IKEKAWA NOBUO (JP)
TANAKA YOKO (US)
| US4202829A | 1980-05-13 | |||
| US4313942A | 1982-02-02 | |||
| US4448721A | 1984-05-15 | |||
| US4225596A | 1980-09-30 |
| 1. | Compounds having the formula wherein R, and R are hydrogen or hydroxyl, except that when R, is hydrogen R. |
| 2. | is hydroxyl and when R. is hydroxyl R. |
| 3. | is hydrogen, and 3 and R. are hydrogen, or acyl having frcm 1 to 4 carbon atoms. *& 22. |
| 4. | (22E,24R)l,24dihydrαxyΔ vitamin D3< 3 The ccmpound of claim 2 in crystalline form.*& 22. |
| 5. | (22E,24S)l,24dihydroxyΔ vitamin D3. |
| 6. | The compound of claim 4 in crystalline form. |
| 7. | Cαrpounds having the formula wherein R. and R2 are hydrogen or hydroxyl except that when R. is hydrogen _ is hydroxyl and when R, is hydroxyl R2 is hydrogen. |
| 8. | and R3 and R. are hydrogen or acyl having fran 1 to 4 carbon atoms. |
| 9. | (22E,24R)cholesta5,7,22trienelα,3β,24triol. 20 . |
| 10. | (22E, 24S) cholesta5, 7, 22trienelα, 3β, 24triol. |
22
1,24-Dihydroxy-Δ -Vitamin P., and Process for Preparing Same
Technical Field
This invention relates to new derivatives of vitamin D. and to a method for their preparation.
More specifically this invention relates to 1,24- dihydroxylated-Δ 22 -vitamin D_ cαtpounds.
Background of the Invention
Since the discovery that the active hormonal form of vitamin * D in the stimulation of intestinal, calcium transport, intestinal phosphate transport, and bone calcium mobilization is 1,25-dihydrσxyvitamin D 3 (1,25-(CH) 2 D_) , considerable interest in the chemical synthesis of analogs of this ccinpαund. has developed with a view toward finding in such analogs either increased biological activity or specific target organ actions. The most potent analogs which have been prepared to date are 26,26,26,27,27,27-hexafluoro-l,25-dihydroxyvitamin D- (26,27-F 6 -l,25-(OH) 2 D 3 ) (U.S. Letters Patent 4,358,406) and 24,24-diflx_Dro-l,25-dil^dro^vitamin D_ (24,24-F 2 -l,25- (CH) 2 D 3 ) (U.S. Letters Patent 4,201,881). These ccπpσunds provide activity at least 10 fold that of the natural hormone. All other modifications of the side-chain appear to reduce biological activity except the ergosterol side-chain which has
22 an unsaturation at the Δ -position and a methyl group in the
24S-position. This cxxπpou d appears to be equally active in binding to the chick intestinal cytosol receptor and in biological activity in maπmalian species, but appears to be one-tenth as active in birds. It is of interest, therefore, to construct various analogs in which each of these modifications is examined separately. Also of interest is the fact that 1,24-dihydroxyvitamin D. (1,24-(0H)_D_) is equally
as active as is 1,25- (0H) 2 D 3 in binding to the chick intestinal receptor, but when given in vivo 1, 24R- (OH) J ) ^ is only one-tenth as active as l,25-(OH) 2 D 3 and that the l,24S-iscmer is even less active than the 1,24R- iscmer. Disclosure of Invention
TVro new vitamin D derivatives have now been prepared. These compounds are the trans-isαners of 1, D- (1,24- (OH) D_) in which a double band has been inserted in the 22-ppsitiαn and an h o^l function substituted in the S- and R-positions on the 24-carbon atcm. The cctπpounds are
22 respectively (22E,24S)-l,24-dihydroκy-Δ -vitamin D 3 and
(22E,24R)-l,24-dihydroxy-Δ 22 ^itamin D 3 .
Both of tiie cxxπpσunds exhibit vitamin D-like activity with the 24-S compound showing the greater activity of the two and approaching, in fact, the activity of 1,25-(OH) Sir * . Best M3de for Carrying Out the Invention
The cαπpαunds of this invention can be synthesized in accordance with the following schematic diagram and description in which like compounds are identified by like numbers.
