1194
J. Org. Chem. 1983, 48, 1194-1197
Potential Bile Acid Metabolites. 8.’ 7,12-Dihydroxy- and 7P-Hydroxy-5P-cholanic Acids2 Takashi Iida3 and Frederic C. Chang*, Department of Biochemistry, College of Medicine, University of South Alabama, Mobile, Alabama 36688 Received September 9, 1982
Syntheses of 70-hydroxy- and the stereoisomeric 7,12-dihydroxycholanic acids, their methyl esters, and some related derivatives are described. 12a-Mesyloxy groups, unlike their 7a analogues, were found to be resistant to K02-crown ether inversion.
As part of a program of synthesis of potential bile acid metabolites, to make available for use as reference standards new or scarce compounds, we have recently reported the preparation of the unreported and uncommon 3,7acids. Since dihydroxy5 and 3,7,12-trihydro~ycholanic~~~ the 3,12-dihydroxy8 and all the monohydroxy acids (substituted a t positions 3, 7, or 12)9910were already known, with the publication of this work all 26 of the theoretically possible 5P-cholanic acids substituted with one to three hydroxyl groups a t positions 3, 7, and 12 have now been prepared, characterized, and recorded in the literature. 7a,12a-Dihydroxycholanicacid (I, Chart I), the starting material in our syntheses of the other 7,12-stereoisomers, was previously prepared by Wolff-Kishner reduction of the 3-keto derivative of cholic acid.’l We have synthesized I by a n alternate route frm cholic acid by a procedure introduced years ago by Barnett and Reichstein12and used successfully in a number of previous syntheses via A3cholenic acid d e r i ~ a t i v e s . ’ ~ - l ~T h e procedure involves dehydrotosylation of a 3a-tosylate of the bile acid and subsequent catalytic hydrogenation of the resulting olefin. T h e feature of this method is the easy and efficient selective tosylation a t C-3 of those 5P-cholanes also having a n a-configurated hydroxy group(s) a t C-7 and/or C-12. In this instance, methyl 3-tosylcholate” on treatment with 2,4-lutidine affords a crystallizable unsaturated product, which could be directly hydrogenated to the desired 7a,12a product Ia in good yield. (1)Paper 7: T. Iida and F. C. Chang, J. Org.Chem., 47,2972 (1982). (2) All cholanic acid derivatives in this work are of the 5 6 series; the 56 designations are subsequently omitted in their names. In uniformity with the nomenculature of the previous papers of this series, the older name “cholanic” is used in place of the newer IUPAC-suggested “cholanoic” acid. (3) Nihon University, Japan. (4) Present address: Department of Chemistry, Harvey Mudd College, Claremont, Ca 91711. (5) T. Iida and F. C. Chang, J. Org. Chem., 47, 2966 (1982). (6) F. C. Chang, J. Org. Chem., 44, 4567 (1979). (7) T. Iida and F. C. Chang, J. Org. Chem., 47, 2972 (1982). (8) F. C. Chang, N. J. Wood, and W. G.Holton, J.Org. Chem., 30,1718 (1965). (9) H. Van Belle, “Cholesterol, Bile Acids and Atherosclerosis”, North Holland Publishing Co, Amsterdam, 1965, pp 33-34. (10) J. T. Matschiner in ”The Bile Acids”, Vol. I, P. P. Nair and D. Kritchevsky, Eds., Plenum Press New York, 1971, pp 16-17. (11) R. Grand and T. Reichstein, Helu. Chem. Acta, 28, 344 (1945). (12) T. Barnett and T. Reichstein, Helu. Chem. Acta, 21, 926 (1938). (13) F. C. Chang, A. Feldstein, J. R. Gray, G. B. McCaleb, and D. H. Sprunt, J . Am. Chem. Soc., 79, 2167 (1957). (14) R. T. Blickenstaff and F. C. Chang, J . Am. Chem. Soc., 80, 2726 (1958). (15) R. T. Blickenstaff and F. C. Chang, J. Am. Chem. Soc., 81, 2835 (1959). (16) R. T. Blickenstaff and E. L. Foster, J.Org. Chem., 26,2883 (1961). (17) R. T. Blickenstaff, K. Atkinson, D. Breaux, E. Foster, Y. Kim, and G . C. Wolf, J. Org. Chem., 36, 1271 (1971).
