Molecules 2015, 20, 21458–21463
4,41-dihydroxy-[1,11-biphenyl]-2-carboxylic acid (
4). Compound 3 (200 g, 1.20 mol) was dissolved in
concentrated H2SO4 (570 mL) at 110–115 ˝C. After 2 h of vigorous stirring, the solution was allowed
to cool and water (1.7 L) was added. Then the solution was filtered. To the filtrate KCl (400 g) was
added and the solution was stirred at room temperature for 30 min. Then the precipitate was filtered
and dried to give a solid (396 g, 82%), which was added in batches to a stirred solution of KMnO4
(208 g, 1.32 mol) in water (7.1 L), maintained at 20–30 ˝C for 2 h, and then filtered. The filtrate was
concentrated in vacuum to 4.5 L, and cooled to 5 ˝C. The precipitate was filtered and dried to obtain
a solid (378 g, 89%), 105 g of which (0.24 mol) was added to a mixture of NaOH (210 g, 5.25 mol) and
KOH (56 g, 1.00 mol). After 1 h of stirring at 270–300 ˝C, the melt was allowed to cool and dissolved
in water (1.5 L). The solution was acidified by addition of concentrated HCl to pH 3.0, and then the
precipitate was filtered and dried. Compound
4
was obtained (52 g, 95%) in an overall yield of 69%;
(cm´1): 3145.4–2491.3 (OH), 1693.3 (C=O); ESI-MS
H]´, C13H10O4 (MW. = 230.1); 1H-NMR (400 MHz, DMSO-d6),
(ppm): 12.60 (br,
˝
˝
m.p. 269–270 C (Lit. 270–273 C [22]); IR (KBr),
(m/z): 229.0 [M
υ
´
δ
1H), 9.73 (s,1H), 9.40 (s, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H),
7.03 (d, J = 2.5 Hz, 1H), 6.91 (dd, J = 8.3, 2.5 Hz, 1H), 6.74 (d, J = 8.4 Hz, 2H).
1
1
Methyl 4,4 -Dihydroxy-[1,1 -biphenyl]-2-carboxylic acid (
5). A mixture of 4 (50 g, 0.22 mol), concentrated
H2SO4 (5 mL), and CH3OH (400 mL) was refluxed for 2 h and then cooled to r.t., precipitated.
˝
5 (52 g, 98%); m.p. 224–226 C; IR (KBr), υ
(cm´1): 3291.0,
The precipitate was filtered and dried to give
3202.8 (OH) 1692.5 (C=O), 1435.5 (CH3); ESI-MS (m/z): 245.0 [M + H]+, C14H12O4 (MW. = 244.1);
1H-NMR (400 MHz, DMSO-d6),
(ppm): 9.80 (s, 1H), 9.42 (s, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.02 (d,
δ
J = 2.0 Hz, 2H), 7.00 (d, J = 2.0 Hz, 1H), 6.95 (dd, J = 8.4, 2.6 Hz, 1H), 6.76 (d, J = 2.0 Hz, 1H), 6.75 (d,
J = 2.0 Hz, 1H), 3.58 (s, 3H).
2,7-Dihydroxy-9H-fluoren-9-one (
2). A mixture of 5 (50 g, 0.20 mol), ZnCl2 (50 g, 0.37 mol), PPA (33 mL)
was stirred at 110–120 ˝C for 2 h and then cooled to r.t., precipitated by addition of water (500 mL).
The precipita˝te was filter˝ed and dried to obtain 40 g red-brown crystallized powder (
2). Yield 95%;
m.p. 336–337 C (Lit. 338 C [24]); IR (KBr),
υ
(cm´1): 3388.9, 3360.6 (OH), 1700.7 (C=O); ESI-MS (m/z):
´
211.0 [M
´
H] , C13H8O3 (MW. = 212.0); 1H-NMR (300 MHz, DMSO-d6),
δ
(ppm): 9.89 (s, 2H), 7.37 (d,
J = 4.8 Hz, 2H), 6.90 (dd, J = 4.9, 2.4 Hz, 2H), 6.86 (d, J = 2.4 Hz, 2H).
2,7-bis[2-(Diethylamino)ethoxy]-9H-fluoren-9-one hydrochloride ( ). A mixture of
1
2 (100 g, 0.47 mol),
2-diethylaminoethylchloride hydrochloride (300 g, 1.74 mol), KOH (225 g, 4.01 mol), water (320 mL)
and toluene (1.5 L) was refluxed for 24 h. The emulsion was cooled, and then organic layer was
separated, washed with KOH aq. (3 mol/L, 500 mL) and concentrated up to dryness in vacuum.
The residue was dissolved in anhydrous ethanol (420 g) and acidified with gasified HCl to pH 3.0.
The orange product was recrystallized with ethanol, filtered, and dried to give 200 g of compound
a yield of 93%; m.p. 231–233 ˝C (Lit. 235–236 ˝C [22]); IR (KBr), (cm´1): 2943.3, 2596.5 (CH), 1708.9
(C=O); ESI-MS (m/z): 411.0 [M + H]+, C25H34N2O3 (MW. = 410.3); 1H-NMR (400 MHz, D2O),
(ppm):
1 in
υ
δ
7.12 (d, J = 6.5 Hz, 2H), 6.99 (dd, J = 6.5, 1.6 Hz, 2H), 6.89 (d, J = 1.6 Hz, 2H), 4.39 (t, J = 3.6 Hz,4H), 3.68
(t, J = 3.6 Hz, 4H), 3.42 (q, J = 5.6 Hz, 8H), 1.44 (t, J = 5.8 Hz, 12H).
3.2. Determination of Serum IFN-α Induced by Tilorone Dihydrochloride by ELISA
SPF mice were housed under conditions of constant temperature and a 12-h light cycle, with food
and water provided ad libitum in the Animal Experimental Center, Nanjing Agricultural University.
Interferon levels were recorded on a Thermo Multiskan Ascent enzyme-labeled instrument (Thermo
Fisher Scientific (Beijing, China). After tilorone dihydrochloride challenge, mice were alive just like the
control group, with normal activity levels, appetite, and growth. Sample serum diluent and different
˝
concentrations of the standard were added to a micro-well plate (96 tests), which was put at 37 C for
2 h. Biotin-conjugate diluent prepared before 30 min was added after dumping the liquid out of the
plate, which would be put at 37 ˝C for another 1 h. The plate was washed five times after dumping
the liquid and Streptavidin-HRP diluent prepared beforehand was added to the plate that would be
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