FR2560597A1 - ANALOGOUS PRODUCTS OF 1A-HYDROXYVITAMIN D2, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS COMPRISING THE SAME - Google Patents

Abstract

THE PRESENT INVENTION PROVIDES NEW SIMILAR PRODUCTS OF VITAMIN D COMPOUNDS WHICH DO NOT HAVE THE METHYL SUBSTITUTE AT POSITION 24 AND WHICH ARE 1-HYDROXY-28-NORVITAMIN D AND 1A, 25-DIHYDROXY-28-NORVITAMINED. THE COMPOUNDS OF THE PRESENT INVENTION ARE CHARACTERIZED BY AN UNEXPECTED STRONG ACTIVITY SIMILAR TO THAT OF VITAMIN D AS WELL AS BY A NEW MODEL OF ACTIVITY AND, AS SUCH, ARE REPLACEMENT PRODUCTS FOR VITAMIN D OR VARIOUS METABOLITES OF VITAMIN D. VITAMIN D KNOWN IN THEIR VARIOUS APPLICATIONS FOR THE TREATMENT OF CALCIUM DISORDERS.

Description

Similar products of lck-hydroxyvitamin D2, their method of

  preparation and pharmaceutical compositions

enclosing them.

  The present invention relates to biologically active 1c <hydroxyvitamin D2 products.

  that manifest unexpected biological properties.

  Because of the well-known and well-established activity of β-hydroxyvitamin D compounds in the regulation of calcium and phosphate homeostasis in animals or humans, they have been shown to be

  interesting in the preparation of natural metabolites

  and the discovery of similar products with useful biological properties. This led to the preparation of a number of gifted compounds

  of a biological activity. Interest in such compounds

  especially because it has been recognized

  now that in addition to their classic function as regular

  of calcium homeostasis, some vitamin D derivatives, in particular lo, 25-dihydroxyvitamin D3 and 1c (, - hydroxyvitamin D3 also influence

  the processes of cellular differentiation and are

  It is possible to prevent the development and proliferation of certain leukemic cells (see U.S. Patent 4,391,802, Proc Natl Acad US 80, 201 (1983);

306, 492-494 (1983)).

  Most of the known vitamin D metabolites and analogues are vitamin D3 series derivatives, that is, they have saturated steroid side chains. However, side chain unsaturated vitamin D compounds are also

  certain hydroxylated derivatives of vita-

  D2 mine such as the 25-hydroxy, 1c, 25-di-

  hydroxy, 24-hydroxy and 24,25-dihydroxy and 1c-hydroxy

  vitamin D2 as well as some 22-trans-dehy-

  related dro not having the substituent

methyl on position 24.

  The present invention provides analog products

  of vitamin D which are characterized by the structure

s ture :.

  wherein R is a hydrogen atom or a hydrogen group

  droxyle. The compound of this invention wherein R is a hydrogen atom can be obtained as an intermediate in the preparation of the compound wherein R is a

hydroxyl group.

  From a structural point of view, the compounds of the present invention are analogous products of hydroxyvitamin D2 compounds which do not contain the 24-methyl substituent, i.e. the compounds which are 1X-hydroxy, respectively. 28-norvitamin D2 and the

  1 ', 25-di-hydroxy-28-norvitamin D2.

  Both the product and the intermediate product

  are highly biologically active and are

  characterized by a new and unexpected type of activity.

  An embodiment of the preparation of

  posed of the present invention is described in the diagram

  In the following description the designation

  compounds by Arabic numerals (for example (1),

  (2), (3), ... (10), (11), (12)) refers to the structures

  numbered on the operative diagram.

  The starting material is the aldehyde having the structure (1), the diene part of which is protected and can be prepared from ergosterol acetate according to the method of Barton et al (J. Chem Soc. 1968

  (1971)). The reaction of aldehyde (1) with 3-methyl-

  1-butylphenylsulfone having the structure indicated below

  below: (CH 3) 2 CHCH 2 CH 2 -SO 2 Ph in an organic solvent and in the presence of a strong organic base gives especially the intermediate hydroxy-sulfone (2). The reduction of the intermediate (2), for example with the amalgam of sodium in an alcoholic solution

  buffered gives the 22,23-trans olefin (22E-olefin) (3).

