1975 1421Synthesis of Active Forms of Vitamin D. Part 1X.l Synthesis of 1a,24-D i hyd roxyc holecalcif erolBy Masuo Morisaki, Naoyuki Koizumi, and Nobuo Ikekawa,' Laboratory of Chemistry for NaturalToru Takeshita and Sachio Ishimoto, Teijin Institute for Biomedical Research, Hino-shi, Tokyo, JapanProducts, Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan24-Oxocholesterol (1 ) (readily available from fucosterol) was converted by three steps into 1 a,24E-dihydroxy-cholesterol (4). From the corresponding triacetate (5), 1 a,24~-dihydroxycholecalciferol (9) was prepared viathe 5,7-diene (8). The C-24 epimers of compound (4) were resolved by silica gel column chromatography of themonohydroxydibenzoates (7). and were separately transformed into the corresponding 1 a.24-dihydroxycholecalci-ferol epimers.VARIOUS metabolites of vitamin D, have been isolatedrecently, some of which may be the biologically activeforms of the vitamin.2 This has stimulated the synthesisof vitamin D analogues with hydroxy-groups at positions25,3* 24 and 25,7*s 25 and 26,9 la and 25,10911 and la,24, and 25.l In addition to these metabolic products,artificial ' analogues such as la-,l2-17 4ct-,l8 22-, l 9 and24-hydroxy- 2o and 20,25-dihydroxy- 21 cholecalciferol,as well as the 1 a-hydroxy-3-deoxy-derivative 22*23 havebeen synthetic targets.We have synthesized severalkey intermediates 24-28 for the preparation of thesecompounds from fucosterol, a marine sterol which isabundant in brown algae.29 From one of these inter-mediates, the epimeric 24-hydroxycholecalciferols t, $t The configuration at C-24 of 24-hydroxycholestero1 has beendetermined by Klyne and Stokes.30 However, van Lier andSmith 31 suggested that this assignment should be reversed, andsome uncertainty still remains.We will therefore retain theoriginal nomenclature 32 for the present paper, referring to theepimers as 24E1- and 24E2-cholesterol (see Experimental section).$ Note added in proof: We have now determined the absoluteconfigurations a t C-24 of 24-hydroxycholestero1 and relatedcompounds. 24f' and 24E2 in this paper are 24s and 24R,respectively (N. Koizumi, M. Morisaki, N. Ikekawa, A. Suzuki,and T. Takeshita, Tetrahedron Letters, in the press).1 Part VIII, N. Ikekawa, M. Morisaki, N.Koizumi, Y . Kato,and T. Takeshita, Chem. and Phavm. Bull. (Japan), 1975, 23, 695;The present paper is also Part XIX of the series ' Studies onSteroids;' Part XVIII, idem., ibid.2 H. F. DeLuca, Amev. J . Med., 1974,57, 1.3 J. W. Blunt and H. F. DeLuca, Biochemistry, 1969, 8, 671.* S. J. Halkes and N. P. van Vliet, Rec. Trav. chiln., 1969,J. A. Campbell, D. M. Squires, and J. C. Babcock, Steroids,T. Suda, H. F. DeLuca, and R. B. Hallick, Biochemistry,7 H-Yat Lam, H. K. Schnoes, H. F. DeLuca, and T. C . Chen,M. Seki, N. Koizumi, M. Morisaki, and N. Ikekawa, Tetra-J. Redel, P. Bell, F. Delbarre, and F. Kodicek, Compt. rend.,10 E. J. Semmler, M. F. Holick, H. I. Schnoes, and H. F.l1 D. H. R. Barton, R. H. Hesse, M. M. Pechet, and E.Riz-l2 M. F. Holick, E. J. Semmler, H. K. Schnoes, and H. F.l3 D. H. R. Barton, R. H. Hesse, M. M. Pechet, and E. Riz-l4 R. G. Harrison, B. Lythgoe, and P. W. Wright, Tctrahedronl5 A. Fiirst, L. Labler, W. Meier, and