Chem. Res. Toxicol. 1988,1, 343-348
343
Enantioselective Synthesis and Preliminary Metabolic Studies of the Optlcal Isomers of 2-n -Propyl-4-pentenoic Acid, a Hepatotoxic Metabolite of Valproic Acid David J. Porubek, Hope Barnes, Louis J. Theodore, and Thomas A. Baillie* Department of Medicinal Chemistry, School of Pharmacy, BG-20, University of Washington, Seattle, Washington 98195 Received May 23, 1988 The enantiomers of 2-n-propyl-4-pentenoic acid (A4-VPA),a known hepatotoxic metabolite of 2-n-propylpentanoic acid (valproic acid, VPA), were synthesized with the aid of the chiral auxiliaries (4S)-4-(2-propyl)-2-oxazolidoneand (4R,5S)d-methyl-5-phenyloxazolidone.Alkylation of the n-valeryl derivatives of these oxazolidones with allyl bromide, followed by reductive cleavage and chromic acid oxidation of the product, afforded the desired acids, (R)-and (S)-A4-VPA. Greater than 93% enantiomeric excess was achieved in the preparation of both enantiomers. Preliminary studies on the metabolic fate of (R)-and (S)-A4-VPA in freshly isolated rat hepatocytes revealed striking differences in the biotransformation of the two enantiomers. Quantification of two major metabolites of A4-VPA, viz., 4,5-diOH-VPA y-lactone and 2-npropyl-2(E) ,Cpentadienoic acid (Asp4-VPA), indicated that larger amounts of the y-lactone were formed in incubations utilizing (R)-A4-VPAas substrate, whereas production of the diene was greater in incubations with (S)-A4-VPA. On the basis of the premise that A4-VPA serves as a mechanism-based irreversible inhibitor of enzymes of the fatty acid P-oxidation complex, these differences in metabolism suggest that the two enantiomers of A4-VPA may differ in their hepatotoxic potential.
Introduction 2-n-Propyl-4-pentenoic acid [A4-VPA1( l ) ,Figure 13, a hepatotoxic metabolite of the antiepileptic drug valproic acid [VPA (2), Figure 11,has been the focus of considerable toxicological interest in recent years in view of ita putative role in the etiology of VPA-induced liver injury (1-4). Thus, the compound has been shown to be cytotoxic, in a dose-dependent fashion, to rat hepatocytes in culture (5, 6 ) )to be a potent inhibitor of fatty acid @-oxidationin vitro (7) and in vivo ( 8 ) ,and to produce marked elevations in blood urea and SGOT levels when given to rats (9). The mechanism by which A4-VPA causes liver toxicity is not understood. However, the close similarity between its structure and that of 4-pentenoic acid, a mechanismbased inhibitor of mitochondrial ,&oxidation enzymes (10, l l ) ,together with the fact that both agents induce hepatic steatosis when administered to rats (12,14),suggests that these unsaturated carboxylic acid derivatives share a common mechanism of action. Experimental support for this view derives from recent metabolic studies in which it was demonstrated that A4-VPAundergoes further biotransformation, both in the isolated perfused rat liver (15) and in rhesus monkeys (16),by a 0-oxidation pathway which would lead to the generation of 3-oxo-A4-VPA,a highly reactive electrophilic compound (15). It may be surmised, therefore, that metabolic activation of A4-VPA by mitochondrial enzymes leads, in a fashion similar to that established for 4-pentenoic acid, to a reactive metabolite that alkylates, and thereby specifically inhibits, key components of the fatty acid @-oxidationcomplex. Unlike VPA, A4-VPA is an asymmetric molecule whose metabolic formation and subsequent biotransformation may be subject to stereoselective processes. However, all previous studies on the metabolism and toxicology of
* To whom correspondenceshould be addressed. 0893-228~/88/2701-0343$01.50/0
A4-VPA have employed the racemate, and nothing is known about the biological fate of the individual enantiomers. The primary objective of the present study, therefore, was to develop a practical enantioselective synthesis of (R)-and (S)-A4-VPAfor biological investigations. An additional objective of this work was to conduct preliminary studies on the metabolism of the individual enantiomers of A4-VPA in order to determine whether biotransformation of this olefin to potentially toxic products occurs by processes that are dependent upon the absolute configuration at C-2. To this end, Aml4-VPA and 4,5-diOH-VPA y-lactone, two metabolites of A4-VPAthat have been found to represent major products of biotransformation by enzymes of 0-oxidation and cytochrome(s) P-450, respectively, were quantified by GC-MS techniques.