  The reaction of 22E-olefin (3) with a reagent

  strong hydride reducer (eg LiAlH4) in a soil

  ether gives the intermediate 5,7-diene (4). This inter

  medium is then irradiated with ultraviolet light

  in an organic solvent to obtain the corresponding previtamin D derivative which can be isolated and heated in an organic solvent at a temperature from room temperature to the reflux temperature to isomerize the previtamin D chromophore to a vitamin D chromophore, giving so the compound

  22E-dehydrovitamin D3 having the structure (5). The composition

  Se (5) is a known vitamin D analog product which has been previously prepared by a less

  Convenient (J.Gen.Chem.USSR 48 (4), 828 (1978)).

  Intermediate (5) can then be hydroxylated on carbon 1, according to the general method of Deluca and

  - coll. (U.S. Patent Nos. 4,195,027 and 4,260,549).

  These reaction stages typically involve

  only the reaction of the compound (5) with toluenesulfonyl chloride in a conventional manner to obtain the corresponding derivative 3 (-tosylate which is directly subjected to the solvolysis in the buffered methanol to obtain the intermediate 3,5-cyclovitamin D (6), the

  reaction with different lower alkanols, for example

  wherein 1 to 4 carbon atoms will give the corresponding lower alkoxy compound. By treating the latter with selenium dioxide and hydroperoxide

  tert.-butylon obtains after purification by chromato-

  the corresponding 1-hydroxylated product comprising

  mainly the compound 1O-hydroxycyclovitamin D (7).

  A small amount of the corresponding 1-hydroxy epimer may also be present in the product but although the separation of the epimers is possible by chroma-

  it is not necessary at this stage. The heater

  This intermediate 1-hydroxycyclovitamin D in, say, glacial acetic acid at 40 ° -60 ° C. then gives a mixture of the 3-acetylated solvolysis products from which the following can be isolated by chromatography:

  5,6-cis and 5,6-trans (8) and (9) compounds, respectively.

  If the 1-hydroxycyclovitamin D obtained which has been subjected to the solvolysconcrete a little 1/3 -hydroxy epimer, the solvolysis mixture will also contain the p1-hydroxy epimers corresponding to the compounds (8) or (9) and, if it is desires, these can also be isolated by chromatrography. Conventional hydrolysis by a base of function

  acetate in the compound (8) gives the diol having the

  This (10) diol (10) can then be subjected to in vitro enzymatic hydroxylation at the carbon level, using a liver homogenizer prepared from vitamin D deficient rats (as described in US Pat. 4,307,025) to obtain, after separation by chromatography of the product mixture, the desired product 1Co <25Dihydroxyl (12).

in the pure state.

  In the following detailed description of the

  synthesis, the physicochemical results have been

  obtained by using the methods and appliances mentioned

  below. High performance liquid chromatography (HPLC) is performed on a model

  LP / GPC "204 of Waters Associates using a colon

  of "Zorbax-Sil" (trade mark of Dupont (

  6.2mm x 25cm; flow rate 4 ml / min; 105 bars). The chroma-

  Column tography is performed on "Silica Gel 60"

  trademark of Merckmaille ASTM 70-230 (212-63 mm).

  Preparative thin layer chromatography (TLC) is carried out on "Silica 60 PF-254" (registered trademark)

  (20 x 20 cm plates, 1 mm silica gel). The irradiation

  This is done using a "Hanovia 608A36" mercury vapor arc lamp (trademark) with a "Vycor" (trademark) filter. All reactions are preferably carried out under an inert atmosphere

(eg argon)

  3-methyl-1-butylphenylsulfone (Isopentylphenyl sulfone). PhSO2Na (1.97 g, 12 mmol) was added to a stirred solution of 3-methyl-1-bromobutane (1.51 g, 1.2 mL, mmol) in DMF (20 mL) at 75 ° C. is heated at 75 C for 5 hours, then cooled, poured

  in water and extracted with benzene. The organic layer

  washed with 5% HCl, 5% NaHCO 3 and water, dried over Na 2 SO 4 and evaporated. The sulfone obtained as an oily product (1.69 g, 80%) is almost pure and is used without further purification; 0.88 NMR (6H, d, J = 6.5 Hz, 2xCH3), 1.61 (3H, m, -CH2-CH), 3.08 (2H, m, SO2-CH2-), 7.50. -7.95 (5H, m, Ar-H); IR: 1300 (broad), 1147, 1088, 745, 662, 564, 539 cm -1; Mass spectrum, m / e 212 (M +, 3), 143 (92), 77 (57), 71 (73), 70

(73), 55 (42), 43 (100), 41 (47). -

  (22E) -5α, 8α - (4-phenyl-1,2-urazolo) -cholestate

6,22-diene-3 / -ol (3).

