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Synthesis and biological activity of 25,26,27-trisnor-vitamin D3 24-oic acid and its 1α-hydroxyl analog
Journal oJ Steroid Biochemisrry. Vol. 10. pp. 261 to 266 Pergamon Press Ltd. 1979. Printed in Great Britain
SYNTHESIS AND BIOLOGICAL ACTIVITY OF 25,26,27-TRISNOR-VITAMIN D, 24-OIC ACID AND ITS la-HYDROXYL ANALOG NAOYUKI
KOIZUMI*,MASUOMORISAKI*,NOBUOIKEKAWA*, YOKOTANAKA?and HECTORF. DELUCA~ * Laboratory of Chemistry for Natural Products, Tokyo Institute of Technology, t Department
Midori-ku, Yokohama, 227, Japan and of Biochemistry, College of Agricultural and Life Science, University of Wisconsin-Madison, Madiso?, WI 53706, U.S.A. (Received 8 June 1978)
SUMMARY Because 1,25-dihydroxyvitamin D undergoes side-chain cleavage and oxidation in uivo, an anticipated metabolite is la-hydroxy-trisnor-vitamin DJ 24-oic acid. A synthesis for this metabolite and trisnorvitamin D, 24-oic acid has been devised. Baeyer-Villager rearrangement of 5,6-dibromo-24-oxo-cholesteryl acetate and its la-acetoxyl analog gave the corresponding cholenoate derivatives, and those were converted to trisnor-vitamin D, 24-oic acid and its la-hydroxyl analog, respectively. The la-hydroxy-25,26,27-trisnor-vitamin D3 24-oic acid (la-OH-24-COOH trisnor-vitamin D3) was tested for biological activity using rats fed a low calcium, vitamin D-deficient diet. It stimulated intestinal calcium transport 6 h after intravenous administration although the response is significantly less than that to an identical base of 1,25_dihydroxyvitamin D, (1,25-(OH),D,). This compound given at 125 ng per rat did not stimulate bone calcium mobilization.
EXPERIMENTAL
INTRODUCTION It is now well accepted that physiologically vitamin D must be converted to 1,25-(OH),D, before it can stimulate calcium transport and bone calcium mobilization [l]. Besides these functions, 1,25-(OH),D3 induces a 25-hydroxyvitamin D-ZChydroxylase yield system. This system then hydroxylates 1,25-(OH)2D, to 1,24,25-trihydroxyvitamin D3 (1,24,25-(OH),-D,). The physiological function of this compound is unknown [Z]. It has also been shown that [26,27-14C]-1,25-(0H)~D~ undergoes side chain oxidation in chickens and in rats to produce radiolabeled 14C02 [3-61 and a metabolite missing a portion of the side-chain. Side-chain cleavage of 25-hydroxycholesterol to give the 24-oic acid derivative by Nocardia restrictus has been observed [7], and cholic acid biosynthesis from cholesterol via 24-hydroxylation followed by oxidation has been reported [S]. These’ metabolic pathways might involve a 24-hydroxylation with the resulting 24,25-glycol becoming oxidatively cleaved to yield the 24-carboxylic acid derivative. Although neither the functional significance of the side-chain cleavage of 1,25-(OH),D, nor the structure of the resulting compound is known, it is distinctly possible that the metabolite is a 24-carboxylic of I ,25-(OH)2D,. The present paper describes chemical synthesis of trisnor-vitamin D3 24-oic acid (15) and its lx-hydroxyl analog (16) from 24-oxocholesterol and the biological activity of la-hydroxy-25,26,27-trisnor-vitamin D3 24-oic acid (la-OH-24-COOH trisnor vitamin D3). Send correspondence S.B. 1013 -I,
to: Dr. N. Ikekawa.
General
Melting points were determined with a hot-stage microscope apparatus. U.V. spectra were recorded with a Shimadzu UV-200 instrument for solutions in ethanol unless otherwise stated. N.M.R. spectra were obtained with a Hitachi R-24A or a JEOL JNM+H-100 spectrometer for solutions in deuteriochloroform, with tetramethylsilane as internal standard. Mass spectra were run with a Shimadzu LKB-9000s spectrometer. Column chromatography was carried out with silica gel (Wakogel C-200). T.L.C. was carried out on Merck silica gel F2+, (0.25 mm thick). Silver nitrateimpregnated t.1.c. plates were prepared by dipping the plates into 2%AgNO, solution in acetonitrile, followed by activation at 80°C for 1 h. High pressure liquid chromatography was achieved with a Shimadzu 830 instrument equipped with Zorbax SIL (DuPont Instruments, Wilmington, Del.) (2.1 mm x 25 cm) column. Treatment of 24-oxo-cholesterol acetate with triJuoroperacetic acid
To a solution of 24-oxo-cholesterol acetate (1) [lo] (91 mg) in methylene
chloride
(1.5 ml), were added di-
basic sodium phosphate (204 mg), trifluoroacetic anhydride (160$), and 90”/, hydrogen peroxide (16~1)[9]. The reaction mixture was stirred for 1 h at room temperature and then extracted with methylene chloride to give a crude product (204 mg), which was chromatographed on silica gel (5 g). Elution with benzene:ethyl acetate @:I, v/v) gave 3&acetoxy-5a261
262
NAOYUK~KOIZUMIet ai.
