Introduction

Top Abstract Introduction Methods Results Discussion References

Extracellular volume expansion is involved in several diseases including hypertension, congestive heart failure and cirrhosis of the liver. It is believed that a "natriuretic hormone" exists that controls sodium excretion and thereby regulates extracellular fluid volume (de Wardener et al., 1961). Many investigators in this field believe that this putative humoral substance may be responsible for hypertension and natriuresis, owing to inhibition of sodium transport. The inhibition of sodium transport is reflected in inhibition of the Na⁺/K⁺-ATPase. The search for the putative natriuretic hormone has led to the isolation of several compounds, some of which have natriuretic activity (Wechter and Benaksas, 1990; Benaksas et al., 1995), and include atrial natriuretic factor (de Bold et al., 1981), 20-hydroxy eicosatetraenoic acid (20-HETE) (Wang and Lu, 1995), "iso-ouabain" (Ludens et al., 1991; Mathews et al., 1991; Tymiak et al., 1993; Zhao et al., 1995) and digoxin or an isomer (Goto et al., 1990). The latter two do not induce natriuresis, however.

We have identified a natriuretic substance, 2,7,8-trimethyl-2-(-carboxyethyl)-6-hydroxy chroman (LLU-, 3a), isolated from human uremic urine (Murray et al., 1995; Wechter et al., 1996), that is the most potent known inhibitor of the 70 pS ATP-sensitive K⁺ channel in the thick ascending limb cells of the kidney. Consequently, it is assumed to be natriuretic by virtue of inhibiting K⁺ excretion and thus K⁺ cycling via the Na⁺/K⁺/2Cl cotransporter. The isolated compound appears to be the result of in vivo side-chain oxidative degradation of a metabolite of -tocopherol, a member of the vitamin E complex (Wechter et al., 1996; Murray et al., accompanying paper, 1997).

Simon et al. (1956a, b) identified an oxidized product of radiolabeled -tocopherol from the urine of -tocopherol-fed rabbits. Isolation of chroman metabolites analogous to 8 from - and -tocopherols has been reported (Chiku et al., 1984; Watanabe et al., 1974). In none of those cases was the stereochemistry at C-2 determined, although it was reasonably assumed, based on biooxidation, to have the same configuration as the parent tocopherol. The -tocopherol metabolite (3b) is the 5-methyl homolog of the -tocopherol metabolite.

We herein assign the absolute stereochemistry of 3a and by inference 3b at C-2 for all tocopherols (). This assignment was made on the basis of X-ray crystallographic analysis. The stereochemistry of the metabolite 3b of -tocopherol is assumed to be the same as 3a, because both are derived from the vitamin E family and are subject to the same oxidation sequence that does not involve opening of the chroman ring. We can therefore assign the S stereochemistry to 3b isolated by Simon et al. (1956a, b) as well. Small-scale resolution of the enantiomers is described as well as the synthesis of derivatives to be used for biological testing (Murray et al., accompanying paper,1997).

	Methods

Top Abstract Introduction Methods Results Discussion References

Synthetic chemistry. Chemicals were obtained from Aldrich Chemical Co. (Milwaukee, WI). All solvents were of analytical grade or better. Solvents were removed under reduced pressure at 40°C. Gravity and medium pressure chromatography was carried out on Silica gel (0.040-0.063 mm) (Bodmann, Aston, PA). Melting points were determined on Fisher-Johns block and are uncorrected.

Chromatography. Thin-layer chromatography was performed on Analtech (Newark, DE) HPTLC plates. The spots were visualized with 3% ceric sulfate in 3 N sulfuric acid and heating to 150-200°C. A Beckman System Gold (126 pump, 168 diode array detector) HPLC, controlled by System Gold software v 5.1 on a PC, was used for purification of synthetic compounds. The eluent was monitored by UV at 295 nm unless otherwise stated. Preparative RP-HPLC was performed on a SPHEREX 10 ODS column (21.2 × 250 mm; Phenomenex) eluted at 6 ml/min using a gradient of 0.05 M acetic acid(aq) (solvent A) and 0.045 M acetic acid in acetonitrile (solvent B) (60:40 [A:B] for 5 min, then a linear gradient to 20:80 [A:B] over 20 min followed by a linear gradient to 0:100 [A:B] over 5 min, and then 10 min at 0:100 [A:B]). Analytical chiral chromatography was conducted isocratically [hexane/2-propanol/acetic acid (80:20:0.5); solvent C] on a S, S Whelk O 1, column (4.6 × 250 mm; Regis Inc., Morton Grove, IL) at 1 ml/min, [hexane/2-propanol/acetic acid (9:1:0.5); solvent D] on a S, S Whelk O 1, column (4.6 × 250 mm; Regis Inc.) at 0.8 ml/min. Preparative chiral chromatography was also performed isocratically [hexane/2-propanol/acetonitrile/acetic acid (75:20:15:0.5); solvent E] on a S, S Whelk O 1, column (10 × 250 mm; Regis Inc.) eluted at 1 ml/min. The chromatograms were monitored at 295 nm unless otherwise stated.