Experimental Procedures Materials. The following chemicals were purchmed from the Aldrich Chemical Co. (Milwaukee, WI): (S)-(+)-valine, (1S,2R)-(+)-norephedrine hydrochloride, boron trifluoride etherate, borane-methyl sulfide complex (10.0-10.2 M), diethyl carbonate, diphenyl carbonate, n-butyllithium (1.6 or 2.5 M in hexane), diisopropylamine, n-valeryl chloride, allyl bromide, lithium aluminum hydride, deuteriochloroform,and the internal standard for GC and GC-MS analyses, l-methyl-l-cyclohexanecarboxylic acid. The silylating agent BSTFA was obtained from Supelco, Inc. (Bellafonte, PA). All other chemicals used in the synthetic procedures were of reagent grade. Dry THF was Abbreviations: VPA, 2-n-propylpentanoic acid; A'-VPA, 2-npropyl-4-pentenoicacid Ame4-VPA,2-n-propy1-2(E),I-pentadienoic acid; 4,5-diOH-VPAy-lactone or 4,5-dihydroxy-VPAy-lactone, 3-n-propyl5-(hydroxymethyl)tetrahydro-2-furanone; 3-oxo-A4-VPA,2-n-propyl-3oxo-4pentenoic acid; BSTFA, N,O-bis(trimethylsily1)trifluoroacetamide; MU, methylene unit; TMS, trimethylsilyl; LDA, lithium diisopropylamide; THF, tetrahydrofuran;SGOT, serum glutamic-oxaloacetictransaminase; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry. 0 1988 American Chemical Society
344 Chem. Res. Toxicol., Vol. 1, No. 6, 1988
A L
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Porubek et al. Scheme I. Synthetic Pathways for ( R ) -and (S)-A4-VPA (9 and 16, Respectively)
2
Figure 1. Structures of A4-VPA (1) and VPA (2). obtained by distillation from sodium benzophenone ketyl prior to use. The chromic acid solution (Na2Crz0,in H2S04)used for oxidations was prepared according to Brown et al. (17). Reference samples of the metabolites, A2E,4-VPAand 4,5-diOH-VPA ylactone, were prepared as described by Rettenmeier et al. (15). @Glucuronidase (Bacterial, type VII) was obtained from the Sigma Chemical Co. (St. Louis, MO). Instrumentation. Melting points were determined in open capillary tubes with a Thomas Hoover melting point apparatus and are uncorrected. Specific rotations were determined with a Jasco DIP-4 digital polarimeter (Jasco, Inc., Easton, MD). 'H (300-MHz) and 13C NMR (75-MHz) spectra were recorded (in CDC13)with a Varian VXR-300 spectrometer (Varian Associates, Inc., Palo Alto, CA). Proton chemical shifts are reported in ppm (6) downfield from internal tetramethylsilane (0.00 ppm), while carbon chemical shifts are reported relative to the central chloroform line (77.0 ppm). Elemental analyses were carried out by Galbraith Laboratories (Knoxville, TN). Gas chromatography was performed with a Hewlett-Packard 5890 instrument equipped with a fused silica capillary column 60 m x 0.32 mm i.d., DB-5, J & W Scientific, Ventura, CA), using helium (20 psi head pressure) as both carrier and detector makeup gas. Samples werre injected by using the splitless mode of operation (250 "C injection port temperature) and "cold-tapped" on the column at 50 "C. The oven temperature was then raised linearly at 20 "C min-' to 280 "C. Detection of eluting compounds was by flame ionization, and retention times were measured relative to a homologous series of n-alkanes coinjected with each sample and are expressed as methylene unit (MU) values. Low-resolution mass spectra of synthetic compounds were obtained with a Hewlett-Packard 5985 GC-MS instrument interfaced with a Hewlett-Packard 5840 gas chromatography adapted for capillary column chromatography. Maas spectra were recorded in the E1 mode, employing an ionizing energy of 70 eV and an ion source temperature of 200 "C. Quantitative GC-MS analyses were carried out with a Hewlett-Packard 5970A mass selective detector (MSD), equipped with a capillary splitless injector and Hewlett-Packard 7673A autosampler. A fused silica capillary GC column (60 m X 0.32 mm i.d., 0.25-pm film thickness) was used, coated with the bonded stationary-phase DB-1 (J & W Scientific, Ventura, CA), and operated with helium (10 psi head pressure) as carrier gas with a 60 mL min-' split flow and 3 mL min-l septum purge through the injector. Samples were injected in the splitless mode (injector temperature 250 "C) and cold-trapped on the column at 40 "C. The column oven temperature was raised rapidly to 80 "C and programmed linearly as follows: 2 "C m i d to 120 "C and then 4 "C m i d to 180 "C, followed by a rapid rise to 250 "C. The mass spectrometer was operated in the selected ion monitoring (SIM) mode, and the filament emission current and electron energy were fixed at 220 pA and 70 eV, respectively. The ion source temperature was 200 "C, and the GC interface was held a t 280 "C. The MSD data system, a Hewlett-Packard 59970C Chem Station, was used to control the selection of the ion windows for monitoring (to 10.2 dalton) and to perform daily tuning of the MSD by means of the Autotune software. Quantification of individual compounds was based upon peak areas in the respective selected ion current chromatograms,measured relative to the peak area of the internal standard, 1-methyl-1-cyclohexanecarboxylic acid. The ions chosen for monitoring purposes were the [M CH3]+ fragments of the TMS derivatives. Biological Experiments. Male Sprague-Dawley rats (160-200 g) were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and were allowed free access to food (Rodent Blox, Wayne Pet Food Division, Continental Grain Co., Chicago, IL) and water prior to use. Hepatocytes were prepared according to MoldGus et al. ( l a ) , and greater than 90% cell viability was achieved routinely as assessed by trypan blue exclusion. Cells were incubated under an atmosphere of 95% 02/5% COz in 50-mL round-bottom flasks, rotated continuously in an incubator at 37 "C. Incubations were
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conducted at a concentration of 2 X lo6 cells mL-' and with a substrate concentration of 1 mM. At appropriate time points, aliquots (2 mL) were removed from the incubation flasks, added to concentration tubes at 0 "C containing the internal standard 1-methyl-1-cyclohexanecarboxylicacid (1.86 pg), and treated with 5% HCl(50 pL) to terminate metabolic activity. Standard curves were prepared by adding 0, 2,4,6,8, and 10 pg of Ams4-VPAor 4,5-diOH-VPA y-lactone to tubes containing 5% HC1 (50 pL), untreated hepatocytes (2 mL, 2 X lo6 cells mL-'), and the internal standard (1.86 pg). The samples were then neutralized (to ca. pH 7) and treated with @-glucuronidase(1mL, 100 units, freshly prepared). Hydrolysis was allowed to proceed overnight (ca. 14 h) at 37 "C, following which the samples were acidified by addition of 5% HCl(50 pL). The samples were then extracted with ethyl acetate (3 X 2 mL), dried (MgS04),concentrated under a gentle stream of dry Nz to a small volume (ca. 0.5 mL), and then transferred to conical Reacti-Vialswhere the volume was reduced further (to ca. 50 pL). BSTFA (100 pL) was then added, the vials were capped, and the samples were heated at 90 "C for 30 min. After cooling to ambient temperature, the derivatized extracts were transferred to 200-pL disposable glass liners which were sealed in autosampler vials for subsequent GC-MS analysis. The latter was performed automatically, with injection, data acquisition, integration, calibration, and quantitative report functions being carried out during unattended operation by the computer system using Sequence software. Synthetic Methods. (A) (2R)-2-n-Propyl-4-pentenoic Acid [(R)-A4-VPA (9)]. The R enantiomer of A4-VPAwas prepared, as outlined in Scheme I, in a five-step sequence starting from (5')-valinol (3) as follows. (4S)-4-(2-Propyl)-~-oxazolidone [(S)-Valinol Oxazolidone (4)]. This chiral auxiliary was synthesized by methods that have been reported previously (19-21) and exhibited the following constants: mp 74 "C; 'H NMR 6 6.15 (b s, 1 H, NH),4.45 (dd, J = 8.7 and 8.7 Hz, 1 H, C,-H), 4.11 (dd, J = 8.7 and 6.3 Hz, 1 H, C,-H), 3.61 (m, 1 H, C,-H), 1.73 [m, 1 H, -CH(CH&], 0.98 [d, J = 6.6 Hz, 3 H, -CH(CHs),], and 0.93 [d, J = 6.6 Hz, 3 H, -CH(CH,),]; 13C NMR 6 17.72 [CH(CH3)2], 18.07 [CH(CH3)2], 32.74 [-CH(CH,),], 58.39 (-C-N-), 68.62 (-C-0-), and 160.29
Enantiomers of 2-n-Propyl-4-pentenoic Acid