hydroxy - 68 - t~~uoro~toxy - cholestan - 24 - one (30mg), m/e 572(M*), 554, 539, 529, 51 I, S 068(3H, s, 13.Me), l.O9(6H, d, J = 7.5 Hz, 25-Me,), l.l4(3H, s, IO-Me), 2.03(38, s, OAc), 4.83(1H, broad, s, 4H), m, 3u-H). (Found: M+ 572.3273, 5/20(W C3rH4,06FJ requtres M, 572.3325). 3fi-Acetoxy-.5,6-dibromocholescun-24-one (2)
To a mixture of 24.oxocholesterof acetate (I)[fOJ, (1.6 g), potassium acetate (4 g), ether (64 ml) and acetic acid (40 ml), was added bromine (0.3 ml) dissolved in acetic acid (20 ml), dropwise at 0°C using an ice bath. After stirring for 2 h, water (150 ml) was added and the resulting precipitate was collected by filtration, and washed with sodium bicarbonate solution and water to give the dibromide 2 (2.02 g), m,p. 87-89°C (from methanol), S 0.70(3H, s, 13.Me), l.O8(6H, d, J = 7.5 Hz, 25-Me,), 146(3H, s, lO-Me), 2.03(38, s, OAc), 4.86(1H, broad, s, 6-H), 5.5O(IH, m, 3a-H). Anal. Calc. for Cz9HOb03Br2 : C, 57.81; H, 7.70. Found: C, 57.83; H, 7.70. IsoFro~~~ 38-acetoxy-5,6-dibromocholanoate (3) To a solution of the dibromide (2) (2.02g) in dry methylene chloride (28 ml), were added dibasic sodium phosphate (4.08 g), trifluoroacetic anhydride (3.2 ml) and 90% hydrogen peroxide (0.32 ml) at 0°C. The reaction mixture was stirred for 80min. at room temperature and extracted with ethyl acetate. The characteristics of the cholanoate formed were: (3) (2.34 g), m.p. 9698°C (from methanol), 6 0.70(3H, s, 13-Me), 1.22(6H, d, J = 7.5 Hz, 25-Me,), 1.47(38, s, 10-Me), 2.04(3H, s, OAc), 4.88(1H, m, 6-H), 5.02(lH, heptet, J = 7.5 Hz, -OCYMe&, 5.5O(lH, m, k-H). Isopropyl 3/I-acetoxycholenoate (4)
The dibromide (3) (2.03 g) was stirred with zinc powder (0.4 g) in a mixture of ether (40 ml) and acetic acid (20 ml) at room temperature for 2 h. The mixture was filtered and the filtrate was extracted with ethyl acetate. The characteristics of the oily product formed were (4) (1.33 g), m.p. lOl--103°C (from methanol), 6 069(3H, s, IO-Me), 10,2(3H, s, lo-Me), 1.22(68, d, J = 7.5 Hz, -OCHJ&), 2.02(3H, s, OAc), 4.65(1H, m, 3x-H), 5.02(1H, heptet, J = 7.5 Hz, -OCflMe& 5.4O(lH, m, 6-H). (Found: Mf-AcOH, 398.3160. C2,Ho202 requires M-AcOH, 398.3185). 24,24-Ethylenedioxycholesta-2,4.&riene-3.one
(5)
A mixture of cholesta-~,4,~t~en~3,24-d~one (5.9 g) [l 11, ethylene glycol (6 ml), a catalytic amount of gtoluene sulfonic acid and benzene (200ml) was heated for 15 h under reflux through molecular sieves in a Soxhlet apparatus. Extraction with ethyl acetate gave an oily product with the characteristics (S) (6.35 g), m/e 438(M+), 395(M+-isopropyl, base peak), 6 0.77(3H, s, 13-Me), 093(6H, d, J = 7 Hz, 25.Me& I.2I(3H, s, IO-Me), 3.96(48, s, -OCH2CHIO-), 6&6.2(4H, m, 2-, 4-, 6- and 7-H). 7.11(1N, d,
J = 10 Hz, 1-H). (Found: M+, 438.3118. C29H4203 requires M, 438.3234). lo1,2a-Epoxy-24,24-ethylenedioxycholesta-4,6-diene-3otie (6)
To a solution of the trienone (5) (6.3 g) in methanol (200 ml), were added 10% sodium hydroxyde in methanol (1.8 ml) and 30% hydrogen peroxide (11.6 ml). The mixture was kept at room tem~rature for 3 days, concentrated, and extracted with ether. The usual work-up gave a pale yellow amorphous powder (5.2 g), which was chromatographed on silica gel (6Og). Elution with benzene-hexane (3:l) gave a colorless crystalline 6 (3.5 g), m-p. 149-150°C 6 0.77(3H, s, 13.Me), 0.92(6H, d, J = 7 Hz, 25-Me& l.l8(3H, s, IO-Me), 3,42(1H, dd, J = 4, l.SHz, 2&H), 3.58(1H, d, J = 4Hz, I/?-H), 3.93(4H, s, --OCHz CH@--), 5.63(1H, d, J = 1.5 Hz, 4-H), 6.07(28, s, 6and 7-H), m/e 454(M’), 439(M+-Me), 4.11(M+-isopropyl, base peak), 396(M+-Me-isopropyl). Anal. Calc. for C29H4204: C, 76.61; H, 9.31. Found C, 76.61; H, 9.35. 24,24-~~hylenediox~ 1ups-dihydroxycholesr-5.ene
(7)
A three-necked flask was fitted with a sealed mechanical stirrer, a cold finger filled with solid COr , and an inlet tube connected to an anhydrous ammonia source. Nitrogen was blown through the system for IOmin, and then ammonia (140 ml) was trapped in the flask. Lithium wire (2.5 g) was cut into short pieces and added. After stirring for 1Omin the epoxide (6) (1.2 g) in tetrahydrofuran (140mI) was added dropwise for 20 min. Stirring under cooling for 30min and then the cooling bath was removed. The mixture was allowed to reflux for 30 min and the flask was dipped in a cooling bath. Anhydrous ammonium chloride (20.8 g) was added for 4 h; the mixture which turned white and pasty was kept at room temperature overnight. Extraction with ethyl acetate gave the crude product (1.34 g). Colorless crystals (940 mg) were obtained by re~ystai~zation from methanol, m.p. 164165°C 6 0.67(38, s, 13-Me), 0.91(6H, d, J = 7 Hz, 2S-Me2), 1.01(3H, s, IO-Me), 3.91(4)3, s, -OCH,CH#-), 3.7-4.2(28, m, l/7- and 3a-H), 5.58(1H, m, 6-H). (Found: M+, 460.3527. C29H4804 requires M, 460.3553). lee-3&Dihydroxy-24-oxocholest-5-ene (8)
A solution of the ketal (7) (2.4 g) and p-toluene sulfonic acid (20mg) in acetone (250ml) was stirred at room temperature overnight. This gave a substance with the characteristics 8, (2.2g), m.p. 150-151°C (from acetone), 6 0.67(3H, s, 13-Me), 0.99(6H, d, J = 7Hz, 2%Me*), l.O1(3H, s, IO-Me), 3.84(1H, m, l&H), 4.05(1H, m, 3cr-H), 5.58(1H, m, 6-H). (Found: M+, 416.3289. C2,H4+03 requires M, 416.3290). la,3fLDiacetoxy-chofesr-5-ene24-one
(9)
The dial(8) (1.9 g) was treated with pyridine (20 mt) and acetic anhydride (20ml) for 3.5 h at 90°C and
Synthesis and biological activity of 25,26,27-trisnor-vitamin
water was added. Extraction with ethyl acetate gave an oily product 9 (2.0 g), S 0.68(38, s, 13-Me), l.O7(6H, d, .J = 7.5 Hz, 2%MeJ, l.O8(3H, s, l@Me), 2.03(38, s, OAc), 2.05(3H, s, OAc), 4.65.2(2H, m, l/3- and 3x-H) 5.54(1H, m, 6-H). la,3/?-Diacetoxy-5,6dibromocholestan-24-one
(10)
To a mixture of 9 (1.7 g), potassium acetate (4 g), ether (64 m?) and acetio acid (40 ml), was added drop wise bromine (0.3 ml) in acetic acid (20 ml) at 0°C. After stirring for 2 h, water (150 ml) was added. Extraction with ethyl acetate gave the dibromide 10 (2g), 6 0.70(3H, s, 13-Me), l.O8(6H, d, J = 7.5 Hz, 25-Me&, l-49(38, s, IO-Me), 2.05(38, s, OAc), 2.07(3H, s, OAc), 4.82(18, M, 6-H), 5.58(1H, m, 3a-H). Isopropyl
(11)
Ja,3/?-diacetoxy-5,6dibromo-cholanoate