0022-326318311948-1194$01.50/0
Chart I a
I, R, = &-OH;R, = a-OH 11, R, = a-OH; R, = P-OH 111, R, = P-OH; R, = a-OH IV, V, VI, VII, VIII,
IX, X, XI, XII, XIII, XIV, XV, XVI,
R, R, R, R, R, R, R, R, R, R, R, R, R,
P-OH; R, = P-OH = a-OH; R, = 0 = P-OH; R, = 0 = a-OAc; R, = a-OH = a-OAc; R, = 0 = a-OMs; R, = a-OH = a-OMs; R, = 0 = a-OMs; R, = a - O M s = H; R, =,a-OMs = 0-OH; R, = H = 0; R, = H = @-OH;R, = H = @-OMS;R, = H =
XVII, R, = a-OTs; R, = o-OH; R, = &-OH XVIII, R, = A 3 ; R, = a-OH;
R, = a-OH An asterisk indicates t h a t the corresponding C-24 methyl esters are designated “a”. a
Syntheses of the stereoisomers 11-IV involve the 7hydroxy-12-oxo epimers V and VI as key intermediates, as summarized in Scheme I. T h e 7a-monoacetate VIIa was oxidized to VIIIa, hydrolyzed to the hydroxy keto acid V, and reesterified to the methyl ester Va. Reduction of Va by tert-butylamine-borane as previously describedI8 yielded stereoselectively the 7cu,l2P-stereoisomer IIa. T h e other two acids I11 and IV were obtained analogously by reduction of the 70-hydroxy-12-oxo ester VIa, prepared by inversion via the mesylate of its epimer Xa. Both the borane complex and N&H4 are stereoselective reducing agents on 12-0xocholanes;~~ the former yields the 120-hydroxy compound, whereas NaBH, affords the 12ahydroxy epimer. T h e 12/3/12a ratos obtained were 67:33 and 37:63, respectively. (18) F. C. Chang, Synth. Commun., 11, 875 (1981).
0 1983 American Chemical Society
J. Org. Chem., Vol. 48, No. 8, 1983
Potential Bile Acid Metabolites
1195
Scheme I a n
VIIa
Va
VIIIa
VI
Xa
i
VIa a
IVa
IIIa
R = -CH(Me)CH,CH,COOMe; R' = corresponding C-24 acid.
Attempts to selectively mesylate the 7a,l2a-ester Ia at C-7, aimed at obtaining the necessary intermediate for the direct preparation of 7@,12a acid I11 were unsuccessful. Under the usual mesylation conditions,19 no monomesylate IXa was found; instead, crystalline dimesylate XIa was readily obtained. Attempts to achieve selective reaction by gradually moderating t h e mesylating conditons only resulted in products containing proportionally higher amounts of t h e starting compound. Direct converson of t h e dimesylate XIa to t h e 7P,12P acid was not pursued when it was found that methyl 12a-(mesy1oxy)cholanate ( X I I a P in the KO,-crown ether reaction did not undergo conversion at C-12 b u t was merely hydrolyzed to 12a-(mesyloxy)cholanic acid (XII).21 T h e four 7,12-~tereoisomericmethyl esters are well resolved by HPLC with a C-18 reverse-phase column, t h e compounds emerging in t h e order 7@,12/3,7@,12a,7cu,12P, a n d 7a,12a, with t h e last much more slowly t h a n t h e others. 7P-Hydroxycholanic acid (XIII) has been prepared by sodium-propanol reduction of the corresponding keto acid XIV.22 We describe an alternative synthesis consisting of t h e K02-crown ether inversionlg of methyl 7a-(mesyl0xy)cholanate (XVIa). The absence of interfering groups in t h e monohydroxy ester XVa enables both t h e mesylation a n d subsequent inversion reaction to proceed smoothly; t h e mesylate XVIa and the desired acid XI11 a r e both obtained crystalline in good yields. T h e 7Phydroxy ester XIIIa was successfully crystallized.22 (19) T. Iida and F. C. Chang, Lipids, 16,863 (1981). (20) F. C. Chang, J. Chin, Chem. Soc. (Taipei),Ser. 2 , 9 , 53 (1962). (21) Thus,of the three a-mesylates at positions 3,7, and 12, the axial 7-mesyloxy group is inverted by K02-crown ether, while both the axial 12-mesylate and the equatorial 3-mesylate' are inert. (22) L. W. Wells, Doctoral Dissertation, St. Louis University, 1964, p 68. The methyl ester prepared with diazomethane (not crystalline) was analyzed for purity by gas chromatography. Methyl ester XIVa has been included in the extensive bile acid TLC and GLC analyses tables compiled by P. Eneroth and J. SjbvaU T h e Bile Acids", Vol. I, P. P. Nair and D. Kritchevsky, Eds., 1971, p 121.