General procedure for synthesis of racemic chromans. To the solution of substituted hydroquinone (1, 10 mmol) and borontrifluoride diethyl etherate (20 mmol) in dioxane (20 ml, dried over sodium) was added -methyl--vinyl-butyrolactone (2, 15 mmol) in dioxane (4.0 ml) via syringe over 60 min at 120°C (oil bath, reflux) under nitrogen. The reaction mixture was cooled to room temperature and diluted with ether (300 ml), then washed with water (3 × 100 ml), dried (Na₂SO₄) and the ether removed under vacuum. The brown oily residue was dissolved in methanol (30 ml) and the methanol removed under vacuum. The resulting brown oil was purified on a flash silica-gel column. The residual yellow oil was crystallized from ether/hexane (1:1) at 5°C or methanol/water. White crystals were recovered by filtration and washed with hexane, dried in air, then in a vacuum desiccator. The preparative HPLC purification was used with solvents A and B as described under "Chromatography."

Separation of racemic 2,7,8-trimethyl-2-(-carboxyethyl)-6-hydroxy chroman (3a). 3a was prepared as described previously (Wechter et al. , 1996). Analytical chiral HPLC was performed as described above with solvent C. The enantiomers eluted at Rt (R) = 7.62 min, Rt (S) = 9.66 min. 3a (200 mg) was resolved on preparative chiral chromatography as described above using solvent C. Polarimetry, (R)-enantiomer []_D = 6.5° (c 1.00 MeOH), (S)-enantiomer []_D = +5.1° (c 1.27 MeOH).

Preparation and separation of racemic 2,5,7,8-tetramethyl-2-(-carboxyethyl)-6-hydroxy chroman (3b). Eluent EtOAc/hexane/acetic acid (500:300:1), yield 47%; m.p. 173-174°C; IR (KBr): 3700-2700, 1702, 1616, 1454, 1414, 1384, 1332, 1304, 1292, 1262, 1234, 1200, 1178, 1114, 1104, 1082 cm¹; ¹H NMR, (CD₃OD) : 2.63-2.58 (m, 2H, CH₂), 2.47-2.39 (m, 2H, CH₂), 2.0-1.84 (m, 2H, CH₂), 1.82-1.76 (m, 2H, CH₂), 2.11 (s, 3H, CH₃), 2.08 (s, 3H, CH₃), 2.04 (s, 3H, CH₃), 1.20 (s, 3H, CH₃); UV (CH₃OH),  (): 207 (40394), 225 (7512), 292 (2906) nm; EIMS, m/z: 105, 111, 121, 136, 149, 164, 165 (100%), 166, 278 (16%, M⁺) Da; Anal. (C₁₆H₂₂O₄) Calc. 69.04%C, 7.97%H, Found 68.76%C, 7.88%H. Analytical chiral chromatography was performed using solvent C, Rt = 7.75 and 11.24 min.

Preparation and separation of racemic 2,7,8-trimethyl-2-(-carboxyethyl) chroman (3c). Eluent EtOAc/hexane (1:1), yield 24%; m.p. 93-94°C; IR (KBr): 3000-2600, 1694, 1578, 1490, 1452, 1426, 1412, 1382, 1352, 1328, 1294, 1264, 1250, 1236, 1216, 1190, 1174, 1156, 1102, 1088 cm¹; ¹H NMR (CD₃OD) : 6.80 (d, 1H, J 7.68Hz, CH), 6.65 (d, 1H, J 7.68Hz, CH), 2.80-2.73 (m, 2H, CH₂), 2.6-2.54 (m, 2H, CH₂), 2.22 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.1-1.90 (m, 2H, CH₂), 1.89-1.72 (m, 2H, CH₂), 1.26 (s, 3H, CH₃); UV (CH₃OH),  (): 207 (39451), 225 (5960), 284 (1230) nm; EIMS, m/z: 105, 122, 134, 135 (100%), 136, 173, 175, 248 (7%, M⁺) Da; Anal. (C₁₅H₂₀O₃) Calc. 72.55%C, 8.12%H, Found 72.18%C, 8.18%H. Analytical chiral chromatography (solvent C), Rt = 4.85 and 5.53 min.