  N-Butyllithium (1.7 M solution in hexane, 4.12 mL, 7 mmol) was added to a stirred solution of di-isopropylamine (707 mg, 1 mL, 7 mmol) in dry THF (14 mmol). ml) and the mixture is stirred for 15 minutes at room temperature. The sulfone prepared above (1.50 g, 7.07 mmol) in dry THF (11 mL) is added dropwise over 10 minutes. The solution is stirred at room temperature for a further min and then cooled to 0 ° C. and the aldehyde (1) (545 mg, 1 mmol) in dry THF (7 ml) is added. Stirring is continued for 2 hours at 0 ° C. and the solution is

  slowly warmed to room temperature (30 min).

  The mixture (containing the hydroxy-sulfone (2)) is poured into the saturated Na 2 HPO 4 solution in methanol (200 ml), sodium amalgam (5.65%, 10 g) is added and the reaction mixture is stirred. at 4 C for 17 hours. The precipitated mercury is filtered off and after concentrating the reaction mixture to about 5 ml, diluted with water and extracted with methylene chloride. The organic extract is washed with water, dried (Na 2 SO 4), concentrated in vacuo and the oily residue is treated by silica gel column chromatography. The excess sulfone reagent is eluted with benzene-ether (7: 3). Elution with benzene-ether (6: 4) gives the product

  pure addition (3) (375 mg, 67%) as a foam.

  RM4N0.81 (3H, s, 18-H3), 0.86 (6Had, J = 6.7 Hz, 26-H3 and

  27-H3), 0.97 (3H, s, 19-H3), 1.03 (3H, d, J = 6.8 Hz, 21-

  H3), 3.16 (1H, dd, J1 = 4.4 Hz, J2 = 14Hz, 9-H), 4.44 (1H, m, 3-H), 5.25 (2H, broad m, 22 -H and 23-H), 6.22 and 6.39 (2H, AB q, J = 8.5 Hz, 6-H and 7-H), 7.40 (5H, broad m, Ar-H) ; IR: 3444, 1754, 1701, 1599, 1402, 969, 577 cm -1; mass spectrum, m / e 557 (M +, 1%), 382 (70), 349 (51),

  253 (28), 251 (45), 119 (PhNC0, 83), 55 (100).

  (22E) -Cholesta-5,7,22-trien-3-ol, (4).

  The adduct (3) (330 mg, 0.6 mmol) and lithium aluminum hydride (700 mg) in

  Dry THF (40 mL) is refluxed for 18 hours.

  Excess reagent is broken down with a few drops

  of water. Anhydrous Na2SO4 is added and the organic phase

  that is decanted and evaporated to obtain a crystalline residue

  tallin which is purified on a column of silica gel. The chosen one-

  with benzene-ether (94: 6) gives pure diene (4) (180 mg, 80%) which is crystallized from methanol: mp 119.5-122.5 ° C; /: 7-4 = 118 (c = 1.2, CHCl3); NMR S 0.63 (3H, s, 18-H3), 0.87 (6H, d, J = 6.7 Hz, 26-H3 and 27-H3), 0.95 (3H, s, 19-H3). ), 1.03 (3H, d, J = 6.8 Hz, 21-H3), 3.64 (1H, m, 3-H), 5.25 (2H, broad m, 22-H and 23- H), 5.38 and 5.57 (2H, AB q, J = 6 Hz, 7-H and 6-H); UV / max 281nm; IR; 3436, 1461, 1382, 136, 1062, 1036, 968 cm -1; mass spectrum m / e 382 (M +, 100), 349 (M-H 2 O -Me, 71), 323 (34), 271 (M-side-chain,

  16), 253 (M - side chain -H 2 O, 32).

  (5Z, 7E, 22E) -9,10-Secocholesta-5,7,10 (19), 22-

tetraen-3-ol (5).

