2-alkyl-19-nor-vitamin d compounds

(74) Agent

Wisconsin Alumni Research Foundation, PO Box 7365, WI53707-7365 MADISON, US Hector F. Deluca, Deerfield, WI, US

Rafal R. Sicinski, Warsaw, PL

Oslo Patentkontor AS, PO Box 7007 Majorstua, 0306 OSLO, NO

<td> (54)</td><td>Designation</td><td>2-Alkyl-19-nor-vitamin D compounds and their use in the preparation of pharmaceutical compositions and such compositions</td>

<td> (56)</td><td>Cited publications</td><td>EP 619 306 Pg</td>

<td> (57)</td><td>Summary</td><td></td>

This invention relates to a new class of vitamin D-related compounds, namely the 2-alkyl-19-nor-vitamin D derivatives, as well as a general method for their chemical synthesis. The compounds have formula (I): where Yi and Y₂, which may be the same or different, each selected from the group consisting of hydrogen and a hydroxy protecting group, R e is selected from the group consisting of alkyl, hydroxyalkyl and fluoroalkyl, and wherein the group R represents any of the typical side chains known for Vitamin D. compounds These 2

<img file="NO321925B1_D0001.tif" />

Substituted compounds are characterized by low intestinal calcium transport activity and high bone calcium mobilizing activity, providing new therapeutic agents for the treatment of diseases where bone formation is desirable, especially osteoporosis due to low bone mass formation. These compounds also exert significant activity in stopping the proliferation of undifferentiated cells and inducing their differentiation into monocytes, suggesting that they may be used as anticancer agents and to treat diseases such as psoriasis.

The invention relates to vitamin D compounds as claimed in claim 1, and more particularly to vitamin D derivatives substituted in the carbon 2 position.

The natural hormone, la, 25-dihydroxyvitamin D₃ and its analogue in the ergosterol series, that is, la, 25-dihydroxyvitamin D₂, ar e known to be very potent regulators of calcium homeostasis in animals and humans, and very recently their activity in cellular differentiation has been demonstrated, Ostrem et al., Proc. Natl. Acad. Sci., USA, 84, 2610 (1987). Many structural analogues of these metabolites have been prepared and tested, including 1α-hydroxyvitamin D₃, 1a-hydroxyvitamin D₂, various side-chain homologous vitamins and fluorinated analogs. Some of these compounds exert interesting separation of activities regarding cell differentiation and calcium regulation. This difference in activity can be useful in the treatment of many diseases, such as renal osteodystrophy, vitamin D-resistant rickets, osteoporosis, psoriasis and certain malignant diseases.

Recently, a new class of vitamin D analogues has been discovered, that is, the so-called 19-nor-vitamin D compounds, which are characterized by replacement of the exocyclic methylene group in the A-ring (carbon 19), which is typical of vitamin D system, with two hydrogen atoms. Biological testing of such 19-nor analogs (e.g., 1α, 25-dihydroxy19-nor vitamin D₃) showed a high potency selective activity profile to induce cellular differentiation, and very low calcium mobilizing activity. Thus, these compounds are potentially useful as therapeutic agents for the treatment of malignant diseases or the treatment of various skin diseases. Two different synthesis methods of such 19-nor vitamin D analogues have been described (Perlman et al., Tetrahedron Lett., 31, 1823 (1990); Perlman et al., Tetrahedron Lett., 32, 7663 (1991); and DeLuca et al., U.S. Patent No. 5,086,191).

In U.S. Patent No. 4,666,634, 2β-hydroxy and alkoxy analogs (e.g., ED-71) are of 1α, 25-dihydroxyvitamin D₃ have been described and investigated by the Chugai Group as potential drugs for osteoporosis and as anti-tumor agents. See also Okano et al., Biochem. Biophys. Res. Commun., 163, 1444 (1989). Other 2-substituted (with hydroxyalkyl, e.g. ED-120, and fluoroalkyl groups) A-ring analogues of 1α, 25-dihydroxyvitamin D₃ have also been prepared and tested (Miyamoto et al., Chem. Phann. Bull., 41, 1111 (1993); Nishii et al., Osteoporosis Int., Suppl. 1, 190 (1993); Posner et al., J Org. Chem., 59, 7855 (1994), and J. Org. Chem., 60, 4617 (1995)).

Recently, 2-substituted analogs of Ia, 25-dihydroxy-19nor vitamin D₃ has also been synthesized, that is, compounds substituted at the 2-position with hydroxy or alkoxy groups (DeLuca et al., U.S. Patent No. 5,536,713), which exhibit interesting and selective activity profiles. All of these studies indicate that the binding sites of the vitamin D receptors may incorporate different substituents at C-2 in the synthesized vitamin D analogues.

In a continuous endeavor to utilize the 19-nor class of pharmacologically important vitamin D compounds, their analogs, which are characterized by the presence of an alkyl substituent (especially methyl) at carbon 2 (C-2), nor vitamin D compounds, and especially 2-methyl19-nor vitamin D compounds, have now been synthesized and tested. Such vitamin D analogues appear to be interesting targets, because the relatively small alkyl group (especially methyl) at C-2 should not interfere with the vitamin D receptor. On the other hand, it is obvious that a change in the conformation of the cyclohexanediol ring A for these new analogues can be expected .

Summary of the Invention

One class of 1-hydroxylated vitamin D compounds that has not been known so far are the 19-nor-vitamin D analogues which have an alkyl group (especially methyl) at the 2-position, that is, 2-alkyl-19-nor. vitamin D compounds, especially 2-methyl-19-nor-vitamin D compounds.

Structurally, these new analogues are characterized by the general formula I shown below:

<img file="NO321925B1_D0002.tif" />

where Yi and Y₂ is hydrogen, R₆ is methyl, and wherein the group R represents one of the substituent groups shown in claim 1 and wherein the stereochemical center at carbon 20 may have R or S configuration, wherein the double bond may have cis or trans geometry.

Specific, important examples of side chains of the group R having a natural 20R configuration are the structures represented by formulas (a), (b), (c), (d) and (e) below, that is, the side chain as present in Figs. hydroxyvitamin D₃ (A); vitamin D₃ (B); 25-hydroxyvitamin D₂ (C); vitamin D₂ (D); and the C-24 epimer of 25-hydroxyvitamin D₂ (e):

<img file="NO321925B1_D0003.tif" />

ΑΛΛΓ (a)

<img file="NO321925B1_D0004.tif" />

(b) (c) (&lt;Π (e)

The above new compounds exert a desired, and very advantageous, pattern of biological activity. These compounds are characterized by little, if any, intestinal calcium transport activity compared with the activity of 1α, 25-dihydroxyvitamin D3, and they exert relatively high activity compared with 1α, 25-dihydroxyvitamin D₃ when it comes to mobilizing calcium from the bone mass. Therefore, these compounds are very specific in their calcium activity. Their preferential activity in mobilizing calcium from bone mass and their reduced intestinal calcium transport activity make it possible to administer these compounds in vivo to treat metabolic bone diseases where bone loss is the major concern. Because of its preferential calcific activity on bone, these compounds will be preferred therapeutic agents for the treatment of diseases where bone formation is desirable, such as osteoporosis, especially osteoporosis with low bone turnover, steroid-induced osteoporosis, senile osteoporosis or postmenopausal osteoporosis, as well as osteomalacia and osteomalacia. The treatment may be transdermal, oral or parenteral. The compounds may be present in a mixture in an amount of from ca. 0.1 pg / g to approx.

pg / g of the mixture, and can be administered in dosages of from ca. 0.1 pg / day to approx. 50 pg / day.