Experimental Section Melting points were determined on an electric micro hot stage and are uncorrected. Infrared spectra were obtained on a Perkin-Elmer infrared spectrophotometer. NMR spectra were determined with a Perkin-Elmer R-32 instrument, with deuteriochloroform containing Me4Sias the solvent except where otherwise indicated. For TLC the development solvent used in the trisubstituted derivatives was CHC1,-EtOAc-HOAc (45:45:10). Solvents were evaporated on a Rotavap at 50 OC under reduced pressure. All bile acid derivatives were dried by azeotropic distillation [benzeneor benzeneCH2C12,CHzC12or CH2ClZ-MeOH (acids)]before use in reactions. HPLC was performed on a Waters Associates assembly with a septumless injector, a refractive index detector, and C-18 reverse-phase columns [Radial Compression (Waters)or Excalibar (Applied Science)];the solvent system used was methanol-water (80:20; flow rate, 0.5 mL/min). Methyl 3-Tosylcholate (XVIIa)?, Methyl cholate (20 g) was dissolved in benzene containing enough dry pyridine to effect complete solution, evaporated to a clear residual oil, redissolved in 120 mL of pyridine, and treated in one portion with a solution of 12 g of tosyl chloride in 12 mL of pyridine. The mixture after being allowed to stand overnight at room temperature was very slowly dripped into a stirred slush of dilute-HC1 and ice chips (ca. 2 L). The filtered colorless prcipitate, washed with HzO, was allowed to air-dry (ca. 25 "C, 48 h) and was crystallized from MeOH-H,O (9:l) to give prismatic needles: 13.4 g (91%, in two crops); mp 132.5-133.5 "C [lit. 133-134 OC,17131-133 OC12 (from MeOH)]; IR 1730 (C=O), 3534,1034,1020, and 985 (OH), 1351 and 1170 (SOz) cm-'; NMR 6 0.64 (3 H, s, C-18 Me), 0.83 (3 H, (23) In contrast with the earlier preparations of this c o m p ~ u n d ~we~ J ~ have found that the reported required conditions [(a) molecular equivalence of tosyl chloride to ester, (b) 0 "C temperature of reacton, and (c) column chromatographic separation] are superfluous. Excess tosyl chloride and room temperature reaction proved to be satisfactory, and the precipitated product was essentially homogenous. The key factors involved in obtaining a clean product are (a) the quality of the starting methyl cholate (ester prepared from commercial grade cholic acid, although nicely crystalline, is impure), (b) freshly crystallizedtosyl chloride, and (c) a very slow drip of the reaction product into the ice-cold stirred HCl solutions. (Ice chips and HCl should be added as the mixture nears neutrality. If the pyridine solution is added b o rapidly, the precipitate will clump and become gummy.)
