Journal of Medicinal Chemistry
ARTICLE
A to 95% B; 2 min hold; then recycle. UV Detection @ 210 and 254 nm
with no reference.
d, 6-H). 13C NMR [125 MHz, (CD3)2SO] 116.5, 117.1, 118.5, 119.7,
130.3, 134.6, 135.4, 155.8, 157.3, and 164.7. m/z (ES þve ion mode) 277
(MNaþ, 100%). Found: m/z, 276.9806; C10H735ClN2O2SNa requires
m/z, 276.9809.
Antiviral assays were performed as described previously.37,65 In
summary, confluent cultures of the chronically HBV-producing cell line,
2.2.15, were maintained on 96-well flat-bottomed tissue culture plates
(confluence in this culture system is required for active, high levels of
HBV replication equivalent to that observed in chronically infected
individuals37,65). Cultures were treated with nine consecutive daily doses
of the test compounds. HBV DNA levels were assessed by quantitative
blot hybridization 24 h after the last treatment. Cytotoxicity was assessed
by uptake of neutral red dye 24 h following the last treatment.
General Procedures for Acid Chloride Couplings and O-
Acetate Deprotections. Acetylsalicyloyl chloride was used directly;
substituted versions were made from the O-acetates as indicated below.
(1) Two-Phase Method. To a stirred solution of a protected salicylic
acid (1 mmol) in dry Et2O (10 mL) at 0 ꢀC was added pyridine (1.2
mmol) followed by dropwise addition of thionyl chloride (1.2 mmol).
Stirring was continued at 0 ꢀC for 4 h. Then the white precipitate formed
was filtered off, and the filtrate was concentrated under vacuum to give
the acid chloride as an oil which was used without further purification.
The appropriate 2-aminothiazole (1 mmol) was added to a vigorously
stirred two-phase mixture of NaHCO3 (3 mmol for a free base, 4 mmol
for an HCl or HBr salt) in H2O (3 mL/mmol) and EtOAc (3 mL/
mmol). A solution of the above acid chloride in EtOAc (2 mL/mmol)
was then added with vigorous stirring. The reaction mixture was stirred
at 20 ꢀC for 12 h. The layers were separated, and the aqueous layer was
extracted once with EtOAc. The combined organic extracts were washed
with 0.5 N HCl (2ꢁ), followed by brine. The organic layer was dried and
concentrated under vacuum to give a pale yellow solid which was
chromatographed on silica, eluting with EtOAcꢀhexane mixtures. In
favorable cases trituration of crude product with Et2O followed by
drying the resulting solid gave the O-acetyl intermediate directly. This
material was heated in concentrated aqueous HCl (3 mL/mmol) at
50 ꢀC for 24 h. The reaction mixture was cooled to ambient temperature,
then filtered and the solid washed with H2O (distilled) until the
washings were at neutral pH. The solid was dried under vacuum to give
the product. On a small scale it was more efficient to extract the final
product into EtOAc, followed by washing with H2O (3ꢁ) and brine,
then drying and evaporation. Chromatography of the final product, if
necessary, was again carried out using EtOAcꢀhexane mixtures.
2-Hydroxy-3-methylbenzoyl-N-(5-bromothiazol-2-yl)amide (7).
Yield: 75 mg, 66%. Mp 187ꢀ188 ꢀC. Anal. (C11H9BrN2O2S) C, H,
N. 1H NMR [400 MHz, (CD3)2SO] 2.21 (3H, s, CH3Ar), 6.86 (1H, d,
J = 7.7 Hz, ArH), 7.39 (1 H, d, J = 7.2 Hz, ArH), 7.73 (1 H, s, 40-H), and
7.95 (1 H, d, J = 7.8 Hz, ArH). 13C NMR [100 MHz(CD3)2SO] 15.6,
101.9, 114.3, 118.8, 126.2, 126.6, 135.7, 136.3, 158.4, 159.9, and 168.8.
MS (CI) m/z 313 and 315 (Mþ for 79Br and 81Br, respectively). HRMS,
found, m/z 312.9641, C11H10BrN2O2S (MHþ for 79Br) requires m/z,
312.9646.
3-Chloro-2-hydroxybenzoyl-N-(5-bromothiazol-2-yl)amide (8). Yield:
0.116 g, 48%. Mp 200 ꢀC. Found: m/z, 332.90930. C10H7BrClN2O2S
(MHþ) requires m/z, 332.91003. 1H NMR [400 MHz, (CD3)2SO] 7.02
(1H, t, J=7.9Hz, ArH), 7.69(1H, dd, J=7.9and1.4Hz, ArH), 7.84(1H, s,
40-H) and 8.02 (1 H, dd, J = 7.9 and 1.4 Hz, ArH). m/z (CI, NH3) 333
(MHþ, 35%).
2-Hydroxy-5-methylbenzoyl-N-(5-chlorothiazol-2-yl)amide (12).
Mp 212ꢀ213 ꢀC. Anal. (C11H9ClN2O2S) C, H, N. 1H NMR
[(CD3)2SO] 2.27 (3 H, s, CH3Ar), 6.94 (1 H, d, J = 8.3 Hz, ArH),
7.29 (1 H, d, J = 8.1 Hz, ArH), 7.59 (1 H, s, 40-H), and 7.78 (1 H, s, ArH).
13C NMR [(CD3)2SO] 19.9, 99.1, 115.7, 117.0, 118.5, 128.5, 130.1,
135.3, 135.7, 155.0, and 164.1. MS (CI) m/z 269, 271 (MHþ for 35Cl,
37Cl, respectively). HRMS, found, m/z 269.0149, C11H10ClN2O2S
(MHþ for 35Cl) requires m/z, 269.0151.