The compounds of the invention are also particularly suitable for the treatment and prophylaxis of human disorders characterized by an imbalance in the immune system, e.g. in autoimmune diseases, including multiple sclerosis, diabetes mellitus, cough versus graft reactions, and graft rejection; and further for the treatment of inflammatory diseases, such as rheumatoid arthritis and asthma, as well as improvement of bone fracture healing and improved bone implants. Acne, alopecia, skin conditions such as dry skin (lack of dermal hydration), excessive skin laxity (insufficient skin firmness), insufficient sebum secretion and wrinkles, and hypertension are other conditions treatable with the compounds of the invention.

The above compounds are also characterized by high cell differentiation activity. Thus, these compounds also provide therapeutic agents for the treatment of psoriasis, or as an anticancer agent, especially against leukemia, colon cancer, breast cancer and prostate cancer. The compounds may be present in a composition for the treatment of psoriasis in an amount of from ca. 0.01 pg / g to approx. 100 µg / g of the mixture, and can be administered topically, transdermally, orally or parenterally in doses ranging from approx. 0.01 pg / day to approx. 100 pg / day.

Brief description of the drawings

Figure 1 is a graph showing the relative activity of a mixture of 2α and 2β-methyl-19-nor-20S-1α, 25-dihydroxyvitamin D3, a mixture of 2α and 2β-methyl-19-nor-1α, 25 -dihydroxyvitamin D₃ and la, 25-dihydroxy-vitamin D3 to compete for binding of [3H] -1,25- (OH) 2-D3 to the intestinal nuclear receptor. vitamin D from pigs; and Figure 2 is a graph illustrating the percentage of HL-60 cell differentiation as a function of the concentration of a mixture of 2α and 2β-methyl-19-nor-20Sla, 25-dihydroxy vitamin D₃, a mixture of 2α and 2β-methyl-19-nor-1α, 25-dihydroxy vitamin D 3 and 1α, 25-dihydroxyvitamin Da.

Detailed description of the invention

As used in the specification and claims, the term hydroxy protecting group means "any group commonly used for the temporary protection of hydroxy functions, such as e.g. alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafter simply referred to as silyl groups) and alkoxyalkyl groups. Alkoxycarbonyl protecting groups are alkyl-O-CO groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. The term acyl means an alkanoyl group of 16 carbon atoms, in all their isomeric forms, or a carboxyalkanoyl group of 1-6 carbon atoms, such as an oxalyl, malonyl, succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, or a halogen. -, nitro- or alkyl-substituted benzoyl group. The term "alkyl" as used in the specification or claims, means a straight-chain or branched alkyl radical of 1-10 carbon atoms, in all its isomeric forms. Alkoxyalkyl protecting groups are groups such as methoxymethyl, ethoxymethyl, methoxy ethoxymethyl, or tetrahydrofuranyl and tetrahydropyranyl. Preferred silyl protecting groups are trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl and analogous alkylated silyl radicals. The term aryl means a phenyl group or an alkyl, nitro or halogen substituted phenyl group.

A protected hydroxy group is a hydroxy group which is derivatized or protected with one of the above groups which is usually used for temporary or permanent protection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl or alkoxycarbonyl groups, as previously defined. The terms hydroxyalkyl, deuteroalkyl and fluoroalkyl refer to an alkyl radical substituted by one or more hydroxy, deuterium and fluorine groups, respectively.

Specific and preferred examples of the 2-alkyl compounds of structure I are:

2 (S) -methyl-19-nor-1α, 25-dihydroxyvitamin D3,

2 (R) -methyl-19-nor-1α, 25-dihydroxyvitamin D₃,

2 (S) -methyl-19-nor-20 (S) -la, 25-dihydroxyvitamin D₃, 2 (R) -methyl-19-nor-20 (S) -la, 25-dihydroxyvitamin D₃.

The preparation of 1α-hydroxy-2-alkyl-19-nor-vitamin D compounds, especially 1α-hydroxy-2-methyl-19-nor-vitamin D compounds of basic structure I, can be carried out by a conventional general method, that is, condensation of a bicyclic Windaus-Grundmann ketone II with the allylic phosphine oxide III to the corresponding 2-methylene-19nor vitamin D analogues IV, followed by a selective reduction of the exomethylene group at C-2 in the latter compounds:

R

<img file="NO321925B1_D0005.tif" />

Ο

II

<img file="NO321925B1_D0006.tif" />

<img file="NO321925B1_D0007.tif" />

In structures II, III and TV, the groups Yi and Y represent₂and R groups as defined above; Yi and Y₂ are preferably hydroxy protecting groups, and it should also be understood that any functionality of R which may be sensitive, or which interferes with the condensation reaction, should be suitably protected, as is well known in the art. The process shown above represents an embodiment of the convergent synthesis concept, which has been used effectively for the preparation of vitamin io D compounds [e.g. Lythgoe et al., J. Chem. Soc. Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev., 9, 449 (1983); Toh et al., J. Org. Chem., 48, 1414 (1983);

Baggiolini et al., J. Org. Chem., 51, 3098 (1986); Sardina et al., J. Org. Chem., 51, 1264 (1986); J. Org. Chem., 51, 1269 (1986); DeLuca et al., U.S. Patent No. 5,086,191; DeLuca et al., U.S. Patent No. 5,536,713].

The hydrin anions of general structure II are known or can be prepared by known methods. Specific important examples of such known bicyclic ketones are the side chain structures (a), (b), (c) and (d) described above, i.e. 25-hydroxy-Grundmann ketone (f) [Baggiolini et al. J. Org. Chem., 51, 3098 (1986)]; Grundmann ketone (g) [Inhoffen et al., Chem. Ber., 90, 664 (1957)]; 25-hydroxy-Windaus ketone (h) [Baggiolini et al., J. Org. Chem., 51, 3098 (1986)] and Windaus ketone (i) [Windaus et al., Ann., 524, 297 (1936)]:

<img file="NO321925B1_D0008.tif" />

To prepare the necessary phosphine oxides of general structure III, one synthetic route has been developed starting from the methyl kinicate derivative 1, which is readily obtained from commercial (IR, 3R, 4S, 5R) - (-) - quinic acid, as described by Perlman et al. , Tetrahedron Lett., 32, 7663 (1991), and DeLuca et al., U.S. Patent No. 5,086,191. The overall process of converting the starting methyl ester 1 into the desired A-ring syntheses is summarized in Scheme I . Thus, the secondary 4-hydroxyl group in 1 was oxidized with

Ruo₄ (a catalytic method with RUCI3 and NaIO₄ as a cooker). The use of such a strong oxidizing agent is necessary for an efficient oxidation process of this highly hindered hydroxyl. However, other common oxidizing agents may also be used (e.g., pyridinium dichromate), although the reactions usually require a much longer time to complete. The second step in the synthesis involves Wittig reaction of the sterically hindered 4-keto compound 2 with ylide prepared from. methyl triphenylphosphonium bromide and n-butyllithium. Other bases may also be used to generate the reactive methylene phosphorus, such as t-BuOK, NaNH2, NaH, K / HMPT, NaN (TMS)₂ etc. For the preparation of the 4-methylene compound 3, some described modifications of the Wittig process can be used, e.g. reaction of 2 with activated methylene triphenylphosphorane [Corey et al., Tetrahedron Lett., 26, 555 (1985)]. Alternatively, other methods commonly used for methylation of unreactive ketones may be used, e.g. Wittig-Horner's reaction with the PO ylid obtained from methyldiphenylphosphine oxide by deprotonation with n-butyllithium [Schosse et al., Chimia, 30, 197 (1976)], or reaction of ketone with sodium methylsulfinate [Corey et al., J. Org. Chem., 28, 1128 (1963)] and potassium methyl sulfinate [Greene et al., Tetrahedron Lett., 3755 (1976)]. Reduction of the ester 3 with lithium aluminum hydride or another suitable reducing agent (e.g. DIBALH) gave the diol 4, which was then oxidized with sodium periodate to the cyclohexanone derivative 5. The next step in the process involves Peterson's reaction of the ketone 5 with methyl (trimethylsilyl) acetate. The resulting allyl ester 6 was treated with diisobutyl aluminum hydride and the resulting allyl alcohol 7 was in turn converted to the desired A-ring phosphine oxide 8. Conversion of 7 to 8 included three steps, namely in situ tosylation with n-butyllithium and p-toluenesulfonyl chloride, followed by reaction with diphenylphosphine lithium salt and oxidation with hydrogen peroxide.

