yield compound 2a12 in 61%
yield.
General procedure for the synthesis of 14: Bromoacetyl bromide
(5.3 mmol) was added dropwise to the corresponding intermediate
13 (4.4 mmol) in CH2Cl2 (30 mL) at 08C. After addition, the reaction
mixture was stirred at room temperature for 1 h. The product that
precipitated from the reaction mixture was filtered, washed with
CH2Cl2 (3ꢁ15 mL), and dried overnight under vacuum at 508C to
provide compound 14 in 73–86% yield.
General procedure for the synthesis of 10: Carbon disulfide
(5.5 mmol) was added to a mixture of KOH (5.5 mmol) in 80%
aqueous EtOH (7.5 mL), and the reaction mixture was stirred at
room temperature for 15 min. Corresponding aniline 9 (5 mmol)
was added, and the reaction mixture was irradiated under micro-
wave at 1308C (with the heating program starting at 150 W) for
15 min and then cooled to room temperature. The reaction mix-
ture was then diluted with water (10 mL), adjusted to pH 5 with
glacial acetic acid, and cooled to 08C for 1 h to complete the crys-
tallization. The shiny precipitate that formed was filtered and dried
overnight under vacuum at 508C to yield compound 10 in 85–
94% yield.
General procedure for the synthesis of 1a: The corresponding tri-
azole compound 2a (0.22 mmol) was dissolved in a mixture of
KOH (0.66 mmol) in 60% aqueous EtOH (16 mL). The correspond-
ing intermediate 14 (0.22 mmol) was added, and the reaction mix-
ture was stirred for 2 h at room temperature. The EtOH was then
removed by rotary evaporation, and the residual mixture was neu-
tralized with dilute acetic acid, followed by extraction with EtOAc/
H2O. The combined organic layer was concentrated and purified
by reverse phase HPLC using solvent A (0.1% TFA in water) and
solvent B (0.1% TFA in CH3CN) as the mobile phase. The mobile
phase was a gradient, with solvent B increasing from 15% to 95%
over 30 min at a flow rate of 8.0 mLminꢁ1. Detection was conduct-
ed at 254 nm. Fractions containing the pure product were com-
bined, concentrated, and dried overnight under vacuum at 508C
to give compound 1a in 56–83% yield.
General procedure for the synthesis of 11: The corresponding
azole derivative 10 (3.0 mmol) was slowly dissolved in concentrat-
ed H2SO4 (1.4 mL, 25.7 mmol), cooling from 08C to ꢁ58C. A mix-
ture of fuming HNO3 (0.4 mL, 9.6 mmol) and concentrated H2SO4
(0.3 mL, 5.6 mmol) was added dropwise, and the reaction mixture
was stirred for 1 h at 08C to ꢁ58C before pouring into ice. The re-
sulting precipitate was filtered and dissolved in a minimum
volume of 25% aqueous ammonia solution. The resulting solution
Enzyme inhibition evaluation
All of the compounds used in biological testing were at least 95%
pure. Compounds were tested against the WNV NS2B-NS3 pro-
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 994 – 1001 999