(c=o);[.Iz6D = -16.6" (c = 1.19 g/100 mL of EtOH; 1 = 1cm). It should be noted that this specific rotation is of similar magnitude, but of opposite sign, to that reported previously for this compound (I9). (4S)-3-Valeryl-4-(2-propyl)-2-oxazolidone (5). To (S)valinol oxazolidone (4,2.69 g, 20.9 mmol) in dry THF (70 mL) was added n-butyllithium (10.8 mL, 23.0 mmol) dropwise at -78 "C. After stirring at -78 "C for 30 min, valeryl chloride (2.84 g, 23.6 mmol) was added rapidly, and the resulting solution was allowed to warm to 0 "C and stirred. After 3 h, the reaction mixture was treated with aqueous 1 M NaHC03 (10 mL), concentrated in vacuo, and extracted with CHzClz(3 X 20 mL). The organic extracts were combined, washed with 1 M NaHC03 (2 X 10 mL) and once with brine (10 mL), and dried over MgSO,. The organic layer was concentrated to yield a crude oily product which was purified by flash chromatography employing a mobile phase of ether/petroleum ether (1:lv/v). Yield = 2.64 g (60%). 'H NMR 6 4.43 (ddd, J = 8.4, 3.7, and 3.4 Hz, 1H, C4-H), 4.21 (m, 2 H, C5-H,), 2.90 [m, 2 H, -CO-CH2-], 2.36 [m, 1H, -CH(CH3)zl,1.63 (m, 2 H, -COCHz-CHz-), 1.38 [m, 2 H, -CHz-CH3], 0.94 (t, J = 7.2 Hz, 3 H, -CHz-CH3), 0.93 [d, J = 6.9 Hz, 3 H, -CH(CHJZ], and 0.88 [d, J = 7.0 Hz, 3 H, CH(CH3)zI; 13C NMR 6 13.91 (-CH,CH,), 14.72 [-CH(CH,)Z], 18.04 [-CH(CH3)2], 22.31 (-CHZCH,), 26.59 (-CH&H&H3), 28.43 [-CH(CH,)Z], 35.29 (-CO-CHz-), 58.37 (-C-N-), 63.29 (-C-0-), 153.92 ( G O ) , and 173.23 (C=O); MS m/z 213 (M", 1.5%), 198 ([M - CH$, 1.5%), 184 ([M - CZHs]', lo%), 171 ([M - C3Hs]'+, 22%), and 85 (100%); GC 15.1 MU. (4S)-3-(2-Allylvaleryl)-4-(2-propyl)-2-oxazolidone(6). (4S)-3-Valeryl-4-(2-propyl)-2-oxazolidone (5, 2.64 g, 12.4 mmol) was added to a solution of LDA generated from n-butyllithium (6.2 mL, 13.7 mmol) and diisopropylamine (1.39 g, 13.7 mmol) at -78 "C in dry T H F (100 mL). The solution was stirred for 30 min, and then allyl bromide (1.51 g, 12.5 mmol) was added all at once. The resulting solution was allowed to warm to 0 "C and after 90 min, the reaction was estimated to be 90% complete as judged by GC. The solution was acidified with 5% HC1 and concentrated in vacuo. The concentrate was treated with HzO (125 mL) and extracted with ether (125 mL). The ether extract was washed once with HzO (75 mL) and once with brine (75 mL) and dried over MgSO,. Removal of the ether in vacuo left a viscous liquid product. Yield = 2.25 g (75%). 'H NMR 6 5.78 (m, 1 H, -CH=CHz), 5.02 (m, 2 H, -CH=CHz), 4.45 (ddd, J = 8.4, 3.7, and 3.4 Hz, 1 H, C4-H), 4.21 (m, 2 H, C5-Hz), 3.98 (m, 1 H, -CO-CH-), 2.48-2.22 [m, 3 H, -CHz-CH=CHz,-CH(CH,),], 1.82-1.43 (m, 2 H, -CO-CH-CH,-), 1.33 (m, 2 H, -CHz-CH3), 0.97 (t, J = 7.3 Hz, 3 H, -CHz-CH&, 0.93 [d, J = 7.0 Hz, 3 H, -CH(CH3)2], and 0.88 [d, J = 7.0 Hz, 3 H, -CH(CH3)2]; "C NMR 6 14.20 (-CHZCH,), 14.68 [-CH(CH,)Z], 18.09 [-CH(CH,)Z], 20.54 (-CH&H,), 28.42 [-CH(CH3)2], 33.69 (-CHzCH&H3), 37.00 (-CHzCH=CHd, 42.12 (40-CH-), 58.57 (4-N-), 62.99 (C-0-), 116.87 (-CH=CHz), 135.20 (-CH=CHJ, 153.90 (C=O), and 175.89 (C=O). MS m / z 253 (M", 3.5%), 224 ([M- CzH5]+,21%), 211 ([M - C3H6]'+,42%), and 130 (100%); GC 15.5 MU. (2R)-2-n -Propyl-4-pentenoicAcid [ (R)-A'-VPA (9)]. (4S)-3-(2-Allylvaleryl)-4-(2-propyl)-2-oxazolidone (6,2.25 g, 8.89 mmol) was dissolved in dry ethyl ether (50 mL) and cooled to 0 "C. Lithium aluminum hydride (895 mg, 23.6 mmol) was then added to the stirred solution which was quenched after 5 h by successive additions of HzO (4 mL), 15% NaOH (4 mL), and HzO (12 mL). The reaction product was stirred for a further 1-h period, during which time a white precipitate (LiOH) formed and was filtered from the solution. The product from this reaction (8) was not isolated, but the ether phase from the above reduction step was used directly for the following oxidation. To the etheral solution (50 mL) containing (2R)-2-n-propyl4-pentenol(8) was added the chromic acid reagent (26.5 mL, 52.9 mmol,2 mmol of oxidant mL-'). The reaction mixture was stirred for 10 h, the ether phase was separated, and the aqueous layer was extracted with ether (2 X 20 mL). The ether extracts were combined and washed with 5% HCl (4 X 10 mL) and with 1 N NaOH (3 X 10 mL). The alkaline washes were combined and treated cautiously with 5% HC1 until judged to be acidic to litmus paper. This acidified aqueous solution was then extracted with ether (3 X 10 mL), and the combined ether extracts were dried over MgS04. Evaporation of the organic extracts in vacuo afforded