In the description which follows pbysico-chemical measurements were determined as follows: Melting points were determined on a hot stage microscope and were uncorrected. ϋV spectra were obtained in ethanol solution with a Shi adzu UV-200 double beam spectrαnater. TI-NMR spectra were run on a Hitachi R-24A spectrometer, a JEOL PS-100 spectrometer or a EOL FX-400 spectrometer. All NMR spectra were taken in DCD1 3 solution with tetramethylsilane as internal reference. Mass spectra were obtained with a Shimadzu KB 9000S spectrcπeter at 70 eV. Column chrαnatography was effected with silica gel (Merck, 70-230 mesh) . Preparative thin layer chrc atography was carried out on precoated plates of silica gel (Merck, silica gel 60 „ 4 ) . The usual work-tap refers to dilution with water, extraction with an organic solvent, washing to
neutrality, drying over magnesium sulfate, filtration, and removal of the solvent under reduced pressure.
Schematic
Legend: THP = tetrahydropyranyl
MPTA. - α-methoxy-α-trifluoromethylpenylacetyl ph - phenyl
Synthesis
22-Hydroxy-23,24-dinorchol-l,4,6-triene-3-one { 2) . To a solution of 3β-acetoxydinorcholenic acid (1) (7.0 g, 18.04 πnle) in THF (20 mL) lithium aluminum hydride (3.0 g, 78.95 mπole) was added. This mixture was stirred at 60 e C for 14 h.
To this reaction mixture water and ethyl acetate were carefully added. Filtration and removal of the solvent gave the residue (5.2 g) . This in dioxane (140 mL) was treated with dichlorodicyanobaizoquinone (11.7 g, 51.54 irπole) under reflux for 14 h. After cooling to rocm temperature the reaction mixture was filtered and the filtrate was evaporated to leave the residue, which was applied to a column of alumina
(200 g). Elution with dichlorαnethane provided the trienαne
(—2) (2.8 g, 47%): πp 156-157° (frαn et-.her). IN nm (ε) :
299 (13000), 252 (9200), 224 (12000), TΪ-NM (CDC1 3 ) : 0.80 (3H, s, 18-H 3 ), 1.04 (3H, d, J=6 Hz, 21-H. j ) , 1.21 (3H, s, 19-H 3 ), 3.10-3.80 (3H, m, 22-H 2 and OH), 5.90-6,40 (4H, m, 2-H, 4-H, 6-H, and 7-H) , 7.05 (IH, d, J=10 Hz, 1-fi) , MS m/z: 326 (M + ), 311, 308, 293, 267, 112.
22-Ttetrahydrσpyranyloxy-23,24-dinorchol-lα,2α-epoxy-4 ,6- dien-3-one (3). The alcohol (2) (2.7 g, 8.28 itπole) in dichloromethane (50 mL) was treated with dihydropyrane (1.5 L, 16.42 πmole) and p-toluenesulfonic acid (50 mg) at room temperature for 1 h. The usual work-up (ethyl acetate for extraction) gave a crude product. To a solution of this product in MsOH (70 mL) , 30% H 2 0 2 (4.8 mL) and 10% NaOH/tfeOH (0.74 mL) were added and this mixture was stirred at room teπperature for 14 h. The usual work-up (ethyl acetate for extraction) gave a crude product, which was applied to a column of silica gel (50 g) . Elution with benzene-ethyl acetate (100:1) provided the epoxide (3) (1.45 g, 41%): p 113-115° (hexane) . 07 λ^ H nm (ε) : 290 (22000) , 1 H-NMR (CDC1 3 ): 0.80 (3H, s, 18-H 3 ) , 1.07 (3H, d, J=6 Hz, 21-Hg) , 1.18 (3H, s, 19-H 3 ), 3.38 (IH, dd, J=4 and 1.5 Hz, 1-H) , 3.55 (IH, d, J=4 Hz, 2-H), 3.30-4.10 (4H, m, 22-H 2 and THP), 4.50
(IH, m, THP), 5.58 (IH, d, J=1.5 Hz, 4-H), 6.02 (2H, s, 6-H and 7-H) , MS m/z: 342 (M + - DHP), 324 (M + - THPOH) , 309, 283, 85.