K-€3. Phoertner, Helv.88, 1080.1969, 13, 667.1971, 43, 139.Biochemistry, 1973, 12, 4851.hedron Letters, 1975, 16.1973, 276D, 2907.DeLuca, Tetrahedvun Letters, 1972, 4147.zardo, J.C.S. Chem. Comm., 1974, 203.DeLuca, Science, 1973, 180, 190.zards, J . Amer. Chew. Soc., 1973, 95, 2745.Letters, 1973, 3649.Chim. Actn, 1973, 56, 1708.were prepared, and were found to be active in stimulatingintestinal calcium transport in rats deficient in vitaminD.20 A significant difference of biological activities wasnoted between the 24-i~omers.~~ That nephrectomyabolishes the intestinal calcium responses suggestedthat these compounds required a l-hydroxy-group forbiological activity.We now report a synthesis of 1~,24~-dihydroxychole-calciferol.Both 24-epimers have also been prepared, inorder to elucidate the effect of configuration on vitaminD activity.24-Oxocholesterol (1), readily available from fucos-ter01,~' was oxidized with 3.3 equiv. of dichlorodicyano-benzoquinone (DDQ) 34 in refluxing dioxan to afford, the1,4,6-triene-3,24-dione (2) in 52% yield. The sametrienedione (2) was also prepared from 2koxocholes-tenone,,Z an ozonolysis product of fucostenone, by16 C. Kaneko, S. Yamada, A. Sugimoto, Y. Eguchi, M. Ishi-kawa, T. Suda, M. Suzuki, S. Kakuta, and S.Sasaki, Steroids,1974, 23, 75; Tetrahedron, 1974, 30, 2701.l7 A. Saiga, K. Bannai, M. Sawamura, J. Rubio-Lightbourn,T. Tachibana, N. Koizumi, M. Morisaki, N. Ikekawa, in prepara-tion.l8 B. Pelc, J.C.S. Perkin I, 1974, 1436.19 D. R. CrumD. D. H. Williams, and B. Pelc, T.C.S. PerRin I, 1.- . "1973, 2731.20 N. Ikekawa. M. Morisaki. N. Koizumi, M. Sawamura. Y.Tanaka, and H. F. DeLuca, Biochern. Biophys. Res. Cornm., 1975,62, 485.21 J. S. Bonetkoe, A. Wignall, M. P. Rappoldt, and J. R.Roborgh, Internat. J . Vitamin Res., 1970, 40, 689.Z t H. Y. Lam, B. L. Onisko, H. K. Schnoes, and H. F. DeLuca,Biochem. Biophys. Res. Comm., 1974, 59, 846.23 W. H. Okamura, M. N. Mitra, R. M. Wing, and A. W. Nor-man, Biochem. Biophys. Res. Comm., 1974,60, 179.zr M.Morisaki, 3. Rubio-Lightbourn, and N. Ikekawa, Chem.and Pharm. Bull. (Japan), 1973, 21, 457.25 M. Morisaki, K. Bannai, and N. Ikekawa, Chem. and Pharm.Bull. (Japan), 1973, 21, 1863.26 J. Rubio-Lightbourn, M. Morisaki, and N. Ikekawa,Chem. and Phavm. Bull. (Japan), 1973, 21, 1854.27 M. Morisaki, J. Rubio-Lightbourn, N. Ikekawa, and T.Takeshita, Chem. and Pharm. Bull. (Japan), 1973, 21, 2568.28 At. Seki, J. Rubio-Lightbourn, M. Morisaki, and N. Ikekawa,Chem. and Pharm. Bull. (Japan), 1973,21,2783.29 N. Ikekawa, N. Morisaki, K. Tsuda, and T. Yoshida,Steroids, 1968, 12, 41.30 TV. Klyne andW. M. Stokes, J . Chem. SOC., 1954, 1979.31 J. E. van Lier and L. L. Smith, J . Pharm. Sci., 1970, 59,a2 A. Ercoli and P. deRuggieri, J .Amer. Chem. Soc., 1953, 75,33 Y. Tanaka, H. Frank, H. F. DeLuca, N. Koizumi, and N.34 A. B. Turner, J . Chem. SOC. (C), 1968, 2568.719.3284.Ikekawa, Biochemistry, in the press1422 J.C.S. Perkin Isuccessive dehydrogenations with chloranil-t -bu t yl alco -hol 35 and DDQ-di~xan.