Preparation and separation of racemic 2,5,7,8-tetramethyl-2-(-carboxyethyl) chroman (3d). Eluent EtOAc/hexane (1:1), yield 42%; m.p. 148-149°C; IR (KBr): 3700-2700, 1700, 1484, 1464, 1448, 1426, 1412, 1382, 1354, 1330, 1306, 1292, 1282, 1254, 1234, 1196, 1172, 1140, 1106 cm¹; ¹H NMR, (CD₃OD) : 6.48 (s, 1H, CH), 2.63-2.57 (m, 2H, CH₂), 2.50-2.40 (m, 2H, CH₂), 2.15 (s, ³H, CH₃), 2.12 (s, 3H, CH₃), 2.02 (s, 3H, CH₃), 2.0-1.93 (m, 1H, HCH), 1.90-1.84 (m, 1H, HCH), 1.84-1.76 (m, 2H, CH₂), 1.22 (s, 3H, CH₃); UV (CH₃OH),  (): 208 (42334), 225 (6807), 283 (520) nm; EIMS, m/z: 105, 148, 149 (100%), 150, 262 (7%, M⁺) Da; Anal. (C₁₆H₂₂O₃) Calc. 73.25%C, 8.45%H, Found 72.71%C, 8.55%H. Analytical chiral chromatography (solvent C), Rt = 5.01 and 6.19 min.

Preparation of 1,8-dioxa-2,7,9,10-tetramethyl-2,7-di-(3-propionic acid)-3H, 4H, 5H, 6H phenanthrene dimethyl ester (4b). 4a (1.0 g) was suspended in methanol (10 ml) and etheral diazomethane solution added until the yellow color of diazomethane remained. The clear solution was left at room temperature for 1 h, then the solvent removed. The residue was purified on a silica-gel column with hexane/acetone (3:1). Solvent was removed and the product was crystallized from hexane, yield 0.59 g (55%). m.p. 75-76°C; IR (KBr): 3000-2800, 1730, 1440, 1414, 1380, 1354, 1334, 1324, 1288, 1260, 1210, 1168, 1138, 1118, 1102, 1090 cm¹; ¹H NMR, (CDCl₃) : 3.66 (s, 6H, 2×OCH₃), 2.57-2.46 (m, 4H, 2×CH₂), 2.07 (s, 6H, 2×CH₃), 2.05-1.75 (m, 12H, 6×CH₂), 1.22 (s, 6H, 2×CH₃); ¹³C NMR (CDCl₃) : 174.37 (C-1,1, CO), 144.23 (C-11,14), 123.51 (C-9,10), 115.46 (C-12,13), 73.27 (C-2,7), 51.56 (OCH₃), 34.60 34.27 (C-3,6), 31.41 31.36 (C-3,3), 28.53 (C-2,2), 23.52 23.31 (C-2a,7a), 19.52 (C-4,5), 11.65 (C-9a,10a); UV (CH₃OH),  (): 207 (53348), 225 (7314), 299 (3093) nm; EIMS, m/z: 105, 107, 109, 111, 113, 115, 119, 129, 135, 141, 149, 150, 151, 163, 167, 175, 189, 203, 216, 217, 418 (17%, M⁺) Da; Anal. (C₂₄H₃₄O₆) Calc. 68.88%C, 8.19%H, Found 68.77%C, 8.40%H. Analytical chiral chromatography (solvent C), Rt = 14.12, 17.38 and 18.44 min.

General procedure for oxidation (Mayer et al., 1964). 3 (100 mg) was dissolved in methanol (2.5 ml) and iron-(III)-chloride solution [1.0 g FeCl₃·6H₂O dissolved in water (4.0 ml), then methanol (4.0 ml) added; 2.5 ml] was added at room temperature with vigorous stirring in the dark. The stirring was continued for 30 min in the dark. Methanol was removed under vacuum and the residue dissolved in ether (70 ml), the etheral solution was washed with water (3 × 20 ml), dried (Na₂SO₄), then the solvent removed. Preparative RP-HPLC purification was done as described under "Chromatography"; however, a different gradient was used: 50:50 [A:B] for 5 min, then a linear gradient to 10:90 [A:B] over 30 min. followed by a linear gradient to 0:100 [A:B] over 5 min, and 5 min at 0:100 [A:B]. Eluant was monitored at 265 nm. A yellow-to-brown oily product resulted.

Preparation and separation of racemic 4-methyl-6-(5,6-dimethylbenzochinoyl)-4-hexanolid (5a). Yield 60%; IR (KBr): 1770, 1650, 1616, 1382, 1320, 1290, 1260, 1208, 1174, 1144, 1088 cm¹; ¹H NMR, (CDCl₃) : 6.52 (s, 1H, CH), 2.70-2.55 (m, 2H, CH₂), 2.55-2.48 (m, 2H, CH₂), 2.25-2.00 (m, 2H, CH₂), 2.02 (s, 3H, CH₃), 2.01 (s, 3H, CH₃), 1.88-1.80 (m, 2H, CH₂), 1.45 (s, 3H, CH₃); UV (CH₃OH),  (): 257.2 (20551), 262.0 (20118), 336.6 (434) nm; EIMS, m/z: 100, 122, 149, 150, 151, 164, 189, 202, 262 (7%, M⁺) Da. Analytical chiral chromatography (solvent D) Rt = 56.76 and 58.46 min.