Chem. Res. Toricol., Vol. 1, No. 6,1988 345 the product, 9, as a clear oil. Yield = 459 mg (37%). This product was purified by passage through a silica gel column (5 cm X 5 cm), eluted with 10% ethyl acetate in hexane. 'H NMR 6 5.80 (m, 1 H, -CH=CH2), 5.08 (m, 2 H, -CH=CHz), 2.20-2.50 (m, 3 H, -CH-CHz-CH=CHd, 1.15-1.70 (m, 4 H, -CHz-CHz-), and 0.90 (m, 3 H, -CH3); 13CNMR 6 14.02 (-CHzCH3), 20.47 (-CHzCH3), 33.74 (-CH&H&HJ, 36.19 (-CHzCH=CHz), 44.96 (CO-CH-), 116.84 (-CH=CH,), 135.13 (-CH=CHz), and 181.29 (C=O). GC (TMS ester) 11.5 Mu; [(Y]%D= +6.2 0.4" (N = 3) (C = 0.99 g/IOO mL of EtOH; 1 = 1 cm). Anal. Calcd for CSH14O6 C, 67.60; H, 9.86. Found: C, 67.56; H, 10.24. (B)(2S)-2-n-Propyl-4-pentenoicAcid [(S)-A4-VPA (16)]. The S enantiomer of A4-VPA was prepared, as outlined in Scheme I, in a five-step sequence starting from (lS,Z)-norephedrine (10) as follows. (4R,5S)-4-Methyl-5-phenyloxazolidone (1 1). This chiral auxiliary was synthesized by published methods (17-19) and exhibited the following constants mp 110 "C; 'H NMR 6 7.2-7.4 (m, 5 H, A r m , 6.43 (bs, 1 H, NH), 5.71 (dd, J = 7.0 and 2.3 Hz, 1 H, C5-H), 4.22 (dq, J = 7.8 and 7.0 Hz, 1 H, C4-H), and 0.81 (dd, J = 7 and 2.3 Hz, 3 H, -CH3); 13C NMR 6 17.49 (-CHCHJ, 52.39 (-C-N-), 80.93 (-C-0-), 125.78, 128.30, 134.77 (Ar), and 159.65 (C=O). One aromatic carbon was not observed. [a]25D = +153" (c = 0.95 g/100 mL of CHC1,; 1 = 1 cm). (4R,5S)-3-Valeryl-4-methyl-5-phenyloxazolidone (12). To (4R,5S)-4-methyl-5-phenyloxazolidone (11,227 mg, 1.28 mmol) in dry T H F (20 mL) was added 0.9 equiv of n-butyllithium at 78 OC. After stirring at -78 "C for 30 min, valeryl chloride (1.74 gm,1.45 "01) was added rapidly. The solution was then warmed to 0 "C and stirred. After 3 h, the reaction mixture was quenched by addition of aqueous 1 M NaHC03 (5 mL), the mixture was concentrated in vacuo, and the solution was extracted with CHzClz (3 X 10 mL). The organic extracts were combined, washed with 1 M NaHC03 (2 X 5 mL) and once with brine (5 mL), and dried over MgS04. The solvent was removed under reduced pressure to give the desired product. Yield = 302 mg (90%). 'H NMR 6 7.3-7.5 (m, 5 H, Arm, 5.67 (d, 1H, J = 7.5 Hz, C5-H), 4.78 (dq, J = 7.5 and 6.5 Hz, C4-H), 2.95 (m, 2 H, -CO-CH,-), 1.70 (m, 2 H, -CO-CHz-CHz-), 1.43 (m, 2 H, -CHz-CH3), 0.95 (t,J = 7.6 Hz, 3 H, -CHz-CH3), and 0.89 (d, J = 6.5 Hz, 3 H, -CH-CH3); 13CNMR 6 13.95 (CHZCHB), 14.66 (-CHCH3), 22.33 (-CHZCH,), 26.46 (-CH&H&H3), 35.41 (-CO-CHz-), 54.76 (-C-N-), 78.93 (4-0-), 125.54, 128.60, and 133.26 (Ar),152.92 (C=O), and 173.02 ( G O ) . One aromatic carbon was not observed. MS m / z 261 (M', 5%), 219 ([M - C3&]*+, 93%), and 85 (100%);GC 20.9 MU.
*
(4R,5S)-3-(2-Allylvaleryl)-4-methyl-5-phenyloxzolidone (13). (4R,5S)-3-Valeryl-3-methyl-5-phenyloxazolidone (12,1.0 g, 3.83 mmol) was added to a solution of LDA generated from n-butyllithium (2.5 mL, 4.0 mmol) and diisopropylamine (0.41 g, 4.0 mmol) at -78 "C in dry THF (50 mL). The solution was stirred at -78 "C for 15 min, after which allyl bromide (4.0 mmol, 88 mg) was added all at once. The solution was then held at 0 "C for 60 min and allowed to warm to 25 "C. After 4 h, the reaction was estimated to be 98% complete as judged by GC. The solution cooled to 0 "C, acidified with 5% HC1 (15 mL), concentrated in vacuo, and treated with ether (100 mL). The ether phase was washed successively with 5% HCl(2 X 25 mL), HzO (2 X 25 mL), and brine (25 mL) and dried over MgSO,. Removal of the ether in vacuo left a viscous liquid product, which was purified by flash chromatography on silica gel and eluted with petroleum ether/ether (4:l v/v). Yield = 1.0 g (87%). 'H NMR 6 7.30-7.55 (m, 5 H, A r m , 5.86 (m, 1 H, -CH=CHz), 5.69 (d, J = 7.4 Hz, 1H, C5-H), 5.10 (m, 2 H, -CH=CHz), 4.84 (dq, J = 7.5 and 6.7 Hz, 1H, C4-H), 4.01 (m, 1 H, - CO-CH-), 2.41 (m, 2 H, -CH2-CH=CH2), 1.46-1.82 (m, 2 H, -CO-CH-CHz-), 1.38 (m, 2 H, CO-CH-CH,-CHz-), 0.97 (t,J = 7.4 Hz, 3 H, -CH,CH3), and 0.90 (d, J = 6.6 Hz, 3 H, -CH-CH3); 13C NMR 6 14.24 (-CHZCHJ, 14.69 (CHCH,), 20.57 (-CHZCH3), 33.89 (-CH&H&HJ, 36.87 (-CH&H=CHz), 42.21 (-COCH-), 54.98 (-C-N-), 78.68 (-C-0-), 116.89 (-CH=CHz), 125.55, 128.60, and 133.18 (Ar), 135.13 (-CH=CH,), 152.60 (C=O), and 175.77 (C=O). One aromatic carbon was not observed. MS m / z 301 ( M + ,7%), 272 ([M - C2H5]+,7%), and 259 ([M - C3He]'+,50%), and 118 (100%); GC 22.3 MU. (2S)-2-n -Propyl-4-pentenoic Acid [(S)-A4-VPA (16)]. This compound was synthesized by first reducing (4R,5S)-3-(2-allyl-
346 Chem. Res. Toxicol., Vol. 1, No. 6, 1988
Porubek et al.