~~ Treatment of the trienedione(2) with alkaline hydrogen peroxide gave the la,2a-epoxide (3) (71%), which was reduced with lithium-ammonium chloride in liquid ammonia-tetrahydro-furan,ll. l3 yielding 1 a,24E-dihydroxycholesterol (4) (68%yield).Compound (4) was converted either into the triacetate(5), for transformation into la,24~-dihydroxycholecalci-ferol (9), or into the Ia-hydroxy-3,24-dibenzoates (7) forIrradiation39 of the 5,7-diene (8) in ether with amedium-pressure mercury lamp gave a mixture of photo-isomers which was refluxed in benzene to effect thermalisomerization of precalciferol into calciferol.Thedesired vitamin D (9) was isolated (12% yield) bypreparative t .l.c. on silver nitrate-impregnated silica gel.For the preparation of the individual C-24 epimers, theseparated dibenzoates (7) were converted into l-acetates,which were then treated with N-bromosuccinimide-carbon tetrachloride. The products were dehydro-(4)( 5 ) R1 = R2 = A c( 6 ) R' = R 2 = BZR' = R2 = H(7) ~1 = H , R ~ = B Zthe resolution of the 24-epimers. When the trio1 (4) wastreated with 2.5 equiv. of benzoyl chloride in pyridine,the product showed three spots on t.1.c. (extendedascending irrigation method 37).The least polar com-pound was identified as the tribenzoate (6) and otherswere found to be the epimeric dibenzoates (7) by n.m.r.analysis, after separation by silica gel column chroma-tography. The lH n.m.r. spectra of the isomers werealmost identical, in contrast with those of 24,25-dihy-droxy-derivatives, in which the 13-methyl signals of(24R)-compounds appeared at lower field (AS N 0.06p.p.m.) than those of (24S)-isomers.lThe triacetate (5) was brominated with dibromodi-met h ylh y dant oin-hexane 3* and the resulting 7- bromo-compound was dehydrobrominated with s-collidine-xylene. U.V. analysis of the crude product indicatedthe presence of 5,7- and 4,g-dienes. The 5,7-diene (8)(37% yield) was isolated by saponification followed byt.1.c.on silver nitrate-impregnated silica gel.35 E. J. Agnello and G. D. Lanbach, J . Amer. Chem. SOL, 1960,36 K. Igarashi, Chem. and Pharm. Bull. (Japan), 1962, 9, 722.37 J. E. van Lierand L. L. Smith, J . Chromatog., 1970, 49, 666.82, 4293.( 8 )brominated with trimethyl phosphite-xylene and theindividual 5,7-dienes (8) were isolated by way of the cyclicadduct with 4-phenyl- 1,2,4-t riazoline-3,5-dione .@By essentially the same method as described for the24E-compound (8), the individual C-24 epimers wereconverted into the corresponding la,24-dihydroxy-cholecalciferols (9). The biological activity of the pro-ducts and their absolute configuration at C-24 are underinvestigation,EXPERIMENTALM.p.s. were determined with a hot-stage microscopeapparatus.U.V. spectra were obtained with a HitachiESP-3T instrument for solutions in ethanol. N.m.r.spectra were. determined with a Varian T-60 or a JEOLPS/PFT- 100 spectrometer for solutions in deuteriochloro-form unless otherwise stated, with tetramethylsilane asinternal standard. Mass spectra were run with a ShimadzuLKB-9000s spectrometer.38 F. Hunziker and F. X. Muller, Helv. Chim. Ada, 1958, 41, 70.39 E. Havinga, Experientia, 1973, 29, 1181.40 D. H. R. Barton, T. Shioiri, and D. A. Widdowson, J . Chern.SOG. ( C ) , 1971, 19681975 1423Column chromatography was effected with silica gel(Wakogel C-200). T.1.c. was carried out on Merck silicagel F,,, (0.25 mm thick). Silver nitrate-impregnated t.1.c.plates were prepared by dipping into 2% AgNO, solutionin acetonitrile, followed by activation a t 80 "C for 1 h.Cholesla- 1,4,6-triene-3,24-dio~e (2) .