Several 2-methylene-19-nor-vitamin D compounds of general structure IV can be synthesized using

The A-ring synthet 8 and the suitable Windaus-Grundmann ketone II, which have the desired side chain structure. , prepared according to the published procedure [Sicinski et al., J. Med. Chem., 37, 3730 (1994)], the expected protected vitamin compound 10. This, after unblocking with AG 50W-X4 cation-exchanging resin Ia, 25dihydroxy-2-methylene-19-nor-vitamin D₃ (11).

The final step of the process was selective, homogeneous, catalytic hydrogenation of the exomethylene unit at Carbon 2 of vitamin 11 performed efficiently in the presence of tris (triphenylphosphine) rhodium (I) chloride [Wilkinson's catalyst, (Ph₃P)₃RhCl]. Such reduction conditions make it possible to reduce only the C (2) = CH2 moiety and do not affect the C (5) -C (8) -butadiene group. The isolated material is an epimeric mixture (about 1: 1) of 2-methyl-19-nor vitamins 12 and 13 that differs in configuration at C-2. The mixture can be used without separation or, if desired, the individual 2α and 2β isomers can be separated by an efficient HPLC system.

The C-20 epimerization was performed by analog coupling of the phosphine oxide 8 with protected (20S) -25-hydroxy-Grundmannketone 15 (Scheme II) to give 19-nor-vitamin 16, which after hydrolysis of dihydroxy protecting groups gave (20S) -l , Dihydroxy-2-methylene-19-norvitamin D₃ (17). Hydrogenation of 17 using Wilkinson's catalyst gave the expected mixture of 2-methyl-19-nor-vitamin D analogues 18 and 19. As indicated above, other 2-methyl-19-norvitamin D analogs can be synthesized by the method described herein . For example, 1α-hydroxy-2-methylene-19-nor-vitamin D₃obtained by providing Grundmann ketone (g); subsequent reduction of the A-ring exomethylene group in the compound formed may give the corresponding epimeric mixture of 1α-hydroxy-2-methyl-19-nor-vitamin D₃compounds.

This invention is described in the following illustrative examples. In these examples, specific products indicated by Arabic numerals (eg, 1, 2, 3, etc.) refer to the specific structures defined in the foregoing description and in Schedule I and Schedule II.

Example 1

Preparation of 1α, 25-dihydroxy-2β-xa and 1α, 25-dihydroxy2β-methyl-19-nor-vitamin P₃ (12 and 13)

Referring first to Scheme I, the starting methyl kinicate derivative 1 was obtained from commercial (-) quinic acid, as previously described [Perlman et al., Tetrahedron Lett., 32, 7663 (1991); and DeLuca et al., U.S. Pat. 5,086,191].

1: mp 82-82.5^QC (from hexane), ¹H-NMR (CDCl₃) δ 0.098, 0.110, 0.142 and 0.159 (each 3 H, each s, 4 x SiCH₃), 0.896 and 0.911 (9H and 9H, each s, 2 x Si-t-Bu), 1.820 (1H, dd, J = 13.1, 10.3 Hz), 2.02 (1H, ddd, J = 14.3, 4.3, 2.4 Hz) ), 2.09 (1H, dd, J = 14.3, 2.8 Hz), 2.19 (1H, ddd, J = 13.1, 4.4, 2.4 Hz), 2.31 (1H, d, J = 2.8 Hz, OH), 3.42 (1H, m; after D₂O dd, J = 8.6, 2.6 Hz), 3.77 (3H, s), 4.12 (1H, m), 4.37 (1H, m), 4.53 (1H, br s, OH).

(a) Oxidation of the 4-hydroxy group in methyl kinicate derivative (3R, 5R) -3,5-bis [(tert-butyldimethylsilyl) oxy] -1-hydroxy

4-oxocyclohexanecarboxylic acid methyl ester (2)

To a stirred mixture of ruthenium (III) chloride hydrate (434 mg, 2.1 mmol) and sodium periodate (10.8 g, 50.6 mmol) in water (42 ml) was added a solution of methyl kinicate 1 (6.09 g, 14 mmol) in CC1₄/ CH₃CN (1: 1, 64 ml). Vigorous staring continued for 8 hours. A few drops of 2-propanol were added and the mixture was poured into water and extracted with chloroform. The organic extracts were combined, washed with water, dried (MgSCM and evaporated to give a dark oily residue (about 5 g) which was purified by flash chromatography.xane / ethyl acetate (8: 2) gave pure, oily). 4-ketone 2 (3.4 g, 56%).

^XH-NMR (CDCl 3) δ 0.054, 0.091, 0.127 and 0.132 (each 3 H, each s, 4 x SiCH₃), 0.908 and 0.913 (9H and 9H, each s, 2x

Si-t-Bu), 2.22 (1H, dd, J = 13.2, 11.7 Hz), 2.28 (1H, ~ dt, J = 14.9, 3.6 Hz), 2.37 (1H, dd, J = 14.9, 3.2 Hz),

2.55 (1H, ddd, J = 13.2, 6.4, 3.4 Hz), 3.79 (3.H, s), 4.41 (1H, t, J - 3.5 Hz), 4.64 (1H, s, OH), 5.04 (1H, dd, J = 11.7, 6.4 Hz); MS: m / z (relative intensity), no M +, 375 (M + - t-Bu, 32), 357 (M + - t-Bu - H₂O, 47), 243 (31), 225 (57), 73 (100).

(b) Wittiq reaction of the 4-ketone 2 (3R, 5R) -3,5-bis [(tert-butyldimethylsilyl) oxy] -1-hydroxy42methenecyclohexanecarboxylic acid 2-ethyl ester (3L

To the methyl triphenylphosphonium bromide (2.813 g, 7.88 mmol) in anhydrous THF (32 ml) at 0 ° C was added dropwise nBuLi (2.5 M in hexane, 6.0 ml, 15 mmol) under argon with stirring. Another portion of MePh₃P + Br- (2.813 g, 7.88 mmol) was then added and the solution stirred at 0 ^ttC for 10 minutes and at room temperature for 40 minutes. The orange-red mixture was again cooled to 0 ° C and a solution of 4-ketone 2 (1.558 g, 3.6 mmol) in anhydrous THF (16 + 2 ml) was added via a siphon to the reaction flask over 20 minutes. The reaction mixture was stirred at 0 ° C for 1 hour and then at room temperature for 3 hours. The mixture was then carefully poured into brine containing 1% HCl and extracted with ethyl acetate and benzene. The combined organic extracts were washed with dilute NaHCO₃ and brine, dried (MgSO₄) and evaporated to give an orange oily residue (about 2.6 g) which was purified by flash chromatography. Elution with hexane / ethyl acetate (9: 1) gave the pure 4-methylene compound 3 as a colorless oil (368 mg, 24%).