To a solution of 10 (1.9 g) in methylene chloride (28 ml), were added dibasic sodium phosphate (4.08 g), trifluoroacetic anhydride (3.2 ml) and 90% hydrogen peroxide (0.32ml) at 0°C. The reaction mixture was stirred at room temperature for 90min and sodium bicarbonate solution was added. Extraction with ethyl acetate gave 11 (2.1 g), 6 0.70(3H, s, Me-18), l-22(68, d, J = 7.5 Hz, -O-CHh&,), 1.52(3H, s, 10-Me), 4.85(1H, m, 6-H), 5.02(1H, heptet J = 7.5Hz, -OCIIMe,), 5.66.0(2H, m, l/I- and 3a-H). Isopropyl
fu,3/Ldiacetoxy-cholenoate
(12)
A mixture of the compound 11 (2.0 g), ether (40 ml) and acetic acid (10ml) was stirred with zinc powder (0.4g) at room temperature for 6 h. Extraction with ethyl acetate gave an oily product (1.32 g), which was purified by column chromatography on silica gel (28g). Elution with the mixed solvent of benzeneethyl acetate (50:1) yielded 12 (500 mg), S 0.67(3H, s, 13-Me), l.O9(3H, s, IO-Me), 1.23(6H, d, J = 7 Hz, -OCH&,), 2.03(38, s, OAc), 2.05(3H, s, OAc), 4.95(1H, m, 3a-H), 5.02(1H, heptet, J = 7Hz, -OCHMe,), 5.09(1H, m, l/Y-H), 5.54(lH, m, 6-H). (Found: M+-2AcOH, 396.3014. C2,H4002 requires M-2AcOH, 396.3028). Isopropyl 38-acetoxy-chola-5,7-diene-24-oate
(13)
To a refluxing solution of 4 (50.7 mg) in carbon tetrachloride (5 ml), N-bromosuccinimide (27 mg) was added in one portion, Stirring under reflux was continued for 25 min. The mixture was cooled and the resulting imide was filtered off. Evaporation of the filtrate gave a pale yellow syrup. The residue in xylene (1 ml) was added dropwise to a refluxing mixture of trimethyl phosphite (150 ~1) and xylene (0.5 ml). After heating for 2 h, the solvent was evaporated to give a yellow oil. The product was purified with preparative t.1.c. developed with benzene 4 times and the main U.V. absorbing band was scraped off. Elution with ethyl acetate gave 13 (5 mg), ,I,,,, 262, 272, 282, 294nm, 6 0.62(38, s, 13-Me), 0.95(3H, s, lo-Me), 1.22(68, d, .I = 7 Hz, -OCHJ&,), 1.23(3H, s, OAc), 4.2(1H, m, 3a-H), 5.02(1H, heptet J = 7 Hz,
D5 2Aoic acid and its la-hydroxyl analog
263
-OCHMe& 5.50(2H, ABq, J = 6 Hz, 6- and 7-H) m/e 456(M+), 496(M+-AcOH), 481(M+-AcOH-Me). Isopropyl
Ja,3&diacetoxy-chola-5,7-diene-24-oate
(14)
A mixture of the compound 12 (51 mg), N-bromosuccinimide (25 mg) and carbon tetrachloride (4 ml) was heated under reflux for 15 min. The precipitate was removed by filtration and the filtrate was evaporated to dryness. The residue in xylene (1.5 ml) was added to a refluxing mixture of trimethyl phospite (0.2 ml) and xylene (1.5 ml). After refluxing for 90 min, the solution was evaporated in uacuo to yield a yellow oil. Purification by preparative t.1.c. gave 14 (8 mg), ,I,,,,, (ether) 262, 271, 282, 294 nm, 6 0.62(3H, s, 13-Me), 1.24(6H, d, .J = 7 Hz, -OCH&z), 2.04(38, s, OAc), 2.09(38, s, OAc), 5.01(1H, heptet J = 7 Hz, -OCYMe2), 4.7-5.2(2H, m, l/3- and 3a-H), 5.54(2H, ABq, J = 6Hz, 6-Land 7-H). (Found: M+, 514.3326. C31H460L requires M, 514.3295). 3~-Hydroxy-9,J(Fseco-chola-5,7,10(J9)-triene-24-oic acid (15)
A solution of the 5,7-diene 13 (5 mg) in a mixture of benzene (IOOml) and ethanol (50 ml) in a quartz apparatus cooled in an ice bath was agitated with a stream of argon for 10min and then irradiated with medium-pressure mercury lamp (Hanovia 654A36; 200 W) immersed in the vessel for 4 min. The mixture was refluxed for 2 h under argon. The solvent was evaporated off and the residue was applied to a silver nitrate-impregnated silica gel plate. Development with a mixed solvent of hexane-benzene (5: 1, V/V) 3 times and elution of the least polar U.V.-absorbing band gave the diester of 15 (1 mg). Stirring of the ester (1 mg) in 10% KOH-MeOH at room temperature for 2.5 h and the usual work-up afforded crude 15, which was purified by high pressure liquid chromatography with 2.5% MeOHCHrCl, to give 25,26,27-trisnor-vitamin D, 24oic acid (0.1 mg), A,,,,, 265nm, m/e 372(M+), 354(M+-H,O), 339(M+-H,O-Me), 136, 118. (Found M+, 372.2748, C24H3(rOJ requires M, 372.2664). 9,1O-Seco-chola-5,7,l~J9)-triene-Ju,3fl-diol-24-oic
acid
(16)
The 5,7-diene 14 was irradiated in a mixture of benzene (1OOml) and ethanol (50 ml) for 2 min under argon with medium pressure mercury lamp. After refluxing for 60min under argon, the mixture was concentrated in uacuo and the residue was chromatographed on preparative t.1.c. The chromatograph was developed with CHCl,-hexane (2: 1, V/V) 3 times and the main U.V.-absorbing band was scraped off. Further purification was performed by high pressure liquid chromatography to give the triester of 16 (1 mgb A,,, 265 nm, m/e 514(M+), 454(M+-AcOH), 412(McLafferty), 394(M+-2AcOH). The ester was stirred in 10% KOH-MeOH at 40°C for 4 h and the mixture was extracted with ethyl acetate. Purification was achieved by high pressure liquid
264
KOIZUMIet aI
NAOYUKI
Table 1. Response of intestinal calcium transport to la-OH-24COOH D, (%a mucosal/4sCa serosal)
h after dose 1 3 6 24
EtOH
1.25(OHbD,
1.46 + 0.25t 1.76 f 0.18 1.50 & 0.30 1.60 + 0.32
trisnor vitamin
1a-OH-24-COOH trisnor vitamin D3
2.18 + 0.32 3.12 k 0.50’ 4.64 + 0.74*1 -
2.06 + 1.79 f 2.81 + 2.10 f
0.28 0.30 0.69*$ 0.71
Rats were fed a low calcium, vitamin D-deficient diet for 3 weeks. One group of rats was given 125 ng of la-OH-24-COOH trisnor vitamin D, intravenously while another group received 125 ng of 1,25-(OH),D,. The control group received ethanol alone. At the indicated time, animals were killed and intestinal calcium transport was measured as described in the text. Each group had 6 rats. * Significantly different from control (P < 0.005). t Standard deviation. $ Significantly different from 8 (P < 0.001).
chromatography (Solv. 4.5% MeOH-CH,Cl,) to give la-hydroxy-25,26,27-t&or-vitamin D, 24oic acid (16) (50 pg), I,,, 265 nm, m/e 388(M+), 37O(M+-H,O), 352(M+-2HzO), 287, 269, 251, 152, 134 (Found: M+, 388.2636. C24H3604 requires M, 388.2614). Measurement
mals were killed by decapitation, the duodenum was removed and blood was collected. Intestinal calcium transport was measured by the everted gut sac technique as described by Martin and DeLuca[14]. The blood was centrifuged to yield serum. Calcium was determined in the serum in the presence of 0.1% lanthanum chloride by means of a Perkin Elmer Model No. 403 atomic absorption spectrometer.