Racemic 4-methyl-6-(3,5,6-trimethylbenzochinoyl)-4-hexanolid (5b). Yield 82%; IR (KBr): 1770, 1642, 1456, 1376, 1290, 1252, 1208, 1188, 1150, 1126, 1088 cm¹; ¹H NMR, (CDCl₃) : 2.67-2.52 (m, 4H, 2×CH₂), 2.28-2.21 (m, 1H, HCH), 2.20-2.00 (m, 1H, HCH), 2.03 (s, 3H, CH₃), 2.00 (s, 6H, 2×CH₃), 1.75-1.68 (m, 2H, CH₂), 1.46 (s, 3H, CH₃); UV (CH₃OH),  (): 207 (15691), 261 (21967), 268 (22553) nm; EIMS, m/z: 105, 107, 121, 134, 136, 149, 163, 175, 178, 203, 276 (11%, M⁺) Da.

Preparation and separation of racemic 2,7,8-trimethyl-2-(-carboxyethyl)-6-O-acetyl chroman (6). 2,7,8-Trimethyl-2-(-carboxyethyl)-6-hydroxy chroman (3a, 500 mg) was dissolved in pyridine (10 ml) at room temperature and acetic anhydride (5 ml) added. The solution remained at room temperature for 5 h. The solvent was then removed under vacuum, methanol added and removed (4 × 10 ml) under reduced pressure. The residual oil was dissolved in ethyl acetate (100 ml) and the organic phase washed with water (50 ml), aq. HCl (1 N, 50 ml), water (50 ml), dried (Na₂SO₄) and the solvent removed. The residual oily material was purified on a silica gel column with hexane/ethyl acetate (1:1), yield 0.45 g (78%) of oily product which crystallized from acetone/hexane; m.p. 105-107°C; IR (KBr): 3500-2100, 1754, 1712, 1580, 1480, 1442, 1416, 1370, 1320, 1211, 1162, 1102 cm¹; ¹H NMR, (CD₃OD) : 6.55 (s, 1H, CH), 2.74-2.72 (m, 2H, CH₂), 2.47-2.43 (m, 2H, CH₂), 2.25 (s, 3H, CH₃), 2.09 (s, 3H, CH₃), 1.99 (s, 3H, CH₃), 2.0-1.85 (m, 2H, CH₂), 1.84-1.76 (m, 2H, CH₂), 1.26 (s, 3H, CH₃); UV (CH₃OH),  (): 207 (41684), 225 (5407), 287 (1674) nm; EIMS, m/z: 105, 107, 121, 122, 123, 138, 150, 151 (100%), 152, 189, 191, 218, 246, 264, 265, 306 (5%, M⁺) Da, Anal. (C₁₇H₂₂O₅) Calc. 66.65%C, 7.24%H, Found 66.33%C, 7.32%H. Analytical chiral chromatography (solvent C), Rt = 11.93 and 20.53 min.

Preparation and separation of racemic 2,7,8-trimethyl-2-(-carboxyethyl)-6-hydroxy chroman methyl ester (7). 3a (0.5 g) was dissolved in methanol (10 ml) and etheral diazomethane solution was added until the yellow color of diazomethane remained. The solution was stirred at room temperature for 1 h, then the mixture was taken to dryness under reduced pressure. The residue was purified on a silica-gel column with hexane/acetone (3:1), yield 0.48 g (91%), crystallized from methanol/water; m.p. 87-88°C; IR (KBr): 3900-2800, 1708, 1620, 1504, 1458, 1438, 1426, 1380, 1346, 1324, 1260, 1244, 1232, 1210, 1176, 1106, 1082 cm¹; ¹H NMR, (CDCl₃) : 6.37 (s, 1H, CH), 3.67 (s, 3H, OCH₃), 2.72-2.67 (m, 2H, CH₂), 2.53-2.47 (m, 2H, CH₂), 2.13 (s, 3H, CH₃), 2.09 (s, 3H, CH₃), 2.1-1.8 (m, 2H, CH₂), 1.8-1.71(m, 2H, CH₂), 1.23 (s, 3H, CH₃); UV (CH₃OH),  (): 208 (40180), 225 (4948), 297 (3505) nm; EIMS, m/z: 100, 105, 107, 108, 109, 110, 111, 112, 113, 119, 121, 123, 124, 125, 126, 135, 150, 151, 278 (1%, M⁺) Da; Anal. (C₁₆H₂₂O₄) Calc. 69.04%C, 7.97%H, Found 68.60%C, 8.02%H. Analytical chiral chromatography (solvent C), Rt = 8.64 and 11.56 min.