^XH-NMR (CDCl3) δ 0.078, 0.083, 0.092 and 0.115 (each 3 H, each s, 4 x S 1 CH 3), 0.889 and 0.920 (9 H and 9 H, each s, 2 x Si-t-Bu), 1.811 (1H, dd, J = 12.6, 11.2 Hz), 2.10 (2H, m), 2.31 (1H, dd, J = 12.6, 5.1 Hz), 3 , 76 (3H, s), 4.69 (1H, t, J = 3.1 Hz), 4.78 (1H, m), 4.96 (2H, m; after D₂0.1 H, br s), 5.17 (1 H, t, J = 1.9 Hz); MS: m / z (relative intensity), no M +, 373 (M + - t-Bu, 57), 355 (M + - t-Bu - H₂O, 13), 341 (19), 313 (25), 241 (33), 223 (37), 209 (56), 73 (100).

(c) Reduction of the ester group in 4-methylene compound 3 [(3R, 5R) -3,5-bis [(tert-butyldimethylsilyl) oxy] -1-hydroxy4-methylene cyclohexyl] methanol (4) (i) To a stirred solution of the ester 3 (90 mg, 0.21 mmol) in anhydrous THF (8 ml) was lithium aluminum hydride (60 mg,

1.6 mmol) added at 0 ° C under argon. The cooling bath was removed after 1 hour and stirring was continued at 6 ° C for 12 hours at room temperature for 6 hours. The excess reagent was digested with saturated aqueous Na₂SO4, and the mixture was extracted with ethyl acetate and ether, dried (MgSO4)₄) and evaporated. Flash chromatography of the residue with hexane / ethyl acetate (9: 1) gave unreacted substrate (12 mg) and a pure crystalline diol 4 (35 mg, 48%, based on the yield of ester 3).

¹1 H-NMR (CDCl 3 + D₂O) 8 0.079, 0.091, 0.100 and 0.121 (each 3 H, each s, 4 x SiCH₃), 0.895 and 0.927 (9H and 9H, each s, 2 X Si-t-Bu), 1.339 (1H, t, J ~ 12 Hz), 1.510 (1H, dd, J = 14.3, 2.7 Hz), 2.10 (2H, m), 3.29 and 3.40 (1H and 1H, each d, J = 11.0 Hz), 4.66 (1H, t, J - 2.8 Hz), 4.78 (1 H,

m), 4.92 (1H, t, J = 1.7 Hz), 5.13 (1H, t, J - 2.0 Hz); MS: m / z (relative intensity), no M +, 345 (M + - t-Bu, 8),

327 (M + - t-Bu - H₂O, 22), 213 (28), 195 (11), 73 (100).

(ii) Diisobutylaluminum hydride (1.5 M in toluene, 2.0 ml, 3 mmol) was added to a solution of the ester 3 (215 mg, 0.5 mmol) in anhydrous ether (3 ml) at -78 ^eC under argon. The mixture was stirred at -78^QC for 3 hours and at -24 ° C for 1.5 hours and diluted with ether (10 mL), and the reaction was suppressed by the slow addition of 2N potassium sodium tartrate. The solution was warmed to room temperature and stirred for 15 minutes, then poured into brine and extracted with ethyl acetate and ether. The organic extracts were combined, washed with dilute (about 1%) HCl and brine, dried (MgSO₄) and evaporated. The crystalline residue was purified by flash chromatography. Elution with hexane / ethyl acetate (9: 1) gave the crystalline diol 4 (43 mg, 24%).

(d) Cleavage of the vicinal diol 4 (3R, 5R) -3,5-bis [(tert-butyldimethylsilyl) oxy] -4-methylenecyclohexanone (5)

Sodium periodate saturated water (2.2 ml) was added to a solution of the diol 4 (146 mg, 0.36 mmol) in methanol (9 ml) at 0 ° C. The solution was stirred at 0 ° C for 1 hour, poured into brine and extracted with ether and benzene. The organic extracts were combined, washed with brine, dried (MgSO4) and evaporated. An oily residue was dissolved in hexane (1 ml) and transferred to a silica Sep-Pak cartridge. The pure 4-methylene cyclohexanone derivative 5 (110 mg, 82%) was eluted with hexane / ethyl acetate (95: 5) as a colorless oil.

¹H-NMR (CDCl 3) δ 0.050 and 0.069 (6 H and 6 H, each s, 4 x S 1 CH 3), 0.881 (18 H, s, 2 x Si-t-Bu), 2.45 (2 H, ddd, J 14.2, 6.9, 1.4 Hz), 2.64 (2H, ddd, J = 14.2, 4.6, 1.4 Hz), 4.69 (2H, dd, J = 6.9, - 4.6 Hz), 5.16 (2 H, s); MS: m / z (relative intensity), no M +, 355 (M + - Me, 3), 313 (M + t-Bu, 100), 73 (76).

(e) Preparation of the Allyl Ester 6 [(3⁽R, 5 * R) -3¹¹5 '-bis [(tert -butyldimethylsilyl) oxy] -4 * methylene cyclohexylidene] acetic acid methyl ester (6)

To a solution of diisopropylamine (37 μΐ, 0.28 mmol) in anhydrous THF (200 μΐ) was added n-BuLi (2.5 M in hexane, 113 μΐ, 0.28 mmol) under argon at -78 ° C with stirring, and methyl (trimethylsilyl) acetate (46 μΐ, 0.28 mmol) was then added. After 15 minutes, the keto compound 5 (49 mg, 0.132 mmol) was added dropwise. The solution was stirred at -78 ° C for 2 hours and the reaction was quenched with saturated NH 4 Cl, the mixture was poured into brine and extracted with ether and benzene. The combined organic extracts were washed with brine, dried (MgSCM and evaporated. The residue was dissolved in hexane (1 mL) and transferred to a silica Sep-Pak cartridge. Elution with hexane and hexane / ethyl acetate (98: 2) gave a pure allyl ester 6 (50 mg, 89%) as a colorless oil.

¹H-NMR (CDCl 3) δ 0.039, 0.064 and 0.076 (6H, 3H and 3H, each s, 4x SiCHl ·}), 0.864 and 0.884 (9H and 9H, each s, 2x Si-t -Bu), 2.26 (1H, dd, J = 12.8, 7.4 Hz), 2.47 (1H, dd, J = 12.8, 4.2 Hz), 2.98 ( 1 H, dd, J = 13.3, 4.0 Hz), 3.06 (1 H, dd, J = 13.3, 6.6 Hz), 3.69 (3 H, s), 4, 48 (2H, m),

4.99 (2H, s), 5.74 (1H, s); MS: m / z (relative intensity), 426 (M +, 2), 411 (M + - Me, 4), 369 (M + - t-Bu, 100), 263 (69).

(f) Reduction of the allyl ester 6

2- [(3 'R, 5' R) -3¹, 5'-Bis [(tert -butyldimethylsilyl) oxy] -4¹methylene cyclohexylidene] ethanol (7)

Diisobutyl aluminum hydride (1.5 M in toluene, 1.6 ml,

2.4 mmol) was slowly added to a stirred solution of the allyl ester 6 (143 mg, 0.33 mmol) in toluene / methylene chloride (2: 1, 5.7 ml) at -78 ° C under argon. Stirring was continued at -78 ° C for 1 hour and at -46 ° C (cyclohexanone-Ztrisris bath) for 25 minutes. The reaction was suppressed by the slow addition of potassium sodium tartrate (2 N, 3 ml), aqueous HCl (2 N, 3 ml) and H 2 O (12 ml), and then the mixture was diluted with methylene chloride (12 ml) and extracted with ether and benzene. The organic extracts were combined, washed with dilute (about 1%) HCl and brine, dried (MgSO₄) and evaporated. The residue was purified by flash chromatography. Elution with hexane / ethyl acetate (9: 1) gave the crystalline allyl alcohol 7 (130 mg, 97%).