1196
J. Org. Chem., Vol. 48, No. 8, 1983
s, C-19 Me), 2.41 (3 H, s, Ar Me), 3.63 (3 H, s, COOMe), 3.82 (1 H, m, C-7 CHOH), 3.92 (1H, m, (2-12 CHOH), 4.29 (1H, br m, C-3 CHOTs), 7.30 and 7.77 (each 2 H, d, J = 8 Hz, para-disubstituted phenyl). Methyl 7a,l2a-Dihydroxy-3-cholenate (XVIIIa).z4The tosylate XVIIIa (4.49 g) was refluxed in 80 mL of 2,4-lutidinez5 under Nz for 5 h. The mixture was dripped slowly into stirred, ice-cold,dilute (0.5 N) HzS04. The suspension was extracted with CH,Clz, and the CHzClzexract was washed with HzOto neutrality. The slightly colored extract, after Norite treatment, was evaporated to a nearly colorless viscous oil (2.84 g), which according to HPLC and NMR was essentially homogeneous. An aliquot of the oil was crystallized from aqueous MeOH, affording dense 3425,1040, triangular prisms: mp 121.0-123.5 "C; IR 1724 (M), and 987 (OH) cm-'; NMR 6 0.72 (3 H, s, C-18 Me), 0.97 (3 H, s, C-19 Me), 3.67 (3 H, s, COOMe), 3.78 (1 H, m, C-7 CHOH), 3.99 (1H, m, C-12 CHOH), 5.60-5.73 (2 H, br m, C-2, C-3, C-4 olefinic). Anal. Calcd for C25H4004: C, 74.21; H, 9.97. Found: C, 74.14; H, 10.12. Methyl 7a,l2a-Dihydroxycholanate(Ia).The oil XVIIIa (2 g) in 100 mL of MeOH, from the dehydrotosylation (above) without crystallization, was catalytically hydrogenated in a Parr apparatus with 100 mg of PtOz at a 2-atm pressure for 4 h. The methanol solution on evaporaton yielded solid which was crystallized from aqueous methanol to form colorless needles: 1.7 g in the first crop (65% from tosylate XVIIIa); mp 157.5-158.0 "C (lit.%mp 155-156 "C); IR 1724 (C=O), 3425,1024, and 984 (OH) cm-'; NMR 6 0.68 (3 H, s, C-18 Me), 0.89 (3 H, s, C-19 Me), 3.63 (3 H, s, COOMe), 3.84 (1H, m, C-7 CHOH), 3.96 (1 H, m, C-12 CHOH). Methyl 7a-Acetoxy-12-oxocholanate (VIIIa). Methyl 7acetoxy-7a,l2a-dihydroxycholanate (VIIa,275.5 g) was oxidized by the method of Fieser and Rajagopalanz8in the preparation of the corresponding 3,7-diacetate. The crystalline product (5.06 g, 92%) was recrystallizedfrom aqueous acetone as colorless stout prisims which melted at 174-175 "C, resolidified, and remelted at 181-182 "C (lit.27mp 172-173 "C): IR 1721 and 1695 (C=O), 1245 and 1016 (acetate) cm-'; NMR 6 1.01 (6 H, s, (2-18 and C-19 Me), 1.99 (3 H,s, OCOMe), 3.64 (3H, s,COOMe),4.97 (1 H, m, C-7 CHOAC). 7a-Hydroxy-12-oxocho1anicAcid (V). A solution of 4.5 g of VIIIa in 120 mL of 10% methanolic KOH was refluxed for 12 h. After evaporation of the solvent, the residue was dissolved in ethanol-water (1:3), and the cooled solution was acidified with 5N HzSOI. The precipitated solid was crystallized from aqueous ethanol as colorless leaflets 3.4 g; mp 98-100 "C. A second crop obtained form the mother liquor weighed 0.46 g (total yield, 3.86 g 97%): IR 1706 and 1678 (C=O), 3390,1032,1017,1000, and 985,909 cm-'; NMR (CHCl, + 10% MezSO-d6)6 1.00 (3 H, s, C-18 Me), 1.02 (3 H, s, C-19 Me), 3.84 (1H, m, C-7 CHOH). Anal. Calcd for Cz4H3804~Hz0: C, 70.55; H, 9.87. Found: C, 70.19; H, 10.21. Methyl 7a-hydroxy-12-oxocholanate (Va),obtained from V by methanol-HC1 esterification and processed in the usual way, on crystallization yielded colorless fine needles: mp 113.5-1 14.5 R 1721 and 1695 (C=O),3390,1031,1014,1000,and 982 (OH) "C; I (24) Although the product is designated A3, olefins prepared by this mnthod of dehydrotosylation are known to have varying proportions of the Az isomer. Issidorides, Fieser, and Fieser [J.Am. Chem. SOC.,82,2002 (1960)l established that the nicely crystalline 'methyl A3-cholenate"obtained by lutidine dehydrotosylation of methyl lithocholate tcaylate was a mixture of Az and A3 and estimated by IR that 25% of the Az isomer is present, on the basis of a comparison of ita IR spectrum with that of authentic Az isomer in the 15-pm region. However, Blickenstaff and Foster,I6using the same IR criteria, concluded that a related compound prepared by collidine dehydrotosylationwas essentially free of A* isomer. Since our goal was to obtain an olefin which could give the C-3 methylene analogue, at present we do not know to what extent our product is a A3-Az mixture. (25) We have found that use of the pyridine base 2,4-lutidine (Eastman practical grade), instead of the more costly 2,6-lutidine,I3 is satisfactory, although if the reaction is not carried out under Nz, the reaction mixture darkens considerably, and the residual oil product obtained had much color. (26) F. Nakada, Steroids, 2, 45 (1963). (27) R. T. Blickenstaff and B. Orwig, J . Org. Chem., 34,1377 (1969). (28) L. F. Fieser and S. Rajagopalan, J . Am. Chem. SOC.,72, 5530 (1950).