Preparation and separation of 2,7,8-trimethyl-2-{-carboxyethylamide[N-2-(1S,2R)-1,2-diphenylethanol]}-6-O-acetyl chroman (10). 6 (306 mg, 1 mmol) was dissolved in methylene chloride (10 ml) and molecular sieves 4A (0.5 g) added. The mixture was stirred for 2 h at room temperature then cooled in an ice-water bath. Triethylamine (130 mg, 1.3 mmol) followed by (1S,2R)-(+)-2-amino-1,2-diphenylethanol (220 mg, 1.1 mmol) and diethylcyano phosphonate (200 mg, 1.3 mmol) were added. The reaction mixture was stirred for 2 h at 0-5°C. After removal of volatiles under reduced pressure, the residue was purified on a silica-gel column with hexane/ethyl acetate (5:3) as eluent, yield 421 mg (84%). m.p. 89-90°C from acetone-hexane. ¹H NMR, (CDCl₃) : 7.23-7.18 (m, 6H, aromat), 7.0-6.9 (m, 4H, aromat), 6.58 (d, 1H, J 2.6 Hz, CH), 6.19 (t, 1H, J 6.8 Hz, NH), 5.28 (ddd, 1H, J 2.1 Hz, CH), 5.05 (dd, 1H, J 9.7 Hz, OH), 2.85 (2d, 1H, J 5 Hz, H), 2.72 (m, 1H, CH₂), 2.39 (m, 1H, CH₂), 2.92, 2.90 (2s, 3H, CH₃-acetyl), 2.10, 2.09 (2s, 3H, CH₃), 2.03, 2.02 (2s, 3H, CH₃), 1.93 (m, 2H, CH₂), 1.74 (m, 2H, CH₂), 1.24, 1.22 (2s, 3H, CH₃); EIMS, m/z: 103, 105, 106 (100), 107, 151, 189, 193, 195, 196, 247, 250, 501 (0.5%, M⁺) Da; Anal. (C₃₁H₃₅NO₅) Calc. 74.23%C, 7.03%H, 2.79%N, Found 74.11%C, 7.30%H, N 2.63%N. Analytical chiral chromatography (Solvent C), Rt(R, S,R) = 9.87 min, Rt(S, S,R) = 11.61 min. 8 (100 mg) was resolved by preparative chiral chromatography (solvent E). Each diastereoisomer (50 mg) was dissolved in acetone (1 ml) in a small vial that was then placed in a larger vial containing isopropyl ether. The larger vial was loosely capped and left at room temperature. No crystallization occurred.

Preparation and separation of 2,7,8-trimethyl-2-{-carboxyethylamide[N-(1S)-1-methyl-1-phenyl methane]}-6-O-acetyl chroman (11). Compound 6 (3.1 g, 10.1 mmol) was dissolved in methylene chloride (30 ml) and molecular sieve 4A (2 g) was added. The mixture was stirred for 2 h at 0-5°C. Triethylamine (1.3 g, 13 mmol) followed by (S)-()-phenetylamine (1.3 g, 10.6 mmol) and diethylcyano phosphonate (2.1 g, 13 mmol) were added. The reaction mixture was stirred for 2 h at 0-5°C. The reaction mixture was diluted with methylene chloride (200 ml), filtered, and washed with aqueous. citric acid (10%, 70 ml), water (70 ml), saturated aqueous NaHCO₃ solution (70 ml), then dried (Na₂SO₄). After solvent removal, the residue was purified on a silica gel column using hexane/ethyl acetate (7:3) as eluent, yield 3.28 g (80.2%). m.p. 136-137°C from acetone/hexane. ¹H NMR, (CDCl₃) : 7.35-7.25 (m, 5H, Ph), 6.57 (s, 1H,), 5.71 (d, 1H, J 7.7 Hz, NH), 5.12 (m, 1H, CH), 2.71 (m, 2H, CH₂), 2.34 (m, 2H, CH₂), 2.29 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.01 (s, 3H, CH₃), 1.94 (m, 2H, CH₂), 1.74 (m, 2H, CH₂), 1.47 (d, 3H, J 6.3 Hz, CH₃), 1.22 (s, 3H, CH₃); ¹³C NMR, (CDCl₃) : 171.84 (CO), 170.06 (CO), 149.14, 143.17, 141.81, 128.64, 127.34, 126.21, 118.97, 75.14, 48.74, 35.51, 31.14, 30.90, 23.79, 22.06, 21.60, 20.80, 12.66, 12.02; EIMS, m/z: 105, 120, 163, 176, 216, 245, 246, 263, 367, 368, 409 (100%, M⁺), 410 (M+H⁺) Da; Anal. (C₂₅H₃₁NO₄) Calc. 73.32%C, 7.63%H, 3.42%N, Found 73.47%C, 7.75%H, 3.41%N. Analytical chiral chromatography, Solvent C, Rt(R, S) = 12.93 min, Rt(S, S) = 15.46 min. 9 (100 mg) was resolved on preparative chiral chromatography (Solvent E). Each diastereoisomer (50 mg) was dissolved in acetone (1 ml) in a small vial that was then placed in a larger vial containing isopropyl ether. The larger vial was loosely capped and left at room temperature. Only one diastereoisomer crystallized (see "Results" and "Discussion").