¹1 H-NMR (CDCl 3) δ 0.038, 0.050 and 0.075 (3H, 3H and 6H, each s_t 4 x S 1 CH 3), 0.876 and 0.904 (9 H and 9 H, each s, 2 x Si-t-Bu), 2.12 (1 H, dd, J = 12.3, 8.8 Hz), 2, 23 (1H, dd, J = 13.3, 2.7 Hz), 2.45 (1H, dd, J = 12.3, 4.8 Hz), 2.51 (1H, dd, J = 13.3, 5.4 Hz), 4.04 (1H, m; after D₂O dd, J = 12.0, 7.0 Hz), 4.17 (1H, m; after D₂O dd, J = 12.0, 7.4 Hz), 4.38 (1H, m), 4.49 (1H, m), 4.95 (1H, br s), 5.05 ( 1 H, t, J = 1.7 Hz), 5.69 (1 H, - t, J = 7.2 Hz); MS: m / z (relative intensity) 398 (M +, 2), 383 (M + - Me, 2), 365 (M + -Me - H 2 O, 4), 341 (M + - t-Bu, 78), 323 (M + - t-Bu - H₂O,

10), 73 (100).

(g) Conversion of allyl alcohol 7 to phosphine oxide 8 [2- [(3'R, 5 * R) -3 ', 5'-bls [(tert-butyldimethylsilyl) oxy] -4¹ methylene cyclohexylidene] ethyl] diphenylphosphine oxide (8)

To the allyl alcohol 7 (105 mg, 0.263 mmol) in anhydrous THF (2.4 ml) was added n-BuLi (2.5 M in hexane, 105 μΐ, 0.263 mmol) under argon at 0 ° C. Recently recrystallized tosyl chloride (50.4 mg, 0.264 mmol) was dissolved in anhydrous THF (480 μΐ) and added to the allyl alcohol-BuLi solution. The mixture was stirred at 0 ° C for 5 minutes and set aside at 0 ° C. In another dry flask with the air replaced with argon, n-BuLi (2.5 M in hexane, 210 μΐ, 0.525 mmol) was added to Ph2PH (93 μΐ, 0.534 mmol) in anhydrous THF (750 μΐ) at 0 ° C below stirring. The red solution was applied via a siphon under argon pressure to the solution of tosylate until the orange color persisted (about half of the solution was added). The resulting mixture was stirred for a further 30 minutes at 0 ° C, and the reaction was suppressed by adding H₂O (30 μΐ). The solvents were evaporated under reduced pressure and the residue was redissolved in methylene chloride (2.4 ml) and stirred with 10% H₂O₂at 0 ° C for 1 hour. The organic layer was separated, washed with cold, aqueous sodium sulfite and H₂O, dried (MgSO₄) and evaporated. The residue was flash chromatographed. Elution with benzene / ethyl acetate (6: 4) gave semicrystalline phosphine oxide 8 (134 mg, 87%).

^XH-NMR (CDCl3) δ 0.002, 0.011 and 0.019 (3H, 3H and 6H, each s, 4 x SiCH₃), 0.855 and 0.860 (9H and 9H, each s, 2 x Si-t-Bu), 2.0-2.1 (3H, br m), 2.34 (1H, m), 3 , 08 (1H, m), 3.19 (1H, m), 4.34 (2H, m), 4.90 and 4.94 (1H and 1H, each s), 5.35 (1 H, - q, J = 7.4 Hz); 7.46 (4H, m), 7.52 (2H, m), 7.72 (4H, m); MS: m / z (relative intensity), no M +, 581 (M + - 1, 1), 567 (M + - Me, 3), 525 (M + - tBu, 100), 450 (10), 393 (48).

(h) Wittig-Horner coupling of protected 25-hydroxy-

Grundmann ketone 9 with the phosphine oxide 8α, 25-dihydroxy-2-methylene-19-nor vitamin P₃ (11)

To a solution of phosphine oxide 8 (33.1 mg, 56.8 pmol) in anhydrous THF (450 μΐ) at 0 ° C, nBuLi (2.5 M in hexane, 23 μΐ, 57.5 μιηοΐ) was slowly added below argon with stirring. The solution turned deep orange. The mixture was cooled to -78 ° C and a pre-cooled (-78 ° C) solution of the protected hydroxy ketone 9 (9.0 mg, 22.8 μπιοί) prepared according to the published procedure [Sicinski et al. , J. Med. Chem., 37, 3730 (1994)], in anhydrous THF (200 + 100 μΐ) was slowly added. The mixture was stirred under argon at -78 ° C for 1 hour and at 0 ° C for 18 hours. Ethyl acetate was added and the organic phase was washed with brine, dried (MgSO₄) and evaporated. The residue was dissolved in hexane and transferred to a silica Sep-Pak cartridge and washed with hexane / ethyl acetate (99: 1, 20 ml) to give the 19-norvitamin derivative 10 (13.5 mg, 78%). The Sep-Pak cartridge was then washed with hexane / ethyl acetate (96: 4, 10 ml) to recover some of the unchanged C, D-ring ketone 9 (2 mg), and with ethyl acetate (10 ml) to recover the diphenylphosphine oxide (20 mg). For analytical reasons, a sample of the protected vitamin 10 was further purified by HPLC (6.2 mm x 25 cm Zorbax-Sil column, 4 ml / minute) using hexane / ethyl acetate (99.9: 0.1) as solvent system. The pure compound 10 was eluted at R_v 26 ml as a colorless oil.

UV (in hexane) λ max 244, 253, 263 nm; ¹H-NMR (CDCl₃) δ 0.025, 0.049, 0.066 and 0.080 (each 3 H, each s, 4 x SiCH 2), 0.546 (3 H, s, 18-H 3), 0.565 (6 H, q, J = 7.9 Hz, 3 x sich₂), 0.864 and 0.896 (9H and 9H, each s, 2 x Si-t-Bu), 0.931 (3H, d, J = 6.0 Hz, 2I-H3), 0.947 (9H, t, J = 7.9 Hz, 3 x SiCH₂CH₃), 1,188 (6H, s, 26- and 27-H₃), 2.00 (2H, m), 2.18 (1H, dd, J = 12.5, 8.5 Hz, 4β-Η), 2.33 (1H, dd, J =

13.1, 2.9 Hz, 10β-Η), 2.46 (1H, dd, J = 12.5, 4.5 Hz, 4aH), 2.52 (1H, dd, J = 13, 1, 5.8 Hz, 10a-H), 2.82 (1H, br d, J = 12 Hz, 9β-H), 4.43 (2H, m, 1β and 3a-H), 4 , 92 and 4.97 (1H and 1H, each s, = CH₂), 5.84 and 6.22 (1H and 1H, each d, J = 11.0 Hz, 7- and 6-H); MS: m / z (relative intensity) 758 (M +, 17), 729 (M + - Et, 6), 701 (M + - t-Bu, 4), 626 (100), 494 (23), 366 (50) , 73 (92).

The protected vitamin 10 (4.3 mg) was dissolved in benzene (150 μΐ) and the resin (AG 50W-X4, 60 mg; pre-washed with methanol) in methanol (800 μΐ) was added. The mixture was stirred at room temperature under argon for 17 hours, diluted with ethyl acetate / ether (1: 1, 4 ml) and decanted. The resin was washed with ether (8 ml) and the combined organic phases were washed with brine and saturated NaHCO₃, dried (MgSOJ and evaporated. The residue was purified by HPLC (6.2 mm x 25 cm Zorbax-Sil column, 4 mL / min) using hexane / 2-propanol (9: 1) as solvent system. Analytically pure 2 -Methylene-19-nor-vitamin 11 (2.3 mg, 97%) was collected at R_v 29 ml (1α, 25-dihydroxyvitamin D3 was eluted at R_v 52 ml in the same system) as a white solid.