23,24-Dinorchol-5-ene-lα,3β,22-triol-l,3-diacetate (4). Lithium (3.25 g) was added in small portions to liquid ammonia (13CtriL) at -78°C under argon atmosphere during 30 min. After stirring for 1 H at -78°C, the epoxide (3) (1.33 g, 3.12 πmole) in dry THF (100 mL) was added dropwise at -78°C during 30 min. and this mixture was stirred for 1 h at -78 e C. To this reaction mixture anhydrous NH.C1 (40 g) was added in small portions at -78°C during 1 h. After 1.5 h the cooling bathwas removed and the most of arππonia was removed with bubbling argcn. The usual work-up (ether for extraction) gave a crude product (1.23 g). This was treated with acetic anhydride (3 mL) and pyridine (4mL) at room temperature for 14 h. The usual work-up (ethyl acetate for extraction) gave a crude product (1.3 g). This in ethanol (4 mL) and THF (5 iriL) was treated with 2 drops of 2 M HC1 at rocm temperature for 2 h. The usual work-up (ether for extraction) gave a crude product (1.1 g) , which was applied to a column of silica gel (40 g). Elution with benzene-ethyl acetate (10:1) provided the 1,3-diacetate (4) (575 g, 42%): oil, ^-NM (CDC1 : 0.68 (3H, s, 18-H 3 ), 1.07 (3H, s, 19-H 3 ), 1.99 (3H, s, acetyl), 2.02 (3H, s, acetyl), 3.02-3.72 (2H, m, 22-H 2 ), 4.79 (IH, m, 3-H), 4.98 (IH, m, 1-H), 5.46 (IB, , 6-H), MS m/z: 372 (M + - CH 3 CO0H), 313, 312, 297, 279, 253. lα,3β-Diacetoxy-23,24-dinorcholan-22-al (5). The 22-alcohol (4) (550 mg, 1.27 romole) in dichloromethane (20 mL) was treated with pyridinium chlorochrcmate (836 mg, 3.85 iπnole) and sodium acetate (100 mg) at ro n temperature for 1 h. To this reaction mixture ether (100 mL) was added and this mixture was filtrated through a short Florisil colunm. The filtrate was concentrated to leave the residue, which was applied to a column of silica gel (20 g). Elution with benzene-ethyl acetate (20:1) provided the 22-aldehyde (5) (448
irg, 82%): oil, ^-NMR (CDC1 3 ) : 0.70 (3H, s, 18-iy , 1.07 (3H, s, 19-H 3 ), 1.09 (3H, d, J=7 Hz, 21-H 3 ) , 1.99 (3H, s, acetyl), 2.02 (3H, s, acetyl), 4.79 (IH, m, 3-H) , 4.98 (IH, , 1-H), 5.45 (IH, m, 6-H) , 9.45 (IH, d, J=4 Hz, 22-H) , MS m z: 310 (M + - 2 x CH 3 C00H) , 295, 253.
(22E)-lα,3g-Diacetoxy-cholesta-5,22-dien-24-one (6) . Tb a solution of the 22-aldehyde (5) (420 mg, 0.977 mmole) in dimethyl sulfoxide (30 mL) isc utyrylmet_ lenetxiphenylphos- phorane (2.03 g, 5.87 mmole) was added. This mixture was stirred at 95°C for 72 h. The usual work-iφ (ether for extraction) gave a crude product, which was applied to a column of silica gel (lO g) . Elution with benzene-ethyl acetate (10:1) provided the enone (6) (392 g, 81%) : oil, " Sl-NMR (CDC_ 3 ): 0.71 (3H, s, 18-H j ) , 1.08 (3H, s, 19-&-.) , 1.09 (9H, d, J=7 Hz, 21-H 3 , 26-H 3 , and 27-H 3 ) , 1.99 (3H, s, acetyl), 2.02 (3H, s, acetyl), 4.79 (IH, m, 3-H) , 4.98 (IH, m, 1-H), 5.45 (IH, m, 6-H) , 5.96 (IH, d, J=16 Hz, 23-H) , 6.65 (IH, dd, J=16 and 8 Hz, 22-H) , MS m/z: 438 (M + - CH 3 CCOH) , 378 (M + - 2 x CH 3 C00H) , 363, 335, 307/ 253, 43.
(22E)-lα,3β-Diacetoxy-5α,8 -(3,5-dioxo-4-phenyl-l,2,4- triazolidino)-cholesta-6,22-dien-24-one (1) . Tb a solution of the enone (6) (385 mg, 0.773 mmole) in carbontetrachloride (20 mL) , N-br iDSuccinimide (193 mg, 1.4 eq.) was added and this mixture was refluxed for 25 min under argon atmosphere. After cooling to 0 β C, the resulting precipitate was filtered off. The filtrate was concentrated below 40°C to leave the residue. This in THF (15 mL) was treated with a catalytic amount of tetra-n-butylaπstionium bromide at room temperature for 50 min. Then, to this reaction mixture a solution of tetra-n-butyl- ammonium fluoride in THF (3.5 mL, 3.5 mmole) was added and this mixture was stirred at room temperature for 30 min. The usual work-up (ethyl acetate for extraction) gave a crude 5,7-diene (380 mg) . This in chloroform (15 mL) was treated with a solution of l-phenyl-l,2,4-triazoline-3,5-dione (95 g, 0.54 mmole) in chloroform (10 mL) at room temperature for 1 h.