of biological activity
Weanling male rats were obtained from Holtzman Co., Madison, Wis. and were fed a vitamin D deficient-low calcium diet [13] and watered ad libitum. After the animals had been fed the diet for 3 weeks, they were considered vitamin D and calcium deficient as shown by severe hypocalcemia. At this stage, rats were injected intrajugularly with 125 ng of either 1,25-(OH)2D, or la-OH-24-COOH trisnor-vitamin D, dissolved in 0.05 ml ethanol. Control animals received the ethanol at the appropriate time. The ani-
RESULTS The synthesis of trisnor-vitamin Dj 24-oic acid (15) and its la-hydroxyl analog (16) is outlined in Scheme 1. The starting material was 24-oxocholesterol acetate 1, which was readily obtained from fucosterol as described previously [lo]. Cleavage of the 24,25-bond of 1 was attempted by Baeyer-Villiger reaction. How-
tio& *co)..j+/ Aco& 1R’= H,
RI AC
2R?OH,R2’H
3
&
3R=H
IOR-
QAc
11 R-OAc
R’=OAc,RiAc
Acog
Aco&+.$fOt_
4R=H
12
R=OAc
$roon
13R=H ldR=OAc
HO’ I5R=H 16R=OH
Scheme 1.
Synthesis and biological activity of 25,26,27-trisnor-vitamin
DJ 24oic acid and its la-hydroxyl analog
Table 2. Lack of response of serum calcium to la-OH-24COOH D, (Ca mg/lCO ml serum)
h after dose 1 3 6 24
EtOH 4.3 4.2 3.6 3.7
+ + + *
0.4t 0.4 0.3 0.3
1.25-(OH)zD, 4.4 + 0.2 5.2 + 0.3* 5.1 k 0.7* -
265
trisnor vitamin
la-OH-24-COOH trisnor vitamin Ds 4.9 4.7 4.1 3.8
* 0.7 * 0.7 + 0.3 & 0.3
Rats were fed a low calcium, vitamin D-deficient diet for 3 weeks. One group of rats was given 125 ng of la-OH-24COOH trisnor vitamin Ds while another group received 125ng of 1,25-(OH)2D, dissolved in ethanol. The control group received ethanol alone. At the indicated time, animals were killed, blood was collected and centrifuged to yield serum. Serum calcium concentration was determined by means of an atomic absorption spectrometer as described in the text. Each group had 6 rats. t Standard deviation. * Significantly different from control (P < 0.005).
ever, the reaction of 1 with m-chloroperbenzoic acid or trifluoroper acetic acid occurred preferentially on the C-5(6) double bond, and in the latter case the major product was found to be the 5x-hydroxy-6fitrifluoroacetate derivative. Therefore, the C-5(6) double bond of 1 was protected as its dibromide 2, which was subjected to oxidation with trifluoroperacetic acid to yield the desired cholenic acid derivative 3. The structure of 3 is consistent with the proton magnetic resonance spectrum indicating a hepted at 5.0ppm (25-H) which on irradiation of the doublet at 1.22 ppm (25Me,) turned to a singlet. The subsequent debromination of 3 with zinc in acetic acid furnished the isopropyl cholenoate 4 in an over-all yield of 50% from 1. The la-acetoxy analog 12 was prepared by a similar successive reactions of bromination, BaeyerVilliger reaction and debromination of the 24-0x0la,3/Ldiacetate (9) which was synthesized as follows. The ethylene ketal of cholesta-1,4,6-triene-3,24dione (5) prepared by the procedure described earlier [ 1I], was converted to the la,2a-epoxide 6 by treatment with hydrogen peroxide under basic conditions. According to Barton’s procedure [ 123, the epoxide 6 was treated with lithium-ammonium chloride in liquid ammonia to afford the lu,3/?-diol 7 in 77% yield. Deketalization followed by acetylation gave the diacetate 9. Conversion of the isopropyl cholenoate 4 and 12 to the vitamin D form involved the usual allylic bromination with N-bromosuccinimide and dehydrobromination with trimethyl phosphite sequence to the 5,7-diene derivatives 13 and 14, respectively, which upon irradiation and then refluxing in benzene-ethanol (2: 1) yielded a vitamin D system. Subsequent hydrolysis produced the target compound 15 and 16 after purification by high pressure liquid chromatography. The vitamin D analogs 15 and 16 exhibited the expected ultra-violet and mass spectra. As shown in Table 1, la-OH-24COOH trisnorvitamin D, will significantly stimulate intestinal calcium transport although not as well as 1,25-(OH),D,. This compound gives a siower response than 1,25-(OH)2DJ and its effectiveness is short-lived, while
the stimulation of intestinal calcium transport by 1,25-(OH),D, is maintained for days in rats [15]. As shown in Table 2, la-OH-24COOH trisnor vitamin D3 does not stimulate bone calcium mobilization at least at this dosage level used. DISCUSSION
Previous work by Holick et aL[16] suggested that reduced biological activity of side chain altered vitamin D analogs is due to, at least in part, failure of their la-hydroxylation. However, despite chemically attached la-hydroxyl groups to the present compound, modification of the side chain decreases biological activity. Although other possible functions of the compound cannot be ruled out, la-OH-24 COOH trisnor vitamin D, does not appear to be a biologically important compound, at least as far as calcium metabolism is concerned. Acknowledgements-This work was supported by grants from the Japanese Society of Promotion of Science No. 5R-080 and Grant AM-14881 from the National Institutes of Health, contract EY-76-S-02-1668 from the United
States Energy Research and Development Administration, Grant INT76-05793 from the National Science Foundation, and the Harry Steenbock Research Fund. REFERENCES
1. DeLuca H. F.: Vitamin D: The vitamin and hormone. Fed. Proc. 33 (1974) 221 l-2219. 2. Tanaka Y., Castillo L., DeLuca H. F. and Ikekawa N. : The 24-hydroxylation of 1,25-dihydroxyvitamin D1. J. biol. Chem. 252 (1977) 1421-1424. 3. Kumar R. and DeLuca H. ‘F.: Side chain oxidation of 25-hydroxy-[26,27-14C] vitamin D, and 1,25-dihydroxy-[26,27-r4C] vitamin D, in uiuo by chickens. Biothem. biophys. Res. Commun. 69 (1976) 197-200. 4. Kumar R., Harnden D. and DeLuca H. F.: Metabolism of 1,25-dihydroxyvitamin D,: Evidence for side-
chain oxidation:
Biochemistry
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2420-2423.
Harnden D., Kumar R.. Holick M. F. and DeLuca H. F.: Side chain metabolism of 25-hydroxy[26,27-‘4C] vitamin Da and 1,25-dihydroxy[26,27-“‘Cl vitamin D, in viuo. Science 193 (1976) 493494. 6. Kumar R. and DeLuca H. F.: Side chain oxidation of 1,25-dihydroxyvitamin D3 in the rat: Effect of remo5.
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NAOYUKIKOIZUMIet al. val of the intestine. Biochem.
biophys.
Res. Commun.
76 (1977) 253-258.