  A solution of the compound (4) (100 mg, 0.26 mmol) in a mixture of ether (120 ml) and benzene (30 ml)

  is degassed with argon for 40 minutes. The solution

  tion is irradiated at 0 ° C. for 13 minutes in a

  quartz immersion equipped with a UV lamp and a filter. The solvent is removed under reduced pressure and the residue is separated by HPLC using a 1% solution of 2-propanol in hexane as eluent to give the pure avitamin D derivative (40.4 mg,

  %) which is collected at 24 ml. NMR? 0.72 (3H, s, 18-

  H3), 0.87 (6H, d, J = 6.7 Hz, 26-H3 and 27-H3), 1.04 (3H, d, J = 6.8 Hz, 21-H3), 1.65 (3H, s, 19-H3), 3.91 (1H, m, 3-H), 28 (2H, broad m, 22-H and 23-H), 5.50 (1H, m, 11- H), 69 and 5.96 (2H, AB q, J = 12.5 Hz, 7-H and 6-H); UVA max 260.5 nm, X min 234 nm. Avitamine (40 mg, 0.1 mmol) is refluxed for 3 hours in

  100 ml of ethanol. After expelling the solvent, the

  The mixture obtained is separated by HPLC (elution with a 1% solution of 2-propanol in hexane). The yield of vitamin (5) (collected at 34 ml) is 30.8 mg (77%); mp (hexane) 99-101 ° C; NMR 0.56 (3H, s, 18-H3), 0.88 6H, d, J = 6.7 Hz, 26-H3 and 27-H3), 1.02 (3H, d, J = 6.6). Hz, 21-H3), 3.96 (1H, m, 3-H), 4.82 and 5.05 (2H, each narrow m, 19-H2), 5.27 (2H, broad m, 22- H and 23-H), 6.03 and 6.24 (2H, ABq J = 11.4 Hz, 7-H and 6-H); UVA max 265 nm, min 228 nm; IR: 3420,1458,1441,1378,1366,1050, 970,943,891, 862 cm1; mass spectrum, m / e (M +, 22), 349 (M'-H 2 O -Me, 4), 271 (M-side chain, 8), 253

  (M-side chain-H20, 13), 136 (100), 118 (80).

  Z, 7E, 22E) -3e-Acetoxy-9,10-secocholesta-5,7,10

(19), 22-tetraen-1CX-ol (8).

  P-Toluenesulfonyl chloride freshly

  recrystallized (50 mg, 0.26 mmol) is added to a solution

  vitamin (5) (30 mg, 0.08 mmol) in 300 μl of

  dry pyridine. After 30 hours at 4 ° C., the reaction mixture

  is poured into a saturated ice-NaHCO3 mixture

  while waving. The mixture is stirred for 15 minutes.

  and extracted with benzene. The organic extract is washed with a saturated solution of NaHCO 3, a saturated solution of copper sulphate and dried water (Na 2 SO 4)

  * and concentrated under vacuum to obtain the derivative 35-

  oily tosyl.The crude tosylate is treated with NaHCO3 (150 mg) in 10 ml of absolute methanol and the mixture

  is stirred for 8.5 hours at 55 C. After cooling

  concentration and concentration to about 2 ml the mixture is diluted with 80 ml of benzene, washed with water, dried

  (Na2SO4) and evaporated under reduced pressure.

  The resulting oily 3,5-cyclovitamin D (6) analog product is sufficiently pure to be used in the following oxidation stage without having

  been further purified. To an excited restless suspension

  SeO 2 (4 mg, 0.036 mmol) in 5 ml of dry CH 2 Cl 2 was added, tert-butyl hydroperoxide (13.2 μl, 0.094 mmol) was added. After 30 min, 40 ul of dry pyridine is added and the mixture is shaken.

  for an additional 25 min at room temperature

  The crude 3,5-cyclovitamin (6) product in 4.5 ml of CH 2 Cl 2 is then added and the reaction is allowed to proceed at 0 ° C. for 15 minutes. We then leave

  the mixture warm up slowly (30 min) at the temperature

  ambient temperature. The mixture is transferred to a decanter and shaken with 30 ml of 10% NaOH. 150 ml of ether are added and the separated organic phase is washed with 10% NaOH, water and dried over Na 2 SO 4. Concentration to dryness in vacuo gives a yellow oily residue which is purified on TLC silica gel plates developed in a 7: 3 mixture of hexane and ethyl acetate (Rf 0.35) to give 1 hydroxycyclovitamin (14.4 mg, 45%): NMR δ 0.55 (3H, s, 18-H3), 0.64 (1H, m, 3-H), 0.88 (6H, d, J = 6); , 9 Hz, 26-H3 and 27-H3), 1.03 (3H, d, J = 6.9 Hz, 21-H3), 3.26 (3H, s, -OCH3), 4.2 (2H), , m, 1-H and 6-H), 4.95 (1H, d, J = 9.3 Hz, 7-H), 5.1-5.4 (4H, broad m, 19-H2, 22 -H and 23-H); mass spectrum, m / e 412 (M, 27),

  380 (m -MeOH, 46), 339 (22), 269 (M-side-chain

  MeOH, 29), 245 (18), 135 (100). The product above

  mainly includes the like product of the -

  β-hydroxycyclovitamin D having the structure (7) and a small amount of the corresponding 125-epimer. A solution of 12 mg of this 1hydroxycyclovitamin D in 0.5 ml of glacial acetic acid is heated to

   C for 15 minutes. The mixture is poured with

  deposit in a saturated ice / NaHCO3 mixture and extracted with benzene and ether. Combined extracts

  are washed with water (Na2SO4) and evaporated.