Rstoval of the solvent under reduced pressure gave the residue, which was applied to a column of silica gel (10 g) . Elution with benzene-ethyl acetate (5:1) provided the triazoline adduct ( ) (191 mg, 37%): oil, " .H-NMR. (CDC1 3 ) : 0.83 (3H, s r 18-H 3 ) , 1.01 (3H, s, 10-H 3 ) , 1.08 (9H, d, J=7 Hz, 21-H 3 , 26-H 3 , and 27-H 3 ) , 1.97 (3H, s, acetyl), 1.98 (3H, s, acetyl), 5.03 (IH, , 1-H) , 5.84 (IH, , 3-H) , 5.96 (IH, d, J=16 Hz, 23-H), 6.28 (IH, d, J=8.5 Hz, 6-H or 7-H) , 6.41 (IH, d, J=8.5 Hz, 6-H or 7-H) , 6.65 (IH, dd, J=16 and 8 Hz, 22-H) , 7.20-7.60 (5H, m, -ph) M m/z: 436 (M + - hC 2 3 0 2 - O^COOH) , 376 (436 - CH 3 C00H) , 333, 305, 251, 43.
(22E,24R)- and(22E,24S)-lα,3β-Diacetoxy-5α,8α(3,5-dioxo -4-phenyl-l,2 ,4-triazolidino)-cholesta-6,22-dien-24-ol (9a and 8a) . The enone (2) (150 mg, 0.224 rrmole) in THF (6 L) and methanol (6iriL) was treated with sodium borohydride (17 mg, 0.448 irmole) at room temperature for 10 min. The usual work-up (ether for extraction) gave a crude product (150 mg) , which was submitted to preparative TLC (benzene-ethyl acetate, 3:1, developed seven times) . The band with an Rf value 0.53 was scraped off and eluted with ethyl acetate. Removal of the solvent under reduced pressure gave the less polar (24S)-24-alcchol (8a) (43.2 rag, 28.7%): mp 142-144°C (ether-hexane) , MS m/z: 438 (M + - phC 2 N 3 0 2 - CH 3 CO0H) , 420, 378 (438 - CH 3 O00H) , 360, 363, 345, 335, 318, 109, 43. The band with an Rf value 0.50 was scraped off and eluted with ethyl acetate to give the more polar (24R)-24-alcohol (9a) (64.8 mg, 43.1%): mp 140-142°C (ether-hexane). Mass spectrum of (9a) was identical with that of (8a) .
(22E,24S)-lα,3β-Diacetoxy-5α,8α-(3,5-dioxo-4-phenyl- 1,2,4-triazolidino)-cholesta-6,22-dien-24-ol (+)-MTPA ester (8b) . The 24 alcohol (8a) (8.3 mg, 0.0123 irmole) in pyridine (ImL) was treated with 3 drcps of (+)-MTPA-Cl at room teπperature for 1 h. The usual work-up (ethyl acetate) provided the MTPA ester (8b) (10.4 mg, 95%): 'Η-NMR (CDC , 100 MHz): 0.85 (3H, s, 18-H 3 ) , 0.88 (3H, d, 7= Hz, 26-E j ) ,
0.92 (3H, d, J=7 Hz, 27-H 3 > , 1.04 (3H, d, J=7 Hz, 21-H 3 ) , 1.08 (3H, s, 19-H 3 ), 2.03 (3H, s, acetyl), 2.06 (3H, s, acetyl), 3.27 (IH, m), 3.54 (3H, s, -O y , 6.28 (IH, d, J=8 Hz, 6-H or 7-H), 6.41 (IH, d, J=8 Hz, 6-H or 7-H) , 7.24-7.56 (5H, m, -ph).