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valeryl)-4-methyl-5-phenyloxazolidone (13) and then oxidizing directly, as described the resulting (2s)-2-n-propyl-4-pentenol(l5) above for the synthesis of the R enantiomer. Yield = 100 mg (45%). 'H NMR 6 5.78 (m, 1 H, -CH=CH2),5.05 (m, 2 H, -CH=CH2),2.20-2.50 (m, 3 H, -CH-CH2-CH=CH2), 1.15-1.70 (m, 4 H, -CH2-CH,-CH-) and 0.90 (m, 3 H, -CH3); I3C NMR 6 14.03 (CH,-CHd, 20.49 (-CH,CH3), 33.74 (-CH2CH&H3), 36.20 (-CH2CH=CH2), 44.93 (-CO-CH-), 116.89 (-CH=CH2), 135.11 (-CH=CH&,and 181.25 ( G O ) . GC (TMS ester) 11.5 Mu; = -5.7 0.47' ( N = 3) (c = 0.87 g/100 mL of EtOH; 1 = 1 cm). Anal. Calcd for CBH1402: C, 67.60;H, 9.86. Found: C, 67.76; H, 10.14. (C)Recondensation of (R)and (S)-A4-VPA (9 and 16) with (4S)-4-(2-Propyl)-2-oxazolidone (4). In a 1-mL Reacti-Vial was placed (R)-or (S)-A4-VPA(5pL, 32 pmol) and thionyl chloride (5pL, 68 pmol). The vial was capped, swirled gently, and allowed to stand at room temperature for 90 min. To a separate 2-mLReacti-Vial containing 1 mL of dry THF was added 4 (8.7mg, 68 pmol). The mixture was stirred mechanically and cooled to 0 'C, and n-butyllithium (30pL, 68 pmol) was added. The latter mixture, which turned cloudly, was stirred for 10 min before introducing the acid chloride of (R)- or (S)-A'-VPA (as prepared above) all at once. The cloudy solution immediately turned clear, and stirring was continued for an additional 15 min at 0 'C. An aliquot (2pL) of the reaction mixture was then added to 1mL of ethyl acetate, and a 1-pL portion of this solution was taken directly for GC analysis.
*
Results Synthesis. Enantioselective synthesis of (R)- and (S)-A4-VPAwas accomplished through utilization of the chiral auxiliaries (4S)-4-(2-propyl)-2-oxazolidone (4) and (4R,5S)-4-methyl-5-phenyloxazolidone (11) (Scheme I). According to the general procedures developed by Evans et al. (19-21), the oxazolidones 4 and 11 were prepared by reaction of valinol and 2-amino-1-phenylpropanol with diethyl carbonate and diphenyl carbonate, respectively. Reaction of the oxazolidones with valery chloride produced the valeryloxazolidones 5 and 12. Alkylation of the lithium enolates of 5 and 12 with allyl bromide produced the desired alkylated intermediates 6 and 13. Minor amounts of the undesired diastereomers 7 and 14 were also produced in these reactions, the quantities of which, determined by capillary GC (Figure 21, corresponded to approximately 1.8 and 2.470, respectively, of the reaction product. Thus, the diastereomeric excess of 6 was 96.4% while that of 13 was 95.2%. No attempts were made to enrich chromatographically the composition of these products. Reductive cleavage of 6 and 13 with lithium aluminum hydride afforded the chiral alcohols 8 and 15 which were then oxidized to the target compounds 9 and 16 by treatment with chromic acid. Biological Studies. When incubated individually with freshly isolated rat hepatocytes, (R)-and (S)-A4-VPAunderwent metabolism to a variety of products characteristic
.
, 2
, 3
,
, 4
. 5
Time (h)
12
Figure 2. GC separation of the diastereomers 6 and 7.
.
Figure 3. Comparison of the time course of AZv4-VPAformation during the metabolism of (R)- or (S)-A4-VPA.Hepatocytes (2 x lo6mL?) were incubated with substrate (1.0mM), and aliquots were removed at 0, 1, 2,and 4 h. Metabolites were isolated, derivatized, and analyzed by automated GC-MS. (0) A**'-VPA formed from (S)-A4-VPA;(B) AZp4-VPAformed from (R)-A4-VPA. ***P = 0.01.
-
10 hepatocytes
4,5-Dihydroxy-VPA
4i
***
**
-y-lactone
A
Time (h)
Figure 4. Comparison of the time course of 4,5-diOH-VPA y-lactone formation during the metaboliim of (R)-or (S)-A4-VPA. Hepatocytes (2X lo6 mL-') were incubated with substrate (1.0 mM), and aliquots were removed at 0, 1,2,and 4 h. Metabolites were isolated, derivatized, and analyzed by automated GC-MS. (0) Lactone formed from (S)-A4-VPA;(B) lactone formed from (R)-A4-VPA.*P = 0.05; **P= 0.025;***P = 0.01, of @-oxidation,the major component of which was the diene metabolite, AWy4-VPA (15). The level of this metabolite was found to increase with time during incubation of both (R)- and (S)-A4-VPA (Figure 3). Indeed, concentrations of Azp4-VPA in the incubations of (R)-A4-VPA increased in an almost linear fashion over a period of 4 h, whereas levels of AWp4-VPAin the incubations of (S)A4-VPA approached a plateau during this interval. At all time points other than zero, the level of Azi4-VPA was greater in incubations containing (S)-A4-VPAthan in the corresponding experiments with the R enantiomer. (R)- and (S)-A4-VPA also underwent metabolism in isolated hepatocytes to 4,5-diOH-VPA y-lactone, which was shown in earlier studies with rat liver microsomal preparations to be a major product of cytochrome P-450 mediated olefin oxidation (22). The formation of this metabolite in hepatocytes was also time-dependent and increased steadily during the 4-h incubation of both (R)and (S)-A4-VPA (Figure 4). At all time points other than zero, the level of 4,5-diOH-VPA y-lactone was greater in incubations containing (R)-A4-VPAthan those in which the S enantiomer served as substrate.