Iida and Chang cm-'; NMR 6 0.99 (3 H, s, C-18 Me), 1.01 (3 H, s, C-19 Me), 3.63 (3 H, s, COOMe), 3.92 (1 H, m, C-7 CHOH). Anal. Calcd for CZ5H4004: C, 74.21; H, 9.97. Found: C, 73.96; H, 9.88. Methyl 7a,l2@-Dihydroxycholanate(IIa).To a magnetically stirred solution of 1.0 g of the hydroxyoxo ester Va in 50 mL of CHZC1, was added 0.50 g of tert-butylamine-borane.'8 The clear solution, after some effervescence, was allowed to stand at room temperature for 3 h and acidified with 3 N HCl. The CH,Cl, solution was shaken with 10% NaHC03, filtered through phase-separating paper, and evaporated to 0.83 g of an oil, which by HPLC was estimated18 to be a 62:38 mixture of the epimeric esters IIa (7a,12p) and Ia (7a,120),respectively. Chromatography over a column of Florisil (50:l ratio) and elution with CHzClzMeOH (99:l) easily separated the epimers. Early fractions consisted of IIa which crystallized from aqueous MeOH in the form of fine needles: 519 mg (52%); mp 129.5-131.0 "C; IR 1724 (C=O); 3413,1004, and 983 (OH) cm-'; NMR 6 0.72 (3 H, s, C-18 Me), 0.91 (3 H, s, C-19 Me), 3.47 (1 H, m, (3-12 CHOH), 3.64 (3 H, s, COOMe), 3.84 (1 H, m, C-7 CHOH). Anal. Calcd for C25H4204: C, 73.85; H, 10.41. Found: C, 73.55; H, 10.19, The late fractions eluted from the column yielded 300 mg of the 70,120-dihydroxy ester Ia, identical with authentic product according to TLC, HPLC, and NMR comparisons. 7a,l2,9-Dihydroxycholanic acid (11) was obtained by methanolic KOH hydrolysis and processed in the usual manner. Crystallized out of ethyl acetate-hexane as thin plates, it melted at 150.0-152.5 "C: IR (KBR) 1701 (C=O), 3448,1018,1005,992 (OH); NMR (CDC13+ 10% MezSO-ds)6 0.72 (3 H, s, C-18 Me), 0.91 (3 H, s, C-19 Me), 3.51 (1 H, br m, (2-12 CHOH), 3.82 (1 H, m, C-7 CHOH). Anal. Calcd for C24H4004: C, 73.43; H, 10.27. Found: C, 72.89; H, 10.30. Methyl 7a-(Mesyloxy)-l2-oxocholanate(Xa).Methyl ester Va (2.9 g in 50 mL of pyridine) was treated with 3 mL of methanesulfonyl chloride and left to stand overnight at room temperature. After the usual processing5an oil was obtained, which was crystallized as stout prisms from isopropyl ether containing a small amount of CHZClz:2.69 g (78%);mp 123.0-123.5 "C; IR 1724 and 1701 (C=O), 1330 and 1170 (SOz), 966,943, and 894 (mesylate) cm-'; NMR 6 1.01 (6 H, s, C-18 and C-19 Me), 2.97 (3 H, s, OSOzMe), 3.63 (3 H, s, COOMe), 4.96 (1 H, m, C-7 CHOMs). Anal. Calcd for Cz6H4206S:C, 64.70; H, 8.77. Found: C, 64.84; H, 8.91. 7~-Hydroxy-l2-oxocholanic Acid (VI). The methyl oxomesylate Xa [2.65 g in 30 mL of MezSO-1,2-dimethoxyethane (1:1)]was added to a suspension of KO2 (1.4 g) and 18-crown-6 (0.88 g) in 70 mL of MezSO, as previously de~cribed.