(22E,24R)-lα,3β-Diacetoxy-5α,8α-(3,5-doxo-4-phenyl-l, 2,4 -triazolidino)-cholesta-6,22-dien-24-ol 24-(+)-MCPA ester (9b). The 24-alcohol (9a) (7.9 mg, 0.0H7 mmole) was converted, as described for (8b) , into the MTPA ester (9b) (9.3 mg, 89%): "Ha-NMR (CDC1 3 , 100 MHz) : 0.83 (3H, s, 18-H 3 ) , 0.88 (6H, d, J=7 Hz, 26-H 3 and 27-H 3 ) , 1.04 (3H, d, J=7 Hz; 21-H 3 ), 1.08 (3H, s, 19-H 3 ), 2.03 (3H, s, acetyl), 2.05 (3H, s, acetyl), 3.27 (IH, m) , 3.54 (3H, s, -O y , 6.28 (IH, d, J=8 Hz, 6-H or 7-H), 6.41 (IH, d, J=8 Hz, 6-H or 7-H), 7.24-7.56 (5H, m, -ph) .
(22E,24S)-6β-M5thoxy-3α,5-cyclo-5α-cholestø-22-en-24- ol 24-(+)-MIPA ester (14b). The known (24S)-24-alcohol (14a) (10.1 mg, 0.0244 mmole) was converted, as described for (8b) , into the (24S)-MIPA ester (14b) (8.2 mg, 54%): " H-N R (CDC1 3 , 100 MHz): 0.72 (3H, S, 18-R-.) , 0.89 (3H, d, J=7 Hz, 26-H j ) , 0.93 (3H, d, J=7 Hz, 27-HJ , 1.02 (3H, d, J=7 Hz, 21-H , 1.04 (3H, s, 19-H 3 ), 2.75 (IH, m, 6-H) , 3.33 (3H, s, -O y , 3.54 (3H, s, "0CH 3 .
(22E,24R)-6β-Msthoxy-3α,5-cyclo-5α-cholesta-22-en-24-o l 24-(+)-MTPA ester (15b) . The known (24R)-24-alcohol (15a) (11.0 mg, 0.0266 mmole) was converted, as described for (8b) , into the (24R)-MTPA ester (15b) (9.4 mg, 56%): ^-N (CDC j , 100 MHz): 0.76 (3H, s, 18-H 3 ) , 0.88 (6H, d, J=7 Hz, 26-H 3 and 27-H 3 ), 1.04 (3H, d, J=7 Hz, 21-H 3 ) , 1.05 (3H, s, 19-H 3 ) , 2.77 (IH, m, 6-H), 3.36 (3H, s, -O y , 3.57 (3H, s, -O y .
(22E,24R)-Cholesta-5,7,22-triene-lα,3β ,24-triol (10). The triazoline adduct (9a) (15.0 mg, 0.0223 mmole) in THF (5mL) was treated with lithium aluminum hydride (5 mg, 0.132 mmole) under reflux for 2 h. To this reaction mixture water was added and filtered. The filtrate was concentrated under
10
reduced pressure to leave the residue, which was submitted to preparative TLC (benzene-ethyl acetate, 1:1, developed three times). The band with an Rf value 0.35 was scraped off and eluted with ethyl acetate. Removal of the solvent provided the 5,7-diene (10) (3.3 mg, 36%) , UV λ ! ? : 294, 282, 272, MS m/z: 414 (M + ) , 396, 381, 378, 363, 353, 335, 317, 287, 269, 251, 127, 109.
(22E,24S)-Cholesta-5,7,22-triene-lα,3β,24-triol (11). The triazoline adduct (8a) (16.5 g, 0.0245 irmole) was converted, as described for (10) , to the 5,7-diene (11) (3.5 mg, 35%) . The UV and MS spectra of (11) were identical with those of (10).
(22E,24R)-lα,24-Dihydroxy-Δ 22 -vitamin P., (12). A solution of the (24R)-5,7-diene (10) (3.3 mg, 7.97 mole) in benzene (9CtriL) and ethanol (40 mL) was irradiated with a medium pressure mercury lamp through a Vycor filter for 2.5 min. with ice-cooling under argon atmosphere. Then the reaction mixture was refluxed for 1 h under argon atmosphere. Ranoval of the solvent under reduced pressure gave a crude product, vriich was submitted to preparative TLC (benzene- ethyl acetate, 1:1, develcped three times) . The band with an Rf value 0.40 was scraped off and eluted with ethyl acetate. Removal of ths solvent under reduced pressure provided the vitamin D 3 analogue (12) (0.59 g, 18%). This was further purified by high performance liquid ciircmatography on a Zorbax-SIL column (4.6 mm x 15 cm) at a flow rate of 2 ml/min with 2% methanol in dichlorαπethane as an eluent. The retention tine of
228 nm, MS m/z: 41 287, 269, 251, 249 5 MHz): 0.57 (3H, s, 18-H 3 ) , 0.87, (3H, d, J=6.7 Hz, 26-H 3 ) , 0.92 (3H, d, J=6.7 Hz, 27-H 3 ) , 1.04 (3H, d, J=6.6 Hz, 21-H 3 ) , 2.32 (IH, dd, J=13.7 and 6.6 Hz), 2.60 (IH, dd, J=13.4 and 3.4 Hz), 2.83 (IH, dd, J=12.6 and 4.0 Hz), 4.23 (IH, m, 3-H) , 4.43 (IH, m, 1-H), 5.00 (IH, bs, W χ/2 =4.3 Hz, 19-H) , 5.33 (IH, bs.