  The resulting product mixture was subjected to HPLC (1.5% propanol in hexane as eluent) to obtain 4.9 mg of the compound (8) (elution at 42 ml) and

  3.1 mg of the compound (9) (elution at 50 ml).

1560S97

  Compound (8): 0.56 NMR (3H, s, 18-H3), 0.88 (6H, d, J = 7.0Hz, 26-H3 and 27H3), 1.02 (3H, d, J) = 6.8 Hz, 21-H3), 2.04 (3H, s, -OCOCH3), 4.41 (1H, m, 1H), 5.02 (1H, narrow m, 19-H), 5, 1-5.4 (4H, broad m, 3-, 19-, 22- and 23H), 6.03 and 6.35 (2H, AB q, J = 11.4 Hz, 7-H and 6-H) ); UVAmax2 264 nm, min 227.5 nm; mass spectrum m / e 440 (M, 15), 380 (M-HOAc, 84), 362 (M HOAc-H20, 9), 269 (M + side-chain HOAc, 40), 251 (15), 135 (100) ,,

134 (94).

  Compound (9): NMR 0.57 (3H, s, 18-H3), 0.89 (6H, d, J = 7.0Hz, 26-H3 and 27H3), 1.03 (3H, d, J) = 6.8 Hz, 21-H3), 2.04 (3H, s, -OCOCH3), 4.49 (1H, m, 1H), 5.00 and 5.14 (2H, each narrow m, 19- H2), 5.25 (3H, broad m, 3-, 22- and 23-H), 5.81 and 6.58 (2H, ABq, J = 12.0 Hz, 7-H and 6-H) ; UV ax 269.5 nm; min 228 nm; mass spectrum,

  m / e 440 (M, 6), 380 (47), 269 (15), 135 (100), 134 (62).

  A small amount (0.87 mg of the 1-hydroxy epimer corresponding to the compound (8)) is also obtained from the solvolysis of the product mixture, characterized by the following results: MN S0.55 (3H, s, 18- H3), 0.87 (6H, d, J = 6.9 Hz, 26-H3 and 27-H3), 1.01 (3H, d, J = 6.9 Hz, 21-H3), 2.06 (3H, s, -OCOCH3), 4.17 (1H, m, 1-H), 4.99 (2H, m, 3-H and 19-H), 5.1-5.4

  (3H, broad m, 19-, 22- and 23-H), 6.00 and 6.38 (2H, AB q.

  J = 11.3 Hz, 7-H and 6-H); UVW max 262.5 nm, t min 227 nm; mass spectrum, m / e 440 (M, 27), 380 (78), 362 (12),

  269 (28), 251 (20), 135 (100), 134 (78).

  Hydrolysis of group 3 (-acetoxy in compounds

(8) and (9).

  A solution of the 3α-acetoxyvitamin derivative (8) (0.76 mg) in 0.1 ml of ethanol is treated with% KOH in methanol (0.8 ml) and the mixture is heated for 1 hour at 50 ° C. After the usual work and the final purification by HPLC (8% solution of 2-propanol in hexane as eluent) the lo (", 3-diol having the structure (10) is obtained in a yield of 84%. : PRNb0.56 (3H, s, 18-H3), 0.87 (6H, d, J = 7.0 Hz, 26-H3 and 27-H3), 1.02 (3H, d, J = 6, 8 Hz, 21-H 3), 4.22 (1H, m, 3-H), 4.42 (1Hm, 1-H), 5.00 (1H, narrow m, 19-H), 5.1 5.4 (3H, broad m, 19-, 22-, and 23-H), 6.01 and 6.38 (2H, AB q, J = 11.4 Hz, 7-H and 6-H); UV / Aax 264.5 nm, + min 227.5 nm, ax + M ,, in mass spectrum, m / e 398 (M, 21), 380 (M -H 2 O, 9), 2.87 (m.p. M side chain, 6), 269 (M + side chain-H 2 O, 8) 251 (5), 152 (38), 134 (100) Compound (10) is eluted

  to 40 ml in the HPLC system above.