UV (in EtOH) λmacg 243.5, 252, 262.5 nm; ^XH-NMR (CDCl 3) δ 0.552 (3H, s, 18-H3), 0.941 (3H, d, J = 6.4 Hz, 21-H₃), 1.222 (6H, s, 26- and 27-H₃), 2.01 (2H, m), 2.27-2.36 (2H, m), 2.58 (1H, m), 2.80-2.88 (2H, m), 4.49 (2H, m, 1β and 3α-H), 5.10 and 5.11 (1H and 1H, each s, = CH₂), 5.89 and 6.37 (1H and 1H, each d, J = 11.3 Hz, 7- and 6-H); MS: m / z (relative intensity) 416 (M +, 83), 398 (25), 384 (31), 380 (14), 351 (20), 313 (100).

(i) Hydrogenation of 2-methylene-19-nor-vitamin 11a, 25-dihydroxy-2a and 1a, 25-dihydroxy-28-methyl-19-norvitamin D3 (12 and 13)

Tris (triphenylphosphine) rhodium (I) chloride (2.3 mg, 2.5 μπιοί) was added to dry benzene (2.5 ml) which was previously saturated with hydrogen. The mixture was stirred at room temperature until a homogeneous solution was formed (about 45 minutes). A solution of vitamin 11 (1.0 mg, 2.4 pmol) in dry benzene (0.5 ml) was then added and the reaction allowed to proceed under a continuous stream of hydrogen for 3 hours. The benzene was removed in vacuo and hexane / ethyl acetate (1: 1, 2 ml) was added to the residue. The mixture was transferred to a silica-Sep-Pak and both 2-methyl vitamins were eluted with the same solvent system (20 ml). Further purification was achieved by HPLC (6.2 mm x 25 cm Zorbax-Sil column, 4 ml / minute) using hexane / 2-propanol (9: 1) as solvent system. The mixture (about 1: 1) of 2-methyl19-noramines (2a and 2p epimers 12 and 13; 0.80 mg, 80%) gave a single peak at R_v 33 ml.

and 13: UV (in EtOH) 243, 251, 261.5 nm; ^XH-NMR (CDCl 3) δ 0.536 and 0.548 (3H and 3H, each s, 18-H3), 0.937 (6H, d, J = 6.3 Hz, 2 x 21-H₃), 1,133 and 1,144 (3 H and 3 H, each d, J - 6 Hz, 2 x 2-CH₃), 1.219 [12H, s, 2x (26 and 27-H3)], 2.60 (1H, dd, J = 13.0, 4.6 Hz), 2.80 (3H, m) , 3.08 (1H, dd, J = 12.6, 4.0 Hz), 3.51 (1H, dt, J = 4.6, 10.2 Hz), 3.61 (1H, dt, J = 4.5, 9.1 Hz), 3.90 (1H, narrow m), 3.96 (1H, narrow m), 5.82, 5.87, 6.26 and 6, 37 (each 1 H, each d, J = 11.2 Hz); MS: m / z (relative intensity) 418 (M +, 100), 400 (25), 385 (15), 289 (30), 245 (25).

Example 2

Preparation of (20S) -la, 25-dihydroxy-2a and (20S) -la, 25dihydroxy-2P-methyl-19-nor-vitamin P₃ (18 and 19)

Scheme II illustrates the preparation of the protected (20S) -25-hydroxy-Grundmann ketone 15, its coupling with the phosphine oxide 8 (obtained as described in Example 1), and the selective hydrogenation of the exomethylene group in the 2-methylene compound 17.

(a) Silylation of the hydroxy ketone 14 (20S) -25 - [(triethylsilyl) oxy] -des-A, B-cholestan-8-one (15)

A solution of the ketone 14 (Tetrionics, Inc .; 56 mg,

0.2 mmol) and imidazole (65 mg, 0.95 mmol) in anhydrous DMF (1.2 ml) were treated with triethylsilyl chloride (95 μΐ, 0.56 mmol) and the mixture was stirred at room temperature under argon for 4 hours. . Ethyl acetate and water were added and the organic layer was separated. The ethyl acetate layer was washed with water and brine, dried (MgSO 4) and evaporated. The residue was passed through a silica Sep-Pak cartridge in hexane / ethyl acetate (9: 1), and after evaporation it was purified by HPLC (9.4 mm x 25 cm Zorbax-Sil column, 4 ml / min) under using hexane / ethyl acetate (9: 1) as a solvent system. Pure protected hydroxy ketone 15 (55 mg, 70%) was eluted at R_v 35 ml as a colorless oil.

¹H-NMR (CDCl 3) δ 0.566 (6 H, q, J = 7.9 Hz, 3 x SiCH₂), 0.638 (3H, s, 18-H3), 0.859 (3H, d, J = 6.0 Hz, 21-H₃),

0.947 (9 H, t, J = 7.9 Hz, 3 x SiCH₂CH₃), 1,196 (6H, s, 26- and 27-H₃), 2.45 (1H, dd, J = 11.4, 7.5 Hz, 14a-H).

(b) Wittig-Horner coupling of the protected (20S) -25hydroxy-Grundmann ketone 15 with the phosphine oxide 8 (20S) -la, 25-dihydroxy-2-methylene-19-nor-vitamin P₃ (17)

To a solution of phosphine oxide 8 (15.8 mg, 27.1 pmol) in anhydrous THF (200 μΐ) at 0 ° C, nBuLi (2.5 M in hexane, 11 μΐ, 27.5 μπιοί) was slowly added below argon with stirring. The solution turned deep orange. The mixture was cooled to -78 ° C and a pre-cooled (-78 ° C) solution of the protected hydroxy ketone (8.0 mg, 20.3 μιηοΐ) in anhydrous THF (100 μΐ) was slowly added. The mixture was stirred under argon at -78 ° C for 1 hour and at 0 ° C for hours. Ethyl acetate was added and the organic phase was washed with brine, dried (MgSOJ and evaporated. The residue was dissolved in hexane and transferred to a silica Sep Pak cartridge and washed with hexane / ethyl acetate (99.5: 0.5, 20 ml ), giving the 19-nor-vitamin derivative 16 (7 mg, 45%) as a colorless oil, then the Sep-Pak cartridge was washed with hexane / ethyl acetate (96: 4, 10 ml) to recover some of the unchanged C , D-ring ketone 15 (4 mg), and with ethyl acetate (10 ml) to recover the diphenylphosphine oxide (9 mg). For analytical reasons, a sample of the protected vitamin 16 was further purified by HPLC (6.2 mm x 25 cm Zorbax Silk column, 4 ml / minute) using hexane / ethyl acetate (99.9: 0.1) as solvent system.

16: UV (in hexane) λmax 244, 253.5, 263 nm; ^XH-NMR (CDCl₃) δ 0.026, 0.049, 0.066 and 0.080 (each 3 H, each s, 4 x SiCH₃), 0.541 (3H, s, 18-H₃), 0.564 (6 H, q, J = 7.9 Hz, 3 x

sich₂), 0.848 (3H, d, J = 6.5 Hz, 21-H₃), 0.864 and 0.896 (9 H and 9 H, each s, 2 x Si-t-Bu), 0.945 (9 H, t, J = 7.9 Hz, 3 x SiCH₂CH₃), 1,188 (6H, s, 26- and 27-H₃), 2.15-2.35 (4H, br m), 2.43-2.53 (3H, br m), 2.82 (1H, br d, J =

12.9 Hz, 9β-Η), 4.42 (2H, m, 1β and 3α-H), 4.92 and 4.97 (1

H and 1H, each s, = CH₂), 5.84 and 6.22 (1H and 1H, each d, J = 11.1 Hz, 7- and 6-H); MS: m / z (relative intensity) 758 (M +, 33), 729 (M + - Et, 7), 701 (M + - t-Bu, 5), 626 (100), 494 (25), 366 (52) , 75 (82), 73 (69).

