Journal of Medicinal Chemistry
ARTICLE
131.94, 135.87, 137.99, 138.48, 139.68, 140.06, 140.75, 144.78. Elemental
7-H benzoxazine), 7.33 (d, J = 8.32 Hz, 2H, biphenyl-H), 7.40ꢀ7.44
(m, 3H, biphenyl-H), 7.47 (d, J = 8.32 Hz, 2H, biphenyl-H). Elemental
analysis (C, H): C21H19NO 0.25H2O. Calculated C 82.45, H 6.27. Found C
3
82.07, H 6.21.
analysis (C, H): C21H17NOS 0.1CH2Cl2. Calculated C 73.94, H 5.09,
3
2-(4-Biphenyl)-4-methyl-3,4-dihydro-2H-1,4-benzothiazine (4). Com-
pound 13 (0.45 mmol) and Ca(BH4)2 were reacted under the
same conditions described for 3 to give 4 as a white solid (50%):
mp = 130.5ꢀ131.5 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.94 (s,
3H, ꢀNCH3), 3.55ꢀ3.63 (m, 2H, 2 ꢁ 3-H benzothiazine), 4.40ꢀ4.43
(2d, J = 4.60 Hz, 1H, 2-H benzothiazine), 6.65 (t, J = 7.44 Hz, 1H, 7-H
benzothiazine), 6.71 (d, J = 8.22 Hz, 1H, 5-H benzothiazine), 7.01 (t, J =
7.82 Hz, 1H, 6-H benzothiazine), 7.07 (d, J = 7.63 Hz, 1H, 8-H
benzothiazine), 7.27ꢀ7.31 (m, 1H, biphenyl-H), 7.37ꢀ7.40 (m, 4H,
biphenyl-H), 7.52 (d, J = 7.82 Hz, 4H, biphenyl-H). 13C NMR (200
MHz, CDCl3) δ (ppm): 40.17, 43.64, 58.56, 112.65, 118.05, 125.84,
126.96, 127.08(2C), 127.48 (4C), 128.34 (2C), 128.81 (2C), 138.53,
Found C 73.74, H 5.16.
2-(4-Biphenyl)-2-hydroxy-4-methyl-4H-benzo[1,4]thiazin-3-one (14).
A stirred solution of 2-(4-biphenyl)-4-methyl-2H-1,4-benzothiazin-3-
(4H)-one 13 (0.9 mmol) in dry CHCl3 was treated with m-chloroper-
benzoic acid, 77% (0.9 mmol), and the mixture was stirred for 3 h at room
temperature.26 The solution was washed with water and saturated NaH-
CO3 solution. The organic layer was dried and concentrated in vacuum.
The residue was purified by silica gel flash chromatography (n-hexane/
ethyl acetate, 8:1) to afford 14 as a yellow solid (64%): mp = 138.0ꢀ
140.5 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.57 (s, 3H, ꢀNCH3),
5.13 (brs, 1H, ꢀOH), 6.90ꢀ6.96 (m, 2H, 5,6-H benzothiazine), 7.11
(t, J = 7.83 Hz, 1H, 7-H benzothiazine), 7.23ꢀ7.26 (m, 2H, 8-H benzo-
thiazine and biphenyl-H), 7.30ꢀ7.37 (m, 4H, biphenyl-H), 7.41ꢀ7.44 (m,
140.62, 140.91, 143.96. Elemental analysis (C, H): C21H19NS 0.25H2O.
3
4H, biphenyl-H). Elemental Analysis (C, H): C21H17NO2S 0.5 hexane.
Calculated C 78.34, H 6.12. Found C 78.48, H 6.12.
3
2-Hydroxyphenylmethylamine (6)21. A solution of 2-aminophenol 5
(18 mmol) in dry DMF was treated with NaHCO3 (19 mmol) and CH3I
(22 mmol) and stirred at room temperature for 5 h. After the usual
workup the residue was purified by silica gel flash chromatography
(petroleum ether/ethyl acetate, 5:1) to afford 6 as a white solid (50%):
mp = 102.5ꢀ105 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.61 (s,
3H, CH3), 4.65 (brs, 1H, NH), 6.29ꢀ6.34 (m, 2H, 3,5-H Ar),
6.54ꢀ6.58 (m, 2H, 4,6-H Ar), 9.07 (s, 1H, ꢀOH).