11
1/2 =4.3 Hz, 19-H), 5.39 (IH, dd, J=15.2 and 7.1 Hz, 22-H) , 5.51 (IH, dd, J=15.2 and 8.3 Hz, 23-H) , 6.01 (IH, d, J=11.4 Hz, 6-H), 6.38 (IH, d, J=11.4 Hz, 7-H) .
(22E,24S)-lα,24-Dihydroxy-Δ 22 -vitamin D, (13). The (24S)-5,7-diene (11) (3.5 g, 8.45 mole) was transformed, as described for (12) , into the vitamin D 3 form (13) (0.56 mg, 16%) . The retention time of (13) under the above described HPLC condition was 4.7 min. The UV and MS spectra of (13) were identical with those of (12) . " hi-NMR (CDC1 3 , 400.5 MHz): 0.57 (3H, s, 18-H 3 ), 0.87 (3H, d, J=6.7 Hz, 26-H 3 ) , 0.92 (3H, d, J=6.7 Hz, 27-H 3 ), 1.05 (3H, d, J=6.6 Hz, 21-H 3 ) , 2.32 (IH, dd, J=13.7 and 6.6 Hz), 2.60 (IH, dd, J=13.4 and 3.4 Hz), 2.83 (IH, dd, J=12.6 and 4.0 Hz), 4.23 (IH, m, 3-H) , 4.43 (IH, m, 1-H), 5.00 (IH, bs, 1/2 =4.3 Hz, 19-H) , 5.33 (IH, bs, W 1/2 =4.3 Hz, 19-H), 5.37 (IH, dd, J=15.4 and 7.5 Hz, 22-H) , 5.46 (IH, dd, J=15.4 and 8.3 Hz, 23-H) , 6.01 (IH, d, J=11.4 Hz, 6-H) , 6.38 (IH, d, J=11.4 Hz, 7-H) .
To determine the configuration at the C-24 position the 24-alcohols 8a and 9a were converted into the corresponding (+)-MPTA ester 8b and 9b. The Tϊ- MR spectra of 8b and 9b were compared with those of the (+)-MTPA esters 14b and 15b, which were derived frcm the known (24S)-24-alcohol 14a and its (24R)-isσmer 15a, respectively. The " H-NMR data of methyl groups of 8b, 9b, 14b, and 15b are shown in Table 1.
As shown in Table 2, the TH-NMR data of C-22, and C-23 protons of the (24R)-vitamin D 3 analog 12 and those of the known (24S)-isomer 13 were in good agreement with those of the known (24R)-allylic alcohol 15a and its (24S)-isσmer 14a, respectively. These Tϊ-NMR data (as shown in Table 1 and 2) confirmed the assignment of the synthetic vitamin D_ analogs 12 and 13.
Table 1
Tϊ-NMR (100 MHz) spectral data of methyl groups in 8b, 9b, 14b, and 15b
Chemical shift
Compound 18-Ms 19-Ma 21-t 26-Efe and 27-Me
8b 0.85 1.08 1.04 (J=7) 0.88 (J=7), 0.92 (J=7)
9b 0.83 1.Q8 1.04 (J=7) 0.88 (J=7)
14b 0.72 1.04 1.02 (J=7) 0.89 (J=7), 0.93 (J=7)
15b 0.76 1.05 1.04 (J=7) 0.88 (J=7)
a Shifts are given in ppm and J values in Hz
Table 2
Tϊ-NMR spectra data of C-22 and C-23 proton in 12, 13 (400 MHz) and 14a, 15a (360 MHz)
Chemical shift
Compound 22-H 23-H
12 5.39 (dd, J=15.2, 7.1) 5.51 (dd, J=15.2, 8.3) 15a 5.374 (dd, J=15.39, 6.80) 5.494 (dd, J=15.40, 8.23)
13 5.37 (dd, J=15.4, 7.5) 5.46 (dd, J=15.4, 8.3) 14a 5.353 (dd, J=15.38, 7.06) 5.448 (dd, J=15.03, 8.20)
Shifts are given in ppm and J values in Hz
Biological Activity
The biological activity of the compounds of this invention was measured in accordance with well known procedures as indicated below.