The protected vitamin 16 (5.0 mg) was dissolved in benzene (160 μΐ) and the resin (AG 50W-X4, 70 mg; pre-washed with methanol) in methanol (900 μΐ) was added. The mixture was stirred at room temperature under argon for 19 hours, diluted with ethyl acetate / ether (1: 1, 4 ml) and decanted. The resin was washed with ether (8 ml) and the combined organic phases were washed with brine and saturated NaHCO₃, dried (MgSOJ and evaporated. The residue was purified by HPLC (6.2 mm x 25 cm Zorbax-Sil column, 4 mL / min) using hexane / 2-propanol (9: 1) as solvent system. Analytically pure 2 -Methylene-19-nor-vitamin 17 (2.6 mg, 95%) was collected at R_v 28 ml of the [(20R) analog was eluted at R_v 29 ml and 1a, 25-dihydroxyvitamin D₃ at R_v 52 ml in the same system] as a white solid.

UV (in EtOH) λ max 243.5, 252.5, 262.5 nm; ¹H-NMR (CDCl₃) δ 0.551 (3H, s, 18-H3), 0.858 (3H, d, J = 6.6 Hz, 21-H₃), 1,215 (6H, s, 26- and 27-H₃), 1.95-2.04 (2H, m), 2.27-2.35 (2H, m), 2.58 (1H, dd, J = 13.3, 3.7 Hz) , 2.80-2.87 (2H, m), 4.49 (2H, m, 1β and 3a-H), 5.09 and 5.11 (1H and 1H, each s, = CH₂), 5.89 and 6.36 (1H and 1H, each d, J = 11.3 Hz, 7- and 6-H); MS: m / z (relative intensity) 416 (M +, 100), 398 (26), 380 (13), 366 (21), 313 (31).

(c) Hydrogenation of 2-methylene-19-nor vitamin 17 (20S) -la, 25-dihydroxy-2a and la, 25-dihydroxy-28-methyl19-nor-vitamin P₃ (18 and 19)

Tris (triphenylphosphine) rhodium (I) chloride (2.3 mg, 2.5 pmol) was added to dry benzene (2.5 ml) which was pre-saturated with hydrogen. The mixture was stirred at room temperature until a homogeneous solution was formed (about 45 minutes). A solution of vitamin 17 (1.0 mg, 2.4 pmol) in dry benzene (0.5 ml) was then added and the reaction allowed to proceed under a continuous stream of hydrogen for 3 hours. The benzene was removed in vacuo and hexane / ethyl acetate (1: 1, 2 ml) was added to the residue. The mixture was transferred to a silica-Sep-Pak and both 2-methyl vitamins were eluted with the same solvent system (20 ml). Further purification was achieved by HPLC (6.2 mm x 25 cm Zorbax-Sil column, 4 ml / minute) using hexane / 2-propanol (9: 1) as solvent system. The mixture (about 1: 1) of 2-methyl19-noramines (2α and 2β epimers 18 and 19; 0.43 mg, 43%) gave a single peak at R_v 31 ml.

and 19: UV (in EtOH) 243, 251, 261 nm; ^XH-NMR (CDCl₃) δ 0.534 and 0.546 (3H and 3H, each s, 2x18-H3), 0.852 and 0.857 (3H and 3H, each d, J = 6.5 Hz, 2 x 21-H₃), 1,133 (3H, d, J = 6.7 Hz, 2-CH3), 1.143 (3H, d, J = 6.5 Hz, 2CH₃), 1,214 [12H, s, 2x (26- and 27-H₃) J, 2.60 (1H, dd, J = 12.7, 4.5 Hz), 2.80 (3H, m), 3.08 (1H, dd, J = 13.1,

4.3 Hz), 3.51 (1H, br m; after D₂0 dt, J = 4.5, 10.0 Hz), 3.61 (1H, br m; after D₂O dt, J = 4.4, 9.2 Hz), 3.90 (1H, narrow m), 3.96 (1H, narrow m), 5.82, 5.87, 6.26 and 6 , 37 (each 1 H, each d, J = 11.3 Hz); MS: m / z (relative intensity) 418 (M +, 100), 400 (45), 385 (20), 289 (38), 245 (47).

Biological activity of 2-methyl-substituted 19-nor-1,25 (OH) ₂P3 compounds and their 20S isomers

Introduction of a methyl group to the 2-position of 19-nor1,25- (OH)₂D₃ or its 20S isomer had little or no effect on the binding to the porcine intestinal vitamin D receptor. All compounds were bound equally well to the porcine receptor including the standard 1.25- (OH)₂D₃ (Figure 1). It should be expected from these results that all of these compounds will have equivalent biological activity. But surprisingly, the 2-methyl substitutions gave very selective analogues with their primary effect on bone mass. When given 7 days in chronic mode, the most potent compounds tested were a mixture of the S and R isomers of 2-methyl-19-nor-20S-1,25- (OH)₂D₃ (Table 1). When given at a concentration of 130 pmol / day, the activity of this mixture of compounds on bone calcium mobilization (serum calcium) was much higher than the activity of the native hormone, possibly as high as 10 or 100 times higher. Under identical conditions, twice the dose of 1.25 (OH) gave 2D₃ a serum calcium value of 7.2 mg / 100 ml, while a mixture of 2-methyl- (S and R) -19-nor-20S-1,25- (OH)₂D₃ gave a io value of 9.6 mg / 100 ml of serum calcium at the concentration

130 pmol / dose. When given in a concentration of

260 pmol / day, this mixture gave the astonishing value 12.2 mg / 100 ml of serum calcium at the expense of bone mass. To show selectivity, these compounds produced no significant change in intestinal calcium transport at the 130 pmol dose level and had a strong bone calcium mobilizing activity. At the higher dose, the 2-methyl20S mixture actually produced an intestinal transport response but produced a tremendous bone mobilizing response. A mixture of S- and

The R-isomers of 2-methyl-19-nor-1,25- (011) 2,3 also had strong bone calcium mobilization at both dose levels, but showed no intestinal calcium transport activity. Thus, when given as a mixture, the 2-methyl-S and -R derivatives showed strong preferential bone calcium immobilizing activity, especially when the side chain was in 20S configuration.

These results illustrate that the 2-methyl and 20S-2-methyl derivatives of 19-nor-1,25- (OH) 2D₃ are selective when it comes to mobilizing calcium from bone mass. Table 2 shows the response of both intestinal calcium and serum calcium to a single large dose of the various compounds; which in turn supports the conclusions derived from Table 1.

The results in Figure 2 illustrate that a mixture of the S and R derivatives of 2-methyl-19-nor-20S-1,25- (OH) 2D₃ is extremely potent in inducing differentiation of HL-6035 cells to the monocytes. The 2-methyl-S and -R compounds had a similar activity as 1,25- (OH)₂D₃. These results illustrate the potential of 2-methyl-19-nor-20S-1.25 (ΌΗ)₂D₃-the compounds as anticancer agents, especially against leukemia, colon cancer, breast cancer and prostate cancer, or as agents for the treatment of psoriasis.

