1,25-Dihydroxyergocalciferol
United States Patent De Luca et al.  
 [451 Apr. 29, 1975 l,25-DIHYDROXYERGOCALCIFEROL Inventors: Hector&#39;F. De Luca; Glenville Jones,  
 both of Madison; Heinrich K. Schnoes, Waunakee, all of Wis.  
 Assignee: Wisconsin Alumni Research Foundation, Madison, Wis.  
 Filed: May 24, 1974 Appl. No.: 472,953  
 US. Cl. 260/3972 Int. Cl. c07c 171/08 Field of Search 260/3972 Primary Examiner-Elbert L. Roberts Attorney, Agent,-0r FirmHoward W. Bremer [57] ABSTRACT l,25-Dihydroxyergocalciferol. The compound is characterized by antirachitic activity substantially greater than that exhibited by vitamin D 1 Claim, No Drawings l .25-DIHYDROXYERGOCALCIFEROL The invention described herein was made in the course of work under a grant or award from the Department of Health, Education, and Welfare.  
  This invention relates to a compound characterized by antirachitic activity substantially greater than that exhibitied by vitamin D More specifically, this invention relates to a derivative of vitamin D The character and activity of the vitamins, D and D are well known and well documented. ln recent years various derivatives of vitaminD and D have been found (see, for example, U.S. Pat. Nos. 3,565,924 and 3,585,221) which exhibit antirachitic activity greater than those D-vitamins and which exhibit other and more specific activity in inducing or promoting various functions at certain sites in the animal body.  
  A derivative of vitamin D has now been found which exhibits substantially greater antirachitic activity than the universally used vitamin D This derivative has been identified as l,25-dihydroxyergocalciferol (1,25- dihydroxy-vitamin D ISOLATION Radioactive (3a- H) vitamin D 1.2 Ci/mMole) was prepared by the method of Callow, Kodicek and Thompson, Proc. Roy. Soc. B. 164, 1 (1966) using sodium H) borohydride (7.5 Ci/mMole) (supplied by New England Nuclear, Boston, Massachusetts). (3a- H)25-hydroxyergocalciferol was isolated from rats given (3a- H) vitamin D and unlabeled 25- hydroxyergocalciferol was isolated from pigs given vitamin D according to the procedure of Suda et al., Biochemistry 8, 3515 1969). A mixture oflabeled and unlabeled 25-hydroxyergocalciferols gave a (3al-l)25 hydroxygergocalciferol of specific radioactivity 3.53 mCi/mMole. This material was dissolved in ethanol at a concentration of 500 ng per ul (as determined by ultraviolet absorption spectrophotometry assuming e =l8,20O M cm).  
  (3a-H)2S-Hydroxyergocalciferol (3.53 mCi/m- Mole) was incubated with kidney mitochondria from rachitic chicks in the system used by Ghazarian and DeLuca, Arch. Biochem. Biophys. 160, 63 (1974) to study the production of 1,25-dihydroxycholecalciferol.  
  One hundred flasks, each containing 500 ng (3a- H )25-hydroxyergocalciferol incubated for 30 minutes, were pooled and extracted with methanol-chloroform (ratio 2:1) by the method of Bligh and Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). The total extract was dissolved in 7 ml of a 65:35 mixture of chloroform:- Skellysolve B (straight run aliphatic napthas (essentially normal hexane) derived from petroleum oil marketed by Skelly Oil Company and having a boiling range of 6068). The extract was divided into 7 equal parts each of which was subjected to chromatography on columns (60 cm X 1.0 cm id.) of 16 g of Sephadex LH-20 (Pharmacia Fine Chemicals) swelled and eluted with chloroform:Skellysolve B (ratio 65:35). Fifty fivemilliliter fractions were collected from each column and 50 pl samples from each fraction were taken for counting in a liquid scintillation counter (Nuclear Chicago Model lsocap 300). Fractions 21-32 from each of the 7 columns contained the peak of radioactivity under investigation, whereas fractions 6-1 1 contained unchanged (3a- H)25-hydroxyergocalciferol. Fractions 21-32 inclusive from each of the 7 columns were pooled and evaporated to dryness under a stream of nitrogen. The resulting dry material was redissolved in 0.5 ml chloroform:Skellysolve B (ratio :70) and sub- 5 jected to the following 4 chromatographic steps in succession to remove remaining impurities. i. Hydroxyalkoxypropyl Sephadex* straight phase column chromatography Eluting solvent: ChloroformzSkellysolve B 30270 Room temp. Column size: 52 X 2 cm containing 50 g gel 5 ml fractions: Derivative infractions -115 (peak radioactivity) *Made from Sephadex LH-20 (Pharmacia Fine Chemicals) by the method of Ellingboe, Nystrom, and Sjovall, J. Lipid. Res. 11. 266, 1976.  
 ii. Celite column chromatography. (Suda et al., Biochemistry 9, 2917 (1970).)  