Enzymatic hydrolysis of 7. A sample solution of 7 (HPLC purified, 13 mg) was prepared by addition of 2-propanol (0.5 ml). To each of the vials in the ChiroScreen-EH Kit (Altus Biologics Inc., Cambridge, MA) was added 1.0 ml of 0.1 M sodium potassium phosphate buffer pH 7.0, stirred with a magnetic stirrer for 15 min at room temperature. Sample solution (10 µl) was added into each vial. The solution/suspension in vials was stirred with a magnetic stirrer at room temperature. Aliquots (20 µl) were removed at 4, 8 and 24 h. Each aliquot was transferred into a test tube containing 40 µl of 2-propanol and mixed on a vortex shaker. The samples were stored at 20°C before HPLC analysis.

	Results

Top Abstract Introduction Methods Results Discussion References

The synthesis of racemic 2,7,8-trimethyl-2-(-carboxyethyl)-6-hydroxy chroman (3a) was accomplished by boron trifluoride-mediated coupling of 2,3-dimethyl hydroquinone and -methyl--vinyl butyrolactone (Scheme 1). The oxidation part of reductive-oxidative cycle of 3a (Scheme 2) was examined with FeCl₃-mediated oxidation and found to proceed without racemization. Analysis of the oxidation mechanism suggested retention of configuration with the chroman ring opening between C-9 and oxygen (Scheme 3). The stereochemistry of natural 3a, and subsequently of all of the LLU- type metabolites of the Vitamin E family, was proven to be S by crystal analysis of a diastereoisomeric amide (11) (fig. 1). We have also investigated the enantioselective enzyme hydrolysis of the methyl ester of 3a (7) to prepare a larger amount of the enantiomers (table 1). We achieved selective hydrolysis of the R-enantiomer by H. languinosa lipase.

View larger version (15K): [in this window] [in a new window]   Scheme 1.

View larger version (16K): [in this window] [in a new window]   Scheme 2.

View larger version (15K): [in this window] [in a new window]   Scheme 3.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   The X-ray structure of 12. Crystal structure data for 12 (from acetone/isopropyl ether): formula = C25H31NO4, FW = 409.52, m.p. 138.5-139.5°C, monoclinic, space group P21, a = 10.387(1)Å, b = 9.939(2)Å, c = 11.300(2)Å,  = 106.388(9)°, V = 1119.2(3)Å3, Z = 2, density(calc) = 1.215 g cm3, T = 298 K. Anisotropic refinement of all nonhydrogen atoms (271 parameters, H riding model, fixed temperature factors) using 1368 unique reflections with I > 3 sigma (I) from 1895 total data, gave R = .055 and Rw = .067. The authors have deposited the atomic coordinates for this structure with the Cambridge Crystallographic Data Center. The coordinates can be obtained, on request, from the Director, Cambridge Crystallographic Data Center, 12 Union Road, Cambridge, CB2 1EZ, UK.

View this table: [in this window] [in a new window]   TABLE 1 Progress of hydrolysis of LLU- methyl ester (7) by various enzymes as represented by the enantiomeric excess of the S-isomer of that substrate at various time points and at the end of the reaction

	Discussion

Top Abstract Introduction Methods Results Discussion References

Oxidation of tocopherols occurs primarily on the chroman ring producing tocopheronic acid, dimers, trimers and quinones followed by biliary excretion of the oxidation products. Some -oxidation has been presumed to occur with -tocopherol followed by -oxidation of the side chain. -oxidation terminates as the propionic acid side chain as in 3a. Because the isolated LLU- was a single enantiomer (8) with the same absolute stereochemistry as the parent -tocopherol, it is apparent that the lipophilic side-chain oxidation proceeds without chroman ring oxidation (Schultz et al., 1995; Kamal-Eldin and Appelqvist, 1996). Because 3a is natriuretic but not kaliuretic it might be involved in the regulation of Na⁺-K⁺ balance at the cellular level. Most important is that our biologic activities for 3a (Wechter et al., 1996; Murray et al., accompanying paper, 1997) are the first described for -tocopherol.