Competitive binding of the analogs to the porcine intestinal receptor was performed b and the method described by Dame et al. (Biochemistry, 25, 4523-4534, 1986).

The differentiation of HL-60 promyelocytic cells into monocytes was determined as described by Ostrem et al.

(J. Biol. Chem., 262, 14164-14171, 1987).

Table 1

Intestinal calcium transport activity and serum calcium activity (bone calcium mobilization) in response to chronic doses of the 2-methyl derivatives of 19-nor-25- (OH)₂D₃and its 20S isomers

<td>Group</td><td>Dose (pmol / day / 7 days)</td><td>Intestinal calcium transport (S / M)</td><td>Serum calcium (mg / 100 ml)</td>

<td>Vitamin Deficiency</td><td>vehicle</td><td> 5,5 ± 0,2</td><td> 5,1 ± 0,1'6</td>

<td>1.25- (OH) ₂D₃treated</td><td> 260</td><td> 6,2 ± 0,4</td><td> 7,2 ± 0,5</td>

<td>2-methyl- (S and R) -19-nor1,25- (OH)₂D₃</td><td> 130 260</td><td> 5,0 ± 0,3 5,3 ± 0,6</td><td> 6,1 ± 0,1 6,7 ± 0,4</td>

<td>2-methyl- (S and R) -19-nor-20S1,25- (OH)₂D₃</td><td> 130 260</td><td> 5,0 ± 0,9 6,9 ± 0,5</td><td> 9,6 ± 0,1 12,2 ± 0,3</td>

Pending male rats were obtained from Sprague Dawley Co. (Indianapolis, IN), and they were fed a 0.47% calcium, 0.3% phosphorus-containing vitamin D deficiency diet for 1 week, and then received the same diet containing 0.02% calcium, 0.3% phosphorus for 2 weeks. During the last week, they received the indicated dose of the compound by intraperitoneal injection in 0.1 ml of 95% propylene glycol and 5% ethanol every day for days. The control animals received only 0.1 ml of 95% propylene glycol, 5% ethanol. 24 Hours after the last dose, the rats were sacrificed and intestinal calcium transport was determined by the inverted sac technique, as previously described, and serum calcium was determined by atomic absorption spectrometry on a Perkin Elmer instrument, Model 3110 (Norwalk, CT). There were 5 rats per day. group, and the values ​​represent the mean ± SEM.

Table 2

Intestinal calcium transport activity and serum calcium activity (bone calcium mobilization) in response to a single dose of the 2-methyl derivatives of 19-nor-1 , 25- (OH)₂D₃ and its 20S isomers

<td>Group</td><td>Intestinal calcium transport (S / M)</td><td>Serum calcium (mg / 100 ml)</td>

<td>-D Control</td><td> 4,2 ± 0,3</td><td> 4,7 ± 0,1</td>

<td>1.25- (OH)₂D₃</td><td> 5,8 ± 0,3</td><td> 5,7 ± 0,2</td>

<td>2-methyl (S and R mixture) 19-nor-1,25- (OH)₂D₃</td><td> 3,6 ± 0,4</td><td> 5,4 ± 0,1</td>

<td>2-methyl (S and R mixture) - 19-nor-20S-1,25- (OH) ₂D₃</td><td> 6,7 ± 0,6</td><td> 8,1 ± 0,3</td>

Pending male rats of the Holtzman breed were obtained from Sprague Dawley Co. (Indianapolis, IN), and were fed the 0.47% calcium, 0.3% phosphorus-containing diet described by Suda et al. (J. Nutr., 100, 1049-1052, 1970) for 1 week, and then received the same diet containing 0.02% calcium and 0.3% phosphorus for a further 2 weeks. At this point, they received a single intrajugular injection of the indicated dose dissolved in 0.1 ml of 95% propylene glycol / 5% ethanol. 24 Hours later, they were sacrificed and intestinal calcium transport and serum calcium were determined as described in Table 1. The dose of the compounds was 650 pmol and there were 5 animals per day. group. The data are expressed as mean ± SEM.

For use in therapeutic treatment, the novel compounds of this invention, defined by formula I, may be formulated pharmaceutically as a solution in non-hazardous solvents, or as an emulsion, suspension or dispersion in suitable solvents or carriers, or as pills, tablets or capsules. , together with solid carriers, according to conventional methods known in the art. Any such formulation may also contain other pharmaceutically acceptable and non-toxic excipients, such as stabilizers, antioxidants, binders, dyes or emulsifying or flavor modifiers.

The compounds may be administered orally, topically, parenterally or transdermally. The compounds are advantageously administered by injection or by intravenous infusion or suitable sterile solutions, either in the form of liquid or solid doses via the digestive tract, or in the form of c.reams, ointments, patches or similar vehicles suitable for transdermal applications. Doses ranging from 0.1 µg to 50 µg per day. The day of the compounds is appropriate for treatment purposes, and such dosages are adjusted according to the disease to be treated, its severity and the patient's response, as is well known in the art. Since the new compounds exert specificity in their effect, each of them may advantageously be administered alone or together with graduated doses of another active vitamin D compound - e.g. 1-hydroxyvitamin D₂ or D₃, or la, 25-dihydroxyvitamin D₃ - in situations where different degrees of bone mineral mobilization and calcium transport stimulation have been found to be advantageous.

Compositions for use in the above treatment of psoriasis and other malignant diseases comprise an effective amount of one or more 2-substituted 19-nor-vitamin D compounds, as defined by the above formula, as active ingredient, and a suitable carrier. An effective amount of such compounds for use in accordance with this invention is from ca. 0.01 pg to approx. 100 pg per day. grams of the composition, and it may be administered topically, transdermally, orally or parenterally in doses ranging from ca. 0.1 pg / day to approx. 100 pg / day.

The compounds can be formulated as creams, lotions, ointments, topical patches, pills, capsules or tablets, or in liquid form as solutions, emulsions, dispersions or suspensions in pharmaceutically harmless and acceptable solvents or oils, and such preparations may additionally contain other pharmaceutically harmless or beneficial components, such as stabilizers, antioxidants, emulsifiers, dyes, binders, or flavor modifiers.

The compounds are advantageously administered in amounts sufficient to cause differentiation of promyelocytes to normal macrophages. The dosages described above are appropriate, but it should be understood that the amounts indicated can be adjusted according to the severity of the disease and the condition and response of the patient, as is well known in the art.

The formulations of the present invention comprise an active ingredient in admixture with a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The carrier must be acceptable in the sense that it is compatible with the other ingredients of the formulation and not harmful to the recipient.

Formulations of the present invention suitable for oral administration may be in the form of separate units such as capsules, odor bags, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion.

Rectal administration formulations may be in the form of a suppository comprising the active ingredient and a carrier, such as cocoa inserter, or in the form of an enema.

Formulations suitable for parenteral administration advantageously comprise a sterile, oily or aqueous preparation of the active ingredient which is preferably isotonic with the patient's blood.

Formulations suitable for topical administration include liquid or semi-liquid preparations such as io liniments, lotions, applicators, oil-in-water or water-in-oil emulsions, such as creams, ointments or pastes; or solutions or suspensions, such as drops; or as sprays.

For asthma treatment, inhalation of powdery, self-propelled or spray formulations released with a spray box, atomizer or atomizing device can be used. The formulations, when released, preferably have a particle size in the range 10-100 µ.

The formulations may advantageously be in dosage unit form and may be prepared by any method well known in the pharmaceutical art. By the term dosage unit is meant a unitary, that is, a single dose that can be administered to a patient as a physically or chemically stable unit dose comprising either the active ingredient as such or a mixture of it with solid or liquid pharmaceutical diluents or carriers.

Schedule I

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Schedule II

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