  An analogous treatment of the acetoxy derivative (9), given after purification by HPLC the 5,6-trans-1 "o, 315-diol having structure (11) with a yield of 72%: RIMN 0.58 (3H, s , 18-H3), 0.87 (6H, d, J = 7.0 Hz, 26-H3 and 27-H3), 1.03 (3H, d, J = 6.8 Hz, 21-H3) , 4.24 (1H, m, 3-H), 4.49 (1H, m, 1-H), 4.97 and 5.13 (2H, each narrow m, 19-H2), 5.25 ( 2H, broad m, 22-H and 23-H), 5.88 and 6.58 (2H, AB q, J = 11.5 Hz, 7-H and 6-H), UVAmax 273 nm, λ min 229.5 nm, mass spectrum, m / e 398 (M *, 21), 380 (5), 287 (6), 269 (5), 251 (4), 152 (33), 134 (100). The

compound (11) is eluted at 38 mi.

  Hydroxylation at the position of the compound (10) The 5,6-cis-10, 3-diol having the structure (10) as

  obtained above is then hydroxylated on the posi-

  by the following method: young male weaned rats are subjected to a diet deficient in vitamin D

  and poor in calcium as described by Suda et al.

  J. Nutr. 100, 1049 (1970) for two weeks. They

  are killed by decapitation and their liver is removed.

  A homogenization product at 20% (w / v) is prepared in a 0.25 M sucrose solution cooled with ice. The incubation is carried out in 10 ml of an incubation medium, contained in a 125 ml Erlenmayer flask containing an aliquot of liver homogenate representing 1 g of tissue, 0.125 M sucrose, 50 EMI. phosphate buffer (pH 7.4), 22.4 μl glucose-6-phosphate, 20 μl PTA, mM nicotinamide, 25 ml. of succinate, 0.4 mM

  NADP, 5 μMgCl 2, 0.1 M KCl and 0.5 glucose units.

  6-phosphate dehydrogenase. The reaction is initiated by adding 400 μg of compound (10) dissolved in 100 μl of 95% ethanol. The mixture is incubated at 37 ° C. with stirring at a rate of 80 oscillations / minute for 3 hours.

  hours. The reaction is stopped by adding 20 ml of

  thanol and 10 ml of dichloromethane. After adding another 10 ml of dichloromethane, the organic phase is collected while the aqueous phase is extracted from

  again with 10 ml of dichloromethane. The organizational phases

  from a total of three extractions are combined

  and evaporated on a rotary evaporator. The residue contains

  The desired product was dissolved in 1 ml of CHCl 3: hexane (65:35) and applied on a column (0.7 cm x 14 cm) packed with balanced and eluted "Sephadex LH-20" (Trade Mark). with the same solvent. The first 10 ml are discarded while the next 40 ml are collected

  and evaporated. The residue is then dissolved in a solution

  8% 2-propanol in hexane and subjected to chromium

  high performance liquid chromatography (Model

  LC / GPC 204 HPLC, Waters Associates, Medford, MA) using

  a column (4.6 mm x 25 cm, filled with "Zorbax-Sil"

  (Dupont, Inc., Wilmington, DE) operating under a

  70 bar with a flow rate of 2 ml / min. The hydroxylated product at the desired position is eluted at 42 ml. The product is further purified by high performance liquid chromatography using a reversed phase column (4.6 mm × 25 cm) packed with "Richrosorb Rp-18" (trademark of E. Mercis, Darmstadt, Germany). West) operating at a pressure of 84 bar and with a flow rate of 2 ml / min. The column is eluted with 22% H 2 O solution in methanol and eluted at 50 ml. The product is further purified by HPLC

  using "Zorbax-Sil" 'and under the conditions described above.

  above. The resulting product is characterized by the

  following results: UV absorption (95% ethanol), max 265 nm, min 228 nm; mass spectrum, m / z 414

max min - ±

  (Molar ion M), 396 (M -H 2 O), 378 (M 2 × H 2 O), 287 (M-side chain), 269 (M-side-chain-H 2 O), 251 (M

  lateral chain-2xH 2 O), 152 (cycle A, cleavage of the link

  C 7/8), 134 (152-H 2 O) and 59 ((CH 3) 2 C = OO resulting from

cut-off of the C24 / 25 link).