13
Rats. Weanling male rats were purchased frcm Holtzman (Madison, WI) and fed either a low phosphorus (0.1%) , high calcium (1.2%) vitamin D-deficient diet as described by Tanaka and DeLuca (Proc. Nat'l. Acad. Sci. USA (1974) 71, 1040) (Table 3) or a low calcium (0.02%) , adequate phosphorus (0.3%) vitamin D-deficient diet as described by Suda et al (J. Nutrition (1970) 100, 1049) (Table 4) for 3 weeks.
Determination of Serum Calcium and Inorganic Phosphorus. Serun calcium was determined by atonic absorption spectrαnetry using saπples diluted in .1% lanthanum chloride. The instrument used was a Perkin-Elmer atonic absorption spectrometer model 403. Serum inorganic phosphorus was determined by the method of Chen et al (Anal. Chem. (1956) 28, 1756) .
Msasurement of Bone Ash. Bone ash measurements were made on femurs. Connective tissue was removed, the femurs extracted successively for 24 h with 100% ethanol followed by 24 h with 100% diethyl ether using a Soxhlet extractor. The fat-free bone was dried 24 h and ashed in a muffle furnace at 650°for 24 H.
Msasurement of Intestinal Calcium Transport Activity. Intestinal calcium transport was measured using the everted duodenal sac method described by Martin and DeLuca (Am. J. Physiol. (1969) 216, 1351).
Displacement of l,25-(OH) 2 -[26,27-Tϊ]D, frcm Chick
Intestinal Cytosol Receptor Protein by Either Compound.
3 Displacement of l,25-(CH) 2 -[26,27- HJD.. frcm chick intestnal receptor was determined according to the method of Shepard et al (Biochem. J. (1979) 182, 55-69).
The results obtained in these measurements are shown in
Figure 1 and in Tables 3 and 4.
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Table 3
Increase of serum inorganic phosphorus concentration and bone ash in response to either (22E,24R)-l,24-(OH) 2 ~Δ 22 D 3 , - (22E,24S)-l,24-(OH) 2 -Δ 22 -D 3 or l,25-(OH) 2 D 3 .
compound given serum inorganic phosphorus bone ash
None 2.4 + 0.1* a) 35.0 + 4.6 e)
1,25-(QH) 2 D 3 3.3 + 0.4 b) — f) 53.2 + 6.9 ' 4R)-1,24- ■ (0H) 2 - 2.7 + 0.4 c) 35.0 + 6.7
4S)-1,24- ■ (CB) 2 - 2.9 + 0.4 d) 46.5 + 4.2 g)
ϊfeanling male rats were fed a rachitogenic diet for 3 weeks. They were then given 32.5 p mol/day of either compound dissolved in a 0.1 ml mixture of 95% ethanol/propylene glycol (5/95) subcutaneously daily for 7 days. Rats in a control group were given the vehicle. Each group had 6-7 rats.
♦ Standard deviation of tie irean.
Significantly different: a) from b) p<0.001 c) p <0.025 d) p<0.005 e) frcm f) & g) p <0.001 f) frcm g) p<0.05
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Table 4
Increase of intestinal calcium transport and serum calcium concentration in response to either (22E,24R)-l,24-(OH) 2 - Δ 22 -D 3 , (22E,24S)-l,24-(OH) 2 -(OH) 2 -Δ 22 -D 3 or 1, 25- (CiΑ) *P 3 -
cαrrpound given intestinal calcium transport serum calcium (Ca serosal/Ca mucosal) (mg/lCO ml)
Weanling male rats were fed a low calcium-vitamin D deficient diet for 3 weeks. They were then given 32.5 p mol/day of either compound dissolved in a 0.1 ml mixture of 95% ethanol/propylene glycol(5/95) subcutaneously daily for 7 days- Rats in a control group received the vehicle. Each group had 7 rats.
♦ Standard deviation of the mean.