I. Sih C. J., Tai H. H., Tsong Y. Y., Lee S. S. and Coombe
R. G.: Mechanisms of steroid oxidation by microorganisms. XIV. Pathway of cholesterol side-chain degradation. Biochemistry 7 (1968) 808-818. 8. Gustafsson J.: Biosynthesis of cholic acid in rat liver: ZCHydroxylation bf 3a,7a,l2ol-trihydroxy+cholestanoic acid. J. biol. Chem. 250 (1975) 8243-8247 and . references cited therein. 9. Hawthorne M. F., Emmons W. D. and McCallum K. S.: A re-examination of the peroxy acid cleavage of ketones, I. Relative migratory aptitudes. J. Am. Chem. Sot. 80 (1958) 6393-6398. 10. Takeshita T., Ishimoto S. and Ikekawa N.: Preparation of desmosterol from fucosterol. Chem. Pharm. Bull. 24 (1976) 1928-1931. 11. Morisaki M., Koizumi N., Ikekawa N., Takeshita T.
12.
13.
I
14.
15. 16.
and Ishimoto S.: Synthesis of active forms of vitamin D. Part IX. Synthesis of la,24_dihydroxycholecalciferol. J. Chem. Sot., Per&in I. (1975) 1421-1424. Barton D. H. R., Hesse R. H., Pechet M. M. and Rizzardo E.: A convenient synthesis of lu-hydroxy-vitamin Da. J. Am. Chem. Sot. 95 (1973) 2748-2749. Suda T., DeLuca H. F. and Tanaka Y.: Biological activity of 25.hydroxyergocalciferol in rats. J. Nutr. 100 (1970) 1049-1052. Martin D. L. and DeLuca H. F.: Influence of sodium on calcium transport by the rat small intestine. Amer. J. Physiol. 216 (1969) 1351-1359. DeLuca H. F.: Vitamin D today. Disease-a-Month (1975) l-37. Holick M. F., Garabedian M., Schnoes H. K. and DeLuca H. F.: Relationship of 25-hydroxyvitamin Ds side-chain structure to biological activity. J. biol. Chem. 2!tO (1975) 226230.
SYNTHESIS AND BIOLOGICAL ACTIVITY OF 25,26,27-TRISNOR-VITAMIN D, 24-OIC ACID AND ITS la-HYDROXYL ANALOG NAOYUKI
KOIZUMI*,MASUOMORISAKI*,NOBUOIKEKAWA*, YOKOTANAKA?and HECTORF. DELUCA~ * Laboratory of Chemistry for Natural Products, Tokyo Institute of Technology, t Department
Midori-ku, Yokohama, 227, Japan and of Biochemistry, College of Agricultural and Life Science, University of Wisconsin-Madison, Madiso?, WI 53706, U.S.A. (Received 8 June 1978)
SUMMARY Because 1,25-dihydroxyvitamin D undergoes side-chain cleavage and oxidation in uivo, an anticipated metabolite is la-hydroxy-trisnor-vitamin DJ 24-oic acid. A synthesis for this metabolite and trisnorvitamin D, 24-oic acid has been devised. Baeyer-Villager rearrangement of 5,6-dibromo-24-oxo-cholesteryl acetate and its la-acetoxyl analog gave the corresponding cholenoate derivatives, and those were converted to trisnor-vitamin D, 24-oic acid and its la-hydroxyl analog, respectively. The la-hydroxy-25,26,27-trisnor-vitamin D3 24-oic acid (la-OH-24-COOH trisnor-vitamin D3) was tested for biological activity using rats fed a low calcium, vitamin D-deficient diet. It stimulated intestinal calcium transport 6 h after intravenous administration although the response is significantly less than that to an identical base of 1,25_dihydroxyvitamin D, (1,25-(OH),D,). This compound given at 125 ng per rat did not stimulate bone calcium mobilization.
EXPERIMENTAL
INTRODUCTION It is now well accepted that physiologically vitamin D must be converted to 1,25-(OH),D, before it can stimulate calcium transport and bone calcium mobilization [l]. Besides these functions, 1,25-(OH),D3 induces a 25-hydroxyvitamin D-ZChydroxylase yield system. This system then hydroxylates 1,25-(OH)2D, to 1,24,25-trihydroxyvitamin D3 (1,24,25-(OH),-D,). The physiological function of this compound is unknown [Z]. It has also been shown that [26,27-14C]-1,25-(0H)~D~ undergoes side chain oxidation in chickens and in rats to produce radiolabeled 14C02 [3-61 and a metabolite missing a portion of the side-chain. Side-chain cleavage of 25-hydroxycholesterol to give the 24-oic acid derivative by Nocardia restrictus has been observed [7], and cholic acid biosynthesis from cholesterol via 24-hydroxylation followed by oxidation has been reported [S]. These’ metabolic pathways might involve a 24-hydroxylation with the resulting 24,25-glycol becoming oxidatively cleaved to yield the 24-carboxylic acid derivative. Although neither the functional significance of the side-chain cleavage of 1,25-(OH),D, nor the structure of the resulting compound is known, it is distinctly possible that the metabolite is a 24-carboxylic of I ,25-(OH)2D,. The present paper describes chemical synthesis of trisnor-vitamin D3 24-oic acid (15) and its lx-hydroxyl analog (16) from 24-oxocholesterol and the biological activity of la-hydroxy-25,26,27-trisnor-vitamin D3 24-oic acid (la-OH-24-COOH trisnor vitamin D3). Send correspondence S.B. 1013 -I,
to: Dr. N. Ikekawa.
General
Melting points were determined with a hot-stage microscope apparatus. U.V. spectra were recorded with a Shimadzu UV-200 instrument for solutions in ethanol unless otherwise stated. N.M.R. spectra were obtained with a Hitachi R-24A or a JEOL JNM+H-100 spectrometer for solutions in deuteriochloroform, with tetramethylsilane as internal standard. Mass spectra were run with a Shimadzu LKB-9000s spectrometer. Column chromatography was carried out with silica gel (Wakogel C-200). T.L.C. was carried out on Merck silica gel F2+, (0.25 mm thick). Silver nitrateimpregnated t.1.c. plates were prepared by dipping the plates into 2%AgNO, solution in acetonitrile, followed by activation at 80°C for 1 h. High pressure liquid chromatography was achieved with a Shimadzu 830 instrument equipped with Zorbax SIL (DuPont Instruments, Wilmington, Del.) (2.1 mm x 25 cm) column. Treatment of 24-oxo-cholesterol acetate with triJuoroperacetic acid
To a solution of 24-oxo-cholesterol acetate (1) [lo] (91 mg) in methylene
chloride
(1.5 ml), were added di-
basic sodium phosphate (204 mg), trifluoroacetic anhydride (160$), and 90”/, hydrogen peroxide (16~1)[9]. The reaction mixture was stirred for 1 h at room temperature and then extracted with methylene chloride to give a crude product (204 mg), which was chromatographed on silica gel (5 g). Elution with benzene:ethyl acetate @:I, v/v) gave 3&acetoxy-5a261
262
NAOYUK~KOIZUMIet ai.