Discusslon Elegant procedures have been developed by Evans et al. (19-21, 23, 24) for the enantioselective synthesis of chiral a-substituted carboxylic acids and proved to be effective in the present studies for the preparation of the
Enantiomers of 2-n-Propyl-4-pentenoic Acid Scheme 11. Condensation of the Acid Chlorides of (R)and (S)-A‘-VPA with the Lithium Salt of 4
//
O
Chem. Res. Toxicol., Vol. 1, No. 6,1988 347
mediated hepatotoxicity does depend on absolute configuration at C-2 remains to be established, although future experiments that make use of the synthetic methodology presented in this report will allow this intriguing hypothesis to be tested directly. Such studies will include an assessment of the respective toxicities of the individual enantiomers of A4-VPA, and a determination of their relative rates of formation during metabolism of VPA itself.
Acknowledgment. We thank Ms. Charlotte Widener, Mr. Robert Gollehon, and Mr. Terry Armadei for assistance in preparation of the manuscript. These studies were supported by National Institutes of Health Research Grants GM 32165 and DK 30699. Registry No. 4, 17016-83-0; 5, 117039-57-3; 6,117039-58-4;
n
7, 117039-59-5;8,117039-60-8; 9,117039-61-9; 11,77943-39-6; 12, 105311-39-5; 13, 117039-62-0; 14, 117039-63-1; 15, 117039-64-2; 16,117039-65-3; 4,5-diOH-VPA ylactone, 25093-79-2; ABv4-VPA, 90830-41-4; valeryl chloride, 638-29-9; allyl bromide, 106-95-6. 6
1
References (1) Zafroni, E. S., and Berthelot, P. (1982) “Sodium valproate in the
enantiomers of A4-VPA. The key step in the synthetic sequence is diastereoselective alkylation of the chiral oxazolidone imide enolates of 5 and 12. Indeed, alkylation of these enolates with allyl bromide was found to proceed quite rapidly at 0 “C and to be highly diastereoselective (greater than 97% in both cases). As observed previously (19-21), the selectivity achieved with auxiliary 4 is somewhat greater than that achieved with 11. The chiral auxiliaries were cleaved from their respective acyl moieties by treatment with lithium aluminum hydride, and the chiral alcohols (8 and 15) thus formed were subjected to chromic acid oxidation to obtain the desired acids (9 and 16). In order to verify that the above reduction and oxidation steps did not lead to appreciable epimerization at the chiral centers, the acid chlorides of 9 and 16 were recondensed with the chiral auxiliary 4 (Scheme 11). It was found by capillary GC analysis that diastereomer 6 was formed in 94.6% excess from the acid chloride of (R)-A4-VPA,while diastereomer 7 was generated in 93.3 % excess from the corresponding derivative of (S)-A4-VPA. Thus, negligible epimerization occurred in the final steps of the synthesis which involved reductive cleavage of the acyloxazolidones and oxidation of the resultant chiral alcohols. With the chiral acids, (R)-and (S)-A4-VPA,in hand, it was possible to investigate the metabolism of each compound in isolated rat hepatocytes. Quantification of the two major metabolites of this terminal olefin, viz., VPA and 4,5-diOH-VPA y-lactone, which may be taken as indices of A4-VPAmetabolism by @-oxidationand cytochrome P-450 enzymes, respectively, revealed marked differences in the conversion of substrate to products in the two experiments. Thus, while (S)-A4-VPA underwent metabolism to Awp4-VPAmore rapidly than (R)-A4-VPA, the latter was transformed more effectively to 4,5-diOHVPA y-lactone. This observation indicates that, during the course of the 4-h incubation period, the enzymes of @-oxidation select preferentially the S enantiomer of A4-VPA,whereas cytochrome P-450 catalyzed olefin oxidation exhibits the reverse stereoselectivity in turnover of A4-VPA. This finding may have important toxicological consequences, in that it implies that one enantiomer of A4-VPAmay be largely responsible for the hepatotoxicity associated with this olefin if, indeed, bioactivation of A4-VPA via the &oxidation pathway represents a key event in the process leading to liver injury. Whether A4-VPA-