  The above results especially the absorp-

  characteristic in the ultraviolet and the dominant peaks

  The mass spectra, 152 and 134 and 59, show that the product is the hydroxylated compound at the desired position represented by structure (12).

  If desired, the compounds of the present invention

  They can be obtained in the crystalline state by crystallisation in suitable solvents, for example hexane, ethers, alcohols or mixtures of these solvents.

  as the specialist can see.

  The biological efficacy of dehydrovitamin D of the present invention is determined by in vivo tests in rats and by comparison with 10-hydroxyvitamin D compounds having known efficacy. We

  both the product (12) and the compound (10)

  key intermediary and we find that they are highly active.

  Biological activity of the compound (10). The test is carried out as follows: young male weaned rats are purchased from Holtzman Co., Madison, WI and supplied with water at will and put on a diet deficient in vitamin D and low in calcium and therefore in phosphorus as described by Suda and Cdl (J. Nutr.1010, 1049 (1970)) for three weeks. The rats are then divided into groups of 6 each and administered 650 mg of either compound (10) or 1E-hydroxyvitamin D3 (1C (-OH-D3)).

  dissolved in 0.05 ml of 95% ethanol intravenously

  18 hours before killing them. To the rats of the control group an ethanol vehicle is administered in the same way. The rats are killed by decapitation and their blood is collected. Serum

  obtained by centrifugation of the blood is diluted with a solution

  0.1% lanthanum chloride (1:20) and the concentration of

  Calcium in serum is measured with an atomic absorption spectrophotometer. The results are shown in Table I below:

Table I

  Increased serum calcium concentration in response to a single dose of 650 pmol of the compound (10) or 10-OH-D3, given 18 hours before killing the rats Compound given Serum calcium concentration (mg / 100ml) + standard deviation ethanol 3.2 + 0.1 a) compound (10) 4.5 + 0.4 b) 1I- 0H-D3 4.7 + 0.5 b)

  (b) is significantly different from (a) p0,001.

  Biological activity of the compound (12). The test is

  performed as follows: young male rats se-

  are fed with a vitamin D

  and low in calcium for 3 weeks as described above.

  above. They are then divided into groups of 5 rats each

  cun. The rats in a control group receive 0.05 ml of 95% ethanol intrajugularly while the rats in the test groups receive 325 pmol of the compound (12) or 1,1,25-dihydroxyvitamin D3 (1c, 25). - (OH) 2D3) dissolved in 0.05 ml of 95% ethanol. 18 hours later they are killed by decapitation and their blood is collected, the concentration of calcium in the serum is determined

  with an atomic spectrophotometer as described above.

  The results are shown in Table II below:

Table II

  Increased serum calcium concentration in response to a single dose of 325 pmol of the compound (12) or 1e, 25- (OH) 2D3 given 18 h before killing the rats Compound given Serum calcium concentration (mg / ml) 100ml) + standard deviation ethanol 4.2 + 0.1 a) compound (12) 5.0 + 0.4 b) 51, 25- (OH) 2D3 5.4 + 0.4.b) i5 b) is The above results show that both the compound (10) and the final product (12) of this

  invention are highly active by causing an increase in

  the amount of calcium in the serum of vitamin D-deficient rats.

  equivalent to saturated side chain compounds

  These are well-known compounds, such as 1y-OH-D3 and 1X, 25- (OH) 2D3, the high efficacy and usefulness of which are well known in many biographical reports.

  patent, for example U.S. Patent 4,225,596. This is particularly remarkable and unexpected.

  tense is the activity of the higher compound (10) in the middle

  Néralisation of the bones of an animal (chicken) which makes establish

  a distinction with the physiological use of compounds

  vitamin D2. This is amply illustrated by the

results below.

  Process One day old White Leghorn male chickens are provided by Northern Hatcheries (Beaver Dam, WI). They are subjected to a low vitamin D soy protein diet described by Omdahl et al. (Biochemistry 10, 2935-2940, 1971) for 3 weeks during which they remain deficient in vitamin D.

  They are then divided into groups of 6 chickens each.

  One group receives an oily vehicle from Wesson through the beak; the other groups receive the indicated compounds (see Table III) dissolved in the same amount of Wesson oil each day for 7 days. Twenty four hours after the last dose all chickens are killed by

  cervical dislocation. Their shins are removed and

  collected from the soft tissues and adherent connectives, and extracted for 24 hours in the alcohol and then for 24 hours in diethyl ether using a Soxhlet extractor. The bones are then dried at 37.8 C until

  constant weight and weighed. They are then incinerated e.