hydroxy - 68 - t~~uoro~toxy - cholestan - 24 - one (30mg), m/e 572(M*), 554, 539, 529, 51 I, S 068(3H, s, 13.Me), l.O9(6H, d, J = 7.5 Hz, 25-Me,), l.l4(3H, s, IO-Me), 2.03(38, s, OAc), 4.83(1H, broad, s, 4H), m, 3u-H). (Found: M+ 572.3273, 5/20(W C3rH4,06FJ requtres M, 572.3325). 3fi-Acetoxy-.5,6-dibromocholescun-24-one (2)
To a mixture of 24.oxocholesterof acetate (I)[fOJ, (1.6 g), potassium acetate (4 g), ether (64 ml) and acetic acid (40 ml), was added bromine (0.3 ml) dissolved in acetic acid (20 ml), dropwise at 0°C using an ice bath. After stirring for 2 h, water (150 ml) was added and the resulting precipitate was collected by filtration, and washed with sodium bicarbonate solution and water to give the dibromide 2 (2.02 g), m,p. 87-89°C (from methanol), S 0.70(3H, s, 13.Me), l.O8(6H, d, J = 7.5 Hz, 25-Me,), 146(3H, s, lO-Me), 2.03(38, s, OAc), 4.86(1H, broad, s, 6-H), 5.5O(IH, m, 3a-H). Anal. Calc. for Cz9HOb03Br2 : C, 57.81; H, 7.70. Found: C, 57.83; H, 7.70. IsoFro~~~ 38-acetoxy-5,6-dibromocholanoate (3) To a solution of the dibromide (2) (2.02g) in dry methylene chloride (28 ml), were added dibasic sodium phosphate (4.08 g), trifluoroacetic anhydride (3.2 ml) and 90% hydrogen peroxide (0.32 ml) at 0°C. The reaction mixture was stirred for 80min. at room temperature and extracted with ethyl acetate. The characteristics of the cholanoate formed were: (3) (2.34 g), m.p. 9698°C (from methanol), 6 0.70(3H, s, 13-Me), 1.22(6H, d, J = 7.5 Hz, 25-Me,), 1.47(38, s, 10-Me), 2.04(3H, s, OAc), 4.88(1H, m, 6-H), 5.02(lH, heptet, J = 7.5 Hz, -OCYMe&, 5.5O(lH, m, k-H). Isopropyl 3/I-acetoxycholenoate (4)
The dibromide (3) (2.03 g) was stirred with zinc powder (0.4 g) in a mixture of ether (40 ml) and acetic acid (20 ml) at room temperature for 2 h. The mixture was filtered and the filtrate was extracted with ethyl acetate. The characteristics of the oily product formed were (4) (1.33 g), m.p. lOl--103°C (from methanol), 6 069(3H, s, IO-Me), 10,2(3H, s, lo-Me), 1.22(68, d, J = 7.5 Hz, -OCHJ&), 2.02(3H, s, OAc), 4.65(1H, m, 3x-H), 5.02(1H, heptet, J = 7.5 Hz, -OCflMe& 5.4O(lH, m, 6-H). (Found: Mf-AcOH, 398.3160. C2,Ho202 requires M-AcOH, 398.3185). 24,24-Ethylenedioxycholesta-2,4.&riene-3.one
(5)
A mixture of cholesta-~,4,~t~en~3,24-d~one (5.9 g) [l 11, ethylene glycol (6 ml), a catalytic amount of gtoluene sulfonic acid and benzene (200ml) was heated for 15 h under reflux through molecular sieves in a Soxhlet apparatus. Extraction with ethyl acetate gave an oily product with the characteristics (S) (6.35 g), m/e 438(M+), 395(M+-isopropyl, base peak), 6 0.77(3H, s, 13-Me), 093(6H, d, J = 7 Hz, 25.Me& I.2I(3H, s, IO-Me), 3.96(48, s, -OCH2CHIO-), 6&6.2(4H, m, 2-, 4-, 6- and 7-H). 7.11(1N, d,
J = 10 Hz, 1-H). (Found: M+, 438.3118. C29H4203 requires M, 438.3234). lo1,2a-Epoxy-24,24-ethylenedioxycholesta-4,6-diene-3otie (6)
To a solution of the trienone (5) (6.3 g) in methanol (200 ml), were added 10% sodium hydroxyde in methanol (1.8 ml) and 30% hydrogen peroxide (11.6 ml). The mixture was kept at room tem~rature for 3 days, concentrated, and extracted with ether. The usual work-up gave a pale yellow amorphous powder (5.2 g), which was chromatographed on silica gel (6Og). Elution with benzene-hexane (3:l) gave a colorless crystalline 6 (3.5 g), m-p. 149-150°C 6 0.77(3H, s, 13.Me), 0.92(6H, d, J = 7 Hz, 25-Me& l.l8(3H, s, IO-Me), 3,42(1H, dd, J = 4, l.SHz, 2&H), 3.58(1H, d, J = 4Hz, I/?-H), 3.93(4H, s, --OCHz CH@--), 5.63(1H, d, J = 1.5 Hz, 4-H), 6.07(28, s, 6and 7-H), m/e 454(M’), 439(M+-Me), 4.11(M+-isopropyl, base peak), 396(M+-Me-isopropyl). Anal. Calc. for C29H4204: C, 76.61; H, 9.31. Found C, 76.61; H, 9.35. 24,24-~~hylenediox~ 1ups-dihydroxycholesr-5.ene
(7)
A three-necked flask was fitted with a sealed mechanical stirrer, a cold finger filled with solid COr , and an inlet tube connected to an anhydrous ammonia source. Nitrogen was blown through the system for IOmin, and then ammonia (140 ml) was trapped in the flask. Lithium wire (2.5 g) was cut into short pieces and added. After stirring for 1Omin the epoxide (6) (1.2 g) in tetrahydrofuran (140mI) was added dropwise for 20 min. Stirring under cooling for 30min and then the cooling bath was removed. The mixture was allowed to reflux for 30 min and the flask was dipped in a cooling bath. Anhydrous ammonium chloride (20.8 g) was added for 4 h; the mixture which turned white and pasty was kept at room temperature overnight. Extraction with ethyl acetate gave the crude product (1.34 g). Colorless crystals (940 mg) were obtained by re~ystai~zation from methanol, m.p. 164165°C 6 0.67(38, s, 13-Me), 0.91(6H, d, J = 7 Hz, 2S-Me2), 1.01(3H, s, IO-Me), 3.91(4)3, s, -OCH,CH#-), 3.7-4.2(28, m, l/7- and 3a-H), 5.58(1H, m, 6-H). (Found: M+, 460.3527. C29H4804 requires M, 460.3553). lee-3&Dihydroxy-24-oxocholest-5-ene (8)
A solution of the ketal (7) (2.4 g) and p-toluene sulfonic acid (20mg) in acetone (250ml) was stirred at room temperature overnight. This gave a substance with the characteristics 8, (2.2g), m.p. 150-151°C (from acetone), 6 0.67(3H, s, 13-Me), 0.99(6H, d, J = 7Hz, 2%Me*), l.O1(3H, s, IO-Me), 3.84(1H, m, l&H), 4.05(1H, m, 3cr-H), 5.58(1H, m, 6-H). (Found: M+, 416.3289. C2,H4+03 requires M, 416.3290). la,3fLDiacetoxy-chofesr-5-ene24-one
(9)
The dial(8) (1.9 g) was treated with pyridine (20 mt) and acetic anhydride (20ml) for 3.5 h at 90°C and
Synthesis and biological activity of 25,26,27-trisnor-vitamin
water was added. Extraction with ethyl acetate gave an oily product 9 (2.0 g), S 0.68(38, s, 13-Me), l.O7(6H, d, .J = 7.5 Hz, 2%MeJ, l.O8(3H, s, l@Me), 2.03(38, s, OAc), 2.05(3H, s, OAc), 4.65.2(2H, m, l/3- and 3x-H) 5.54(1H, m, 6-H). la,3/?-Diacetoxy-5,6dibromocholestan-24-one
(10)