~ After the mixture was processed, the resulting product crystallized from ethyl acetate to give 1.41 g (66%)of stout prisms: mp 185.5-187.5 "C; IR (KBr) 1706 (C=O), 3333 and 10.18 (OH) cm-'; NMR 6 1.04 (6 H, s, C-18 and C-19 Me), 3.59 (1 H, br m, C-7 CHOH). Anal. Calcd for Cz4&04: C, 73.80; H, 9.81. Found: C, 73.56; H, 9.73. Methyl 7~-hydroxy-12-oxocholanate (VIa)was obtained by the usual MeOH-HCl esterification. The ester crystallized from aqueous methanol as fine needles: mp 114-115 "C; IR 1721 and 1692 (C=O), 3425,1014, and 980 (OH) cm-'; NMR 6 1.04 (6 H, s, C-18 and C-19 Me), 3.56 (1 H, br m, C-7 CHOH), 3.63 (3 H, s, COOMe). Anal. Calcd for C,Ha04: C, 74.21; H, 9.97. Found: C, 74.48; H, 10.01. Methyl 7,9,12,9-Dihydroxycholanate(IVa). To a stirred solution of methyl ester VIa (0.95 g in 50 mL of CHzCl,) was added 0.45 g of tert-butylamine-borane. Processed as in the preparation of the 7a,120 ester IIa, the reduction product examined by HPLC showed two predominant components in the ratio of 6535. The material (0.94 g) was chromatographed on a column of alumina (activity II). Early fractions eluted by benzene-ethyl acetate (9:l) yielded homogenous product (476 mg) which crystallized from aqueous acetone as fine needles (mp 110-110.5 "C) and was characterized as the 76,12@-dihydroxyester IVa: IR 1724 ( C 4 ) 3425, 1015, 995, and 952 (OH) cm-'; NMR 6 0.76 (3 H, s, C-18 Me), 0.96 (3 H, 3, C-19 Me), 3.41 (2 H, br m, C-7 and (2-12 CHOH), 3.64 (3 H, s, COOMe). Anal. Calcd for C25H4204: C, 73.85; H, 10.41. Found: C, 74.18; H, 10.68. Methyl 7,9,12a-Dihydroxycholanate(IIIa). The later fractions eluted from the column (above) afforded 245 mg of an essentially homogeneous oil which crystallized from aqueous
J. Org. Chem. 1983,48, 1197-1202 acetone as fine needles (mp 131-132 "C) and was characterized as the 7P,12a-dihydroxy ester IVa: IR 1730 (C=O), 3436,1026, 1015, and 942 (OH) cm-'; NMR 8 0.70 (3 H, s, (2-18 Me), 0.93 (3 H, s, C-19 Me), 3.57 (1H, br m, C-7 CHOH), 3.63 (3 H, s, COOMe), 3.98 (1H, m, C-12 CHOH). Anal. Calcd for C Z ~ H ~ ZC,O73.85; ~: H, 10.41. Found: C, 74.11; H, 10.66. 7B,12B-Dihydroxycholanicacid (IV) was obtained from IVa by the usual hydrolysis procedure and crystallized from ethyl acetate as fine needles: mp 179-182 "C; IR 1642 (C=O), 3413, 1026,1015, and 956 (OH) cm-'; NMR (CDC13 10% MezSO-d6) 6 0.72 (3 H, s, C-18 Me), 0.93 (3 H, s, (2-19 Me), 3.41 (2 H, br m, C-7 and C-12 CHOH). Anal. Calcd for C24H4004: C, 73.43; H, 10.27. Found: C, 73.45; H, 10.23. 7~,12a-Dihydroxycholanic acid (111) was similarly obtained from the corresponding ester IIIa. The crude acid crystallized from EtOAehexane as fine needles: mp 174.5-176.0 "C: IR (KBr) 1689 (C=O), 3356,1026,1015, and 943 (OH) cm-l; NMR (CDC13 + 10% MezSO-d6)8 0.67 (3 H, 8 , C-18 Me), 0.90 (3 H, s, C-19 Me), 3.49 (1H, br m, C-7 CHOH), 3.88 (1H, m, (2-12 CHOH). Anal. Calcd for CNHa04: C, 73.43; H, 10.27. Found C, 73.17; H, 10.14. Reduction of t h e Ester VIa by NaBH4. Methyl 7phydroxy-12-oxocholanate (VIa; 1.84 g in 160 mL of MeOH) was treated with 