Calculated C 73.81, H 6.21, Found C 73.62, H 6.30.
Methyl (4-Biphenyl)-R-bromoacetate (15)27. Compound 15 was
synthesized according to the literature.27 Yellow oil (96%). H NMR
1
(400 MHz, CDCl3) δ (ppm): 3.83 (s, 3H, ꢀOCH3), 5.42 (d, J =
2.56 Hz, 1H, ꢀCHBr), 7.38ꢀ7.51 (m, 3H, biphenyl-H), 7.54ꢀ7.66
(m, 6H, biphenyl-H).
2-(4-Biphenyl)-2H-1,4-benzoxazine-3(4H)-one (16). A mixture of
2-aminophenol 5 (5.30 mmol), methyl (4-biphenyl)-R-bromoacetate
15 (4.82 mmol), K2CO3 (5.78 mmol), and acetone (25 mL) was re-
fluxed for 2 h.27 The reaction mixture was diluted with water and
extracted with EtOAc. The organic phase was washed with water, dried,
and concentrated in vacuum, and cold CHCl3 was added to the residue.
The precipitated solid was triturated with acetone, filtered off, and dried
to afford 16 as a white solid (31%): mp = 250 °C (dec). 1H NMR (400
MHz, DMSO-d6) δ (ppm): 5.74 (s, 1H, 2-H benzoxazine), 6.28ꢀ6.55
(m, 1H, 6-H benzoxazine), 6.85ꢀ6.87 (m, 3H, 5,7,8-H benzoxazine),
6.93ꢀ6.95 (m, 1H, biphenyl-H), 7.38 (d, J = 7.34 Hz, 2H, biphenyl-H),
7.52ꢀ7.60 (m, 6H, biphenyl-H), 10.90 (s, 1H, NH).
Ethyl (4-Biphenyl)acetate (9)22. Compound 9 was synthesized ac-
cording to the procedure described previously.22 Colorless oil (89%). 1H
NMR (400 MHz, CDCl3) δ (ppm): 1.21 (t, J = 7.05 Hz, 3H, ꢀCH3),
3.60 (s, 2H, ꢀCH2CO), 4.12 (q, J = 7.05 Hz, 2H, OCH2), 7.26ꢀ7.31
(m, 3H, biphenyl-H), 7.36ꢀ7.39 (m, 2H, biphenyl-H), 7.49ꢀ7.54 (m,
4H, biphenyl-H).
Ethyl (4-Biphenyl)-R-bromoacetate (10)23. Compound 10 was syn-
thesized according to the procedure described previously.23 Dark yellow
oil (91%). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.25 (t, J = 7.14 Hz,
3H, ꢀCH3), 4.20 (q, J = 7.24 Hz, 2H, ꢀOCH2), 5.33 (s, 1H, ꢀCHBr),
7.29ꢀ7.34 (m, 1H, biphenyl-H), 7.37ꢀ7.42 (m, 2H, biphenyl-H),
7.51ꢀ7.58 (m, 6H, biphenyl-H).
2-(4-Biphenyl)-4-methyl-2H-1,4-benzoxazin-3(4H)-one (17). 2-(4-
Biphenyl)-2H-1,4-benzothiazin-3(4H)-one 16 (1.9 mmol) and methyl
iodide (2.8 mmol) were reacted under the same conditions25 described
2-(4-Biphenyl)-2H-1,4-benzothiazin-3(4H)-one (12). A solution of
ethyl (4-biphenyl)-R-bromoacetate 10 (2.7 mmol) and 2-aminobenze-
nethiol 11 (4.1 mmol) in dry DMF was stirred at room temperature for
25 h.24 The reaction mixture was then poured into iceꢀwater. The
precipitated solid was filtered off, triturated with acetonitrile, filtered off,
and dried to afford 12 as a white solid (70%): mp = 235ꢀ236.5 °C. 1H
NMR (400 MHz, CDCl3) δ (ppm): 4.68 (s, 1H, 2-H benzothiazine),
6.81 (d, J = 8.02 Hz, 1H, 8-H benzothiazine), 6.96 (dt, J1 = 1.17 Hz, J2 =
7.62 Hz, 1H, 7-H benzothiazine), 7.11 (dt, J1 = 1.18 Hz, J2 = 7.83 Hz, 1H,
6-H benzothiazine), 7.25ꢀ7.29 (m, 2H, 5-H benzothiazine and biphe-
nyl-H), 7.34 (d, J = 7.83 Hz, 2H, biphenyl-H), 7.38 (d, J = 8.22 Hz, 2H,
biphenyl-H), 7.45ꢀ7.48 (m, 4H, biphenyl-H), 8.55 (s, 1H, NH).