induction of unusual hepatotoxicity”. Hepatology (Baltimore) 2, 648-649. (2) Zimmerman, H. J., and Ishak, K. G. (1982) “Valproate-induced hepatic injury: analysis of 23 fatal cases”. Hepatology (Baltimore) 2, 591-597. (3) Dreifuss, F. E., Santilli, N., Langer, D. H., Sweeney, K. P., Moline, K. A., and Menander, K. B. (1987) “Valproic acid hepatic fatalities: a retrospective review”. Neurology 37, 379-385. (4) Baillie, T. A., and Rettenmeier, A. W. (1988) “Valproate: biotransformation”. In Antiepileptic Drugs (Levy, R. H., Dreifuss, F. E., Mattson, R. H., Meldrum, B., and Penry, J. K., Eds.) 3rd ed., Raven, New York, in press. (5) Kingsley, E., Tweedale, R., and Tolman, K. G. (1980) “Hepatotoxicity of sodium valproate and other anticonvulsants in rat hepatocyte cultures”. Epilepsia (N.Y.)21, 699-704. (6) Kingsley, E., Gray, P., Tolman, K. G., and Tweedale, R. (1983) “The toxicity of metabolites of sodium valproate in cultured hepatocytes”. J. Clin. Pharmacol. 23, 178-185. (7) Bjorge, S. M., and Baillie, T. A. (1985) “Inhibition of mediumchain fatty acid &oxidation by valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoicacid”. Biochem. Biophys. Res. Commun. 132, 245-252. (8) Turnbull, D. M., Bone, A. J., Bartlett, K., Koundakjian, P. P., and Sherratt, S. A. (1983) “The effects of valproate on intermediary metabolism in isolated rat hepatocytes and intact rats”. Biochem. Pharmacol. 32, 1887-1892. (9) Schiifer, H., and Liihrs, R. (1984) “Responsibility of the metabolite pattern for potential side effects in the rat being treated with valproic acid, 2-propylpenten-2-oicacid, and 2-propylpenten-4-oic acid”. In Metabolism of Antiepileptic Drugs (Levy, R. H., Pitlick, W. H., Eichelbaum, M., and Meijer, J., Eds.) pp 73-83, Raven, New York. (10) Fong, J. C., and Schulz, H. (1978) “On the rate-determining step of fatty acid oxidation in heart. Inhibition of fattty acid oxidation by 4-pentenoic acid”. J. Biol. Chem. 253, 6917-6922. (11) Schulz, H. (1983) “Metabolism of 4-pentenoic acid and inhibition of thiolase by metabolites of 4-pentenoic acid”. Biochemistry 22, 1827-1832. (12) Glasgow, A. M., and Chase, H. P. (1975) “Production of the features of Reye’s syndrome in rats with 4-pentenoic acid”. Pediatr. Res. 9, 133-138. (13) Kesterson, J. W., Granneman, G. R., and Machinist, J. M. (1984) “The hepatotoxicity of valproic acid and its metabolites in rats. I. Toxicologic, biochemical and histopathologic studies”. Hepatology (Baltimore) 4, 1143-1152. (14) Granneman, G. R., Wang, &-I., Kesterson, J. W., and Machinist, J. M. (1984) “The hepatotoxicity of valproic acid and ita metabolites in rats. 11. Intermediary and valproic acid metabolism”. Hepatology (Baltimore) 4, 1153-1158. (15) Rettenmeier, A. W., Prickett, K. S., Gordon, W. P., Bjorge, S. M., Chang, S.-L., Levy, R. H., and Baillie, T. A. (1985) “Studies on the biotransformation in the perfused rat liver of 2-n-propyl4-pentenoic acid, a metabolite of the antiepileptic drug valproic acid. Evidence for the formation of chemically reactive
348 Chem. Res. Toxicol., Vol. 1, No. 6, 1988 intermediates”. Drug Metab. Dispos. 13, 81-96. (16) Rettenmeier, A. W., Gordon, W. P., Prickett, K. S., Levy, R. H., and Baillie, T. A. (1986) “Biotransformation and pharmacokinetics in the rhesus monkey of 2-n-propyl-4-pentenoic acid, a toxic metabolite of valproic acid”. Drug Metab. Dispos. 14, 454-464. (17) Brown, H. C., Gary, C. P., and Lin, K.-T. (1971) “The oxidation of secondary alcohols in diethyl ether with aqueous chromic acid. A convenient procedure for the preparation of ketones in high epimeric purity”. J. Org. Chem. 36, 387-390. (18) MoldBus, P., Hogberg, J., and Orrenius,,S. (1978) “The isolation and use of liver cells”. Methods Enzymol. 52,60-71. (19) Evans, D. A., Bartroli, J., and Shih, T. L. (1981) “Enantioselectivealdol condensations. 2. Erythro-selectivechiral 103, aldol condensations via boron enolates”. J. Am. Chem. SOC. 2126-2129. (20) Evans, D. A., Takacs, J. M., McGee, L. R., Ennis, M. D., Mathre, D. J., and Bartroli, J. (1981) “Chiral enolate design”.
Porubek et al. Pure Appl. Chem. 53,1109-1127. (21) Evans, D. A., Ennis, M. D., and Mathre, D. J. (1982)
‘Asymmetric alkylation reactions of chiral imide enolates. A practical approach to the enantioselective synthesis of a-substi104, tuted carboxylic acid derivatives”. J. Am. Chem. SOC. 1737-1739. (22) Prickett, K. S., and Baillie, T. A. (1986) “Metabolism of unsaturated derivatives of valproic acid in rat liver microsomes and destruction of cytochrome P-450”. Drug Metab. Dispos. 14, 221-229. (23) Evans, D. A. (1984) “Studies in asymmetric carbon-carbon bond construction”. In Stereospecificity in Chemistry and Biochemistry, Proceedings of the Robert A. Welch Foundation Conferences on Chemical Research, Vol. 27, pp 13-49, Robert A. Welch Foundation, Houston. (24) Evans, D. A. (1988) “Stereoselectiveorganic reactions: catalysts for carbonyl addition processes”. Science (Washington, D.C.) 240, 420-426.