To a mixture of 9 (1.7 g), potassium acetate (4 g), ether (64 m?) and acetio acid (40 ml), was added drop wise bromine (0.3 ml) in acetic acid (20 ml) at 0°C. After stirring for 2 h, water (150 ml) was added. Extraction with ethyl acetate gave the dibromide 10 (2g), 6 0.70(3H, s, 13-Me), l.O8(6H, d, J = 7.5 Hz, 25-Me&, l-49(38, s, IO-Me), 2.05(38, s, OAc), 2.07(3H, s, OAc), 4.82(18, M, 6-H), 5.58(1H, m, 3a-H). Isopropyl
(11)
Ja,3/?-diacetoxy-5,6dibromo-cholanoate
To a solution of 10 (1.9 g) in methylene chloride (28 ml), were added dibasic sodium phosphate (4.08 g), trifluoroacetic anhydride (3.2 ml) and 90% hydrogen peroxide (0.32ml) at 0°C. The reaction mixture was stirred at room temperature for 90min and sodium bicarbonate solution was added. Extraction with ethyl acetate gave 11 (2.1 g), 6 0.70(3H, s, Me-18), l-22(68, d, J = 7.5 Hz, -O-CHh&,), 1.52(3H, s, 10-Me), 4.85(1H, m, 6-H), 5.02(1H, heptet J = 7.5Hz, -OCIIMe,), 5.66.0(2H, m, l/I- and 3a-H). Isopropyl
fu,3/Ldiacetoxy-cholenoate
(12)
A mixture of the compound 11 (2.0 g), ether (40 ml) and acetic acid (10ml) was stirred with zinc powder (0.4g) at room temperature for 6 h. Extraction with ethyl acetate gave an oily product (1.32 g), which was purified by column chromatography on silica gel (28g). Elution with the mixed solvent of benzeneethyl acetate (50:1) yielded 12 (500 mg), S 0.67(3H, s, 13-Me), l.O9(3H, s, IO-Me), 1.23(6H, d, J = 7 Hz, -OCH&,), 2.03(38, s, OAc), 2.05(3H, s, OAc), 4.95(1H, m, 3a-H), 5.02(1H, heptet, J = 7Hz, -OCHMe,), 5.09(1H, m, l/Y-H), 5.54(lH, m, 6-H). (Found: M+-2AcOH, 396.3014. C2,H4002 requires M-2AcOH, 396.3028). Isopropyl 38-acetoxy-chola-5,7-diene-24-oate
(13)
To a refluxing solution of 4 (50.7 mg) in carbon tetrachloride (5 ml), N-bromosuccinimide (27 mg) was added in one portion, Stirring under reflux was continued for 25 min. The mixture was cooled and the resulting imide was filtered off. Evaporation of the filtrate gave a pale yellow syrup. The residue in xylene (1 ml) was added dropwise to a refluxing mixture of trimethyl phosphite (150 ~1) and xylene (0.5 ml). After heating for 2 h, the solvent was evaporated to give a yellow oil. The product was purified with preparative t.1.c. developed with benzene 4 times and the main U.V. absorbing band was scraped off. Elution with ethyl acetate gave 13 (5 mg), ,I,,,, 262, 272, 282, 294nm, 6 0.62(38, s, 13-Me), 0.95(3H, s, lo-Me), 1.22(68, d, .I = 7 Hz, -OCHJ&,), 1.23(3H, s, OAc), 4.2(1H, m, 3a-H), 5.02(1H, heptet J = 7 Hz,
D5 2Aoic acid and its la-hydroxyl analog
263
-OCHMe& 5.50(2H, ABq, J = 6 Hz, 6- and 7-H) m/e 456(M+), 496(M+-AcOH), 481(M+-AcOH-Me). Isopropyl
Ja,3&diacetoxy-chola-5,7-diene-24-oate
(14)
A mixture of the compound 12 (51 mg), N-bromosuccinimide (25 mg) and carbon tetrachloride (4 ml) was heated under reflux for 15 min. The precipitate was removed by filtration and the filtrate was evaporated to dryness. The residue in xylene (1.5 ml) was added to a refluxing mixture of trimethyl phospite (0.2 ml) and xylene (1.5 ml). After refluxing for 90 min, the solution was evaporated in uacuo to yield a yellow oil. Purification by preparative t.1.c. gave 14 (8 mg), ,I,,,,, (ether) 262, 271, 282, 294 nm, 6 0.62(3H, s, 13-Me), 1.24(6H, d, .J = 7 Hz, -OCH&z), 2.04(38, s, OAc), 2.09(38, s, OAc), 5.01(1H, heptet J = 7 Hz, -OCYMe2), 4.7-5.2(2H, m, l/3- and 3a-H), 5.54(2H, ABq, J = 6Hz, 6-Land 7-H). (Found: M+, 514.3326. C31H460L requires M, 514.3295). 3~-Hydroxy-9,J(Fseco-chola-5,7,10(J9)-triene-24-oic acid (15)
A solution of the 5,7-diene 13 (5 mg) in a mixture of benzene (IOOml) and ethanol (50 ml) in a quartz apparatus cooled in an ice bath was agitated with a stream of argon for 10min and then irradiated with medium-pressure mercury lamp (Hanovia 654A36; 200 W) immersed in the vessel for 4 min. The mixture was refluxed for 2 h under argon. The solvent was evaporated off and the residue was applied to a silver nitrate-impregnated silica gel plate. Development with a mixed solvent of hexane-benzene (5: 1, V/V) 3 times and elution of the least polar U.V.-absorbing band gave the diester of 15 (1 mg). Stirring of the ester (1 mg) in 10% KOH-MeOH at room temperature for 2.5 h and the usual work-up afforded crude 15, which was purified by high pressure liquid chromatography with 2.5% MeOHCHrCl, to give 25,26,27-trisnor-vitamin D, 24oic acid (0.1 mg), A,,,,, 265nm, m/e 372(M+), 354(M+-H,O), 339(M+-H,O-Me), 136, 118. (Found M+, 372.2748, C24H3(rOJ requires M, 372.2664). 9,1O-Seco-chola-5,7,l~J9)-triene-Ju,3fl-diol-24-oic
acid
(16)
The 5,7-diene 14 was irradiated in a mixture of benzene (1OOml) and ethanol (50 ml) for 2 min under argon with medium pressure mercury lamp. After refluxing for 60min under argon, the mixture was concentrated in uacuo and the residue was chromatographed on preparative t.1.c. The chromatograph was developed with CHCl,-hexane (2: 1, V/V) 3 times and the main U.V.-absorbing band was scraped off. Further purification was performed by high pressure liquid chromatography to give the triester of 16 (1 mgb A,,, 265 nm, m/e 514(M+), 454(M+-AcOH), 412(McLafferty), 394(M+-2AcOH). The ester was stirred in 10% KOH-MeOH at 40°C for 4 h and the mixture was extracted with ethyl acetate. Purification was achieved by high pressure liquid
264
KOIZUMIet aI
NAOYUKI
Table 1. Response of intestinal calcium transport to la-OH-24COOH D, (%a mucosal/4sCa serosal)
h after dose 1 3 6 24
EtOH
1.25(OHbD,
1.46 + 0.25t 1.76 f 0.18 1.50 & 0.30 1.60 + 0.32
trisnor vitamin
1a-OH-24-COOH trisnor vitamin D3
2.18 + 0.32 3.12 k 0.50’ 4.64 + 0.74*1 -
2.06 + 1.79 f 2.81 + 2.10 f
0.28 0.30 0.69*$ 0.71
Rats were fed a low calcium, vitamin D-deficient diet for 3 weeks. One group of rats was given 125 ng of la-OH-24-COOH trisnor vitamin D, intravenously while another group received 125 ng of 1,25-(OH),D,. The control group received ethanol alone. At the indicated time, animals were killed and intestinal calcium transport was measured as described in the text. Each group had 6 rats. * Significantly different from control (P < 0.005). t Standard deviation. $ Significantly different from 8 (P < 0.001).