-(a) A solution of3P-hydroxycholest-5-en-24-one ( 1) 26 (8.0 g) and dichloro-dicyanobenzoquinone (15 g) in dioxan (120 nil) was refluxedfor 14 h, cooled, filtered, diluted with methylene dichloride,and applied to a short column of alumina (80 g).Elutionwith niethylene dichloride (600 ml) gave the trienedione(2) (4.1 g, 52%), m.p. 110-112 " (from methanol), A,,223 (E 12 000), 257 (9 ZOO), and 301 nm (13 000), 6 0.80 (3H,s, 13-Me), 0.95 (3H, d, J 6 Hz, 20-Me), 1.10 (6H, d, J 7 Hz,35-Me2), 1.21 (3H, s, 10-Me), 6.0-6.2 (4H, m, 2-, 4-, 6-,and 7-H), and 7.1 (lH, d, J 10 Hz, 1-H) (Found: M+,394.2 888.(b) A solution of cholest-4-ene-3,24-dione 32 (4.72 g)prepared by ozonolysis of fucostenone, and chloranil (4.7 g)in t-butyl alcohol (20 ml) was refluxed for 2 h, cooled,filtered, and evaporated to dryness.The residue wasapplied to a column of alumina (100 g) and eluted withmethylene dichloride to give the crude 4,6-dienone (3.45 g),287 nm. A solution of the dienone and dichloro-dicyanobenzoquinone (3.0 g) in dioxan (30 ml) was refluxedfor 14 h, cooled, filtered, and evaporated to dryness. Theresidue dissolved in methylene dichloride was filteredthrough a column of alumina (50 g) affording the trienedione(2) (2.31 g).la, 2a-E~oxycholesta-4,6-diene-3,24-dioxe (3) .-Hydrogenperoxide (30%; 5 ml) was added to a solution of thetrienedione (2) (3.53 g) in methanol (100 ml), dioxan (50ml), and tetrahydrofuran (20 ml) containing sodium hydr-oxide (80 mg), and the mixture was stirred a t 15 "C over-night. The solution was diluted with ether and washedwith brine.Drying (Na,SO,) and evaporation left a solidwhich was crystallised from acetone to give the epoxide(3) (2.41 g), m.p. 150-151.5", AmZ 291 nm (c 22 000),6 0.78 (3H, s, 13-Me), 0.96 (3H, d, J 6 Hz, 20-Me), 1.08(6H, d, J 7 Hz, 25-Me2), 1.19 (3H, s, 10-Me), 3.40 (lH,dd, J 4 and 1.5 Hz, 2P-H), 3.58 (lH, d, J 4 Hz, lP-H),5.63 (lH, d, J 1.5 Hz, 4-H), and 6.10 (2H, s, 6- and 7-H)(Found: M', 410.2793.la,3~,24~-TrihydroxycJzoZest-5-ene (4) .-A four-neckedflask was fitted with a sealed mechanical stirrer, a droppingfunnel, a cold-finger filled with solid CO,, and an inletconnected to an anhydrous ammonia source.Nitrogen wasswept through the system for 10 min, and then ammonia(10 ml) was trapped in the flask. Lithium wire (150 mg)was cut into short pieces and added. After stirring for10 min, the epoxide (3) (100 mg) in tetrahydrofuran (13 ml)was added dropwise during 20 min. The cooling bath wasremoved and the mixture was allowed to reflux for 20 min.The flask was dipped in a cooling bath and anhydrousammonium chloride (1.5 g) was added during 2 h; themixture turned white and pasty. Most of the ammoniawas removed in a stream of nitrogen and the residue wasdiluted with ethyl acetate, washed with brine, and dried(Ka2S0,). Evaporation left a white solid, which wasapplied to a column of silica gel (4 g).Elution withbenzene-acetone (3 : 1) afforded the triol (4) (68 mg) as anamorphous powder, 6 (C,D,N), 0.70 (3H, s, 13-Me), 3.3(lH, m, 24-H, overlapped with OH), 3.83 (lH, m, lp-H),and 4.05 (lH, m, 3a-H) (Found: M+, 418.3428. C,,H,,O3requires &l, 418.3449).C,,H,80, requires M , 394.2874).C,,H,80, requires M , 410.2821).la, 3p, 24E-TriacetoxychoZest-5-ene (5) .