Direct application of literature procedures for the synthesis of 3b (Weichert et al., 1959; Smith and Ungnade, 1939; Smith et al., 1939) to prepare 3a led to complex mixtures in low yield. The synthesis of 3a initially was improved by use of boron trifluoride to catalyze the coupling of 2,3-dimethyl hydroquinone with -methyl--vinyl butyrolactone (Wechter et al., 1996; Kabbe and Heitzer, 1978). The synthesis of 3a also resulted in the double condensation product, a 1,8-dioxaphenanthrene (4a) which was the major product. The methyl ester of 4a (4b) was prepared to fully characterize the double condensation product. After optimization of reaction time, temperature and order of reagent addition, the yield of 3a was improved from 5 to 52%. Even with the careful exclusion of oxygen, polymeric and oxidative products were formed. After silica-gel chromatography, 3a contained traces of by-products that were removed on preparative RP- HPLC. The absence of a methyl group at C-5 in hydroquinone 1a is the likely explanation for the problems encountered. This would allow for oxidation, polymerization [reaction mixture was dark red (Kantoci, D., unpublished observations)] and formation of the 1,8-dioxaphenanthrene double condensation by-product 4a as well as other side reactions. This improved methodology was used to synthesize several derivatives (3b-d) for biological evaluation (Scheme 1).

The side-chain oxidation mechanism proposed by Simon et al. (1956b) involves the -oxidation of the lipophilic chain subsequent to chroman ring oxidation. The side-chain oxidation continues by -oxidation and terminates at the 3-carbon residue. Schultz et al. (1995) suggested that the - and subsequent -oxidation proceeds without prior oxidation of the chroman ring. Tocopherols are also known as anti-/pro-nitrosating agents (Kamal-Eldin and Appelqvist, 1996) at C-5. It is also possible that nitrogen oxides (NO_x) might react directly with the 4-carbon atom on the lipophilic side chain of tocopherols, which is then easily attacked by peroxide. The resulting reaction would produce a chromanyl aldehyde that is subsequently oxidized to 3a in the case of -tocopherol.

To characterize the oxidized metabolites of Simon et al. (1956a, b) (Scheme 2) and oxidative by-products from synthesis, compounds 5a and 5b were synthesized by FeCl₃ oxidation by use of the procedure of Mayer et al. (1964) of 3a and 3b, respectively (Scheme 4). To establish the retention or inversion of configuration at the C-2 of the chroman ring, 3a and 8 were oxidized with FeCl₃ and analyzed on chiral HPLC. The retention times for enantiomers of 5a were 56.76 and 58.46 min and were produced in equal amounts. When the single enantiomer 8 was oxidized, we observed only one peak at 57.55 min. (slight time difference caused by long retention). This demonstrates that oxidation proceeds without the racemization at C-2. Cohen et al. (1981) investigated the mechanism of oxidation-reduction of various chromanes, and based on optical rotation measurements of the products, concluded that the oxidation with FeCl₃ proceeded likewise with the retention of configuration.

View larger version (20K): [in this window] [in a new window]   Scheme 4.

Kamal-Eldin and Appelqvist (1996) reviewed the chemistry and antioxidant properties of tocopherols and tocotrienols. They suggested a mechanism of radical oxidation of -tocopherol in water and alcohol. We applied the proposed mechanism to the oxidation of 3a and subsequently 8 (Scheme 3) with FeCl₃. Iron-(III)-chloride initiated the reaction by formation of the radical on the oxygen of the hydroxyl group. The radical exists in several resonant forms. Based on isolated products, the radical at C-9 predominates. This radical reacts with iron-(III)-chloride producing a carbocation. The carbocation can then react with water to produce the unstable 9-hydroxy chroman that rearranges into the quinone. This quinone undergoes water abstraction and formation of the hydroquinone lactone 5a. Based on this analysis of the mechanism the configuration at C-2 should be retained; the chroman ring opening occurring between C-9 and the chroman ring oxygen.

From the approximately 600 µg of LLU- (Murray et al., 1995; Wechter et al., 1996) isolated from approximately 800 liters of human uremic urine the direct determination of the absolute configuration at C-2 was not feasible. Therefore we compared the CD spectra from isolated and synthesized 3a. The CD spectrum of each enantiomer of 3a was recorded. One enantiomer exhibited a positive CD at 225 and 295 nm and another a negative CD curve at the same wavelengths (fig. 2). The CD spectrum of natural optically active 3a exhibited a positive CD at 225 and 295 nm (weak +CD expected at 295 nm for n-^*).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   CD spectra of 8 and 9.