chromatography (Solv. 4.5% MeOH-CH,Cl,) to give la-hydroxy-25,26,27-t&or-vitamin D, 24oic acid (16) (50 pg), I,,, 265 nm, m/e 388(M+), 37O(M+-H,O), 352(M+-2HzO), 287, 269, 251, 152, 134 (Found: M+, 388.2636. C24H3604 requires M, 388.2614). Measurement
mals were killed by decapitation, the duodenum was removed and blood was collected. Intestinal calcium transport was measured by the everted gut sac technique as described by Martin and DeLuca[14]. The blood was centrifuged to yield serum. Calcium was determined in the serum in the presence of 0.1% lanthanum chloride by means of a Perkin Elmer Model No. 403 atomic absorption spectrometer.
of biological activity
Weanling male rats were obtained from Holtzman Co., Madison, Wis. and were fed a vitamin D deficient-low calcium diet [13] and watered ad libitum. After the animals had been fed the diet for 3 weeks, they were considered vitamin D and calcium deficient as shown by severe hypocalcemia. At this stage, rats were injected intrajugularly with 125 ng of either 1,25-(OH)2D, or la-OH-24-COOH trisnor-vitamin D, dissolved in 0.05 ml ethanol. Control animals received the ethanol at the appropriate time. The ani-
RESULTS The synthesis of trisnor-vitamin Dj 24-oic acid (15) and its la-hydroxyl analog (16) is outlined in Scheme 1. The starting material was 24-oxocholesterol acetate 1, which was readily obtained from fucosterol as described previously [lo]. Cleavage of the 24,25-bond of 1 was attempted by Baeyer-Villiger reaction. How-
tio& *co)..j+/ Aco& 1R’= H,
RI AC
2R?OH,R2’H
3
&
3R=H
IOR-
QAc
11 R-OAc
R’=OAc,RiAc
Acog
Aco&+.$fOt_
4R=H
12
R=OAc
$roon
13R=H ldR=OAc
HO’ I5R=H 16R=OH
Scheme 1.
Synthesis and biological activity of 25,26,27-trisnor-vitamin
DJ 24oic acid and its la-hydroxyl analog
Table 2. Lack of response of serum calcium to la-OH-24COOH D, (Ca mg/lCO ml serum)
h after dose 1 3 6 24
EtOH 4.3 4.2 3.6 3.7
+ + + *
0.4t 0.4 0.3 0.3
1.25-(OH)zD, 4.4 + 0.2 5.2 + 0.3* 5.1 k 0.7* -
265
trisnor vitamin
la-OH-24-COOH trisnor vitamin Ds 4.9 4.7 4.1 3.8
* 0.7 * 0.7 + 0.3 & 0.3
Rats were fed a low calcium, vitamin D-deficient diet for 3 weeks. One group of rats was given 125 ng of la-OH-24COOH trisnor vitamin Ds while another group received 125ng of 1,25-(OH)2D, dissolved in ethanol. The control group received ethanol alone. At the indicated time, animals were killed, blood was collected and centrifuged to yield serum. Serum calcium concentration was determined by means of an atomic absorption spectrometer as described in the text. Each group had 6 rats. t Standard deviation. * Significantly different from control (P < 0.005).
ever, the reaction of 1 with m-chloroperbenzoic acid or trifluoroper acetic acid occurred preferentially on the C-5(6) double bond, and in the latter case the major product was found to be the 5x-hydroxy-6fitrifluoroacetate derivative. Therefore, the C-5(6) double bond of 1 was protected as its dibromide 2, which was subjected to oxidation with trifluoroperacetic acid to yield the desired cholenic acid derivative 3. The structure of 3 is consistent with the proton magnetic resonance spectrum indicating a hepted at 5.0ppm (25-H) which on irradiation of the doublet at 1.22 ppm (25Me,) turned to a singlet. The subsequent debromination of 3 with zinc in acetic acid furnished the isopropyl cholenoate 4 in an over-all yield of 50% from 1. The la-acetoxy analog 12 was prepared by a similar successive reactions of bromination, BaeyerVilliger reaction and debromination of the 24-0x0la,3/Ldiacetate (9) which was synthesized as follows. The ethylene ketal of cholesta-1,4,6-triene-3,24dione (5) prepared by the procedure described earlier [ 1I], was converted to the la,2a-epoxide 6 by treatment with hydrogen peroxide under basic conditions. According to Barton’s procedure [ 123, the epoxide 6 was treated with lithium-ammonium chloride in liquid ammonia to afford the lu,3/?-diol 7 in 77% yield. Deketalization followed by acetylation gave the diacetate 9. Conversion of the isopropyl cholenoate 4 and 12 to the vitamin D form involved the usual allylic bromination with N-bromosuccinimide and dehydrobromination with trimethyl phosphite sequence to the 5,7-diene derivatives 13 and 14, respectively, which upon irradiation and then refluxing in benzene-ethanol (2: 1) yielded a vitamin D system. Subsequent hydrolysis produced the target compound 15 and 16 after purification by high pressure liquid chromatography. The vitamin D analogs 15 and 16 exhibited the expected ultra-violet and mass spectra. As shown in Table 1, la-OH-24COOH trisnorvitamin D, will significantly stimulate intestinal calcium transport although not as well as 1,25-(OH),D,. This compound gives a siower response than 1,25-(OH)2DJ and its effectiveness is short-lived, while
the stimulation of intestinal calcium transport by 1,25-(OH),D, is maintained for days in rats [15]. As shown in Table 2, la-OH-24COOH trisnor vitamin D3 does not stimulate bone calcium mobilization at least at this dosage level used. DISCUSSION
Previous work by Holick et aL[16] suggested that reduced biological activity of side chain altered vitamin D analogs is due to, at least in part, failure of their la-hydroxylation. However, despite chemically attached la-hydroxyl groups to the present compound, modification of the side chain decreases biological activity. Although other possible functions of the compound cannot be ruled out, la-OH-24 COOH trisnor vitamin D, does not appear to be a biologically important compound, at least as far as calcium metabolism is concerned. Acknowledgements-This work was supported by grants from the Japanese Society of Promotion of Science No. 5R-080 and Grant AM-14881 from the National Institutes of Health, contract EY-76-S-02-1668 from the United
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