-The triol (4) (60mg) was kept a t 95 "C for 3.5 h with acetic anhydride(8 ml) and pyridine (25 ml). Work-up as usual gave thetriacetate (5) (65 mg), an oil, 6 0.67 (3H, s, 13-Me), 2.02(9H, s, 3 x OAc), 4.8 (2H, m, 3a- and 24-H), 5.05 (lH, m,1P-H), and 5.6 (lH, m, 6-H), rn/e 484 (M+ - AcOH), 424(M+ - 2AcOH), and 364 (M+ - 3AcOH).la, 3p,24~-TribenzoyloxychoZest-5-ene (6) .-The triol (4)(1.25 g) was kept a t room temperature overnight withbenzoylchloride (1.05 g) and pyridine (20 ml).After work-up in the usual manner, the product was chromatographedon a column of silica gel (60 g). Elution with benzene gavethe tribenzoate (6) (250 mg), an oil, 6 0.65 (3H, s, 13-Me),5.0 (lH, m, 24-H), 5.2 (lH, m, 3a-H), 5.4 (lH, m, lP-H),5.7 (lH, m, 6-H), and 7.4-8.2 (15H, m, Ph).The la-Hydr0~~-3P, 24E-dibenzoxycholest-5-enes (7) .-Inthe foregoing chromatography, further elution with ben-zene-ethyl acetate (200 : 1) afforded the 3P,24E2-dibenzoate(8) (596 mg), m.p. 168-169' (from methanol), 6 0.67 (3H,s, 13-Me), 3.91 (lH, m, lp-H), 5.00 (lH, ni, 24-H), 5.3(lH, m, 3a-H), 5.65 (lH, m, 6-H), and 7.3-8.2 (lOH, m,Ph) (Found: C, 78.6; H, 8.7.C41H540, requires C, 78.55;€3, 8.7%). Continued elution with the same solvent gavethe 3p,24c1-dibenzoate (684 mg), m.p. 139.5-140.5" (frommethanol), n.m.r. spectrum identical with that of the 24E2-isomer (Found: C , 78.4; H, 8.8%).la, 3p, 24~-Trihydroxycholesta-5,7-diene (8) .-To a reflux-ing solution of the acetate (5) (301 mg) in n-hexane (4.5 ml),dibromodimethylhydantoin (92 mg) was added in oneportion. Stirring under reflux was continued for 15 min.The mixture was cooled and the resulting imide was filteredoff. Evaporation of the filtrate gave a pale yellow syrup(349 mg). The residue in xylene (2.5 ml) was added drop-wise to a mixture of s-collidine (0.85 ml) and xylene (1.90ml) a t 165 "C, during 15 min.After heating for a further10 min, the solution was cooled and the precipitate wasfiltered off. The filtrate was evaporated under vacuum andthe residue was redissolved in ether. The solution waswashed with N-hydrochloric acid and then with brine, dried(Na,SO,) , and evaporated to give a pale orange oil (3 10 mg) .U.V. analysis (Amx 233, 241, 249, 272, 282, and 293 nm)showed the presence of 4,6- and 5,7-dienes in the ratio ca.1 : 2. The mixture was dissolved in methanol (8 ml),benzene (9 ml) , and methanolic 2~-potassium hydroxide(10 ml) and heated a t 65 "C for 1 h. Extraction withethyl acetate and the usual work-up gave an oil (256 mg).A repeated development of the product on a silver nitrate-impregnated silica gel plate with 6 yo methanol-chloroformshowed two distinct u.v.-absorbing bands.The U.V.spectrum of the less polar fraction (Amx 232, 240, and 249nm) was characteristic of the 4,6-diene chromophore.Elution of the more polar band with ethyl acetate affordedthe pure 5,7-diene (8) (85 mg), m.p. 102-103", Amx 271.5( E 11 000), 282 (12 000), and 294.5 nm (7 000), 6 0.63 (3H,s, 13-Me), 3.33 (IH, m, 24-H), 3.75 (lH, m, lp-H), 4.06(lH, m, 3a-H), and 5.5 (2H, AB-type q J 6 Hz, 6- and 7-H)(Found: M+, 416.3357.The la, 3p, 24~-Trihydroxycholesta-5,7-dienes (8) .