Significantly different: a) from b) & d) p<0.001 a) frcm c) p<0.005 b) frcm c) & d) p<0.001 e) frcm f) p<0.005
Figure 1 demonstrates the ability of the two synthetic l,24-(OH) 2 D 3 iscmers to displace radiolabeled 1,25-(OH) J. frcm the chick intestinal receptor. The results demonstrate that the 24S-iscmer is equally potent as unlabeled l,25-(OH) 2 D 3 in displacing radiolabeled l,25-(OH) 2 D 3 from the receptor. The 24R-isσmer proved to be approximately one-tenth as active as either l,25-(OH) 2 D 3 or the S-isσner. In the stimulation of intestinal calcium transport of rats on a low calcium vitamin D-deficient diet, it is apparent that neither
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iscxrer equalled l,25-(OH) 2 D 3 in this capacity (Table 4). This contrasts with the results obtained with the chick intestinal receptor in which the S-iscmer equalled 1,25-(0H) 2 D 3 in its ability to displace radiolabeled l,25-(OH) 2 D 3 frcm the receptor. Neither iscmer at the doses administered was able to elicit a bone calcium mobilization response as revealed by elevation of serum calcium of rats on a low calcium diet. In contrast, 1,24-(OH)-D- did stimulate this response to a minimal degree at this dosage.
Table 3 illustrates the ability of the isomers to mineralize feπair of rachitic rats. The dosage used 1,25-(0H) 2 D 3 was fully able to mineralize rachitic femur within 7 days. On the other hand, the R-iscmer was unable to mineralize significant amounts of bone at this dosage level, whereas the 24S-cαπpound was less active than 1,25-(0H) 2 D 3 but was clearly effective in this capacity.
The rise in serum inorganic phosphorus concentration in animals on a low phosphorus diet is a critical response for mineralization of bone. It is evident that all three forms of vitamin D stimulated serum inorganic phosphorus levels; however, neither iscmer was equal to l,25-(OH) ._ in this capacity.
The measured biological activity of the compounds of this invention point to their use in physiological situations where vitamin D-lϋe activity is indicated. The l,24S-iscmer can, in fact, be regarded as a very potent 1-hydroxylated form of vitamin D that would find application where preferential effectiveness on intestine and bone mineralization, as opposed to bone mobilization, would appear to be in order.
The compounds of this invention, or combinations thereof with other vitamin D derivatives or other therapeutic agents, can be readily administered as sterile parenteral solutions by injection or intravenously, or by alimentary canal in the form of oral dosages, or trans-der ally, or by suppository. Doses of from about 0.5 micrograms to about 25 micrograms per day of
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the compounds, per se, or in combination with other vitamin D derivatives, the proportions of each of the compounds in the cαnbination being dependent upon the particular disease state being addressed and the degree of bone mineralization and/or bone mobilization desired, are generally effective to practice the present invention. Although the actual amount of the cαrpounds used is not critical, in all cases sufficient of the compound should be used to induce bone mineralization. Amounts in excess of about 25 micrograms per day of the cαrpounds, alone, or in cαribination with a bone mobilization- inducing vitamin D derivative, are generally unnecessary to achieve the desired results and may not be economically sound practice. In practice the higher doses are used where therapeutic treatment of a disease state is the desired end while the lower doses are generally used for prophylactic purposes, it being understood that the specific dosage administered in any given case will be adjusted in accordance with the specific cαrrpounds being administered, the disease to be treated, the condition of the subject and the other relevant medical facts that may modify the activity of the drug or the response of the subject, as is well ncwn by those skilled in the art.
Dosage forms of the ccmpounds can be prepared by ccmbining them with non-toxic pharmaceutically acceptable carriers as is well known in the art. Such carriers may be either solid or liquid such as, for example, corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and propylene glycol. If a solid carrier is used the dosage form of the cαrpounds may be tablets, capsules, powders, troches or lozenges. If a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspension, emulsions or solutions may be the dosage form. The dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, etc. They may also contain other therapeutically valuable substances.
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It is to be understood that the acylated derivatives of ccπpounds 10, 11, 12 and 13 are also to be considered within tie scope of the present invention, certain acylates being susceptible to administration as described for cαrpounds 12 and 13, with conversion of the acylates to the hdrojy derivatives being accomplished in vivo. Thus, the cαrpounds
wherein R. and W~ are hydrogen or hydroxy except that when R- is hydrogen - - is hydroxy and when R. is hydroxy 2 is hydrogen and
R-. and 4 are each hydrogen or acyl having from 1 to 4 carbon atoms. Also, if desired, the cαπpounds of this invention may be obtained in crystalline form by dissolution in a suitable solvent or solvent system, e.g. methanol-ether, methanol- hexane and then removing the solvent(s) by evaporation or other means as is well known.
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