Because the biological activity of our isolate is sensitive to the configuration at C-2 (Wechter et al., 1996) it was imperative that it be determined by an unequivocal method. Racemate resolution via salt formation (Cohen et al., 1982; Jacques et al., 1994) with chiral amines was used to prepare sufficient quantity of either diastereoisomer for X-ray analysis. Salts of 3a with the chiral amines [(S)-()--methylbenzylamine or (1S,2R)-(+)-2-amino-1,2-diphenylethanol] were prepared. Resolution by crystallization of these salts was unsuccessful.

To establish the stereochemistry at C-2 unequivocally, the crystal structure of at least one enantiomer needed to be determined. To confirm the stereochemistry of 3a by X-ray crystallographic analysis, the diasteromeric amides 10 and 11 were prepared from 6 and (1S,2R)-(+)-2-amino-1,2-diphenylethanol or (S)-()--methylbenzylamine, respectively (Scheme 5). Crystallization from ethyl acetate/hexane, acetone/hexane or methanol/water resulted in oily or semicrystalline precipitates that did not resolve the diastereoisomers. The diastereoisomers 10 and 11 were therefore separated by preparative chiral HPLC. Crystallization was attempted of all four diastereomers. Only one diastereomer, that of compound 11 (Rt = 15.46 min), crystallized. This monocrystal was subjected to X-ray crystallographic analysis and stereochemistry at C-2 was assigned as S (fig. 1; diastereomer 12, Scheme 5).

View larger version (16K): [in this window] [in a new window]   Scheme 5.

Once a stereochemical assignment was made for diastereomer 12, it was necessary to determine which enantiomer of 3a was the synthetic precursor to that derivative and which enantiomer of 3a corresponded to the natural material. Separation on chiral HPLC of 3a gave two peaks eluting at 7.62 and 9.66 min. The retention time of the second peak (8) corresponded to the elution time of the natural product. The separated enantiomer 8 was acetylated and amidated with (S)-()--methylbenzylamine. The amide 12 that was prepared for X-ray crystallographic analysis was compared with the amide that was prepared from natural 3a enantiomer. The retention times on chiral HPLC for both amides were identical (15.46 min), therefore the absolute stereochemistry of the natural enantiomer at C-2 was assigned as S.

Enzyme Hydrolysis

Because the desired resolution via diastereomeric salts or amides failed, the stereospecific enzymatic cleavage of the methyl ester 7 was investigated. The selective enzymatic hydrolysis of the methyl ester of chromane 3b has been described previously (Horiguchi and Mochida, 1995). To examine the utility of a large number of enzymes for this resolution, the CHIRO-Screen kit (Altus Biologics Inc., Cambridge, MA) was used. The results of this experiment are shown in table 1. The optimal result would be for all of the 7 S-enantiomer to be hydrolyzed (indicated in table 1 as 1.00R). This occurred only for Candida antartica "B" lipase at the 24-h time point. The resulting product was essentially 3a, as would be expected by the earlier time points for this enzyme. The next most advantageous result would be little or no ester hydrolysis of 7 S-enantiomer with complete hydrolysis of 7 R-enantiomer (indicated by 1.00 in table 1). This occurred for many enzymes; however, the most rapid and consistent was Humicola languinosa lipase that hydrolyzed the R-enantiomer of 7 with enantiomeric excess of 1 (Scheme 4). This was used for a future scale-up of the resolution (E. D. Murray, private communication). For some enzymes there appears to be a shift in the activity, depending on the measured time points (table 1).

Conclusions

We have established, by X-ray crystallographic analysis, the absolute configuration of 2,7,8-trimethyl-(S)-2-(-carboxyethyl)-6-hydroxy chroman (8), that arises via side-chain oxidation of -tocopherol, probably in the liver. This assignment can be extended to all members of the vitamin E family (  ) because the process associated with side-chain oxidation does not affect the stereochemistry of the chroman ring. By use of chiral HPLC analysis of the product of chroman ring oxidation of 8, it was established that this oxidation also proceeded without racemization. This suggests that the oxidation cycle (Scheme 2) hypothesized to control extracellular fluid volume proceeds with retention of configuration. We plan to test these intermediates for natriuretic and potassium channel inhibitory activity (Wechter et al., 1996) as well as establish the configuration of the reductive products from this cycle. The separation of the enantiomers of 3a (8 and 9) for biological studies was accomplished by preparative chiral chromatography. The stereoselective enzymatic hydrolysis of methyl ester 7 was also investigated for scale-up preparation of 8. A detailed biological profile of 3a, 8, 9 and derivatives is reported in the accompanying paper (Murray et al., 1997). Our next major study will be the quantitation of 8 and LLU- (Murray et al., 1995) in physiological states associated with volume expansion in humans.