2.0 g of NaBH4 and allowed to stand overnight at room temperature. Ice chips were added to the solution which was extracted wth CHZCl2.The CHZCl2extract was washed with cold dilute-HCl and then water, dried (Drierite),and evaporated to a clear oil (1.84 g). By HPLC the oily product was seen to consist predominantly of the same two components as obtained in the amineborane complex reduction (above) but in the reversed ratio of 37:63 7P,12P/7/3,12a. Column chromatographic separation as in the amineborane reaction yielded the two expected products, IVa (581 mg) and IIIa (973 mg), shown by HPLC, NMR, and melting point comparisons to be identical with the corresponding esters prepared above. Methyl 7a,l%a-Bis(mesy1oxy)cholanate (XIa). To methyl 7a,l2a-dihydroxy ester Ia (500 mg in 10 mL of pyridine) was added dropwise 0.5 mL of methanesulfonyl chloride. The usual workup after overnight standing yielded product which crystaUized from ethyl ether-hexane as thin plates: 567 mg (83%); mp 77.0-78.5 "C; IR 1727 (C=O), 1333 and 1170 (SOZ), 971,943, and 901 (mesylate) cm-'. NMR 6 0.78 (3 H, s, (2-18 Me), 0.92 (3 H, s, C-19 Me), 3.04 and 3.08 (each 3 H, s, C-7 and (2-12 OSOZMe), 3.66 (3 H, s, COOMe), 4.91 (1H, m, C-7, CHOMs), 5.12 (1H, m, C-12 CHOMs). Anal. Calcd for Cz7H608Sz: C, 57.63; H, 8.24. Found: C, 57.60; H, 8.32. 12a-(Mesy1oxy)cholanic Acid (XII). Methyl 12-(mesyl0xy)cholanate (XIIa,zO260 mg) after being subjected to the standard inversion procedure (solvent MezSO-1,2-dimethoxyethane)5 and after processing yielded 201 mg of product which
+
1197
did not crystallize but by TLC showed a single spot, and its NMR spectrum was appropriate for acid XII: NMR 6 0.77 (3 H, s, C-18 Me), 0.91 (3 H, s, C-19 Me), 3.02 (3 H, s, C-7 SOZMe),5.11 (1H, m, C-12 CHOS02Me). Esterification of the oil (MeOH-HC1) after processing and crystallization from MeOH yielded colorless prisms, which according to mixture melting point, NMR, and HPLC comparisons was identical with the starting methyl ester XIIa. Methyl 7a-(Mesy1oxy)cholanate (XVIa). Methyl 7ahydroxy ester XVa2' (1.0 g) was treated with 2.0 mL of methanesulfonyl chloride in 20 mL of pyridine and was processed as described previouslf to yield 1.1g of oil which was crystallized from isopropyl ether as stout prisms: 940 mg (78%); mp 74-75 "C; IR 1730 (C=O), 1332 and 1170 (SOz), 909 and 892 (mesylate) cm-'; NMR 8 0.66 (3 H, s, C-18 Me), 0.91 (3 H, s, C-19 Me), 2.99 (3 H, s, SOZMe),3.63 (3 H, s, COOMe), 4.90 (1 H, m, C-7 ( CHOMs). Anal. Calcd for CZH4O5S: C, 66.64; H, 9.64. Found C, 66.77; H, 9.13. 