  650 C for 24 hours in a foil oven and the percentage of ash for each tibia is calculated. The results are indicated

in Table III below.

Table III

  Root bone bone mineralization Composed Amount Number of animals Ash% (pmol / d / 7 days)

-D 0 6 32.0 + 1.0

1.25- (0H) 2D3 130 6 41.0 + 3.4 O

1I-0H-D3 130 6 42.0 + 3.2

  lo-OH-D2 1300 6 38.0 + 4.0 x Compound (10) 130 6 38.0 + 3.9 x These values do not differ much from each other The results show that 130 pmoles is 1.25- ( OH) 2D3 either l -OH-D3 or the new compound (10) of the present invention are also effective in increasing the mineral content of the rachitic tibias. However, to obtain the same degree of efficiency, 1300 pmoles of 1I-OH-D2 are required. This corresponds to the previous results which show that a dose of 110-OH-D210 times greater than 1 <-OH-D3 is required to obtain the same shin mineralization.

  of rickets. These results show in a way that

  that although compound (10) is a product analogous to 10H-D2, it exhibits a superior and unexpected activity in bone mineralization of an animal that is known to distinguish between vitamin compounds

  D2 in physiological use. This is a characteristic

* Unexpected value of these new analogous and useful products. Since the activity in

  the unexpected vivo of the compound (10) is unmistakably

  after hydroxylation to the compound 1c, 25-dihydroxylated

  corresponding (compound (12)) in vivo, this compound, i.e.

  say, compound (12), which is the methylmethyl product at 24 of the known 10o, 25-dihydroxyvitamin D3, must function to provide mineralization of chicken bones in a manner equivalent to that shown by

  1x <, 25-dihydroxyvitamin D3; thus this new product

  Vitamin D2 will be fully active for chickens. This high efficacy of compounds (10) and (12)

  in an animal that distinguishes

  D2 mine is a new and particular feature

of these substances.

  Because of their marked biological activity,

  compounds of the present invention will be readily

  as substitutes for various vitamin D metabolites known to be used in therapy or prophylaxis

  disorders of calcium metabolism such as rachis

  hypoparathyroidism, osteodystrophy, osteomalacia or osteoporosis in humans or diseases giving a reported calcium deficiency (eg milk fever) in animals. Likewise, these compounds can be used for the treatment of certain virulent diseases such as human leukemia. Given the marked and unexpected activity of the compounds of the present invention for causing mineralization of bones in birds (see Table III) these compounds will be particularly useful in the prevention or control of

  treatment of the states causing an imbalance of the cal-

  in poultry (eg slimness of

  egg keel, weakness of poultry paws) o all known vitamin D2

very weak activity.

  For therapeutic applications, the compounds

  may be administered by an administrative route.

  any conventional form and in any form appropriate to the chosen method of administration. The

  may be formulated with a pharmaceutical vehicle

  acceptable and harmless method in the form, for example,

  tablet pills, capsules or suppositories

  or in the form of solutions, emulsions, dis-

  persions or suspensions in harmless solvents or oils; these formulations may also contain

  other therapeutically active ingredients and

  which are suitable for specific applications.

  For applications in humans, the compounds are advantageously administered in amounts ranging from 0.5 to 1 μg / day, the specific dosage being adjusted according to the specific compound administered, the disease to be treated,

  the state and response of the subject as is well known to the specialist

listing.

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Claims (9)

  1. Compound characterized by having the formula: R -   Wherein R is a hydrogen atom or a hydroxyl group. ] 5 2. Compound according to claim 1, characterized   in that R is a hydrogen atom.   3. Compound according to claim 2, characterized   in that it is in crystalline form.   4. Compound according to claim 2, characterized   in that R is a hydroxyl group.   5. Compound according to claim 4, characterized   in that it is in crystalline form.   6. Process for preparing a compound according to   any of claims 1 to 5, characterized   in that it consists in hydroxylating the position of the compound of formula:   in a known manner and, optionally, to hydroxylate the   sition of the resulting 1c-hydroxy compound in a known manner. 7. Pharmaceutical composition characterized by   the fact that it comprises a compound according to any one   claims 1 to 5, and a pharmaceutical excipient acceptable.   8. Composition according to claim 7, characterized   rised by the fact that it includes the compound of vending 2.   9. A composition according to claim 7, characterized in that it comprises the compound of claim 4.