Elemental analysis (C, H): C20H15NOS. Calculated C 75.68, H 4.76,
Found C 75.35, H 4.71.
1
for 13 to give 17 as a yellow semisolid (80%): H NMR (400 MHz,
CDCl3) δ (ppm): 3.48 (s, 3H, -NCH3), 4.64 (s, 1H, 2-H benzothiazine),
6.96 (t, J = 7.58 Hz, 1H, 6-H benzothiazine), 7.02 (d, J = 8.07 Hz, 1H,
5-H benzothiazine), 7.17 (m, 1H, 7-H benzothiazine), 7.24ꢀ7.36 (m,
6H, 8-H benzothiazine and biphenyl-H), 7.42 (d, J = 8.07 Hz, 2H,
biphenyl-H), 7.46 (d, J = 7.58 Hz, 2H, biphenyl-H).
3.3. In Vitro Microsomal Lipid Peroxidation. Heat-inactivated
hepatic microsomes from untreated rats were prepared as described
previously. The inhibitory effect of compounds on lipid peroxidation
was assessed spectrophotometrically (535 against 600 nm) as TBAR
material.8
3.4. Isolation and in Vitro LDL Oxidation. Blood was collected
from a normolipidemic human volunteer, and LDL was isolated by
discontinuous density gradient (using KBr) ultracentrifugation.28
LDL (56 μg of protein/mL) in PBS was incubated at 37 °C in the
absence or presence of various concentrations of 1 (in 10% DMSOꢀ
H2O). Oxidation was initiated by 10 μM CuSO4. Conjugated dienes
were determined every 10 min for 5 h as the increase in absorbance at
234 nm and calculated using the extinction coefficient of 29 500.13
3.5. In Vitro Squalene Synthase Activity Assay. SQS activity
was evaluated by determining the amount of [3H]FPP converted to
squalene as previously described.9,14
2-(4-Biphenyl)-4-methyl-2H-1,4-benzothiazin-3(4H)-one (13). An
excess of KOH (4.1 mmol) was added to a solution of 2-(4-biphenyl)-
2H-1,4-benzoxazin-3(4H)-one 12 (1.1 mmol) in dry acetone.25 The
mixture was refluxed, while methyl iodide (1.7 mmol) in dry acetone was
added dropwise. The mixture was then stirred at 60 °C for 15 min.
Acetone was filtered, evaporated in vacuum, and diethyl ether was added.
The organic layer was washed with water and brine, dried, and concen-
trated in vacuum. The residue was purified by silica gel flash chroma-
tography (petroleum ether/dichloromethane, 2:3) to afford 13 as a
yellow solid (84%): mp = 170ꢀ171.5 °C. 1H NMR (400 MHz, CDCl3)
δ (ppm): 3.37 (s, 3H, ꢀNCH3), 5.69 (s, 1H, 2-H benzoxazine),
6.69ꢀ6.76 (m, 2H, 5,6-H benzoxazine), 6.89ꢀ6.92 (m, 1H, 8-H
benzoxazine), 6.96ꢀ6.99 (m, 2H, biphenyl-H), 7.01ꢀ7.04 (m, 1H,
3.6. Docking Studies. Preparation of ligands, protein crystal
structure (1EZF), and docking runs were performed with Spartan04
(Wavefunction Inc., Irvine, CA, 2004), Chimera, and Autodock
4/Autodock Tools29,30 and according to previous methodology.9
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dx.doi.org/10.1021/jm200763k |J. Med. Chem. 2011, 54, 5583–5591