7B-Hydroxycholanic Acid (XIII). Methyl 7a-mesylate (XVIa, 1.0 g), in a 2-h reaction with the inverting solution [KOz (60 mg) and 18-crown-6 (33 mg) in 40 mL of Me2SO],as described previo~sly,~ yielded a crude product which crystallized from EtOAc-hexane as colorless needles: 0.46 g (57%); mp 131-133.5 OC; IR (KBr) 1695 (C=O), 3401,1015,990 (OH) cm-'; NMR 6 0.68 (3 H, d, C-18 Me), 0.93 (3 H, s, (2-19 Me), 3.58 (1H, br m, C-7 CHOH). Anal. Calcd for CNHaO3: C, 76.55; H, 10.71. Found C, 76.02; H, 11.12. Methyl 7@-hydroxycholanate(XIIIa) was obtained quantatively from XI11 by the usual MeOH-HCI esterification. The ester crystallized from aqueous methanol as dense prisms: mp 89.0-90.5 "C; IR 1730 (C=O), $436,1015,993 (OH) cm-'; NMR 6 0.84 (3 H, I, C-18 Me), 0.99 (3 H, s, C-19 Me), 3.60 (1 H, br m, C-7 CHOH), 3.68 (3 H, s, COOMe). Anal. Calcd for C25H4203' 0.5MeOH: C, 75.32; H, 10.90. Found: C, 75.58; H, 10.76.
Acknowledgment. This work was supported in p a r t by a grant from the National Large Bowel Cancer Project. We thank the Chemical Research Institute of Non-Aqueous Solutions and the Pharmaceutical Institute, Tohoku University, Japan, for elemental analysis. We are indebted to Ms. Susan Brannan for valuable technical help. Registry No. Ia, 3701-54-0; 11, 84413-82-1; IIa, 84325-10-0; 111,84413-81-0;IIIa, 84895-26-1;IV, 84413-80-9;IVa, 84895-27-2; V, 84895-28-3; Va, 84895-29-4; VI, 84895-30-7;VIa, 84895-31-8; VIIa, 19684-66-3; VIIIa, 19684-67-4; Xa, 84895-32-9; XIa, 84895-33-0;XII, 84895-34-1;XIIa, 84926-46-5;XIII, 10601-78-2; XIIIa, 28050-20-6; XVa, 28050-19-3; XVIa, 84926-47-6; XVIIa, 28192-77-0; XVIIIa, 77731-11-4; methyl cholate, 1448-36-8.
Stereospecific Synthesis of Ether Phospholipids. Preparation of 1-Alkyl-2-(acylamino)-2-deoxyglycerophosphorylc holines Nizal S. Chandrakumar and Joseph Hajdu* Department of Chemistry, Boston College, Chestnut Hill, Massachusettes 02167 Received July 20, 1982
A novel stereospecific synthesis of biologically active ether phospholipids is reported. The synthesis is based upon (1)utilizing L-serine to provide the chiral center, (2) developing the aliphatic ether function by coupling the methanesulfonate of the fatty acid alcohol with an oxazoline-protected deoxyglyceride, and (3) introducing the phosphorylcholine moiety via the 2-chloro-2-oxo-l,3,2-dioxaphospholane-trimethylamine sequence. The synthetic alkoxyphospholipids have been shown to exhibit potent platelet activation, antihypertensive properties, and cytotoxicity against HL-60 cells. Microcalorimetric studies of one compound have revealed a unique phase-transition behavior resulting from the presence of a 1-alkyl rather than a 1-acyl substituent in the molecule. The synthetic method developed has a great deal of flexibility, providing a convenient general route to a wide range of ether phospholipids for physicochemical as well as enzymological studies. Ether phospholipids represent an important class of exceptionally potent biologically active phospholipid de0022-3263/83/1948-1l97$01.50/0
rivatives.'V2 Recent studies of a series of naturally occurhg 1-sn-alkoxyglycerophosphorylcholineshave established 0 1983 American Chemical Society