2-Hydroxy-3-methylbenzoyl-N-(4-phenylthiazol-2-yl)amide (20).
1
Mp 180 ꢀC. H NMR (400 MHz, CDCl3) 7.94 (1H, d, J = 8.1, ArH),
7.83 (2H, d, J = 8.5, 2 ꢁ ArH), 7.52ꢀ7.41 (4H, m, ArH), 7.2 (1H, s, ArH),
6.69 (1H, t, J = 7.7, ArH), 2.31 (3H, s, CH3). 13C NMR [125 MHz,
(CDCl3] 15.8, 108.4, 112.0, 118.7, 123.2, 126.0, 128.2, 128.8, 134.1, 136.4,
150.2, 157.2, 160.4, and 167.7. m/z (CI) 311 (35%, [M þ H]þ); (ES þve
ion mode) 333 (MNaþ, 100%). Found: m/z, 333.0659; C17H14N2O2SNa
requires m/z, 333.0668.
See Supporting Information for the characterization of all other
analogues made using either of the above methods. The thiazole pre-
cursors of compounds 17 and 18 are known.66,67
Acetate Deprotection, Basic Conditions. Mild base hydrolysis using
aqueous ammonia is also used for deacetylation, as in the following
example.
2-Hydroxybenzoyl-N-(5-acetamidothiazol-2-yl)amide (23). To a
solution of nitazoxanide 1 (615 mg, 2.0 mmol) in acetic anhydride
(∼70 cm3), Raney Ni was added with vigorous stirring. The reaction
mixture was evacuated and twice refilled with hydrogen, then allowed to
stir at room temperature under an atmosphere of hydrogen until the
theoretical volume (∼135 cm3, 6.0 mmol) was consumed after 30 min.
The reaction mixture was stirred for a few minutes and flushed with N2
to allow the escape of hydrogen present in the flask. The solution was filtered
through a sinter followed by the evaporation of solvent in vacuo. The
crude product was redissolved in EtOAc (50 cm3) and washed with
excess saturated aqueous NaHCO3. The organic fraction was again
evaporated, and the crude product was dissolved in acetone (10 cm3)
and stirred for 2 h with 1 N HCl (5 cm3) to hydrolyze overacetylated
material. The reaction was neutralized with satd. aq. NaHCO3 and
extracted with EtOAc (4 ꢁ 20 cm3). The combined organic fractions
were evaporated, and the O-acetate of 23 was purified through flash
column chromatography as a white amorphous solid (548 mg, 86%). Mp
176.5 ꢀC. 1H NMR (400 MHz, CDCl3) 2.08 (3H, s, CH3), 2.25 (3H, s,
CH3), 7.16 (1H, s, CH), 7.26 (1H, dd, J = 0.6, 8.1 Hz, ArH), 7.39 (1H,
td, J = 1.0, 7.6 Hz, ArH), 7.60 (1H, td, J = 1.6, 8.0 Hz, ArH), 7.75 (1H, dd,
J = 1.6, 7.6 Hz, ArH), 11.0 (1H, s, NH). 13C NMR (100 MHz, CDCl3)
21.1, 22.6, 123.7, 126.1, 127.3, 130.0, 131.0, 132.7, 148.8, 166.8, 169.1,
169.4, 184.5, 201.8. m/z EI [M þ Na]þ 342 [(M þ Na)þ, 100%].
Found: m/z, 342.0524; C14H13N3O4NaS requires 342.0538. To a
solution of this O-acetate (319 mg, 1.0 mmol) in acetone (5 cm3),
20 cm3 of aqueous NH3 was added. Then the mixture was stirred
overnight at room temperature, followed by evaporation. The crude
mixture was dissolved in EtOAc (20 mL) and was washed with 1 M HCl.
The desired product 23 was purified through flash column chromatog-
raphy as a white solid (0.25 g, 90%). 1H NMR (400 MHz, CDCl3) 2.10
(3H, s, CH3), 6.98 (1H, t, J 7.0 Hz, ArH), 7.03 (1H, d, J 8.0 Hz, ArH),
7.18 (1H, s, ArH), 7.45 (1H, td, J = 1.75, 7.0 Hz, ArH), 8.01 (1H, dd,
(2) Anhydrous Conditions. Either the free amine was used directly or
the HBr or HCl salt of the appropriate 2-aminothiazole was partitioned
between dilute aqueous NaOH and EtOAc. Then the organic layer was
separated, dried, and evaporated to dryness. A solution of the thiazole
(1 mmol) in THF (3 mL) was added to a stirred solution of salicyloyl
chloride (1 equiv) in THF (3 mL/mmol) at 0 ꢀC before the addition of
Et3N (1 equiv). The solution was stirred at room temperature until
reaction was complete. Then the reaction mixture was poured into water
and extracted with ethyl acetate (2ꢁ). The organic layer was washed
with 1 M HCl, water, dried, and evaporated. The product was purified by
column chromatography to give the intermediate O-acetate, which was
hydrolyzed as above to deliver the product.
2-Hydroxybenzoyl-N-(5-chlorothiazol-2-yl)amide (3). Mp 227ꢀ
1
228 ꢀC (dec). H NMR [500 MHz, (CD3)2SO] 7.00 (1 H, t, ArH),
7.04 (1 H, d, ArH), 7.48 (1 H, t, 4-H), 7.60 (1 H, s, 40-H), and 7.96 (1 H,
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dx.doi.org/10.1021/jm200153p |J. Med. Chem. 2011, 54, 4119–4132