-The 3p,-24El-dibenzoate (545 mg) was kept a t 15 "C overnight withacetic anhydride (4 ml) and pyridine (4 ml). Work-up asusual gave the corresponding la-acetate (566 mg), 6 0.67(3H, s, 13-Me), 2.06 (3H, S, Ac), 4.8-5.4 (3H, m, lp-, 3a-,and 24-H), 5.55 (lH, m, 6-H), and 7.3-8.2 (lOH, m, Ph).The acetate (545 mg) and N-bromosuccinimide (222 mg) incarbon tetrachloride (20 ml) were heated under reflux forC27H4403 requires N , 416.3293)1424 J.C.S.Perkin I30 min. The precipitate was filtered off and the filtrateevaporated to dryness. The residue in xylene (2 ml) wasadded to a refluxing mixture of trimethyl phosphite (0.52ml) and xylene (6 ml). After refluxing for 90 min the solu-tion was evaporated under mcuum and the residue dissolvedin chloroform (6 ml) . A solution of 4-phenyl- 1,2,4-triazo-line-3,5-dione (290 mg) in chloroform (5 ml) was addeduntil the pink colour persisted for 5 min. The solvent wasevaporated off under vacuum and the residue was applied toa column of silica gel (20 g).Elution with benzene-ethylacetate (25 : 1) afforded the adduct (129 mg), 6 2.06 (3H,s, Ac), 4.7-5.2 (3H, m, I@-, 3a-, and 24-H), 6.41 (2H, AB-type q, J 8 Hz, 6- and 7-H), and 7.2-8.1 (15H, m, Ph).The adduct was refluxed with lithium aluminium hydride(200 mg) in tetrahydrofuran (15 mi) for 8 h. Work-up asusual and column chromatography on silica gel afTorded animpure compound (25 mg). The U.V. spectrum showed Amax248 nm in addition to the expected 272, 282, and 292 nm.Final purification by preparative t.1.c. on silver nitrate-impregnated silica gel gave the 24c1-5,7-diene (10 mg),m.p. 121-124".The 3p,2g2-dibenzoate was converted into the 5,7-dieneJm.p. 96-99', according to the same procedures.la,24~-DihydroxychoZe~uZc~~~~oZ (9) .-A solution of themixture of 5.7-dienes (8) (30 mg) in ether (500 ml) in aquartz apparatus cooled in an ice-bath was agitated by astream of argon for 10 min and then irradiated with amedium-pressure mercury lamp (Hanovia 654A36 ; 200 W)immersed in the vessel for 5.5 min. Most of the ether wasevaporated off and the residue dissolved in benzene (180ml) was refluxed for 2 h under argon. The solvent wasevaporated off and the residue was applied to a silvernitrate-impregnated silica gel plate. Development with6% methanol-chloroform (3 times) revealed three u .v.-absorbing bands. The least polar band was scraped off andeluted with ethyl acetate to give the vitamin (9) (3.7 mg),m.p. 84-85", I.,, 266 nm (E 17 000), 6 [(CD,),CO], 0.57(3H, s, 13-Me), 0.87 (6H, d, J 7 Hz, 25-Me2), 0.96(3H, d, J 5 Hz, 20-Me), 3.19 (lH, m, 24-H), 4.15 (lH, m,lP-H), 4.36 (lH, m, 3a-H), 4.85br (lH, s) and 5.30br (lH,s) (19-Ha), and 6.05 (lH, d, JAB I1 Hz) and 6.26 (lH, d,JAB 11 Hz) (6- and 7-H), m/e 416 (M+), 398 (M - H,O), 380(A4 - 2H,O), 269, 251, 134, and 105.The individual la, 24El- and la, 24~2-dihydroxychoZe-caEciferoZs were similarly obtained from the 24E,l- and 24t2-5,7-dienes. Their spectral (u.v., n.m.r., and mass)properties were practically indistinguishable from thoseof the 24E-mixture.We thank the Ministry of Education for a grant.[5/090 Received, 14th January, 1975
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