 Column size: 60 X 1 containing 20 g Celite (a diatomaceous silica product marketed by Johns Manville Company).  
 Stationary phase: 300 ml of percent methanol 10 percent water Mobile phase: 750 ml of 80 percent Skellysolve B -20 percent chloroform Temperature: 23 C 5 ml fractions: Derivative in fractions 39-42 (peak radioactivity) iii. Hydroxyalkoxypropyl Sephadex reverse phase column chromatography Eluting solvent: Redistilled anhydrous methanol Room temp.  
 Column size: 51 X 1 cm containing 18 g gel 5 ml fractions: Derivative in fractions 5 and 6 (peak radioactivity) iv. Sephadex LH-20 gel filtration column chromatogra- P y Eluting solvent: Redistilled anhydrous methanol Room temp.  
 Column size: X 1 cm containing 30 g gel 1.7 ml fractions: Derivative in fractions 4349 (peak radioactivity) IDENTIFICATION Ultraviolet absorption spectrophotometry showed that the cis-triene structure of 25- hydroxyergocalciferol remained intact ()\,,,,,,=265 nm Mass spectrometry (performed using an A.E.l. MS-9 mass spectrometer using a direct probe inlet at temperatures of 118-130 above ambient) of the metabolite showed that the molecular ion was at m/e 428. The presence of 3 hydroxyl functions was demonstrated by the formation of a tritrimethylsilyl ether derivative of molecular weight 644. The mass fragment of m/e 131 in the case of the tritrimethylsilyl ether derivative, and the fragments m/e 370 and 352 (370-H O) in the case of the metabolite confirms that the 25-hydroxyl function of the 25-hydroxyergocalciferol remains intact during conversion to this new derivative. Small peaks at m/e 287, 269 (287-H O) and 251 (287-214 0) which arise by loss of the entire side chain (C-17-C-20 cleavage) confirm the lack of additional oxygen substituents on the side chain. The other 2 hydroxyl groups must be located in ring A, since the mass spectrum of the derivative exhibited prominent ions at m/e 152 and 134 152-H O) which can only be interpreted as the oxygen analogs of the characteristic ions at m/e 136 and 118 (136-H O) observed in the spectra of 25- hydroxyergocalciferol and vitamin D (Suda et al., Biochemistry 8, 3515 (1969).) The interpretations are further strengthened by the observation of fragments of m/e 296 (152+2 silyl groups) and 206 (296- l-lOSi(CH.-,) in the mass spectrum of the tritrimethyl silyl ether derivative of the derivative. The presence of the triene structure is also confirmed by the peaks m/e 152 and 134.  
  As this derivative was produced from the incubation of 25-hydroxyergocalciferol. it can be assumed with confidence that the C-3 hydroxyl group is still present. This is supported by the likely possibility that an inversion or loss of the 3B-hydroxyl function would have resulted in loss of the 3a-H during production of the derivative. The foregoing results permit the extra hydroxyl function to be present only in the A ring at C-l, C-2, and C-4. Treatment of the derivative with sodium m-periodate (Suda et al., Biochemistry 9, 4776 (1970)) resulted in no loss of tritium from the molecule and no change in its chromatographic position on Sephadex LH- (run as described earlier under isolation). Under identical conditions an analogous compound (24.25-dihydroxycholecalciferol) with vicinal hydroxyl functions was cleaved by periodate treatment. These observations establish that the extra hydroxyl function of the A ring is not at C2 or G4 but at O1. The structure of this derivative is therefore l,25-dihydroxyergocalciferol.  
 BIOLOGICAL ACTIVITY Calcification Activity score IU/yg Standard vitamin D 4.3 40 l.ZS-dihydroxyvitamin D, 4.2 470 Prepared by Ben Venue Laboratories. Bcdfnrd. Ohio.  
  Having thus described the invention what is claimed is:  
 l. l.25-Dihydroxyergocalciferol.